Highly Potent, Stable, and Selective Dimeric Hetarylpropylguanidine-Type Histamine H2 Receptor Agonists.

On the basis of the long-known prototypic pharmacophore 3-(1H-imidazol-4-yl)propylguanidine (SK&F 91486, 2), monomeric, homodimeric, and heterodimeric bisalkylguanidine-type histamine H2 receptor (H2R) agonists with various alkyl spacers were synthesized. Aiming at increased H2R selectivity of the ligands, the imidazol-4-yl moiety was replaced by imidazol-1-yl, 2-aminothiazol-5-yl or 2-amino-4-methylthiazol-5-yl according to a bioisosteric approach. All compounds turned out to be partial or full agonists at the h/gp/rH2R. The most potent analogue, the thiazole-type heterodimeric ligand 63 (UR-Po461), was a partial agonist (Emax = 88%) and 250 times more potent than histamine (pEC50: 8.56 vs 6.16, gpH2R, atrium). The homodimeric structures 56 (UR-Po395) and 58 (UR-Po448) exhibited the highest hH2R affinities (pKi: 7.47, 7.33) in binding studies. Dimeric amino(methyl)thiazole derivatives, such as 58, generated an increased hH2R selectivity compared to the monomeric analogues, e.g., 139 (UR-Po444). Although monomeric ligands showed up lower affinities and potencies at the H2R, compounds with a short alkylic side chain like 129 (UR-Po194) proved to be highly affine hH4R ligands.

Introduction

In humans, the histamine receptor family comprises four subtypes, namely, H1, H2, H3, and H4 receptors.[1−4] They are activated by the biogenic amine histamine (1, Figure )[5] and belong to the superfamily of G-protein-coupled receptors (GPCRs).[6] For more than three decades, 3-(1H-imidazol-4-yl)propylguanidine (SK&F 91486, 2, Figure )[7] has been used as prototypic pharmacophore for the synthesis of highly potent histamine H2-receptor (H2R) agonists of the guanidine class, e.g., compounds such as arpromidine (3, Figure ).[8] The application of the bivalent ligand approach to acylguanidine-type H2R agonists by Birnkammer et al. led to highly potent and selective H2R agonists, e.g., UR-AK 381 (4, Figure ), raising questions about the binding mode and usability of such dimeric ligands as pharmacological tools.[9] Insufficient chemical stability of these acylguanidines, due to hydrolytic cleavage, led to carbamoylguanidine-type H2R agonists, which proved to be stable.[10] As many class A GPCRs were reported to form homo- and heterodimers, bivalent ligands can potentially be used as pharmacological tools to investigate the binding mode.[11,12] Using different spacer lengths, Birnkammer et al. showed that an interaction of the second pharmacophore with an allosteric binding site at the same receptor protomer is more likely than binding to the second orthosteric binding site of an H2R homodimer.[9]

Figure 1

Structures of histamine and selected H2R agonists (2–5).

For a better understanding of the structure–activity relationship of bisalkylguanidine-type dimeric H2R ligands, we prepared and pharmacologically characterized several monomeric and dimeric compounds derived from the recently reported homodimeric H2R agonist SK&F 93082 (5, Figure ).[13] In particular, the influence of different heteroaromatic ring systems and different spacer lengths on histamine receptor subtype selectivity was studied, using radioligand binding assays to investigate the affinities to the respective receptors. Investigations on H2R species selectivity were performed involving recombinant human, guinea pig, and rat H2Rs ([35S]GTPγS binding assay),[14] which gave information about the type, affinity/potency, and efficacy of the receptor ligand. Organ pharmacological studies (gpH1R (ileum), gpH2R (right atrium))[15,16] afforded agonistic (H2R) and antagonistic (H1R) activities under more physiological conditions. Functional activities on the guinea pig ileum and right atrium were measured via the contractility of the tissue and the increase of the heart frequency, respectively. The main focus of this project was the development of H2R agonists. This also includes the characterization of numerous compounds at the other histamine receptors (H1,3,4R), which led to the identification of selective H4R ligands.

