Preparation of Fluorescent Nonpeptidic Neuropeptide Y Receptor Ligands: Analogues of the Quinazoline-type Anti-obesity Y5 Antagonist CGP 71683A

Article abstract of DOI:10.1002/ardp.200300813

As part of a programme to develop fluorescence-based methods for the study of the interactions between G-protein coupled receptors (GPCRs) and their ligands the preparation of low molecular weight fluorescence-labelled neuropeptide Y (NPY) Y₅ a

Full text of DOI:10.1002/ardp.200300813

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 585–590
Fluorescent Nonpeptidic Neuropeptide Y Receptor Ligands 585
Liantao Lia,
Preparation of Fluorescent Nonpeptidic
Matthias Mayerb,
Erich Schneiderb,
Elvira Schreiberb,
Günther Bernhardtb,
Shiqi Penga,
Neuropeptide Y Receptor Ligands: Analogues of the
Quinazoline-type Anti-obesity Y5 Antagonist
CGP 71683A
Armin Buschauerb
As part of a programme to develop fluorescence-based methods for the study of the
interactions between G-protein coupled receptors (GPCRs) and their ligands the
preparation of low molecular weight fluorescence-labelled neuropeptideY (NPY)Y5
antagonists is reported. The naphthylsulfonyl group in the potent quinazoline-type
NPYY5 receptor antagonist CGP 71683A was replaced with a dansyl, nitrobenzox-
adiazole (NBD) or acridine-9-carbonyl group. In radioligand binding studies on hu-
man Y5 receptor expressing HEC-1B cells the substances labelled with acridine
(Ki 311 nM) and NBD (Ki > 1000 nM) proved to be moderately active or inactive, re-
spectively. By contrast, a Ki value of 49 nM was found for the dansyl analogue com-
pared to 2 nM for CGP 71683A.No binding toY1 receptors (SK-N-MC cells, displace-
ment of [3H]propionyl-NPY) was detected for the new compounds at concentrations
Յ 1 µM.
a
College of Pharmaceutical
Sciences, Peking University,
100083, Beijing, PR China
b
Institute of Pharmacy,
University of Regensburg,
D-93040 Regensburg,
Germany
Keywords: Neuropeptide Y; Y5 receptor antagonist; Fluorescence labelling;
Quinazoline; CGP 71683A
Received: May 6, 2003; Accepted July 9, 2003 [FP813]
DOI 10.1002/ardp.200300813
suitable fluorescence dyes conferring additional affinity
may result in highly potent antagonists [2, 3]. Here, we
report on a first approach to prepare fluorescent low mo-
lecular weight NPY antagonists.
Introduction
Fluorescent probes offer attractive alternatives to the ap-
plication of radioligands, e.g., to detect receptors in cells
and tissues, and to study drug-receptor interactions on
soluble or cell-bound targets. As part of a programme to
develop fluorescence-based methods for the study of lig-
and receptor interactions at G-protein coupled receptors
(GPCRs) [1] histamine and neuropeptideY (NPY) recep-
tors were selected as models to design fluorescent
probes. Previously, by using cyanine5-labelled NPY
(cy5-NPY) we demonstrated that the affinity of agonists
and antagonists can be determined in a very convenient
manner by flow cytometry under equilibrium conditions
[1].In contrast to the labelled peptide cy5-NPY that binds
to all NPY receptors the use of selective fluorescent lig-
ands should be of advantage to detect receptor sub-
types in tissue preparations and on cells.Usually, the de-
crease in affinity induced by incorporation of bulky fluor-
ophores into low molecular weight ligands is much more
pronounced than in case of peptides. Nevertheless, as
demonstrated very recently for histamine H1 and H2 re-
ceptor ligands, the combination of pharmacophores with
Among the NPY receptors (for nomenclature see
IUPHAR recommendations [4]) especially theY1 and Y5
receptors have been considered interesting drug tar-
gets, e.g. for the development of new anti-obesity drugs.
