[3H]UR-DE257: Development of a tritium-labeled squaramide-type selective histamine H2 receptor antagonist

Article abstract of DOI:10.1002/cmdc.201402344

A series of new piperidinomethylphenoxypropylamine-type histamine H₂ receptor (H₂R) antagonists with different substituted "urea equivalents" was synthesized and characterized in functional in vitro assays. Based on these data as selection criteria, radiosynthesis of N-[6-(3,4-dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propylamino}cyclobut-1-enylamino)hexyl]-(2,3-³H₂)propionic amide ([³H]UR-DE257) was performed. The radioligand (specific activity: 63Cimmol^-1) had high affinity for human, rat, and guinea pig H₂R (hH₂R, Sf9 cells: K_d, saturation binding: 31 nM, kinetic studies: 20 nM). UR-DE257 revealed high H₂R selectivity on membranes of Sf9 cells, expressing the respective hH_xR subtype (K_i values: hH1R: > 10000 nM, hH₂R: 28 nM, hH₃R: 3800 nM, hH₄R: > 10000 nM). In spite of insurmountable antagonism, probably due to rebinding of [³H]URDE257 to the H₂R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H₂R affinities in competition binding assays.

Full text of DOI:10.1002/cmdc.201402344

DOI: 10.1002/cmdc.201402344
Full Papers
[3H]UR-DE257: Development of a Tritium-Labeled
Squaramide-Type Selective Histamine H2 Receptor
Antagonist
Paul Baumeister, Daniela Erdmann, Sabrina Biselli, Nicole Kagermeier, Sigurd Elz,
Gꢀnther Bernhardt, and Armin Buschauer*[a]
Dedicated to Prof. Dr. Helmut Schçnenberger on the occasion of his 90th birthday.
A series of new piperidinomethylphenoxypropylamine-type
histamine H2 receptor (H2R) antagonists with different substi-
tuted “urea equivalents” was synthesized and characterized in
functional in vitro assays. Based on these data as selection cri-
teria, radiosynthesis of N-[6-(3,4-dioxo-2-{3-[3-(piperidin-1-yl-
methyl)phenoxy]propylamino}cyclobut-1-enylamino)hexyl]-(2,3-
3H2)propionic amide ([3H]UR-DE257) was performed. The radio-
ligand (specific activity: 63 Cimmolꢀ1) had high affinity for
human, rat, and guinea pig H2R (hH2R, Sf9 cells: Kd, saturation
binding: 31 nm, kinetic studies: 20 nm). UR-DE257 revealed
high H2R selectivity on membranes of Sf9 cells, expressing the
respective hHxR subtype (Ki values: hH1R: >10000 nm, hH2R:
28 nm, hH3R: 3800 nm, hH4R: >10000 nm). In spite of insur-
mountable antagonism, probably due to rebinding of [3H]UR-
DE257 to the H2R (extended residence time), the title com-
pound proved to be a valuable pharmacological tool for the
determination of H2R affinities in competition binding assays.
Introduction
The biogenic amine histamine mediates its various (patho)phy-
siological effects via four subtypes of G-protein-coupled recep-
tors (GPCRs), designated H1, H2, H3 and H4 receptors.[1,2] The H2
receptor (H2R) is known as the target of drugs such as cimeti-
dine, ranitidine, famotidine and roxatidine (Figure 1), which
have been used, e.g., for the treatment of gastroduodenal
ulcers.[1,3] Apart from marketed drugs, numerous structurally re-
lated compounds,[2,4] for example, iodoaminopotentidine,[5]
BMY 25368,[6] and tiotidine,[7] are known as H2R antagonists.
Histamine as well as several of the antagonists have been ra-
diolabeled as pharmacological tools (for review, see Refs. [8–
11]). Nevertheless, [3H]histamine, [3H]ranitidine,[12] [3H]cimeti-
dine,[13] and [3H]tiotidine[14] are far from being ideal radio-
ligands. The H2R affinities of the endogenous (nonselective) ag-
onist and the antagonists cimetidine and ranitidine are low.
[3H]Tiotidine,[14,15] although routinely used for a long period of
time, is known for high unspecific binding. [125I]Iodoamino-
potentidine[9] has the highest affinity of all reported H2R li-
gands and was used, for instance, for autoradiography of the
potentidine is not commercially available and can only be
used within 4–6 weeks after preparation.
Aiming at the development of high-affinity tritium-labeled
H2R antagonists, we considered piperidinomethylphenoxyalkyl-
amines most appropriate as precursors. Previous work suggest-
ed that substituents at the amino group are tolerated in this
class of compounds and can even confer additional H2R bind-
ing affinity.[5,17,18] The piperidinomethylphenoxyalkyl portion of
aminopotentidine and related substances was retained, the
polar group (“urea equivalent”) was optimized by exploring cy-
anoguanidine, nitroethenediamine, amide or squaric amide
moieties, and a terminal amino group, connected via a linker
of different length, was introduced. The latter allowed coupling
with commercially available succinimidyl [2,3-3H2]propionate
under standard laboratory conditions,[19–22] in case that the cor-
responding “potential radioligands” (i.e., the “cold” forms)
were identified as high affinity H2R antagonists.
H2R in heart and brain of the guinea pig. Compared with triti- Results and Discussion
um-labeled compounds, iodinated radioligands have the ad-
vantage of higher specific activity.[16] However, [125I]iodoamino-
Chemistry
3-[3-(Piperidin-1-ylmethyl)phenoxy]propan-1-amine (1) was
prepared in a three step synthesis as previously described.[23]
Compound 1 served as key intermediate for the preparation of
w-aminoalkyl-substituted potentidine-like structures by analo-
gy with previously described procedures.[23] For the synthesis
of the cyanoguanidines (Scheme 1), the amine 1 was treated
with diphenyl cyanocarbonimidate[24] by analogy with pub-
lished protocols[5,23] to give the intermediate 2. Aminolysis, per-
[a] Dr. P. Baumeister, Dr. D. Erdmann, S. Biselli, N. Kagermeier, Prof. Dr. S. Elz,
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
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402344.
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formed with an excess of the respective diamine,
yielded the cyanoguanidines 3 and 4. By analogy
with known procedures,[25] condensation of 1 with
1,1-bis(methylthio)-2-nitroethene gave intermediate
5, and subsequent reaction with an excess of pen-
tane-1,5-diamine yielded the nitroethenediamine 6.
For the synthesis of roxatidine-like structures, the w-
phthalimidoalkanoic acids 7 and 8,[26,27] obtained
from the respective w-aminoalkanoic acids and
phthalic anhydride as described previously,[18,26,27]
were treated with compound 1 using carbonyldiimi-
dazole (CDI) as coupling reagent to yield the amides
9 and 10. Cleavage of the phthaloyl protecting group
with hydrazine gave the amines 11 and 12.
The synthesis of the squaramides 14–18
(Scheme 2) was adapted from described proce-
dures.[28–31] The precursor 1 and 3,4-diethoxycyclobut-
3-ene-1,2-dione gave the corresponding squaric acid
ester amide (13) as intermediate, which was treated
with a 5- to 20-fold excess of the pertinent alkanedia-
mine to give the amines 15–17. As an alternative for
the preparation of the squaramides 14 and 18, pre-
venting the formation of symmetric by-products, the
mono-Boc protected alkanediamines were used to
yield the tert-butyl carbamates 14a and 18a, which
were cleaved with trifluoroacetic acid (TFA). The pro-
pionamides 20–23 and 24a were synthesized from
the primary amines 14–18 and succinimidyl propio-
nate (19a, Scheme 2).
Figure 1. Structures of cimetidine, roxatidine, tiotidine, and histamine H2 receptor ago-
nists and antagonists used as reference compounds in this study.
Radiosynthesis
Based on the pharmacological
data presented below, the tritiat-
ed propionamide 24b ([3H]UR-
DE257), the “hot” form of 24a,
was prepared by acylation of an
excess of amine precursor 18 in
the presence of triethylamine
with commercially available triti-
ated succinimidyl propionate
19b (Scheme 2, see Refs. [19–
22]). After purification by HPLC,
radioligand 24b was obtained in
a radiochemical purity of >99%
(Figure 2) with a specific activity
(as) of 63.0 Ci mmolꢀ1. The iden-
tity of the radioligand was con-
firmed by HPLC analysis of la-
beled (24b) and unlabeled (24a)
UR-DE257. Moreover, chemical
stability in ethanol at ꢀ208C
was proven over a period of at
least 24 months (see Supporting
Scheme 1. Synthesis of cyanoguanidines, amides and the nitroethenediamine. Reagents and conditions: a) MeOH,
RT, 5 h; b) ethane-1,2-diamine or hexane-1,6-diamine, MeOH, RT, overnight; c) EtOH, RT, 3 h; d) pentane-1,5-dia-
mine, MeOH, RT, overnight; e) phthalic anhydride, 135–1408C, 30 min; f) 1. 7 or 8, CDI, anhydrous THF, 1 h, RT,
2. 1, RT, overnight; g) 1. hydrazine hydrate, 1 h, reflux, 2. 2n HCl, 30 min, reflux.
