Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2008.09.033

According to a 'bivalent ligand approach' to increase the affinity of the potent argininamide-type NPY Y₁ receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent compound were linked by different spacers via N^G-acylation at the guanidino groups. A synthetic route for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing nanomolar antagonistic activity and binding affinity to the NPY Y₁ receptor (calcium assay on HEL cells, radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226 derivate showed, surprisingly, no affinity to the NPY Y₁ receptor at all.

Full text of DOI:10.1016/j.bmc.2008.09.033

Bioorganic & Medicinal Chemistry 16 (2008) 9858–9866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modular synthesis of non-peptidic bivalent NPY Y1 receptor antagonists
Stefan Weiss a, Max Keller b, Günther Bernhardt b, Armin Buschauer b, Burkhard König a,
*
a Institut für Organische Chemie, Universität Regensburg, D-93040 Regensburg, Germany
b Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2008
Revised 4 September 2008
Accepted 10 September 2008
Available online 13 September 2008
According to a ‘bivalent ligand approach’ to increase the affinity of the potent argininamide-type NPY Y1
receptor antagonist BIBP-3226, dimeric ligands were synthesized in which two molecules of the parent
compound were linked by different spacers via NG-acylation at the guanidino groups. A synthetic route
for the preparation of the title compounds was developed, which includes a copper(I)-catalyzed azide
alkyne cycloaddition as the key step. Three bivalent analogues of BIBP-3226 were prepared showing
nanomolar antagonistic activity and binding affinity to the NPY Y1 receptor (calcium assay on HEL cells,
radioligand binding assay on SK-N-MC cells), but these ligands were not superior to the parent compound
and there was no correlation with the length or the chemical nature of the spacer. A trivalent BIBP-3226
derivate showed, surprisingly, no affinity to the NPY Y1 receptor at all.
Keywords:
Neuropeptide Y
Bivalent ligands
Guanidinium compounds
Binding
Ó 2008 Elsevier Ltd. All rights reserved.
Huisgen reaction
Cycloaddition
1. Introduction
more favourable binding interactions of the dimeric structures
resulting from the bridging of independent recognition sites.5–7
Neuropeptide Y (NPY) is a highly conserved peptide that plays
an important role as a neurotransmitter in the central and periph-
eral nervous system. Together with the pancreatic polypeptide (PP)
and peptide YY (PYY), NPY belongs to the neuropeptide Y family. In
contrast to PP and PYY, which have hormone character, NPY acts as
a neurotransmitter. Five Gi/o protein coupled NPY receptor sub-
types are known.1 In humans, the biological effects of NPY are
mediated by four receptor subtypes, referred to as NPY Y1, Y2, Y4
and Y5 receptors. For instance, in the periphery NPY Y1 receptor
(Y1R) stimulation causes an increase in blood pressure. In the cen-
tral nervous system (CNS) Y1R activation elicits anxiolytic and sed-
ative effects and is involved in the stimulation of food intake.2
Potent and selective antagonists and agonists are required as phar-
macological tools to investigate the numerous biological effects of
NPY and to study the ligand receptor interactions on the molecular
level.
The (R)-argininamide BIBP-3226 (1) is a potent NPY Y1R antag-
onist.8 Previous investigations revealed that acylation at the NG-
position of 1 are tolerated over a wide range of structural varia-
tions including the introduction of bulky fluorophores.9 Compared
to guanidines, the pKa values of the acylguanidines are 4–5 orders
of magnitude lower. Depending on the nature of the NG-substitu-
ent, highly potent and selective Y1R antagonists, superior to the
parent compound and capable of penetrating into the brain, were
obtained. Based on the structure–activity relationships of these
compounds the NG-terminus of 1 has been considered an appropri-
ate position for covalent linkage of two molecules to obtain puta-
tive bivalent Y1R ligands, for instance, of general structure 2
(Fig. 1). The synthesis of such derivatives from 1 is hampered by
the high polarity and basicity of the guanidino group.10 Therefore,
NG-protected or functionalized NG-acylated derivatives of 1 were
preferred as a building block. Here we present two versatile syn-
thetic routes based on copper(I)-catalyzed azide–alkyne cycloaddi-
tion reactions as key steps that facilitate the preparation of
bivalent Y1R antagonists derived from BIBP-3226.
The present study deals with a ‘bivalent ligand approach’3 to
synthesize Y1R antagonists. Bivalent ligands are molecules that
contain two covalently linked biologically active chemical entities;
their potency can exceed the effect of the corresponding mono-
meric ligand at double concentration, as shown for opioid recep-
tors by Portoghese.4 It is a matter of speculation whether such
affinity-enhancing effects may be attributed to thermodynamically
2. Results and discussion
First, the BIBP-3226 derivative 8 with azide functionality was
prepared (Scheme 1). Commercially available S-methylisothiourea
3 was mono-Boc-protected and coupled with 4-azidobutanoic acid
(5) using standard peptide coupling procedures. Azide 6 and amine
* Corresponding author. Tel.: +49 941 943 4576; fax: +49 941 943 1717.
E-mail address: Burkhard.koenig@chemie.uni-regensburg.de (B. König).
7, which is available from D-ornithine in six steps in 32% overall
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.033

S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
9859
H2N
HN
NH
H2N
N
N
NH2
N
N
N
N
spacer
NH
O
O
HN
N
N
OH
OH
O
HO
H
N
O
O
H
N
H
N
N
H
N
H
N
H
O
O
O
1
2
Figure 1. NPY Y1R antagonist BIBP-3226 (1) and general structure of bivalent Y1R antagonists (2) constructed from NG-acylated 1 by ‘click’ reaction.
NH
SMe
NH
SMe
a
O
H2N
BocHN
O
N3
N
b
3
4
BocHN
N
BocHN
SMe
N3
HN
O
O
N3
6
HO
c
OtBu
5
H
N
N
H
H2N
O
OtBu
O
H
N
8
N
H
O
7
Scheme 1. Synthesis of azide building block 8. (a) Boc2O, NaOH, tBuOH, 90%; (b) DIPEA, EDC, HOBT, dichloromethane, 89%; (c) HgCl2, NEt3, DMF, 76%.
yield,11 were smoothly converted into compound 8 by a mercury
chloride mediated guanidinylation reaction.12
antagonist moieties at the end of the synthesis. The cycloaddition
of two equivalents of azide 5 with diynes 9 becomes the first step
(Scheme 3). Diyne 9b was prepared from propargyl bromide and
the respective glycol according to described procedures.14 The
resulting dicarboxylic acids 13a and 13b were obtained in high
yield and good purity using CuSO4 and ascorbate to catalyze the
reaction. Their conversion into bis-isothiourea derivatives 14 by
the dual reaction with compound 4 proceeded in very good yield.