In search for low molecular weight fluorescentY5 recep-
tor [5] ligands we started from the quinazoline derivative
CGP 71683A [6, 7] (Scheme 1) which is a highly potent
and probably the most intensively studied NPYY5 recep-
tor antagonist. Numerous structural modifications of the
arylsulfonamide moiety were shown to be tolerated with-
out loss of theY5 antagonistic activity.Therefore, we used
the
quinazolinylaminomethylcyclohexylmethylamine
scaffold to design several fluorescent Y5 receptor
ligands.
Chemistry
The reference compound 6 a as well as the fluorescent
analogues 6 b–d were prepared according to standard
procedures as shown in Scheme 1. The building block
trans-1,4-bis(aminomethyl)cyclohexane (2) [8] was syn-
thesized from N-Boc-protected tranexamic acid [9] via
formation of the amide 1 by activation with 1,1Ј-carbonyl-
Correspondence: Armin Buschauer, Institute of Pharmacy,
University
of
Regensburg,
Universitätsstraße
31,
D-93040 Regensburg, Germany. Phone: +49 941 943-4827,
Fax: +49 941 943-4820;
e-mail: armin.buschauer@chemie.uni-regensburg.de
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

586 Li et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 585–590
Scheme 1. Synthesis of the quinazoline-type NPY Y5 antagonists.
diimidazole (CDI) and subsequent ammonolysis, fol-
lowed by deprotection with trifluoroacetic acid (TFA) and
reduction with LiAlH4.The diamine 2 was allowed to react
with 4-amino-2-chloroquinazoline (3) [10] to give the
amine 4 which was coupled with naphthalene-1-sulfonyl
chloride (5 a), 5-dimethylaminonaphthalene-1-sulfonyl
chloride (5 b), 4-chloro-7-nitrobenzo[2,1,3]oxadiazole
(5 c) or acridine-9-carbonyl chloride (5 d) to give 6 a–d.
Pharmacology
The NPY Y5 receptor affinities of the compounds 6 a–d
were determined by radioligand binding studies (dis-
placement of [3H]propionyl-NPY) on human endometrial
carcinoma cells stably transfected with the humanY5-re-
ceptor (hY5-HEC-1B cells) [11]. Additionally, the com-
pounds 6 b–d were investigated for Y1 receptor binding
Table 1. NPY receptor binding data of compounds 6 a–d and longest wavelength absorption and fluorescence emis-
sion maxima of the fluorescence-labelled ligands.
Substance No.
λmax Exa
(nm)
λmax Ema
(nm)
Y5 receptor bindingb Y1 receptor bindingc
Ki [nM]
Ki [nM]
6 ad (CGP 71683A)
2.06 0.25
6 b
6 c
6 d
340
483
359
482
537
432
49.05 9.43
>1000
311 127
+ 1000
+ 1000
+ 1000
a
Determined with solutions of the compounds in a buffer (NaCl 120 mM, KCl 5 mM, MgCl2 2 mM, CaCl2 1.5 mM, Hepes
25 mM, glucose 10 mM, adjusted to pH 7.4 with NaOH).
b
c
d
determined from the displacement of [3H]propionyl-NPY (0.8 nM) from human Y5 receptors stably expressed in
HEC-1B cells [11].
Investigated on SK-N-MC cells using [3H]propionyl-NPY (1 nM) as the radioligand according to the procedure de-
scribed elsewhere.
Reported: IC50 2.9 nM [12].
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 585–590
Fluorescent Nonpeptidic Neuropeptide Y Receptor Ligands 587
Figure 1.Y5 receptor binding of compounds 6 a (CGP 71683A), 6 b and 6 d on stably hY5-transfected HEC-1B cells [11]
(displacement of [3H]propionyl-NPY, 0.8 nM); mean SEM, experiments performed in triplicate).