Information).
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of the piperidinomethylphenoxy-
propyl moiety with a squaramide
fragment and an additional basic
center linked via an alkyl chain
(14–18) resulted in a tremendous
increase in H2R antagonist activi-
ty, similar to BMY 25368.[6] The Kb
values (13–210 nm) decreased
with the chain length of the at-
tached alkanediyl spacer. Except
for 22, the data for the corre-
sponding squaramides 20–23
and 24a were similar to those of
the amine precursors (Kb: 38–
200 nm). Only very weak, negligi-
ble inverse agonistic effects
were detected. Highest antago-
nistic activity (Kb values, hH2R:
38 nm, gpH2R: 28 nm) resided in
compound 24a (Figure 3).
Scheme 2. Synthesis of squaramides and [3H]UR-DE257 (24b). Reagents and conditions: a) 3,4-diethoxycyclobut-3-
ene-1,2-dione, EtOH, RT, 3 h; b) for 14 and 18: 1. tert-butyl 2-aminoethylcarbamate (H2N-C2H4-NHBoc) or tert-butyl
6-aminohexylcarbamate (H2N-C6H12-NHBoc), EtOH, RT, overnight, 2. TFA, CH2Cl2, RT, overnight; for 15–17: H2N-
(CH2)n-NH2, EtOH, RT, 5–18 h; c) for 20–23, 24a: 19a, CH3CN or DMSO or MeOH/DMSO (1:1), Et3N, RT, 30 min–18 h;
for 24b: 19b, CH3CN/DMSO (1:1), Et3N, RT, 18 h.
The selectivity of the amides
20–23 and 24a for human H2
over H1, H3, and H4 receptors
was proven by radioligand com-
Pharmacology
petition binding assays using membranes of Sf9 insect cells ex-
pressing the respective histamine receptor subtype (Table 2; Ki
values at hH2R in Table 2 completed with competition binding
data determined with 24b). The affinities determined for the
hH2R were in accordance with the functional data from the
steady-state GTPase assay. Compound 24a, which has a hexam-
ethylene spacer, showed highest affinity with a Ki value of
28 nm, followed by compound 21 (Ki 91 nm). Affinities to the
hH3R ranged between 3800 and 6800 nm for the squaramides
21–23 and 24a, whereas binding of compound 20 was not de-
tectable up to a concentration of 10 mm. The highest selectivity
was found for 24a with 140-fold higher affinity at hH2R than
Amine precursors 3, 4, 6, 11, 12 and 14–18 and the “cold
forms” of potential radioligands 20–23 and 24a were investi-
gated for functional activity in a steady-state GTPase assay on
membranes of Sf9 cells expressing the hH2R-GsaS fusion pro-
tein.[10] Compounds 3, 4, 6, 11, 12 and 14–18 exerted minor in-
verse agonism (Emax: ꢀ0.20 to ꢀ0.06, see Table 1) or, when
studied in the antagonist mode, inhibited histamine stimulated
GTP hydrolysis.
The cyanoguanidines, nitroethenediamines and amides 3, 4,
6, 11 and 12 were only moderate to weak antagonists at the
H2R (Kb values 590–5200 nm, Table 1). By contrast, combination
Figure 2. Identity and purity control of [3H]UR-DE257 (24b). a) Reaction control after 20 h. b) Radiochromatogram after purification. c) UV (l=280 nm) chro-
matogram of 24a, c=100 mm.
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Table 1. Pharmacological data for hH2R antagonistic amines BMY 25368,
8–12, 14–18 and propionic amides 20–23, 24a.
Table 2. Histamine receptor subtype selectivities (hH1–4R binding data) of
propionic amides 20–23, 24a.
[c]
Compd
n[a]
Kb [nm][b]
Emax
Compd
n[a]
Ki [nm]
hH3R[d]
hH1R[b]
hH2R[c]
hH4R[e]
BMY 25368[d]
3
4
6
11
12
14
15
16
17
18
20
21
22
23
–
2
6
5
1
5
2
3
4
5
6
2
3
4
5
6
44ꢁ5
ꢀ0.06ꢁ0.05
ꢀ0.16ꢁ0.17
ꢀ0.06ꢁ0.01
ꢀ0.11ꢁ0.02
ꢀ0.14ꢁ0.01
ꢀ0.12ꢁ0.12
0.18ꢁ0.07
ꢀ0.17ꢁ0.07
0.20ꢁ0.03
ꢀ0.20ꢁ0.06
ꢀ0.19ꢁ0.08
0.11ꢁ0.04
ꢀ0.33ꢁ0.18
ꢀ0.23ꢁ0.05
ND
980ꢁ290
590ꢁ63
20
21
22
23
2
3
4
5
6
>10000 150ꢁ6
>10000
5600ꢁ610
>10000
>10000
>10000
91ꢁ6
>1000
>10000 290ꢁ33
>10000 130ꢁ12
6800ꢁ1400 >10000
1600ꢁ280
5200ꢁ30
210ꢁ11
75ꢁ10
39ꢁ19
13ꢁ9
3900ꢁ380
3800ꢁ380
>10000
>10000
24a (UR-DE257)
>10000
28ꢁ2
[a] Length of the linker expressed as number of atoms connecting the
“urea equivalent” and the w-amino function (for structures, see
Schemes 1 and 2). [b] hH1R binding determined on Sf9 insect cell mem-
branes expressing the hH1R+RGS4 using [3H]pyrilamine (5 nm) as the
radioligand. [c] hH2R binding: Sf9 insect cell membranes expressing the
hH2R–GsaS; radioligand: 24b (30 nm). [d] hH3R binding: Sf9 insect cell
membranes expressing the hH3R+Gia2 +b1g2; radioligand: [3H]Na-methyl-
histamine (3 nm). [e] hH4R binding: Sf9 insect cell membranes expressing
the hH4R+Gia2 +b1g2; radioligand: [3H]histamine (15 nm); values repre-
sent mean ꢁSEM of 2–3 independent experiments each performed in
triplicate.
18ꢁ2
110ꢁ15
59ꢁ13
200ꢁ48
69ꢁ7
24a (UR-DE257)
38ꢁ8
0.08ꢁ0.01
[a] Length of the linker expressed as number of atoms connecting the
“urea equivalent” and the w-amino function (for structures, see
Schemes 1 and 2). [b] Steady-state GTPase assay on Sf9 cell membranes;
[ligands]: 1 nm–100 mm; typical GTPase activities (1 mm histamine), hH2R:
3.5–7 pmolmgꢀ1 minꢀ1. [c] Emax maximal response relative to histamine
(Emax =1.0) determined in the agonist mode; [ligands for efficacy]: 10 mm;
mean values ꢁSEM (n=2–4 independent experiments performed in du-
plicate); ND: not determined.
Table 3. Saturation binding parameters of [3H]UR-DE257 (24b) on mem-
branes of Sf9 cells expressing human, guinea pig, and rat H2R and at
hH2R on HEK293T CRE-Luc hH2R cells.[a]
H2R
orthologue
Kd
[nm]
Kd(scatchard)
[nm]
Bmax
Specific binding
sites per well
[pmol(mg prot.)ꢀ1
]
hH2R[b]
hH2R[c]
gpH2R[b]
rH2R[b]
31ꢁ5
55ꢁ8
37ꢁ13
29ꢁ2
37ꢁ3
52ꢁ9
39ꢁ24
40ꢁ12
0.78ꢁ0.08
2.4ꢂ106[d]
0.77ꢁ0.03
4.07ꢁ0.11
4.8ꢂ1010
2.4ꢂ1011
3.0ꢂ1010
1.4ꢂ1011
[a] Data are mean values ꢁSEM of two independent experiments, each
performed in triplicate. [b] Binding data determined on membranes of
Sf9 insect cells expressing the respective xH2R–GsaS fusion protein (x=h,
gp or r). [c] hH2R binding on HEK293T CRE-Luc hH2R cells. [d] Estimated
Bmax (sites/cell).
Determination of binding constants of [3H]UR-DE257 (24b)
Figure 3. Antagonism of UR-DE257 (24a) on human and guinea pig H2R,
determined in the GTPase assay on membranes of Sf9 cells expressing the
hH2R–GsaS or the gpH2R–GsaS fusion protein. Inhibition curves were recorded
in the presence of 1 mm histamine. The incubation period was 60 min. Data
are mean values ꢁSEM (n=2–3 independent experiments performed in trip-
licate).