Finally, guanidinylation of compound 7 with isothioureas 14 gave
compounds 15a and 15b in good yields, and deprotection with
DCM/TFA resulted in the target bivalent BIBP-3226 derivatives
16a and 16b. The obtained overall yield based on consumption of
compound 7 improved significantly, when compared to the previ-
ously discussed route. Three bivalent BIBP-3226 derivatives 16a–c,
Initial attempts to react azide 8 with 1,4-diethynylbenzene (9a)
in the presence of CuSO4 and ascorbate were unsuccessful: At room
temperature no conversion was detectable, whereas heating above
60 °C destroyed the starting materials. However, by addition of
20 mol% of the Cu(I)-stabilizing tris–triazole ligand 1013 (Scheme
2) the dual cycloaddition product 11a was obtained in 44% yield
at room temperature. Deprotection with TFA/dichloromethane
1:1 yielded the target compound 12a.
Although the synthetic route provides access to bivalent BIBP-
3226 derivatives, the introduction of the BIBP-3226 skeleton with
compound 7 at an early stage of the synthesis is not very efficient.
Therefore, a second route was developed introducing the Y1R
O
N
OH
N
N
O
HO
R1
HN
N
N
R1
NH
N
N
N
N
N
N
N
N
O
O
N
N
N
N
OR2
R2O
N
O
O
8
2
+
H
N
H
N
10
HO
O
N
H
N
H
9a
O
O
a)
11a R1 = NHBoc; R2 = tBu
b)
12a R1 = NH3 , R2 = H
+
Scheme 2. Synthesis of the bivalent BIBP-3226 derivative 12a via a dual ‘click’ reaction. (a) CuSO4, 10, sodium ascorbate, 44%; (b) TFA, DCM, quantitative yield.

9860
S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
HO
OH
a)
b)
N
N
N
N
N
+
2
5
X
X
O
O
N
9a-b
13a-b
c)
BocHN
SMe
N
N
NHBoc
N
N
N
N
N
X
O
O
SMe
N
14a-b
R1
N
N
R1
N
N
N
N
X
O
HN
NH
O
N
N
R2
O
O
R2
O
O
H
N
H
N
N
H
N
H
O
O
15a-b R1 = NHBoc; R2 = tBu
d)
+
16a-b R1 = NH3 , R2 = H
BocHN
O
O
NHBoc
N SMe
O
O
c)
b)
MeS
N
X
HO
X
OH
13c
14c
R1
O
O
R1
O
O
O
N
O
N
NH
HN
R2
R2
O
O
O
H
N
H
N
N
H
N
H
O
O
15c R1 = NHBoc; R2 = tBu
d)
16c R1 = NH3 , R2 = H
+
a
b
c
X =
O
O
X =
X =
O
O
O
3
O
Scheme 3. Synthesis of bivalent BIBP derivatives 16a–c using bis-methylisothioureas 14a–c. (a) CuSO4, sodium ascorbate, MeOH/H2O, 65–75%; (b) EDC, HOBt, DIPEA, DMF or
DCM, 4, 63–71%; (c) HgCl2, NEt3, DMF, 7, 55–65%; (d) TFA/DCM, quantitative yield.
varying in length15 and rigidity of the spacer, monovalent com-
pound 16d (for comparison) and a trivalent ligand 16e, based on
tricarboxylic acid 10, were prepared (Scheme 4).
The compounds 16a–e were investigated for NPY Y1R antago-
nism in a calcium (Fura-2) assay on human erythroleukemia
(HEL) cells. In addition, the Y1R affinities were determined in a
radioligand binding assay on SK-N-MC human neuroblastoma cells
using NG-([2,3-3H]propionyl)-BIBP-3226 as radioligand (prepared
and pharmacologically evaluated in our laboratory, to be reported
elsewhere). Kb and Ki values are summarized in Table 1. The Y1R
antagonistic activities of the BIBP-3226 derivatives 16a–d in the
functional assay remain in the same range as that of the parent
monovalent compound 1, whereas the Ki values of 16a, b, d are
about 5- to 44-fold higher than that of 1. As small changes of the
acyl substituent may result in significantly altered binding affini-
ties, a direct comparison of a monovalent and a bivalent ligand is
only possible for compounds 16a and 16d. While the Kb is slightly
lower for the bivalent compound 16a, the Ki values of 16a and 16d
are identical. Thus a beneficial effect of the second pharmacophore
is not observed in this specific case. The comparison of the varia-

S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
9861
BocHN
SMe
N
HO
a)
b)
N
N
N
N
+
5
O
O
N
N
14d
9d
13d
R1
N
N
N
NH
O
N
OH
O
c)
O
R2
O
H
N
N
H
O
1
2
t
HN
15d
16d
R = NHBoc; R = Bu
NH
O
d)
R = NH3+, R2 = H
1
O
N
NH
N
N
+
NH3
N
H
N
N+
+
N
N
NH3
NH
N
b), c), d)
28 %
N
O
+H3N
HN
10
N
N
O
N
O
O
NH
HN
HN
O
NH
O
16e
OH
HO
Scheme 4. Synthesis of mono- and trivalent BIBP-3226 derivatives 16d and e. (a) CuSO4, sodium ascorbate, MeOH/H2O, 53%; (b) EDC, HOBt, DIPEA, DCM, 4, 66–78%; (c) HgCl2,
NEt3, DMF, 7, 43–72%; (d) TFA/DCM, quantitative yield.
Table 1
IC50 and Ki values for NPY Y1R antagonism on HEL cells (calcium assay) and Y1R binding determined on SK-N-MC cells
Y1R antagonism (Ca2+-assay) Kb [nM]
Y1R binding (SK-N-MC cells) Ki [nM]
a
b
Compound
Number of BIBP moieties
Spacer length (number of atoms)
1c
16a
16b
16c
16d
16e
1
2
2
2
1
3
—
18
29
11
—
1.5 0.3
0.6 0.2
1.8 0.6
7.0 2.5
0.9 0.2
1.3 0.2
43 12
58 12
6.1 1.9
49
6
d
21
—
>50,000
a
Kb values for inhibition of NPY (10 nM) induced calcium mobilization in HEL cells (Fura-2 assay). Mean values SEM from two independent experiments.
Ki values determined from the displacement of NG-([2,3-3H]propionyl)-BIBP-3226 on SK-N-MC cells. Mean values SEM from two independent experiments performed in
b
triplicate.
c
BIBP-3226.
Not determined.
d
tion in spacer length in 16a–c with changes in their binding affin-
ities does not reveal a simple correlation either. The trivalent deriv-
ative 16e proved to be completely inactive.
nificant variation in length and rigidity of the spacer unit, binding
affinities to the NPY Y1 receptor, which are in the same range as
that of the parent ligand. Surprisingly, the trivalent compound
16e is completely inactive. Although of limited scope, the small
study shows that the principle of bivalent ligands, as reported for
opioid receptors, does not follow a simple structure–activity rela-
tionship in the case of NPY Y1 receptors, if transferable at all.