(radioligand:[3H]propionyl-NPY) on human neuroblasto-
ma (SK-N-MC) cells.The data are summarized inTable 1
and displacement curves for 6 a, b, and d are shown in
Figure 1.
only very minor binding to both NPY receptors. Accord-
ing to a ligand-receptor interaction model published by
Islam et al.[16] the sulfonamide-NH ofY5 antagonists re-
lated to CGP 71683A may form a hydrogen bond with
histidine-398 in transmembrane domain 6 of the recep-
tor protein and the terminal aromatic rings are suggest-
ed to increase the affinity by hydrophobic interactions.In
agreement with this model the NBD-labelled compound
6 c, which is lacking an amide group, is only very weakly
binding to Y5 receptors. By contrast, Ki values in the na-
nomolar range were determined for the amides 6 b and
6 d. Although the activity of 6 a (Ki 2 nM) was not
achieved, the dansyl analogue 6b, a very close conge-
ner of 6 a, showed rather highY5 receptor affinity with a Ki
value of 49 nM.Compound 6 d, having a more bulky acri-
dine-9-carbonyl group instead of the naphthalene-1-sul-
fonyl group in CGP 71683A, was about 150-fold less ac-
tive than the reference compound.
Results and discussion
The 2,4-diaminoquinazoline motif was the scaffold of
some early moderately active Y5 antagonists with slight
Y1/Y5 selectivity [12]. Structural modifications led to the
discovery of numerous quinazoline-type Y5 antagonists
with different substitution patterns [7, 12–15]. A consid-
erable increase inY5 receptor affinity and selectivity was
achieved by the combination of the diaminoquinazoline
moiety with a N-(cyclohexylmethyl)arylsulfonamide sub-
structure as in compound CGP 71683A [6, 7]. As the
naphthalene-1-sulfonyl residue is not essential, we se-
lected this portion of the molecule for structural modifica-
tion to obtain several fluorescent analogues of CGP
71683A. The congeners 6 b and 6d proved to be selec-
tive forY5 compared toY1 receptors, whereas 6 c showed
Many attempts have been made to use fluorescent lig-
ands as an alternative to radioligands for the study of re-
ceptors.This approach is hampered especially when re-
ceptors are to be investigated in their natural environ-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

588 Li et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 585–590
ment, i.e.in membranes or in whole cells as in case of G-
protein coupled receptors (GPCR), due to a low signal-
to-noise ratio resulting from autofluorescence.This prob-
lem can be overcome by the use of fluorophores emitting
light of long wavelengths as, for instance, cyanine dyes
such as cy5.In this respect, the dyes used for the prepa-
ration of the fluorescentY5 antagonists 6 b–d are far from
being optimal. On the other hand, the bulk of cyanine
dyes may limit their value as labelling reagents for small
molecules.As an alternative, regardless of fluorescence
at shorter wavelengths, a shift in the emission spectrum
on binding of the ligand or high intensity of the emitted
light compared to autofluorescence could be sufficient to
solve the aforementioned problem of detection.General-
ly, fluorescent GPCR antagonists should be superior to
labelled agonists, as the latter may be internalised after
receptor binding and activation resulting in apparently
non-displaceable binding of the ligand. Also for NPY re-
ceptors agonist-induced β-arrestin interaction and re-
ceptor internalisation was demonstrated [17–19]. The
dansyl group is known to emit fluorescence light with
high intensity and it is a close structural analogue of the
naphthalene-1-sulfonyl group present in CGP 71683A.
The first results obtained with compound 6 b in binding
studies are promising with respect to the development of
highly potent and selective fluorescent probes for Y5 re-
ceptors.The optimization of the fluorescence properties
and the investigation of the applicability of such com-
pounds to study Y5 receptors in tissues and on whole
cells is subject of ongoing work.
