Synthesis and functional characterization of imbutamine analogs as histamine H3 and H4 receptor ligands

Article abstract of DOI:10.1002/ardp.201300316

Imbutamine (4-(1H-imidazol-4-yl)butanamine) is a potent histamine H ₃ (H₃R) and H₄ receptor (H₄R) agonist (EC₅₀ values: 3 and 66 nM, respectively). Aiming at improved selectivity for the H₄R, the imidazole ring in imbutamine was methyl-substituted or replaced by various differently substituted heterocycles (1,2,3-triazoles, 1,2,4-triazoles, pyridines, pyrimidines) as potential bioisosteres. Investigations in [³⁵S]GTPγS binding assays using membranes of Sf9 insect cells expressing the respective human histamine receptor subtype revealed only very weak activity of most of the synthesized hetarylalkylamines at both receptors. By contrast, the introduction of substituents at the 4-imidazolyl ring was most effective regarding H ₄R selectivity. This holds for methyl substitution in position 2 and, especially, in position 5. 5-Methylimbutamine (H₄R: EC₅₀ = 59 nM, α = 0.8) was equipotent with imbutamine at the hH₄R, but revealed about 16-fold selectivity for the hH₄R compared to the hH₃R (EC₅₀ 980 nM, α = 0.36), whereas imbutamine preferred the hH₃R. The functional activities were in agreement with radioligand binding data. The results support the hypothesis that, by analogy with histamine, methyl substitution in histamine homologs offers a way to shift the selectivity in favor of the H₄R. According to a bioisosteric approach, the imidazole ring in the dual histamine H₃/H₄ receptor agonist imbutamine (n = 4) was replaced by various five- and six-membered N-heterocycles. Whereas these structural modifications resulted in a reduction or loss of activity at both receptors, 5-methyl substitution at the imidazol-4-yl ring in imbutamine changed the receptor subtype selectivity in favor of the H₄R.

Full text of DOI:10.1002/ardp.201300316

Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
77
Full Paper
Synthesis and Functional Characterization of Imbutamine
Analogs as Histamine H and H Receptor Ligands
3
4
Roland Geyer, Melanie Kaske, Paul Baumeister, and Armin Buschauer
Faculty of Chemistry and Pharmacy, Department of Pharmaceutical/Medicinal Chemistry II, University of
Regensburg, Regensburg, Germany
Imbutamine (4-(1H-imidazol-4-yl)butanamine) is a potent histamine H (H R) and H receptor (H R)
3
3
4
4
agonist (EC50 values: 3 and 66 nM, respectively). Aiming at improved selectivity for the H R, the
4
imidazole ring in imbutamine was methyl-substituted or replaced by various differently substituted
heterocycles (1,2,3-triazoles, 1,2,4-triazoles, pyridines, pyrimidines) as potential bioisosteres. Inves-
3
5
tigations in [ S]GTPgS binding assays using membranes of Sf9 insect cells expressing the respective
human histamine receptor subtype revealed only very weak activity of most of the synthesized
hetarylalkylamines at both receptors. By contrast, the introduction of substituents at the 4-imidazolyl
ring was most effective regarding H R selectivity. This holds for methyl substitution in position 2 and,
4
especially, in position 5. 5-Methylimbutamine (H R: EC ¼ 59 nM, a ¼ 0.8) was equipotent with
4
50
imbutamine at the hH R, but revealed about 16-fold selectivity for the hH R compared to the hH R (EC
50
4
4
3
9
80nM, a ¼ 0.36), whereas imbutamine preferred the hH R. The functional activities were in agreement
3
with radioligand binding data. The results support the hypothesis that, by analogy with histamine,
methyl substitution in histamine homologs offers a way to shift the selectivity in favor of the H R.
4
3 4
Keywords: Bioisosteres / H receptor / H receptor / Histamine / Imbutamine
Received: August 20, 2013; Revised: September 11, 2013; Accepted: September 12, 2013
DOI 10.1002/ardp.201300316
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
less, recent reports on partial agonistic effects of the standard
H R antagonist JNJ 7777120 [20] at certain H R species
4
4
Since the discovery of the human histamine H
4
receptor
orthologs [21] in vitro and alternative (b-arrestin-mediated)
signaling [22] suggest that ligand effects in vivo should be
interpreted with caution [23, 24]. Therefore, both selective
antagonists and agonists are required as pharmacological
(
hH R) in the years 2000 and 2001 [1–7], this new member of
4
the histamine receptor family [8] has been considered a
potential drug target. The predominant expression of the
H R on various cells of the immune system like eosinophils,
4
tools to further explore the role of the H R [25].
4
T-lymphocytes, dendritic cells, mast cells, and basophils [1, 4,
The H R shares highest sequence similarity with the
4
5, 9–13], and results of in vitro and in vivo studies suggest that
histamine H
3 3
receptor (H R). Therefore, it is not surprising
the H4R plays crucial role in immunological and
a
that the H R is targeted by various imidazole-containing H R
4
3
inflammatory processes [14–16]. Hence, the H R is considered
ligands [1, 4–6]. Histamine (1) and its homologs homohist-
3
amine (2) and imbutamine (3) are agonists with similar hH R
4
a promising drug target for the treatment of inflammatory
and immunological diseases like allergic rhinitis, rheumatoid
arthritis, bronchial asthma, and pruritus [17–19]. Neverthe-
and hH R affinity, whereas the higher homolog impentamine
4
(4) is an almost full hH R agonist but shows no agonistic
3
activity at the hH
with further improved selectivity profile, especially
compared to the H R, by analogy with a successful approach
4 4
R [26, 27]. Therefore, aiming at H R agonists
Correspondence: Prof. Armin Buschauer, Institut für Pharmazie,
Universität Regensburg, Universitätsstr. 31, D-93053 Regensburg,
Germany.
E-mail: armin.buschauer@chemie.uni-regensburg.de
Fax: þ49 941 9434820
a
3
to H
2
R agonists [28, 29], the bioisosteric replacement of the 1H-
imidazol-4-yl ring was considered a promising approach
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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R. Geyer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Figure 1. Replacement of the imidazole ring by potential bioisosteres in histamine analogs.
(
Fig. 1). In the present study, a series of hetarylalkylamines
histamine [30–32]. Furthermore, we tried to cover a wide
range of physico-chemical properties, with emphasis on
different basicity, and focused, in a first approach, on
prototypical compounds that were easily accessible.
related to imbutamine (3), bearing various heterocycles
instead of the imidazol-4-yl ring, were synthesized and
investigated on recombinant human histamine receptor
subtypes. In addition, methyl substitution at the imidazole
ring was performed, since a 5-methyl group in histamine Results and discussion
cf. 5-methylhistamine, often also referred to as 4-methylhist-
amine) has been shown to confer H R selectivity [26]. In the
(
4
Chemistry
present study, the attention was turned to heterocycles that
have already been employed in the development of HR
ligands, especially for the preparation of hetaryl analogs of
The investigated amines were synthesized as depicted in
Schemes 1–4. The v-(2- or 5-methyl-1H-imidazole-4-yl)alkan-1-
amines 13, 14, and 20 (Scheme 1) were prepared by analogy
Scheme 1. Synthesis of the (2- or 5-methyl-1H-imidazol-4-yl)alkan-1-amines 13, 14, and 20. Reagents and conditions: (i) K-phthalimide
1 eq), K CO (1.5 eq), KI (cat.), DMF, 12 h, 100°C; (ii) Br (1 eq), urea (1 eq), MeOH, 24 h, rt; (iii) acetamidinium chloride (4 eq), K CO (4 eq),
DMF, 48 h, 75°C; (iv) N · H (5 eq), K CO (6.1 eq), CHCl , 5 h, 0°C; (vi) K-phthalimide
1 eq), K CO (1.5 eq), KI (cat.), DMF, 12 h, 100°C; (vii) Br
· H
O (6 eq), EtOH, 2 h, reflux ! overnight, rt.
(
2
3
2
2
3
2
H
4
2
O (6 eq), EtOH, 2 h, reflux ! overnight, rt; (v) Br
2
2
3
3
(
2
3
2
(1 eq), glacial acetic acid, 1.5 h, 10°C ! rt; (viii) formamide, 12 h, 170°C; (ix)
N
H
2 4
2
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Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Imbutamine Analogs
79
Scheme 2. Synthesis of 4-(1H-1,2,3-triazol-5-yl)butan-1-amine 24. Reagents and conditions: (i) phthalimide (1.1 eq), PPh
1.1 eq), THF, overnight, 0°C ! rt; (ii) TMSN (2 eq), CuI (cat.), DMF/MeOH 9:1 v/v, microwave, 12 h, 100°C; (iii) N · H
reflux ! overnight, rt.
3
(1.1 eq), DIAD
2
O (6 eq), EtOH, 2 h,
(
3
2
H
4
Scheme 3. Synthesis of the 2-amino-1,2,4-triazole derivatives 30, 38, 39, and 43. Reagents and conditions: (i) Boc
dioxane/water 2:1 v/v, overnight, 0°C ! rt; (ii) (a) TBTU (1.2 eq), DIPEA (2 eq), DCM, 30 min, rt; (b) S-methylisothiuronium iodide (27) (1 eq),
DIPEA (3 eq), DCM, overnight, rt; (iii) N · H O (30 eq), NEt (32.5 eq), EtOH, overnight, reflux; (iv) TFA 50%, MeOH, overnight, rt; (v)
diphenyl cyanocarbonimidate (33) (1 eq), 2-propanol, 2 h, rt; (vi) N · H O (1.5 eq), MeOH, 24 h, rt; (vii) 6 M HCl in 2-propanol, EtOAc/MeOH
:1 v/v, overnight, rt; (viii) 5-amino-4H-1,2,4-triazole-3-thiol (41) (0.8 eq), 1 M NaOH, EtOH, 3 h, reflux; (ix) 6 M HCl in 2-propanol, EtOAc/
MeOH 6:1 v/v, overnight, rt.