3. Conclusion
The copper(I) catalyzed alkyne–azide cycloaddition to triazoles
facilitates the preparation of bivalent argininamide-type NPY Y1R
antagonists derived from BIBP-3226 (1). The affinity-conferring
moiety can be introduced either as NG-(4-azidobutanoyl)-substi-
tuted 1 (8), which is allowed to react with a diyne, or, more effi-
ciently, at the end of the synthesis by guanidinylation of the
corresponding ornithine derivative using bis-methylisothiurea
building blocks. The new BIBP-3226 derivatives retain, despite sig-
4. Experimental
1H NMR spectra were recorded at 300 MHz on a Bruker Avance
300 spectrometer. 13C NMR spectra were recorded at 76 MHz. All
chemical shifts values are reported in ppm. IR-spectra were mea-
sured with a Varian Biorad FT-IR spectrometer Excalibur FTS

9862
S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
3000. UV/vis spectra were recorded with a Varian Cary BIO 50 UV/
vis/NIR spectrophotometer. Mass spectra: Varian CH-5(EI), Finni-
gan MAT 95(CI), Finnigan MAT TSQ 7000(ESI). Compounds 16a–e
were purified as TFA salts using HPLC: Phenomenex Luna C18
(RP-HPLC) column; detection: DAD at 220 nm; temperature:
25 °C; gradient: 3% AcN 97% H2O[0.0059%TFA](t = 0) ? 98% AcN
2% H2O[0.0059%TFA](t = 30 min.); flow: 35.0 mL/min. Melting
points were determined with a Büchi Tottoli apparatus, they are
not corrected. Thin layer chromatography (TLC) was performed
room temperature. DMF was removed completely and the residue
was dissolved in DCM. The precipitate of mercury salts was re-
moved by filtering over celite, the organic phase was washed with
sodium carbonate (50 mL), citric acid (50 mL), brine (50 mL) and
dried over magnesium sulphate. DCM was removed and the crude
product was purified by column chromatography (EA/PE 1:1
Rf = 0.4) yielding compound 8 (223 mg, 76%), as a white solid,
mp > 190 °C. 1H NMR (300 MHz, CDCl3): 1.33 (m, 9H), 1.44 (m,
9H), 1.54–1.99 (m, 6H), 2.37–2.54 (m, 2H), 3.20–3.30 (m, 2H),
3.30–3.40 (m, 2H), 4.20–4.40 (m, 2H), 4.52–4.70 (m, 1H), 4.90–
5.00 (m, 1H), 6.84–6.90 (m, 2H), 7.02–7.09 (m, 2H), 7.18–7.45
(m, 10H). C40H52N8O6: MS (ES): m/z (%) 641 (13), 665 (17), 739
(100, M+).
on alumina plates coated with silica gel (Merck silica gel 60 F245
,
thickness 0.2 mm). Column chromatography (CC) was performed
with Merck Geduran SI 60 silica gel as the stationary phase. PE
means petrol ether with a boiling point range from 60 to 70 °C.
EA is ethyl acetate.
Compounds 5,16 717 and 1013 were prepared according to liter-
ature procedures. The previously described synthesis of compound
417 was improved.
4.4. Di-tert-butyl (1,4-phenylenebis{1H-1,2,3-triazole-4,1-diyl-
(1-oxobutane-4,1-diyl)imino[(Z)-({(4R)-5-[(4-tert-butoxy-
benzyl)amino]-4-[(diphenylacetyl)amino]-5-oxopentyl}-
amino)methylylidene]})biscarbamate (11a)
4.1. tert-Butyl [imino(methylthio)methyl]carbamate (4)
Compound 8 (50.0 mg, 67.5
MeOH and a solution of compound 9a (4.3 mg, 33.7
MeOH (2 mL) was added. Then the solid ligand 10 (4.5 mg, 6.8
mol) was added. solution of sodium ascorbate (5.4 mg,
20.7 mol) in 0.5 mL of H2O and subsequent under vigorous stir-
ring a solution of copper(II) sulphate pentahydrate (1.7 mg, 6.8
mol) in 0.5 mL of H2O was added to the reaction mixture. The
mixture was stirred at room temperature overnight. MeOH was re-
moved completely under reduced pressure. Water (20 mL) was
added and the product was extracted with DCM (3 ꢁ 20 mL). The
organic phase was dried over magnesium sulphate and the solvent
was evaporated. The crude material was purified by column chro-
matography: (EA Rf = 0.6); the product is a yellow solid (24 mg,
44%). For analytical data see compound 15a.
l
mol) was dissolved in 2 mL of
S-Methylisothiourea hemisulphate
3
(5.0 g, 35.9 mmol,
lmol) in
1.0 equiv) was dissolved in 10 mL of aqueous NaOH (1.44 g,
35.9 mmol, 1.0 equiv) and tBuOH (100 mL). Boc2O (6.26 g,
28.7 mmol, 0.8 equiv), dissolved in 50 mL of tBuOH, was added
dropwise under cooling (ice bath) to this solution over a period
of 1 h. The reaction mixture was stirred overnight. Dichlorometh-
ane (100 mL) was added, the solution was washed with aqueous
satd sodium carbonate solution (100 mL), the organic layer was
separated, dried over magnesium sulphate and the solvent was re-
moved under reduced pressure yielding compound 4 (4.9 g, 90%),
as a white solid, mp = 77 °C.
l
A
l
l
1H NMR (300 MHz, DMSO-d6): 1.40 (s, 9H), 2.31 (s, 3H), 8.56 (br
s, 2H). 13C NMR (300 MHz, DMSO-d6): 12.7, 27.8, 77.7, 160.6, 171.4.
C7H14N2O2S: MS (ES): m/z (%): 190(17, M+), 175(50), 134(44),
57(100).
4.5. General procedure for preparation of compounds 13a and
13b
4.2. tert-Butyl [(1Z)-[(4-azidobutanoyl)amino](methylthio)-
methylene]carbamate (6)
To a solution of the appropriate diyne (13a: 3.69 mmol, 13b:
5.55 mmol) in MeOH (20 mL) 4-azidobutanoic acid (13a:
7.75 mmol, 13b: 11.10 mmol) was added under stirring. A solution
of sodium ascorbate (13a: 0.37 mmol, 13b: 0.56 mmol) in water
(1 mL) and a solution of copper(II) sulphate pentahydrate (13a:
0.07 mmol, 13b: 0.11 mmol) in water (1 mL) were added slowly.
The reaction mixture was stirred overnight at room temperature
and then refluxed for 3 h.
4-Azidobutanoic acid (5, 170 mg, 1.32 mmol), HOBTꢀH2O
(242 mg, 1.58 mmol), DIPEA (341 mg, 2.64 mmol), and EDC
(303 mg, 1.58 mmol) were dissolved in cold dichloromethane (ice
bath) under stirring. After 15 min, compound 4 (2.55 g, 1.32 mmol)
was added, the reaction mixture was stirred overnight, diluted
with water and the organic layer was washed with aqueous citric
acid solution (2 ꢁ 50 mL) and aqueous satd sodium hydrogen car-
bonate solution (2 ꢁ 50 mL). The organic layer was dried, the sol-
vent was removed under reduced pressure and the crude product
was purified by column chromatography (EA/PE, 1:1 Rf = 0.7) yield-
ing compound 6 (354 mg, 89% yield), as a white solid, mp = 43 °C.