Acknowledgements
The authors are grateful to the DAAD for a scholarship to
L. Li (DAAD “sandwich PhD programme”), to the Deut-
sche Forschungsgemeinschaft for financial support to
the Graduiertenkolleg“Medicinal Chemistry”(GRK 760),
and to the Fonds der Chemischen Industrie for grants to
G. B. and A. B.
Experimental
Melting points (m.p.; are uncorrected) were determined on a
Büchi 530 apparatus (Uster, Switzerland). Elemental analyses
(indicated by elemental symbols) were performed at the analyt-
ical department of the faculty of chemistry and pharmacy, Uni-
1
versity of Regensburg. The structures were confirmed by H-
NMR spectra using a Bruker WM 250 (250 MHz; δ in ppm rela-
tive to TMS as internal standard; J in Hz) (Bruker, Karlsruhe,
Germany) and mass spectroscopy (EI-MS: Varian MAT 112S.
CI-MS and +FAB-MS:Varian MAT 95. +FAB-MS: xenon, MeOH/
glycerol. CI-MS: desorption chemical ionization (DCI), ammo-
nia) (Varian, Darmstadt, Germany). Excitation/emission spec-
tra were recorded with a Kontron UV-VIS spectrometer
(Kontron, Neufahrn, Germany) and a Perkin-Elmer LS-50B
spectrofluorimeter (Perkin Elmer, Überlingen, Germany).
Chromatographic separations on a preparative scale were car-
ried out on a Chromatotron model 8294 (Harrison Research,
Muttenz, Switzerland) using glass rotors with 4 mm layers of
silica gel 60 PF254 containing gypsum (Merck, Darmstadt, Ger-
many) or on a preparative HPLC system from Knauer (Well-
chrom preparative pump K-1800; UV-detector K-2000, precol-
umn Eurospher 100-C18, column Eurospher 100-C18
B962170 (5 µm, 250 × 40 mm)) (Knauer, Berlin, Germany).
The building blocks 2 [8] (CAS no.10029-07-9), 3 [10] (CAS no.
59870-43-8), 4 [7] (hydrochloride: CAS no. 192323-10-7), and
5 d [20] as well as theY5 antagonist 6 a [7] (hydrochloride: CAS
no. 192321-23-6) were prepared according to or by analogy
with the methods described in the literature.The derivatization
reagents 5 a–c are commercially available. The spectral and
analytical data of synthesized compounds are in agreement
with the structures shown in Scheme 1.
Conclusion
As demonstrated previously by using cy5-NPY, the flow
cytometric determination of receptor affinity represents
an interesting alternative to conventional radioligand
binding studies. Availability of suitable fluorescent
probes provided, this technique may also offer perspec-
tives to develop multiparametric fluorimetric methods to
determine both affinity and functional data.
trans-4-[[(tert-Butoxycarbonyl)amino]methyl]cyclohexanecar-
boxylic amide (1)
