Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y2 receptor antagonists

Article abstract of DOI:10.1002/cmdc.201100241

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug-likeness of numerous biologically active compounds, including ligands of G-protein-coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide-type neuropeptideY (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N^G-acylated argininamides were obtained by guanidinylation with tailor-made mono-Boc-protected N-acyl-S-methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N^G-substituted (S)-argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N^G-acylated or -carbamoylated analogues with a terminal amine were superior (Y₂R: K_i and K_B values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4-5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for invitro studies of the Y₂R. The results support the concept of bioisosteric guanidine-acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.

Full text of DOI:10.1002/cmdc.201100241

DOI: 10.1002/cmdc.201100241
Application of the Guanidine–Acylguanidine Bioisosteric
Approach to Argininamide-Type NPY Y2 Receptor
Antagonists
Nikola Pluym, Albert Brennauer, Max Keller, Ralf Ziemek, Nathalie Pop, Gꢀnther Bernhardt,
and Armin Buschauer*[a]
Strongly basic groups such as guanidine moieties are crucial
structural elements, but they compromise the drug-likeness of
numerous biologically active compounds, including ligands of
G-protein-coupled receptors (GPCRs). As part of a project fo-
cused on the search for guanidine bioisosteres, argininamide-
type neuropeptide Y (NPY) Y2 receptor (Y2R) antagonists relat-
ed to BIIE0246 were synthesized. Starting from ornithine deriv-
atives, NG-acylated argininamides were obtained by guanidiny-
lation with tailor-made mono-Boc-protected N-acyl-S-methyli-
sothioureas. The compounds were investigated for Y2R antago-
nism (calcium assays), Y2R affinity, and NPY receptor subtype
selectivity (flow cytometric binding assays). Most of the NG-
substituted (S)-argininamides showed Y2R antagonistic activi-
ties and binding affinities similar to those of the parent com-
pound, whereas NG-acylated or -carbamoylated analogues with
a terminal amine were superior (Y2R: Ki and KB values in the
low nanomolar range). This demonstrates that the basicity of
the compounds, although 4–5 orders of magnitude lower than
that of guanidines, is sufficient to form key interactions with
acidic amino acids of the Y2R. The acylguanidines bind with
high affinity and selectivity to Y2R over the Y1, Y4, and Y5 recep-
tors. As derivatization of the amino group is tolerated, these
compounds can be considered building blocks for the prepara-
tion of versatile fluorescent and radiolabeled pharmacological
tools for in vitro studies of the Y2R. The results support the
concept of bioisosteric guanidine–acylguanidine exchange as a
broadly applicable approach to retain pharmacological activity
despite decreased basicity.
Introduction
Guanidino groups (including arginine residues) are crucial
structural features of numerous ligands of G-protein-coupled
receptors (GPCRs). Such strongly basic centers (pKa ꢀ13) are
almost quantitatively protonated at physiological pH, adversely
affecting bioavailability and brain penetration. This is true for
many high-affinity agonists and antagonists of aminergic and
peptidergic GPCRs, such as histamine[1,2] and neuropeptide Y
(NPY)[3,4] receptors, respectively. NPY is a highly conserved 36-
residue peptide, related to peptide YY (PYY) and pancreatic
polypeptide (PP), and plays an important role as neurotrans-
mitter in the central and peripheral nervous system.[3,4] In
humans, the biological effects of NPY, PYY and PP (the NPY or
pancreatic polypeptide family) are mediated by four receptor
subtypes, designated Y1, Y2, Y4, and Y5 (YxR, x=1, 2, 4, or 5).
Orally available brain-penetrant antagonists have been devel-
oped, mostly for the Y1R and Y5R, and in the meantime, a few
for the Y2R such as JNJ-5207787,[5] SF-11,[6] SF-21,[6] and JNJ-
31020028.[7]
Arginine derivatives such as the R-configured Y1R antagonist
BIBP3226[8] and the S-configured Y2R antagonist BIIE0246[9] (1,
Figure 1) are still highly valuable pharmacological tools regard-
less of having properties that are far from drug-like. These
compounds are reference compounds for pharmacological in-
vestigations and attractive prototypical molecules for proof-of-
concept studies to explore the range of tolerated structural
modifications of the guanidine group. Using histamine and
NPY receptors as models, our research group is in the process
[a] N. Pluym,+ Dr. A. Brennauer,+ Dr. M. Keller, Dr. R. Ziemek, Dr. N. Pop,
Prof. Dr. G. Bernhardt, Prof. Dr. A. Buschauer
Institut fꢀr Pharmazie, Pharmazeutische/Medizinische Chemie II
Universitꢁt Regensburg, Universitꢁtsstr. 31, 93053 Regensburg (Germany)
Fax: (+49)941-943-4820
E-mail: armin.buschauer@chemie.uni-regensburg.de
[+] These authors contributed equally to this work.
Figure 1. Structures of the Y1R-antagonistic (R)-argininamide BIBP3226 and
Y2R-antagonistic (S)-argininamide BIIE0246 (1).
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100241.
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A. Buschauer et al.
MED
of applying bioisosteric ap-
proaches to biologically active
compounds, with the aim of de-
creasing basicity while retaining
or increasing potency; this ap-
proach could yield prodrugs, bi-
valent ligands, and receptor-
subtype-selective tools, such as
radiolabeled and fluorescent li-
gands. Previously, the replace-
ment of guanidine groups with
acyl- or carbamoylguanidine
moieties proved to be a very
successful bioisosteric approach
for histamine H2, H3, and H4 re-
ceptor ligands,[10–14] and for argi-
Scheme 2. Preparation of carboxylic acids 6 and 7. Reagents and conditions: a) NaN3 (1.2 equiv), H2SO4, CHCl3,
10 h, reflux; b) NaBH4 (2 equiv), EtOH, 1 h, reflux; c) SOCl2, CHCl3, 30 min, reflux; d) piperazine (5 equiv), 1,4-diox-
ane, 30 min, reflux; e) 3,3-tetramethylene glutaric anhydride (1 equiv), CH2Cl2, 18 h, RT.
ninamide-type NPY Y1R antagonists[15–17] derived from
BIBP3226 (Figure 1). The introduction of various electron-with-
drawing substituents at the NG-position of BIBP3226 resulted
in analogues with similar or even increased Y1R antagonistic
condensation of 1,2-diphenylhydrazine and ethyl allophanate,
which was prepared from urea and ethyl chloroformate.[21]
For the synthesis of building block 6,[19] anthraquinone was
converted into 5H-dibenzo[b,e]azepine-6,11-dione (3) by
Schmidt reaction (Scheme 2). The resulting ketone was re-
duced to the alcohol 4 by sodium borohydride in ethanol at
reflux. 11-Hydroxy-5,11-dihydrodibenzo[b,e]azepine-6-one (4)[19]
was converted into the 11-chloro derivative using thionyl chlo-
ride. Subsequently, the alkyl chloride was allowed to react with
activities,[16] and opened
a route toward fluorescence-la-
beled[17,18] and tritiated[15] highly potent Y1R antagonists. The
basicity of these derivatives is 4–5 orders of magnitude lower
than that of the parent molecule, but still high enough to un-
dergo key interactions with acidic amino acids of the receptor
protein.
excess
piperazine
to
yield
11-piperazin-1-yl-5,11-
Encouraged by the aforementioned examples of successful
guanidine–acylguanidine bioisosteric exchange, we synthe-
sized substituted (S)-argininamides related to BIIE0246 with de-
creased basicity, aiming at potent and selective Y2R antago-
nists. Moreover, the attachment of terminal amino groups was
considered, with respect to future conjugation with amine-re-
active fluorescent dyes or radiolabeling reagents for the prepa-
ration of Y2R tracers.[18]
dihydrodibenzo[b,e]azepine-6-one (5).[19] Finally, reaction of pi-
perazine 5 with 3,3-tetramethylene glutaric anhydride afforded
6.[19] In an analogous manner, carboxylic acid 7 was prepared
from 1-benzhydrylpiperazine. The preparation of the reference
compound BIIE0246 (1) and the NG-unsubstituted compound 8
is outlined in Scheme 3.[19,20,22]
The acylguanidine derivative 9 as well as the citrulline and
Nw’-nitroarginine derivatives 10[23] and 11 were prepared from
the corresponding Na-tert-butoxycarbonyl (Boc)-protected
amino acids by activation, coupling with 2, Na deprotection,
and acylation using the succinimidyl ester of 7. For the prepa-
ration of 9, the phthaloyl group was cleaved off by hydrazinol-
ysis, followed by guanidinylation of the amine with S-methyli-
Results and Discussion
Synthesis
The amide nitrogen of the (S)-argininamide-type Y2R antago-
nists bears a 2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)eth-
yl group as in 1.[19] The required building block 2 was prepared
by N-alkylation of deprotonated 1,2-diphenylurazole (1,2-di-
phenyl-1,2,4-triazolidine-3,5-dione) with N-(2-bromoethyl)ph-
thalimide (Scheme 1), followed by hydrazinolysis of the phtha-
loyl protective group.[20] 1,2-Diphenylurazole was obtained by
Scheme 3. Preparation of BIIE0246 (1) and compound 8. Reagents and condi-
tions: a) 2 (1 equiv), HOBt (1 equiv), EDC (1 equiv), DMF, 16 h, 08C!RT;
b) Et2NH/DMF (1:10 v/v), 10 min, RT; c) 6 or 7 (1 equiv), HOBt (1 equiv), EDC
(1 equiv), DIPEA (1 equiv), DMF, 16 h, 08C!RT; d) TFA/CH2Cl2 (1:1 v/v), 2 h,
RT. Pbf=2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl.
Scheme 1. Synthesis of 4-(2-aminoethyl)-1,2-diphenyl-1,2,4-triazolidine-3,5-
dione (2). Reagents and conditions: a) xylene, 24 h, reflux; b) KOtBu
(1.1 equiv), DMF, N-(2-bromoethyl)phthalimide (1.1 equiv), 5 h, reflux;
c) 1. N2H5OH (2.75 equiv), THF/MeOH, 16 h, 2. 2m HCl, 2 h, RT.
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NPY Y2 Receptor Antagonists
toxycarbonyl-1H-pyrazole-1-car-
boxamidines 13–15 as guanidi-
nylation
reagents
(Table 1,
Method A). However, the syn-
thesis of these reagents was
less convenient, and the use of
Boc2O/DMAP, required for pro-
tection of the monoacylated in-
termediate, is incompatible with
various functional groups. The
conditions for synthesis of the
pertinent guanidinylation re-
agents are summarized in
Table 1.