The radioligand 24b was characterized by saturation and ki-
netic binding experiments on membrane preparations of Sf9
insect cells expressing the fusion proteins hH2R–GsaS, gpH2R–
GsaS or rH2R–GsaS or on HEK293T CRE-Luc hH2R cells stably ex-
pressing the hH2R. [3H]UR-DE257 was bound in a saturable
manner (see Figure 4 for the hH2R at Sf9 insect cell mem-
branes; for gpH2R and rH2R at Sf9 insect cell membranes and
hH2R experiments on HEK293 cells, see Supporting Informa-
tion). Up to a radioligand concentration of 200 nm, nonspecific
binding, determined in the presence of famotidine (10 mm),
was low, amounting to 5–20% of total binding. Specific bind-
ing versus 24b ([3H]UR-DE257) concentration was best fitted
by nonlinear regression to a one-site binding model, nonspe-
cific binding to a standard linear curve. The determined Kd
values (29–55 nm, Table 3) were similar to the Kb values from
functional GTPase assays (Kb =38 and 28 nm, Figure 3). The
maximum number of binding sites (Bmax) ranged between 0.8
and 4.1 pmolmgꢀ1 membrane protein in the Sf9 cell mem-
branes and amounted to 2.4ꢂ106 sites on HEK cells. In all
hH3R. Up to a concentration of 10 mm the synthesized squara-
mides revealed binding neither to the hH1R nor to the hH4R re-
ceptor.
Compound 24a was chosen for the preparation of the corre-
sponding radioactive tracer 24b. Furthermore, with respect to
future studies in laboratory animals, the compounds 20–23,
24a, and 24b were characterized on the guinea pig and rat H2
receptor orthologues (gpH2R and rH2R) in membranes of Sf9
insect cells. The Ki values of 24b on gpH2R and rH2R were in
agreement with the data from binding experiments at the
hH2R (Table 3). Thus, there were no relevant differences in affin-
ity between these H2R species orthologues.[32]
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Figure 4. Saturation binding of 24b on the hH2R expressed in Sf9 mem-
branes a) Representative saturation binding curve (dpm); total binding: *;
specific binding: *; nonspecific binding: &. b) Normalized saturation bind-
ing curve. Specific binding: *; nonspecific binding: &; Kd(sat) =31ꢁ5 nm.
c) Scatchard analysis, best fitted by linear regression, Kd =ꢀ1/slope=
37ꢁ3 nm; (mean values ꢁSEM of n=3 independent experiments, each per-
formed in triplicate).
Figure 5. Binding kinetics of the radiolabeled H2R antagonist 24b on H2R-
GsaS fusion proteins (Sf9 cell membranes) and the hH2R expressed in
HEK293T cells. a) Radioligand (c=30 nm) association on hH2R (Sf9). Inset: Lin-
earized plot; kob =0.050 minꢀ1. b) Radioligand (c=30 nm, pre-incubation for
60 min) dissociation determined on hH2R (Sf9) in the presence of a 100-fold
excess of famotidine. Inset: Linearization of the data (mean values ꢁSEM,
n=4) results in two straight lines; initial (fast) phase of dissociation:
cases, within the investigated concentration range, the Scatch-
ard plots were linear, in agreement with the binding of 24b to
a single binding site, following the law of mass action (Fig-
ure 4c and Supporting Information).
The results of kinetic studies of 24b (c=30 nm) at the hH2R
on Sf9 cell membranes at 228C are presented in Figure 5a,b.
Similar data were obtained for gpH2R and rH2R (Sf9 cell mem-
branes) and hH2R expressed in HEK293 cells (Figure 5c, Table 4
and Supporting Information).
k
off =0.020 minꢀ1. Calculated from a) and b): kon =(kobꢀkoff)/[L]=
8.6ꢂ10ꢀ4 minꢀ1 nmꢀ1. c) Dissociation of the radioligand (c=30 nm, pre-incu-
bation for 60 min) determined in the presence of a 100-fold excess of famo-
tidine on membranes of Sf9 insect cells expressing the respective H2R–GsaS
orthologue or on HEK293T CRE-Luc hH2R cells.
Linearization of the association curves revealed kob values
ranging from 0.050 to 0.073 minꢀ1. Dissociation in the presence
of famotidine (Figure 5c) gave similar results for all investigat-
ed H2R orthologues in Sf9 membranes and HEK293 cells. After
90 min the residual specific binding of the tritiated compound
amounted to approximately 60–70% for rat and human H2R,
suggesting (pseudo)irreversible binding (see Ref. [22]). Lineari-
zation revealed biphasic dissociation kinetics with different
slopes (Figure 5b, inset) for rat and human H2R, but monopha-
sic dissociation for the gpH2R (20–30% residual binding after
120 min; see Supporting Information). Regardless of that, the
equilibrium dissociation constant of 24b, calculated from ki-
netics (Kd =koff/kon =13–44 nm; Figure 5, Table 4 and Support-
ing Information), was consistent with the Kd values derived
from saturation binding experiments (Kd =29–55 nm), in
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Table 4. Binding kinetics of 24b to H2R orthologues.
[a]
[b]
[d]
H2R
orthologue
koff
kon
k
off/kon
Kd(sat)
[minꢀ1
]
[minꢀ1 nmꢀ1
]
Kd [nm]
[nm]
[c]
hH2R (Sf9)
0.024
0.025
0.015
0.019
0.00086
0.0016
0.00033
0.0015
20
16
44
13
31
55
37
29
hH2R (HEK293)
gpH2R (Sf9)
rH2R (Sf9)
[a] Dissociation rate constant determined by linear regression from the in-
itial rate. [b] Association rate constant determined by linear regression.
[c] Kinetically determined dissociation constant. [d] Equilibrium dissocia-
tion constant determined by saturation binding experiments.
accordance with the law of mass action,[33] provided that the
koff value of the initial (fast) phase of dissociation was consid-
ered. The percentage of residual specific binding after 30–
60 min of dissociation was highest in case of the hH2R ex-
pressed in HEK293 cells and lowest for the gpH2R in Sf9 cell
membranes. Clearly, the extent of receptor occupation after
this period of time depended on the number of binding sites,
that is, the expression level of the respective receptor. This is
in accordance with the concept of slow dissociation of ligands
due to rebinding as a function of receptor density.[34–36]
The pseudo-irreversibility of binding, apparent from the later
phase of dissociation (>30 min), is reminiscent of the pharma-
cological behavior reported for several classes of H2R antago-
nists, for instance, piperidinomethylphenoxyalkyl-substituted
squaramides, thiadiazolamines, or aminotriazoles.[6,37–39] Squara-
mides such as BMY 25368 (Figure 1) were described as insur-
mountable H2R antagonists, causing a concentration-depen-
dent depression of the maximal response to the agonist and
tight binding to the H2R in the guinea pig right atrium (Kb =
13 nm) relative to standard ligands such as famotidine.[40] Such
a peculiar behavior at the guinea pig right atrium was also
confirmed for 14 (pA2 =7.38ꢁ0.10), 18 (pA2 =7.41ꢁ0.10), 20
(pA2 =7.36ꢁ0.14), and 21 (pA2 =7.22ꢁ0.08) as examples.
Compounds 18 and 20 caused a concentration-dependent de-
crease in the maximum response to histamine (Figure 6), and
in the presence of 1 mm of 14 and 21, Emax of histamine was
78ꢁ4% and 67ꢁ2%, respectively (n=4 each; incubation
period: 60 min; see Supporting Information). Recently, a Y2R
selective radioligand with similar properties in kinetic and func-
tional experiments was reported as an insurmountable
pseudo-irreversible nonpeptide neuropeptide Y receptor an-
tagonist.[22] Possible explanations are a slow rate of dissociation
from the receptor,[41] a slow rate of interconversion between in-
active and active receptor conformations,[42,43] or stabilization
of a ligand (antagonist) specific inactive receptor conforma-
tion.[44]
Figure 6. Histamine H2R antagonism on the isolated spontaneously beating
guinea pig right atrium. Concentration–response curves of histamine alone
(*) and in the presence of 18 a) at 100 nm (&, Emax =94ꢁ7%, n=3),
316 nm (!, Emax =64ꢁ3%, n=5), 1000 nm (~, Emax =17ꢁ4%, n=2), and in
the presence of 20 b) at 316 nm (&, Emax =77ꢁ4%, n=3) and 1000 nm (!,
E
max =54ꢁ7%, n=3); incubation period: 60 min. From the rightward shift
calculated and corrected by the mean desensitization observed in untreated
control organs (0.13ꢁ0.02, n=16), mean apparent pA2 values obtained for
18 and 20 were 7.41ꢁ0.10 (n=10) and 7.36ꢁ0.14 (n=6), respectively. Data
are given as mean ꢁSEM.
