Nω-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([3H]UR-MK299) with Extended Residence Time

Article abstract of DOI:10.1021/acs.jmedchem.5b00925

Analogues of the argininamide-type NPY Y₁ receptor (Y₁R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar K_i values and caused depression of the maximal response to NPY (calcium

Full text of DOI:10.1021/acs.jmedchem.5b00925

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Article
#
N-carbamoylation of the argininamide moiety: an avenue to insurmountable
1
NPY Y receptor antagonists and a radiolabeled selective high
3
affinity molecular tool ([H]UR-MK299) with extended residence time
Max Keller, Stefan Weiss, Christoph Hutzler, Kilian K. Kuhn, Catherine Mollereau, Stefanie
Dukorn, Lisa Schindler, Guenther Bernhardt, Burkhard König, and Armin Buschauer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00925 • Publication Date (Web): 14 Oct 2015
Downloaded from http://pubs.acs.org on October 22, 2015
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N -carbamoylation of the argininamide moiety: an
avenue to insurmountable NPY Y receptor antagonists
1
and a radiolabeled selective high affinity molecular
3
tool ([ H]UR-MK299) with extended residence time
,†
‡
†,#
†
§
Max Keller,* Stefan Weiss, Christoph Hutzler, Kilian K. Kuhn, Catherine Mollereau,
†
†
†
‡
†
Stefanie Dukorn, Lisa Schindler, Günther Bernhardt, Burkhard König, and Armin Buschauer
†
‡
Institute of Pharmacy and Institute of Organic Chemistry, Faculty of Chemistry and Pharmacy,
University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany
§
CNRS/IPBS (Institut de Pharmacologie et Biologie Structurale), 205 route de Narbonne, 31077
Toulouse cedex 5, France
KEYWORDS
neuropeptide Y; NPY Y receptor antagonist; radioligand; target residence time; argininamide;
1
neuropeptide FF; carbamoylguanidine
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ABSTRACT Analogs of the argininamide-type NPY Y
1
receptor (Y
1
R) antagonist BIBP3226,
values and caused
bearing carbamoyl moieties at the guanidine group, revealed subnanomolar K
i
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depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration-
and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the
α
mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N -
ω
3
diphenylacetyl-N -[2-([2,3- H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-
3
3
3
amide ([ H]UR-MK299, [ H]38), was prepared. [ H]38 revealed a dissociation constant in the
picomolar range (K
d
0.044 nM, SK-N-MC cells) and very high Y
1
R selectivity. Apart from
3
superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [ H]38
compared to the higher homolog containing a tetramethylene instead of an ethylene spacer (t1/2
3
3
min, K
d
2.0 nM,). Y
1
R binding of [ H]38 was fully reversible and fully displaceable by
nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism
observed in the functional assay has to be attributed to the extended target-residence time, a
phenomenon of relevance in drug research beyond the NPY receptor field.
Introduction
The 36 amino acid peptide neuropeptide Y (NPY) is involved in the regulation of various
physiological functions and was reported to be implicated in diseases such as obesity, depression
1
-3
and addiction to alcohol. NPY binds to receptors of the NPY family, which, in humans,
comprises four functionally expressed receptor subtypes (Y R, Y R, Y R and Y R). Due to
(over)expression in malignant tumors, the Y R has been suggested as a target for cancer
diagnosis. Complementary to radiolabeled peptidic Y R agonists, highly selective radiolabeled
1
2
4
5
1
4
-8
1
nonpeptidic antagonists are valuable pharmacological and potential diagnostic tools, but
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commercially not available. Apart from high affinity and selectivity, slow target off-rate and
high in vivo stability of the radiotracer are considered favorable.
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9
The tritiated analog of the (R)-argininamide 1a (BIBP3226) (Figure 1) was the first described
1
0
radiolabeled Y
1 d
R antagonist, exhibiting a K value of 2.1 nM. Bioisosteric replacement of the
guanidine group in 1a by acyl- or carbamoylguanidine moieties afforded the radiolabeled Y
1
R
3
3
11
3
3
12
antagonists [ H]UR-MK114 ([ H]2) and [ H]UR-MK136 ([ H]4) (Figure 1) with affinities
comparable to 1a. Aiming at Y R radioligands with higher affinity, potential radioligands
derived from the argininamide 1b (BIBO3304) (Figure 1) were recently described. However,
derivatization of 1b resulted in a pronounced decrease in Y R affinity, leading to ligands with
1
13
14
1
binding constants in the single-digit nanomolar range. In continuation of our work on
ω
argininamide-type Y
1
R antagonists we focused on N -carbamoylated argininamides, as the
carbamoylguanidine moiety was recently identified as a stable bioisostere of the acylguanidine
1
2,15-16
group,
conferring higher affinity than the guanidine group in the parent compound 1a, as
1
7-18
previously reported for 5-7
(Figure 1).
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Figure 1. Structures and Y
1
R affinities (determined at SK-N-MC neuroblastoma or HEL cells)
3
3
ω
of 1a, 1b, the argininamide-type radioligands [ H]2 and [ H]4, and the N -carbamoylated
a
3
10 b
argininamides 3 and 5-7. K value reported for [ H]1a by Entzeroth et al. K value determined
d
i
3
15 c
11 d
with [ H]2 as radioligand.