1H NMR (300 MHz, CDCl3): 1.45 (s, 9H), 1.89 (m, 2H), 2.33 (s,
3H), 2.50 (m, 2H), 3.34 (m, 2H), 12.30 (d, 1H). 13C NMR (75 MHz,
CDCl3): 14.6, 24.1, 28.0, 36.1, 50.6, 82.5, 170.0, 171.1, 185.2.
C11H19N5O3S: MS (CI-MS, NH3): m/z (%) 203 (1), 274 (8), 302
(100, M+).
4.5.1. 4,40-[1,4-Phenylenebis(1H-1,2,3-triazole-4,1-diyl)]-
dibutanoic acid (13a)
The yellow, insoluble product was filtered off and dissolved in
20 mL of NaOH (1 mol/L). The aqueous phase was washed with
dichloromethane (2 ꢁ 20 mL) and PE (1 ꢁ 20 mL), and acidified
with citric acid (pH < 2) yielding a yellow precipitate (0.99 g,
70%), mp > 190 °C. 1H NMR (300 MHz, DMSO-d6): 2.10 (m, 4H),
2.29 (t, J = 7.2, 4H), 4.45 (t, J = 7.0, 4H), 7.93 (s, 4H), 8.64 (s, 2H),
12.12 (br s, 2H). 13C NMR (75 MHz, DMSO-d6): 25.0, 30.4, 48.8,
121.3, 125.5, 130.1, 146.0, 173.5. C18H20N6O4: MS (ES): m/z (%)
385(100, M+). IR (cmꢂ1): 2559, 1685, 1430, 1291, 1215, 1045,
910, 822.
4.3. tert-Butyl [(1Z)-[(4-azidobutanoyl)amino]({(4R)-5-[(4-tert-
butoxybenzyl)amino]-4-[(diphenylacetyl)amino]-5-oxopentyl}-
amino)methylene]carbamate (8)
4.5.2. 4,40-[2,5,8,11,14-Pentaoxapentadecane-1,15-diylbis(1H-
1,2,3-triazole-4,1-diyl)]dibutanoic acid (13b)
Compound
7 (120 mg, 0.40 mmol), compound 6 (194 mg,
0.40 mmol) and HgCl2 (109 mg, 0.40 mmol) were dissolved sepa-
rately in small amounts of DMF (1–2 mL). Solutions of compound
7 and compound 6 were combined in a small flask under nitrogen
atmosphere. NEt3 (404 mg, 4 mmol) was added under stirring, the
HgCl2 solution was added and the mixture was stirred overnight at
The solvent was removed completely under reduced pressure
and the remaining yellow oil was purified by column chromatogra-
phy (EA/MeOH 1:1 Rf = 0.2) yielding compound 13b (1.98 g, 67%)
as a yellow oil. 1H NMR (300 MHz, CD3OD): 2.20 (m, 4H), 2.30 (t,
J = 6.9, 4H), 3.60 (m, 16H), 4.50 (t, J = 6.9, 4H), 4.62 (s, 4H), 7.98

S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
9863
(s, 2H). 13C NMR (75 MHz, CD3OD): 26.9, 31.9, 50.7, 65.0, 70.7, 71.4,
125.3, 146.0, 176.7. C22H36N6O9: MS (ES): m/z (%) 529(100, M+),
551 (12). IR (cmꢂ1): 3458, 1720, 1558, 1219, 1084, 836.
ing compound 14d (313 mg, 78%) as a white solid, mp = 140 °C. 1H
NMR (300 MHz, CDCl3): 1.50 (s, 9H), 2.30 (m, 2H), 2.37 (s, 3H), 2.50
(m, 2H), 4.49 (m, 2H), 7.29–7.44 (m, 3H), 7.81 (m, 2H), 7.83 (s, 1H),
12.30 (d, 1H). 13C NMR (75 MHz, CDCl3): 14.6, 25.9, 28.0, 37.7, 49.5,
83.6, 119.8, 125.7, 128.2, 128.9, 130.6, 147.9, 160.9, 170.8, 184.7.
C19H25N5O3S: MS (ES): m/z (%) 304(35), 404 (100, M+), 807(15).
IR (cmꢂ1): 2978, 1715, 1640, 1557, 1412, 1279, 1226, 1150, 1119,
869, 815, 762, 691.
4.6. General procedure for the preparation of compounds 14a–
14e
The appropriate carboxylic acid 13 (1 mmol), HOBTꢀH2O
(1.2 mmol for monovalent, 2.4 mmol for bivalent and 3.6 mmol
for trivalent ligands), EDC (1.2 mmol, 2.4 mmol, 3.6 mmol) and DI-
PEA (2 mmol, 4 mmol, 6 mmol) were combined in a flask under
nitrogen atmosphere with 10 mL of cold dichloromethane (14d
and 14e) or DMF (14a, 14b and 14c). After 15 min compound 4
(1 mmol, 2 mmol and 3 mmol) was added and the mixture was
stirred overnight at room temperature.
4.6.5. Di-tert-butyl([({1-[4-({[(Z)-[(tert-butoxycarbonyl)imino]
(methylthio)methyl]amino}carbonyl)benzyl]-1H-1,2,3-triazol-
4-yl}methyl)imino]bis{methylene-1H-1,2,3-triazole-4,1-diylm-
ethylene-4,1-phenylenecarbonylimino[(E)-(methylthio)meth-
ylylidene]})biscarbamate (14e)
The DCM phase was washed with sodium carbonate (50 mL)
and sodium hydrogensulphate (50 mL), dried over magnesium sul-
phate and the DCM was removed under reduced pressure. The
crude product was recrystallized with MeOH yielding the product
as white solid (783 mg, 66%), mp > 190 °C. 1H NMR (300 MHz,
CDCl3): 1.52 (s, 27H), 2.55 (s, 9H), 3.71 (s, 6H), 5.57 (s, 6H), 7.30
(d, J = 8.20, 6H), 7.73 (s, 3H), 8.20 (d, J = 8.20, 6H), 12.52 (s, 3H).
13C NMR (75 MHz, CDCl3): 15.0, 28.0, 47.0, 53.7, 83.6, 124.1,
127.7, 130.8, 137.0, 139.3, 144.2, 150.9, 172.6, 175.1.
C54H66N16O9S3: MS (ES): m/z (%) 591(100), 1180(40, M+).
