[3H]UR-PLN196: A Selective Nonpeptide Radioligand and Insurmountable Antagonist for the NeuropeptideY Y2Receptor

Article abstract of DOI:10.1002/cmdc.201200566

Radioing in on NPY: Attachment of a [2,3-³H]propionyl group through an appropriate linker to the guanidine group of an (S)-argininamide-type neuropeptideY (NPY) Y₂ receptor antagonist resulted in a subtype-selective radioligand.

Full text of DOI:10.1002/cmdc.201200566

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DOI: 10.1002/cmdc.201200566
[3H]UR-PLN196: A Selective Nonpeptide Radioligand and
Insurmountable Antagonist for the Neuropeptide Y
Y2 Receptor
Nikola Pluym, Paul Baumeister, Max Keller, Gꢀnther Bernhardt, and Armin Buschauer*[a]
Neuropeptide Y (NPY) has been reported to be involved in the
regulation of numerous physiological processes and implicated
in diseases such as obesity, depression and alcoholism (for
recent Review articles, see. Ref. [1,2]). In humans, the biological
effects of NPY are mediated by four functionally expressed re-
ceptor subtypes, designated Y1, Y2, Y4 and Y5 receptors (Y1R,
Y2R, Y4R, Y5R). Numerous potent and selective Y1R and Y5R
antagonists have been described since 1994, especially aiming
at new drugs for the treatment of obesity. Argininamide
BIIE 0246[3] (Scheme 1), the first reported potent and selective
Y2R antagonist, proved to be a valuable tool for the study of
Y2R. Meanwhile, there is growing interest in the Y2R as a thera-
peutic target, not least stimulated by recently published brain-
penetrant, orally available Y2R antagonists such as JNJ-
31020028,[4] JNJ-5207787[5] and SF-11[6] (Scheme 1), as well as
structural analogues of the latter, such as CYM 9552 or CYM
9484,[7] and imidazolidine-2,4-diones, such as NVP-833.[8]
Radioligands used for the study of Y2R are, for instance,
[3H]NPY,[9,10]
[ [
125I]PYY,[11] and 125I]PYY(3–36),[12,13] which are
devoid of subtype selectivity. Selective nonpeptidic radio-
ligands for Y2R are not available. Aiming at potent and sub-
type-selective tracers for Y2R, we synthesized a series of deriva-
tives of argininamide-type Y2R antagonist BIIE 0246.[3] Previous-
ly, electron-withdrawing substituents, such as acyl and carba-
moyl residues, attached to the guanidine group (NG) of the
parent compound were found to be tolerated despite the de-
crease in basicity by four to five orders of magnitude.[14] Deriv-
atives bearing a terminal amino group (Nw) in the acyl moiety
are of special interest with respect to labeling with fluorescent
dyes or radioisotopes. Starting from these precursors, we syn-
thesized a small library of potential radioligands (“cold” forms)
by propionylation of the terminal amino moiety with respect
to selection of an appropriate candidate for radiolabeling.
NG-Substituted amine precursors 11–20 were obtained by
guanidinylation of the corresponding ornithinamide with dif-
ferently substituted N-acyl-N’-tert-butoxycarbonyl-S-methyliso-
thioureas 1–10 (Scheme 2), followed by acidic tert-butoxycar-
bonyl (Boc) deprotection (Scheme 3). Guanidinylating reagents
1–10 (for 5, 6, 8, 10, see Ref. [14]) were prepared by coupling
the mono-Boc-protected S-methylisothiourea with the corre-
sponding carboxylic acids (Scheme 2), which were either acti-
vated in situ with hydroxybenzotriazole (HOBt)/N,N,N’,N’-tetra-
methyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU)
or used as commercially available succinimidyl esters (2–4, 6).
In the final reaction step, amine precursors 11–20 were acylat-
ed with succinimidyl propionate to yield Y2R antagonists 21a
and 22–30 (Scheme 3). Mono-Boc-protected argininamide 33
was synthesized by guanidinylation with di-Boc-protected S-
methylisothiourea followed by acidic cleavage of only one Boc
group. Deprotection under acidic conditions using mixtures of
acetonitrile and water containing 0.1% trifluoroacetic acid
(TFA) was complete after three hours (monitored by HPLC). By
analogy with the previously described synthesis of a NG-propio-
nylated argininamide-type Y1R antagonist,[15] NG-propionylated
derivative 34 was obtained by acylation of 33 with 31a and
Boc deprotection (Scheme 3).
