Novel bradykinin-1 antagonists containing a (1,2,3,4-tetrahydro-isoquinolin-1-yl)acetic acid scaffold

Article abstract of DOI:10.1016/j.ejmech.2007.10.030

A novel B₁ antagonist core was utilized and the effects of modification of its amide side chain on the biological activity were tested. The imino functional group of isoquinolin-1-ylacetic acid and its 6,7-dimethoxy variant was sulfonylated (4-toluenesulfonyl), while the acetyl side chain was converted to amides. Three of the synthesized compounds exhibited significant activity at the recombinant human B₁ receptors in binding tests and also in a functional assay.

Full text of DOI:10.1016/j.ejmech.2007.10.030

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1552e1558
http://www.elsevier.com/locate/ejmech
Preliminary communication
Novel bradykinin-1 antagonists containing a (1,2,3,4-
tetrahydro-isoquinolin-1-yl)acetic acid scaffold
a
Jozsef Huszar , Zoltan Timar , Krisztina Katalin Szalai ,
a,
b
*
Gyorgy Keseru , Ferenc Fulop , Botond Penke
´
´
´
´
b
c
a
}
¨
¨
¨
a Department of Medical Chemistry, University of Szeged, Do´m te´r 8., Szeged H-6725, Hungary
b Pharmacological and Drug Safety Research, Richter Gedeon Plc., Gyo¨mrTi u´t 19-21., Budapest, Hungary
c Institute of Pharmaceutical Chemistry, University of Szeged, Eo¨tvo¨s utca 6., Szeged, Hungary
Received 11 April 2007; received in revised form 18 October 2007; accepted 29 October 2007
Available online 4 November 2007
Abstract
A novel B1 antagonist core was utilized and the effects of modification of its amide side chain on the biological activity were tested. The
imino functional group of isoquinolin-1-ylacetic acid and its 6,7-dimethoxy variant was sulfonylated (4-toluenesulfonyl), while the acetyl
side chain was converted to amides. Three of the synthesized compounds exhibited significant activity at the recombinant human B1 receptors
in binding tests and also in a functional assay.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Bradykinin-1 receptor; B1 antagonist; Bradykinin; Isoquinoline; Peptidomimetic
1. Introduction
several modes of physiological action [8e10]. They are rela-
tively selective endogenous agonists of B2 receptors and are
known to mediate acute inflammatory pain responses. The
C-terminally truncated des-Arg carboxypeptidase metabolites
of BK and Lys-BK, des-Arg9-BK and des-Arg10-Lys-BK are
selective ligands at the B1 receptors; they are generally absent
in normal tissues, but are induced locally following inflamma-
tion, tissue injury, trauma or stimulation [11,12]. The role of
the particularly important BK B1 receptors (in addition to
the B2 receptors) in the chronic phase of pain responses is
associated with inflammatory conditions [6,13e15].
Accordingly, there is great interest in investigations of the
pathophysiological roles of BK B1 receptors (and ligands)
and in the development of potent, selective and orally bioavail-
able bradykinin B1 antagonists, initially peptides [9,16], and
later non-peptide analogues [17e22] as a novel class of anti-
nociceptive and antiinflammatory drugs [2].
A large number of people suffer from unnecessary persis-
tent pain because, although really effective analgetics (typi-
cally non-steroidal antiinflammatory drugs and opioids) are
available, they cause serious side-effects [1].
However, the recently discovered biological effects of oli-
gopeptide hormones (e.g. kinins), modified peptides [2,3],
pseudopeptides [4,5] and peptidomimetics with novel mecha-
nisms of action might be utilized to counter chronic pain.
In humans, two endogenous peptides are classified as
kinins: bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-
Phe8-Arg9) and kallidin (Lys-BK; Lys1-Arg2-Pro3-Pro4-Gly5-
Phe6-Ser7-Pro8-Phe9-Arg10), which are produced by the
proteolytic cleavage of high- and low-molecular weight kini-
nogen precursor proteins, respectively, by plasma or tissue ser-
ine proteases named kallikrein enzymes [6,7]. Kinins exert
Novel BK1 receptor antagonists containing rigid core mol-
ecules such as benzodiazepine were earlier revealed to demon-
strate in vivo efficacy comparable to that of morphine for the
suppression of hyperalgesia [22,23]. Optimization of a series
* Corresponding author. Tel.: þ36 62 546831; fax:þ36 62 546826.
´
E-mail address: ztimar@yahoo.com (Z. Timar).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.030

J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
1553
of B1 antagonists with 2,3-diaminopyridine [24] or 2-alkyla-
mino-5-sulfamoylbenzamide [1] cores has been reported.
Incorporation of piperidine-3-carboxylic acid and piperidine-
4-carboxylic acid spacers and the use of morpholine and piper-
azine derivatives led to an increased receptor-binding affinity
and oral absorption in animal models [1].
A highly potent and selective member of a new generation
of arylsulfonamide dihydroquinoxalinone B1 receptor antago-
nists has also been reported and characterized [17,23,25,26].
There were primary amines with flexible alkyl linkers of
different lengths. A guanidine-containing alkyl chain was
also incorporated, as was an imidazoline-terminal apolar one
containing a phenyl group [17].