Results and Discussion

Chemistry

The structures of amines 6–9, which were used for the synthesis of compounds 5, 53–63, 127–141, and 145, are depicted in Figure . For the preparation of the dimeric ligands, the diamines 11–16 were treated with benzoyl isothiocyanate (10) to give the corresponding dibenzoylthioureas 17–22 (Scheme ) via nucleophilic addition, followed by alkaline hydrolysis yielding the bisthioureas 23–28 as described.[17,8] After S-methylation using methyl iodide, the bis(S-methylisothioureas) 29–34 were Boc-protected to give the guanidinylation reagents 35–40 (Scheme ) following reported procedures.[8,18] The homodimeric ligands 5, 53–60 were obtained by treating amines 6, 7, 8, or 9 (each 2 equiv) with some of the guanidinylation reagents 35–40 in the presence of HgCl2 (2 equiv) and triethylamine (NEt3),[19] followed by Boc deprotection of the intermediates 41–49. The heterodimeric ligands 61–63 were synthesized by treatment of the guanidinylating reagent 38 with an equimolar mixture of two different amines (6 and 7, 6 and 8, or 7 and 8), followed by deprotection of the intermediates 50–52 using trifluoroacetic acid (TFA, Scheme ). It should be mentioned that the use of more than 2 equiv of HgCl2 led to a decrease in yield, presumably due to conversion of one S-methylisothiourea moiety to the respective carbodiimide as described in the literature.[18,19]

Figure 2

Structures of the building blocks 6–9, which were used for the preparation of the dimeric and monomeric compounds 5, 53–63, 127–141, and 145 (cf. scheme –3).

Scheme 1

Synthesis of the Homodimeric (5, 53–60) and Heterodimeric (61–63) Histamine Receptor (HR) Ligands

Reagents and conditions: (a) diamine (1 equiv), 10 (2 equiv), dichloromethane (DCM), overnight, 0 °C → room temperature (rt); (b) K2CO3 (4.1 equiv), MeOH/H2O (7/3, v/v), 3–5 h, rt; (c) CH3I (2.1 equiv), MeCN, 1 h, reflux; (d) NEt3 (2 equiv), di-tert-butyl dicarbonate (Boc2O) (2 equiv), overnight, rt; (e) 6, 7, 8, or 9 (2 equiv), HgCl2 (2 equiv), NEt3 (6 equiv), DCM, overnight, rt; (f) equimolar mixtures (each 1 equiv) of 6 and 7, 6 and 8, or 7 and 8, HgCl2 (2 equiv), NEt3 (6 equiv), DCM, overnight, rt; (g) 20% TFA, DCM, overnight, reflux.

Scheme 3

Synthesis of N-[3-(1H-Imidazol-4-yl)propyl]-1,4,5,6-tetrahydropyrimidin-2-amine (145)

Reagents and conditions: (a) tetrahydropyrimidine-2(1H)-thione (1 equiv), NaH (4.5 equiv), Boc2O (2.2 equiv), tetrahydrofuran (THF), hexane, 2 h, 0 °C → rt; (b) 6 (1 equiv), HgCl2 (2 equiv), NEt3 (3 equiv), DCM, overnight, rt; (c) 20% TFA, DCM, overnight, reflux.

The synthetic strategy for the dimeric compounds was also used for the synthesis of the monomeric compounds 127–141 (Scheme ). Although the alkylated thioureas 82–87 were commercially available, 76–81 had to be synthesized by nucleophilic addition of the corresponding amine (64–69) with 10 to give 70–75, followed by alkaline hydrolysis yielding the desired compounds (cf. Scheme ). S-methylation (88–99), Boc-protection (100–111), and guanidinylation (112–126) were accomplished as described for the dimeric ligands with adapted amount of substance (Scheme ). Finally, the precursors 112–126 were deprotected using TFA to give 127–141 (Scheme ).

Scheme 2

Synthesis of the Monomeric HR Ligands 127–141

Reagents and conditions: (a) amine (1 equiv), 10 (1 equiv), MeCN, 2 h, 0 °C → rt; (b) K2CO3 (2.1 equiv), MeOH/H2O (7/3, v/v), 3–5 h, rt; (c) CH3I (1.1 equiv), MeCN, 1 h, reflux; (d) NEt3 (1 equiv), Boc2O (1 equiv), overnight, rt; (e) 6, 7, 8, or 9 (1 equiv), HgCl2 (2 equiv), NEt3 (3 equiv), DCM, overnight, rt; (f) 20% TFA, DCM, overnight, reflux.