A solution of 1,1Ј-carbonyldiimidazole (2.0 g, 12.3 mmol) and
trans-4-[[(tert-butoxycarbonyl)amino]methyl]cyclohexanecarbo-
xylic acid [9] (2.27 g, 8.8 mmol) in 30 mL of anhydrousTHF was
stirred at room temperature for 1 h. After cooling to 0 °C, the
mixture was gassed with dry NH3 at 0 °C for 20 min and then
stirred at room temperature for 5 h.The precipitate was filtered
off and washed with THF to obtain 2.23 g (98 %) of 1 as colour-
less crystals, m.p. 195 °C (dec.). – Analysis C13H24N2O3
(256.34): calcd. C 60.91 H 9.44 N 10.93; found C 60.64 H 9.39
N 11.21. – EI-MS: m/z (%) = 256 (<1, M᭹+), 57 (100, [C4H9]+). –
1H-NMR (DMSO-d6): δ (ppm) = 7.14 (s, 1 H), 6.78 (t, J = 5.8,
1 H), 6.62 (s, 1 H), 2.74 (t, J = 6.3, 2 H), 1.97 (m, 1 H), 1.70 (m,
4 H), 1.36 (s, 9 H), 1.24 (m, 2 H), 0.82 (m, 2 H).
The results onY5 antagonists presented in this paper as
well as data presented in the literature on other classes
of compounds, e.g. recent publications on histamine re-
ceptor antagonists [2, 3], demonstrate that, in principle,
the development of low molecular weight fluorescence-
labelled ligands of GPCRs is possible. However, com-
pared to the labelling of peptides or proteins the affinity
of the resulting fluorescent probes is much more affected
by the bulk of the fluorophores.Thus, the spectrum of ap-
propriate dyes is restricted to structures conferring addi-
tional receptor affinity.As predictions are nearly impossi-
ble, the combination of pharmacophore and fluorophore
must be optimized in each individual case.
trans-1,4-Cyclohexanebis(methylamine) (2)
Compound 1 (4.5 g, 17.6 mmol) was stirred with 10 mL of tri-
fluoroacetic acid at room temperature for 48 h. After removal of
the volatiles in vacuo the residue crystallized from EtOAc to
give the trifluoroacetate of trans-4-(aminomethyl)cyclohexane-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 585–590
Fluorescent Nonpeptidic Neuropeptide Y Receptor Ligands 589
carboxylic amide in quantitative yield. M.p. 77–78 °C; EI-MS:
m/z (%) = 157 (<1, M ), 139 (39), 126 (18), 30 (100). Lithium
N2-{trans-4-[(7-Nitrobenzo[2,1,3]oxadiazol-4-ylamino)methyl]-
cyclohexylmethyl}quinazoline-2,4-diamine (6 c)
᭹+
aluminium hydride (2.0 g) was added portionwise to the
suspension of trans-4-(aminomethyl)cyclohexanecarboxylic
amide (4.5 g, 11.8 mmol) in 60 mL of anhydrous THF at room
temperature.The mixture was heated under reflux for 5 h. After
cooling THF was removed by evaporation under reduced pres-
sure, and the residue was treated with 100 mL of CHCl3. Hy-
drolysis was carried out by dropwise addition of water.The solid
was filtered off, the filtrate was dried over NaSO4 and evaporat-
ed to give 1.3 g (78 %) of 2 as colourless oil which was used
without purification for further reactions. C8H18N2 (142.2);
Cl-MS: m/z (%) = 143 (100, [M + H]+), 160 (15, [M + NH4]+).
NBD-chloride (5 c, 20.6 mg, 0.103 mmol) was added to the so-
lution of 4 (26.7 mg, 0.093 mmol) in a mixture of CHCl3 (2 mL),
DMSO (1 mL) and Et3N (50 µL).The mixture was stirred at room
temperature in the dark for 24 h. After evaporation in vacuo the
residue was chromatographed twice (Chromatotron, EtOAc:
MeOH, 10:1) to give 15 mg (36 %) of 6 c as a brown solid, m.p.
65 °C. – HR-+FAB-MS: C22H25N8O3 (M + H)+, calcd. 449.20496,
found: 449.20303. – +FAB-MS: m/z (%) = 449 (100, [M + H]+). –
1H-NMR (acetone-d6): δ = 8.51 (d, J = 8.7, 1 H), 8.40 (br, 1 H),
7.91 (d, J = 8.3, 1 H), 7.51 (m, 1 H), 7.32 (d, J = 8.3, 1 H), 7.04
(m, 1 H), 6.65 (br, 2 H), 6.49 (d, J = 8.7, 1 H), 5.80 (br, 1 H),
3.60–3.50 (m, 2 H), 3.35–3.27 (m, 2 H), 2.03–1.85 (m, 4 H),
1.76–1.36 (m, 2 H), 1.18–1.04 (m, 2 H).