K
d
value described by Keller et al.
K
d
value described by Keller
12 e,f
3
e
17
et al.
i
K value determined with [ H]propionyl-pNPY as radioligand, Brennauer et al.,
f
18
Schneider et al.
Very recent detailed functional studies with 5-7 (synthesis not reported so far; cf. Supporting
Information) revealed insurmountable antagonism of these nonpeptide Y R ligands. To elucidate
1
this phenomenon we synthesized and characterized new analogs of 5-7 including compounds
with bulkier carbamoyl substituents as well as the radiolabeled version of an optimized
antagonist.
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Results and Discussion
Chemistry. The synthesis of argininamides 5-7, which were prepared from amine 15 and the
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S-methylisothiourea derivatives 10, 13 and 14, is outlined in Scheme 1. The guanidinylating
reagents 10, 13 and 14 were obtained by treatment of 9 with the commercially available
isocyanates 8, 11 or 12. Guanidinylation of amine 15 with 10, 13 and 14 in the presence of
mercury(II) chloride gave Boc/tBu-protected intermediates, which were treated with
trifluoroacetic acid (TFA) to afford argininamides 5-7 (Scheme 1).
ω
a
Scheme 1. Synthesis of the N -carbamoylated argininamide-type Y
1
R antagonists 5-7.
a
Reagents and conditions: (a) DIPEA, CH
2 2 2 2 2
Cl , 86-99%; (b) (1) HgCl , DIPEA, CH Cl ; (2) TFA,
CH
2
Cl , H O, 62-75%.
2
2
Scheme 2 shows the synthesis of argininamides 17, 28-30 and 33, which contain a triazole
moiety in the substituent attached to the guanidine group. Palladium-catalyzed hydrogenation of
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the previously described argininamide 16 afforded aniline derivative 17. The synthesis of
argininamides 28-30 started from azides 18 and 19, which were subjected to a copper(I)-
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catalyzed cycloaddition reaction with alkyne 20 or 21 to give the triazole derivatives 22, 23 and
2
4 (Scheme 2). Compounds 22-24 were converted to the guanidinylating reagents 25-27 by
treatment with triphosgene and subsequent reaction with thiourea derivative 9. Guanidinylation
of amine 15 with 25-27 followed by Boc/tBu-deprotection with TFA afforded compounds 28-30.
a
Scheme 2. Synthesis of the triazole-containing argininamides 17, 28-30 and 33.
a
4
Reagents and conditions: (a) 10% Pd/C, hydrogen, MeOH, 99%; (b) (1) ascorbic acid, CuSO ,
MeOH, H O; (2) MeOH/conc. aq HCl 3:1 (v/v) (22, 24) or CH Cl /TFA 2:1 (v/v) (23), 38-86%;
2
2
2
(c) triphosgene, DIPEA, CH
2
Cl
2
(25-27) or acetonitrile (32), 25-79%; (d) (1) HgCl
Cl 21-72%; (e) (1) iodomethane, NaN
tetrabutylammonium tetrafluoroborate, diethyl ether, H O; (2) alkyne 20, ascorbic acid, CuSO
MeOH, diethyl ether, H O; (3) MeOH/conc. aq HCl 3:1 (v/v), 72%.
2
,
3
,
4
,
triethylamine, DMF; (2) TFA, CH
2
2
,
2
2
Methyl azide, required for the preparation of compound 31, was prepared from methyl iodide
and sodium azide in a water/diethyl ether mixture using tetrabutylammonium tetrafluoroborate as
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phase transfer catalyst. To circumvent the isolation of the explosive methyl azide, alkyne 20 and
aqueous solutions of ascorbic acid and copper(II) sulfate were added directly to the separated
diethyl ether phase, containing the azide, to obtain the respective cycloaddition product, which
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2
was chromatographed and treated with hydrochloric acid to give the unprotected amine 31 .
Treatment of 31 with triphosgene and subsequent addition of compound 9 gave the
guanidinylating reagent 32, which was used to convert amine 15 to argininamide 33 (Scheme 2).
For the synthesis of propionamide 38 (UR-MK299) and the fluorescently labeled argininamide
23
4
0, thiourea derivative 9 and amine 34 were converted to guanidinylating reagent 35, using a
1
5
procedure reported for the higher homolog of 35 (Scheme 3). Treatment of amine 15 with 35 in
the presence of mercury(II) chloride and subsequent deprotection yielded amine 36, which was
propionylated to give 38, or treated with Fluorescence Red Mega 480 succinimidyl ester (39) to
afford compound 40 (Scheme 3).
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a
Scheme 3. Synthesis of the Y
1
R antagonist 38 and the fluorescently labeled argininamide 40.
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Reagents and conditions: (a) triphosgene, DIPEA, CH Cl , 66%; (b) (1) HgCl , DIPEA,
CH Cl ; (2) TFA, CH Cl , H O, 65%; (c) DIPEA, DMF, 38: 83%, 40: 69%.
