Structure-activity relationships of neuropeptide Y Y1 receptor antagonists related to BIBP 3226

Article abstract of DOI:10.1016/S0960-894X(00)00292-4

Analogues of BIBP 3226, (R)-N(α)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y₁ antagonism (Ca²⁺-assay, HEl cells) and binding on Y₁, Y₂ and Y₅ receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y₁ activity. Combination with N(G)-phenylpropyl arginine and a N(α)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y₅. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00292-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1597±1600
Structure±Activity Relationships of Neuropeptide Y Y1 Receptor
Antagonists Related to BIBP 3226y
Iris Aiglstorfer, Ingo Hendrich, Christiane Moser, Gunther Bernhardt,
Stefan Dove and Armin Buschauer*
Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany
Received 31 January 2000; accepted 16 May 2000
AbstractÐAnalogues of BIBP 3226, (R)-Na-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated
for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetra-
hydrobenzazepinyl group preserves most of the Y1 activity. Combination with a NG-phenylpropyl arginine and a Na-p-biphenyly-
lacetyl moiety shifted the NPY receptor selectivity towards Y5. # 2000 Elsevier Science Ltd. All rights reserved.
Previously, o-guanidino- and o-aminoalkanamides,1
structurally derived from arpromidine-like histamine H2
receptor agonists, were reported as novel neuropeptide
Y (NPY) Y1 antagonists. Except for the backbone, these
compounds resemble BIBP 3226 (1),2 an argininamide
with both high NPY Y1 receptor anity and selectivity,
with respect to the nature and arrangement of the `term-
inal' diaryl, guanidine, and hydroxyphenyl groups. Hybrid
compounds were synthesized combining the argininamide
backbone of BIBP 3226 or partial structures derived from
the C-terminal dipeptide of NPY with structural elements
of arpromidine- and amide-type NPY antagonists.3 Based
on these investigations and supported by molecular
modeling studies we have suggested that the binding
sites of NPY Y1 antagonists with one or two basic groups
are not identical. In order to get more information about
the interaction of argininamides with the human Y1
receptor we further varied the N- and the C-terminus as
well as the terminal basic group of argininamides.
Additional to the functional assay for Y1 antagonism
(inhibition of the NPY-stimulated increase in [Ca2+]i in
HEL cells), radioligand binding studies with Y1, Y2, and
Y5 receptors were performed for selected compounds.
NG-nitroarginine and (R)-Na-Z-Nd-Boc-ornithine or via
batchwise solid-phase synthesis using Rinke amide resin
and (R)-Na-Fmoc-NG-Pmc-Arg-OH as arginine build-
ing block. (R)-Na-Boc-NG-nitroarginine was activated
with N,N0-carbonyldiimidazole (CDI) and coupled with
the pertinent primary and secondary amines (Scheme 1).
After cleavage of the Na-Boc protecting group with tri-
¯uoroacetic acid (TFA), the substituted (R)-NG-nitro-
argininamides were acylated with arylalkanoic acid N-
hydroxysuccinimidyl esters. The title compounds (Table
1) were obtained after hydrogenation using Pd-C (10%)
as catalyst in acetic acid.4 The phenylpropyl substituted
argininamides 31, 32 (Table 1) were prepared using (R)-
Na-Z-Nd-Boc-ornithine as building block. After amidation
with a secondary amine, the Z group was cleaved by
hydrogenation over Pd-C (10%) catalyst and the obtained
Nd-Boc-ornithinamides were Na-acylated with p-biphenyl-
ylacetic acid N-hydroxysuccinimidyl ester. The Boc group
was cleaved with TFA and the ornithinamides were gua-
nylated with S-methyl-N-(3-phenylpropyl)isothiourea
according to standard procedures.5 Argininamides with
a C-terminal carboxamide group (14±16) were prepared
by batchwise solid-phase synthesis starting from Na-
Fmoc-protected phenylglycine or p-hydroxyphenyl-
glycine anchored to Rinke amide resin. The Fmoc group
was removed with 30% piperidine in anhydrous DMF.