4-Amino-2-[trans-(4-aminomethylcyclohexyl)methylamino]-
quinazoline (4)
Acridine-9-carboxylic acid {trans-4-[(4-aminoquinazolin-2-
The solution of 4-amino-2-chloroquinazoline (3) [10] (200 mg,
1.1 mmol) and 2 (176 mg, 1.2 mmol) in 200 mL of methanol was
heated under reflux for 16 h. After concentration in vacuo the
mixture was chromatographed (Chromatotron, CHCl3 : MeOH,
1:1, ammonia atmosphere) to obtain 150 mg (48 %) of 4 as pale
yellow solid, m.p. 91 °C ([7]: mp. of the monohydrochloride:
189–192 °C). – Analysis: C16H23N5 × H2O (303.41), calcd. C
63.34, H 8.31, N 23.08; found C 63.29, H 8.38, N 23.39. –
Cl-MS: m/z (%) = 286 (13, [M + H]+), 171 (93), 144 (35), 116
(100).
ylamino)methyl]cyclohexylmethyl}amide (6 d)
The mixture of 130 mg (0.58 mmol) of acridine-9-carboxylic
acid and 3 mL of thionyl chloride was heated under reflux for
3 h. After removal of the volatiles under reduced pressure, the
residue was dried in vacuo to give acridine-9-carboxylic acid
chloride (5 d) as a yellow powder which was dissolved in 5 mL
of abs. CHCl3 and added to a solution of 4 (140 mg, 0.49 mmol)
and 200 µL of diisopropylethylamine in 10 mL of abs. CHCl3 at
0 °C (N2 atmosphere).The mixture was stirred at room temper-
ature overnight, then 10 mL of CHCl3 and 5 mL of water were
added, the organic layer was washed with 5 mL of water and
dried over sodium sulfate. After removal of the solvent the resi-
due was purified with HPLC to give 105 mg (24.5 %) of 6 d, as
Naphthalene-1-sulfonic acid {trans-4-[(4-aminoquinazolin-2-
ylamino)methyl]cyclohexylmethyl}amide (6 a)
yellow crystals, m.p. 81 °C.
– Analysis: C30H30N6O ·
Naphthalene-1-sulfonyl chloride (5 a, 260 mg, 1.15 mmol) was
added to the solution of 4 (300 mg, 1.05 mmol) and 200 µL of
Et3N in a mixture of CHCl3 (20 mL) and DMSO (5 mL) at room
temperature under N2 atmosphere. The reaction mixture was
stirred at room temperature in the dark overnight.TLC indicated
complete disappearance of 4. After evaporation of the solvents
in vacuo, the residue was purified twice by chromatography
(Chromatotron, CHCl3 : MeOH, 95:5) to give 410 mg (82 %) of
6 a as a white solid, m.p. 99 °C ([7]: m.p. of the hydrochloride:
155–164 °C).– Analysis:C26H29N5O2S · 1.5 H2O (502.6), calcd.