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Functional studies at the human NPY Y
1
receptor. The carbamoylated argininamide-type
Y R antagonists 5-7 (Figure 1), exhibiting high Y R affinity (K < 1 nM, cf. Figure 1 and Table
1
1
i
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+
1), were investigated in a Fura-2 Ca assay on human erythroleukemia (HEL) cells using pNPY
as agonist. The effect of pNPY, elicited at a concentration of 10 nM, was inhibited by increasing
concentrations of the antagonist. Interestingly, the antagonistic effect turned out to be time-
dependent, increasing with the incubation period of the cells in the presence of the antagonist
prior to the addition of the agonist pNPY (Figure 2 B-D). The maximum shift of the curves was
reached after 5 min (compound 5, Figure 2B) and 15-20 min (6 and 7, Figure 2C and 2D),
respectively. By contrast, this phenomenon was not observed in case of the parent compound 1a
(Figure 2A), indicating that the time-dependent effect has to be attributed to the carbamoyl
substituents attached to the guanidine group in 5-7.
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Figure 2. Concentration- and time-dependent inhibition of the pNPY (10 nM) induced Ca
response in HEL cells by the argininamides 1a (A), 5 (B), 6 (C), 7 (D), 33 (E), and 38 (F). Mean
values ± SEM from at least three independent experiments (performed in singlet).
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As shown for compound 6 (Figure 3A) and 16 (Supporting Information Figure S5) as
examples, the concentration-response curve of pNPY was rightward-shifted and depressed upon
pre-incubation of the cells with increasing concentrations of the antagonists for 15 minutes. The
maximal depression of the concentration-response curve was confirmed by a different
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experimental approach: adding pNPY at a ‘supramaximal effective concentration’ of 300 nM
to cells pre-incubated with compound 6 at increasing concentrations for 15 minutes resulted in a
biphasic curve, indicating the extent of insurmountable antagonism (Figure 3B). Partially
insurmountable antagonism, although less pronounced, was also observed for compounds 5 and
38 (Figure 3B). In a more simplified kinetic experiment, only one (‘supra-effective’)
concentration of antagonist, derived from the respective determined (1a, 5-7, 33 and 38, Figure
11-12
2) or published
IC value (2, 4), was applied to inhibit the response elicited by 300 nM of
50
pNPY (Figure 3C). The plateaus of the curves, reached in a time-dependent manner, correspond
to the maximum depression caused by the antagonist, as exemplified by compound 6 at a
concentration of 1 nM (Figure 3 A,B). Whereas 5-7, 33 and 38 caused a strong depression of the
maximal pNPY effect by up to 90% (Figure 3), a phenomenon characteristic of insurmountable
2
5-26
2+
antagonists,
the argininamides 2 and 4 caused a slight depression of the maximal Ca
response, and the parent compound 1a did not affect the pNPY response within the limits of
experimental error (Figure 3C).
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Figure 3. Investigation of the (insurmountable) antagonism of argininamides 1a, 2, 4, 5-7, 33
and 38 in a Fura-2 Ca assay performed with Y R expressing HEL cells. A: Concentration-
1
response curves of pNPY in the absence and presence of antagonist 6 (15 min pre-incubation).
2+
The presence of 6 led to a rightward shift of the curves and to a strong depression of the
2+
maximum response to pNPY. B: Concentration-dependent inhibition of the Ca response,
elicited by pNPY at a supramaximal concentration of 300 nM, by the antagonists 5, 6 and 38 (15
min pre-incubation). The biphasic course of the curves revealed partially insurmountable
2
4
2+
antagonism. C: Time-dependent depression of the pNPY (300 nM) induced Ca response by
1a, 2, 4, 5-7, 33 and 38. Presented are mean values ± SEM from at least two (A, B) or three (C)
independent experiments (performed in singlet).
The results shown in Figure 3C might be interpreted as a hint to insurmountable antagonism
2
+
but not a proof, because the Fura-2 Ca assay is a non-equilibrium assay, which can produce
depressions of the maximal agonist response due to hemi-equilibrium among receptor, agonist
27
and antagonist. However, this is improbable due to the results of the additional experiments
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7
8
9
shown in Figure 3A,B and due to the fact that the parent compound revealed typical, that is,
surmountable antagonism.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding studies at human NPY receptors. Insurmountable antagonism might be caused, for
example, by stabilization of receptor conformations with low affinity to the agonist (pNPY) or
by a long-lasting (pseudo-irreversible) binding. Therefore, we performed equilibrium
3
competition binding experiments with the radiolabeled agonist [ H]propionyl-pNPY. Two
variants of the competition binding experiment were performed. Firstly, the antagonists were
3
added immediately after the agonist [ H]propionyl-pNPY, and secondly, the cells were pre-
incubated with the antagonists for 30 min. The binding constants (K values) of 5, 6 and 38
i
obtained in these experiments are shown in Table 1, and competition binding curves are depicted
in Figure 4. Pre-incubation of the cells with the antagonist did not result in a marked increase in
1
apparent Y R affinity, that is, the antagonists did not show (pseudo-)irreversible binding after an
incubation period of 2 h (equilibrium conditions). To gain insight into the mechanism causing