The racemic analogues were synthesized by simple chemi-
cal substitutions. Both THIQAA and its 6,7-dimethoxy variant
are b-amino acids. The tosylate moiety was introduced into
both the molecules first. The carboxylic acid function was
then reacted with 5 different amines: cycloheptylamine, 1,4-
diaminobutane, 1,4-diaminohexane, agmatine and 2-[4-(4,5-
dihydro-1H-imidazol-2-yl)phenyl]ethylamine. In each series,
one compound contained only the cycloheptylamine group,
while three compounds included flexible alkyl chains of differ-
ent lengths and a basic side-chain terminal (1,4-diaminobutane,
1,4-diaminohexane and agmatine). The dimethoxy derivative is
rather rigid relative to the other amines used. All racemic
products were purified by preparative HPLC, and appropriate
fractions (>95%) were co-freeze-dried. Amines were gained
as trifluoroacetate salts. Lyophilised products were tested in
biological assays without desalting or further purification
(Scheme 2, Table 1).
The data in Table 1 allow the following conclusions. Mod-
ification of the core with methoxy groups decreased the bio-
logical activity. Incorporation of a cyclohexyl amide side
chain resulted in inactive molecule. Amides of primary alkyl-
amines did not display significant activity. The alkyl guanidino
group increased the inhibitory potency of the molecule in the
[Ca2þ]i assay, but the activity remained low. Significant activ-
ity was observed only for the phenylimidazoline side chain.
The positive effect of this key moiety was also observed
with the dimethoxy core 14, although the activity was still al-
most 2 orders of magnitude lower than that of THIQAA 9.
The imidazoline-containing side chain with the phenyl
group may interact with apolar and aromatic amino acid side
chains and is also a linear spacer that may keep the basic group
in a position suitable for high affinity binding. Moreover, this
side chain is the most rigid (conformationally most restricted)
of our set. The positive effect of the non-basic cycloheptyla-
mide on binding, as compared with the alkyl primary amine
chains, suggests that the role of a bulky apolar part in the
spacer may be important. The primary amine-containing li-
gands are flexible and might bend back on the core. On the
2. Results and discussion
Our goal was to design and synthesize a series of non-
peptide bradykinin antagonists, using derivatives of the
commercially available racemic (1,2,3,4-tetrahydroisoquino-
lin-1-yl)acetic acid (THIQAA) core. THIQAA is already men-
tioned in a patent application [27] as a potential B1 antagonist,
but it has not been utilized as a B1 key moiety.
We performed a comparative analysis of a broad set of B1
antagonists, that has indicated, that the phenylsulfonamide
moiety plays a key role in the binding and in the in vivo anti-
nociceptive efficacy. Thus, we decided to use the THIQAA
core, sulfonylated on its nitrogen atom. The 4-toluenesulfonyl
group was used to form the aromatic sulfonamide moiety.
Changes were made in the THIQAA core at positions 6 and
7, by the insertion of methoxy groups, and in the amide side
chain (Scheme 1).
The first set of synthesized molecules, containing both the
THIQAA and its 6,7-dimethoxy variant as holding scaffold for
the sulfonamide and the basic side chain, included several
inactive compounds. These contained the tosyl (4-toluenesul-
fonyl) sulfonamide moiety and different amides bound to the
carboxyl functional group. There were apolar amides (cyclo-
heptyl) and amides with slightly basic terminal primary amino
groups, on alkyl spacers of different lengths, which had low
activity in the 5 mM [Ca2þ]i assay (several examined exam-
ples, with no activity at all, are not presented in this report).
However, when highly basic amides were applied (pKa of
the amine part >10), significant improvements were observed
in the biological tests. Thus, a set of bifunctional basic amines
were chosen and were used for conversion to the amide moiety
of the THIQAA-containing B1 antagonists.
core modification
R1
R1=H or OMe
R1
amide side-chain
R2=cycloheptyl or basic
O
O
N
NH
R2
N
H
OH
O2S
THIQAA core
sulfonamide
Scheme 1. The THIQAA core and its modifications.

1554
J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
R1 R1
R1
R1
R1
R1
O
O
O
NaHCO3, Tos-Cl
OH
a then b
N
S
OH
N
S
N
H
nNH2
N
H
O
O
O
O
1 R1= H
2 R1=OMe
3 R1= H
4 R1=OMe
6 R1= H, n=4
7 R1= H, n=6
11 R1= OMe, n=4
12 R1= OMe, n=6
d
e
c
R1
R1
R1
R1
R1
O
R1
O
N
N
H
O
H
N
NH
2
N
S
N
N
S
N
H
N
S
N
H
H
O
O
O
O
NH
O
O
5 R1= H
8 R1= H
9 R1=H
10 R1= OMe
13 R1= OMe
14 R1= OMe
Scheme 2. Synthesis of B1 antagonists: (a) DCC/HOBt, (4-aminobutyl)carbamic acid tert-butyl ester or (6-aminohexyl)carbamic acid tert-butyl ester in DMF;
(b) TFA/DCM; (c) DCC/HOBt, cycloheptylamine in DMF; (d) HBTU/HOBt/DIEA in DMF (agmatine was used dissolved in aq. NaHCO3); (e) HBTU/HOBt/
DIEA, 2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethylamine in DMF.
other hand, a more rigid side chain evidently favours long-
distance binding without significant loss of entropy. Further
investigations are under way to prove our hypotheses.