Scheme 1. Structures of nonpeptidic NPY Y2 receptor antagonists.
[a] Dr. N. Pluym, P. Baumeister, Dr. M. Keller, Prof. Dr. G. Bernhardt,
Prof. Dr. A. Buschauer
Institut fꢀr Pharmazie, Pharmazeutische/Medizinische Chemie
Universitꢁt Regensburg
Universitꢁtsstr. 31, 93053 Regensburg (Germany)
E-mail: armin.buschauer@chemie.uni-regensburg.de
Amine precursors 11–20 (for 12, 16, 17, 20, see Ref. [14]) as
well as propionamides 21a, 22–30 and 34 were investigated
for Y2R binding and antagonism (Table 1). The Y2R binding af-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200566.
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All
synthesized
propionic
amides exhibited a decreased af-
finity compared to the corre-
sponding amine precursors,
except for compound 23, which
contains an additional amine
function in the linker that is pos-
itively charged at physiological
pH. Apparently, an additional
polar group in the acyl linker of
the antagonists is preferred by
Y2R. “Masking” the positive
charge by acylation with pro-
pionic acid resulted in a decrease
in affinity, especially in the case
of NG-carbamoylated analogues
25–28. Nevertheless, most of the
synthesized potential radioli-
gands possess Y2R affinities in
the one- to two-digit nanomolar
range, for instance, 21a, 23 and
24 (Ki values around 10 nm). Pro-
Scheme 2. Preparation of guanidinylating reagents 1–10. Reagents and conditions: a) HOBt (1 equiv), TBTU
(1 equiv), DIPEA (2 equiv), DMF, 16 h, RT; b) DIPEA (2 equiv), DMF, 16 h, RT.
finities were determined in a flow cytometric binding assay,
using CHO cells stably expressing the human Y2 receptor
(hY2R),[16] and fluorescence-labeled pNPY (Cy5-pNPY or Dy-635-
pNPY).[17] NPY Y2R antagonistic activities were determined in
a spectrofluorimetric Ca2+ assay (fura-2 assay) in CHO cells
stably expressing hY2R.[18]
pionylation of BIIE 0246 in NG-position yielded 34, the com-
pound with the highest Y2R antagonistic activity (Table 1).
Taking into consideration Y2R affinity, synthetic pathway and
overall yield, amine precursor 11 was selected for labeling.
Nw-[2,3-3H]Propionyl-substituted argininamide 21b ([3H]UR-
PLN196), the “hot” form of 21a, was prepared by acylation
Scheme 3. Synthesis of NG-substituted amine precursors 11–20, corresponding potential radioligands 21a, 22–30, 34, and tritiated ligand 21b. Reagents and
conditions: a) HgCl2 (1 equiv), NEt3 (2 equiv), DMF, 16 h, RT; b) TFA/CH2Cl2 (1:1 v/v), 2 h, RT; c) for 21a, 22–30: 31a (1 equiv), NEt3 (2 equiv), CH3CN, 16 h, RT; for
21b: 31b (0.025 equiv), NEt3 (2 equiv), CH3CN, 20 h, RT; d) CH3CN/0.1% aq. TFA (1:1 v/v), 5 h, RT; e) 1. 31a (1.2 equiv), NEt3 (1 equiv), CH3CN, 21 h, RT; 2. TFA,
2 h, RT.
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(21b) and unlabeled (21a) UR-PLN196. Moreover, chemical sta-
bility in ethanol containing 100 mm TFA at ꢀ208C was proven
over a period of at least 12 months (see the Supporting Infor-
mation).
Table 1. Pharmacological data for Y2R antagonistic amine precursors 11–
20 and propionic amides 21a, 22–30, 34.