the discrepancies between the result from binding studies and the insurmountable antagonism
observed in the functional assay, a radiolabeled form of an insurmountable antagonist such as
compound 38 was considered a useful molecular tool.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Y
1
receptor affinities of pNPY and the argininamides 5-7, 16, 17, 28-30, 33, 36, 38,
3
3
3
40 and 41 determined by equilibrium competition binding with [ H]2, [ H]4, [ H]propionyl-
3
pNPY or [ H]38.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
K
i
[nM] (Y
1
R)
used radioligand:
3
a
3
b
3
c
3
d
[ H]2
[ H]4
[ H]propionyl-pNPY
[ H]38
e
f
g
h
i
pNPY
1a
0.5
0.7
0.6
0.41 ± 0.058 / 0.42 ± 0.054
e
f
g
1.3
1.5
1.5
1.0 ± 0.019
--
j
5
0.17 ± 0.017 0.40 ± 0.082 0.06
k
l
0
.29 ± 0.016 / 0.24 ± 0.017
g
6
0.21 ± 0.059 0.42 ± 0.12
0.3
0
0
0.037 ± 0.009
j
.06
k
l
.28 ± 0.21 / 0.12 ± 0.007
g
7
--
0.49 ± 0.021 0.3
--
1
1
2
6
7
8
4.5 ± 0.3
4.0 ± 0.20
6.9 ± 1.4
3.1 ± 0.63
41 ± 1.7
--
--
--
--
--
--
--
--
--
--
--
--
--
29
--
3
0
--
33
0.56 ± 0.062 0.34 ± 0.014 --
--
3
6
--
22 ± 3
--
--
k
l
38
--
0.31 ± 0.031 0.23 ± 0.026 / 0.10 ± 0.008
0.077 ± 0.021
4
0
--
--
--
--
--
>1,000
--
m
41
400 ± 19
a
3
d
Determined by radioligand competition binding with [ H]2 (K = 1.2 nM, c = 1.5 nM) at SK-
b
3
d
N-MC cells. Determined by radioligand competition binding with [ H]4 (K = 2.0 nM, c = 2.0
nM) at SK-N-MC cells. Determined by radioligand competition binding with [ H]propionyl-
c
3
d
pNPY (K = 1.9 nM, c = 5.0 nM) at SK-N-MC or HEL cells. Determined by radioligand
d
3
e-g
competition binding with [ H]38 (K
reported by: Keller et al., Keller et al.,
immediately prior to [ H]38 (h) or 60 min after pre-incubation of the cells with [ H]38 (i) (K
values not significantly different (P > 0.05)). K
argininamides 5, 6 and 38 were either added to the cells immediately after the radioligand (k) or
pre-incubated with the cells (30 min) prior to the addition of [ H]propionyl-pNPY (l) (5: Ki
d
= 0.044 nM, c = 0.15 nM) at SK-N-MC cells.
K
i
values
e
11
f
12
g
18 h/i
Schneider et al.
pNPY was either added
3
3
i
j
17 k/l
i
value reported by Brennauer et al.
The
3
values not significantly different (P > 0.05), 6 and 38: K
i
values significantly different (P < 0.02))
m
20
K
i
value reported by Weiss et al. Presented are mean values ± SEM from at least two
independent experiments (performed in triplicate). Note: All K values were determined in our
laboratory essentially according to the same experimental procedure. Because of the very high
affinity of [ H]38 for the NPY Y
i
3
1
receptor, the binding assay was performed in a way to ensure
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that the free radioligand concentration was not markedly decreased by binding to the receptor.
Therefore, cells were kept below 80% confluency, and the volume per well was increased (500
µL, instead of 250 µL used for ligands with lower affinity such as [ H]2 and [ H]4) to decrease
3
3
the ratio of receptor-bound to free radioligand.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
Figure 4. Displacement of [ H]propionyl-pNPY (K
6
d
= 1.9 nM, c = 5.0 nM) by argininamides 5,
and 38 at SK-N-MC neuroblastoma cells in a competition binding experiment. The
argininamides were either added immediately after the radioligand (open symbols, dashed lines)
or cells were pre-incubated with the antagonists for a period of 30 min prior to the addition of the
radioligand (filled symbols, solid lines). Pre-incubation with the argininamide resulted in a slight
decrease in the equilibrium binding constant K
8) or did not result in a statistically significant difference in K values (compound 5) (cf. Table
i
(leftward shift of the curves) (compounds 6 and
3
i
1
). Mean values ± SEM from at least three independent experiments (performed in triplicate).
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ω
Figure 5. Structures of the N -carbamoylated argininamides 16, 17, 28-30, 33, 36, 38, 40 and
41.
Y R affinity data of the carbamoylated derivatives of 1a (compounds 5-7, 16, 17, 28-30, 33,
1
3
3
3
6, 38 and 41) (cf. Figure 5) were determined by competition binding with [ H]2 or [ H]4 at SK-
N-MC neuroblastoma cells (K values presented in Table 1). Among the series of triazole
i
derivatives (16, 17, 28-30, 33, 41) the highest Y
compound with the smallest substituent (cf. Figure 5). Argininamide 41, bearing the largest
substituent in this series of compounds, exhibited the lowest Y R affinity, suggesting limited
space in the binding region interacting with the moiety attached to the guanidine. The
1
R affinity resided in derivative 33, the
2
0
1
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propionamide 38, which represents the shorter homolog of the previously reported radioligand
3
[
1 i
H]4 (Figure 1), showed very high Y R affinity (K = 0.31 nM), comparable to that of
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds 5-7 (Table 1). Compound 38 is closely related to argininamide 7: the length of the
chain attached to the guanidine group is the same (8 atoms), the difference between the two
ligands resides in the ester group (7) and amide group (38) (cf. Figures 1 and 5). When
comparing amine 36 and propionamide 38 (Figure 5), propionylation of 36 led to a > 50-fold
increase in Y
1
R affinity (Table 1), whereas, in case of the shorter homologs 3 and 4, respectively,
R affinity (Figure 1). Therefore, it can be speculated
propionylation had almost no effect on Y
1
that the amide group in the substituent attached to the guanidine group in 38 contributes to
receptor binding.