Scheme 4. Preparation of compounds 9–11. Reagents and conditions: for Y=HNC(=X)NH2: a) 1. CDI (1.01 equiv),
CH2Cl2, 30 min, RT, 2. 2, 12 h, RT; b) TFA/CH2Cl2 (1:1 v/v), 1 h, RT; c) succinimidyl 2-{1-[2-(4-benzhydrylpiperazin-1-
yl)-2-oxo-ethyl]cyclopentyl}acetate (1 equiv), DIPEA (2 equiv), MeOH/THF, 16 h, RT; for Y=NPht: a) 2 (1 equiv), DCC
(1 equiv), HOBt (1 equiv), THF, 20 h, 08C!RT; b) HCl in EtOAc, 2 h, RT; c) 7 (1 equiv), EDC (1 equiv), HOBt (1 equiv),
DIPEA (2 equiv), CH3CN, 16 h, 08C!RT; d) 1. N2H5OH (2.5 equiv), MeOH, 16 h, RT, 2. 1m KHSO4, 3 h, RT; e) 16, HgCl2
(1 equiv), DMF, 16 h, RT; f) TFA/CH2Cl2 (1:1 v/v), 2 h, RT.
sothiourea 16 and Boc deprotection of the guanidi-
no moiety (Scheme 4).
The NG-substituted argininamides were prepared
by guanidinylation of the corresponding ornithina-
mide precursor 12 (Scheme 5). The Boc and phthalo-
yl groups were used as orthogonal protecting
groups for Na and Nd in the ornithine moiety, respec-
tively. Acylation with 2 followed by Boc deprotection
and a second acylation step yielded the phthaloyl-
protected ornithine amide building block. The free
amine 12 was obtained after hydrazinolysis
(Scheme 5). The variously substituted NG-acylated ar-
ginine derivatives were prepared in good yield and
high purity by guanidinylation of the primary amine
with the appropriate guanidinylating agent (13–31;
Scheme 5 and Table 1). Finally, the Boc-protected ar-
gininamides obtained by guanidinylation were de-
protected with 50% trifluoroacetic acid in dichloro-
methane to yield the NG-monoacylated arginina-
mides 32–50 (Scheme 5).
To elaborate appropriate protocols for the guanidi-
nylation step, we explored various synthetic routes.
The best results were obtained by analogy with the
frequently applied aminolysis of bis(alkoxycarbonyl)-
protected isothioureas,[24] when one of these protect-
ing groups was replaced with the acyl residue re-
quired for the preparation of the title compounds.
Therefore, we synthesized a collection of various N-
acyl-N’-tert-butoxycarbonyl-S-methylisothioureas (16–
29, Scheme 5; Table 1) as building blocks for the effi-
cient and versatile preparation of N-acyl-N’-alkyl-sub-
stituted guanidines. These guanidinylation reagents
are conveniently accessible by acylation of N-Boc-S-
methylisothiourea (Table 1, Method B). For the prepa-
ration of N,N’-dialkylguanidines, N,N’-bis(tert-butoxy-
carbonyl)-S-methylisothiourea was alkylated under
Mitsunobu conditions (Table 1, Method C) to obtain
N-alkyl-N,N’-bis(tert-butoxycarbonyl)-S-methyliso-
thioureas 30 and 31 as building blocks.[25] Satisfacto-
ry yields were also obtained using N-acyl-N’-tert-bu-
Scheme 5. Preparation of 1H-pyrazole-1-carboxamidines (13–15) and S-methylisothiour-
eas (16–31) and guanidinylation of compound 12 (for R groups, see Table 1). Method A:
Synthesis of the N-acyl-N’-tert-butoxycarbonyl-1H-pyrazole-1-carboxamidines for the
preparation of NG-alkoxycarbonyl- and NG-alkanoyl-substituted Y2R antagonists; Method B:
N-Acyl-N’-tert-butoxycarbonyl-S-methylisothioureas as commonly used guanidinylating
agents; Method C: Preparation of N-alkyl-N,N’-bis(tert-butoxycarbonyl)-S-methylisothiour-
eas under Mitsunobu conditions for the synthesis of alkanoyl-substituted BIIE0246 ana-
logues. a) Acylation conditions are summarized in Table 1). Preparation of NG-substituted
argininamides 32–50. Reagents and conditions: b) 2 (1 equiv), DCC (1 equiv), HOBt
(1 equiv), THF, 20 h, 08C!RT; c) HCl in EtOAc, 2 h, RT; d) 6 (1 equiv), EDC (1 equiv), HOBt
(1 equiv), DIPEA (2 equiv), CH3CN, 16 h, 08C!RT; e) 1. N2H5OH (2.5 equiv), MeOH, 16 h,
RT, 2. 1m KHSO4, 3 h, RT; f) THF, 2 days, RT; g) HgCl2 (1 equiv), NEt3 (2 equiv), DMF, 16 h,
RT; h) TFA/CH2Cl2 1:1 (v/v), 2 h, RT.
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A. Buschauer et al.
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Table 1. Preparation of the guanidinylation reagents 13–31.
Compd
R
Acylation conditions a):
Yield [%]
Method[a]
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
CO2Me
CO2Bn
C(O)Ph
CO2Et
C(O)Bn
C(O)CH2OPh
C(O)(CH2)2Ph
CO2(CH2)3NHBoc
C(O)Et
SO2Me
88
87
80
90
70
71
81
65
95
99
99
85
62
61
80
51
45
31
55
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
C
C
R-Cl (1 equiv), DIPEA (2 equiv), CH2Cl2, 16 h, RT
R-Cl (1 equiv), DIPEA (1 equiv), CH2Cl2, 16 h, RT
[HO(CH2)3NHBoc (1 equiv) + DSC (1.5 equiv)], NEt3 (1.5 equiv), CH2Cl2, 16 h, RT
R2O (1 equiv), DIPEA (1 equiv), CH2Cl2, 16 h, RT
Cl-SO2Me (1 equiv), DIPEA (1 equiv), CH2Cl2, 16 h, RT
OCNEt (1 equiv), CH2Cl2, 16 h, RT
CONHEt
C(O)(CH2)5NHBoc
C(O)(CH2)5NHC(O)-4-F-C6H4
C(O)(CH2)3CONH(CH2)3NHBoc
C(O)(CH2CH2O)4C2H4NHBoc
CONH(CH2)3NHBoc
CONH(CH2)4NHBoc
CH2CH3
R-OH (1 equiv), HOBt (1 equiv), TBTU (1 equiv), DIPEA (2 equiv), DMF, 16 h, RT
R-NHS (1 equiv), DIPEA (2 equiv), DMF, 16 h, RT
R’-OH (1 equiv), DIAD (1.5 equiv), PBu3 (1.5 equiv), THF, 16 h, RT
CH2Ph
[a] Method A: 1H-pyrazole-1-carboxamidines are used as guanidinylation reagents; Methods B and C: S-methylisothioureas are prepared for guanidinylation.
Pharmacology
which lack the strongly basic guanidine group, revealed a dras-
tic decrease in binding affinity by several orders of magnitude
relative to 1 (Table 2). This result substantiates the relevance of
the basic center in the arginine side chain of 1.
Compounds 8–11, 32–50, and the reference substance
BIIE0246 (1) were investigated for Y2R binding and antagonism
as well as for Y2R selectivity over Y1R, Y4R, and Y5R. The Y2R
binding affinities were determined in a flow cytometric binding
assay using CHO cells stably expressing the human Y2 receptor
(hY2R)[23] and fluorescently labeled NPY. NPY Y2R antagonistic
activities were determined in a spectrofluorimetric Ca2+ assay
on CHO cells stably expressing the hY2R. Cell clones giving a
robust [Ca2+]i response upon agonistic activation were ob-
tained by co-transfection of the CHO-hY2 cells with the gene
encoding the chimeric G-protein Gqi5 (CHO-hY2-K9-qi5-K9).[26]
Selected compounds were studied in two additional func-
tional assays: a flow cytometric calcium assay, using fluo-4 as
Ca2+-sensitive fluorescent dye, and a luminescence-based ae-
quorin assay.[23] To study receptor selectivity, all compounds
were investigated in flow cytometric binding assays on human
Y1, Y4, and Y5 receptors using fluorescently labeled pNPY (Y1R,
Y5R) or [K4]hPP (Y4R) according to previously described meth-
ods (Table 2).[18,27,28]
NPY Y2R binding and antagonism
Two basic residues, Arg33 and Arg35, in the C terminus of
NPY were identified to be crucial for high Y2R affinity.[29] The
NPY mimetic argininamides related to BIIE0246 (1) also com-
prise two basic centers, namely the guanidino group in the ar-
ginine side chain and the tertiary amino nitrogen in the pipera-
zine ring. The diphenylmethyl-substituted analogue 8, also
comprising two basic moieties, shows antagonistic Y2R activity
and binding affinity in the same range as 1, whereas its ethyl
ester analogue 9 retains its potency, but exhibits decreased
binding affinity (Figure 2a). In contrast, the displacement
curves of the citrulline Nw’-nitroarginine derivatives 10 and 11,
Figure 2. a) Y2R antagonism in the flow cytometric fluo-4 Ca2+ assay with
~
&
&
*
compounds 1 ( ), 8 ( ), 9 ( ), 10 ( ), and 11 (N). Inhibition of pNPY
(70 nm)-induced calcium response in CHO-hY2-K9-qi5-K9 cells (mean values
ꢁSEM, n=3–7); b) Flow cytometric competition binding assay using 5 nm
~
&
*
*
Cy5–pNPY [for 1 ( ), 33 ( ), 38 ( ), 45 ( ), and 46 (N)], or 10 nm Dy635–
!
pNPY [for 50 ( )] as fluorescent ligand in presence of antagonists at various
concentrations (mean values ꢁSEM, n=3)
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NPY Y2 Receptor Antagonists
ancies between binding and
functional data were also ob-
served for several acylguani-
Table 2. Pharmacological data for argininamide-type Y2R antagonists.
dine-type
Y2R
antagonists
(Table 2), in particular, for com-
pounds bearing aromatic resi-
dues (e.g., 34, 38, and 43). The
discrepancies between Ki and KB
values determined in binding
and functional assays, respec-
tively, may originate from differ-
ent periods of incubation. The
functional assays were per-
formed in a time window of a
few minutes, sufficient to reach
equilibrium of the antagonists
rather than the agonist pNPY,
which is characterized by slow
binding kinetics. In contrast, the
flow cytometric binding assay
was performed after incubation
for 90 min: a time period in
which the agonist peptide also
reaches equilibrium.