Compd
Linker length[a]
Kb[b] [nm]
Ki[c] [nm]
The selectivity of the BIIE0246 derivatives for human Y2 over
Y1, Y4, and Y5 receptors was proven by flow cytometric binding
assays using fluorescence-labeled pNPY (Y1R, Y5R) or [K4]hPP
(Y4R) according to previously described methods (see Table 2
for selectivity data of 21a, the “cold” form of radioligand
21b).[17,19]
11
8
9
11
6
4
5
6
8
8
15
8
9
11
6
4
5
6
8
8
15
–
3.7ꢁ0.1
4.4ꢁ0.5
16 ꢁ2
3.4ꢁ0.1
2.3ꢁ1.4
16ꢁ3
12[d]
13
14
15
16[d]
17[d]
18
1.0ꢁ0.2
7.0ꢁ5.1
2.5ꢁ0.2
1.8ꢁ0.2
2.1ꢁ0.4
6.5ꢁ0.02
6.0ꢁ0.5
16ꢁ8
7.8ꢁ1.0
7.6ꢁ4.2
2.1ꢁ0.3
3.2ꢁ0.3
7.0ꢁ1.2
18ꢁ1
19
20[d]
21a
22
8.1ꢁ3.2
9.9ꢁ1.0[e]
22ꢁ6
Table 2. NPY receptor subtype selectivity of 21a/21b.
8.5ꢁ0.1
16 ꢁ8
hY2R
hY1R
hY4R
hY5R
23
24
25
26
27
28
29
30
9.9ꢁ1.1
9.0ꢁ0.2[e]
64 ꢁ4
Kd [nm]
Ki [nm]
Ki [nm]
Ki [nm]
11ꢁ4
30ꢁ11
22ꢁ2
43[a]
67[b]
>5000[c]
>6500[c]
>5000[c]
32ꢁ0.1
55ꢁ15
84ꢁ64
15ꢁ0.2
48ꢁ14
5.2ꢁ1.8
9.4ꢁ0.1
12ꢁ5
[a] Kinetically determined dissociation constant. [b] Kd from saturation
binding. [c] Flow cytometric binding assays using 10 nm Cy5-pNPY (Y1R),
5 nm Cy5-pNPY (Y5R) or 3 nm Cy5-[K4]-hPP (Y4R) on HEL-Y1, CHO-Y4, and
HEC-1B-Y5 cells, respectively.
16ꢁ2
50ꢁ3
34
8.2ꢁ0.4
[a] Length of the linker expressed as the number of atoms connecting
the argininamide NG and the w-amino function (for structures, see
Scheme 3) [b] Inhibition of pNPY (70 nm)-induced [Ca2+]i mobilization in
hY2R-expressing CHO cells; data represent the mean ꢁSEM (n=2–5).
[c] Ki values were determined in a flow cytometric binding assay from the
displacement of Cy5-pNPY (Kd =5.2 nm, c=5 nm) on CHO-hY2R cells,
unless otherwise indicated; data represent the mean ꢁSEM (n=2–
4).[d] Data are in good agreement with those reported in Ref. [14], [e] Dy-
635-pNPY was used as the fluorescent ligand (10 nm) for competition
binding.
Y2R antagonism of 21a was investigated in a fura-2 based
Ca2+ assay on hY2R-expressing CHO cells under different condi-
tions: (1) simultaneous incubation of the cells with pNPY and
21a; (2) pre-incubation with 21a for 20 minutes prior to stimu-
lation with pNPY (Figure 2a, b). Concentration–response
curves (CRCs) of NPY were constructed in the absence and
presence of different concentrations of the antagonist, and the
data were subjected to Schild analysis.[20]
Simultaneous addition of pNPY and 21a led to a parallel
rightward shift of the CRCs of pNPY, suggesting competitive
antagonism by analogy with previous reports on BIIE 0246.[21,22]
Schild regression, constructed from the set of CRCs obtained
upon co-application of pNPY with 21a (Figure 2a), revealed
a slope of 1.3, which was not significantly different from unity
(t-test, a=0.05). The pA2 value reflects the affinity of the an-
tagonist; the A2 value (43 nm) equals the Kd value determined
from the study of binding kinetics (Table 2). By contrast, pre-
treatment of the cells with 21a strongly decreased the maxi-
mal agonist response in a concentration-dependent manner,
indicating insurmountable antagonism against pNPY (Fig-
ure 2b).[22,23] The depression of the maximal response to pNPY
by 21a was concentration- and time-dependent as shown in
Figure 3.