The cyclic guanidine derivative N-[3-(1H-imidazol-4-yl)propyl]-1,4,5,6-tetrahydropyrimidin-2-amine (145) was prepared in a three-step synthesis starting from tetrahydropyrimidine-2(1H)-thione (142) (Scheme ). Di-Boc-protection of 142 in the presence of sodium hydride resulted in 143 (Scheme ), as described.[20] After guanidinylation with 6, intermediate 144 was treated with TFA to receive 145 (Scheme ).

Stability of Dimeric Alkylguanidines

The chemical stability of the bisalkylguanidine-type dimeric HR ligands was exemplarily investigated for compounds 57 and 58, which were incubated in a binding buffer (BB, pH 7.4) at a concentration of 100 μM at room temperature for 12 months. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis revealed that 57 and 58 exhibited, in contrast to the previously reported acylguanidine-type HR ligands,[10] excellent chemical stabilities (Figure ).

Figure 3

RP-HPLC images of 57 (A) and 58 (B) after incubation in BB (pH 7.4) at rt for 12 months. Both compounds showed no decomposition.

Pharmacology

The synthesized dimeric (5, 53–63) and monomeric (127–141 and 145) ligands were investigated in radioligand competition binding assays (hH1,2,3,4R), in the [35S]GTPγS binding assay (hH2,3,4R, gp/rH2R), in the guinea pig ileum assay (gpH1R), and in the guinea pig right atrium assay (gpH2R). For the radioligand and the GTPγS binding assay, membranes of Sf9 cells, expressing hH1R + RGS4, h/gp/rH2R + Gsαs, hH3R + Giα2 + Gβ1γ2, or hH4R Giα2 + Gβ1γ2, were used.

Receptor Subtype Selectivity

To investigate the affinities of 5, 53–63, 127–141, and 145 at the four hHR subtypes, competition binding studies were performed using the radioligands [3H]mepyramine (hH1R), [3H]tiotidine or [3H]UR-DE257[21] (hH2R), [3H]Nα-methylhistamine (hH3R), and [3H]histamine (hH4R). The imidazol-4-yl-type homodimeric ligands 56 and 57, containing a decamethylene and a dodecamethylene spacer, respectively, exhibited the highest affinities at every HR subtype with the following selectivity profile: pKi H1R < H2R ≈ H3R ≈ H4R (Table ). The same selectivity profile was evident for the homodimeric compounds 5 and 53–55. The 2-amino-4-methylthiazol-5-yl- and 2-aminothiazol-5-yl-type homodimeric ligands 58 and 59, respectively, as well as the 2-amino-4-methylthiazol-5-yl/2-aminothiazol-5-yl-type heterodimeric compound 63 proved to be selective H2R ligands with at least 1.5 log units difference in pKi (H2R) over pKi (H1,3,4R) (Table ). This demonstrated that replacement of the imidazol-4-yl by an 2-aminothiazol-5-yl moiety is bioisosteric with respect to H2R binding, but not in case of H3 and H4 receptor binding, and was in accordance with previous reports on 2-aminothiazol-5-yl-type H2R selective ligands.[22] The binding data at the hH1R showed values, which were dependent from the spacer length, respectively the lipophilicity. The replacement of imidazol-4-yl by a imidazol-1-yl ring (60) leads to a collapse of the affinities at all histamine receptor subtypes. The monomeric reference substances 127–141 and 145 displayed increasing affinities at the hH2R with respect to their chain length, but lower affinities compared to the respective dimeric compounds. Overall, the affinities at the hH3,4Rs were higher, except of the aminothiazole-containing structures 139–140. Molecules with a small side chain like 129 are of special interest as highly affine hH4R ligands. Sigmoidal radioligand displacement curves at all HR subtypes are shown for 58, which exhibited the highest H2R selectivity (Figure ).