4.6.1. Di-tert-butyl (1,4-phenylenebis{1H-1,2,3-triazole-4,1-
diyl(1-oxobutane-4,1-diyl)imino[(Z)-(methylthio)-
methylylidene]})biscarbamate (14a)
DMF was removed completely under reduced pressure and the
crude product was purified by column chromatography (EA/PE
1:1 ? EA Rf = 0.6[EA]) yielding compound 14a (460 mg, 63%) as a
pale yellow solid, mp = 148 °C. 1H NMR (300 MHz, CDCl3): 1.47
(s, 18H), 2.30 (m, 4H), 2.38 (s, 6H), 2.55 (m, 4H), 4.51 (t, J = 6.42,
4H), 7.84 (s, 2H), 7.89 (s, 4H), 12.30 (d, 2H). 13C NMR (75 MHz,
CDCl3): 14.6, 25.9, 28.0, 37.7, 49.2, 83.6, 119.8, 128.4, 130.6,
147.9, 160.4, 170.2, 184.3. C32H44N10O6S2: MS (ES): m/z (%) 653
(5), 727(100, M+). IR (cmꢂ1): 2978, 1725, 1640, 1555, 1409,
1279, 1226, 1130, 810.
4.7. General procedure for preparation of compounds 15a–15e
The appropriate guanidinylation reagent 14 (0.2 mmol), com-
pound 7 (0.2 mmol for monovalent, 0.4 mmol for bivalent or
0.6 mmol for trivalent) and HgCl2 (0.2 mmol, 0.4 mmol, 0.6 mmol)
were dissolved separately in small amounts of DMF (1–2 mL). Solu-
tions of appropriate compound 14 and compound 7 were com-
bined in a small flask under nitrogen atmosphere. NEt3 (2 mmol,
4 mmol and 6 mmol) was added under stirring, the HgCl2 solution
was added and the mixture was stirred overnight at room temper-
ature. DMF was removed completely and the residue was dissolved
in DCM. The precipitate of mercury salts was removed by filtering
over celite, the organic phase was washed with sodium carbonate
(50 mL), citric acid (50 mL), brine (50 mL) and dried over magne-
sium sulphate. DCM was removed and the crude product purified
by column chromatography.
4.6.2. Di-tert-butyl (2,5,8,11,14-pentaoxapentadecane-1,15-
diylbis{1H-1,2,3-triazole-4, 1-diyl(1-oxobutane-4,1-diyl)-
imino[(Z)-(methylthio)methylylidene]})biscarbamate (14b)
DMF was removed completely under reduced pressure; the
remaining crude oil was dissolved in dichloromethane, washed
with satd aqueous sodium carbonate solution (50 mL) and brine
(50 mL), dried over magnesium sulphate and the solvent was evap-
orated. The crude product was purified by column chromatography
[EA ? EA/MeOH 1:1 Rf = 0.8 (EA/MeOH 1:1)] yielding compound
14b (556 mg, 64%) as a viscous oil. 1H NMR (300 MHz, CDCl3):
1.49 (s, 18H), 2.20 (m, 4H), 2.37 (s, 6H), 2.50 (m, 4H), 3.60 (m, 16
H), 4.40 (t, J = 6.81, 4H), 4.66 (s, 4H), 7.59 (s, 2H), 12.30 (d, 2H).
13C NMR (75 MHz, CDCl3): 14.6, 25.4, 28.0, 37.7, 49.5, 64.7, 69.7,
70.5, 84.1, 122.7, 145.3, 160.8, 170.0, 184.6. C36H60N10O11S2: MS
(ES): m/z (%) 873(100, M+), 895(15). IR (cmꢂ1): 2978, 1728, 1644,
1559, 1395, 1277, 1228, 1132, 809.
4.7.1. Di-tert-butyl (1,4-phenylenebis{1H-1,2,3-triazole-4,1-
diyl(1-oxobutane-4,1-diyl)imino[(Z)-({(4R)-5-[(4-tert-butoxy-
benzyl)amino]-4-[(diphenylacetyl)amino]-5-oxopentyl}-
amino)methylylidene]})biscarbamate (15a)
Column chromatography: (EA Rf = 0.6); the product is a yellow
solid (173 mg, 55%), mp > 190 °C. 1H NMR (300 MHz, CDCl3): 1.29
(m, 18H), 1.40–1.47 (m, 18H), 1.50–1.90 (m, 8H), 2.10–2.50 (m,
8H), 3.15–3.45 (m, 4H), 4.20–4.50 (m, 8H), 4.52–4.70 (m, 2H),
4.96 (m, 2H), 6.80–6.90 (m, 4H), 7.00–7.09 (m, 4H), 7.18–7.45
(m, 24H), 7.67–7.85 (m, 6H). C90H110N16O12: MS (ES): m/z (%) 705
(23), 755 (30), 805 (100), 1509 (8), 1608 (16, M+), 1630 (6).
4.6.3. tert-Butyl [(1Z,14Z)-19,19-dimethyl-1,15-bis(methyl-
thio)-3,13,17-trioxo-5,8,11,18-tetraoxa-2,14,16-triazaicosa-1,
14-dien-1-yl]carbamate (14c)
Compound 14c was worked up as described for 14b. The crude
product was purified by column chromatography (PE/EA 3:1
Rf = 0.1) yielding compound 14c (402 mg, 71%) as a white solid,
mp = 103 °C. 1H NMR (300 MHz, CDCl3): 1.38 (s, 18H), 2.26 (s,
6H), 3.64 (s, 8H), 4.02 (s, 4H), 12.30 (d, 2H). 13C NMR (75 MHz,
CDCl3): 14.6, 27.9, 69.7, 70.5, 84.1, 160.8, 170.0, 184.6.
C22H38N4O9S2: MS (ES): m/z 567(100, M+), 589(7), 605(2). IR
(cmꢂ1): 2978, 1720, 1650, 1553, 1425, 1365, 1272, 1228, 1144,
813.
4.7.2. tert-Butyl [(1Z)-({(4R)-5-[(4-tert-butoxybenzyl)amino]-4-
[(diphenylacetyl)amino]-5-oxopentyl}amino)({4-[4-(15-{1-[4-
({(1E)-({(4R)-5-[(4-tert-butoxybenzyl)amino]-4-[(diphenylace-
tyl)amino]-5-oxopentyl}amino)[(tert-butoxycarbonyl)amino]-
methylene}amino)-4-oxobutyl]-1H-1,2,3-triazol-4-yl}-2,5,8,11,-
14-pentaoxapentadec-1-yl)-1H-1,2,3-triazol-1-yl]butanoyl}-
amino)methylene]carbamate (15b)
Column chromatography: (DCM/MeOH 17:3 Rf = 0.4); the prod-
uct is obtained as a pale yellow very viscous oil (218 mg, 64%). 1H
NMR (300 MHz, CD3OD): 1.28 (m, 18H), 1.40–1.50 (m, 18H), 1.50–
2.50 (m, 16H), 3.20–3.40 (m, 4H), 3.50–3.70 (m, 16H), 4.20–4.60
(m, 14H), 5.08 (m, 2H), 6.86–6.89 (m, 4H), 7.05–7.15 (m, 4H),
4.6.4. tert-Butyl((1Z)-(methylthio){[4-(4-phenyl-1H-1,2,3-
triazol-1-yl)butanoyl]amino} methylene)carbamate (14d)
The organic layer was washed with sodium carbonate (50 mL),
citric acid (50 mL) and brine (50 mL), dried over magnesium sul-
phate and the solvent was removed in vacuum. The crude product
was purified by column chromatography (PE/EA 1:1 Rf = 0.3) yield-

9864
S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
7.15–7.30 (m, 20H), 7.90–7.95 (m, 2H). C94H126N16O17: MS (ES): m/
z (%) 827 (20), 877 (100), 1653 (6), 1753 (18, M+).