with commercially available tritiated succinimidyl propionate
31b (Scheme 2). After purification by HPLC, radioligand 21b
was obtained in a radiochemical purity of >99% (Figure 1)
with a specific activity of (as) 80.7 Cimmolꢀ1. The identity of
the radioligand was confirmed by HPLC analysis of labeled
The radioactive form of 21a, compound 21b, was character-
ized by saturation and kinetic binding experiments. Saturation
experiments with 21b using CHO-hY2R cells afforded a Kd
value of 67 nm (Table 3), which was higher than expected from
previous flow cytometric binding assays (Ki =9.9 nm, see 21a
in Table 1), determined by displacement of fluorescence-
labeled pNPY (Dy-635-pNPY). The radioligand showed a low
nonspecific binding (Figure 4a). Scatchard analysis of satura-
tion binding revealed linearity, which is consistent with the
presence of a single binding site (Figure 4b).[24] The maximum
number of binding sites (Bmax) amounted to approximately
Figure 1. HPLC analysis (radiochromatogram) of [3H]UR-PLN196 (21b).
Eluent: mixtures of CH3CN+0.05% TFA (A) and 0.05% aq. TFA (B), gradient:
0!20 min: A/B 30:70!60:40, 20!22 min: 60:40!95:5, 22!25 min: 95:5.
The identity of 21b was confirmed by spiking with “cold form” 21a (UV
detection; spectrum not shown).
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Table 3. Y2R binding and functional characteristics of 21a/21b.
[a]
[b]
[c]
[d]
[e]
koff
kon
k
off/kon
[nm]
Kd
A2
[minꢀ1
0.030
]
[minꢀ1 nmꢀ1
]
[nm]
[nm]
0.000692
43
67
43
[a] Dissociation rate constant from linear regression. [b] Association rate
constant from linear regression. [c] Kinetically derived dissociation con-
stant. [d] Equilibrium dissociation constant determined in saturation bind-
ing experiments. [e] Antagonist dissociation constant of 21a derived
from Schild analysis.
Figure 3. Time course of the pNPY (c=300 nm)-induced Ca2+ signal in the
presence of antagonist 21a. Fura-2 assay on CHO-hY2R cells performed after
&
*
pretreatment of the cells with 12a ( , 30 nm; , 75 nm) for different periods
Figure 2. Concentration–response curves (CRCs) of pNPY in the presence of
21a obtained from fura-2 assays on CHO-hY2R cells. a) Simultaneous addi-
of time.
*
tion of pNPY and antagonist 21a at different concentrations: control ( );
&
&
^
*
^
21a: 30 nm ( ); 100 nm ( ); 300 nm ( ), 1000 nm ( ), 3000 nm ( ). Inset:
Schild regression: log(r–1) plotted against the log of antagonist concentra-
tion; the concentration ratios r (r=10DpEC50) were calculated from the right-
ward shifts (DpEC50) of the CRCs when co-incubated with 21a (dashed line:
slope=1; continuous line: slope=1.31ꢁ0.16). pA2 =7.37; A2 =43 nm. Com-
pletion of the CRCs beyond pNPY concentrations higher than 3 mm was
abandoned for economic reasons. b) CRCs of pNPY constructed after pre-
treatment of the cells with different concentrations of antagonist 21a for
In contrast to the dissociation experiments, competition
binding studies showed that the radioligand was completely
displaceable and revealed tremendous discrepancies, when
competitors of a different chemical nature were used. In case
of nonpeptide Y2R antagonists, the displacement was mono-
phasic and in the expected concentration range. By contrast,
pNPY displaced radioligand 21b in a biphasic manner (Fig-
ure 6a). The high-affinity binding site corresponds to a Ki value
of 2.0 nm, which is close to the data for pNPY determined by
displacement of [3H]propionyl-pNPY (Ki =0.4ꢁ0.1 nm)[16] or
Cy5-labeled pNPY (Ki =0.8ꢁ0.2 nm).[16] Displacement of the
major portion (80%) of specifically bound 21b required ap-
proximately 1000-fold higher concentration of pNPY, corre-
sponding to a Ki value of 1300 nm. The behavior of the non-
peptide antagonist against pNPY was dependent on the order
in which labeled ligand and competitor were added and on
the duration of the incubation period. When the competition
binding experiment was performed in an inverse manner, that
is to say by displacing Dy-635-pNPY with 21a in a flow cyto-
metric assay (Figure 6b), the Ki value of 21a was comparable
with the Kd value of 21b.