Table 1

Binding Data (pKi Values) of Compounds Diphenhydramine (DPH), 1, 2, 5, 53–63, 127–141, and 145 Determined at Human HRs (x = 1–4)a

	hH1Rb		hH2Rc		hH3Rd		hH4Re
compound	pKi	N	pKi	N	pKi	N	pKi	N
DPH	7.62 ± 0.01	4	n.d.g		n.d.		n.d.
1	5.62 ± 0.03[23]	3	6.58 ± 0.04	48	7.59 ± 0.01	42	7.60 ± 0.01	45
2	<4	3	5.39 ± 0.04f	3	7.42 ± 0.04	3	8.13 ± 0.08	3
5	5.50 ± 0.01	2	7.05 ± 0.02	3	7.52 ± 0.01	3	7.06 ± 0.01	3
53	<5	2	6.76 ± 0.03	3	6.95 ± 0.02	3	6.70 ± 0.01	3
54	<5	2	6.39 ± 0.02	3	6.84 ± 0.01	3	6.18 ± 0.04	3
55	<5.5	2	6.82 ± 0.04	3	7.28 ± 0.03	3	6.37 ± 0.02	3
56	5.90 ± 0.01	2	7.47 ± 0.12	3	7.72 ± 0.03	3	7.68 ± 0.04	3
57	6.45 ± 0.01	2	7.41 ± 0.03	3	7.79 ± 0.01	3	7.70 ± 0.01	3
58	<5.5	2	7.33 ± 0.05	3	5.25 ± 0.05	3	5.00 ± 0.05	3
59	<5	2	6.63 ± 0.03	3	4.96 ± 0.05	3	4.28 ± 0.02	3
60	<5	2	5.35 ± 0.03	3	5.56 ± 0.02	3	4.47 ± 0.03	3
61	<6	2	6.93 ± 0.04	3	7.49 ± 0.03	3	7.13 ± 0.04	3
62	<6	2	7.27 ± 0.04	3	7.43 ± 0.03	3	6.97 ± 0.05	3
63	<5.5	2	6.91 ± 0.04	3	5.40 ± 0.05	3	5.14 ± 0.04	3
127	<4.5	2	5.56 ± 0.07	3	6.81 ± 0.03	3	7.58 ± 0.07	3
128	<4.5	2	5.31 ± 0.05	3	7.03 ± 0.04	3	7.87 ± 0.01	3
129	<4.5	2	5.52 ± 0.05	3	7.21 ± 0.02	3	8.04 ± 0.05	3
130	<5	2	5.38 ± 0.07	3	7.04 ± 0.02	3	7.42 ± 0.01	3
131	<5	2	6.11 ± 0.06	3	7.21 ± 0.04	3	8.04 ± 0.02	3
132	<4.5	2	6.12 ± 0.05	3	7.18 ± 0.03	3	7.75 ± 0.03	3
133	<4	2	5.60 ± 0.10	3	6.43 ± 0.03	3	6.66 ± 0.06	3
134	<5	2	6.03 ± 0.06	3	7.04 ± 0.02	3	8.17 ± 0.04	3
135	<5	2	6.10 ± 0.06	3	6.94 ± 0.04	3	7.60 ± 0.01	3
136	<5.5	2	6.96 ± 0.07	3	6.97 ± 0.04	3	6.90 ± 0.01	3
137	5.70 ± 0.01	2	6.85 ± 0.09	3	7.50 ± 0.03	3	7.01 ± 0.03	3
138	5.53 ± 0.01	2	6.22 ± 0.01	3	7.53 ± 0.02	3	7.90 ± 0.03	3
139	<5.5	2	6.33 ± 0.04	2	5.69 ± 0.01	3	5.25 ± 0.03	3
140	<5.5	2	6.57 ± 0.03	2	4.85 ± 0.06	3	4.95 ± 0.05	3
141	<5.5	2	5.90 ± 0.01	2	5.19 ± 0.01	3	4.89 ± 0.06	3
145	<5	2	5.50 ± 0.02	2	6.73 ± 0.05	3	7.42 ± 0.01	3

Data represent mean values ± standard error of the mean (SEM) from at least two independent experiments (N), each performed in triplicate.

Radioligand competition binding experiments performed with [3H]mepyramine (hH1R, Kd 4.5 nM, c = 5 nM) at membranes of Sf9 cells expressing the hH1R + RGS4.