USA). The human B1 receptor-expressing cell line was pur-
chased from Euroscreen (Belgium). For the [Ca2þ]i measure-
ments, we used Chinese hamster ovary (CHO) cells stably
expressing recombinant human B1 receptors. Cells were cul-
tured in Dulbecco’s modified Eagle medium containing 10%
foetal bovine serum, 1% MEM non-essential amino acid solu-
tion, 400 mg/cm3 G418 and antibiotics (0.25 mg/cm3 amphoter-
icin B, 100 U/cm3 penicillin G, and 100 mg/cm3 streptomycin).
Cells were kept at 37 ꢀC in a humidified atmosphere of 5%
CO2/95% air. They were plated into 96-well microplates at
a density of 2.5 ꢁ 104 cells/well and measurements of
[Ca2þ]i were carried out 1 day after cell plating. After removal
of the medium, the cells were loaded with a Ca2þ-sensitive
fluorescent dye, fluo-4 (2 mM), for 40e120 min in the follow-
ing buffer: 2 mM MgCl2, 140 mM NaCl, 5 mM KCl, 5 mM
HEPESeNa, 5 mM HEPES, 2 mM CaCl2, 20 mM D-glucose,
2 mM probenecid. To remove excess dye, the cells were
washed twice with buffer. After washing, the test compounds
were added and the cells were incubated for 20e25 min at
37 ꢀC. The test compounds were diluted in buffer from
a DMSO stock solution; the final DMSO concentration was
<0.1%. After the incubation period, the baseline and the ago-
nist-evoked fluorescence changes were monitored with a plate
reader fluorometer (Fluoroskan Ascent, Thermo-Labsystems,
Finland). The agonist was [Lys-des-Arg9]bradykinin applied
at EC80 concentration. Excitation was at 485 nm, and the fluo-
rescence emission was detected at 538 nm. The whole mea-
surement process was performed at 37 ꢀC and was controlled
3. Conclusions
B1 antagonists containing the THIQAA core and its 6,7-di-
methoxy derivative were synthesized. The 6,7-methoxy substi-
tution was found to lower the receptor-binding affinity
markedly relative to the unsubstituted core. The best Ki ob-
tained was 0.067 mM for the THIQAA core-containing com-
pound and 1.996 mM for the 6,7-dimethoxy compound with
the same substituents. Although the number of examined ef-
fective THIQAA-based BK1 antagonists was not too high,
one preliminary conclusion may be drawn from the results.
The linear, rigid apolar or rather aromatic spacer with a phenyl
group in the amide side chain bearing an imidazolidine basic
terminal group may be important in the recognition of the
B1 antagonist by the BK B1 receptor. Further investigations
on the THIQAA core are under way.
4. Experimental protocols
4.1. Fluorometric measurement of
cytoplasmic Ca2þ ([Ca2þ]i)
All materials and cell culture reagents were from Invitrogen
(Carlsbad, CA, USA) or SigmaeAldrich (St. Louis, MO,

J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
1555
Table 1
Biological assay results on Chinese hamster ovary cells expressing human B1 receptors (CHO-hB1)
R1
R1
O
R2
N
S
N
H
O
O
Compd
R1
H
R2
[Ca2þ]i assay CHO-hB1, Inhibition (%) (at 5 mM)
Binding assay, CHO-hB1 Ki (mM)
5
37.0
e
NH2
6
7
H
H
15.9
16.3
e
e
NH2
H
N
NH2
NH
8
H
44.9
99.0
9.3
e
H
N
9
H
0.067
e
N
10
OMe
NH2
11
12
OMe
OMe
15.3
10.9
e
e
NH2
H
N
NH2
13
14
OMe
OMe
19.0
85.7
e
NH
H
N
1.996
N
by custom software. Raw fluorescence data were expressed as
DF/F values (fluorescence changes normalized to the baseline).
The inhibitory effects of the test compounds were expressed as
percentage inhibition of the control agonist response. Data
analysis was carried out with Microsoft Excel.
homogenized in a glass homogenizer. The crude membrane
fraction was collected through two consecutive centrifugation
steps at 40,000g for 25 min at 4 ꢀC separated by a washing
step in the above buffer. The final pellet was resuspended in
the buffer 75 mM TriseHCl (pH 7.5), 12.5 mM MgCl2,
0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose. The membrane
was divided into aliquots, flash-frozen and stored at ꢂ80 ꢀC
until use. Protein concentrations were determined with the
BCA method.
4.2. Human recombinant bradykinin-1
receptor-binding assay
4.2.1. Preparation of membrane
Membrane was prepared from hB1-A5 cells (expressed in
CHO cells) according to the Euroscreen Technical Data Sheet
(Cat. No.: ES-091). The cells were dissociated in Dulbecco’s
phosphate-buffered saline and centrifuged (1500 rpm, 10 min,
4 ꢀC). The pellet was resuspended in 15 mM TriseHCl (pH
7.5), 2 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, and
4.2.2. Binding conditions
Binding assays were performed in triplicate in DeepWell
plates containing the binding buffer [50 mM TriseHCl (pH
7.4), 5 mM MgCl2], hB1 membrane (20 mg protein/tube),
and [3H]Lys-BK (Perkin Elmer Life Sciences) as radioligand.