Compd
R
NPY receptor binding: Ki [nm][a]
Y2R antagonism
Y2R
Y1R
Y4R
Y5R
KB [nm][b]
1
H
2.6ꢁ1.2
>5000
>7500
>5000
5.6ꢁ0.4
15ꢁ4[c,d]
4.2ꢁ0.6[e]
2.8ꢁ0.3
23ꢁ2[c,d]
1.1ꢁ0.1[e]
11.0ꢁ1
16ꢁ2[c,d]
2.2ꢁ0.2[e]
39ꢁ7
8
6.8ꢁ2.3
97ꢁ24
>5000
>5000
>5000
>5000
>7500
>7500
>7500
>7500
>5000
>5000
>5000
>5000
9
10
11
976ꢁ251
2065ꢁ652
110 ꢁ7[c,d]
21ꢁ4[e]
239ꢁ16[c,d]
35ꢁ13[e]
14ꢁ2
32
33
34
35
36
37
38
39
40
41
42
CO2Me
CO2Et
CO2Bn
50ꢁ8
50ꢁ10
95ꢁ7
7ꢁ2
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
Antagonistic activities and
binding affinities of the NG-sub-
stituted analogues 32–50 in
CHO cells stably expressing the
hY2R are summarized in Table 2.
The results indicate that the Y2R
tolerates electron-withdrawing
substituents at the NG-position,
except for sulfonyl groups (cf.
compound 42). The poor antag-
onistic activities of 42 support
the hypothesis that a certain
level of “residual guanidine ba-
sicity” is required, which is abol-
ished by strong electron-with-
drawing substituents. The NG-
acylated arginine derivatives de-
scribed herein, the parent com-
pound 1 (BIIE0246), and ana-
logues of 1 reported in the
patent literature[20,22] have struc-
tural moieties in common: the
dibenzoazepine and the 3,5-
dioxo-1,2-diphenyl-1,2,4-triazoli-
dine moiety, enabling hydro-
14ꢁ5
8.8ꢁ0.9
11ꢁ1
CO2(CH2)3NH2
CONHEt
12ꢁ2
4.3ꢁ0.6
250ꢁ24
43ꢁ5
43ꢁ11
14ꢁ2
18ꢁ1
C(O)Et
C(O)Ph
C(O)Bn
7.4ꢁ0.1
13ꢁ1
5.3ꢁ0.4
15ꢁ2
C(O)CH2OPh
C(O)(CH2)2Ph
SO2Me
6.8ꢁ2.5
583ꢁ46
ND
43
44
45
46
47
48
49
50
CH2Ph
CH2CH3
103ꢁ34
26ꢁ3
>5000
>5000
>5000
>5000
>5000
>3000
>3000
>3000
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>7500
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
4.9ꢁ0.4
14ꢁ1
C(O)(CH2)5NH2
8ꢁ1
1.2ꢁ0.2
8.4ꢁ1.4
3.9ꢁ0.5
5.3ꢁ0.5
2.1ꢁ0.1
1.6ꢁ0.2
C(O)(CH2)5NHC(O)-4-F-C6H4
C(O)(CH2)3CONH(CH2)3NH2
C(O)(CH2CH2O)4C2H4NH2
CONH(CH2)3NH2
CONH(CH2)4NH2
34ꢁ6
2.3ꢁ1.4
8ꢁ3
1.5ꢁ0.4
3.3ꢁ0.3
[a] Flow cytometric binding assays using fluorescently labeled pNPY or [K4]hPP (Y4R) in CHO-hY2-K9-qi5-K9 cells,
HEL-Y1 cells, CHO-Y4, and HEC-1B-Y5 cells, respectively (n=3–5);[23,27,28] ND: not determined. [b] Unless other-
wise indicated: inhibition of pNPY induced [Ca2+]i mobilization in CHO-hY2-K9-qi5-K9-mt-AEQ-A7 cells (fura-2)
(n=2–3 independent experiments, performed in duplicate).[23] [c] Inhibition of pNPY induced [Ca2+]i mobiliza-
tion in CHO-hY2-K9-qi5-K9-mt-AEQ-A7 cells in the luminescence based aequorin assay (n=3–6).[23] [d] Apparent
discrepancies between data from the bioluminescence-based aequorin assay and those from the spectrofluori-
metric fura-2 and the flow cytometric fluo-4 assay reflect different sensitivities; however, within a series of com-
pounds the results (order of potencies) are consistent. [e] Inhibition of pNPY induced [Ca2+]i mobilization in
CHO-hY2-K9-qi5-K9 cells using the Ca2+-sensitive fluorescent dye fluo-4 in flow cytometry (n=3–7).[23]
phobic interactions, and
a
group that is quantitatively or
The affinity-lowering effect of non-basic groups was not
always reflected to the same extent in functional studies (cf.
compound 9): the negative impact on Y2R antagonistic activity
was less pronounced in the aequorin assay, and in both the
spectrofluorimetric and flow cytometric Ca2+ assays, only a
moderate decrease in activity was observed (Figure 2). Discrep-
at least partially protonated, thereby able to undergo electro-
static interactions with the receptor. Acylguanidine analogues
of 1 and the corresponding Nd-unsubstituted ornithinamides
should exhibit Y2R antagonistic activities and binding affinities
in the same range as compound 1 due to preserved albeit di-
minished basicity. In fact, the Y2R antagonistic properties are
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1731

A. Buschauer et al.
MED
affected by the chemical nature of the acyl residue. Alkoxycar-
bonyl, alkylaminocarbonyl, and alkanoyl substituents have sim-
ilar influence on the potency of the Y2R antagonistic arginina-
mides; the respective compounds display activities ranging
from ~70 to 200% of the KB value of the unsubstituted antag-
onist 1. Compounds 45 and 47–50, bearing a terminal amino
group in the acylguanidine moiety, are more potent in the
spectrofluorimetric Ca2+ assay than 1, and their binding affini-
ties (Ki: 1.5–7.8 nm) are similar to that of 1. Clearly, polar
groups at an appropriate distance from the guanidine group
can confer additional receptor affinity. In contrast, acyl sub-
stituents comprising an aromatic residue (34, 38–41, 43, and
46) are less tolerated as determined by flow cytometry (cf.
compound 38 in Figure 2b). The introduction of a 4-fluoroben-
zoyl residue at the terminal amino group in 45 leads to an ap-
proximate eightfold decrease in both affinity and activity (com-
pound 46). However, Ki and KB values of 46 are still in the low
nanomolar range. Therefore, 46 is an interesting lead com-
pound for the design of radiolabeled (18F) Y2R antagonists.
binding of BIIE0246.[33,34] Other residues were found to be im-
portant for direct antagonist interactions, such as Tyr in TM2
and Leu in TM5.[33,35] Nevertheless, an Asp or Glu residue is
thought to bind to the guanidine moiety of BIIE0246. Clearly,
both receptors tolerate acylguanidines as a less basic replace-
ment of the guanidine motif.
Conclusions
We established a straightforward synthetic route to NG-acylar-
gininamide-type Y2 receptor antagonists. One of the key steps
is the guanidinylation of the respective ornithinamide with N’-
substituted N-Boc-S-methylisothioureas. The resulting NG-sub-
stituted (S)-argininamides related to BIIE0246—except for the
NG-sulfonyl-substituted analogues—showed Y2R antagonistic
activities and binding affinities similar to those of the parent
compound, corroborating the relevance of a basic center in
the arginine side chain. Some NG-acylated or carbamoylated
analogues containing a terminal amino residue show even
higher activities than the parent compound 1. As derivatization
of the amino group is compatible with high Y2R affinity and se-
lectivity, these compounds are considered building blocks for
the preparation of versatile pharmacological tools such as fluo-
rescent and radiolabeled ligands.
Selectivity for human Y2R over human Y1R, Y4R, and Y5R
The affinity for non-Y2R was investigated by flow cytometry
using Cy5-labeled peptides (Cy5–pNPY or Cy5–[K4]hPP) and
cells stably expressing the human Y1, Y4, and Y5 recep-
tors.[23,27,28] All investigated compounds were found to be
highly selective for Y2R. The selectivity for hY2R is 100- to 3000-
fold versus hY1R and hY5R and even higher relative to hY4R
(Table 2). Compounds 32–50 have the same argininamide core
structure and vary only in the acyl substituent at the guanidine
group. Hence, Y2R receptor selectivity is not affected by acyla-
tion of the guanidine regardless of decreased or even loss of
basicity in this part of the molecule.
In summary, the acylguanidine substructure is useful for the
synthesis of arginine-derived nonpeptidic NPY Y2R ligands with
decreased basicity. In view of recent results on Y1R and hista-
mine receptor ligands, our study corroborates the hypothesis
that guanidine–acylguanidine exchange is a promising and
broadly applicable bioisosteric approach.
Experimental Section
General
Acylguanidines as less basic bioisosteric replacements for
guanidines
Chemicals were purchased from the following suppliers: Acros Or-
ganics (Belgium), Advanced ChemTech Inc. (UK), Alfa Aesar GmbH
& Co. KG (Germany), Bachem AG (Switzerland), IRIS Biotech GmbH
(Germany), Mallinckrodt Baker (Netherlands), Merck KGaA and
Merck Biosciences GmbH (Germany), and Sigma–Aldrich Chemie
GmbH (Germany). Deuterated solvents for NMR spectroscopy were
obtained from Deutero GmbH (Germany). All solvents were of ana-
lytical grade or distilled prior to use. If moisture-free conditions
were required, reactions were performed in dried glassware under
inert atmosphere (argon or nitrogen); solvents were dried by hold-
ing at reflux over NaH (Et2O, DME, 1,4-dioxane, MTBE, THF, and tol-
uene) or P2O5 (CH3CN, CHCl3, CH2Cl2, 1,2-dichloroethane). Dried sol-
vents were stored over molecular sieves (4 ꢂ) under protective gas
(Ar). DMF (H2O<0.01%) was purchased from Sigma–Aldrich
Chemie GmbH. Et3N and DIPEA were distilled from CaH2. Parallel
syntheses were performed in a Synthesis 1 Liquid 12 reactor from
Heidolph (Germany). Optical rotations at l 589 nm (NaD line) were
determined on a Optronic P8000T Polarimeter (A. Krꢀss, Germany)
using a quartz microcuvette (layer thickness: 100 mm, volume:
1 mL, thermostated at 208C) and CH3CN/H2O (10:1 v/v) as solvent.