For the amide preparation (R)-Na-Fmoc-NG-Pmc-
arginine and diphenylacetic acid were allowed to react
with the solid-phase bound amine component using 2-
[(1H)-benzotriazol-1-yl]-1,1,3,3-tetramethyluronium-tetra-
¯uoroborate (TBTU) and diisopropylethylamine (DIPEA)
for the coupling process. Deprotection of Arg(Pmc) and
cleavage of the products from the polymeric support
Chemistry
The argininamides were synthesized according to a pre-
viously described procedure3 either from (R)-Na-Boc-
yDedicated to Professor Dr. Dr. Dr.h.c. W. Schunack, Berlin, on the
occasion of his 65th birthday.
*Corresponding author. Tel.: +49-941-9434827; fax: +49-941-9434820;
e-mail: armin.buschauer@chemie.uni-regensburg.de
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00292-4

1598
I. Aiglstorfer et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1597±1600
was carried out with 95% TFA. The arginylphenyl-
glycinamides 14, 15 and the arginyl-p-hydroxy-
phenylglycinamide 16 were puri®ed by preparative
reversed-phase HPLC.
Pharmacology
The compounds were screened for their Y1-antagonistic
activity in human erythroleukemia (HEL) cells by mea-
suring the inhibition of porcine NPY (10 nM) stimulated
increase in the intracellular Ca2+ concentration in a
FURA-assay1 (for structures and biological data see
Scheme 1. (i) CDI, primary or secondary amines, THF, rt; (ii) TFA,
rt; (iii) arylalkanoyl-N-hydroxysuccinimidyl ester (acyl group cf.
compds 1±30, R1=Ph2CH-, Ph-C6H4-CH2-, Ph3C-, p-Cl-C6H4-CH-
Ph, (p-Cl-C6H4)2CH-), DIPEA, THF, 40 ꢀC; (iv) H2/Pd-C (10%), 5
bar, HOAc, rt.
Table 1. Structures and NPY Y1 antagonistic activities of argininamides in HEL cells (calcium assay)a,1
No.
R2
R4
R2 con®g.
Y1-antag.a
IC50 [mM]
No.
R2
R4
R2 con®g.
Ð
Y1-antag.a
IC50 [mM]
1b
2c,d
OH
H
Ð
Ð
0.017
0.11
20
21.8
76
3c
4c
H
OH
Ð
Ð
2.20
0.28
21
22
23
24
25
26
27
28
29
30
Ð
S
5c
6c
7
H
H
OH
R
S
RS
21
1.4
1.0
inact.
inact.
51
Ð
Ð
Ð
Ð
S
8
9
10
H
H
OH
R
S
RS
0.20
2.5
0.06
11
12
R
S
22.3
0.9
0.081
1.1
13
1R, 3S
inact.
3.0
14
15
16
H
H
OH
R
S
R
4.6
7.5
8.1
Ð
S
0.17
> 10e
1.1
17
18
19
RS
inact.
3.75
Ð
31
32
H
OH
Ð
Ð
inact.
11.9
11.5
aInhibition of NPY (10 nM) induced increase in [Ca2+]i; antagonist concentrations used: 0.01, 0.025, 0.1, 0.5, 1, 10, 50 and 100 mM depending on
activity; compounds designated inactive produced less than 20% inhibition at 100 mM.
bBIBP 3226.
cCompounds 2±6 cf. ref 3.
dSee also ref 7.
e28.7% Inhibition at 10 mM; the compound could not be tested in higher concentrations due to induction of cytolysis.

I. Aiglstorfer et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1597±1600
1599
Table 1). Experiments were performed in triplicate at
antagonist concentrations between 0.01 and 100 mM, and
IC50 values were calculated.1 The SEM of the percentual
inhibition at a single concentration was always less than
10%, resulting in an error of pIC50 (-log IC50) of less
than 0.35 log units. Additionally, selected substances
were investigated in radioligand binding studies on Y1-
(SK-N-MC cells),3 Y2- (SMS-KAN cell membranes)1 and
Y5-receptors (Y5-transfected HEC-1B cells)6 by displace-
ment of [3H]propionyl-NPY (1 nM) according to the
procedures described elsewehere1,3,6 in detail (Table 2).