4.8.2. (E)-{[4-(4-{15-[1-(4-{[(1Z)-Ammonio({(4R)-4-[(dipheny-
lacetyl)amino]-5-[(4-hydroxybenzyl)amino]-5-oxopentyl}-
amino)methylene]amino}-4-oxobutyl)-1H-1,2,3-triazol-4-yl]-2,
5,8,11,14-pentaoxapentadec-1-yl}-1H-1,2,3-triazol-1yl) butan-
oyl]amino}({(4R)-4-[(diphenylacetyl)amino]-5-[(4-hydroxy-
benzyl)amino]-5-oxopentyl}imino)methanaminium
4.7.3. tert-Butyl{(1E,14E,20R)-20-{[(4-tert-butoxybenzyl)-
amino]carbonyl}-1-({(4R)-5-[(4-tert-butoxybenzyl)amino]-4-
[(diphenylacetyl)amino]-5-oxopentyl}amino)-15-[(tert-
butoxycarbonyl)amino]-3,13,22-trioxo-23,23-diphenyl-5,8,11-
trioxa-2,14,16,21-tetraazatricosa-1,14-dien-1-yl}carbamate (15c)
Column chromatography: (EA Rf = 0.6); the product is a white
solid (190 mg, 65%), mp > 190 °C. 1H NMR (300 MHz, CDCl3): 1.31
(m, 18H), 1.42–1.48 (m, 18H), 1.50–1.90 (m, 8H), 3.20–3.50 (m,
4H), 3.65–3.85 (m, 8H), 4.20–4.40 (m, 8H), 4.52–4.70 (m, 2H),
4.87–4.98 (m, 2H), 6.80–6.90 (m, 4H), 7.00–7.10 (m, 4H), 7.18–
7.30 (m, 20H). C80H104N10O15: MS (ES): m/z (%) 623(40), 673(30),
734(100), 1346(7), 1446(48, M+).
bis(trifluoroacetate) (16b)
The product is obtained as a white solid (77 mg, 100%),
mp > 190 °C. 1H NMR (300 MHz, DMSO-d6): 1.30–1.75 (m, 8H),
2.00 –2.20 (m, 4H), 2.40–2.60 (t, J = 6.87, 4H), 3.15–3.30 (dd,
J1 = 5.89, J2 = 11.55, 4H), 3.45–3.60 (m, 16H), 4.15–4.30 (m, 4H),
4.35–4.50 (m, 6H), 4.55–4.60 (br s, 4H), 5.13 (s, 2H), 6.65–6.70
(br d, J = 8.36, 4H), 6.95–7.05 (br d, J = 8.32, 4H), 7.15–7.30 (m,
20H), 7.96 (s, 2H), 8.35–8.40 (t, J = 5.49, 2H), 8.45–8.55 (d,
J = 7.97, 2H), 8.50–8.90 (br s, 4H), 9.14 (br s, 2H), 11.81 (s, 2H).
13C NMR (75 MHz, DMSO-d6): 24.2, 29.3, 33.0, 40.5, 41.5, 48.2,
52.2, 55.8, 63.4, 68.9, 69.6, 114.9, 123.8, 126.5, 128.1–128.9,
140.2, 143.9, 152.6, 156.2, 170.9, 174.1. C76H94N16O13: MS (ES):
m/z (%) 494 (11), 721 (100), 1440 (5, M+). IR (cmꢂ1): 3286, 2926,
4.7.4. N-(4-tert-Butoxybenzyl)-N5-((Z)-[(tert-butoxycarbonyl)-
imino]{[4-(4-phenyl-1H-1,2,3-triazol-1-yl)butanoyl]amino}-
methyl)-N2-(diphenylacetyl)-D-ornithinamide (15d)
Column chromatography: (EA Rf = 0.6); the product is a white
solid (122 mg, 72%), mp > 190 °C. 1H NMR (300 MHz, CDCl3): 1.31
(m, 9H), 1.40 (m, 9H), 1.50–1.90 (m, 4H), 2.10–2.50 (m, 4H),
3.15–3.45 (m, 2H), 4.20–4.50 (m, 4H), 4.52–4.70 (m, 1H), 4.96
(m, 1H), 6.84–6.90 (m, 2H), 7.02–7.09 (m, 2H), 7.18–7.45 (m,
13H), 7.68–7.71 (m, 1H), 7.78–7.83 (m, 2H). C48H58N8O6: MS
(ES): m/z (%) 843(100, M+). C48H58N8O6: calc. C 68.4 H 6.9 N 13.3,
found: C 67.2 H 7.6 N 13.0.
1656, 1515, 1450, 1362, 1198, 1127, 700. UV (MeOH): k(
e)
269(3 ꢁ 103). HPLC: tr = 12.6 min.
4.8.3. ((14Z,20R)-15-Ammonio-1-({(4R)-4-[(diphenylacetyl)-
amino]-5-[(4-hydroxybenzyl)amino]-5-oxopentyl}amino)-20-
{[(4-hydroxybenzyl)amino]carbonyl}-3,13,22-trioxo-23,23-
diphenyl-5,8,11-trioxa-2,14,16,21-tetraazatricos-14-en-1-
ylidene)ammonium bis(trifluoroacetate) (16c)
The product is obtained as a white solid (70 mg, 100%),
mp > 190 °C. 1H NMR (300 MHz CD3OD): 1.40–1.90 (m, 8H),
3.20–3.30 (br m, 4H), 3.65–3.80 (m, 8H), 4.15–4.30 (m, 4H),
4.40–4.50 (m, 2H), 5.07 (s, 2H), 6.65–6.75 (br d, J = 8.37, 4H),
7.00–7.05 (br d, J = 8.37, 4H), 7.20–7.30 (m, 20H). 13C NMR (75
MHz, DMSO-d6): 25.5, 30.3, 42.1, 43.8, 54.3, 58.7, 71.3, 71.4,
72.2, 116.4, 128.2, 129.6–130.3, 140.9, 154.7, 157.9, 173.3–
174.9. C62H72N10O11: MS (ES): m/z (%) 567(100), 1133(13, M+).
IR (cmꢂ1): 3284, 2928, 1655, 1515, 1451, 1358, 1199, 1130,
4.7.5. tert-Butyl [(Z)-[(4-{[4-({bis[(1-{4-[({(1Z)-({5-[(4-tert-but-
oxybenzyl)amino]-4-[(diphenylacetyl)amino]-5-oxopentyl}-
amino)[(tert-butoxycarbonyl)amino]methylene}amino)car-
bonyl]benzyl}-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-
1,2,3-triazol-1-yl]methyl}benzoyl)amino]({5-[(4-tert-butoxyb-
enzyl)amino]-4-[(diphenylacetyl)amino]-5-oxopentyl}imino)-
methyl]carbamate (15e)
Column chromatography: (EA/MeOH 9:1 Rf = 0.7); the product
is
a
pale yellow solid (208 mg, 43%), mp > 190 °C.