&
*
^
*
^
20 min: control ( ); 21a: 30 nm ( ); 100 nm ( ); 300 nm ( ), 1000 nm ( ),
&
3000 nm ( ). Data represent the mean ꢁSEM (n=3–8).
175000 per cell. The results from kinetic studies of 21b are
presented in Figure 5. Association was almost complete after
30 minutes (Figure 5a). Dissociation experiments in the pres-
ence of pNPY (data not shown), the low-molecular-weight Y2R
antagonist SF-11[6] (data not shown) or BIIE 0246 (Figure 5b)
led to comparable results. After 90 minutes, the residual specif-
ic binding of the tritiated compound amounted to approxi-
mately 25%, suggesting in part irreversible binding. However,
the equilibrium dissociation constant of 21b, calculated from
the linearization of the kinetics (Kd =koff/kon =43 nm; Figure 4),
was in agreement with the Kd value derived from saturation
binding experiments (Kd =67 nm) proving that radioligand 21b
follows the law of mass action.[24]
Recently, a Y5R-selective radioligand with similar behavior in
kinetic and functional experiments was reported as an insur-
mountable pseudo-irreversible nonpeptide antagonist.[25] Possi-
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Figure 5. Association and dissociation kinetics of the specific Y2R binding of
21b at CHO-hY2R cells. a) Radioligand (c=75 nm) association as a function
of time. Inset: Linearization ln[Beq/(Beq–B)] versus time of the association ki-
netics for the determination of kob, slope=kob =0.082 minꢀ1, kon =(kobꢀkoff)/
[L]=6.92ꢂ10ꢀ4 minꢀ1 nmꢀ1. b) Radioligand (c=75 nm, pre-incubation for
30 min) dissociation as a function of time, monophasic exponential fit, t1/
2 =9.3 min, dissociation performed with 100-fold excess of BIIE 0246. Inset:
Linearization ln(B/B0) versus time of the dissociation kinetic for the determi-
nation of koff =slopeꢂ(ꢀ1)=0.030 minꢀ1. Data represent the mean ꢁSEM
(n=3).
Figure 4. a) Saturation binding of 21b at CHO-hY2R cells. Data represent the
mean ꢁSEM from four independent experiments, performed in triplicate.
&
*
Specific binding: ; nonspecific binding: ; Kd(sat) =67ꢁ14 nm; Inset: Repre-
sentative saturation binding curve (dpm), one experiment, performed in trip-
licate. b) Scatchard plot from data shown in panel a), best fitted by linear
regression, Kd =ꢀ1/slope=87 nm.
labeled agonists (Ki =24 nm[32] and 36 nm[21]). Furthermore, the
dissociation constants of the other investigated compounds
are in good agreement with data from flow cytometric binding
studies (Table 1). The Ki value of 21a (36 nm), determined by
competition binding experiments with the labeled analogue
21b, corresponds very well to the Kd value of compound 21b
(43 nm).
ble explanations for such a behavior are a slow rate of dissoci-
ation from the receptor,[26] a slow rate of interconversion be-
tween inactive and active receptor conformations,[27,28] stabili-
zation of an inactive ligand (antagonist)-specific receptor con-
formation,[29] or binding to a site distinct from the peptide ago-
nist binding site.[30] The presented data suggest that the Y2R
binding sites of BIIE 0246-derived antagonists and NPY are dif-
ferent or overlap only partially.