IR spectra were measured on an FTS 3000 MX spectrometer (Excali-
bur Series) from Bio-Rad (USA) equipped with an attenuated total
reflectance (ATR) unit (Specac Golden Gate Diamond Single Reflec-
tion ATR System). 1H and 13C NMR spectra were recorded on
Avance-300, -400, or -600 NMR spectrometers from Bruker BioSpin
As known from supramolecular chemistry, acylguanidines (like
guanidines) have the ability to form high-affinity complexes
with carboxylate groups.[30] Similar to guanidines, acylguani-
dines are capable of forming charge-assisted hydrogen bonds
with carboxylates. NMR studies on acylguanidine-type hista-
mine receptor and NPY Y1R ligands using bisphosphonate
tweezers as “biomiometic receptors” confirmed key interac-
tions with anions and suggested conformational preferen-
ces.[31] Recently, Keller et al. characterized a tritium-labeled NG-
propionylargininamide as a highly potent and selective radioli-
gand for the Y1R, prepared according to the concept of guani-
dine–acylguanidine bioisosterism.[15] Furthermore, in the field
of histamine receptor agonists, acylguanidines were identified
as bioisosteres of guanidines or mimics of amino groups.[10,14]
Receptor mutagenesis and molecular modeling studies re-
vealed that the guanidino group of the Y1R antagonistic (R)-ar-
gininamide BIBP3226 and its NG-acylated analogues interact
with a highly conserved Asp residue in TM6 of the Y1R.[32] In
the Y2R an Asp residue at the corresponding position is crucial
for agonist binding, but does not substantially contribute to
1732
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ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1727 – 1738

NPY Y2 Receptor Antagonists
GmbH (Germany). (CH3)4Si was added as internal standard (d=
0 ppm) to all samples. Multiplicities are specified with the following
abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (mul-
tiplet), br (broad signal), as well as combinations thereof. If atoms
are specified with numbers, for example, 4-H or C-2, the number-
ing is relative to substituent/heteroatom with highest priority
within denoted subsystem. In certain cases 2D NMR techniques
yl-1,2,4-triazolidine-3,5-dione 2 (3.54 g, 11.9 mmol). The reaction
mixture was stirred and kept at 08C for 2 h; the cooling bath was
removed, and stirring was continued for 20 h. Dicyclohexylurea
(DCU) was removed by filtration, and the filtrate concentrated in
vacuum. The residue was re-dissolved in EtOAc and washed with
H2O, 5% aq. KHCO3, 5% aq. KHSO4, and brine. The organic layer
was dried over anhydrous Na2SO4 and evaporated to dryness. Pu-
rification by vacuum flash chromatography eluting with CHCl3/
CH3CN gave the product as a white powder (6.3 g, 83%): mp:
1
(HSQC, HMQC, HMBC, and COSY) were used to assign H and 13C
chemical shifts. MS analyses were performed on Finnigan MAT 95
(PI-EIMS 70 eV, HRMS), Finnigan SSQ 710A (PI-EIMS 70 eV, CIMS
(NH3)), and Finnigan ThermoQuest TSQ 7000 (ESI-MS, APCI-MS)
spectrometers. Melting points (mp) were measured on a Bꢀchi 530
instrument or on an electrically heated copper block apparatus
from Pefra (Germany) using open capillaries and are uncorrected.
Merck silica gel 60 (particle size 0.040–0.063 mm) was used for
flash column chromatography; vacuum flash chromatography was
carried out using Merck silica gel 60 H (particle size 90%<
0.045 mm). The department of microanalysis Regensburg carried
out elemental analysis. Compounds were dried in vacuum (0.1–
1 Torr) at room temperature or with heating up to 508C for at least
24 h prior to submission for elemental analysis. Reactions were
routinely monitored by thin-layer chromatography (TLC) on Merck
silica gel 60 F254 aluminum sheets, and spots were visualized with
UV light at l 254 nm, and/or iodine vapor, ninhydrin spray, or am-
monium molybdate/cerium(IV) sulfate solution. Preparative HPLC
was performed with a pump model K-1800 (Knauer, Berlin, Germa-
ny); the column was a Eurosphere-100 (250ꢃ32 mm) (Knauer),
which was attached to a UV detector model K-2000 (Knauer). UV
detection was done at l 220 nm, with temperature at 258C and at
a flow rate of 37 mLminꢂ1. The mobile phase was 0.1% trifluoro-
acetic acid (TFA) in Millipore-purified H2O and CH3CN. Analytical re-
versed-phase HPLC (RP-HPLC) was performed on a system from
Thermo Separation Products equipped with an SN 400 controller, a
P4000 pump, an AS3000 auto sampler, and a Spectra Focus UV/Vis
detector. The stationary phase was either a Nucleodur 100-5 C18
(250ꢃ4.0 mm, 5 mm) column (Macherey–Nagel), or a Eurosphere-
100 C18 (250ꢃ4.0 mm, 5 mm) column (Knauer), thermostated at
308C. Absorbance was detected at l 210 or 220 nm; t0 (Nucleodur-
100 C18)=2.30 min, t0 (Eurosphere-100 C18)=2.70 min; k’=(tRꢂt0)/
t0. As mobile phase, the following gradient of CH3CN and 0.05%
aq. TFA was used for the Nucleodur 100-5 C18 column: 0 min 15:85,
35 min 95:5, 40 min 95:5, 42 min 15:85, 50 min 15:85. As mobile
phase, the following gradient of CH3CN and 0.05% aq. TFA was
used for the Eurosphere 100-5 C18 column: 0 min 20:80, 30 min
95:5, 40 min 95:5, 41 min 20:80, 49 min 20:80. (flow rate=
0.7 mLminꢂ1). Relative molecular weights (Da) are given in paren-
theses following the formula. See the Supporting Information for a
detailed description of the preparation of compounds 1–8, 10, 11,
and 20–31, analytical characterization of compounds 9a, 13–19,
and 32a–50a, and HPLC purity data for 1 and 32–50. The NMR
data for 9 and 32 are included in the Experimental Section, as
both compounds may be considered representative examples of
the Y2R antagonists with benzhydryl and dibenzo[b,e]azepinyl moi-
eties described herein. See the Supporting Information for NMR
data of the structurally related compounds 33–50.
1
1308C (dec., sintering >908C); H NMR (300 MHz, CDCl3): d=1.41
(s, 9H, Boc), 1.18–1.71 (m, 4H, CbH2CgH2), 3.50–3.75 (m, 2H, CdH2
3
and 2H, NHCH2CH2N), 3.82 (t, J=5.3 Hz, 2H, NHCH2CH2N), 4.24 (m,
3
3
1H, CaH), 5.08 (d, J=8.4 Hz, 1H, NH), 6.80 (t, J=5.7 Hz, 1H, NH),
4
7.12–7.23 (m, 2H, Ph), 7.25–7.37 (m, 8H, Ph), 7.71 (dd, J=3.1 Hz,
3J=5.5 Hz, 2H, Pht), 7.84 ppm (dd, J=3.1 Hz, J=5.5 Hz, 2H, Pht);
13C NMR (75 MHz, CDCl3): d=24.7 (Cg), 28.3 (Boc), 30.0 (Cb), 36.8
(NHCH2CH2N), 38.0 (Cd), 40.1 (NHCH2CH2N), 53.0 (Ca), 79.9 (Boc),
121.9 (C-2/6 Ph), 123.2 (Pht), 126.7 (C-4 Ph), 129.1 (C-3/5 Ph), 132.2
(Pht), 134.0 (Pht), 136.4 (C-1 Ph), 152.9 (C=O urazol), 155.8 (Boc),
168.7 (Pht), 172.6 ppm (C=O amide); MS (ES, +p) m/z (%): 663 (30)
[M+Na]+, 641 (95) [M+H]+, 585 (20) [MHꢂC4H8]+, 541 (100)
[MHꢂBoc]+; Anal. calcd (%) for C34H36N6O7·H2O: C 61.99, H 5.82, N
12.76, found: C 62.35, H 5.68, N 12.85%. C34H36N6O7 (640.69).
4
3
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]-Nd-phthaloylornithin-
amide:
1,2,4-triazolidin-4-yl)ethyl]-Nd-phthaloylornithinamide
(S)-Na-tert-Butoxycarbonyl-N-[2-(3,5-dioxo-1,2-diphenyl-
(7.00 g,
10.9 mmol) was dissolved in a saturated solution of HCl in EtOAc
(25 mL). After stirring for 2 h the reaction mixture was poured into
300 mL petroleum ether (PE). The solid product was separated by
filtration, washed with Et2O and n-pentane, and dried in vacuum.
Yield: 6.18 g (98%) Na-deprotected ornithinamide.
6 (4.85 g,
10.5 mmol), HOBt·H2O (1.61 g, 10.5 mmol) and DIPEA (1.80 mL,
10.5 mmol) were dissolved in 50 mL CH3CN and cooled to 08C.