1
Argininamides 5-7, 33, 36 and 38 were characterized in terms of Y R selectivity by flow
cytometric competition binding studies on CHO-hY
fluorescently labeled peptides Cy5-pNPY (Y R and Y
these compounds proved to be highly selective Y
R selectivity, the presented argininamides harbor potential ‘off-target effects’ in terms of
binding to neuropeptide FF receptors as the parent compound 1a was reported to bind to the
2
, CHO-hY
4
and HEC-1B-hY
5
cells using the
1
8,28
4
29
2
5
R)
and Cy5-[K ]hPP (Y
4
R). All of
1
R ligands (Table 2). However, despite high
Y
1
3
0
NPFF receptor (K = 12 nM) and the NPFF receptor (K = 84 nM). Therefore, selected
1
i
2
i
argininamides with high Y
1
R affinity (6, 33 and 38) as well as 1a were investigated in
and CHO-hNPFF cells using the
competition binding studies at membranes of CHO-hNPFF
1
2
3
31
3
31
radiolabeled peptides [ H]NPVF and [ H]EYF . In contrast to the parent compound 1a, the
ω
N -carbamoylated derivatives 6, 33 and 38 exhibited low NPFF
1
2
and NPFF receptor affinities
(
K
i
> 500 nM, Table 2, Supporting Information Figure S6) showing that the introduction of
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
carbamoyl residues at the guanidine group in 1a leads to increased Y R specificity with respect
to NPFF receptors.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. NPY receptor subtype selectivity data of argininamides 5-7, 33, 36 and 38 and K
i
values of selected compounds determined at NPFF and NPFF receptors.
1
2
K [nM]
i
Compound
a
b
c
b
d
e
hY
1
R
hY
2
R
hY
4
R
hY
5
R
hNPFF
1
R
2
hNPFF R
f
1
5
6
7
a
1.5
n.d.
n.d.
n.d.
18 ± 1.5
n.d.
250 ± 21
n.d.
0.40 ± 0.082 >3,000
0.42 ± 0.12 >3,000
0.49 ± 0.021 >3,000
0.34 ± 0.014 >10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1,100 ± 260 >3,000
n.d.
n.d.
33
630 ± 62
n.d.
1,200 ± 240
n.d.
36
22 ± 3
>10,000
3
8
0.31 ± 0.031 >3,000
>10,000
3
1,000 ± 130 >3,000
a
b
Determined by radioligand competition binding with [ H]4 (data from Table 1). Determined
by flow cytometry in a competition binding assay on CHO-hY and HEC-1B-hY cells using
2
5
c
Cy5-pNPY as fluorescent ligand (K
by flow cytometry in a competition binding assay on CHO-hY
d
= 5.2 nM (Y
2
R) and 4.4 nM (Y
5
R), c = 5 nM). Determined
4
4
cells with Cy5-[K ]hPP (K
nM, c = 3 nM) as fluorescent ligand. Determined by radioligand competition binding with
cell membranes. Determined by radioligand
competition binding with [ H]EYF (c = 0.93 nM) at CHO-hNPFF
d
= 5.6
d
3
e
[
H]NPVF (c = 0.54 nM) at CHO-hNPFF
1
3
f
2
cell membranes. K
i
value
1
2
reported by Keller et al. Data represent mean values ± SEM from at least three independent
experiments (performed in triplicate (Y R) or duplicate (NPFF R and NPFF R)), or the results
R and Y R).
1
1
2
from at least two independent experiments (performed in duplicate) (Y
2
R, Y
4
5
3
Preparation and characterization of the radioligand [ H]38. Aiming at the preparation of
the high affinity propionamide 38 in its tritiated form, the stability of 38 in aqueous solution was
investigated at pH 7 over 48 h. The compound showed excellent chemical stability (Figure 6).
Stability studies were also performed with argininamides 5, 6 and 33. Whereas 5 and 33 showed
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no decomposition, compound 6 proved to be unstable due to hydrolysis of the ester group (see
Supporting Information Figures S2−S4).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Reversed-phase HPLC analysis of argininamide 38 after incubation in PBS (pH 7.0) at
21 °C for up to 48 h. Compound 38 showed no decomposition.
The tritiated form of 38 was prepared by treating an excess of amine 36 with succinimidyl
3
3
[
H]propionate (Figure 7A) to afford [ H]38 with a radiochemical yield of 94% and a specific
activity of 1.81 TBq/mmol. The radioligand was obtained with a radiochemical purity of 97%
(Figure 7B) and proved to be stable when stored in ethanol at -20 °C (Figure 7C).
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
Figure 7. A: Synthesis of the radioligand [ H]38 by [ H]propionylation of amine 36.
Radiochemical yield: 94%. B/C: RP-HPLC analysis (conditions see experimental section) of
H]38 (0.7 µM), spiked with ‘cold’ 38 (20 µM), analyzed three days after synthesis (B) and after
seven months of storage at -20 °C (C). Radiochemical purity: 97% and 95%, respectively. The
3
[
minor shifts in t
R
result from serial detection of the UV and the radiometric signal.