Non-specific binding was determined in the presence of

1556
J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
10 mM Lys-des-Arg9-BK. The samples were incubated in a fi-
nal volume of 0.25 cm3 for 15 min at 25 ꢀC. Binding reactions
were terminated by rapid filtration through a 96-well GF/B
Unifilter presoaked for at least 1 h in 0.5% PEI. The filters
were washed four times with 1 cm3 of ice-cold washing buffer
(same composition as the binding buffer), using a Brandel har-
vester. The filters were allowed to dry, Perkin Elmer Micro-
scint 20 scintillant was added (20 ml/well) and the bound
radioactivity was determined with a Packard TopCount scintil-
lation counter for 3 min. Data were analysed with Prism,
Graph Pad Software.
(1040 mg, 75%). MS (APCI) Calcd for C18H19NO4S: m/z
1
345.41 (Mþ). Found: m/z 345.97 (Mþ). H NMR (500 MHz,
DMSO-d6) d 7.6 (2H, d, J ¼ 8 Hz), 7.25 (2H, d, J ¼ 8 Hz),
7.15 (1H, m), 7.09 (2H, m), 6.97 (1H, m), 5.33 (1H, t,
J ¼ 7 Hz), 3.63 (2H, m), 3.49 (2H, m), 2.6 (2H, m), 2.3 (3H, s).
4.3.2. [6,7-Dimethoxy-2-(toluene-4-sulfonyl)-
1,2,3,4-tetrahydroisoquinolin-1-yl]acetic acid 4
Compound 4 (1.035 g, 63%) was prepared from 2 accord-
ing to the method for the preparation of 3. MS (ESI) Calcd
for C20H23NO6S: m/z 406.46 (M þ Hþ). Found: m/z 409.02
(M þ Hþ). 1H NMR (500 MHz, DMSO-d6) d 7.61 (2H, d,
J ¼ 8 Hz), 7.26 (2H, d, J ¼ 8 Hz), 6.75 (1H, s), 6.52 (1H, s),
5.28 (1H, t, J ¼ 7 Hz), 3.68 (3H, s), 3.66 (2H, m), 3.64 (3H,
s), 3.42 (2H, m), 2.64 (2H, t, J ¼ 6 Hz), 2.31 (3H, s).
4.3. Chemistry
Commercial chemicals and solvents were of reagent grade
and used without further purification. The following abbre-
viations are used: DCC, N,N0-dicyclohexylcarbodiimide;
DCM, dichloromethane; DIEA, ethyldiisopropylamine; DCU,
N,N0-dicyclohexylurea; DMF, dimethyl formamide; HBTU,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate; HOBt, 1-hydroxybenzotriazole; and TFA, trifluoro-
acetic acid. Preparative reverse-phase HPLC was performed
on a Shimadzu SPD-6AV 3000 liquid chromatograph, using
a C18 reverse-phase silica gel column (Phenomenex, Jupiter
4.3.3. N-Cycloheptyl-2-[1-(toluene-4-sulfonyl)-
1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide 5
To a solution of [1-(toluene-4-sulfonyl)-1,2,3,4-tetrahydroi-
soquinolin-2-yl]acetic acid (3, 100 mg, 0.29 mmol) in DMF
(1 cm3), DCC (72 mg, 0.35 mmol) and HOBt (50 mg,
0.35 mmol) were added, the reaction mixture was stirred for
30 min, and DCU was then filtered off. Cycloheptylamine
(44 ml, 0.35 mmol) was next added to the solution, which
was subsequently stirred for 4 h at room temperature. DMF
was removed under vacuum and the residue was dissolved in
ethyl acetate (20 cm3), washed three times with saturated
NaHSO4, and with saturated NaHCO3 and once with brine,
dried with Na2SO4, filtered and concentrated. The crude resi-
due was purified by HPLC to give 5 (79 mg, 62%). HRMS
(EI) Calcd for C25H32N2O3S: m/z 441.2212 (M þ Hþ). Found:
˚
250 ꢁ 21.2 mm, 15 m, 300 A). Eluent: acetonitrile (20e100%,
v/v) in water containing 0.1% (v/v) TFA. Peak detection was
at 254 nm. NMR: with a Bruker Avance DRX 500 Spectrometer
(1H: 500.13 MHz), CDCl3 or DMSO-d6 solutions, d (ppm), J
(Hz). Spectral patterns: s, singlet; d, doublet; dd, double
doublet; t, triplet; m, multiplet; br, broad. Mass spectra (MS)
were measured on Bruker Esquire 3000þ and Waters Q-Tof
Premier instruments. Low-resolution MS were acquired on
a Bruker Esquire 3000þ ion trap mass spectrometer, equipped
with an electrospray ionization source. High-resolution exact
mass measurements were performed on a hybrid, quadrupole-
orthogonal acceleration time-of-flight mass spectrometer
(Waters Q-Tof Premier), equipped with an electrospray ioniza-
tion source. Samples were dissolved in acetonitrile:water ¼ 1:1,
0.1% acetic acid (v/v) solvent mixture. High-resolution experi-
ments were performed in the presence of 0.2 ng/ml leucine
enkephalin peptide as lock mass calibration standard. Chemical
yields were not optimized.
1
m/z 441.2224 (M þ Hþ). H NMR (500 MHz, CDCl3) d 7.61
(2H, d, J ¼ 8 Hz), 7.09 (5H, m), 6.88 (1H, d, J ¼ 8 Hz), 6.13
(1H, d, J ¼ 7 Hz), 5.38 (1H, d, J ¼ 7 Hz), 3.96 (1H, m), 3.8
(1H, m), 3.52 (1H, m), 2.81 (1H, dd, J ¼ 8 Hz), 2.61 (1H, d,
J ¼ 6 Hz), 2.57 (2H, m), 2.32 (3H, s), 1.94 (2H, m), 1.61
(4H, m), 1.44 (6H, m).