EDC·HCl (2.00 g, 10.5 mmol) and subsequently a solution of (S)-N-
[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nd-phthaloy-
lornithinamide·HCl (6.18 g, 10.7 mmol) and DIPEA (1.80 mL,
10.5 mmol) in 25 mL CH3CN were added. The reaction mixture was
stirred for 16 h, during which time the cooling bath was allowed
to come up to room temperature. CH3CN was rotary evaporated
and the residue was re-dissolved in CH2Cl2. The solution was
washed with H2O, 5% aq. KHSO4, and brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude
product was purified using vacuum flash chromatography (eluent:
CHCl3/MeOH 100:1 v/v) to yield a white solid (9.66 g, 93%): mp:
1608C with sintering and gas evolution >1208C; 1H NMR
(400 MHz, [D6]DMSO, HMQC, COSY): d=1.34–1.52 (m, 4H, 2/5-H
cyclopentyl), 1.51 (m, 4H, 3/4-H cyclopentyl), 1.41 and 1.60 (m, 2H,
CbH2), 1.53 (m, 2H, CgH2), 2.01 (m, 4H, 3/5-H piperazine), 2.20 (m,
2H, CH2C(O)NaH), 2.42 (m, 2H, NC(O)CH2), 3.25 and 3.28 (m, 4H, 2/
6-H piperazine), 3.25 and 3.40 (m, 2H, NHCH2CH2N), 3.47 (t, 3J=
3
6.5 Hz, 2H, CdH2NPht), 3.58 (t, J=6.0 Hz, 2H, NHCH2CH2N), 4.14 (m,
1H, CaH), 4.22 (s, 1H, 11-H dibenzazepine-11-yl), 7.05 (m, 1H, Har),
7.09 (m, 1H, Har), 7.22 (m, 2H, Ph), 7.24 (m, 1H, Har), 7.32 (m, 1H,
Har), 7.36 (m, 8H, Ph), 7.39 (m, 2H, Har), 7.48 (m, 1H, Har), 7.72 (m,
1H, Har), 7.83 (m, 2H, Pht), 7.84 (m, 2H, Pht), 7.94 (d, 3J=8.1 Hz,
Preparation of building block 12
3
1H, NaH), 8.17 (t, J=6.0 Hz, 1H, C(O)NHCH2CH2), 10.33 ppm (s, 1H,
(S)-Na-tert-Butoxycarbonyl-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-tri-
azolidin-4-yl)ethyl]-Nd-phthaloylornithinamide: Boc-Orn(Pht)-OH
(4.33 g, 11.9 mmol) and HOBt·H2O (1.84 g, 12.0 mmol) were dis-
solved in 50 mL THF and cooled to 08C. DCC (2.46 g, 11.9 mmol)
was added in one batch, followed by 4-(2-aminoethyl)-1,2-diphen-
C(O)NH lactam); 13C NMR (100 MHz, [D6]DMSO, HMQC): d=23.1 (C-
3/4 cyclopentyl), 24.7 (Cg), 29.1 (Cb), 36.0 (NHCH2CH2N), 37.0 (Cd),
37.0 (C-2/5 cyclopentyl), 38.3 (NCOCH2-cyclo-C5H8), 39.6
(NHCH2CH2N), 42.8 (CH2C(O)NaH), 43.9 (C-1 cyclopentyl), 45.4 (C-2/6
piperazine), 50.7 (C-3/5 piperazine), 51.9 (Ca), 73.6 (C-11 dibenzaze-
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1733

A. Buschauer et al.
MED
pine-11-yl), 121.2 (CarH), 122.5 (Ph), 122.9 (Pht), 123.6 (CarH), 126.5
(Ph), 127.6 (CarH), 127.8 (CarH), 128.3 (CarH), 128.9 (Ph), 129.8 (CarH),
130.4 (CarH), 131.1 (Car), 131.2 (CarH), 131.5 (Pht), 131.6 (Car), 134.3
(Pht), 136.1 (Car), 136.5 (Ph), 142.0 (Car), 152.5 (N(C(O)NPh)2), 167.7
(C=O Pht), 168.0 (C=O lactam), 169.8 (CON), 171.1 (CONaH),
171.8 ppm (CONHC2H4N); MS (ES, +p): m/z (%): 985 (100) [M+H]+;
Anal. calcd (%) for C56H57N9O8·CHCl3: C 62.04, H 5.30 N 11.46,
found: C 61.66, H 5.49, N 11.54. C56H57N9O8 (984.11).
evaporated. The solid product, which precipitated from the cold
solution, was collected on a filter and dried in vacuum.
Preparation of the guanidinylating agents 28 and 29: The succi-
nimidyl ester of the corresponding carbamic acid (1.5 mmol) in
DMF (1 mL) was added to a stirred solution of N-tert-butoxycarbon-
yl-S-methylisothiourea (0.29 g, 1.5 mmol) and DIPEA (0.51 mL,
3.0 mmol) in DMF (2 mL), and stirring was continued for 16 h. The
solution was diluted with EtOAc (30 mL) and washed with 5% aq.
KHSO4, 5% aq. KHCO3 and brine, dried over anhydrous Na2SO4 and
evaporated. The crude products were purified by flash chromatog-
raphy (eluent: CH2Cl2/EtOAc 20:1 v/v).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]ornithinamide (12): The
corresponding phthalimide (3.94 g, 4.0 mmol) was dissolved in
MeOH (40 mL) and treated with H2NNH2·H2O (0.48 mL, 10.0 mmol).
After stirring for 16 h a solution of KHSO4 (2.72 g, 20.0 mmol) in
H2O (20 mL) was added, and stirring was continued for additional
3 h. The precipitated solid was filtered off and washed with MeOH.
The filtrates were pooled, and the solvent was removed under re-
duced pressure. The residue was made alkaline with 5% aq. K2CO3
and extracted with CH2Cl2 (3ꢃ75 mL). The combined extracts were
dried over anhydrous K2CO3 and evaporated to dryness. The crude
product was purified by vacuum flash chromatography eluting
with CHCl3/MeOH mixtures containing 1% formic acid. The collect-
ed fractions were combined and reduced to a volume of 200 mL;
the solution was washed with aq. K2CO3, H2O, and brine. The or-
ganic layer was dried over anhydrous K2CO3 and evaporated. Yield:
General procedure for the alkylation of N,N’-bis(tert-butoxycar-
bonyl)-S-methylisothiourea with alcohols under Mitsunobu con-
ditions (30 and 31): A solution of tri-n-butylphosphine in toluene
(2m, 1.5 mL, 3.0 mmol) was added to a stirred solution of N,N’-bis(-
tert-butoxycarbonyl)-S-methylisothiourea (0.58 g, 2.0 mmol) and
the pertinent alcohol (2.0 mmol) in anhydrous THF (10 mL) was
added under argon. A solution of DIAD in toluene (2m, 1.5 mL,
3.0 mmol) was introduced dropwise by syringe. Stirring was contin-
ued for 16 h, and the resulting solution was diluted with EtOAc.
The organic phase was washed with 5% KHSO4, 5% KHCO3, and
brine, dried over anhydrous Na2SO4, and evaporated under re-
duced pressure. The residual raw product was subjected to flash
chromatography (eluent: PE/EtOAc 20:1, v/v).
1
4.40 g (87%); H NMR (400 MHz, [D6]DMSO, HMQC, COSY): d=1.26
(m, 2H, CgH2), 1.41–1.53 (m, 4H, 2/5-H cyclopentyl), 1.54 (m, 4H, 3/
4-H cyclopentyl), 1.37 and 1.60 (m, 2H, CbH2), 2.03 (m, 4H, 3/5-H pi-
perazine), 2.23 (m, 2H, CH2C(O)NaH), 2.41 (m, 2H, CdH2NH2), 2.47
(m, 2H, NC(O)CH2), 3.29 and 3.31 (m, 4H, 2/6-H piperazine), 3.25
General procedure for the guanidinylation with 1H-pyrazole-
1-carboxamidines (32a, 34a, and 38a)
The 1H-pyrazole-1-carboxamidines 13–15 (0.30 mmol), synthesized
according to standard procedures (see Supporting Informa-
tion),[36–38] were dissolved in CH2Cl2 (1 mL) and treated with 1m
Boc2O in CH2Cl2 (0.60 mL) and DMAP (0.03 mmol) in CH2Cl2 (1 mL).
The reaction mixtures were shaken for 18 h at room temperature.
The solutions were washed with 0.6% aq. AcOH and brine, dried
over anhydrous Na2SO4, and evaporated. The residues were re-dis-
solved in THF (3 mL) and treated with Mg(ClO4)2 (7 mg, 0.03 mmol).
After shaking for 2 h at 558C, the mixtures were allowed to cool to
room temperature and a solution of 12 (213 mg, 0.25 mmol) in
2.5 mL THF was added. The reaction mixtures were shaken for
2 days. EtOAc (25 mL) was added, and the solutions were washed
with 5% aq. KHCO3 and brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The products were purified
by flash chromatography (eluent: CHCl3/MeOH).
3
and 3.44 (m, 2H, NHCH2CH2N), 3.60 (t, J=6.0 Hz, 2H, NHCH2CH2N),
4.09 (m, 1H, CaH), 4.23 (s, 1H, 11-H dibenzazepine-11-yl), 7.05 (m,
1H, Har), 7.09 (m, 1H, Har), 7.22 (m, 2H, Ph), 7.24 (m, 1H, Har), 7.32
(m, 1H, Har), 7.38 (m, 8H, Ph), 7.39 (m, 2H, Har), 7.48 (m, 1H, Har),
3
3
7.72 (m, 1H, Har), 7.94 (d, J=8.1 Hz, 1H, NaH), 8.17 (t, J=6.0 Hz,
1H, C(O)NHCH2CH2), 10.33 ppm (brs, 1H, C(O)NH lactam); 13C NMR
(100 MHz, [D6]DMSO, HMQC): d=23.1 (C-3/4 cyclopentyl), 29.1
(Cb), 29.2 (Cg), 35.8 (NHCH2CH2N), 37.1 (C-2/5 cyclopentyl), 38.4
(NC(O)CH2), 39.6 (NHCH2CH2N), 40.8 and 45.4 (C-2/6 piperazine),
40.9 (Cd), 42.7 (CH2C(O)NaH), 44.0 (C-1, cyclopentyl), 50.7 (C-3/5 pi-
perazine), 52.0 (Ca), 73.7 (C-11 dibenzazepine-11-yl), 121.2 (CarH),
122.5 (Ph), 123.6 (CarH), 126.5 (Ph), 127.6 (CarH), 127.8 (CarH), 128.3
(CarH), 128.9 (Ph), 129.8 (CarH), 130.4 (CarH), 131.1 (Car), 131.2 (CarH),
131.6 (Car), 136.1 (Car), 136.5 (Ph), 142.0 (Car), 152.5 (N(CONPh)2),
168.0 (C=O lactam), 169.8 (CON), 171.1 (CONaH), 172.3 ppm
(CONHC2H4N); MS (ES, +p): m/z (%): 854 (100) [M+H]+, 876 (6)
[M+Na]+. C48H55N9O6 (854.01).
General procedure for the synthesis of 9a, 33a, 35a–37a,
39a–50a using S-methylisothioureas as guanidinylating re-
agents
For the synthesis of 33a, 35a–37a, and 39a–50a, HgCl2 (68 mg,
0.25 mmol) was added to a solution of ornithinamide 12 (213 mg,
0.25 mmol), substituted S-methylisothiourea (0.25 mmol), and Et3N
(69 mL, 0.50 mmol) in 3 mL DMF. After stirring overnight, the result-
ing mixture was diluted with EtOAc (25 mL). Insoluble Hg salts
were separated by vacuum filtration through a short bed of Celite
in a Pasteur pipette plugged with cotton wool and washed with
EtOAc. The combined filtrates were washed with 5% aq. KHSO4
and brine and dried over anhydrous Na2SO4. The products were
purified by flash chromatography (eluent: CHCl3/MeOH or CH2Cl2/
MeOH).