3
Saturation binding experiments with [ H]38 at SK-N-MC neuroblastoma cells and MCF-7-Y1
breast cancer cells yielded binding constants of 0.044 nM and 0.14 nM, respectively (Figure 8,
1 1 1
Table 3). A slightly lower affinity to Y receptors expressed by MCF-7-Y cells, compared to Y
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receptors at SK-N-MC cells, was previously observed for the radioligands [ H]2 and [ H]4,
1
1-12
too.
The determined numbers of binding sites per cell (SK-N-MC: approx. 32,000 sites/cell,
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0-12
MCF-7-Y
1
: approx. 130,000 sites/cell) were consistent with previously reported data.
3
Figure 8. Representative saturation isotherms and Scatchard transformations of specific [ H]38
binding to SK-N-MC cells (panel A) and MCF-7-Y cells (panel B). Experiments were
performed in triplicate. Error bars of ‘specific binding’ and error bars in the Scatchard plot
represent propagated errors calculated according to the Gaussian law of errors. Error bars of
1
‘nonspecific binding’ represent the SEM.
3
The association and dissociation kinetics of [ H]38 was determined at SK-N-MC cells
3
3
11-12
analogous to the experiments performed previously with [ H]2 and [ H]4.
Compared to the
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
3
higher homolog [ H]4, reaching a plateau after 15 min, the association of [ H]38 was slower
(
plateau reached after approx. 40 min; Figure 9A). Likewise, with a half-life of 95 min, the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
dissociation of [ H]38 was markedly slower compared to [ H]4 (t1/2 = 3 min; Figure 9B).
3
Figure 9. Association and dissociation kinetics of [ H]38 (in comparison to reference compound
3
12
[
H]4 ), determined at SK-N-MC neuroblastoma cells at 22 °C. A: Radioligand (c = 0.3 nM)
association to the Y R as a function of time. Inset: ln[Beq/(Beq−B)] versus time, slope = kobs
.092 min . Mean values ± SEM from three independent experiments (performed in triplicate).
B: Radioligand (pre-incubation: 1 nM, 45 min) dissociation from the Y R as a function of time,
monophasic exponential decay: t1/2 = 93 min. Inset: ln(B/B ) versus time, slope·(−1) = koff
1
=
−1
0
1
0
=
−
1
0.0074 min , t1/2 = 95 min. Mean values ± SEM from four independent experiments (performed
in triplicate).
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The kinetically derived dissociation constant K
d
(kin), calculated according to K
d
(kin) = koff/kon
,
amounted to 0.026 nM being in good agreement with the K
d
value obtained from saturation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
binding experiments (K
d
= 0.044 nM). Complete dissociation of [ H]38, best fitted by an
equation describing a two-parameter mono-exponential decline (Figure 9B), is incompatible with
the hypothesis of irreversible binding.
3
Table 3. NPY Y
1
receptor binding data of [ H]38.
Saturation binding
Binding kinetics (SK-N-MC cells)
a
b
−1
−1 c
−1 d
e
K
d
[nM]
K
d
[nM]
k
on [min ·nM ] koff [min ]
d
K (kin) [nM]
t1/2 [min]
0.044 ± 0.003
0.14 ± 0.017
0.28 ± 0.026
0.0074 ± 0.0007 0.026 ± 0.005
95 ± 8
a
Equilibrium dissociation constant determined at SK-N-MC neuroblastoma cells; mean ± SEM
b
from 10 independent experiments (performed in triplicate). Equilibrium dissociation constant
determined at MCF-7-Y cells; mean ± SEM from 10 independent experiments (performed in
triplicate). Association rate constant ± propagated error, calculated from kobs (0.092 ± 0.0072
min , mean ± SEM from 3 independent experiments (performed in triplicate)), koff (0.0074 ±
.0007 min ) and the applied radioligand concentration (0.3 nM). Dissociation rate constant
1
c
−
1
−
1
d
0
and derived half-life; mean ± SEM from 4 independent experiments (performed in triplicate).
Kinetically derived dissociation constant ± propagated error (K (kin) = koff/kon).
d
e
3
In competition binding experiments with [ H]38, the K
i
values of compounds 1a, 6, 33, the
fluorescently labeled argininamide 40 and pNPY were determined at SK-N-MC cells (Figure 10,
Table 1). The K value of 1.0 nM obtained for the parent compound 1a was in excellent
i
3
3
agreement with previously determined K
i
values using the radioligands [ H]2, [ H]4 and
3
[ H]propionyl-pNPY (cf. Table 1). The K
i
value of 0.077 nM determined for 38 is in good
3
accordance with the K value of [ H]38 (Table 1). The fluorescently labeled compound 40 was
d
3
incapable of displacing [ H]38 (K
i
> 1,000 nM), which, at first sight, appears consistent with the
ω
aforementioned decrease in Y
1
R affinity by the introduction of bulky moieties in the N -
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2
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
substituent (compound 41, cf. Figure 5 and Table 1). However, the length of the spacer and the
chemical nature of both the spacer and the fluorophore, are critical for Y
1
R affinity as becomes
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
obvious from previously reported structure-activity relationships of fluorescent ligands derived
1
5,32
from 1a.