As summarized in Table 5, the hH2R binding affinity of the
agonists histamine (Ki =534 nm), amthamine (Ki =244 nm), UR-
Bit24[46] (Ki =7.1 nm) and UR-AK480[47] (Ki =1.9 nm), and of the
antagonists famotidine (Ki =136 nm), ranitidine (Ki =1730 nm),
BMY 25368 (Ki =19 nm) and iodoaminopotentidine (Ki =
0.31 nm) were consistent with reported data from functional
and binding experiments.[40,48] The Ki value of 24a on the hH2R
(28 nm), determined by competition binding experiments with
the labeled analogue 24b, corresponds very well to the Kd
value of compound 24b (31 nm), and the data for the rat and
guinea H2R species orthologues were in the same range (Ki
values: 16 and 43 nm, respectively).
As reported from binding studies with [125I]iodoamino-
potentidine, H2R binding sites were detected in the cerebral
cortex in layers I–III (I: molecular layer; II: external granular
layer; III: layer of medium-sized pyramidal cells), in the thala-
mus, and in the hippocampus in guinea-pig brain.[9,48,49] To ex-
plore the applicability of [3H]UR-DE257 to autoradiography, cry-
osections of a guinea-pig brain were incubated with the new
radioligand. However, there were no significant differences be-
tween the autoradiographs representing specific and non-
specific binding (data not shown).
Regardless of the slow dissociation (Figure 5b), the radioli-
gand 24b was completely displaceable in competition binding
studies and proved to be a very useful tool to determine bind-
ing data of H2R agonists and antagonists as shown for several
reference compounds in Figure 7 and in the Supporting Infor-
mation. The Kd value determined from saturation binding was
applied for the calculation of Ki values by means of the
Cheng–Prusoff equation.[45]
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Conclusions
Potent and selective propionylated H2R antagonists were ob-
tained by the derivatization of amino-functionalized precursors
structurally related to potentidine. Compound 24a proved to
be a high affinity H2R antagonist with 140-fold selectivity over
the hH3R. Neither affinity to the hH1R nor at the hH4R was de-
tected. The Kd value (20 nm) of compound 24b, the tritiated
form of 24a, determined from kinetic experiments was in
good agreement with the dissociation equilibrium constant
(31 nm) from saturation analysis. In contrast to investigations
for antagonism in GTPase assays on Sf9 membranes, functional
studies performed with selected substances related to 24a on
the isolated guinea pig right atrium revealed insurmountable
antagonism. This may be explained by rebinding of these li-
gands to the H2R rather than irreversible binding, as reflected
by the different dissociation curves of 24b depending on the
expression level of the receptor protein. Such a behavior has
been reported as a possible reason for extended residence
times of GPCR ligands.
Nonetheless, 24b proved to be a useful tool to determine
the H2R binding affinities of unlabeled compounds in competi-
tion binding experiments, as confirmed for a set of reference li-
gands. [3H]UR-DE257 (24b) is an attractive alternative to
[3H]tiotidine,[14,15] which showed a high degree of unspecific
binding. Relative to [125I]iodoaminopotentidine,[9] [3H]UR-DE257
has a longer half-life, which is advantageous for performing
both synthesis and pharmacological studies, as well as being
more convenient to handle safely.
Figure 7. Displacement of the radioligand 24b (c=30 nm, Kd =31 nm) by
the H2R ligands UR-AK480 (r, Ki =1.9ꢁ0.5 nm), UR-DE257 (24a, *, Ki =
28ꢁ2 nm), famotidine (*, Ki =136ꢁ17 nm), histamine (~, Ki = 534 ꢁ
^
52 nm), UR-Bit24 ( , Ki =7.1ꢁ1.2 nm), amthamine (!, Ki = 244ꢁ46 nm), and
^
ranitidine ( , Ki =1730ꢁ220 nm). The assay was performed on Sf9 insect cell
membranes expressing the hH2R–GsaS fusion protein. The incubation period
was 60 min. Data are mean values ꢁSEM (2–3 independent experiments,
each performed in triplicate).
Experimental Section
Table 5. Binding of reference compounds (agonists, antagonists) and
propionamides 20–23 and 24a to the human (h), rat (r) and guinea pig
(gp) H2 receptor.
For general information, experimental details regarding synthesis,
analytical characterization of compounds 1, 7–10, 13, 14a and
18a, determination of purity of 3, 4, 6, 14–18, 20–23, 24a and
long-term stability of 24b (HPLC) see the Supporting Information.
For the synthesis of compound 5, see Ref. [23,25].
Compd[a]
Ki [nm]
rH2R[b]
hH2R[b]
gpH2R[b]
Histamine
Amthamine
UR-AK480
UR-Bit24
Famotidine
Tiotidine
Ranitidine
Iodoamino potentidine
BMY 25368
20
534ꢁ52
244ꢁ46
1.9ꢁ0.5
7.1ꢁ1.2
136ꢁ17
267ꢁ82
1730ꢁ220
0.31ꢁ0.04
19ꢁ1
486ꢁ106
ND
150ꢁ29
General procedure for the synthesis of cyanoguanidines 3 and
ND
1.7ꢁ0.5
54ꢁ3
0.15ꢁ0.02
9.5ꢁ2
4: Compound
1 (1 equiv) and diphenyl cyanocarbonimidate
(1 equiv) were dissolved in MeOH (50–60 mL) and stirred at RT for
5 h. Subsequently, the respective diamine (20 equiv), dissolved in
MeOH (10–15 mL), was added and the solution was stirred over-
night at RT. The organic solvent was evaporated in vacuo and the
residue was dissolved in chloroform (60–80 mL). The solution was
washed with water (two times, 20 mL) and with sodium hydroxide
solution (10% mmꢀ1, twice, 20 mL). The organic layer was dried
over sodium sulfate and the solvent was removed in vacuo. The re-
sulting product was dried over phosphorus pentoxide in vacuo.
423ꢁ33
377ꢁ65
ND
107ꢁ5
50ꢁ4
ND
0.28ꢁ0.05
ND
0.24ꢁ0.03
ND
150ꢁ6
140ꢁ6
62ꢁ1
72ꢁ0
21
22
23
91ꢁ6
45ꢁ4
290ꢁ33
130ꢁ12
28ꢁ2
49ꢁ11
320ꢁ95
16ꢁ1
57 ꢁ5
320ꢁ95
43ꢁ30
UR-DE257 (24a)
1-(2-Aminoethyl)-2-cyano-3-{3-[3-(piperidin-1-ylmethyl)phen-
oxy]propyl}guanidine (3): Compound 3 was synthesized from
[a] For structures, see Figure 1 and Scheme 2. [b] Binding determined on
membranes of Sf9 insect cells expressing the H2R–GsaS fusion protein of
the respective receptor orthologue; radioligand: [3H]UR-DE257 (30 nm); Ki
values represent means ꢁSEM of 2–4 independent experiments each per-
formed in triplicate; ND: not determined.
1
(812 mg, 3.30 mmol, 1 equiv), diphenyl cyanocarbonimidate
(826 mg, 3.50 mmol, 1.1 equiv) and ethane-1,2-diamine (4.00 g,
660 mmol, 20 equiv). Yield: 900 mg (76%) as yellow oil: RP-HPLC:
98% (tR =13.1 min, k=4.2); 1H NMR (300 MHz, CD3OD): d=1.45–
1.46 (m, 2H), 1.55–1.62 (m, 4H), 2.00–2.08 (m, 2H), 2.41 (m, 4H),
2.74 (t, J=6.3 Hz, 2H), 3.24 (t, J=6.3 Hz, 2H), 3.42 (t, J=6.7 Hz,
2H), 3.45 (s, 2H), 4.05 (t, J=5.9 Hz, 2H), 6.85–6.90 (m, 2H), 6.94–
6.95 (m, 1H), 7.21 ppm (t, J=7.8 Hz, 1H); 13C NMR (75 MHz,
CD3OD): d=25.2, 26.5, 30.1, 40.3, 41.9, 45.2, 55.4, 64.8, 66.7, 114.7,
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Full Papers
116.9, 120.1, 123.5, 130.3, 140.0, 160.3, 161.7 ppm; IR: n˜ =3265,
MeOH containing 1% NH3) yielding a sticky yellow oil (250 mg,
47%): 1H NMR (300 MHz, CDCl3): d=1.25–1.60 (m, 12H), 2.02 (m,
2H), 2.17 (m, 2H), 2.42 (m, 4H), 2.75–2.80 (m, 2H), 3.37–3.46 (m,
2H), 3.49 (s, 2H), 4.00–4.04 (m, 2H), 6.74–6.78 (m, 1H), 6.87–6.93
(m, 2H), 7.17–7.22 (m, 1H), 7.62 ppm (s, 1H); 13C NMR (75 MHz,
CDCl3): d=24.2, 25.2, 25.6, 26.1, 27.9, 29.2, 36.0, 36.8, 39.9, 54.3,
+
2931, 2163, 1580, 1441, 1343, 1258 cmꢀ1; HRMS-EI m/z [M] calcd
C
for C19H30N6O: 358.2481, found: 358.2474.