General procedure for the preparation of S-methylisothio-
ureas 16–31
S-Methylisothiourea and N-tert-butoxycarbonyl-S-methylisothiourea
were prepared as previously described.[14,16]
N’-Substituted N-tert-butoxycarbonyl-S-methylisothioureas 16–
27: DIPEA (4.0 mmol) in CH2Cl2 (10 mL) was added to a solution of
N-tert-butoxycarbonyl-S-methylisothiourea (0.76 g, 4.0 mmol) in
10 mL anhydrous CH2Cl2. A solution of the respective acylating
agent (4.0 mmol) in CH2Cl2 (5 mL) was added, and the reaction
mixture was shaken overnight. The solution was washed with H2O,
5% aq. KHSO4, 5% aq. KHCO3, and brine and dried over anhydrous
Na2SO4. The solution was diluted with n-hexane, and CH2Cl2 was
Compound 9a was synthesized in the same manner by starting
from (2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-
1734
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1727 – 1738

NPY Y2 Receptor Antagonists
Na-(2-{1-[2-(4-benzhydrylpiperazin-1-yl)-2-oxoethyl]cyclopentyl}ace-
tyl)ornithinamide, which was synthesized from 7 in analogy to the
procedure for the preparation of 12 and used for guanidinylation
without prior purification; C47H56N8O5 (813.00).
(NHCH2CH2N), 37.3 (C-2/5 cyclopentyl), 38.3 (NCOCH2-cyclo-C5H8),
39.6 (NHCH2CH2N), 40.7 (Cd), 40.6 and 45.5 (C-2/6 piperazine), 42.8
(CH2CONaH), 44.0 (C-1 cyclopentyl), 50.8 and 51.3 (C-3/5 pipera-
zine), 51.8 (Ca), 53.3 (OCH3), 73.6 (C-11 dibenzazepine-11-yl), 121.4
(CarH), 122.5 (Ph), 123.7 (CarH), 126.6 (Ph), 127.9 (CarH), 128.1 (CarH),
128.5 (CarH), 129.0 (Ph), 130.0 (CarH), 130.5 (CarH), 131.4 (CarH), 131.6
(Car), 136.1 (Car), 136.5 (Car), 141.8 (Car), 152.6 (N(CONPh)2), 152.9
(XC(X)N), 153.2 (XC(X)N), 168.0 (C=O lactam), 169.9 (CON), 171.2
(CONaH), 171.9 ppm (CONHC2H4N); IR (neat): n˜ =3255, 2941, 1649,
1443, 1257, 1143, 1132 cmꢂ1; MS (ES, +p): m/z (%): 955 (100) [M+
H]+; RP-HPLC: k’=6.7 (tR =17.8 min); Anal. calcd for
C51H59N11O8·H2O·2HCl: C 58.61, H 6.08, N 14.75, found: C 58.69, H
6.37, N 14.76; C51H59N11O8 (954.08).
Preparation of the deprotected acylguanidines 9 and 32–50
Deprotection was carried out in TFA/CH2Cl2 (1:1 v/v; ~5 mL per
0.2 mmol substrate). After stirring for 2 h, CCl4 or toluene (10 mL)
was added, and the volatiles were removed under reduced pres-
sure. The products were purified by preparative HPLC in CH3CN/
0.1% aq. TFA. The final products were obtained as white to pale-
yellow powders in yields ranging from 59 to 99%. For combustion
analysis, samples (~20 mg) were re-dissolved in 0.5 mL MeOH and
treated with 0.25 mL 2m HCl in Et2O. After 20 min, Et2O (2.5 mL)
was added, and the resulting suspension was centrifuged. The pre-
cipitated HCl salts were separated by decanting the supernatant
and washed twice with 2.5 mL Et2O. Prior to analysis the samples
were dried in vacuum at 50–608C for 24 h.
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
ethoxycarbonyl-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
l]argininamide (33): ½aꢃ2D0 =ꢂ5.1 cm3 gꢂ1 dmꢂ1 (c=1.12 in CH3CN/
H2O 10:1); IR (neat): n˜ =3285, 2954, 1645, 1445, 1250, 1179,
1131 cmꢂ1; MS (ES, +p): m/z (%): 969 (100) [M+H]+; RP-HPLC: k’=
7.03 (tR =18.5 min); Anal. calcd for C52H61N11O8·H2O·2HCl: C 58.97, H
6.19, N 14.55, found: C 58.88, H 6.18, N 14.50. C52H61N11O8 (968.11).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
ethoxycarbonyl-Na-(2-{1-[2-(4-benzhydrylpiperazin-1-yl)-2-oxoe-
thyl]cyclopentyl)acetyl}argininamide (9): ½aꢃ2D0 =ꢂ1.6 cm3 gꢂ1 dmꢂ1
(2S)-Nw-Benzyloxycarbonyl-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-tri-
azolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
l]argininamide (34): ½aꢃ2D0 =+13.3 cm3 gꢂ1 dmꢂ1 (c=0.85 in CH3CN/
H2O 10:1); IR (neat): n˜ =3310, 2954, 1646, 1446, 1246, 1178,
1131 cmꢂ1; MS (ES, +p): m/z (%): 1031 (60) [M+H]+, 516 (100)
[M+2H]2+; RP-HPLC: k’=8.1 (tR =20.8 min); Anal. calcd (%) for
C57H63N11O8·H2O·2HCl: C 61.06, H 6.02, N 13.70, found: C 61.03, H
6.32, N 13.70; C57H63N11O8 (1030.18).
1
(c=0.93 in CH3CN/H2O 10:1); H NMR (600 MHz, [D6]DMSO, HSQC):
3
d=1.23 (t, J=7.1 Hz, 3H, OCH2CH3), 1.46 (m, 2H, CgH2), 1.44 and
1.62 (m, 2H, CbH2), 1.43–1.56 (m, 4H, 2/5-H cyclopentyl), 1.55 (m,
4H, 3/4-H cyclopentyl), 2.23 and 2.26 (m, 4H, 3/5-H piperazine),
2.20 and 2.31 (m, 2H, CH2CONaH), 2.43 and 2.52 (m, 2H, NCOCH2-
cyclo-C5H8), 3.16 (m, 2H, CdH2), 3.47 and 3.50 (m, 4H, 2/6-H pipera-
zine), 3.31 and 3.39 (m, 2H, NHCH2CH2N), 3.59 (t, 3J=6.1 Hz, 2H,
3
NHCH2CH2N), 4.17 (m, 1H, CaH), 4.20 (q, J=7.1 Hz, 2H, OCH2CH3),
4.28 (m, 1H, Ph2CH), 7.20 (m, 2H, Ph), 7.22 (m, 2H, Ph), 7.29 (m,
(2S)-Nw-(3-Aminopropyloxycarbonyl)-N-[2-(3,5-dioxo-1,2-diphen-
yl-1,2,4-triazolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-di-
hydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopen-
tyl)acetyl]argininamide (35): MS (ES, +p): m/z (%): 998 (60) [M+
H]+, 791 (60) [M+Hꢂ207]+, 500 (100) [M+2H]2+; RP-HPLC: k’=
5.44 (tR =14.8 min); Anal. calcd for C53H64N12O8·5H2O·3HCl·CHCl3: C
49.28, H 5.90, N 12.78, found: C 49.17, H 6.90, N 12.65%;
C53H64N12O8 (997.15).
3
4H, Ph), 7.37 (m, 8H, Ph), 7.43 (m, 4H, Ph), 8.00 (d, J=8.0 Hz, 1H,
3
NaH), 8.24 (t, J=5.8 Hz, 1H, CONHC2H4N), 8.38–8.65 (m, 2H, NwH2),
8.68 (brs, 1H, NdH), 11.44 (s, 1H, Nw’H) ppm; 13C NMR (150 MHz,
[D6]DMSO, HSQC): d=13.8 (CH2CH3), 23.2 (C-3/4 cyclopentyl), 24.2
(Cg), 28.6 (Cb), 36.0 (NHCH2CH2N), 37.3 (C-2/5 cyclopentyl), 38.3
(NCOCH2-cyclo-C5H8), 39.5 (NHCH2CH2N), 40.7 (Cd), 40.7 and 45.5 (C-
2/6 piperazine), 42.8 (CH2CONaH), 44.0 (C-1 cyclopentyl), 51.1 and
51.7 (C-3/5 piperazine), 51.7 (Ca), 62.5 (OCH2CH3), 74.5 (Ph2C), 122.5
(Ph), 126.5 (Ph), 126.9 (Ph), 127.6 (Ph), 128.5 (Ph), 128.8 (Ph), 152.5
(N(CONPh)2), 152.7 (NC(X)X), 152.9 (NC(X)X), 169.9 (CON), 171.2
(CONaH), 171.8 ppm (CONHC2H4N); IR (neat): n˜ =3294, 2954, 1667,
1443, 1250, 1193, 1132 cmꢂ1; MS (ES, +p): m/z (%): 928 (100) [M+
H]+; Anal. calcd (%) for C51H62N10O7·2H2O·2HCl: C 59.12, H 6.62, N
13.52, found: C 59.13, H 6.89, N 13.32. C51H62N10O7 (926.48).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
ethylaminocarbonyl-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
l]argininamide (36): ½aꢃ2D0 =ꢂ2.9 cm3 gꢂ1 dmꢂ1 (c=1.05 in CH3CN/
H2O 10:1); IR (neat): n˜ =3307, 2952, 1651, 1443, 1178, 1131 cmꢂ1
;
MS (ES, +p): m/z (%): 968 (100) [M+H]+; RP-HPLC: k’=6.89 (tR =
18.1 min); Anal. calcd for C52H62N12O7·1.5H2O·2HCl: C 58.53, H 6.33,
N 15.76, found: C 58.54, H 6.57, N 15.77; C52H62N12O7 (967.12).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
methoxycarbonyl-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]-Nw-propanoylarginina-
mide (37): ½aꢃD20 =ꢂ1.1 cm3 gꢂ1 dmꢂ1 (c=0.58 in CH3CN/H2O 10:1);
IR (neat): n˜ =3298, 2955, 1650, 1443, 1288, 1180, 1129 cmꢂ1; MS
(ES, +p): m/z (%): 953 (100) [M+H]+; RP-HPLC: k’=7.1 (tR =
18.1 min); Anal. calcd for C52H61N11O7·1.5H2O·2HCl: C 59.36, H 6.32,
N 14.65, found: C 59.48, H 6.48, N 14.65; C52H61N11O7 (952.11).