1023, 1000, 823, 700. UV (MeOH): k(
e
) 277(2 ꢁ 103). HPLC:
C141H165N25O18: MS (ES): m/z (%) 834 (20), 1250 (100), 2400 (2),
tr = 12.6 min.
2500 (8, M+).
4.8.4. ({(4R)-4-[(Diphenylacetyl)amino]-5-[(4-hydroxybenzyl)-
amino]-5-oxopentyl}amino){[4-(4-phenyl-1H-1,2,3-triazol-1-
yl)butanoyl]amino}methaniminium trifluoroacetate (16d)
The product is obtained as a white solid (117 mg, 100%),
mp > 190 °C. 1H NMR (400 MHz, DMSO-d6): 1.35–1.49 (m, 2H),
1.49–1.75 (m, 2H), 2.12–2.21 (p, J1 = 7.04, J2 = 7.14, 2H), 2.47–
2.53 (m, 2H), 3.17–3.26 (dd, J1 = 6.59, J2 = 13.01, 2H), 4.09–4.20
(m, 2H), 4.30–4.36 (dd, J1 = 7.87, J2 = 14.15, 1H), 4.43–4.49 (t,
J = 6.91, 2H), 5.12 (s, 1H), 6.65–6.69 (d, J = 8.49, 2H), 6.98–7.02
(d, J = 8.51, 2H), 7.16–7.24 (m, 2H), 7.25–7.30 (m, 8H), 7.31–
7.35 (m, 1H), 7.40–7.46 (m, 2H), 7.80–7.85 (m, 2H), 8.30–8.36
(t, J = 5.80, 1H), 8.45–8.48 (d, J = 8.06, 1H), 8.56 (s, 1H), 8.60–
8.90 (m, 2H), 9.12–9.20 (m, 1H), 11.85 (s, 1H). 13C NMR (101
MHz, DMSO-d6): 24.3, 29.4, 33.0, 40.5, 41.7, 48.6, 52.3, 56.0,
115.0, 116.0, 121.4, 125.1, 126.6, 127.9–129.2, 130.8, 140.4,
146.4, 152.8, 156.3, 159.1, 171.0, 174.3. C39H42N8O4: MS (ES):
m/z (%) 687(100, M+). IR (cmꢂ1): 3285, 2930, 1628, 1516,
4.8. General procedure for preparation of compounds 16a–16e
The appropriate Boc- and tBu-protected ligand (15) (0.03–
0.15 mmol) was dissolved in 5 mL of DCM/TFA 1:1 and stirred for
2 h. The solvent was removed completely under reduced pressure
and the oily residue was repeatedly dissolved in DCM and the sol-
vent evaporated to remove TFA traces. The deprotected ligands
were purified by HPLC for the cell assays.
4.8.1. (E)-{[4-(4-{4-[1-(4-{[(1Z)-Ammonio({(4R)-4-
[(diphenylacetyl)amino]-5-[(4-hydroxybenzyl)amino]-5-
oxopentyl}amino)methylene]amino}-4-oxobutyl)-1H-1,2,3-
triazol-4-yl]phenyl}-1H-1,2,3-triazol-1-yl)butanoyl]amino}-
({(4R)-4-[(diphenylacetyl)amino]-5-[(4-hydroxybenzyl)amino]-
5-oxopentyl}imino)methanaminium bis(trifluoroacetate) (16a)
The product is obtained as a pale yellow solid (75 mg, 100%),
mp > 190 °C. 1H NMR (300 MHz, CD3OD): 1.40–1.90 (m, 8H),
2.20–2.60 (m, 8H), 3.10–3.25 (m, 4H), 4.15–4.25 (m, 4H), 4.30–
4.40 (m, 2H), 4.40–4.60 (m, 4H), 5.06 (s, 2H), 6.70 (d, J = 8.53,
4H), 7.00 (d, J = 8.49, 4H), 7.20–7.35 (m, 20H), 7.85–7.90 (m, 2H),
8.35–8.40 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 24.3, 29.4, 33.0,
40.5, 41.7, 48.6, 52.3, 56.0, 115.0, 116.0, 121.4, 125.1, 126.6,
127.9, 129.2, 130.8, 140.4, 146.4, 152.8, 156.3, 159.1, 171.0,
174.3. C72H78N16O8: MS (ES): m/z (%) 649(100), 1296(6, M+). IR
(cmꢂ1): 3285, 2934, 1638, 1514, 1445, 1359, 1194, 1128, 699. UV
1448, 1379, 1199, 1132, 694. UV (MeOH): k(
e
) 235(15ꢀ103).
HPLC: tr = 13.8 min.
4.8.5. (Ammoniotris{methylene-1H-1,2,3-triazole-4,1-diylmeth-
ylene-4,1-phenylenecarbonylimino[({4-[(diphenylacetyl)amino]-
5-[(4-hydroxybenzyl)amino]-5-oxopentyl}amino)methylylidene]})
triammonium tetrakis(trifluoroacetate) (16e)
The product is a pale yellow solid (82 mg, 100%), mp > 190 °C.
1H NMR (300 MHz DMSO-d6): 1.40–1.70 (m, 12H), 3.30–3.40 (br
m, 6H), 4.15–4.50 (m, 15H), 5.13 (s, 3H), 5.82 (s, 6H), 6.65–6.70
(MeOH): k(
e
) 277 (16 ꢁ 103). HPLC: tr = 13.3 min.

S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
9865
(br d, J = 8.46, 6H), 7.00–7.05 (br d, J = 8.47, 6H), 7.15–7.30 (m, 30
H), 7.45–7.55 (br d, 6H), 7.95–8.00 (br d, 6H), 8.35–8.55 (br m,
9H), 8.85–8.95 (br s, 5H), 9.26 (br s, 3H), 11.58 (br s, 2H). 13C
NMR (101 MHz, DMSO-d6): 24.4, 29.3, 33.2, 41.5, 45.9, 52.2, 52.3,
55.8, 114.9, 126.5, 128.1–129.0, 131.2, 140.2, 140.4, 141.5, 153.2,
156.2, 167.2, 170.9. C114H117N25O12: MS (ES): m/z (%) 509(30),
678(100), 1015(40), 2030(8, M+). IR (cmꢂ1): 3284, 2930, 1642,
1515, 1451, 1358, 1170, 1132, 699. HPLC: tr = 13.5 min.
to a value of 106/ml, cells were incubated for at least 15 min at
20 °C in the dark.