2
O (1.1 eq), 1 M NaOH,
2
H
4
2
3
H
2 4
2
6
with a pathway for the preparation of homohistamine [33].
The v-phthalimidoalkan-2-ones (7, 8, and 17) were obtained
from the reaction of the corresponding v-chloroalkan-2-one
19, formamide was used instead of acetamidine, again
followed by hydrazinolysis to yield 20.
The preparation of 4-(1H-1,2,3-triazol-5-yl)butan-1-amine 24
(Scheme 2) started from hex-5-yn-1-ol (21), which was
converted to the corresponding 2-(hex-5-yn-1-yl)isoindoline-
1,3-dione (22) under Mitsunobu conditions [39]. 2-[4-(1H-1,2,3-
Triazol-5-yl)butyl]isoindoline-1,3-dione (23) was synthesized,
using click-chemistry according to Jin et al. [40], and subse-
quently converted to the amine 24 with the help of hydrazine.
The N-Boc protected 5-aminopentanoic acid (26) was
coupled to 27, as described by Adang et al. [41], to yield 28.
Cyclization of 28 with hydrazine and deprotection under
acidic conditions gave 5-(4-aminobutyl)-1H-1,2,4-triazol-3-
amine (30) (Scheme 3).
(5 and 6) or 7-bromoheptan-2-one (16) with potassium
phthalimide [34]. 7-Bromoheptan-2-one (16) was prepared
from 1-methylcyclohexanol (15) in a retro-Barbier reaction [35].
Regioselective bromination of 7 and 8 was achieved in
methanol as solvent in the presence of urea according to
Zav’yalov and Kravchenko [36]. For the regioselective
bromination of 17, glacial acetic acid was used as solvent
according to Black et al. [37]. Cyclization of acetamidine with
the a-bromoketone 9 or 10 in a Bredereck synthesis [38] gave the
imidazoles 11 and 12, which were converted to 13 and 14 by
hydrazinolysis of the phthalimide group. For the synthesis of
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R. Geyer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
The 3,5-diamino-1,2,4-triazole derivatives 38 and 39
were synthesized using diphenyl cyanocarbonimidate (33) as
a synthon [42, 43]. Reaction of mono-protected diaminoalkanes
1 and 32 with 33 and subsequent ring closure with hydrazine
ammonia. The introduction of the second amino substituent
was performed in a similar manner, using the respective
Boc-protected alkanediamine (31 or 51). Subsequent depro-
tection under acidic conditions gave the hydrochlorides
of 52–55.
3
yielded the 3,5-diaminotriazoles 36 and 37. Deprotection under
acidic conditions gave the hydrochlorides of 38 and 39.
Substitution of tert-butyl 3-bromopropylcarbamate (40) with 5-
amino-4H-1,2,4-triazole-3-thiol (41) and deprotection under
acidic conditions gave 43 [44].
The aminopyridine derivatives 45 and 46 [45] (Scheme 4)
were prepared by reaction of the pertinent alkanediamine
and 2-chloropyridine (44) under microwave irradiation. The
diaminopyrimidines 52–55 were prepared from 2,4-dichlor-
opyrimidine (47). Heating with ammonia under microwave
irradiation yielded the isomers 48 and 50, which can be
easily separated by flash chromatography. Compound 49
was synthesized from 47 using methylamine instead of
Pharmacology
The synthesized compounds described above were investigat-
ed for agonism and antagonism at hH R and hH R subtypes in
4
3
3
5
[ S]GTPgS binding assays using membrane preparations of
Sf9 insect cells co-expressing the hH R plus Gai2 plus Gb or
co-expressing the hH R plus Ga plus Gb g . Moreover, the
4
1 2
g
3
i2
1 2
4
reference H R agonist 5-methylhistamine (56) [46], 2-methyl-
imidazole-1-pentanamine (57) [47], and 2-[(5-methylimidazol-
4-yl)methylsulfanyl]ethanamine (58) [48], a thia-analog of 20,
known as a building block for the preparation of cimetidine,
3 4
were included (Fig. 2). In addition, the hH R and hH R binding
data of selected compounds were determined on the
corresponding Sf9 membrane preparations. In the following,
agonistic potencies are expressed as EC50 values. Intrinsic
activities (a) refer to the maximal response induced by the
standard agonist histamine. Compounds identified to be
inactive as agonists (a < 0.1 or negative values, respectively,
determined in the agonist mode; cf. Table 1) were investigated
B
in the antagonist mode. The corresponding K values of
neutral antagonists and inverse agonists (Table 1) were
determined from the concentration-dependent inhibition of
3
5
the histamine-induced increase in [ S]GTPgS binding.
Binding data represent K values determined by displacement
i
3
a
3
of [ H]N -methylhistamine (hH R) or [ H]histamine (hH R)
3
4
(Table 2).
Prior to the discovery of the H
3
R and H
4
R, in search for the
structural determinants of H R or H R agonism, numerous
1
2
derivatives and heterocyclic analogs of histamine were
investigated. Although even minor structural modifications
of the endogenous ligand led to less active compounds at the
H R and H R, these studies revealed possible bioisosteric
1 2
replacements of the imidazole ring [30]. Decades later,
numerous histamine analogs proved to be potent agonists
3 4
at the H R and the H R [26]. For instance, 5-methylhistamine,
2
used by Black et al. as a weak agonist preferring the H R
Scheme 4. Synthesis of aminopyridines 45, 46 and diaminopyr-
imidines 52, 53, 54, and 55. Reagents and conditions: (i)
alkanediamine (6 eq), pyridine (1.3 eq), microwave, 30 min, 190°C;
(
ii) for 48 and 50: 7 M NH
THF 3:1 v/v, microwave, 10 min, 100°C, separation of isomers by
flash chromatography; for 49: 40% methylamine in H O (1 eq),
3
in MeOH (1.5 eq), DIPEA (2 eq), EtOAc/
2
DIPEA (1.5 eq), EtOAc/MeOH 1:1 v/v, 36 h, rt; (iii) (a) for 52, 54, 55:
tert-butyl 2-aminoethylcarbamate (51) (1 eq), DIPEA (1.5 eq), EtOH,
microwave, 30 min, 120°C, (b) acetyl chloride (3 eq), MeOH, 4 h,
0
1
°C ! rt; for 53: 31 (1 eq), DIPEA (1.5 eq), EtOH, microwave, 30 min,
20°C, (b) acetyl chloride (3 eq), MeOH, 4 h, 0°C ! rt.
Figure 2. Structures of amines 56–58.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Imbutamine Analogs
a)
81
3
5
Table 1. Potencies and efficacies of the synthesized amines at the hH
3
R and hH
4
R in the [ S]GTPgS assay.
3
hH R
4
hH R
Compound
EC50 or (K
B
, nM)
a
N
EC50 or (K
B
, nM)
a
N
b,c
d,e
1
2
3
4
1
1
2
2
3
3
3
4
4
4
5
5
5
5
5
5
5
, Histamine
, Homohistamine
, Imbutamine
, Impentamine
3
4
0
4
0
8
9
3
5
6
2
3
23.2 ꢀ 7.2
103 ꢀ 63
1.00
2
3
3
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
3
3
3
10.2 ꢀ 1.8
19.3 ꢀ 2.2
1.00
4
3
4
3
2
2
5
3
2
2
2
2
2
2
3
2
2
3
3
2
3
f
g
0.81 ꢀ 0.06
0.82 ꢀ 0.13
0.13 ꢀ 0.02
0.45 ꢀ 0.04
0.09 ꢀ 0.04
0.36 ꢀ 0.11
0.36 ꢀ 0.04
n.d.
0.77 ꢀ 0.06
0.69 ꢀ 0.07
ꢁ0.01 ꢀ 0.02
0.39 ꢀ 0.05
0.39 ꢀ 0.01
0.80 ꢀ 0.08
0.20 ꢀ 0.04
ꢁ0.15 ꢀ 0.03
ꢁ0.30 ꢀ 0.1
ꢁ0.11 ꢀ 0.09
n.d.
ꢁ0.22 ꢀ 0.08
ꢁ0.22 ꢀ 0.09
n.d.
n.d.
n.d.
0.87 ꢀ 0.04
0.98 ꢀ 0.03
n.d.
0.70 ꢀ 0.01
h
i
3.13 ꢀ 1.1
66.1 ꢀ 24.6
j
(57.6 ꢀ 28.8)
>10,000
(>10,000)
980
(n.d.)
346 ꢀ 7
1700 ꢀ 186
k
59.4 ꢀ 20.2
>10,000
(>10,000)
(>10,000)
(>10,000)
Inactive
>10,000
Inactive
Inactive
Inactive
Inactive
(2790 ꢀ 164)
(1430 ꢀ 164)
Inactive
n.d.
n.d.
n.d.