4.3.4. N-(4-Aminobutyl)-2-[1-(toluene-4-sulfonyl)-1,2,3,4-
tetrahydro-isoquinolin-2-yl]acetamide 6
To a solution of [1-(toluene-4-sulfonyl)-1,2,3,4-tetrahydroi-
soquinolin-2-yl]acetic acid (3, 75 mg, 0.22 mmol) in DMF
(1 cm3), DCC (54 mg, 0.26 mmol) and HOBT (35 mg,
0.26 mmol) were added, the reaction mixture was stirred for
30 min, and DCU was then filtered off. (4-Aminobutyl)carba-
mic acid tert-butyl ester (62 mg, 0.33 mmol) was added to the
solution, which was next stirred for 4 h at room temperature.
DMF was removed under vacuum and the residue was dis-
solved in ethyl acetate (20 cm3), washed three times with sat-
urated NaHSO4, NaHCO3 and brine, dried over Na2SO4,
filtered and concentrated. The crude residue was dissolved in
50% TFA/DCM (5 cm3), the reaction mixture was stirred for
20 min at room temperature and the solvent was then removed.
The crude product was purified by HPLC to give 6 (39 mg,
43%). HRMS (EI) Calcd for C22H29N3O3S: m/z 416.2008
(M þ Hþ). Found: m/z 416.2021 (M þ Hþ). 1H NMR
(500 MHz, DMSO-d6) d 7.87 (1H, m), 7.73 (3H, m), 7.59
4.3.1. [1-(Toluene-4-sulfonyl)-1,2,3,4-
tetrahydroisoquinolin-2-yl]acetic acid 3
NaHCO3 (672 mg, 8 mmol) was added to a solution of
(1,2,3,4-tetrahydroisoquinolin-2-yl)acetic acid (1, 765 mg,
4 mmol) in distilled water (3 cm3), followed by the dropwise
addition of 4-methylbenzenesulfonyl chloride (915 mg,
4.8 mmol) in acetone (5 cm3) and the reaction mixture was
then stirred for 4 h at room temperature at pH 8. Acetone
was removed under vacuum and the residue was extracted
with NaHCO3/diethyl ether (5 cm3). The basic solution was
acidified with solid NaHSO4 to pH 4e5 and extracted with
ethyl acetate (3 ꢁ 10 cm3). The combined organic phases
were dried, filtered and concentrated. The crude product was
crystallized from ethyl acetate/diethyl ether and gave 3

J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
1557
1
(2H, d, J ¼ 8 Hz), 7.25 (2H, d, J ¼ 8 Hz), 7.08 (3H, m), 6.97
(1H, m), 5.35 (1H, t, J ¼ 7 Hz), 3.66 (2H, m), 2.98 (2H, m),
2.76 (2H, m), 2.63 (2H, m), 2.5 (2H, m), 2.29 (3H, s), 1.48
(2H, m), 1.37 (2H, m).
Found: m/z 517.2265 (M þ Hþ). H NMR (500 MHz, CDCl3)
d 10.46 (2H, s), 7.96 (1H, t, J ¼ 7 Hz), 7.84 (2H, d, J ¼ 8 Hz),
7.61 (2H, d, J ¼ 8 Hz), 7.45 (2H, d, J ¼ 8 Hz), 7.26 (2H, d,
J ¼ 8 Hz), 7.1 (3H, m), 7.0 (1H, t, J ¼ 5 Hz), 5.37 (1H,
t, J ¼ 7 Hz), 4.01 (4H, s), 3.69 (2H, m), 3.5 (2H, m), 3.27
(2H, t, J ¼ 4 Hz), 2.77 (2H, m), 2.66 (2H, m), 2.22 (3H, s).
4.3.5. N-(6-Aminohexyl)-2-[1-(toluene-4-sulfonyl)-1,2,3,4-
tetrahydroisoquinolin-2-yl]acetamide 7
Compound 7 (28 mg, 29%) was prepared from 3 according
to the method for the preparation of 6 using (4-aminohexyl)-
carbamic acid tert-butyl ester instead of (4-aminobutyl)carba-
mic acid tert-butyl ester. HRMS (EI) Calcd for C24H33N3O3S:
m/z 444.2321 (M þ Hþ). Found: m/z 444.2306 (M þ Hþ).
1H NMR (500 MHz, DMSO-d6) d 7.77 (1H, m), 7.59 (2H,
d, J ¼ 8 Hz), 7.25 (2H, d, J ¼ 8 Hz), 7.09 (3H, m), 6.98 (1H,
m), 5.35 (1H, t, J ¼ 7 Hz), 3.66 (2H, m), 3.00 (1H, m), 2.92
(1H, m), 2.76 (2H, m), 2.65 (2H, m), 2.3 (3H, s), 1.5 (2H,
m), 1.26 (5H, m), 1.16 (3H, m) (NHþ3 signal is likely under
the water peak).