Radioligand competition binding experiments performed with [3H]tiotidine (hH2R, Kd 19.7 nM, c = 10 nM) at membranes of Sf9 cells expressing the hH2R + Gsαs.

Radioligand competition binding experiments performed with [3H]Nα-methylhistamine (hH3R, Kd 8.6 nM, c = 3 nM) at membranes of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2.

Radioligand competition binding experiments performed with [3H]histamine (hH4R, Kd 16.0 nM, c = 15 nM) at membranes of Sf9 cells expressing the hH4R + Gαi2 + Gβ1γ2.

Displacement of [3H]UR-DE257 (hH2R, Kd 31.3 nM, c = 20 nM) instead of [3H]tiotidine.

n.d. = not determined.

Figure 4

Radioligand displacement curves from radioligand competition binding experiments performed with compound 58 and [3H]mepyramine (hH1R, Kd 4.5 nM, c = 5 nM), [3H]tiotidine (hH2R, Kd 19.7 nM, c = 10 nM), [3H]Nα-methylhistamine (hH3R, Kd 8.6 nM, c = 3 nM), or [3H]histamine (hH4R, Kd 16.0 nM, c = 15 nM) at membranes of Sf9 cells expressing the respective hHR. Data represent mean values ± SEM from at least two independent experiments, each performed in triplicate.

Functional Characterization at the hH2,3,4R ([35S]GTPγS Binding Assay)

The compounds 5, 57–59, 61–63, 136, and 139 were chosen to be investigated in the [35S]GTPγS binding assay to determine their agonistic or antagonistic activities at the hH2,3,4R (Table ). All compounds proved to be partial agonists at the hH2R and silent antagonists at the hH3R and hH4R, except the monomeric ligands 136 and 139, which exhibited inverse agonistic activity at the hH3R and the hH4R, respectively. The homodimeric ligand 58, containing a C12-spacer, showed the highest H2R potency with a pEC50 of 7.27 and a maximal response of 52% relative to histamine. At the H3R, the antagonistic activities of imidazole-containing ligands (5, 57, 61, 62, and 136) were considerably higher than those of compounds with an amino(methyl)thiazole moiety (58, 59, 63, and 139) (e.g., pKB values of 57 and 59: 7.33 vs 4.05; Table ). Antagonistic activities at the hH4R were throughout low (pKB < 4).

Table 2

Agonistic (pEC50) and Antagonistic (pKB) Activities of 1, 2, 5, 57–59, 61–63, 136, and 139 at the hH2,3,4R Determined in the [35S]GTPγS Binding Assaya

	hH2Rb			hH3Rc			hH4Rd
compound	pEC50e	Emaxf	N	pEC50e (pKB)g	Emaxf	N	pEC50e (pKB)g	Emaxf	N
1	6.01 ± 0.07	1.00	7	8.52 ± 0.10	1.00	6	8.20 ± 0.08	1.00	3
2	5.59 ± 0.01h,[24]	0.66 ± 0.02h,[24]	3	8.12 ± 0.10h,[24]	0.69 ± 0.04h,[24]	3	8.09 ± 0.04h,[24]	0.83 ± 0.01h,[24]	3
5	6.78 ± 0.01	0.50 ± 0.03	6	(6.87 ± 0.05)	0	3	(3.39 ± 0.02)	0	3
57	7.27 ± 0.05	0.52 ± 0.03	6	(7.33 ± 0.07)	0	3	(3.49 ± 0.01)	0	3
58	6.61 ± 0.03	0.33 ± 0.03	5	(4.53 ± 0.05)	0	3	(3.83 ± 0.03)	0	3
59	6.53 ± 0.08	0.40 ± 0.05	3	(4.05 ± 0.10)	0	3	(3.58 ± 0.05)	0	3
61	6.23 ± 0.09	0.54 ± 0.05	3	(7.18 ± 0.02)	0	3	(3.69 ± 0.02)	0	3
62	6.51 ± 0.03	0.45 ± 0.04	3	(7.09 ± 0.01)	0	3	(3.43 ± 0.01)	0	3
63	6.60 ± 0.02	0.47 ± 0.03	6	(4.67 ± 0.05)	0	3	(3.73 ± 0.03)	0	3
136	6.86 ± 0.08	0.48 ± 0.02	3	7.54 ± 0.06	–0.53 ± 0.03	3	(3.68 ± 0.01)	0	3
139	5.16 ± 0.03	–0.43 ± 0.01	3	(5.37 ± 0.04)	0	3	4.86 ± 0.03	–0.97 ± 0.01	3