1-(6-Aminohexyl)-2-cyano-3-{3-[3-(piperidin-1-ylmethyl)phen-
oxy]propyl}guanidine (4): Compound 4 was synthesized from
1
(500 mg, 2.00 mmol, 1 equiv), diphenyl cyanocarbonimidate
63.5, 65.8, 113.1, 115.3, 121.8, 129.1, 139.5, 158.8, 173.8 ppm; MS
(CI, NH3) m/z (%): 362.2 (100) [M+H]+, C21H36N3O2
.
+
(477 mg, 2.00 mmol, 1 equiv) and hexane-1,6-diamine (4.60 g,
40.0 mmol, 20 equiv). Yield: 690 mg (84%) as oil: RP-HPLC: 96%
(tR =15.1 min, k=4.9); 1H NMR (600 MHz, [D6]DMSO): d=1.22 (m,
4H), 1.31–1.43 (m, 6H), 1.45–1.48 (m, 4H), 1.87–1.92 (m, 2H), 2.28
(m, 4H), 2.50 (m, 2H), 3.06–3.07 (m, 2H), 3.25–3.26 (m, 2H), 3.36 (s,
2H), 3.95 (t, J=6.1 Hz, 2H), 6.24 (s, 1H), 6.77–6.78 (m, 1H), 6.84 (s,
1H), 6.94–6.99 (m, 1H), 7.19 ppm (t, J=8.0 Hz, 1H); 13C NMR
(150 MHz, [D6]DMSO): d=24.0, 25.6, 26.1, 28.7, 29.0, 32.8, 38.3,
40.0, 41.0, 41.3, 53.9, 62.8, 65.0, 112.7, 114.6, 118.2, 120.9, 129.0,
3-(2-Aminoethylamino)-4-{3-[3-(piperidin-1-ylmethyl)phenoxy]-
propylamino}cyclobut-3-ene-1,2-dione bis(hydrotrifluoroacetate)
(14): Compound 14a (100 mg, 206 mmol) was dissolved in CH2Cl2,
and TFA in CH2Cl2 (10% v/v) was added. The solution was stirred
overnight at RT. The solvent was evaporated and a mixture of
EtOH and Et2O (1:10, v/v) was added. After storage at 4–68C for
16 h, the yellow precipitate was filtered off and dried in vacuo to
give a yellow semi-solid as di-TFA-salt. RP-HPLC: 96%. A sample of
26.4 mg was purified by preparative HPLC. CH3CN was removed in
vacuo. After lyophilization, 3.25 mg were obtained as yellow oil:
140.3, 158.5, 159.3 ppm; IR: n˜ =3265, 2930, 2162, 1580, 1443, 1341,
+
1259, 1039 cmꢀ1; HRMS-EI m/z [M] calcd for C23H38N6O: 414.3107,
C
found: 414.3098.
1
RP-HPLC: 99% (tR =9.5 min, k=2.7); (measured as amine). H NMR
Preparation of N1-(2-nitro-1-{3-[3-(piperidin-1-ylmethyl)phen-
oxy]propylamino}ethenyl)pentane-1,5-diamine bis(hydrotrifluor-
oacetate) (6): N-[1-(Methylthio)-2-nitroethen-1-yl]-3-[3-(piperidin-1-
ylmethyl)phenoxy]-propan-1-amine[25] (5, 50.0 mg, 137 mmol,
1 equiv) was dissolved in 10 mL MeOH, pentane-1,5-diamine
(70.0 mg, 685 mmol, 5 equiv) was added and the solution was
stirred at RT for 12 h. The solvent was evaporated and the residue
purified by preparative HPLC yielding the product as a brown oil
(42.0 mg, 47%): RP-HPLC: 100%; (tR =8.6 min, k=2.4); 1H NMR
(600 MHz, CD3OD): d=1.29–1.35 (m, 4H), 1.47–1.81 (m, 8H), 1.93–
2.03 (s, 2H), 2.75–2.76 (m, 2H), 2.84 (m, 2H), 3.08–3.09 (m, 2H) 3.16
(s, 1H), 3.29 (m, 4H), 4.01–4.06 (m, 2H), 4.22 (s, 2H), 7.03–7.09 (m,
3H), 7.37 (t, J=7.9 Hz, 1H), 7.63 (m, 4H), 9.50 ppm (s, 0.6H);
13C NMR (150 MHz, CD3OD): d=21.3 (2C), 22.3, 22.9, 26.6, 28.0,
38.6, 40.0, 45.7, 48.7, 51.8 (2C), 58.9, 64.9, 117.1, 119.7, 123.4, 128.0,
130.1, 138.0, 155.6, 158.5 ppm; HRMS-FAB m/z [M+H]+ calcd for
C22H38N5O3: 420.2975, found 420.2980.
(300 MHz, CD3OD): d=1.46–1.48 (m, 2H), 1.56–1.64 (m, 4H), 2.10
(qui, J=6.3 Hz, 2H), 2.43 (m, 4H), 2.80 (t, J=6.1 Hz, 2H), 3.48 (s,
2H), 3.63–3.65 (m, 2H), 3.82 (m, 2H), 4.09 (t, J=6.0 Hz, 2H), 6.84
(dd, J=2.1 Hz, J=7.9 Hz, 1H), 6.89 (d, J=7.6 Hz, 1H), 6.92 (s, 1H),
7.21 ppm (t, J=7.8 Hz, 1H); 13C NMR (150 MHz, CD3OD): d=25.1,
26.4, 31.8, 42.5, 43.4, 47.4, 55.3, 64.7, 66.6, 114.7, 116.9, 123.5, 130.3,
139.7, 160.3, 169.8, 183.78, 183.83 ppm; HRMS-EI m/z [M] +, calcd
C
for C21H30N4O3: 386.2318, found: 386.2315.
General procedure for preparation of 15–17: 3-Ethoxy-4-{3-[3-(pi-
peridin-1-ylmethyl)phenoxy]propylamino}cyclobut-3-ene-1,2-dione
(13) (1 equiv) was dissolved in EtOH (15–30 mL). After addition of
the respective diamine (18–20 equiv dissolved in 5–15 mL EtOH),
the solution was stirred overnight at RT. The solvent was removed
in vacuo. The residue was dissolved in a small amount of EtOH or
MeOH (1–3 mL), before Et2O was added to precipitate the product.
The suspension was stored at 4–68C for 2 h to 1 d. The white or
yellow crystals were collected on glass filter crucibles and dried in
vacuo. Products were obtained with purities of 54–89%. These
compounds were further purified with preparative HPLC.
2-Amino-N-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl}aceta-
mide (11): To a solution of 9 (230 mg, 0.530 mmol, 1 equiv) in
MeOH (15 mL) hydrazine hydrate (90 mL, 1.80 mmol, 3.3 equiv) was
added and the mixture was heated at reflux for 1 h. After addition
of HCl (2m, 1.5 mL) the solution was heated at reflux for another
30 min. The insoluble material was filtered off and washed with
water. The filtrate was concentrated, adjusted to pH 9–10 and ex-
tracted with CH2Cl2 (3ꢂ20 mL). The organic layers were combined,
dried over MgSO4, and the solvent was evaporated to give a sticky
3-(3-Aminopropylamino)-4-{3-[3-(piperidin-1-ylmethyl)phenoxy]-
propylamino}cyclobut-3-ene-1,2-dione bis(hydrotrifluoroacetate)
(15): The reaction of compound 13 (140 mg, 0.380 mmol, 1 equiv)
and propane-1,3-diamine (0.64 mL, 7.56 mmol, 20 equiv) produced
yellow crystals (100 mg, 66%), mp: 1158C (dec.); 40 mg were puri-
fied by preparative HPLC. CH3CN was removed in vacuo. Lyophili-
zation gave 32.2 mg of a pale-yellow oil: RP-HPLC: 100% (tR =
1
yellow oil (108 mg, 68%): H NMR (300 MHz, CDCl3): d=1.42–1.60
1
(m, 6H), 2.02 (qui, J=6.1 Hz, 2H), 2.37 (m, 4H), 3.35 (s, 2H), 3.43 (s,
2H), 3.47–3.51 (q, J=6.2 Hz, 2H), 4.05 (t, J=5.9 Hz, 2H), 6.75–6.80
(m, 1H), 6.88–6.91 (m, 2H), 7.17–7.23 (m, 1H), 7.62 ppm (s, 1H);
13C NMR (75 MHz, CDCl3): d=24.4, 26.0, 29.1, 36.9, 44.9, 44.8, 54.5,
9.1 min, k=2.6); H NMR (300 MHz, CD3OD): d=1.46–2.01 (m, 8H),
2.10 (q, J=6.3 Hz, 2H), 2.90–3.03 (m, 4H), 3.46 (m, 2H), 3.68 (t, J=
6.6 Hz, 2H), 3.84 (m, 2H), 4.12 (t, J=5.9 Hz, 2H), 4.23 (s, 2H), 7.02–
7.06 (m, 3H), 7.37 ppm (t, J=7.8 Hz, 1H); 13C NMR (75 MHz,
CD3OD): d=22.8, 24.1, 30.4, 31.7, 37.9, 41.9, 42.5, 54.1, 61.7, 66.2,
63.8, 66.2, 112.8, 115.1, 121.8, 129.1, 140.4, 158.7, 172.8 ppm; MS
(CI, NH3) m/z (%): 306.3 (100) [M+H]+, C17H28N3O2
.