In summary, the guanidine–acylguanidine bioisosteric ap-
proach was applied to the preparation of a tritiated Y2R-selec-
tive radioligand starting from the highly potent argininamide-
type Y2R antagonist BIIE 0246. Acylation of the amine precur-
Regardless of the special binding characteristics in competi-
tion with the peptide, radioligand 21b proved to be very
useful as a standard ligand in competition binding studies
with nonpeptide Y2R antagonists as shown in Figure 7. The Kd
value determined from binding kinetics was applied for the
calculation of Ki values by means of the Cheng–Prusoff equa-
tion.[31] The binding affinity of BIIE 0246 (Ki =17 nm) is consis-
tent with reported data from competition binding using radio-
sor,
NG-[5-(2-aminoethylamino)-5-oxopentanoyl]-substituted
BIIE 0246 (11), with succinimidyl [2,3-3H]propionate (as =
80.7 Cimmolꢀ1) afforded desired radioligand 21b. Nonpeptidic
radioligand 21b ([3H]UR-PLN196) is superior to Y2R-addressing
radiolabeled native peptides due to NPY-receptor-subtype se-
lectivity, resistance against cleavage by peptidases, long-term
stability, mode of action (antagonist vs agonist), and costs of
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preparation. [3H]UR-PLN196 can be used for the detection and
quantification of Y2R binding sites. Detailed investigations in
binding experiments (with 21b) and functional assays (with
21a) revealed insurmountable antagonism versus pNPY and
pseudoirreversible binding at the Y2R. Moreover, the tritiated
compound was successfully applied as a standard ligand in
competition binding experiments with several Y2R antagonists.
In conclusion, the prepared radioligand (21b) is a valuable
pharmacological tool for the detection of Y2R, for investiga-
tions on ligand binding modes, and for the characterization of
nonpeptidic Y2 receptor antagonists.
Experimental Section
Chemicals and solvents were purchased from commercial suppliers
and used without further purification. Porcine NPY (pNPY) was
kindly provided by Prof. Dr. Chiara Cabrele (Paris-Lodron University,
Salzburg, Austria). SF-11 was purchased from Tocris Bioscience
(Bristol, UK). All solvents were of analytical grade or distilled prior
to use. Succinimidyl [2,3-3H]propionate was from Hartmann Analyt-
ic (Braunschweig, Germany) and provided as a solution in hexane/
EtOAC (9:1 v/v) (as =3.07 TBqmmolꢀ1 =83 Cimmolꢀ1
; 185 MBq/
2.5 mL).
Analytical and preparative HPLC were performed on a Waters
system (two pumps 510, pump control module, 486 UV detector,
Packard Radiomatic Flow-1 beta series A-500 detector, liquid scintil-
lator (Rotiszint eco plus, Carl Roth GmbH & Co. KG, Karlsruhe, Ger-
many) flow rate: 4 mLminꢀ1) using a Synergi C-18 column (250ꢂ
4.6 mm, 5 mm). See the Supporting Information for a detailed de-
scription of the preparation and analytical characterization of com-
pounds 1–20, 21a, 22–30, 32–34, purity (determined by HPLC) of
11–20, 21a, 22–30 and 32–34, long-term stability 21b controlled
by HPLC, and pharmacological methods.
Figure 6. a) Displacement of tritiated Y2R antagonist 21b (c=75 nm) by the
agonist pNPY. Ki1 =2.0ꢁ1.1 nm, Ki2 =1300ꢁ700 nm. b) Flow cytometric com-
petition binding. Displacement of Dy-635-pNPY (c=5 nm, Kd =5.2 nm) by
21a, the “cold form” of the radioligand, Ki =9.9ꢁ3.0 nm. The binding was
determined after an incubation period of 120 min on CHO cells stably ex-
pressing the human Y2R. Data represent the mean ꢁSEM (n=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]-Nw-[4-(2-[2,3-3H]-prop-
anoylaminoethyl)aminocarbonylbutanoyl]argininamide
(21b):
Compound 11 (1.23 mg, 0.964 mmol) was dissolved in anhydrous
CH3CN (140 mL) yielding a concentration of 6.9 mm, and NEt3 was
diluted in CH3CN to a concentration of 2.7 mL NEt3/100 mL. Com-
pound 11 (6.9 mm in CH3CN, 50 mL, 0.344 mmol) and NEt3 (10 mL of
the above described solution in CH3CN, 1.928 mmol) were added to
succinimidyl [2,3-3H]propionate (4.22 mg, 0.0241 mmol) in 1 mL
hexane/EtOAc (9:1 v/v) in a 1.5 mL Eppendorf reaction vessel
(screw top). The solvent was removed in a vacuum concentrator
(408C) over a period of 20 min and additional 11 (6.9 mm in
CH3CN, 90 mL, 0.620 mmol) was added. Again, the solvent was re-
moved in a vacuum concentrator (408C) over a period of 40 min.