3
Competition binding experiments at the Y R with [ H]38 and pNPY resulted in a complete
1
displacement of the labeled antagonist (Figure 10B) affording a K
expected range (0.41 nM). This is in contrast to a recently reported competition binding
experiment at the Y R with an insurmountable radiolabeled argininamide-type Y R antagonist
and pNPY, where the displacement curve was biphasic and dramatically shifted to approximately
i
value of pNPY in the
2
2
2
3
d
1000-fold higher pNPY concentrations than expected from the K value of the peptide. The fact
3
that a pre-incubation of the Y receptor with [ H]38 prior to the addition of the competitor pNPY
1
did not affect the K
i
value of the peptide (Figure 10B) further support fully reversible binding of
argininamide-type Y
1
1
R antagonists such as 5-7, 33 and 38 to the Y receptor.
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8
9
10
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13
14
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18
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
Figure 10. Competition binding experiments performed with [ H]38 at SK-N-MC cells. A:
3
Displacement of [ H]38 (K = 0.044 nM, c = 0.15 nM) by argininamides 1a, 6 and 38. Mean
values ± SEM from at least three independent experiments (performed in triplicate). B:
Displacement of [ H]38 by pNPY. The peptide was either added immediately prior to the
radioligand (open squares, dashed line) or 60 min after pre-incubation of the cells with the
radioligand (filled squares, solid line). The affinity of pNPY was not affected by pre-incubation
of the cells with [ H]38. Mean values ± SEM from at least four independent experiments
d
3
3
(
performed in triplicate).
Conclusion
ω
1
Unlike the parent compound, argininamide 1a, N -carbamoylated argininamide-type Y R
antagonists caused a concentration- and time-dependent depression of the concentration-response
2+
curve of pNPY, indicative of insurmountable antagonism, in the Fura-2 Ca assay. In contrast to
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
the functional assay, radioligand binding studies using the tritiated agonist [ H]propionyl-pNPY
1
gave no hint to irreversible Y R binding. Inversely, the radiolabeled form of such an antagonist,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
[ H]38, proved to be completely displaceable by pNPY (K
i
= 0.41 nM) under equilibrium
3
conditions, irrespective of pre-incubation of the cells with [ H]38, suggesting the peptidic agonist
and the nonpeptide antagonist to compete for overlapping binding sites. With a K of 0.044 nM
d
3
(
[
[
[
SK-N-MC cells), [ H]38 proved to be by far superior to the previously reported higher homolog
3
3
3
12
H]4 (K
H]38 exhibited a strongly increased target residence time (half-life of 95 min) compared to
H]4 (t1/2 = 3 min). As Y R binding was fully reversible, the strong depression of the
d
= 2.0 nM), bearing a tetramethylene instead of an ethylene spacer. Most interestingly,
1
concentration-response curves in the functional assay, suggesting insurmountable antagonism,
3
has to be attributed to the slow dissociation of [ H]38. With respect to affinity, NPY receptor
subtype selectivity, and specificity with respect to NPFF receptors, this new radioligand
1
represents an outstanding molecular tool for the study of the Y R. In combination with related
3
argininamide-type antagonists, [ H]38 may be useful to gain deeper insight into the structural
determinants of increased drug-target residence time, a generally important issue for lead
1
optimization today. The presented high affinity Y R antagonists will be studied in more detail,
e.g., in binding studies at receptor mutants to explore the binding mode, and harbor the potential
of being used for the crystallization of receptor-ligand complexes.
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9
Experimental Section
General experimental conditions. Solvents and reagents (analytical grade), isocyanates 8 and
11 (Sigma-Aldrich Chemie, Munich, Germany), isocyanate 12 (ABCR, Karlsruhe, Germany),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
,4-diaminobenzene (Sigma-Aldrich), propargylbromide (Sigma-Aldrich) and amine 34 (ABCR)
were purchased from commercial suppliers and used without further purification. Technical
grade solvents (EtOAc), light petroleum (40-60 °C) and dichloromethane) were distilled before
use. Acetonitrile for HPLC (gradient grade) was from Merck (Darmstadt, Germany). Compounds
33
11
34
35
36
37
11
9,
15, 20, 18, 19, 21 and 37 were prepared according to published procedures.
Porcine NPY (pNPY) and human pancreatic polypeptide (hPP) were a gift from Prof. Dr. C.
Cabrele (University Salzburg, Salzburg, Austria). The syntheses of the fluorescently labeled
18
4
29
3
peptides Cy5-pNPY and Cy5-[K ]hPP were described elsewhere. [ H]propionyl-pNPY
(specific activity: 27.4 Ci/mmol) was prepared in house by labeling of pNPY with commercially
3
available succinimidyl [2,3- H]propionate (see Supporting Information). Millipore water was
used throughout for the preparation of buffers and HPLC eluents. 1.5- or 2-mL polypropylene
reaction vessels with screw cap (Süd-Laborbedarf, Gauting, Germany) were used for the
3
preparation and storage of stock solutions, for the synthesis of the radioligand [ H]38 and the
fluorescently labeled compound 40, and for the investigation of chemical stabilities. Thin layer
chromatography was performed on Merck silica gel 60 F254 TLC aluminum plates. For column
chromatography silica gel Geduran 60 (0.063-0.200 µm, Merck) or Silica Gel 60 (40-63 µm,
Merck) (flash chromatography) was used. In case of the purification of acid sensitive compounds
(e.g. Boc protection group) the stationary phase was conditioned with the respective solvent
containing 0.5% triethylamine. The purity of final compounds was determined by RP-HPLC and
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was ≥ 95% throughout. In addition, the purity of compounds used for detailed pharmacological
studies was proven by elemental analysis (compounds 5-7, 33, 36 and 38).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Melting points were determined with a Büchi 510 apparatus (Büchi, Essen, Germany)
(
compounds 10, 13, 14, 35) or with a Lambda Photometrics Optimelt MPA100 apparatus
Lambda photometrics, Harpenden, UK) (compounds 22, 24-27, 31, 32) and are uncorrected.