117.2, 118.4, 124.5, 131.5, 131.8, 160.8, 183.8 ppm (2C); 2C cyclobu-
+
tenyl not detected; HRMS-FAB m/z [M+H]+ calcd for C22H33N4O3
:
+
6-Amino-N-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl}hexana-
mide (12): To a solution of 10 (730 mg, 1.48 mmol, 1 equiv) in
MeOH (20 mL) hydrazine hydrate (240 mL, 4.90 mmol, 3 equiv) was
added and the mixture was heated at reflux for 1 h. After addition
of HCl (2m, 2 mL) the solution was heated at reflux for another
30 min. The insoluble material was filtered off and washed with
water. The filtrate was concentrated, adjusted to pH 8–9 and ex-
tracted with CH2Cl2 (3ꢂ20 mL). The organic layers were combined,
dried over MgSO4, and the solvent was evaporated. The product
was purified by column chromatography on silica gel (elution with
401.2553, found: 401.2551.
3-(4-Aminobutylamino)-4-{3-[3-(piperidin-1-ylmethyl)phenoxy]-
propylamino}cyclobut-3-ene-1,2-dione bis(hydrotrifluoroacetate)
(16): 16 was synthesized in 30 mL EtOH from 13 (140 mg, 376
mmol, 1 equiv) and butane-1,4-diamine (480 mg, 6.40 mmol,
14.5 equiv) yielding 140 mg (89%) of a white solid. RP-HPLC: 94%,
mp: 1388C (dec.). A sample (13 mg) was purified by preparative
HPLC. CH3CN was removed in vacuo. Lyophilization gave 8.47 mg
of the product as white semi-solid: RP-HPLC: 99.8%. (tR =9.7 min,
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ꢁ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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+
k=2.8); 1H NMR (300 MHz, CD3OD): d=1.50–1.52 (m, 1H), 1.70–
1.76 (m, 10H), 1.92–1.95 (m, 2H), 2.10 (qui, J=6.4 Hz, 2H), 2.92–
2.97 (m, 4H), 3.42–3.44 (m, 2H), 3.60 (m, 2H), 3.83 (m, 2H), 4.12 (t,
J=5.9 Hz, 2H), 4.23 (s, 2H), 7.02–7.04 (m, 2H), 7.06 (s, 1H),
7.37 ppm (t, J=7.9 Hz, 1H); 13C NMR (150 MHz, CD3OD): d=22.7,
24.1, 25.3, 29.2, 31.7, 40.2, 42.5, 44.3, 54.1, 61.7, 66.2, 117.2, 118.5,
124.5, 131.4, 131.7, 160.8, 183.6 ppm (2C); 2C cyclobutenedione
not detected; HRMS-EI m/z [M]+ calcd for C23H34N4O3: 414.2631,
found: 414.2636.
(2C), 177.1, 183.7 ppm (2C); HRMS-EI m/z [M]
C24H34N4O4: 442.2580, found: 442.2584.
calcd for
C
N-[3-(3,4-Dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino}cyclobut-1-enylamino)propyl]propionamide
hydrotri-
fluoroacetate (21): Compound 21 was prepared from 15 (35.0 mg,
87.0 mmol, 1 equiv) and succinimidyl propionate (12.0 mg,
70.0 mmol, 0.8 equiv) in DMSO (general procedure). The solution
was stirred for 45 min at RT and the reaction was stopped by addi-
tion of 10% TFA in CH3CN (250 mL). Yield: yellow oil (22 mg, 55%):
RP-HPLC: 99% (tR =11.6 min, k=3.6); 1H NMR (300 MHz, CD3OD):
d=1.12 (t, J=7.6 Hz, 3H), 1.49–1.97 (m, 8H), 2.10 (qui, J=6.2 Hz,
2H), 2.12 (q, J=7.6 Hz, 2H), 2.89–2.94 (m, 2H), 3.23 (t, J=7.6 Hz,
2H), 3.42–3.46 (m, 2H), 3.58–3.61 (m, 2H), 3.82–3.85 (m, 2H), 4.14
(t, J=5.8 Hz, 2H), 4.23 (s, 2H), 7.02–7.04 (m, 3H), 7.36 ppm (m,
1H); 13C NMR (150 MHz, CD3OD): d=10.5, 22.8, 24.2 (2C), 30.3, 31.6,
32.1, 37.1, 42.6, C propyl, superimposed by solvent, 54.1 (2C), 61.8,
3-(5-Aminopentylamino)-4-{3-[3-(piperidin-1-ylmethyl)phenoxy]-
propylamino}cyclobut-3-ene-1,2-dione bis(hydrotrifluoroacetate)
(17): Compound 17 was synthesized from 13 (130 mg, 349 mmol,
1 equiv) and pentane-1,5-diamine (700 mg, 6.60 mmol, 18.9 equiv)
in 15 mL EtOH yielding 80.0 mg (54%) as white solid: mp:>1308C
(dec.); RP-HPLC: 94%; 55 mg were purified by preparative HPLC.
The organic solvent was removed in vacuo. After lyophilization the
product was obtained as yellow oil (11.9 mg). RP-HPLC: 99% (tR =
9.3 min, k=2.7); 1H NMR (300 MHz, CD3OD): d=1.44–1.52 (3H),
1.66–1.81 (m, 7H), 1.74–1.81(m, 2H), 2.10 (qui, J=6.3 Hz, 2H),
2.90–2.96 (m, 4H), 3.42–3.44 (m, 2H), 3.60 (m, 2H), 3.83 (m, 2H),
4.12 (t, J=5.9 Hz, 2H), 4.23 (s, 2H), 7.02–7.06 (m, 3H), 7.36 ppm (t,
J=7.9 Hz, 1H); 13C NMR (150 MHz, CD3OD): d=22.7, 24.1, 24.2,
66.3, 117.1, 118.5, 120.9, 124.5, 131.5, 131.7, 160.8, 170.0 (2C), 177.3,
183.6 ppm (2C); HRMS-EI m/z [M] calcd for C25H36N4O4: 456.2737,
+
C
found: 456.2749.
N-[4-(3,4-Dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino}cyclobut-1-enylamino)butyl]propionamide hydrotrifluoro-
acetate (22): Compound 16 (29.7 mg, 72.0 mmol, 1 equiv) and suc-
cinimidyl propionate (20.0 mg, 116 mmol, 1.6 equiv) in DMSO were
stirred for 45 min at RT, and the reaction was stopped by addition
of 10% TFA in CH3CN (250 mL) (General procedure). The product
was obtained as yellow oil (28.0 mg, 69%): RP-HPLC: 99% (tR =
28.0, 31.7, 31.7, 40.5, 42.5, 44.8, 54.1, 61.7, 66.3, 117.2, 118.4, 124.5,
+
C
131.4, 131.7, 160.8, 169.5 (2C), 183.6 ppm (2C); HRMS-EI m/z [M]
calcd for C24H36N4O3: 428.2787, found: 428.2783.
3-(6-Aminohexylamino)-4-{3-[3-(piperidin-1-ylmethyl)phenoxy]-
1
propylamino}cyclobut-3-ene-1,2-dione bis(hydrotrifluoroacetate)
(18): Compound 18a (185 mg, 0.341 mmol, 1 equiv) was stirred for
4 h in a mixture of CH2Cl2 (30 mL) and HCl (6 n in 2-propanol,
13 mL) at RT. The solvent was removed in vacuo and the crude
product was obtained as hygroscopic yellow solid (188 mg, 107%).