4.10.4. Fluorimetric determination of intracellular Ca2+
One-millilitre aliquots of loading buffer were filled into dis-
posable acrylic cuvettes (Sarstedt, No. 67.755). Immediately
after addition of 1 ml of the Fura-2/AM loaded cell suspension
and a magnetic stirrer, the cuvette was placed into the thermo-
statted (25 °C) stirred cell holder of a LS 50 B Luminescence
Spectrometer (Perkin Elmer, Überlingen, Germany), equipped
with a fast filter accessory. Fluorescence signals were registered
(instrument settings: excitation 340/380 nm, emission 510 nm,
4.9. Radioligand binding
Y1R radioligand binding studies on SK-N-MC cells were per-
formed as described elsewhere,18 except for [3H]NG-propionyl-
BIBP-3226 as radioligand (specific activity 3.6 TBq/mmol,
Kd = 1.1 nM, c = 1.5 nM) and an incubation period of 20 min. Ki val-
ues were calculated using the Cheng–Prusoff equation.19
slits 10 nm, resolution 0.1, stirrer low) after addition of 10
ll
of 20 M porcine NPY (Bachem Biochemica GmbH, Heidelberg,
l
Germany), dissolved in 10 mM HCl, supplemented with 0.1%
BSA, for 300 s.
4.10. NPY Y1 receptor antagonist activity in human
erythroleukemia (HEL) cells
4.10.5. Calculation of Ca2+ concentrations
Calcium concentrations were calculated from dual wavelength
fluorescence intensities according to the Grynkiewicz equation:21
The title compounds were screened for NPY Y1 receptor antag-
onism by measuring the inhibition of the NPY-stimulated increase
in intracellular [Ca2+].
ðR ꢂ Rmin
Þ
½Ca2þꢃ ¼ Kd
ꢀ
ꢀ SFB
ð1Þ
ðRmax ꢂ RÞ
were Kd (224 nM)22 is the dissociation constant of the Fura-2–
4.10.1. Cell culture
Ca2+-complex,
R
is the experimental fluorescence ratio value
The human HEL erythroleukemia cell line was kindly provided
by Dr. M.C. Michel, Department of Pharmacology and Pharmaco-
therapy, University of Amsterdam, The Netherlands. Cell line bank-
ing and quality control were performed according to the ‘seed
stock concept’.20 Cells were maintained as suspension cultures in
antibiotic-free RPMI 1640 medium (Sigma, Deisenhofen, Germany)
(F340/F380), Rmin and Rmax are the fluorescence value ratios (F340
/
F380) under Ca2+-free and Ca2+-saturation conditions, respectively,
and SFB is the ratio of fluorescence intensities for Ca2+-bound/
Ca2+-free indicator, measured at 380 nm. Rmin, Rmax and SFB were
determined by calibration experiments, performed in every test
series.
containing 0.3 g/l L-glutamine, 2 g/l NaHCO3 and 5% foetal calf ser-
To measure Rmax, 10
ll of an aqueous solution of 2% digitonin
um (Biochrom AG, Berlin, Germany) using 75-cm2-culture flasks
(Thermo Fisher Scientific (Nunc GmbH & Co. KG), Langenselbold,
Germany) in a water saturated atmosphere (95% air / 5% CO2) at
37 °C. The cells were passaged weekly by 1:10 dilution with fresh
culture medium. Cells were routinely monitored for, and shown to
be free of, Mycoplasma contamination with the VenorTMGeM-
Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).
(Sigma), were pipetted into the cuvette, whereas Rmin was deter-
mined after subsequent addition of 50
(in 1 M Tris/HCl, pH 8.7).
ll of a 0.6 M EGTA solution
4.10.6. Determination of NPY Y1 receptor antagonist activity
The cells were pre-incubated with 10 l of the putative antago-
l
nists (dissolved either in 50% ethanol or DMSO) for 1 min, prior to
the stimulation with porcine NPY at a final concentration of 10 nM.
The Ca2+-signal in every fifth cuvette was taken as the non-inhib-
ited reference (100%), that is, the cells were only exposed to the
respective solvent, before NPY stimulation.
4.10.2. Preparation of the cells
The day before testing, 25 ml of a plateau-phase culture were
transferred to a 175-cm2 flask Thermo Fisher Scientific (Nunc
GmbH & Co. KG), Langenselbold, Germany) containing 125 ml of
fresh culture medium. After 24 h, the suspension (2–
4 ꢁ 105 cells/ml), was centrifuged for 10 min at 200 g and room
temperature. After resuspension in 10 ml of loading buffer
(25 mM HEPES (Sigma, Deisenhofen, Germany), 120 mM NaCl,
5 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose), pH 7.4, cell
number was determined with a hemocytometer (Neubauer, im-
proved), and the cells were adjusted to a density of 1.3 ꢁ 106/ml
by addition of an appropriate volume of loading buffer.
4.10.7. Calculation of IC50- and Kb-values
IC50- values were calculated from at least two antagonist con-
centrations, inhibiting the NPY-stimulated increase in intracellu-
lar [Ca2+] between 20% and 80%. The mean percentual inhibition
values (P), determined from at least two independent experi-
ments, performed on different days, were logit transformed,
and IC50-values (logit P = 0) were determined according to the
equation
4.10.3. Loading of the cells with the Ca2+ indicator Fura-2/AM
To three volumes of the prepared cell suspension, one volume of
loading dispersion22 was added, before the cells were incubated in
the dark at room temperature for 30 min. The loading dispersion
was freshly made by mixing 10 ml of loading buffer, containing
P
logitðPÞ ¼ log
ð2Þ
100 ꢂ P
by linear regression (SigmaPlot 8.0 software).
Kb-values were calculated according to the Cheng–Prusoff equa-
tion19 with an EC50-value for pNPY of 1.8 nM.
20 mg/ml bovine serum albumin (BSA), with 50 ll of Pluronic-F-
127 (Invitrogen, Karlsruhe, Germany) (20% in DMSO), and 40
ll
Acknowledgements
of Fura-2/AM (Invitrogen) (1 mM in anhydrous DMSO).
Cells were centrifuged (200g, 7 min), resuspended in fresh load-
ing buffer and allowed to stand for another 30 min at room tem-
perature in the dark. After two washing/centrifugation cycles
(loading buffer, 200g, 7 min) and adjustment of the cell number
The authors are grateful to Mrs. Elvira Schreiber for expert tech-
nical assistance. This work was supported by the Graduate Training
Program (Graduiertenkolleg) GRK 760, ‘Medicinal Chemistry:

9866
S. Weiss et al. / Bioorg. Med. Chem. 16 (2008) 9858–9866
Buschauer,
Neuropeptide
A.
Y
Structure–Activity
Receptor Antagonists. In Handbook of Experimental
Relationships
of
Nonpeptide
Molecular Recognition—Ligand–Receptor Interactions’, of the
Deutsche Forschungsgemeinschaft.
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2004; Vol. 162, pp 505–546; (c) Schneider, E.; Keller, M.; Brennauer, A.;
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Buschauer, A. Chembiochem 2007, 8, 1981.
Supplementary data
10. (a) Suhs, T.; König, B. Chem. Eur. J. 2006, 12, 8150; (b) Suhs, T.; König, B. Mini-
Rev. Org. Chem 2006, 3, 315.
11. To be reported elsewhere.
12. DeMong, D. E.; Williams, R. M. Tetrahedron Lett. 2001, 42, 3529.
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