4.3.8. N-Cycloheptyl-2-[6,7-dimethoxy-1-(toluene-4-
sulfonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide 10
Compound 10 (81 mg, 44%) was prepared from 4 accord-
ing to the method for the preparation of 5. HRMS (EI) Calcd
for C27H36N2O5S: m/z 501.2423 (M þ Hþ). Found: m/z
501.2426 (M þ Hþ). 1H NMR (500 MHz, CDCl3) d 7.61
(2H, d, J ¼ 8 Hz), 7.13 (2H, d, J ¼ 8 Hz), 6.62 (1H, s), 6.38
(1H, d, J ¼ 8 Hz), 6.32 (1H, s), 5.32 (1H, t, J ¼ 5 Hz), 3.97
(2H, m), 3.87 (3H, s), 3.83 (3H, s), 3.46 (1H, m), 2.82 (1H,
dd, J ¼ 8 Hz), 2.65 (1H, dd, J ¼ 5 Hz), 2.44 (2H, m), 2.34
(3H, s), 1.94 (2H, m), 1.61 (4H, m), 1.51 (6H, m).
4.3.6. N-(4-Guanidinobutyl)-2-[1-(toluene-4-sulfonyl)-
1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide 8
4.3.9. N-(4-Aminobutyl)-2-[6,7-dimethoxy-2-(toluene-
To a solution of [1-(toluene-4-sulfonyl)-1,2,3,4-tetrahydroi-
soquinolin-2-yl]acetic acid (3, 150 mg, 0.43 mmol) in DMF
(2 cm3), HBTU (330 mg, 0.86 mmol), HOBt (117 mg,
0.86 mmol) and DIEA (139 ml, 0.86 mmol) were added. The
solution was next added dropwise to a solution of agmatine sul-
fate (200 mg, 0.86 mmol) in aqueous NaHCO3 (147 mg,
1.72 mmol in 2 cm3 water), and the reaction mixture was
then stirred at room temperature overnight. The solvents
were removed under vacuum and the residue was dissolved
in ethyl acetate (20 cm3), washed three times with saturated
NaHSO4, NaHCO3 and brine, dried over Na2SO4, filtered and
concentrated. The crude residue was purified by HPLC to
give 8 (98 mg, 45%). HRMS (EI) Calcd for C23H31N5O3S:
m/z 458.2226 (M þ Hþ). Found: m/z 458.2229 (M þ Hþ).
1H NMR (500 MHz, DMSO-d6) d 7.83 (1H, t, J ¼ 6 Hz),
7.59 (2H, d, J ¼ 8 Hz), 7.49 (1H, t, J ¼ 5 Hz), 7.25 (2H, d,
J ¼ 8 Hz), 7.08 (3H, m), 6.97 (1H, t, J ¼ 4 Hz), 6.9 (4H, m),
5.35 (1H, t, J ¼ 7 Hz), 3.65 (2H, m), 3.05 (2H, m), 2.95 (2H,
m), 2.65 (2H, m), 2.5 (2H, m), 2.29 (3H, s), 1.36 (4H, m).
4-sulfonyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]acetamide 11
Compound 11 (44 mg, 25%) was prepared from 4 accord-
ing to the method for the preparation of 6. HRMS (EI) Calcd
for C24H33N3O5S: m/z 476.2219 (M þ Hþ). Found: m/z
1
476.2217 (M þ Hþ). H NMR (500 MHz, DMSO-d6) d 7.89
(1H, t, J ¼ 5 Hz), 7.73 (3H, m), 7.58 (2H, d, J ¼ 8 Hz), 7.26
(2H, d, J ¼ 8 Hz), 6.66 (1H, s), 6.52 (1H, s), 5.28 (1H, t,
J ¼ 7 Hz), 3.66 (3H, s), 3.64 (3H, s), 3.5 (2H, m), 3.43 (2H,
m), 2.98 (2H, m), 2.76 (2H, m), 2.52 (2H, m), 2.3 (3H, s),
1.47 (2H, m), 1.38 (2H, m).
4.3.10. N-(6-Aminohexyl)-2-[6,7-dimethoxy-2-(toluene-4-
sulfonyl)-1,2,3,4-tetrahydroisoquinolin-1-yl] acetamide 12
Compound 12 (50 mg, 27%) was prepared from 4 accord-
ing to the method for the preparation of 7. HRMS (EI) Calcd
for C26H37N3O5S: m/z 504.2532 (M þ Hþ). Found: m/z
1
504.2533 (M þ Hþ). H NMR (500 MHz, DMSO-d6) d 7.78
(3H, m), 7.59 (2H, d, J ¼ 8 Hz), 7.26 (2H, d, J ¼ 8 Hz), 6.65
(1H, s), 6.53 (1H, s), 5.28 (1H, t, J ¼ 7 Hz), 3.67 (5H, m),
3.64 (3H, s), 3.38 (2H, m), 2.97 (2H, m), 2.75 (2H, m), 2.53
(2H, m), 2.31 (3H, s), 1.49 (2H, m), 1.17 (6H, m).
4.3.7. N-{2-[4-(4,5-Dihydro-1H-imidazol-
2-yl)phenyl]ethyl}-2-[1-(toluene-4-sulfonyl)-
1,2,3,4-tetrahydroisoquinolin-2-yl]acetamide 9
To a solution of [1-(toluene-4-sulfonyl)-1,2,3,4-tetrahydroi-
soquinolin-2-yl]acetic acid (3, 138 mg, 0.40 mmol) in DMF
(1 cm3), HBTU (303 mg, 0.8 mmol), HOBt (108 mg,
0.80 mmol) and DIEA (128 ml, 0.80 mmol) were added and the
reaction mixture was stirred for 15 min. 2-[4-(4,5-Dihydro-1
H-imidazol-2-yl)phenyl]ethylamine dihydrochloride (126 mg,
0.48 mmol) was then added, and the solution was stirred for
4 h at room temperature. DMF was removed under vacuum
and the residue was dissolved in ethyl acetate (20 cm3), washed
three times with saturated NaHSO4, saturated NaHCO3 and once
with brine, dried over Na2SO4, filtered and concentrated. The
crude residue was purified by HPLC to give 9 (60 mg, 29%).