(
Specific optical rotations at 589 nm (Na-D line) were measured on a Polarimeter P8000-T
equipped with an electronic Peltier thermostat PT31 (A. KRÜSS Optronic, Hamburg, Germany)
using a micro-cuvette (layer thickness: 100 mm, volume: 1 mL, thermostated at 20 °C) and
2
acetonitrile/H O 70:30 (v/v) as solvent. IR spectra were measured on a NICOLET 380 FT-IR
spectrophotometer (Thermo Scientific, Waltham, MA). NMR spectra were recorded on a Bruker
1
13
Avance 300 instrument (7.05 T, H: 300 MHz, C: 75 MHz), a Bruker Avance 400 instrument
1
13
(
9.40 T, H: 400 MHz, C: 100 MHz) or a Bruker Avance 600 instrument with cryogenic probe
1
13
(14.1 T, H: 600 MHz, C: 150 MHz) (Bruker, Karlsruhe, Germany). Elemental analysis was
performed with a Vario MICRO Cube elemental analyzer (Elementar Analysensysteme, Hanau,
Germany). Low-resolution mass spectrometry (MS) was performed on a Finnigan ThermoQuest
TSQ 7000 instrument (Thermo Finnigan, San Jose, CA) equipped with an electrospray ionization
(
ESI) source and coupled to an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara,
CA). The following LC method was used: Column: Luna C18, 2.5 µm, 50 × 2 mm HST
Phenomenex, Aschaffenburg, Germany); column temperature: 40 °C; flow: 0.40 mL/min;
(
solvent A: MeCN, solvent B: 0.1% aq formic acid; gradient program: 0–1 min: A/B 5:95, 1–8
min: 5:95–98:2, 8–11 min: 98:2, 11–12 min: 98:2–5:95, 12–15 min: 5:95. High-resolution mass
spectrometry (HRMS) analysis was performed on an Agilent 6540 UHD Accurate-Mass Q-TOF
LC/MS system (Agilent Technologies, Santa Clara, CA) using an ESI source. Preparative HPLC
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was performed with a system from Knauer (Berlin, Germany) consisting of two K-1800 pumps
and a K-2001 detector. A Nucleodur 100-5 C18, 5 µm, 250 × 21 mm (Macherey-Nagel, Dueren,
Germany), a Kinetex XB C18, 5 µm, 250 × 21 mm (Phenomenex, Aschaffenburg, Germany) or a
Puriflash C18HQ Flash Column, 15 µm, 120 g (Interchim, France) served as RP-columns at flow
rates of 18, 20 and 50 mL/min, respectively. Mixtures of acetonitrile and 0.1% or 0.2% aq TFA
were used as mobile phase and a detection wavelength of 220 nm was used throughout.
Acetonitrile was removed from the eluates under reduced pressure (final pressure: 60 mbar) at 40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
°C prior to lyophilisation (Christ alpha 2-4 LD lyophilisation apparatus (Martin Christ
Gefriertrocknungsanlagen, Osterode am Harz, Germany) equipped with a Vacuubrand RZ 6
rotary vane vacuum pump (Vacuubrand, Wertheim, Germany)). Analytical HPLC analysis of
compounds 5-7, 17, 28, 29, 33, 36 and 38 (concentrations ca 50 µM) was performed with a
system from Thermo Separation Products composed of a SN400 controller, a P4000 pump, a
degasser (Degassex DG-4400, Phenomenex), an AS3000 autosampler and a Spectra Focus UV-
VIS detector. A Eurospher-100 C18, 5 µm, 250 × 4 mm (Knauer, Berlin, Germany) served as
RP-column at a flow rate of 0.8 mL/min. Mixtures of acetonitrile (A) and 0.05% aq TFA (B)
were used as mobile phase. The following linear gradient was applied: 0–30 min: A/B 20:80–
9
5:5, 30–40 min: 95:5. An oven temperature of 30 °C, an injection volume of 100 µL and a
detection wavelength of 220 nm were used throughout. Analytical HPLC analysis of compounds
0 and 40 (concentration ca 100 and 70 µM, respectively) was performed on a system from
Agilent Technologies composed of a 1290 Infinity binary pump equipped with a degasser, a
290 Infinity Autosampler, a 1290 Infinity Thermostated Column Compartment and a 1260
3
1
Infinity Diode Array Detector. A Kinetex XB C18, 2.5 µm, 100 × 3 mm (Phenomenex) served as
stationary phase at a flow rate of 0.6 mL/min. Mixtures of acetonitrile (A) and 0.05% aq TFA
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