After addition of 100 mL of CH3CN, the reaction mixture was vigo-
rously blended (vortexer) for 1 min, briefly centrifuged and stirred
with a magnetic microstirrer at RT overnight.
For HPLC analysis, 0.5 mL of the reaction mixture (reaction control)
were diluted (1:200) with CH3CN/0.05% aq. TFA (30/70, 97.5 mL)
and spiked with “cold” radioligand 21a (1 mm in CH3CN, 2 mL) to
a total volume of 100 mL. This solution was completely injected
into the HPLC system and analyzed by means of UV and radiomet-
ric detection. Eluent: CH3CN+0.05% aq. TFA (A) and 0.05% aq.
TFA (B), gradient: 0 to 25 min: A/B 30/70 to 55/45, 25 to 27 min:
55/45 to 95/5, 27 to 35 min: 95/5. Afterwards, the reaction was
stopped with 10% aq. TFA (2.9 mL, corresponds to ꢂ160 eq of
Figure 7. Displacement of the radioligand 21b (c=75 nm, Kd =43 nm) by ar-
&
*
gininamide-type Y2R antagonists BIIE 0246 ( , Ki =17ꢁ2.6 nm), 16 ( ,
^
Ki =16ꢁ3.0 nm), 21a ( , Ki =47ꢁ7.3 nm) and the low-molecular-weight
~
Y2R-selective antagonist SF-11 ( , Ki =2750ꢁ710 nm). The assay was per-
formed on CHO cells stably expressing the Y2R with an incubation period of
30 min. Data represent the mean ꢁSEM (n=2–3).
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ChemMedChem 2013, 8, 587 – 593 592

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TFA), and the reaction mixture was purified with analytical HPLC.
Therefore, the reaction mixture was diluted with 0.1% aq. TFA
(350 mL), and the product was isolated (three injections) using the
conditions specified for the reaction control. In this instance, radio-
metric detection was not performed. The product was eluted at
18.8 to 19.3 min and collected in a 1.5 mL Eppendorf reaction
vessel (screw top), which was put into the vacuum concentrator
between injections. The combined product fractions were evapo-
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to a clean 3 mL Amersham glass vial together with the washings
(2ꢂ100 mL).
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(0.25, 0.5, 1.0, 1.5 and 3.0 mm; injection volume: 100 mL). Eluent:
CH3CN+0.05% aq. TFA (A) and 0.05% aq. TFA (B), gradient: 0 to
20 min: A/B 30/70 to 60/40, 20 to 20 min: 60/40 to 95/5, 22 to
30 min: 95/5 (tR =15.72 min). The solutions for injection were pre-
pared in CH3CN/0.05% aq. TFA (20/80) less than 5 min prior to in-
jection. All standard solutions were prepared from a 40 mm solution
of 21a (in CH3CN/0.05% aq. TFA 20/80), which was freshly made
from a 1 mm stock solution of 21a in CH3CN. Two aliquots (2.0 mL)
of the ethanolic solution (total volume: 500 mL) of the product
were diluted with 100 mL of CH3CN/0.05% aq. TFA (20/80), and
100 mL were analyzed by HPLC. Whereas one sample was only
used for quantification of the product by UV detection, the second
sample was additionally monitored radiometrically to determine ra-
diochemical purity. The molarity of the ethanolic solution of 21b
was calculated from the mean of the peak areas, and the linear cal-
ibration curve was obtained from the peak areas of the standards.
Yield: 9.1 mg (7.41 nmol, 31%).
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The authors thank Elvira Schreiber and Brigitte Wenzl for expert
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Keywords: acylguanidines · bioisosterism · neuropeptide Y ·
radiochemistry · radioligands
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Received: December 10, 2012
Published online on January 29, 2013
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 587 – 593 593