Data represent mean values ± SEM from at least three independent experiments (N), each performed in triplicate. Data were analyzed by nonlinear regression and were best-fitted to sigmoidal concentration–response curves (CRCs).

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the hH2R + Gsαs.

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the hH3R + Gαi2 + Gβ1γ2.

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the hH4R + Gαi2 + Gβ1γ2.

pEC50: −log EC50.

Emax: maximal response relative to histamine (Emax = 1.00).

For determination of antagonism, reaction mixtures contained histamine (1) (100 nM), and ligands were at concentrations from 10 nM to 1 mM; pKB = −log KB.

Determined in a steady-state [32P]GTPase assay on Sf9 cells expressing the related receptors.

H2R Species Selectivity

To study the H2R species selectivity of the bisalkylguanidine-type dimeric ligands, the agonistic activities of compounds 5, 57, 58, and 63 were also investigated at the gpH2R and rH2R in the GTPγS binding assay. The studied compounds exhibited slightly higher agonistic potencies at the gpH2R and rH2R compared to the hH2R, with pEC50 values >7.2 and >6.8, respectively (Table ). Compound 5 showed the highest gpH2R potency (pEC50 = 7.60) with a high efficacy (Emax = 0.95), and 57 displayed the highest rH2R potency (pEC50 = 7.61) with an efficacy of 0.80. In general, the efficacies were considerably higher at the gpH2R and rH2R (Emax > 0.7) compared to the hH2R (Emax < 0.52), reaching full agonism in case of compound 63 (gpH2R, Emax = 1.02). In sum, these investigations revealed that the highest potencies and efficacies were observed at the gpH2R followed by the rH2R and the hH2R.

Table 3

Agonistic Activities of 1, 5, 57, 58, and 63 at the h/gp/rH2R Determined in the [35S]GTPγS Binding Assaya

	hH2Rb,g			gpH2Rc			rH2Rd
compound	pEC50e	Emaxf	N	pEC50e	Emaxf	N	pEC50e	Emaxf	N
1	6.01 ± 0.07	1.00	7	5.82 ± 0.02	1.00	3	5.97 ± 0.02	1.00	3
5	6.78 ± 0.01	0.50 ± 0.03	6	7.60 ± 0.02	0.95 ± 0.05	3	7.18 ± 0.07	0.80 ± 0.01	3
57	7.27 ± 0.05	0.52 ± 0.03	6	7.53 ± 0.03	0.89 ± 0.07	3	7.61 ± 0.10	0.80 ± 0.03	3
58	6.61 ± 0.03	0.33 ± 0.03	5	7.28 ± 0.05	0.97 ± 0.06	3	6.83 ± 0.09	0.70 ± 0.04	3
63	6.60 ± 0.02	0.47 ± 0.03	6	7.33 ± 0.03	1.02 ± 0.04	3	6.85 ± 0.07	0.87 ± 0.02	3

Data represent mean values ± SEM from at least three independent experiments (N), each performed in triplicate. Data were analyzed by nonlinear regression and were best-fitted to sigmoidal CRCs.

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the hH2R + Gsαs.

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the gpH2R + Gsαs.

[35S]GTPγS binding assay at membranes of Sf9 cells expressing the rH2R + Gsαs.

pEC50: −log EC50.

Emax: maximal response relative to histamine (Emax = 1.00).

Data taken from Table .