HRMS (EI) Calcd for C29H32N4O3S: m/z 517.2273 (M þ Hþ).
4.3.11. 2-[6,7-Dimethoxy-2-(toluene-4-sulfonyl)-1,2,3,
4-tetrahydroisoquinolin-1-yl]-N-(4-guanidinobutyl)
acetamide 13
Compound 13 (71 mg, 32%) was prepared from 4 accord-
ing to the method for the preparation of 8. HRMS (EI) Calcd
for C25H35N5O5S: m/z 518.2437 (M þ Hþ). Found: m/z
518.2437 (M þ Hþ). 1H NMR (500 MHz, DMSO-d6) 7.85
(1H, t, J ¼ 6 Hz), 7.59 (2H, d, J ¼ 8 Hz), 7.48 (1H, t,
J ¼ 5 Hz), 7.26 (2H, d, J ¼ 8 Hz), 6.9 (4H, m), 6.65 (1H, s),
6.52 (1H, s), 5.28 (1H, t, J ¼ 7 Hz), 3.69 (2H, m), 3.66 (3H,
s), 3.64 (3H, s), 3.42 (2H, m), 3.06 (2H, m), 3.00 (2H, m),
2.5 (2H, m), 2.3 (3H, s), 1.41 (2H, m), 1.36 (2H, m).

1558
J. Husza´r et al. / European Journal of Medicinal Chemistry 43 (2008) 1552e1558
[11] F. Marceau, J.F. Hess, D.R. Bachvarov, Pharmacol. Rev. 50 (1998) 357e
386.
4.3.12. N-{2-[4-(4,5-Dihydro-1H-pyrrol-2-yl)phenyl]ethyl}-
2-[6,7-dimethoxy-2-(toluene-4-sulfonyl)-1,2,3,4-
tetrahydroisoquinolin-1-yl] acetamide 14
[12] M. Siebeck, M. Schorr, E. Spannagl, M. Lehner, H. Fritz, J.C. Cheronis,
E.T. Whalley, Immunopharmacology 40 (1998) 49e55.
Compound 14 (36 mg, 29%) was prepared from 4 accord-
ing to the method for the preparation of 9. HRMS (EI) Calcd
for C31H36N4O5S: m/z 577.2485 (M þ Hþ). Found: m/z
577.2488 (M þ Hþ). 1H NMR (500 MHz, DMSO-d6)
d 10.43 (2H, s), 7.96 (1H, t, J ¼ 6 Hz), 7.83 (2H, d,
J ¼ 8 Hz), 7.59 (2H, d, J ¼ 8 Hz), 7.44 (2H, d, J ¼ 8 Hz),
7.25 (2H, d, J ¼ 8 Hz), 6.66 (1H, s), 6.53 (1H, s), 5.29 (1H,
t, J ¼ 7 Hz), 4.00 (4H, s), 3.68 (2H, m), 3.67 (3H, s), 3.64
(3H, s), 3.52 (2H, s), 3.41 (2H, m), 3.26 (2H, m), 2.76 (2H,
m), 2.21 (3H, s).
[13] P. Belichard, M. Landry, P. Faye, D.R. Bachvarov, J. Bouthillier,
D. Pruneau, F. Marceau, Immunopharmacology 46 (2000) 139e147.
[14] N.M.J. Rupniak, S. Boyce, J.K. Webb, A.R. Williams, E.J. Carlson,
R.G. Hill, J.A. Borkowski, J.F. Hess, Pain 71 (1997) 89e97.
[15] N.M.J. Rupniak, J. Longmore, R.G. Hill, in: J. Wood (Ed.), Molecular
Basis of Pain Induction, John Wiley Press, 2000, pp. 149e174.
[16] D. Regoli, J. Barabe, W.K. Park, Can. J. Physiol. Pharmacol. 55 (1977)
855e867.
[17] D.S. Su, M.K. Markowitz, R.M. DiPardo, K.L. Murphy, C.M. Harrell,
S.S. O‘Malley, R.W. Ransom, R.S.L. Chang, S. Ha, F.J. Hess,
D.J. Pettibone, G.S. Mason, S. Boyce, R.M. Freidinger, M.G. Bock,
J. Am. Chem. Soc. 125 (2003) 7516e7517.
[18] M.G. Bock, J.F. Hess, D.J. Pettibone, Annu. Rep. Med. Chem. 38 (2003)
111e120.
Acknowledgments
[19] M.G. Bock, J. Longmore, Curr. Opin. Chem. Biol. 40 (2000) 401e406.
[20] J. Gougat, B. Ferrari, L. Sarran, C. Planchenault, M. Poncelet,
J. Maruani, R. Alonso, A. Cudennec, T. Croci, F. Guagnini, K. Urban-
The authors thank for the financial support to Richter Gedeon
´
Plc.; for B1 binding experiments to Andrea Marta Papp; for the
NMR measurements to Dr. Peter Forgo; for accurate mass deter-
´
Szabo, J.-P. Martinolle, P. Soubrie, O. Finance, G.G. Le Fur, J. Pharma-
col. Exp. Ther. 309 (2004) 661e669.