1
l]argininamide (32): H NMR (600 MHz, [D6]DMSO, HSQC): d=1.44
(m, 2H, CgH2), 1.40 and 1.65 (m, 2H, CbH2), 1.44–1.54 (m, 4H, 2/5-H
cyclopentyl), 1.54 (m, 4H, 3/4-H cyclopentyl), 2.04 and 2.06 (m, 4H,
3/5-H piperazine), 2.21 and 2.31 (m, 2H, CH2CONaH), 2.41 and 2.49
(m, 2H, NCOCH2-cyclo-C5H8), 3.17 (m, 2H, CdH2), 3.24–3.28 (m, 4H,
3
2/6-H piperazine), 3.29 and 3.41 (m, 2H, NHCH2CH2N), 3.59 (t, J=
5.9 Hz, 2H, NHCH2CH2N), 3.74 (s, 3H, OCH3), 4.15 (m, 1H, CaH), 4.29
(m, 1H, 11-H dibenzazepine-11-yl), 7.07 (m, 1H, Har), 7.11 (m, 1H,
Har), 7.23 (m, 2H, Ph), 7.25 (m, 1H, Har), 7.34 (m, 1H, Har), 7.37 (m,
8H, Ph), 7.40 (m, 2H, Har), 7.50 (m, 1H, Har), 7.74 (m, 1H, Har), 8.04
(2S)-Nw-Benzoyl-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-
yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo-
[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]arginina-
mide (38): ½aꢃD20 =+6.9 cm3 gꢂ1 dmꢂ1 (c=0.77 in CH3CN/H2O 10:1);
IR (neat): n˜ =3292, 2953, 1645, 1444, 1272, 1177, 1130 cmꢂ1; MS
(ES, +p): m/z (%): 1001 (100) [M+H]+, 501 (30) [M+2H]2+; RP-
3
3
(d, J=8.0 Hz, 1H, NaH), 8.28 (t, J=5.9 Hz, 1H, CONHC2H4N), 8.64
(m, 2H, NwH2), 8.83 (t, 3J=5.6 Hz, 1H, NdH), 10.40 (s, 1H, C(O)NH
lactam), 11.65 (s, 1H, Nw’H) ppm; 13C NMR (150 MHz, [D6]DMSO,
HSQC): d=23.2 (C-3/4 cyclopentyl), 24.2 (Cg), 28.7 (Cb), 36.0
ChemMedChem 2011, 6, 1727 – 1738
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1735

A. Buschauer et al.
MED
HPLC:
k’=7.6
(tR =19.7 min);
Anal.
calcd
(%)
for
51.54, H 6.53, N 13.12, found: C 51.23, H 6.53, N 13.00. C55H68N12O7
(1009.20).
C56H61N11O7·H2O·2HCl: C 61.64, H 6.01, N 14.12, found: C 61.51, H
6.27, N 14.07; C56H61N11O7 (1000.15).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
[6-(4-fluorobenzamido)hexanoyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-
6,11-dihydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cy-
clopentyl)acetyl]argininamide (46): ½aꢃ2D0 =ꢂ4.7 cm3 gꢂ1 dmꢂ1 (c=
0.71 in CH3CN/H2O 10:1); IR (neat): n˜ =3300, 2943, 1641, 1443,
1289, 1172 cmꢂ1; MS (ES, +p): m/z (%): 1132 (100) [M+H]+; RP-
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]-Nw-(phenylacetyl)argi-
ninamide (39): ½aꢃ2D0 =+5.5 cm3 gꢂ1 dmꢂ1 (c=0.78 in CH3CN/H2O
10:1); IR (neat): n˜ =3292, 2952, 1650, 1443, 1180, 1128 cmꢂ1; MS
(ES, +p): m/z (%): 1015 (100) [M+H]+; RP-HPLC: k’=7.8 (tR =
20.3 min); Anal. calcd for C57H63N11O7·H2O·2HCl: C 61.95, H 6.11, N
13.94, found: C 62.13, H 6.37, N 13.96; C57H63N11O7 (1014.18).
HPLC:
k’=7.8
(tR =20.3 min);
Anal.
calcd
(%)
for
C62H71FN12O8·2H2O·2HCl: C 60.04, H 6.26, N 13.56, found: C 60.27,
H 6.55, N 13.51; C62H71FN12O8 (1131.30).
(2S)-Nw-[4-(3-Aminopropyl)aminocarbonylbutanoyl]-N-[2-(3,5-
dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-
[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-
yl]ethyl}cyclopentyl)acetyl]argininamide (47): IR (neat): n˜ =3277,
2948, 1648, 1442, 1192, 1130 cmꢂ1; MS (ES, +p): m/z (%): 1066
(100) [M+H]+; HRMS (PI-LSI-MS): m/z [M+H]+ calcd for
C57H72N13O8: 1066.5621, found: 1066.5642; RP-HPLC: k’=4.46 (tR =
14.75 min); Anal. calcd for C57H71N13O8·1.75H2O·3.25TFA: C 51.95, H
5.32, N 12.40, found: C 51.86, H 5.10, N 12.44; C57H71N13O8
(1065.55).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]-Nw-(phenoxyacetyl)ar-
gininamide (40): ½aꢃ2D0 =+14.8 cm3 gꢂ1 dmꢂ1 (c=0.68 in CH3CN/H2O
10:1); IR (neat): n˜ =3309, 2955, 1645, 1445, 1178, 1131 cmꢂ1; MS
(ES, +p): m/z (%): 1031 (100) [M+H]+; RP-HPLC: k’=7.8 (tR =
20.3 min); Anal. calcd for C57H63N11O8·1.5H2O·2 HCl: C 60.58, H 6.07,
N 13.64, found: C 60.52, H 6.31, N 13.62; C57H63N11O8 (1030.18).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-
[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-
yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]-Nw-(3-phenylpropa-
noyl)argininamide (41): ½aꢃ2D0 =ꢂ5.9 cm3 gꢂ1 dmꢂ1 (c=1.02 in
CH3CN/H2O 10:1); IR (neat): n˜ =3307, 2947, 1652, 1444, 1175,
1131 cmꢂ1; MS (ES, +p): m/z (%): 1029 (100) [M+H]+; RP-HPLC:
k’=8.2 (tR =21.2 min); Anal. calcd for C58H65N11O7·H2O·2HCl: C
62.25, H 6.21, N 13.77, found: C 61.89, H 6.58, N 13.73%;
C58H65N11O7 (1028.21).
(2S)-Nw-(15-Amino-4,7,10,13-tetraoxapentadecanoyl)-N-[2-(3,5-
dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-
[4-(6-oxo-6,11-dihydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-
yl]ethyl}cyclopentyl)acetyl]argininamide (48): PI-LSI-MS (MeOH/
gly) m/z (%): 1144 (100) [M+H]+; RP-HPLC: k’=4.70 (tR =
15.39 min); purity: 96%; C60H78N12O11 (1142.59).
(2S)-Nw-(3-Aminopropyl)aminocarbonyl)-N-[2-(3,5-dioxo-1,2-di-
phenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-
6,11-dihydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cy-
clopentyl)acetyl]argininamide (49): IR (neat): n˜ =3252, 2949, 1650,
1443, 1180, 1129 cmꢂ1; PI-LSI-MS (MeOH/gly): m/z (%): 997 (100)
[M+H]+; HRMS (PI-LSI-MS): m/z [M+H]+ calcd for C53H66N13O7:
996.5208, found: 996.5200; RP-HPLC: k’=4.54 (tR =14.97 min);
Anal. calcd for C53H65N13O7S·1.75H2O·2.75TFA: C 52.38, H 5.37, N
13.57, found: C 52.36, H 5.32, N 13.56; C53H65N13O7 (995.51).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
methanesulfonyl-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
l]argininamide (42): IR (neat): n˜ =3314, 2954, 1646, 1443, 1170,
1131 cmꢂ1; MS (ES, +p): m/z (%): 975 (100) [M+H]+; RP-HPLC: k’=
7.3 (tR =19.1 min); Anal. calcd for C50H59N11O8S·0.5H2O·HCl: C 58.90,
H 6.03, N 15.11, found: C 58.77, H 6.02, N 14.96%; C50H59N11O8S
(974.14).
(2S)-Nw-(4-Aminobutyl)aminocarbonyl)-N-[2-(3,5-dioxo-1,2-di-
phenyl-1,2,4-triazolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-
6,11-dihydro-5H-dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cy-
clopentyl)acetyl]argininamide (50): ½aꢃ2D0 =ꢂ1.7 cm3 gꢂ1 dmꢂ1 (c=
0.98 in CH3CN/H2O 10:1); IR (neat): n˜ =3284, 2958, 1652, 1445,
1175, 1131 cmꢂ1; MS (PI-LSI, MeOH/gly): m/z (%): 1011 (100) [M+
H]+; HRMS (PI-LSI-MS): m/z [M+H]+ calcd for C54H68N13O7:
1010.5365, found: 1010.5361; RP-HPLC: k’=4.57 (tR =15.03 min);
Anal. calcd for C54H67N13O7·2H2O·2.5TFA: C 53.21, H 5.60, N 13.67,
found: C 53.24, H 5.87, N 13.77; C54H67N13O7 (1009.53).
(2S)-Nw-Benzyl-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-
yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo-
[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]arginina-
mide (43): ½aꢃD20 =ꢂ3.0 cm3 gꢂ1 dmꢂ1 (c=1.04 in CH3CN/H2O 10:1);
IR (neat): n˜ =3294, 2960, 1638, 1447, 1176, 1130 cmꢂ1; MS (ES, +p)
m/z (%): 987 (100) [M+H]+; RP-HPLC: k’=7.5 (tR =19.6 min); Anal.
calcd for C56H63N11O6·3 H2O·2HCl: C 60.42, H 6.43, N 13.84, found: C
60.36, H 6.57, N 13.83; C56H63N11O6 (986.17).
(2S)-N-[2-(3,5-Dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]-Nw-
ethyl-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-dibenzo-
[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acetyl]arginina-
mide (44): MS (ES, +p) m/z (%): 925 (100) [M+H]+; RP-HPLC: k’=
6.7 (tR =17.7 min); Anal. calcd for C51H61N11O6·5H2O·5HCl: C 51.19,
H 6.40, N 12.88%, found: C 51.46, H 6.40, N 12.59%; C51H61N11O6
(952.11).