The crude product was directly used in the next step. For pharma-
cological investigations 40 mg were purified by preparative HPLC
yielding the product as di-TFA-salt (45 mg): RP-HPLC: 99% (tR =
10.3 min, k=3.4); 1H NMR (400 MHz, CD3OD): d=1.42–1.44 (m,
4H), 1.48–1.57 (m, 1H), 1.62–1.68 (m, 4H), 1.70–1.84 (m, 3H), 1.91–
1.96 (m, 2H), 2.07–2.14 (qui, J=6.3 Hz, 2H), 2.90–2.98 (m, 4H),
3.43–3.46 (m, 2H), 3.60 (br, 2H), 3.83 (br, 2H), 4.13 (t, J=5.9 Hz,
2H), 4.23 (s, 2H), 7.02–7.07 (m, 3H), 7.37 ppm (t, J=7.9 Hz, 1H);
13C NMR (100 MHz, CD3OD): d=22.7, 24.1, 26.8, 26.9, 28.4, 31.6,
32.0, 40.6, 42.4, 45.0, 54.1, 61.7, 66.3, 117.2, 118.3, 124.5, 131.4,
131.7, 160.8, 169.7 (2C), 183.6, 183.9 ppm; HRMS-ESI m/z [M+H]+
calcd for C25H39N4O3+: 443.3017, found: 443.3018.
11.5 min, k=3.5); H NMR (600 MHz, CD3OD): d=1.10 (t, J=7.7 Hz,
3H), 1.52–1.61 (m, 4H), 1.71–1.95 (m, 6H), 2.07–2.11 (m, 2H), 2.17
(q, J=7.7 Hz, 2H), 2.94 (m,2H), 3.18 (t, J=6.8 Hz, 2H), 3.43–3.45
(m, 2H), 3.60–3.61 (m, 2H), 3.83 (m, 2H), 4.13 (t, J=5.8 Hz, 2H),
4.22 (s, 2H) 7.02–7.03 (m, 3H), 7.36 ppm (t, J=8.0 Hz, 1H); 13C NMR
(150 MHz, CD3OD): d=10.6, 22.7, 24.1 (2C), 27.4, 29.6, 30.2, 31.6,
39.8, 42.6, 44.9, 54.1 (2C), 61.7, 66.3, 117.1, 118.5, 124.5, 131.5,
131.7, 160.7, 169.6 (2C), 177.1, 183.6 ppm (2C); HRMS-EI m/z [M]
+
C
calcd for C26H38N4O4: 470.2893, found: 470.2891.
N-[5-(3,4-Dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino}cyclobut-1-enylamino)pentyl]propionamide
hydrotri-
fluoroacetate (23): Compound 17 (33.0 mg, 77.0 mmol, 1 equiv)
and succinimidyl propionate (9.70 mg, 57.0 mmol, 0.7 equiv), in
MeOH/DMSO (1:1) were prepared according to general procedure.
The solution was stirred for 30 min at RT, and the reaction was
stopped by addition of 10% TFA in CH3CN (250 mL). Yield: yellow
1
oil (20.7 mg, 61%): RP-HPLC: 100% (tR =12.0 min, k=3.7); H NMR
General procedure for the propionylation of primary amines:
The respective amine (1 equiv) was dissolved in 2–3 mL of solvent
in a small flask. 2–3 drops Et3N were added and succinimidyl propi-
onate (0.7–1.6 equiv), dissolved in CH3CN or MeOH, was added and
the solution stirred at RT for 30 min to 18 h. The product was puri-
fied by preparative HPLC.
(600 MHz, CD3OD): d=1.10 (t, J=7.6 Hz, 3H), 1.34–1.39 (m, 2H),
1.49–1.60 (m, 4H), 1.72–1.95 (m, 6H), 2.07–2.11 (m, 2H), 2.17 (q, J=
7.6 Hz, 2H), 2.94 (m, 2H), 3.15 (t, J=7.1 Hz, 2H), 3.43–3.45 (m, 2H),
3.57 (m, 2H), 3.83 (m, 2H), 4.13 (t, J=5.8 Hz, 2H), 4.22 (s, 2H) 7.02–
7.03 (m, 3H), 7.40 ppm (t, J=7.6 Hz, 1H); 13C NMR (150 MHz,
CD3OD): d=10.6, 22.7, 24.1 (2C), 24.6, 30.0, 30.2, 31.6, 31.9, 40.1,
42.5, 45.1, 54.1 (2C), 61.7, 66.3, 117.1, 118.5, 124.5, 131.5, 131.7,
+
N-[2-(3,4-Dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino}cyclobut-1-enylamino)ethyl]propionamide hydrotrifluoro-
acetate (20): Compound 14 (33.2 mg, 86.0 mmol, 1 equiv) and suc-
cinimidyl propionate (16.0 mg, 94.0 mmol, 1.1 equiv) in CH3CN were
allowed to react according to the general procedure. Product:
yellow oil (11.9 mg, 25%): RP-HPLC: 98% (tR =10.9 min, k=3.3);
1H NMR (600 MHz, CD3OD): d=1.07 (t, J=7.6 Hz, 3H), 1.78–1.93 (m,
6H), 2.09–2.11 (m, 2H), 2.17 (m, 2H), 2.94 (m, 2H), 3.36 (m, 2H),
3.42 (m, 2H), 3.64 (m, 2H), 3.82 (m, 2H), 4.14 (t, J=5.8 Hz, 2H),
4.22 (s, 2H) 7.01–7.06 (m, 3H), 7.36 ppm (t, J=7.9 Hz, 1H); 13C NMR
(150 MHz, CD3OD): d=10.33, 22.7, 24.1 (2C), 30.2, 31.6, 41.5, 42.5,
49.6, 54.1, 61.1, 66.4, 117.1, 118.5, 124.5, 131.4, 131.7, 160.8, 169.6
C
160.7, 169.7 (2C), 177.1, 183.6 ppm (2C); HRMS-EI m/z [M] calcd
for C27H40N4O4: 484.3050, found: 484.305.
N-[6-(3,4-Dioxo-2-(3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino)cyclobut-1-enylamino)hexyl]propionamide hydrotrifluoro-
acetate (24a): 18 (140 mg, 0.272 mmol, 1 equiv) was dissolved in
Et3N (110 mg, 1.09 mmol, 4 equiv) and CH2Cl2 (5 mL). The reaction
mixture was stirred for 5 min, succinimidyl propionate (69.7 mg,
0.407 mmol, 1.5 equiv) was added, and stirring was continued at
RT for 16 h. The solvent was removed in vacuo, and the product
was purified by preparative HPLC. Yield: white semi-solid (143 mg,
86%): RP-HPLC: 100% (tR =13.90 min, k=5.0); 1H NMR (400 MHz,
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[D6]DMSO): d=0.97 (t, J=7.6 Hz, 3H, (CO)CH2CH3), 1.26–1.27 (m,
4H, NH(CH2)2(CH2)2(CH2)2NH-), 1.34–1.40 (m, 3H, C4-H pip, CH2
hexyl), 1.47–1.51 (m, 2H, CH2 hexyl), 1.57–1.70 (m, 3H, C3,4,5-H
pip), 1.97–2.07 (m, 4H, (CO)CH2CH3, C3,5-H pip), 2.82–2.90 (m, 2H,
C2,6-H pip), 2.98–3.03 (m, 2H, OCH2CH2CH2NH), 3.29–3.32 (m, 2H,
C2,6-H pip), 3.47 (br, 2H, NHCH2(CH2)5NH), 3.67–3.68 (m, 2H,
OCH2CH2CH2NH), 4.04–4.07 (t, J=6.1 Hz, 2H, OCH2CH2CH2NH),
4.22–4.24 (m, 2H, pip CH2), 7.01–7.08 (m, 3H, C2,4,6-H phenoxy),
7.35–7.39 (t, J=7.9 Hz, 1H, C5-H phenoxy), 7.54 (br, 1.7H, -NH- cy-
clobutenedione), 7.68–7.70 (m, 1H, HN+ pip), 9.36 ppm (br, 1H,
NH(CO)CH2CH3); 1H NMR (400 MHz, CD3OD):=1.08–1.14 (t, J=
7.6 Hz, 3H, (CO)CH2CH3), 1.35–1.37 (m, 4H, NH(CH2)2(CH2)2-
(CH2)2NH), 1.45–1.54 (m, 3H, C4-H pip, CH2 hexyl), 1.58–1.62 (m,
2H, CH2 hexyl), 1.69–1.84 (m, 3H, C3,4,5-H pip), 1.91–1.96 (m, 2H,
C3,5-H pip), 2.06–2.22 (m, 4H, OCH2CH2CH2NH, (CO)CH2CH3), 2.90–
2.99 (m, 2H, C2,6-H pip), 3.12–3.17 (t, J=6.9 Hz, 2H, NH-
(CH2)5CH2NH), 3.42–3.46 (m, 2H, C2,6-H pip), 3.58 (br, 2H, NHCH2-
(CH2)5NH), 3.82–3.85 (br, 2H,OCH2CH2CH2NH), 4.10–4.14 (t, J=
5.8 Hz, 2H, OCH2CH2CH2NH-), 4.23 (s, 2H, pip CH2), 7.01–7.05 (m,
3H, C2,4,6-H phenoxy), 7.33–7.38 ppm (t, J=7.8 Hz, 1H, C5-H
phenoxy); 13C NMR (100 MHz, CD3OD): d=10.6 ((CO)CH2CH3), 22.7
(C4 pip), 24.1 (C3,5 pip), 27.0 (CH2 hexyl), 27.4 (CH2 hexyl), 30.2
(-(CO)CH2CH3), 30.3 (CH2 hexyl), 31.6 (OCH2CH2CH2NH), 32.1 (CH2
hexyl), 40.2 (NH(CH2)5CH2NH), 42.4 (OCH2CH2CH2NH), 45.1 (NHCH2-
(CH2)5NH), 54.1 (C2,6 pip), 61.7 (pip CH2), 66.2 (OCH2CH2CH2NH),
117.1 (C6 phenoxy), 118.3 (C2 phenoxy), 124.5 (C4 phenoxy), 131.4
(C5 phenoxy), 131.7 (C3 phenoxy), 160.7 (C1 phenoxy), 169.5 (2C,
Monitoring of reaction by HPLC: Reaction mixture (0.5 mL) was
added to 105 mL of unlabeled compound 24a solution (50 mm), the
“cold” version of 24b, to allow UV detection in addition to radio-
detection; injection volume: 100 mL, see Figure 2). For purification,
the reaction mixture was diluted with 1060 mL of a mixture of
CH3CN/H2O (20:80 v/v) containing 0.05% TFA and the product was
isolated (seven injections, 180 mL each) with analytical HPLC. In this
instance, radiometric detection was not performed. Fractions con-
taining the radioligand were collected at ꢂ20 min (gradient:
0.05% TFA in CH3CN/0.05% TFA in H20: 0 min: 20:80 (v/v), 37 min:
30:70 (v/v), 38 min 90:10 (v/v), 48 min 90:10 (v/v)). The solvent of
the combined fractions was evaporated in vacuo, the product dis-
solved in 1000 mL EtOH and transferred to an Amersham glass vial.