The assays were run in triplicate.
Figure 1. NPY-induced increase in intracellular [Ca2+] (HEL cells).
Concentration±response curves of (&) pNPY alone and in presence of
10 nM (&), 25 nM (~) and 100 nM (*) of compound 10. The right-
ward shift of the concentration±response curves for NPY in the
presence of compound 10 corresponds to a KB value of 3.01 Æ 0.44
nM (pKB=8.52, slope constrained to 1.0) or a pA2 value of 8.38 (slope
1.17) determined by Schild plot.
Structure±Activity Relationships
In order to investigate in which spatial position a certain
bulk is tolerated by the Y1-receptor, argininamides 3±7
with conformationally constrained benzylamide groups
were synthesized (2±6: ref. 3, 2 and other analogues of 1
see also ref. 7. Among these bicyclic compounds3 the 7-
hydroxybenzazepine derivative 4 is the most active one.
Introduction of a `para-equivalent' OH group in the
indane moiety slightly increases Y1-antagonistic activity
(7 versus 6; note that 7 is a mixture of diastereomers).
Among the diastereomers 8 and 9, the (R,R)-con®gurated
N-[1-phenylethyl]argininamide 8 is 13 times more active
than 9 (R,S). Introduction of an OH group in the phenyl-
ethylamide moiety further increases Y1-receptor anity,
but 10 (mixture of diastereomers)8 is 4 times less potent
than 1 (BIBP 3226). Representativeconcentration response
curves for NPY in the presence of 10 are shown in Fig. 1.
suggested that the dipeptide Arg-Phg-NH2 interacts with
the Y1 receptor in a di€erent way than the C-terminus of
NPY. Among the pyridylalkyl substituted arginina-
mides 18 and 19, the shorter homolog is the more active
one, whereas the piperidine derivative 17 shows no Y1
antagonism. Comparing the benzylamide 2 and its pyridine
analogue 18, the reduced activity of the latter may be
due to the basicity or an unfavorable charge distribution
of the pyridine ring.
Previous investigations regarding the N-terminus of argi-
ninamides have shown, that homologization and vicinal
arrangement of the phenyl rings reduce Y1-receptor
antagonistic activity.3 Incorporation of a p-biphenyl-yl-
acetyl (20±22) or a triphenylacetyl group (23, 24) results in
inactive or only weakly active compounds. Interestingly,
p-Cl monosubstitution in the diphenylacetyl moiety may
increase Y1-antagonistic activity (25 versus 2, 26 versus
3), indicating that hydrophobic and/or van-der-Waals
interactions might contribute to binding in this position.
The e€ect of p,p0-di-Cl substitution is varying (28±30).
A hydroxymethyl group (11±13) results in decreased Y1
antagonistic activity compared to the parent compounds
2 and 6. The absolute con®guration of R2 in the more
active indane diastereomer 6 (S) does not correspond to
the eutomers of the methyl and the hydroxymethyl
derivatives 9 (R) and 12 (S), respectively. Thus, overlap
of the `additional' bulk of 6 with that of 9 and 12 on
binding is unlikely.
Compounds containing the C-terminal dipeptide Arg35-
Tyr36-NH2 of NPY were nearly inactive.3 The phenyl-
glycinamide derivatives 14±16 are moderate Y1 antago-
nists. In contrast to 2, 6 and 8, introduction of an OH
group decreases Y1-receptor anity (16 versus 14). It is
Replacement of the diphenylacetyl by a p-biphenylyl-
acetyl group together with phenylpropyl substitution of
the terminal guanidine (31, 32) decreases Y1-antagonistic
activity (31 versus 21) but leads to a change in receptor-
subtype selectivity: 32 has 29 times higher anity to the
Y5-receptor than to the Y1-receptor (see Table 2).
Compound 32, in some respect resembling a recently
published NPY-antagonistic amidine9 (selectivity data
not given in ref. 9), shows submicromolar anity to the
Y5-receptor.