ꢁ0.51 ꢀ 0.09
ꢁ0.50 ꢀ 0.05
n.d.
ꢁ0.28 ꢀ 0.14
ꢁ0.13 ꢀ 0.11
ꢁ0.16 ꢀ 0.03
0.70 ꢀ 0.11
ꢁ0.97 ꢀ 0.12
0.49 ꢀ 0.05
(>10,000)
(>10,000)
Inactive
Inactive
inactive
(>10,000)
(>10,000)
(91 ꢀ 1)
4
5
500 ꢀ 150
70.3 ꢀ 44.1
Inactive
k
6, 5-Me-histamine
7
8
12,400 ꢀ 3130
(2460 ꢀ 58)
751 ꢀ 303
1590 ꢀ 1300
a)
35
Functional [ S]GTPgS binding assays using membrane preparations of Sf9 cells expressing the hH
hH
R þ Gai2 þ Gb
response) of histamine was set to 1.00 and a values of other compounds were referred to this value. a values of neutral antagonists and
inverse agonists were determined at 10 mM. n.d., no agonistic activity detected. The K values of neutral antagonists and inverse agonists
were determined in the antagonist mode versus histamine (100 nM) as the agonist. [49]: EC50 25 nM. [26]: EC50 13 nM. [49]: EC50
3
R þ Gai2 þ Gb
1
g
2
or the
4
1 2
g . N gives the number of independent experiments performed in duplicate. The intrinsic activity (a, maximal
B
b)
c)
d)
e)
f)
g)
h)
i)
j)
k)
1
2 nM. [26]: EC50 11 nM. [26]: EC50 40 nM. [26]: EC50 200 nM. [26]: EC50 0.6 nM. [49]: EC50 32 nM. [26]: EC50 4 nM. [49]: EC50 70 nM.
b,d,k)
Data from the literature:
hH
hH
EC50 values determined in the steady-state GTPase assay on membranes of Sf9 cells expressing the human
EC50 values determined from the inhibition of 1 mM forskolin-induced CRE-b-galactosidase activity in SK-N-MC/
cells [26].
e–h,j)
3
R or hH
4
R [49],
or SK-N-MC/hH
3
4
compared to the H
1
R to pharmacologically define the H
2
R,
4
efficient to increase the potency of cyanoguanidine-type H R
turned out to be a potent agonist at the human H R [26].
agonists [49, 50].
4
Extension of the carbon chain between imidazole and amino
group from two carbon atoms (histamine, 1) was tolerated up
to four methylene groups (imbutamine, 3), and the incorpo-
ration of imbutamine as a building block proved to be highly
Stimulated by these previous results we substituted the
imidazole ring in imbutamine with a methyl group at different
positions and prepared several structural analogs. The
introduction of a 2-methyl substitution at the imidazole ring
a)
Table 2. Binding data of selected compounds at the hH
3 4
R and hH R.
3
hH R
4
hH R
Compound
K
i
(nM)
N
K
i
(nM)
N
1
2
3
4
2
5
5
, Histamine
18.3 ꢀ 3.5
94.4 ꢀ 0.4
4.27 ꢀ 0.8
36.9 ꢀ 7.0
639 ꢀ 227
9940 ꢀ 1400
1670
3
2
2
2
2
2
1
12.7 ꢀ 1.5
31.8 ꢀ 11.3
12.7 ꢀ 0.8
627 ꢀ 63
68.8 ꢀ 14.5
28.5 ꢀ 4.4
1460
3
3
2
3
3
3
1
, Homohistamine
, Imbutamine
, Impentamine
0, 5-Me-imbutamine
6, 5-Me-histamine
8
a)
Determined on membrane preparations of Sf9 insect cells expressing the hH
3
R þ Gai2 þ Gb
1
g
2
or the hH
4
R þ Gai2 þ Gb
1
g
2
, the
3
a
3
hH
3
R þ Gai2 þ Gb
1
g
2
; radioligands: for hH
3
R: [ H]N -methylhistamine (3 nM), for hH
4
R: [ H]histamine (10 nM). N gives the number of
independent experiments performed in duplicate.
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R. Geyer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
in 4 decreased potency and efficacy at the H R and, much more
and H R activation [53, 54]. This is not possible in the case of
4
4
3
pronounced, at the H R. Compound 13 was a partial agonist at
4
compound 57. Furthermore, as impentamine is devoid of H R
the H R with an EC of 346 nM. The same tendency became
agonistic properties as well, the spacer length of five
4
50
obvious for the 2-methyl analog of homohistamine (14, H R:
methylene groups obviously prevents optimal orientation
4
EC50 ¼ 1700 nM). Hence, both compounds showed a changed
4
of the ligand in the binding pocket of the H R. Interestingly,
compound 58, the thia-analog of compound 20, was
selectivity profile and preferred the H R compared to the
4
respective parent compounds (2 and 3). This is in accordance
with data for 2-methylhistamine [26]. Thus, regardless of
lowering the potency, substituents at position 2 of the
imidazole ring can be taken into consideration to increase
equipotent with 20 at the H R, but proved to be about 30
times less potent as an H R agonist.
4
The functional data are in accordance with the affinities
determined for selected compounds (1–4, 20, 56, and 58) at the
3
the selectivity of imidazole-type H
substitution in position 5 of the imidazole ring in 3 did not
markedly influence the potency and the intrinsic activity at the
4
R agonists. A methyl
hH
Essentially, the results are in agreement with data from
previous studies on imidazole-type H R agonists, indicating
3 4
R and the hH R (Table 2).
4
H
4
R, but slightly reduced the intrinsic activity (EC50 ¼ 59nM,
that both ethylamine and butylamine side chains are
equivalent substructures. The search for bioisosteres of the
imidazole ring was not successful in terms of H R agonism.
4
Obviously, the basicity of the heterocycle, the H-bond donor
and acceptor properties, and the spatial demand should be
comparable to those of imidazole. By analogy with histamine,
methyl substitution at the 4-imidazolyl ring resulted in a
a ¼ 0.8). At the H R a 300-fold decrease in activity and partial
3
agonism (a ¼ 0.36) was observed for compound 20.
The replacement of the imidazole in 3 by the less basic 1,2,3-
triazole ring (24) decreased potency and intrinsic activity in
3
5
the [ S]GTPgS assay and yielded a very weakly active partial
agonist at both receptor subtypes. A drastic decrease in
potency compared to the imidazole series was also observed
for the 3-amino-1,2,4-triazole derivatives 30, 38, 39, and 43. At
preference for the H R. This holds for methyl substitution in
4
position 2 and, especially, in position 5 (cf. 56 vs. 1, 14 vs. 2, 20
vs. 3). 5-Methylimbutamine (20) was the most potent and
4
the H R very weak inverse agonism was detected for 30, 38,
and 39. Compound 43 was inactive (up to 100 mM, the highest
concentration tested). The results suggest that the basicity of
the heterocycle should be comparable to that of imidazole to
obtain histamine H R activity. At the H R, neither agonistic
selective H R agonist among the ring-substituted higher
homologs of histamine.
In conclusion, the search for bioisosteres of the imidazole
4
ring in H R agonists related to histamine remains challeng-
4
3
4
nor antagonistic activity was detectable for 30, 38–39, and 43
in the [ S]GTPgS assay. The two pyridine derivatives 45 and 46
were weak inverse agonists at both receptor subtypes. The KB
ing. A different scaffold as in oxime-type agonists derived from
H R antagonists with an indole or benzimidazole moiety, e.g.,
4
JNJ 28610244 [55], may offer an alternative approach to H4R
selectivity. Moreover, simple structures as that of S-(2-
guanidinoethyl)isothiourea (VUF 8430) [56] suggest that a
heterocyclic core is not required at all to obtain potent and
3
5
3
values for antagonism at the H R were in the low micromolar
range, and the concentration–response curves revealed
inverse agonism (a ¼ ꢁ0.5). The antagonist activities at the
H
4
R were even lower. The spatial demand of the six-membered
4
selective H R agonists.
ring and again the lower basicity of the heterocycle might
contribute to this dramatic decrease in potency.
Experimental
Amino-substituted pyrimidines are known as core struc-
tures of numerous high-affinity H R antagonists [32, 51],
4
Chemistry
Commercial reagents and chemicals were purchased from Acros
Organics (Geel, Belgium), IRIS Biotech GmbH (Marktredwitz,
Germany), Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany),
Merck KGaA (Darmstadt, Germany), Sigma–Aldrich Chemie
GmbH (Munich, Germany), TCI Europe (Zwijndrecht, Belgium)
and used without further purification. Deuterated solvents for
NMR spectroscopy were from Deutero GmbH (Kastellaun,
Germany). 5-(2-Methyl-1H-imidazol-1-yl)pentan-1-amine (57) was
a gift from Dr. Birgit Striegl, Department of Pharmaceutical/
Medicinal Chemistry I, University of Regensburg. All solvents
were of analytical grade or distilled prior to use. Millipore water
was used throughout for the preparation of buffers and HPLC
eluents. If moisture-free conditions were required, reactions were
performed in dried glassware under inert atmosphere (argon or
including those compounds that entered clinical trials (e.g.,
ZPL-38937887; for a recent review, cf. [52]). To explore the
diaminopyrimidine moiety as a replacement of the imidazole
ring in histamine analogs, we synthesized the aminoalkylpyr-
2
imidine-2,4-diamines 52–55. The three N -substituted deriva-
4
tives 52–54 were inactive or only very weakly active at the H R
4
and H R. Interestingly, the N -substituted derivative 55 was a
3
rather potent H
some selectivity over the H
Compound 57, a methyl-substituted structural isomer of
impentamine (4), was inactive at the H R but revealed inverse
agonism at the H value of 2460 nM.