Pharmacological methods
Flow cytometric binding assays: These assays were performed es-
sentially as described.[23] Cells were grown to 80–90% confluence,
trypsinized, and resuspended in Ham’s F12 medium containing
10% fetal calf serum (FCS). Cells were counted, centrifuged for
5 min at 1200 rpm (Minifuge RF, Heraeus, Fellbach, Germany) and
re-suspended at a density of 106 cellsmLꢂ1 in binding buffer
(pH 7.4) containing 25 mm HEPES, 2.5 mm CaCl2 and 1 mm MgCl2
in Millipore (Schwalbach, Germany) filtered water, supplemented
with 1% bovine serum albumin (BSA) and 0.1 mgmLꢂ1 bacitracin
(Sigma, Deisenhofen, Germany). Cells were incubated at room tem-
perature in 1.5 mL polypropylene vessels, which were “siliconized”
using Sigmacoteꢄ (Sigma). Incubation was carried out with gentle
(2S)-Nw-(6-Aminohexanoyl)-N-[2-(3,5-dioxo-1,2-diphenyl-1,2,4-tri-
azolidin-4-yl)ethyl]-Na-[2-(1-{2-oxo-2-[4-(6-oxo-6,11-dihydro-5H-
dibenzo[b,e]azepin-11-yl)piperazin-1-yl]ethyl}cyclopentyl)acety-
l]argininamide (45): ½aꢃ2D0 =+12.0 cm3 gꢂ1 dmꢂ1 (c=0.49 in CH3CN/
H2O 10:1); IR (neat): n˜ =3298, 2954, 1665, 1447, 1179, 1130 cmꢂ1
;
MS (ES, +p) m/z (%): 1010 (32) [M+H]+, 803 (70) [MHꢂ207]+; RP-
HPLC (gradient CH3CN/0.05% aq. TFA: 0 min 15:85, 30 min 50:50):
k’=9.6 (tR =24.4 min); Anal. calcd for C55H68N12O7·5H2O·5HCl: C
1736
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1727 – 1738

NPY Y2 Receptor Antagonists
shaking (120 rpm) in the dark. Samples were measured without
further processing with a Becton Dickinson (Heidelberg, Germany)
FACSCaliburꢄ flow cytometer; instrument settings were: FSC, E-1;
SSC, 280 V; FL4, 800 V; flow, high; measurement stopped when
20000 gated events were counted. The cells were gated and the
geometric means of the fluorescence were calculated using
WinMDI software. Competition experiments were performed using
485 mL cell suspension, 10 mL Cy5–pNPY (final concentration 5 nm)
and 5 mL test compound. Incubation time was 120 min at room
temperature. The Ki value for inhibition of Cy5–pNPY binding by
unlabeled competitors was calculated from the concentration of
unlabeled competitor, producing 50% inhibition (IC50) of the spe-
cific Cy5–pNPY binding using the following relation Ki =IC50-
[Kd/(Kd+[L])], for which Kd is the dissociation constant and [L] is the
concentration of Cy5–pNPY.[39]
cell suspension was connected with the flow cytometer under per-
manent stirring, and recording was started. Instrument settings
were: FSC, E-1; SSC, 280 V; FL-1, 350 V; flow, high.
After 30 s measurement of basal fluorescence, pNPY solution
(10 mL, 70 nm) was injected with a Hamilton syringe, and data were
recorded for another 90 s. The needle of the flow cytometer was
washed with Millipore water after each measurement. Raw data
were averaged with WinMDI software and then exported to Sigma-
Plot 9.0 and smoothed. The level of increase in fluorescence was
calculated from the difference between the baseline (mean fluores-
cence of the first 25 s) and the highest value of the averaged
curve. These amplitudes of the averaged signals were used to con-
struct concentration–response curves.
Dilutions of Y2R antagonists were made in DMSO, and 10 mL antag-
onist solution were pre-incubated with 990 mL cell suspension for
1 min before measurement. Calcium response was triggered with
10 mL 70 mm pNPY in 10 mm HCl containing 0.1% BSA. The 100%
reference signal was induced in every third measurement as well.
In this case, cells were pre-incubated in the presence of the solvent
without antagonist. IC50 values were calculated with SigmaPlot 9.0
using the equation of the four-parameter logistics. KB values were
calculated using the Cheng–Prusoff equation.[39]
Spectrofluorimetric Ca2+ assays: Y2R antagonistic activities (KB
values) were determined by spectrofluorimetric measurement of
the inhibition of pNPY-induced (70 nm) mobilization of intracellular
[Ca2+]i in CHO-hY2-K9-qi5-K9-mtAEQ-A7 cells. The spectrofluorimet-
ric calcium assay with the ratiometric Ca2+ indicator fura-2 was
performed as previously described.[26] The ratio of fluorescence in-
tensities at l 510 nm after excitation at l 340 and 380 nm was
used for the calculation of the calcium concentration according to
the Grynkiewicz equation.[40] IC50 values were calculated from at
least two antagonist concentrations [B], inhibiting the NPY-stimu-
lated increase in intracellular [Ca2+]i between 20 and 80%. The
mean percent inhibition values P with SEM<10%, determined
from 2–3 independent experiments performed on different days,
were logit transformed, according to the equation logit(P)=log
[P/(100ꢂP)], and IC50 values were obtained by plotting logit(P)
versus log[B] with the slope n according to log[P/(100ꢂP)]=
nlog[B]ꢂnlogIC50 by linear regression.[5] The KB value for the inhibi-
tion of pNPY-induced Ca2+ mobilization was calculated from the
concentration of competitor, producing 50% inhibition (IC50) using
the following relation KB =IC50[EC50/(EC50+[L])], where EC50 is the
agonist concentration that induces 50% of the maximum re-
sponse, and [L] is the concentration of pNPY.[26]
Aequorin assay: The bioluminescent functional assay for the de-
termination of Y2R antagonistic activity was performed essentially
as described[23] using CHO cells expressing the hY2 receptor, a chi-
meric G-protein (Gqi5), and mitochondrially tagged aequorin. Cells
were grown to 80–90% confluence, trypsinized, and detached with
Ham’s F12 medium supplemented with 10% FCS. After centrifuga-
tion at 300 g for 5 min, the cells were resuspended in Dulbecco’s
modified Eagle’s medium (DMEM) without phenol red, supple-
mented with 1% FCS at a density of 107 cellsmLꢂ1. Coelenterazine
(Biotrend, Cologne, Germany; 1 mm stock solution in MeOH) was
added to a final concentration of 2 mm, and reconstitution of the
holoenzyme was accomplished by incubation for 2 h at room tem-
perature under gentle stirring in the dark. Cell suspensions were
diluted with loading buffer to 5ꢃ105 cellsmLꢂ1 and incubated at
room temperature for 3 h in the dark. Cell suspension (162 mL) was
added to 18 mL peptide agonist in loading buffer supplemented
with 1% BSA and 100 mgmLꢂ1 bacitracin in a white 96-well plate,
and the emitted luminescence (peak 1) was recorded in the kinetic
mode for 43 s as a series of 200 ms integrations with a GENios Pro
plate reader (Tecan, Grçdig, Austria). Subsequently, 20 mL of a 1%
Triton X-100 solution were injected to discharge the remaining ae-
quorin, and light emission was recorded for an additional 22 s
(peak 2). For the characterization of antagonists, the cells were in-
cubated in the presence of the antagonist for 1 h. 18 mL 700 nm
pNPY were added to 162 mL of the cell suspension, and lumines-
cence (peak 1) was recorded for 43 s before the cells were lysed by
the injection of 20 mL of Triton X-100 (1% in binding buffer)
(peak 2).
Flow cytometric Ca2+ assay (Y2R antagonism): The assay was per-
formed with CHO-hY2-K9-qi5-K9 cells and 70 nm pNPY to trigger in-
tracellular Ca2+ mobilization. The non-ratiometric Ca2+ indicator
fluo-4 was used for the measurement of Ca2+ responses by flow
cytometry as described previously.[23,27] Cells were grown for 2 days
to 70–90% confluence, trypsinized, and detached with Ham’s F12
supplemented with 10% FCS. Cells were counted in a hemocytom-
eter, centrifuged for 5 min at 300 g at room temperature, and re-
suspended at a density of 2.7ꢃ106 cellsmLꢂ1 in loading buffer con-
taining 120 mm NaCl, 5 mm KCl, 2 mm MgCl2, 1.5 mm CaCl2, 25 mm
HEPES, and 10 mm glucose at pH 7.4. For the preparation of the
loading suspension, 3 mL fluo-4-AM (Invitrogen, Darmstadt, Germa-
ny; 1 mm stock solution in anhydrous DMSO) were added to 5 mL
pluronicTM F-127 (Invitrogen, 20% stock solution in DMSO) and
mixed carefully before addition of 1 mL loading buffer containing
2% BSA; 330 mL loading suspension were added to 1 mL cell sus-
pension, resulting in a cell number of 2ꢃ106 cellsmLꢂ1 and a dye
concentration of 0.7 mm. The cells were incubated in the dark for
30 min at room temperature and re-centrifuged at 300 g for 5 min.
After resuspension in loading buffer at a density of 0.5–1ꢃ
106 cellsmLꢂ1, the cells were incubated for another 30 min at room
temperature in the dark. Measurements were performed in a
custom-built glass tube closed by a silicon septum as described.[41]
This instrumentation allows injections into the samples during con-
tinuous flow cytometric measurements. A tube containing 1 mL
The fractional luminescence was calculated by dividing the area of
peak 1 by the total area under peaks 1 and 2 using SigmaPlot 9.0
software. For the calculation of IC50 values, the logit transformation
was used (as described above). The maximum signal was induced
by 300 nm pNPY, and antagonist concentrations reducing the
pNPY-induced luminescence signal to 20–80% were used. KB
values were calculated using the Cheng–Prusoff equation.[39]
Determination of selectivity for Y2R relative to Y1, Y4, and Y5R:
The selectivity of the new Y2R antagonists for human NPY Y2 over
Y1, Y4, and Y5 receptors was proven by flow cytometric binding
assays on fluorescence-labeled pNPY, and fluorescence-labeled
ChemMedChem 2011, 6, 1727 – 1738
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www.chemmedchem.org
1737

A. Buschauer et al.
MED
[K4]hPP according to previously described methods.[23,27,28] All flow
cytometric measurements were performed on a FACSCaliburTM flow
cytometer equipped with an argon laser (l 488 nm) and a red
diode laser (l 635 nm). The compounds were tested at two con-
centrations (1 and 10 mm).
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Graduate Training Program (Graduiertenkolleg) GRK 760, “Medici-
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Keywords: acylguanidines
chemistry · neuropeptide Y · receptors
·
bioisosterism
·
medicinal
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Received: May 13, 2011
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Published online on June 20, 2011
1738
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ChemMedChem 2011, 6, 1727 – 1738