Quantification: A four-point calibration was performed with the
unlabeled ligand 24a (1.0, 2.5, 5.0 and 7.5 mm, inj. vol.: 100 mL, gra-
dient: 0.05% TFA in CH3CN/0.05% TFA in H2O: 0 min: 20:80 (v/v),
15 min: 52.5:47.5 (v/v), 16 min: 90:10 (v/v), 21 min: 90:10 (v/v), tR
ꢂ14 min, see Figure 2). The solutions for injection were prepared
in CH3CN/0.05% aq. TFA (20/80) less than 5 min prior to injection.
All standard solutions were prepared from a 100 mm solution of
24a (in CH3CN/0.05% aq. TFA 20:80), which was freshly made from
a 10 mm stock solution of 24a in CH3CN. Two aliquots (2.0 mL) of
the ethanolic solution 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 prod-
uct by UV detection, the second sample was additionally moni-
tored radiometrically to determine radiochemical purity (see
Figure 2). The molarity of the ethanolic solution of 24b was calcu-
lated from the mean of the peak areas, and the linear calibration
curve was obtained from the peak areas of the standards. Yield:
7.01 mg (14.0 nmol, 44.6%).
C=C cyclobutenedione), 177.0 ((CO)CH2CH3), 183.4 ppm (2C, C=O
cyclobutenedione); HRMS-ESI m/z [M+H]+ calcd for C28H43N4O4
:
+
499.3279, found: 499.3287.
General conditions for radiosynthesis: Chemicals and solvents
were purchased from Merck KGaA (Darmstadt, Germany) and
Sigma Aldrich GmbH (Munich, Germany) and were used without
further purification unless otherwise stated. All solvents were of an-
alytical grade, and DMSO was distilled prior to use and stored over
4 ꢃ molecular sieves. Succinimidyl [2,3-3H]-propionate was pur-
chased from Hartmann Analytic GmbH (Braunschweig, Germany)
Determination of the specific activity: An aliquot (1.5 mL) of the
ethanolic solution was diluted with 448.5 mL of a mixture of
CH3CN and H2O (20:80) in duplicate, and 50 mL of the 1:300 dilu-
tions were counted three times in RotiszintTM eco plus (3 mL) in
a LS 6500 liquid scintillation counter (Beckmann Coulter, Munich,
Germany). The total activity in 1000 mL stock solution amounted to
32.5 MBq (0.877 mCi), resulting in a calculated specific activity of
2.33 TBqmmolꢀ1 (63.0 Cimmolꢀ1). The activity of the stock solution
was adjusted to 9.25 MBqmLꢀ1, (0.25 mCimLꢀ1, c=3.97 mm) by
adding EtOH. HPLC analysis showed a radiochemical purity of
>99%. The identity of the radioligand was confirmed by HPLC
analysis of the labeled and the corresponding unlabeled com-
pound (24b, 24a) under the same conditions, resulting in identical
retention times. The radioligand 24b was stored at ꢀ208C.
and provided in EtOAc (specific activity as =2.96 TBqmmolꢀ1
,
80 Cimmolꢀ1; av =185 MBqmLꢀ1, 5 mCimLꢀ1). Scintillation cocktail
RotiscintTM Eco Plus was from Carl Roth (Karlsruhe, Germany). Ana-
lytical HPLC was performed on a system from Waters (Eschborn,
Germany), equipped with a pump control module, a 510 HPLC
pump, a 486 UV/VIS detector, and a Flo-One beta series A-500
radiodetector (Packard, Meriden, USA). The stationary phase was
a Synergi Hydro-RP (250ꢂ4.6 mm, 4 mm) column equipped with
a Luna C18 (4ꢂ3.0 mm) column guard (Phenomenex, Aschaffen-
burg, Germany). Linear gradients of CH3CN/TFA 0.05% (v/v) and
H2O/TFA 0.05% (v/v) were used as mobile phases at a flow rate of
0.8 mLminꢀ1. Absorbance was detected at 280 nm, and radioactivi-
ty was measured with the radiodetector by liquid scintillation
counting (liquid scintillator: RotiscintTM Eco Plus, flow rate:
3.8 mLminꢀ1).
Pharmacological methods
Functional studies in the steady-state GTPase assay[10,50,51] on mem-
branes of Sf9 cells (for preparation, see Refs. [52,53]) expressing
the hH2R-GsaS or the gpH2R-GsaS and on the isolated spontaneously
beating guinea pig right atrium[46,54] were performed as previously
described, except that the incubation period of the guinea pig
atrium in the presence of the antagonists was extended to 60 min.
Binding data on recombinant histamine receptor subtypes and or-
thologues expressed in Sf9 cells (hH1R+RGS4; hH2R–GsaS, gpH2R–
GsaS, rH2R–GsaS; hH3R+Gia2 +b1g2; hH4R+Gia2 +b1g2) or HEK293T
CRE-Luc cells[55] (hH2R) using the following radioligands:
H1R,[3H]pyrilamine (Hartmann Analytics, Braunschweig, Germany);
H2R, 24b; H3R,[3H]Na-methylhistamine (Hartmann Analytics);
H4R,[3H]histamine (Hartmann Analytics). Saturation binding, binding
kinetics and competition binding studies were performed by analo-
N-[6-(3,4-Dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propyl-
amino}cyclobut-1-enylamino)hexyl]-[2,3-3H2]propionamide (24b):
Succinimidyl [2,3-3H2]propionate (5.35 mg, 0.0313 mmol, 1 equiv,
2.5 mCi) in EtOAc (500 mL) was transferred to a 1.5 mL Eppendorf
reaction vessel (screw top) and the solvent was removed in
a vacuum concentrator (408C) for 30 min. Compound 18 (553 mg,
1.25 mmol, 40 equiv) in DMSO (69 mL) and NEt3 (253 mg, 2.50 mmol,
80 equiv) in CH3CN (69 mL) was added. After addition of CH3CN
(62 mL), the reaction mixture was vortexed, briefly centrifuged and
stirred with a magnetic microstirrer for 18 h at RT.
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Full Papers
gy with the previously reported protocols,[20,51,53,56] using Micro
Beta2 1450 scintillation counter (PerkinElmer, Rodgau, Germany) in
the 96-well plate format.
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Published online on October 15, 2014
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