R (a ¼ ꢁ0.97) with a K
According to Yao et al. the imidazole N–H in histamine is
3
R antagonist (K
B
¼ 91 nM, a ¼ ꢁ0.16) with
4
R (EC50 ¼ 500 nM, a ¼ 0.87).
4
3
B
nitrogen). Anhydrous DMF was purchased from Sigma–Aldrich
ꢂ
Chemie GmbH and stored over 3 A molecular sieves. Flash
chromatography was performed in glass columns on silica gel
3
essential to form a hydrogen bond, which is necessary for H R
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Imbutamine Analogs
83
(Merck silica gel 60, 40–63 mm). Automated flash chromatography
2-[3-(2-Methyl-1H-imidazol-4-yl)propyl]isoindoline-
was performed on a Varian IntelliFlash 310 using pre-packed
Varian Superflash columns (Varian, Darmstadt, Germany).
Reactions were monitored by TLC on aluminum plates coated
with silica gel (Merck silica gel 60 F254, thickness 0.2 mm). The
compounds were detected by UV light (254 nm), a 0.3% solution of
ninhydrine in n-butanol (amines), a 1.0% solution of Fast Blue B
1
,3-dione (12) [57]
A mixture of 10 (8.0 g, 25.8 mmol), acetamidine hydrochloride
9.8 g, 103.2 mmol), and K CO (14.3 g, 103.2 mmol) in 50 mL DMF
was stirred at 75°C for 48 h. The solvent was evaporated, the
residue taken up in DCM and extracted with saturated NaHCO
(
2
3
3
.
Evaporation of the solvent and flash chromatography (DCM/
MeOH 100:0–90:10 v/v) gave a yellow oil (1.0 g, 15%); H NMR
salt (imidazole containing compounds) in EtOH/H
2
O ¼ 30:70 v/v
1
or iodine staining. All melting points are uncorrected and were
measured on a Büchi 530 (Büchi GmbH, Essen, Germany)
apparatus. Lyophilization was done with a Christ alpha 2-4 LD
equipped with a vacuubrand RZ 6 rotary vane vacuum pump.
Microwave-assisted reactions were performed on an Initiator 2.0
synthesizer (Biotage, Uppsala, Sweden).
(
300 MHz, CD
s, 3H, CH
), 2.56 (t, 2H, J ¼ 7.2 Hz, Phthal-(CH
J ¼ 6.9 Hz, Phthal-CH ), 6.66 (s, 1H, Im-H-5), 7.75–7.99 (m, 4H,
Phthal-H). C NMR (75 MHz, CD ),
OD): d [ppm] ¼ 13.21 (þ, CH
), 38.49 (ꢁ, CH ), 116.98 (þ, Im-C-5),
24.05 (þ, Phthal-C-4,7), 133.39 (Cquat, Phthal-C-3a,7a), 135.33 (þ,
3
OD): d [ppm] ¼ 1.93 (m, 2H, Phthal-CH
2
–CH
2
), 1.98
) –CH ), 3.70 (t, 2H,
3
(
3
2 2 2
3
2
13
3
3
2
1
5.11 (ꢁ, CH
2
), 29.02 (ꢁ, CH
2
2
1
13
Nuclear magnetic resonance spectra ( H NMR and C NMR)
Phthal-C-5,6), 136.75 (Cquat, Im-C-4), 145.26 (Cquat, Im-C-2), 169.87
1
were recorded with Bruker Avance 30013 ( H: 300.1 MHz,
–
(
(
C
quat, 2 Phthal-C–O). ES-MS (DCM/MeOH þ NH
4
OAc) m/z (%): 270
13
1
C: 75.5 MHz), Avance 400 ( H: 400.1 MHz, C: 100.6 MHz), or
þ
100) [MþH] . C15
15 3 2
H N O (269.30).
1
13
Bruker Avance 600 ( H: 600.1 MHz,
C: 150.9 MHz) NMR
spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany).
Chemical shifts are given in d (ppm) relative to external standards.
Abbreviations for the multiplicities of the signals: s (singlet),
d (doublet), t (triplet), m (multiplet), brs (broad singlet), and
4
-(2-Methyl-1H-imidazol-4-yl)butan-1-amine (13)
A solution of 11 (0.8 g, 2.8 mmol) and hydrazine monohydrate
0.7 mL, 14.1 mmol, 5 eq) in EtOH (50 mL) was refluxed for 2 h
(
13
and cooled to room temperature overnight. The precipitate was
filtered off and washed with cold ethanol. The filtrate was
concentrated in vacuo and subjected to flash chromatography
combinations thereof. The multiplicity of carbon atoms ( C NMR)
was determined by DEPT 135 (distortionless enhancement by
polarization transfer): “þ” primary and tertiary carbon atom
(DCM/MeOH/32% NH3(aq) 80:18:2 v/v/v) to yield a yellow oil (0.31 g,
(
positive DEPT 135 signal), “ꢁ” secondary carbon atom (negative
1
7
2%); H NMR (300 MHz, CD
m, 2H, CH ), 2.29 (s, 3H, CH
2H, J ¼ 7.0 Hz, CH ), 6.58 (s, 1H, Im-H-5). C NMR (75 MHz,
CD ), 27.35 (ꢁ, CH ), 27.82 (ꢁ, CH ),
OD): d [ppm] ¼ 13.51 (þ, CH
–NH
3
OD): d [ppm] ¼ 1.53 (m, 2H, CH
2
), 1.62
), 2.68 (t,
DEPT 135 signal), “Cquat” quaternary carbon atom. In certain
3
(
2
3
), 2.52 (t, 2H, J ¼ 7.6 Hz, CH
2
cases, 2D-NMR techniques (COSY, HMQC, HSQC, HMBC, and
3
13
1
13
2
NOESY) were used to assign H and C chemical shifts. Mass
spectra (MS) were recorded on a Finnigan MAT 95 (EI-MS 70 eV,
3
3
2
2
3
2.47 (ꢁ, CH
2
), 42.05 (ꢁ, CH
2
2
), 117.25 (þ, Im-C-5), 137.59
3
HR-MS), Finnigan SSQ 710A (CI-MS (NH )), and on a Finnigan
(C
quat, Im-C-4), 145.10 (Cquat, Im-C-2). HRMS (EI-MS) calcd. for
ThermoQuest TSQ 7000 (ES-MS) spectrometer. The peak-intensity
in % relative to the strongest signal is indicated in parenthesis.
Elemental analysis (C, H, N, Heraeus Elementar Vario EL III) was
performed by the Analytical Department of the University
Regensburg and are within ꢀ0.4% unless otherwise noted. The
purity of tested compounds was >95% as determined by HPLC.
For the synthesis and the experimental data of compounds
þ•
C
8
H
N
15 3
[M ] 153.1266; found 153.1262. Anal. (C
8
H
15
N
3
· 0.85
2 8 15 3
H O) C, H, N. C H N (153.22).
3
-(2-Methyl-1H-imidazol-4-yl)propan-1-amine (14) [58]
A solution of 12 (0.5 g, 1.9 mmol) and hydrazine monohydrate
0.54 mL, 11.1 mmol, 5 eq) in EtOH (20 mL) was refluxed for 2 h
(
and cooled to room temperature overnight. The precipitate was
filtered off and washed with cold ethanol. The filtrate was
concentrated in vacuo and subjected to flash chromatography
7
–10, 16, 17, 22, 26, 27, 31, 32, 40, 45, 46, and 48–51, conditions of
preparative and analytical HPLC, HPLC purity data, and elemental
analyses cf. Supporting Information.
(
DCM/7 M NH1
3
in MeOH 90:10–70:30 v/v), yielding a yellow oil
OD): d [ppm] ¼ 1.75 (m, 2H,
13
(0.21 g, 79%); H NMR (300 MHz, CD
3
2
1
-[4-(2-Methyl-1H-imidazol-4-yl)butyl]isoindoline-
,3-dione (11)
3
CH
2
), 2.29 (s, 3H, CH
3
), 2.54 (t, 2H, J ¼ 7.5 Hz, CH
2
), 2.64 (t, 2H,
OD): d
3
J ¼ 7.2 Hz, CH ), 6.59 (s, 1H, Im-H-5). C NMR (75 MHz, CD
2
3
A mixture of 9 (5 g, 15.4 mmol), acetamidine hydrochloride (5.8 g,
[
ppm] ¼ 13.54 (þ, CH
3
), 25.02 (ꢁ, CH
2
), 33.43 (ꢁ, CH ), 41.99 (ꢁ,
2
6
2 3
1.7 mmol), and K CO (8.5 g, 61.7 mmol) in 50 mL DMF was
CH
2
–NH
2
), 117.19 (þ, Im-C-5), 137.43 (Cquat, Im-C-4), 145.20 (Cquat
,
stirred for 72 h at 50°C. After evaporation of the solvent, the
residue was taken up in DCM and extracted with saturated
NaHCO
þ•
Im-C-2). HRMS (EI-MS) calcd. for C
7
H
N
13 3
[M ] 139.1109; found
139.1113. Anal. (C
7 13 3 2 3 7 13 3
H N · 0.5H O · 0.4CH OH) C, H, N. C H N
3
. Removal of the solvent in vacuo and flash chromatogra-
(139.20).
phy (DCM/MeOH 95:5–85:15 v/v) yielded a yellow oil (0.84 g, 20%);
1
H NMR (300 MHz, CD
CH –CH –CH ), 2.27 (s, 3H, CH
–CH
), 3.58 (t, 2H, J ¼ 6.7 Hz, Phthal-CH
), 7.67 (m, 4H, Phthal-H).