´
´
[21] R.A. Horlick, M.H. Ohlmeyer, I.L. Stroke, B. Strohl, G. Pan,
A.E. Schilling, V. Paradkar, J.G. Quintero, M. You, C. Riviello,
M.B. Thorn, B. Damaj, V.D. Fitzpatrick, R.E. Dolle, M.L. Webb,
J.J. Baldwin, N.H. Sigal, Immunopharmacology 43 (1999) 169e177.
[22] M.R. Wood, J.J. Kim, W. Han, B.D. Dorsey, C.F. Homnick,
R.M. DiPardo, S.D. Kuduk, T. MacNeil, K.L. Murphy, E.V. Lis,
R.W. Ransom, G.L. Stump, J.J. Lynch, S.S. O’Malley, P.J. Miller,
T.-B. Chen, C.M. Harrel, R.S.L. Chang, P. Sandhu, J.D. Ellis,
P.J. Bondiskey, D.J. Pettibone, R.M. Freidinger, M.G. Bock, J. Med.
Chem. 46 (2003) 1803e1806.
´
mination to Dr. Karoly Vekey and Dr. Gitta Schlosser.
´
References
[1] T.J. Ritchie, E.K. Dziadulewicz, A.J. Culshaw, W. Muller, G.M. Burgess,
¨
G.C. Bloomfield, G.S. Drake, A.R. Dunstan, D. Beattie, G.A. Hughes,
P. Ganju, P. McIntyre, S.J. Bevan, C. Davis, M. Yaqoob, J. Med.
Chem. 47 (2004) 4642e4644.
[2] D. Regoli, S.N. Allogho, A. Rizzi, F.J. Gobeil, Eur. J. Pharmacol. 348
(1998) 1e10.
[23] R.W. Ransom, C.M. Harrell, D.R. Reiss, K.L. Murphy, R.S.L. Chang,
J.F. Hess, P.J. Miller, S.S. O’Malley, P.J. Hey, P. Kunapuli, D.S. Su,
M.K. Markowitz, M.A. Wallace, C.E. Raab, A.N. Jones, D.C. Dean,
D.J. Pettibone, R.M. Freidinger, M.G. Bock, Eur. J. Pharmacol. 499
(2004) 77e84.
[24] S.D. Kuduk, C. Ng, D.-M. Feng, J.M.-C. Wai, R.S.L. Chang,
C.M. Harrell, K.L. Murphy, R.W. Ransom, D. Reiss, M. Ivarsson,
G. Mason, S. Boyce, C. Tang, T. Prueksaritanont, R.M. Freidinger,
D.J. Pettibone, M.G. Bock, J. Med. Chem. 47 (2004) 6439e6442.
[25] D.S. Su, M.K. Markowitz, K.L. Murphy, B.L. Wan, M.M. Zrada,
C.M. Harrell, S.S. O’Malley, F.J. Hess, R.W. Ransom, R.S. Chang,
M.A. Wallace, C.E. Raab, D.C. Dean, D.J. Pettibone, R.M. Freidinger,
M.G. Bock, Bioorg. Med. Chem. Lett. 14 (2004) 6045e6048.
[26] S.N. Ha, P.J. Hey, R.W. Ransom, C.M. Harrell Jr., K.L. Murphy,
R. Chang, T.-B. Chen, D.S. Su, M.K. Markowitz, M.G. Bock,
R.M. Freidinger, F.J. Hess, Biochem. Biophys. Res. Commun. 331
(2005) 159e166.
[3] J.M. Stewart, L. Gera, E.J. York, D.C. Chan, E.J. Whalley, P.A. Bunn,
R.J. Vavrek, Biol. Chem. 382 (2001) 37e41.
[4] M. Pellegrini, M. Tancredi, P. Rovero, D.F. Mierke, J. Med. Chem. 42
(1999) 3369e3377.
[5] M. Amblard, I. Daffix, P. Bedos, G. Berge, D. Pruneau, J.-L. Paquet,
´
J.-M. Luccarini, P. Belichard, P. Dodey, J. Martinez, J. Med. Chem. 42
(1999) 4185e4192.
[6] R. Couture, M. Harrisson, R.M. Vianna, F. Cloutier, Eur. J. Pharmacol.
429 (2001) 161e176.
[7] K.D. Bhoola, C.D. Figueroa, K. Worthy, Pharmacol. Rev. 44 (1992)
1e80.
[8] E.K. Dziadulewicz, T.J. Ritchie, A. Hallett, C.R. Snell, J.W. Davies,
R. Wrigglesworth, A.R. Dunstan, G.C. Bloomfield, G.S. Drake,
P. McIntyre, M.C. Brown, G.M. Burgess, W. Lee, C. Davis,
M. Yaqoob, S.B. Phagoo, E. Phillips, M.N. Perkins, E.A. Campbell,
A.J. Davis, H.P. Rang, J. Med. Chem. 45 (2002) 2160e2172.
[9] D. Regoli, J. Barabe, Pharmacol. Rev. 32 (1980) 1e46.
[10] S.G. Farmer (Ed.), The Kinin System, Academic Press, London, 1997.
[27] B. Ferrari, J. Gougat, C. Muneaux, Y. Muneaux, P. Perreaut, C. Planche-
nault, WO 9725315, 1997, 98.