Table 2. Radioliganda binding data (IC50ÆSEM [mM]) of selected
substances in SK-N-MC cells (Y1), SMS-KAN cell membranes (Y2)
and HEC-1B cells (Y5)
Compds
Y1
Y2
Y5
1
4
8
10
12
14
25
27
32
0.0059 (Æ0.001)
0.67 (Æ0.1)
0.15 (Æ0.03)
0.019 (Æ0.003)
0.184 (Æ0.009)
2.4 (Æ0.2)
>100
247 (Æ22)
177 (Æ43)
575 (Æ105)
540 (Æ120)
>400
97 (Æ11)
59 (Æ17)
78 (Æ9)
80 (Æ7)
25 (Æ3)
Radioligand binding data of selected substances on SK-N-
MC cells (Y1), SMS-KAN cell membranes (Y2) and HEC-
1B cells (Y5) show that the substituted argininamides
(except for 32, see above) preferentially bind to the Y1
receptor (see Table 2). Surprisingly, increasing hydro-
phobicity in the diphenylacetyl portion (25, 27) enhances
Y5-anity. For the nine compounds considered, Y1
antagonistic activity in the functional test (pIC50=-log
IC50) is very highly correlated with pIC50 values in the
binding assay:
48 (Æ2)
75 (Æ2)
68 (Æ7)
163 (Æ36)
2.6 (Æ0.3)
11.9 (Æ0.6)
0.77 (Æ0.08)
0.17 (Æ0.01)
5.3 (Æ0.4)
22 (Æ2)
aRadioligand: [3H]propionyl-NPY (1 nM); assays were run in tripli-
cate; the procedures are described elsewhere in detail.1,3,6

1600
I. Aiglstorfer et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1597±1600
A.; Merz, S.; Malawska, B.; Bernhardt, G.; Dove, S.;
Buschauer, A. Regul. Pept. 1998, 75±76, 9.
pIC50ꢀCa2‡ ˆ 0:78ꢀÆ0:11†pIC50ꢀbind:† ‡ 1:31ꢀÆ0:70†
†
4. (R)-Na-Diphenylacetyl-N-[(RS)-1-(4-hydroxyphenyl)-ethyl]
argininamide (10): (R)-Na-Diphenylacetyl-NG-nitro-N-[(RS)-1-
(4-hydroxyphenyl)ethyl]argininamide (0.26 g, 0.49 mmol) was
dissolved in 60% acetic acid and hydrogenated at room tem-
perature in an autoclave (5 bar, H2/Pd-C 10%). The catalyst was
®ltered o€, washed with 60% acetic acid and the ®ltrate was
evaporated in vacuo. The crude product was puri®ed chroma-
tographically on silica gel with CHCl3:MeOH (1:1) as eluent.
Yield: 0.20 g (0.37 mmol; 62%) hygroscopic yellow solid;
r ˆ 0:94 s ˆ 0:35 F ˆ 53:6
Conclusions
The C-terminal 4-hydroxybenzamide moiety of BIBP
3226 (1) seems to be optimal for Y1 antagonism although
a certain bulk, which may be also part of a bicyclic struc-
ture (e.g., of a benzazepinyl group), is well tolerated. At
the N-terminus, compounds with a diphenylacetyl or
even more with a (RS)-4-chlorodiphenylacetyl group are
most active. This suggests to prepare Cl-substituted posi-
tional isomers and derivatives with other substituents.
Interestingly, a p-biphenylylacetyl moiety as N-terminus
in combination with a phenylpropyl substituent at the
guanidino group and with a C-terminal hydroxybenza-
zepine leads to Y5-receptor anity in the submicro-
molar range. Further work is ongoing to investigate whe-
ther this structure indeed represents a promising lead for
new Y5 receptor ligands. Possibly, compounds with com-
bined Y1 and Y5 antagonism are interesting as potential
drugs, as both NPY receptor subtypes appear to be
associated with increased food intake.
C28H32N5O3 CH3CO2H (547.65); mp 76±80 ꢀC (CHCl3:
.