), 27.08 (ꢁ, CH
3
OD): d [ppm] ¼ 1.60 (m, 4H, Phthal-
3
2-(5-Bromo-6-oxoheptyl)isoindoline-1,3-dione (18)
2
2
2
3
), 2.50 (t, 2H, J ¼ 7.1 Hz, Phthal-
), 6.58 (s, 1H, Im-H-
C NMR (75 MHz, CD OD): d
), 27.81 (ꢁ, CH ), 29.21
3
Compound 17 (14.0 g, 54.0 mmol) was dissolved in 150 mL glacial
acetic acid. After the addition of bromine (2.7 g, 52.0 mmol) at
ꢃ10°C, the mixture was allowed to warm to room temperature
and stirred for 1.5 h. Subsequently the solution was poured in 1 L
(CH
2
)
3
2
2
1
3
5
3
[
(
4
ppm] ¼ 13.68 (þ, CH
3
2
2
ꢁ, CH
2
), 38.68 (ꢁ, CH ), 117.45 (þ, Im-C-5), 124.10 (þ, Phthal-C-
2
3
of ice-cold water and extracted with CHCl . The organic layer was
evaporated and the residue was crystallized from 100 mL MeOH.
The precipitated product was filtered, washed with MeOH
,7), 133.26 (Cquat, Phthal-C-3a,7a), 135.33 (þ, Phthal-C-5,6), 137.13
(C
quat, Im-C-4), 145.18 (Cquat, Im-C-2), 169.68 (Cquat, 2 Phthal-C–O).
þ
ES-MS (DCM/MeOH þ NH
4
OAc) m/z (%): 284 (100) [MþH] .
(10 mL), and dried in vacuo to yield a white solid (11.3 g, 62%);
16 17 3 2
C H N O
(283.33).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
4
R. Geyer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
1
m.p. 82°C. H NMR (300 MHz, CDCl
3
): d [ppm] ¼ 1.51 (m, 2H, CH
), 2.34 (s, 2H, CH ), 3.68 (t, 2H,
), 4.21 (t, 1H, J ¼ 6.9 Hz, CH–Br), 7.70 (m,
2
),
3a,7a), 133.99 (þ, Phthal-C-5,6), 134.29 (þ, Triazol-C), 141.76
(Cquat, Triazol-C), 168.55 (Cquat, 2 C–O). ES-MS (DCM/MeOH þ
1
.71 (m, 2H, CH
2
), 2.00 (m, 2H, CH
J ¼ 7.1 Hz, Phthal-CH
H, Phthal-H), 7.82 (m, 2H, Phthal-H). C NMR (75 MHz, CDCl
2
3
3
3
þ
þ
2
NH
4
OAc) m/z (%): 271 (90) [MþH] , 312 (100) [MþHþMeCN] .
1
3
2
[
CH
1
3
): d
), 32.79 (ꢁ,
), 53.86 (þ, CH–Br), 123.25 (þ, Phthal-C-4,7),
14 14 4 2
C H N O (270.29).
ppm] ¼ 24.55 (ꢁ, CH
2
), 26.36 (þ, CH
3
), 27.92 (ꢁ, CH
2
2
), 37.47 (ꢁ, CH
2
4-(1H-1,2,3-Triazol-5-yl)butan-1-amine (24)
A solution of 23 (4.0 g, 14.8 mmol) and hydrazine monohydrate
32.05 (Cquat, Phthal-C-3a,7a), 133.99 (þ, Phthal-C-5,6), 168.39
(
3
C
quat, 2 Phthal-C–O), 201.81 (Cquat, C–O). CI-MS (NH
3
) m/z (%): 355,
(3.6 mL, 75 mmol, 5 eq) in EtOH (50 mL) was refluxed for 1.5 h and
þ
þ
57 (50) [MþNH
4
] , 277 (100) [MꢁBrþHþNH
4
] . Anal.
cooled to room temperature overnight. The precipitate was
filtered off and washed with cold ethanol. The filtrate was
concentrated in vacuo and the residue taken up in 30 mL water.
(C
15
H
16BrNO
3
) C, H, N. C15
H
16BrNO
3
(338.20).
2
-[4-(5-Methyl-1H-imidazol-4-yl)butyl]isoindoline-1,3-
Lyophilization yielded a white solid (2.0 g, 97%); m.p. 119–120°C.
1
dione (19)
H NMR (300 MHz, CD
), 2.74 (m, 4H, 2 CH
(75 MHz, CD
OD): d [ppm] ¼ 25.13 (ꢁ, CH
(ꢁ, CH ), 41.18 (ꢁ, CH –NH ), 129.14 (þ, triazol-C), 145.47 (Cquat
OAc) m/z (%): 141 (100)
3
OD): d [ppm] ¼ 1.58 (m, 2H, CH
2
), 1.72 (m,
13
Compound 18 (1.0 g, 3.0 mmol) was dissolved in formamide
2H, CH
2
2
), 7.52 (s, 1H, Triazol-H). C NMR
), 27.54 (ꢁ, CH ), 30.25
(
30 mL) and the mixture was stirred for 12 h at 170°C. The
mixture was taken up in 100 mL saturated NaHCO and extracted
. The organic layer was dried over MgSO
3
2
2
2
2
2
,
3
with 3 ꢄ 80 mL CHCl
3
4
triazol-C). ES-MS (DCM/MeOH þ NH
4
þ
þ
and evaporated. Flash chromatography (DCM/MeOH 95:5 v/v)
[MþH] , 182 (25) [MþHþMeCN] . HRMS (EI-MS) calcd. for
1
þ•
yielded a yellow solid (0.58 g, 68%); m.p. 115°C. H NMR (300 MHz,
C
6
H
N
12 4
[M ] 140.1062; found 140.1058. Anal. (C
6
H
12
N
4
) C, H,
CD
3
OD): d [ppm] ¼ 1.62 (m, 4H, Phthal-CH
2
–CH
), 2.55 (t, 2H, J ¼ 6.9 Hz, Phthal-(CH –CH
J ¼ 6.7 Hz, Phthal-CH ), 7.44 (s, 1H, Im-H-2), 7.80–8.04 (m, 4H,
2
–CH
2
), 2.13 (s, 3H,
6 12 4
N. C H N (140.19).
3
CH
3
2
)
3
2
), 3.66 (t, 2H,
3
2
tert-Butyl 5-[imino(methylsulfanyl)methylamino]-5-
oxopentylcarbamate (28)
TBTU (8.8 g, 28 mmol, 1.2 eq) was added to a solution of 26 (5.0 g,
13
Phthal-H). C NMR (75 MHz, CD
3
OD): d [ppm] ¼ 10.42 (þ, CH
3
2
),
),
2
1
1
5.53 (ꢁ, CH
2
), 28.09 (ꢁ, CH
2
), 28.96 (ꢁ, CH ), 38.57 (ꢁ, CH
2
24.10 (þ, Phthal-C-4,7), 127.32 (Cquat, Im-C), 131.71 (Cquat, Im-C),
23 mmol, 1 eq) and DIPEA (8.1 mL, 46 mmol, 2 eq) in DCM (50 mL)
33.40 (Cquat, Phthal-C-3a,7a), 133.94 (þ, Im-C-2), 135.37 (þ,
and the mixture was stirred for 30 min. Subsequently a solution
of 27 (5.0 g, 23 mmol, 1 eq) and DIPEA (12.2 mL, 69 mmol, 3 eq) in
DCM (20 mL) was slowly added. The mixture was stirred
overnight. After adding 50 mL ice-cold water the mixture was
extracted with 3 ꢄ 30 mL DCM. The combined organic layers were
Phthal-C-5,6), 169.90 (Cquat, 2 Phthal-C–O). ES-MS (DCM/MeOH þ
þ
NH
4
OAc) m/z (%): 284 (100) [MþH] . C16
17 3 2
H N O (283.33).
4
-(5-Methyl-1H-imidazol-4-yl)butan-1-amine (20) [48]
A solution of 19 (2.5 g, 8.8 mmol) and hydrazine monohydrate
2.14 mL, 44.1 mmol, 5 eq) in EtOH (50 mL) was refluxed for 2 h
4
dried over MgSO and the solvent was removed in vacuo. Flash
(
chromatography (PE/EtOAc 60:40 v/v) yielded a yellow oil (3.0 g,
1
and cooled to room temperature overnight. The precipitate was
filtered off and washed with cold ethanol. The filtrate was
concentrated in vacuo and directly subjected to flash chromatog-
raphy (Superflash SF15-30g, RP C18 pre-packed column, H
MeCN 100:0–80:20 v/v) yielding a yellow oil (1.1 g, 82%); m.p.