C 62.13, H 6.42, N 13.93; found C 62.31, H 6.50, N 13.96. –
FAB-MS:m/z (%) = 476 (100, [M + H]+).– 1H-NMR (CD3OD):δ =
8.70 (d, J = 8.7, 1 H), 8.16 (dd, J = 7.1 and 1.2, 1 H), 8.08 (d, J =
8.3, 1 H), 7.95 (dd, J = 7.9 and 1.2, 1 H), 7.84 (dd, J = 8.3 and
1.2, 1 H), 7.69–7.48 (m, 4 H), 7.33 (d, J = 8.3, 1 H), 7.08 (m,
1 H), 3.15 (d, J = 6.7, 2 H), 2.64 (d, J = 6.7, 2 H), 1.75–1.53 (m,
4 H), 1.46–1.10 (m, 2 H), 0.85–0.55 (m, 4 H).
3 CF3COOH · 2 H2O (868.70), calcd. C 49.77, H 4.29, N 9.67;
found C 50.16, H 4.28, N 9.65.– Chromatographic HPLC sepa-
ration on analytical scale:Eurospher 100-C18 B971185 [Knau-
er], precolumn: 100-C18 B952163 [Knauer], eluent: MeOH/
0.1 % TFA (50/50), flow rate: 1 mL/min, UV: 254 nm, EX: 360
nm, EM: 460 nm. Retention time of 6 c: 10.6 min. Preparative
HPLC: Eurospher 100-C18 B962170 [Knauer], precolumn:
100-C18 [Knauer]; eluent: MeOH/0.1 % TFA (50/50), flow rate:
60 mL/min, UV:254 nm.Retention time of 6 c:11.5 min.– +FAB-
MS: m/z (%) = 491 (100, [M + H]+). – H-NMR (CD3OD): δ =
1
12.51 and 11.60 (br, 1 H), 9.05 (t, J = 5.6, 1 H), 9.00–8.50 (br,
2 H), 8.21 (d, J = 8.7, 2 H), 8.19 (d, J = 7.9, 1 H), 7.99 (d, J = 8.7,
2 H), 7.91 (dd, J = 6.7 and 1.2, 2 H), 7.80 (t, J = 7.7, 1 H), 7.69
(dd, J = 6.7 and 1.2, 2 H), 7.55–7.30 (m, 2 H), 3.46–3.20 (m,
4 H), 1.90 (s, 4 H), 1.64 (s, 2 H), 1.05 (m, 4 H).
Receptor binding data
SK-N-MC and HEC-1B cells were obtained from the American
Type Culture Collection (Rockeville, MD, USA). The HEC-1B
cells were stably transfected with the humanY5 receptor as de-
scribed previously [11].The cultivation of the cells and the per-
formance of the radioligand binding studies using
[3H]propionyl-NPY (Amersham, Braunschweig, Germany) as
the radioligand on Y1 (SK-N-MC cells) and Y5 receptors (hY5-
HEC-1B cells) followed the procedures described elsewhere in
detail [11, 21]. Ki values were calculated from IC50 values ac-
cording to the Cheng-Prusoff equation [22]. The experiments
were done in triplicate.
5-(Dimethylamino)naphthalene-1-sulfonic acid {trans-4-[(4-ami-
noquinazolin-2-ylamino)methyl]cyclohexylmethyl}amide (6 b)
Dansyl chloride (5 b, 30 mg, 0.11 mmol) was added to the solu-
tion of 4 (421 mg, 0.073 mmol) and 40 µL of Et3N in a mixture of
2 mL of CHCl3 and 0.5 mL of DMSO.The mixture was stirred at
room temperature in the dark for 10 h, then another 29 mg of
dansyl chloride was added.The mixture was stirred for 20 h.Af-
ter evaporation of the solvent in vacuo the residue was purified
twice with a Chromatotron (CHCl3 : MeOH, 3:1) to give 30 mg
(79 %) of 6 b, as hygroscopic pale green solid. HR-+FAB-MS:
C28H35N6O2S (M + H+), calcd.: 519.2524, found: 519.2517;
+FAB-MS:m/z (%) = 519 (100, [M + H]+).– 1H-NMR( DMSO-d6):
δ (ppm) = 8.43 (d, J = 8.7, 1 H), 8.29 (d, J = 8.5, 1 H), 8.06 (d, J =
7.1, 1 H), 7.94 (d, J = 7.9, 1 H), 7.87 (t, J = 5.5, 1 H), 7.65–7.40
(m, 4 H), 7.35–7.10 (m, 3 H), 6.99 (t, J = 7.3, 1 H), 6.80–6.20 (br,
1 H), 3.08 (t, J = 7.1, 2 H), 2.80 (s, 6 H), 2.59 (t, J = 7.1, 2 H),
1.75–1.50 (m, 4 H), 1.48–1.05 (m, 2 H), 0.85–0.55 (m, 4 H).
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