MeOH); [a]2d5=+9.4Æ2ꢀ (c=0.16 g/100 ml; MeOH); IR
(KBr): n 3408 br (OH, NH), 3061w, 3013w (Ar-H), 2958w,
1
2883w (CH), 1652s (CO); H NMR (300 MHz, DMSO-d6): d
1.27 (d, J=6.7Hz, 3H, CH3), 1.39 71 (m, 4H, CH2CH2CH2),
3.00 17 (m, 2H, CH2CH2CH2), 4.29±4.31 (m, 1H, CH), 4.76±
4.81 (m, 1H, CHCH3), 5.11 and 5.13 (2s, 0.6/0.4H, Ph2CH),
6.04 (br, 3H, NH), 6.66 (d, J=8.3 Hz, 2H, Ar-OH), 7.03 (d,
J=8.3Hz, 2H, Ar-OH), 7.21±7.30 (m, 10H, Ph), 7.99±8.29 (br,
1H, OH), 8.31±8.37 (m, 1H, NHCH), 8.40±8.46 (m, 1H,
NHCH), 8.61±8.68 (m, 1H, NHCH2), 9.32 (br, 1H, NH);
+FAB±MS m/z (relative intensity): 975 (>1, [2M+H]+), 488
(100, [M+H]+), 167 (62, [Ph2CH]+).
5. Taniguchi, K.; Katsura, Y.; Ueda, I.; Matsuo, M. Chem.
Pharm. Bull. 1992, 40, 240.
6. Moser, C.; Bernhardt, G.; Michel, J.; Schwarz, H.;
Buschauer, A. Can. J. Physiol. Pharmacol. 2000, 78, 134.
7. Rudolf, K.; Eberlein, W.; Engel, W.; Beck-Sickinger, A.G.;
Wittneben, H.; Wieland, H. A.; Doods, H. N. In Neuropeptide
Y and Drug Development; Grundemar, L.; Bloom S. R., Eds.;
Academic Press: San Diego, 1997; Chapter 9, pp 175.
8. Compound 10 was among a series of substances presented by
our group on a poster at the 5th NPY meeting, Grand Cayman,
1999. At the same meeting Gedda et al. (ASTRA-Hassle, Moln-
dal, Sweden) presented the (R,R)-con®gurated stereoisomer of 10,
(R)-Na-diphenylacetyl-N-[(R)-1-(4-hydroxyphenyl)ethyl]arginin-
amide (H 409/22), as a potent and selective Y1-antagonist (Gedda,
K.; Berglund, M.-L.; Larefalk, A.; Nilson, A.-K.; Ebstand, J.;
Vauquelin, G.; Chkajlani, V. Abstracts of Papers, 5th Interna-
tional NPY Meeting, April 17±22, 1999; Grand Cayman; p 25).
The reported IC50 value of 13.6 nM (radioligand binding, SK-N-
MC cells using [3H]propionyl-NPY (0.5 nM)) is in good agree-
ment with our data (19 nM, cf. Table 2).
Acknowledgements
The authors are grateful to Mrs. S. Dechant and Mrs.
E. Schreiber for excellent technical assistance and to the
Fonds der Chemischen Industrie for a grant.
References and Notes
1. Muller, M.; Knieps, S.; Gessele, K.; Dove, S.; Bernhardt,
G.; Buschauer, A. Arch. Pharm. Pharm. Med. Chem. 1997,
330, 333.
2. Rudolf, K.; Eberlein, W.; Engel, W.; Wieland, H. A.;
Willim, K. D.; Entzeroth, M.; Wienen, W.; Beck-Sickinger, A.
G.; Doods, H. N. Eur. J. Pharmacol. 1994, 271, R11.
3. Aiglstorfer, I.; U€recht, A.; Gessele, K.; Moser, C.; Schuster,
9. Ito, S.; Sagara, T.; Koito, K.; Nishioka, T.; Ouchi, K.;
Fukuroda, N. Jpn. Kokai Tokkyo Koho JP 10287637 A2,
1998; Chem. Abstr. 1998, 129, 330479.