45%); H NMR (300 MHz, CDCl
(m, 2H, CH ), 1.68 (m, 2H, CH
S-CH ), 3.12 (m, 2H, CH -NHBoc). C NMR (75 MHz, CDCl
[ppm] ¼ 13.26 (þ, CH ), 22.45 (ꢁ, CH ), 28.39 (þ, C(CH ), 29.50
(ꢁ, CH ), 39.23 (ꢁ, CH ), 78.37 (Cquat, C(CH ),
, C–O). CI-MS
3
): d [ppm] ¼ 1.44 (s, 9H, Boc), 1.52
), 2.45 (m, 2H, CH ), 2.48 (s, 3H,
): d
2
2
2
13
3
2
3
2
O/
3
2
3 3
)
2
2
), 40.12 (ꢁ, CH
2
3 3
)
1
156.08 (Cquat, C–O), 165.72 (C , C–S), 182.49 (C
(NH
–
quat
quat
–
(
(
dihydrochloride) 215°C (AcOH) ([48]: 206–208°C). H NMR
300 MHz, CD ), 2.29 (s, 3H,
OD): d [ppm] ¼ 1.71 (m, 4H, CH
), 2.72 (t, 2H, J ¼ 6.9 Hz, CH
þ
3
) m/z (%): 290 (100) [MþH] . C12
H
23
N
3
O
3
S (289.39).
3
2
3
3
CH
8
3
2
), 2.97 (t, 2H, J ¼ 7.2 Hz, CH
2
),
13
.53 (s, 1H, Im-H-2). C NMR (75 MHz, CD
3
OD): d [ppm] ¼ 9.16 (þ,
tert-Butyl 4-(3-amino-1H-1,2,4-triazol-5-yl)-
butylcarbamate (29)
A solution of 28 (3.6 g, 12.4 mmol), hydrazine monohydrate
CH
CH
3
2
), 24.07 (ꢁ, CH
2
), 27.12 (ꢁ, CH
2
), 27.86 (ꢁ, CH
2
), 40.45 (ꢁ,
–NH
2
), 126.63 (Cquat, Im-C), 130.08 (Cquat, Im-C-4), 133.19 (þ,
þ•
Im-C-2). HRMS (EI-MS) calcd. for C
8
H
N
15 3
[M ] 153.1266; found
(18 mL, 374 mmol), and NEt
3
(56 mL, 404 mmol) in ethanol
1
53.1265.
(150 mL) was heated at reflux overnight. The solvent was
evaporated and subjected to flash chromatography (DCM/MeOH
1
2
-[4-(1H-1,2,3-Triazol-5-yl)butyl]isoindoline-1,3-dione (23)
95:5 v/v) yielding a white oily solid (0.7 g, 22%); H NMR (300 MHz,
CDCl
CH ), 2.69 (m, 2H, CH
75 MHz, CDCl
): d [ppm] ¼ 28.71 (ꢁ, CH
C(CH
3
): d [ppm] ¼ 1.44 (s, 9H, Boc), 1.54 (m, 2H, CH
), 3.14 (m, 2H, CH -NHBoc). C NMR
), 30.66 (ꢁ, CH ), 32.12 (þ,
), 83.10 (Cquat, C(CH ),
2
), 1.72 (m, 2H,
To a solution of 22 (1.5 g, 6.6 mmol) in DMF/MeOH (10 mL, 9:1 v/v),
13
azidotrimethylsilane (1.73 mL, 13.2 mmol) and CuI (0.63 g,
2
2
2
(
3
2
2
0
.33 mmol) were added [40]. The mixture was stirred under
3
)
3
), 32.98 (ꢁ, CH
2
), 43.74 (ꢁ, CH
2
3 3
)
microwave irradiation at 100°C for 12 h and then cooled to room
temperature. The solvents were evaporated and the residue was
taken up in 50 mL EtOAc. The solution was filtered through a
short pad of silica gel and washed with 3 ꢄ 30 mL water. Flash
1
60.70 (Cquat, C–O), 162.91 (Cquat, triazol-C), 171.27 (Cquat, triazol-
þ
C). ES-MS (DCM/MeOH þ NH
4
OAc) m/z (%): 256 (100) [MþH] .
11 21 5 2
C H N O
(255.32).
chromatography (DCM/MeOH 95:5 v/v) yielded a yellow solid
1
(1.5 g, 84%); m.p. 113–114°C. H NMR (300 MHz, CDCl
3
): d
-triazol), 3.71 (t,
-Phthal), 7.51 (s, 1H, Triazol-H), 7.70 (m, 2H,
5-(4-Aminobutyl)-1H-1,2,4-triazol-3-amine (30) [59]
[
2
ppm] ¼ 1.73 (m, 4H, 2 CH
2
), 2.79 (m, 2H, CH
2
Compound 29 (0.7 g, 2.9 mmol) was dissolved in 5 mL MeOH and
5 mL TFA was added. The mixture was stirred overnight and the
solvents were removed in vacuo. The crude product was purified by
3
H, J ¼ 6.4 Hz, CH
2
1
3
Phthal-H), 7.80 (m, 2H, Phthal-H). C NMR (75 MHz, CDCl
3
): d
), 37.52 (ꢁ,
-Phthal), 123.26 (þ, Phthal-C-4,7), 132.04 (Cquat, Phthal-C-
[
ppm] ¼ 24.38 (ꢁ, CH
2
), 26.44 (ꢁ, CH
2
), 28.04 (ꢁ, CH
2
preparative HPLC to yield
a white solid (0.59 g, 53%,
trifluoroacetate); m.p. 135–137°C. H NMR (300 MHz, CD OD,
1
CH
2
3
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Imbutamine Analogs
85
trifluoroacetate): d [ppm] ¼ 1.77 (m, 2H, 2 CH
2
), 2.73 (t, 2H,
according to the general procedure yielding a white solid (1.25 g,
3
3
13
1
J ¼ 6.9 Hz, CH
2
), 2.96 (t, 2H, J ¼ 7.0 Hz, CH
2
). C NMR (75 MHz,
), 25.81 (ꢁ, CH ),
71%); m.p. 84–85°C. H NMR (300 MHz, CD
3
OD): d [ppm] ¼ 1.43 (s,
3
CD
3
OD, trifluoroacetate): d [ppm] ¼ 24.40 (ꢁ, CH
2
2
9H, Boc), 1.70 (m, 2H, CH
2
), 3.11 (t, 2H, J ¼ 6.7 Hz, CH
2
-NHBoc),
–NH). C NMR (75 MHz, CD OD): d
–N), 41.39
3
13
2
1
7.74 (ꢁ, CH
2
), 40.21 (ꢁ, CH
2
–NH
2
), 152.25 (Cquat, triazol-C),
3.16 (t, 2H, J ¼ 6.8 Hz, CH
[ppm] ¼ 28.80 (þ, C(CH
(ꢁ, CH –N), 79.97 (Cquat, C(CH
(DCM/MeOH þ NH
20 6 2
) C, H, N. C10H N O (256.30).
2
3
53.33 (Cquat, triazol-C), 163.32 (Cquat, C–O). ES-MS (DCM/MeOH
)
), 30.97 (ꢁ, CH
), 38.77 (ꢁ, CH
3
3
2
2
þ
2þ
þ NH
4
OAc) m/z (%): 156 (45) [MþH] , 161 (100) [Mþ2Hþ4MeCN]
.
2
3
)
3
), 158.68 (Cquat, C–OO). ES-MS
þ•
þ
HRMS (EI-MS) calcd. for
C
6
H
13
N
5
[M
]
155.1171; found
4
OAc) m/z (%): 257 (100) [MþH] . Anal.
1
55.1172. Anal. (C
6
H
13
N
5
·1.9 TFA·0.4 H
2
O) C, H, N. C
6
H
N
13 5
·2
(C10
H
20
N
6
O
2
TFA (383.25).
tert-Butyl 4-(5-amino-1H-1,2,4-triazol-3-ylamino)-
butylcarbamate (37)
The title compound was prepared from 35 (2.3 g, 6.9 mmol) and
hydrazine monohydrate (0.5 mL, 10 mmol) in MeOH (40 mL)
according to the general procedure yielding a white solid (1.6 g,
General procedure for the preparation of the isoureas
(
34 and 35)
A solution of the pertinent amine (1 eq) and diphenyl cyanocar-
bonimidate (33, 1 eq) in 2-propanol was stirred for 2 h. After
evaporation of the solvent, the product was crystallized from Et
1
3
3
2
O.
86%); m.p. 138°C. H NMR (300 MHz, CD OD): d [ppm] ¼ 1.42 (s,
9
3
H, Boc), 1.53 (m, 4H, CH
2
), 3.05 (t, 2H, J ¼ 6.6 Hz, CH
2
-NHBoc),
OD): d
), 41.09
), 158.59 (Cquat
3
13
.13 (t, 2H, J ¼ 6.4 Hz, CH
), 28.40 (ꢁ, CH
–N), 43.80 (ꢁ, CH –N), 79.88 (Cquat, C(CH
OAc) m/z (%): 271 (100) [MþH] .
(270.33).
2
–NH). C NMR (75 MHz, CD
3
tert-Butyl 3-[(cyanoimino)(phenoxy)methylamino]-
propylcarbamate (34)
A solution of 31 (1.5 g, 8.4 mmol) and 33 (2.0 g, 8.4 mmol) in
[
ppm] ¼ 28.08 (ꢁ, CH
2
2
), 28.83 (þ, C(CH
3 3
)
(
ꢁ, CH
2
2
3
)
3
,
þ
C–OO). ES-MS (DCM/MeOH þ NH
4
2
-propanol (50 mL) was stirred for 2 h. Flash chromatography (PE/
Anal. (C11 ) C, H, N. C11H N O
H
22
N
6
O
2
22 6 2
EtOAc 60:40–40:60 v/v) yielded a white solid (2.26 g, 85%); m.p.
1
1
13°C (Et
2
O). H NMR (300 MHz, CDCl
), 3.24 (m, 2H, CH
m, 2H, Ph-H), 7.26 (m, 1H, Ph-H-4), 7.40 (m, 2H, Ph-H). C NMR
75 MHz, CDCl ), 29.87 (ꢁ, CH ), 37.33
): d [ppm] ¼ 28.39 (þ, C(CH
ꢁ, CH –N), 39.65 (ꢁ, CH –N), 79.48 (Cquat, C(CH ), 115.88 (Cquat
3
): d [ppm] ¼ 1.43 (s, 9H,
3
Boc), 1.80 (m, 2H, CH
2
2
), 3.46 (m, 2H, CH ), 7.08
2
General procedure for the preparation of N -aminoalkyl-
H-1,2,4-triazole-3,5-diamines (38 and 39)
13
(
(
(
1
3
3
)
3
2
Thirty milliliters of 6 M HCl in 2-propanol was added to a solution
of the Boc-protected precursor in 30 mL EtOAc and 5 mL MeOH.
The mixture was stirred overnight; the precipitate was filtered
off, washed, and dried in vacuo.
2
2
3
)
3
,
CN), 121.49 (þ, 2 Ph-C), 126.62 (þ, Ph-C-4), 129.52 (þ, 2 Ph-C),
1
51.07 (Cquat, Ph-C-1), 156.33 (Cquat, C–O), 163.90 (Cquat, C–N). ES-
þ
MS (DCM/MeOHþNH
4
OAc) m/z (%): 319 (65) [MþH] , 637 (100)
þ
þ
[
2Mþ] , 654 (90) [2MþNH
4
] . Anal. (C16
H
22
N
4
O
3
) C, H, N.
3
N -(3-Aminopropyl)-1H-1,2,4-triazole-3,5-diamine (38)
16 22 4 3
C H N O (318.37).
Compound 38 was prepared from 36 (1.2 g, 4.7 mmol) according
to the general procedure yielding a white solid (0.95 g, 72%); m.p.
tert-Butyl 4-[(cyanoimino)(phenoxy)methylamino]-
butylcarbamate (35)
A solution of 32 (1.58 g, 8.4 mmol) and 33 (2.0 g, 8.4 mmol) in
1
1
CH
17–118°C. H NMR (300 MHz, DMSO-d
6
): d [ppm] ¼ 1.83 (m, 2H,
), 3.23 (t, 2H, J ¼ 6.6 Hz, CH ), 7.68 (brs, 1H,
NH), 8.22 (brs, 3H, NH), 10.58 (brs, 4H, NH). C NMR (75 MHz,
DMSO-d
3
2
), 2.81 (m, 2H, CH
2
2
13
2
-propanol (50 mL) was stirred for 2 h. Flash chromatography (PE/
6
): d [ppm] ¼ 26.17 (ꢁ, CH
2
), 36.31 (ꢁ, CH –N), 39.15 (ꢁ,
2
EtOAc 60:40–40:60 v/v) yielded a white solid (2.24 g, 80%); m.p. 99°C
CH
C
2
–N), 151.55 (Cquat, 2 triazol-C). HRMS (EI-MS) calcd. for
1
þ•
(
(
Et
m, 2H, CH
), 7.07 (m, 2H, Ph-H), 7.26 (m, 1H, Ph-H-4), 7.40 (m, 2H, Ph-H).
C NMR (75 MHz, CDCl ), 27.21 (ꢁ, CH ),
): d [ppm] ¼ 26.71 (ꢁ, CH
8.41 (þ, C(CH ), 39.98 (ꢁ, CH –N), 42.22 (ꢁ, CH –N), 79.26
quat, C(CH
), 115.87 (Cquat, CN), 121.50 (þ, 2 Ph-C), 126.57
þ, Ph-C-4), 129.55 (þ, 2 Ph-C), 151.09 (Cquat, Ph-C-1), 156.07 (Cquat
OAc) m/z (%):
2
O). H NMR (300 MHz, CDCl
3
): d [ppm] ¼ 1.42 (s, 9H, Boc), 1.56
5
H
12
N
6
[M ] 156.1123; found 156.1126. Anal. (C
5
H
N
12 6
·2.5
2
), 1.65 (m, 2H, CH
2
), 3.14 (m, 2H, CH ), 3.41 (m, 2H,
2
2 5 12 6
HCl·0.3 H O) C, H, N. C H N ·3 HCl (265.57).
CH
2
1
3
3
2
2
3
N -(4-Aminobutyl)-1H-1,2,4-triazole-3,5-diamine (39)
2
3
)
3
2
2
Compound 39 was prepared from 37 (1.6 g, 5.9 mmol) according
(C
3 3
)
to the general procedure yielding a white solid (1.6 g, 96%); m.p.
(
,
1
C–O), 163.94 (Cquat, C–ON). ES-MS (DCM/MeOH þ NH
1
CH
20–121°C. H NMR (300 MHz, DMSO-d
6
): d [ppm] ¼ 1.57 (m, 4H, 2
), 3.14 (t, 2H, J ¼ 6.4 Hz, CH ), 7.72 (brs, 1H,
NH), 8.19 (brs, 3H, NH), 11.31 (brs, 4H, NH). C NMR (75 MHz,
DMSO-d
CH
–N), 41.50 (ꢁ, CH
calcd. for C
· 2.5HCl · 0.5H
4
3
þ
þ
þ
4
), 2.75 (m, 2H, CH
2
2
3
33 (70) [MþH] , 665 (80) [2Mþ] , 682 (100) [2MþNH
) C, H, N. C17 (332.40).
] . Anal.
2
13
(C
17
H
24
N
4
O
3
24 4 3
H N O
6
): d [ppm] ¼ 24.09 (ꢁ, CH
2
), 25.52 (ꢁ, CH ), 38.14 (ꢁ,
2
–N), 151.70 (Cquat, 2 triazol-C). HRMS (EI-MS)
General procedure for the preparation of 3,5-diamino-
triazoles (36 and 37)
Hydrazine monohydrate was added to a solution of the isourea in
2
2
þ•
6
H
N
14 6
[M ] 170.1280; found 170.1279. Anal.
(C
6
H
N
14 6
2 6 14 6
O) C, H, N. C H N · 3HCl (279.60).
4
2
3
0 mL MeOH and the mixture was stirred at room temperature for
4 h. The solvent was evaporated, the residue was taken up in
0 mL water and 50 mL DCM was added. The precipitated product
tert-Butyl 3-(5-amino-1H-1,2,4-triazol-3-ylsulfanyl)-
propylcarbamate (42)
A solution of 40 (3.0 g, 12.6 mmol) in 50 mL EtOH was added
to a mixture of 12.6 mL 1 M NaOH and 5-amino-4H-1,2,4-triazole-3-
thiol (41) (1.22 g, 10.5 mmol). The solution was refluxed for 3 h,
concentrated in vacuo, diluted with 10 mL water, and extracted
was filtered off and washed with water and DCM.
tert-Butyl 3-(5-amino-1H-1,2,4-triazol-3-ylamino)-
propylcarbamate (36)
The title compound was prepared from 34 (2.2 g, 6.9 mmol) and
hydrazine monohydrate (0.5 mL, 10 mmol) in MeOH (40 mL)
with 3 ꢄ 100 mL DCM. The organic layer was dried over MgSO
4
and the solvent was removed in vacuo. Flash chromatography
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
6
R. Geyer et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
þ•
(
7
1
CH
DCM/MeOH 95:5 v/v) yielded a colorless foam-like solid (2.25 g,
calcd. for C
7
H
13
N
5
[M ] 167.1171; found 167.1172. Anal.
1
8%); H NMR (300 MHz, CDCl
3
): d [ppm] ¼ 1.41 (s, 9H, Boc),
(C · 2.5HCl · 0.8H
7
H
N
13 5
2
O) C, H, N. C
7
H
13
N
5
· 2HCl (240.13).
3
.84 (m, 2H, CH
2
), 3.04 (t, 2H, J ¼ 6.0 Hz, CH
–N). C NMR (75 MHz, CDCl
): d [ppm] ¼ 28.47 (þ, C(CH
), 30.08 (ꢁ, CH –S), 39.03 (ꢁ, CH –N), 79.48 (Cquat
), 155.50 (Cquat, triazol-C), 156.46 (Cquat, C–OO), 158.24
2
–S), 3.19 (m, 2H,
13
)
3
),
2
4
2
3
3
N -(2-Aminoethyl)-N -methylpyrimidine-2,4-diamine (54)
Compound 54 was prepared from 49 (0.215 g, 1.5 mmol) and 51
0.24 g, 1.5 mmol) according to the general procedure. Crystalli-
zation yielded a pale green solid (0.2 g, 55%), m.p. 217–219°C
dec.) H NMR (300 MHz, CD
2
9.55 (ꢁ, CH
2
2
2
,
C(CH
3 3
)
(
(C
quat
,
Triazol-C-3). ES-MS (DCM/MeOH þ NH
4
OAc) m/z (%):
þ
2
71 (100) [MþH] . Anal. (C10
H
19
N
5
O
2
S) C, H, N. C10
H
19
N
5
O
2
S
1
(
(
1
3
OD): d [ppm] ¼ 3.03 (s, 3H, CH
3
), 3.25
–N), 6.14 (d,
(273.36).
3
3
t, 2H, J ¼ 6.0 Hz, CH
2
–N), 3.79 (t, 2H, J ¼ 6.0 Hz, CH
2
3
3
13
H, J ¼ 7.3 Hz, Py-H-5), 7.58 (d, 1H, J ¼ 7.3 Hz, Py-H-6). C NMR
3
-(3-Aminopropylsulfanyl)-1H-1,2,4-triazol-5-amine (43)
(75 MHz, CD OD): d [ppm] ¼ 28.21 (þ, CH ), 39.68 (ꢁ, CH –N),
3
3
2
Thirty milliliters of 6 M HCl in 2-propanol was added to a solution
of 42 (2.2 g, 8 mmol) in 30 mL EtOAc and 5 mL MeOH. The mixture
was stirred over-night; the precipitate was filtered off, washed,
and dried in vacuo. The crude product was recrystallized from
40.01 (ꢁ, CH –N), 99.59 (þ, Py-C-5), 141.19 (þ, Py-C-6), 155.73
2
(Cquat, Py-C), 164.77 (Cquat, Py-C). HRMS (EI-MS) calcd. for C
7
H
N
13 5
þ•
[M ] 167.1171; found 167.1170. Anal. (C
7
H
13
N
5
· 2HCl) C, H, N.
7 13 5
C H N · 2HCl (240.13).
1
EtOH to yield a white solid (1.67 g, 85%); m.p. 137°C. H NMR
(
300 MHz, DMSO-d
6
): d [ppm] ¼ 1.96 (m, 2H, CH
2
), 2.85 (m, 2H,
), 8.28 (brs, 5H, NH), 13.22 (brs,
H, NH). C NMR (75 MHz, DMSO-d ),
4
3
N -(2-Aminoethyl)pyrimidine-2,4-diamine (55) [61]
CH
2
), 3.19 (t, 2H, J ¼ 7.0 Hz, S–CH
2
13
Compound 55 was prepared from 50 (0.155 g, 1.2 mmol) and 51
0.192 g, 1.2 mmol) according to the general procedure. Yield
.16 g (60%) white solid, m.p. 239–241°C (dec.) H NMR (300 MHz,
CD
2
2
1
1
6
): d [ppm] ¼ 26.60 (ꢁ, CH
2
(
0
8.33 (ꢁ, CH
2
–S), 37.08 (ꢁ, CH
2
–N), 146.91 (Cquat, triazol-C-5),
1
þ•
52.19 (Cquat, triazol-C-3). HRMS (EI-MS) calcd. for C
73.0735; found 173.0734. Anal. (C
S · 2HCl (246.16).
5
H
N
11 5
S [M
]
3
3
OD): d [ppm] ¼ 3.22 (t, 2H, J ¼ 5.9 Hz, CH
2
–N), 3.77 (t, 2H,
5 11 5
H N S · 1.9HCl) C, H, N.
3
3
J ¼ 5.9 Hz, CH
2
–N), 6.20 (d, 1H, J ¼ 7.3 Hz, Py-H-5), 7.64 (d, 1H,
5 11 5
C H N
3
13
J ¼ 7.3 Hz, Py-H-6). C NMR (75 MHz, CD
3
OD): d [ppm] ¼ 39.54 (ꢁ,
CH
2
–N), 40.22 (ꢁ, CH –N), 99.83 (þ, Py-C-5), 141.85 (þ, Py-C-6),
2
General procedure for the preparation of the
N-(v-aminoalkyl)pyrimidine-2,4-diamines (52–55)
1
57.05 (Cquat, Py-C), 165.71 (Cquat, Py-C). HRMS (EI-MS) calcd. for
þ•
C
6
H
N
11 5
[M ] 153.1014; found 153.1016. Anal. (C
6
H
N
11 5
· 2HCl) C,
The respective chloropyrimidine (1 eq) was dissolved in 10 mL
ethanol. The pertinent mono-Boc-protected diamine (1 eq) and
DIPEA (1.5 eq) were added and the mixture was heated under
microwave irradiation at 120°C for 30min. The reaction mixture
was diluted with EtOAc, and washed with water and brine; the
6 11 5
H, N. C H N · 2HCl (226.11).
Pharmacology
3
5
[
S]GTPgS binding assays on hH R and hH R
3 4
35
4
organic layer was dried over MgSO and evaporated. Flash
[
S]GTPgS binding assays were performed as previously
chromatography (DCM/MeOH 90:10 v/v) gave the Boc-protected
precursors, which were taken up in 10mL methanol. Acetylchloride
described [49, 62, 63]. For details cf. Supporting Information.
All data are presented as mean of N independent experiments
(3eq) was slowly added at 0°C and the mixture was stirred at room
ꢀ
SEM. Agonist potencies were given as EC50 values (molar
temperature for 4 h. Evaporation of the solvent and crystallization
from MeOH/EtOAc yielded 52–55 as hydrochlorides.
concentration of the agonist causing 50% of the maximal
response). Maximal responses (intrinsic activities) were expressed
as a values. The a value of histamine was set to 1.00, a values of
other compounds were referred to this value. IC50 values were
2
N -(2-Aminoethyl)pyrimidine-2,4-diamine dihydrochloride
converted to K
EC50 and
B
values using the Cheng–Prussoff equation [64].
values from the functional GTPgS assays
(
52) [60]
K
B
Compound 52 was prepared from 48 (0.155 g, 1.2 mmol) and 51
0.192 g, 1.2 mmol) according to the general procedure. Yield
were analyzed by nonlinear regression and best fit to
sigmoidal dose–response curves (GraphPad Prism 5.0 software,
San Diego, CA).
(
0
1
.13 g (49%) white solid, m.p. 228–230°C (dec.) H NMR (300 MHz,
3
CD
3
OD): d [ppm] ¼ 3.22 (t, 2H, J ¼ 5.7 Hz, CH
2
–N), 3.74 (t, 2H,
3
3
J ¼ 5.7 Hz, CH
2
–N), 6.17 (d, 1H, J ¼ 7.2 Hz, Py-H-5), 7.69 (d, 1H,
3
13
J ¼ 7.2 Hz, Py-H-6). C NMR (75 MHz, CD
3
OD): d [ppm] ¼ 39.70 (ꢁ,
Histamine H and H radioligand binding assays on hH R
3
4
3
CH
1
C
2
–N), 40.48 (ꢁ, CH –N), 98.67 (þ, Py-C-5), 143.08 (þ, Py-C-6),
2
and hH R
4
55.81 (Cquat, Py-C), 166.88 (Cquat, Py-C). HRMS (EI-MS) calcd. for
Competition binding experiments were performed on membrane
preparations of Sf9 insect cell expressing the hH
R þ Gai2 þ Gb
. General procedures for the
þ•
6
H
N
11 5
[M ] 153.1014; found 153.1016. Anal. (C
6
H
N
11 5
· 2HCl) C,
3
1 2
g
6 11 5
H, N. C H N · 2HCl (226.11).
þ RGS4 or the hH
4
R þ Gai2 þ Gb
1 2
g
generation of recombinant baculoviruses, culture of Sf9 cells, and
membrane preparation have been described elsewhere [65, 66].
The respective membranes were thawed and sedimented by
centrifugation at 4°C and 13,000g for 10 min. Membranes were
2
N -(3-Aminopropyl)pyrimidine-2,4-diamine (53)
Compound 53 was prepared from 48 (0.155 g, 1.2 mmol) and 31
0.209 g, 1.2 mmol) according to the general procedure. Yield:
(
1
0
.13 g (44%) white solid, m.p. 193–195°C (dec.) H NMR (300 MHz,
CD ), 3.02 (m, 2H, CH –N), 3.53 (t,
OD): d [ppm] ¼ 1.99 (m, 2H, CH
–N), 6.12 (d, 1H, J ¼ 7.2 Hz, Py-H-5), 7.64 (d, 1H,
resuspended in binding buffer (12.5 mM MgCl
75 mM Tris/HCl, pH 7.4). Each tube (total volume 100mL)
contained 40 mg (hH R) or 60 mg (hH R) of membrane protein
and increasing concentrations of unlabeled ligands. Radioligands:
2
, 1 mM EDTA, and
3
2
3
2
3
2
H, J ¼ 6.5 Hz, CH
2
3
4
3
13
J ¼ 7.3 Hz, Py-H-6). C NMR (75 MHz, CD
CH –N), 38.81 (ꢁ, CH –N), 98.33 (þ, Py-C-5), 142.89
), 38.35 (ꢁ, CH
þ, Py-C-6), 155.50 (Cquat, Py-C), 166.85 (Cquat, Py-C). HRMS (EI-MS)
3
OD): d [ppm] ¼ 28.36 (ꢁ,
3
a
2
2
2
3
H R [ H]N -methylhistamine (Hartmann Analytic, Braunschweig,
(
Germany), specific activity 85.3 Ci/mmol, K
d
¼ 8.6nM, c ¼ 3 nM,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 77–88
Imbutamine Analogs
87
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[
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