Organ Bath Studies

In addition to the pharmacological characterization by radioligand competition binding (cf. Table ) and by functional studies using the [35S]GTPγS binding assay (cf. Tables and 3), organ bath experiments at the guinea pig ileum (gpH1R) and at the spontaneously beating guinea pig right atrium (gpH2R) were carried out. All ligands (5, 53–63, 127–141, and 145) displayed antagonistic activities at the gpH1R (Table ). The imidazol-4-yl-type homodimeric ligands 57 and the monomeric compound 137, containing a dodecamethylene spacer and a decamethyl side chain, respectively, showed the highest pA2 values (6.91 and 6.74, respectively, cf. Table ). A variation of the heteroaromatic moieties did almost not affect the pA2 value (gpH1R), as shown for compounds containing a C8-spacer (5 and 58–63). The experiments at the gpH2R provided interesting information about the structure–activity relationship from monomeric and dimeric HR ligands. Regarding the imidazole-containing ligands (5, 53–57, and 127–138), longer alkyl spacers resulted in higher agonistic potencies (e.g., pEC50 of 53 (C3-spacer) and 57 (C12-spacer): 7.31 vs 8.11, pEC50 of 127 (methyl) and 138 (dodecyl): 5.10 vs 6.63). By contrast, an inverse correlation was observed for the dimeric compounds with respect to the efficacy: compound 53, containing a C3-spacer, acted nearly as a full agonist (Emax = 0.96), and 57, containing a C12-chain, exhibited a maximum response of 0.63 compared to histamine (Table ). With the aim of getting compounds of highest potency and efficacy, 58–63 were equipped with a C8-spacer with 5 (pEC50 = 7.98, Emax = 0.91) as a prototype. The replacement of imidazol-4-yl (5 and 138) by imidazol-1-yl (60 and 141) led to a drastic decrease in potency and efficacy (5 vs 60: pEC50: 7.98 vs 5.31, Emax: 0.91 vs 0.20; 138 vs 141: pEC50: 6.63 vs not active, Emax: 0.91 vs 0). The introduction of amino(methyl)thiazole moieties (58, 59, and 61–63) resulted in potent gpH2R agonists (pEC50: 7.69–8.56, Emax: 0.78–1.02, Table ). Overall, a switch from monomeric to dimeric ligands revealed compounds, which are approximately 100 times more potent than their monomeric analogues. The concentration–response curve (CRC) of 63 (atrium, gpH2R) is exemplarily shown in Figure A.

Table 4

Agonistic (pEC50) and Antagonistic (pA2) Activities of 1, 2, 5, 53–63, 127–141, and 145 Determined by Organ Bath Studies at the gpH1R (Ileum) and the gpH2R (Atrium)a

	gpH1R		gpH2R
compound	pA2b (pEC50)	N	pEC50c,d	Emaxe	N
1	(6.68 ± 0.03)	255	6.16 ± 0.01	1.00	225
2	n.a.f	24	5.16 ± 0.04	0.75 ± 0.03	5
5	5.77 ± 0.04	9	7.98 ± 0.05	0.91 ± 0.05	3
53	6.20 ± 0.03	9	7.31 ± 0.03	0.96 ± 0.01	3
54	5.89 ± 0.03	9	7.42 ± 0.07	0.94 ± 0.01	3
55	5.69 ± 0.04	9	7.78 ± 0.04	0.90 ± 0.04	3
56	6.64 ± 0.05	8	7.60 ± 0.08	0.71 ± 0.04	3
57	6.91 ± 0.04	9	8.11 ± 0.08	0.63 ± 0.03	3
58	5.88 ± 0.03	9	8.38 ± 0.05	0.78 ± 0.01	3
59	6.08 ± 0.03	9	7.69 ± 0.06	0.94 ± 0.03	3
60	5.37 ± 0.05	9	5.31 ± 0.06	0.20 ± 0.02	3
61	5.85 ± 0.04	6	7.99 ± 0.05	0.91 ± 0.03	3
62	6.01 ± 0.05	6	8.25 ± 0.06	1.02 ± 0.06	3
63	6.00 ± 0.04	6	8.56 ± 0.06	0.88 ± 0.03	3
127	4.23 ± 0.02	4	5.10 ± 0.06	0.58 ± 0.07	3
128	5.08 ± 0.03	10	5.14 ± 0.05	0.55 ± 0.07	3
129	4.83 ± 0.05	8	5.40 ± 0.11	0.83 ± 0.03	3
130	5.27 ± 0.04	10	5.81 ± 0.08	0.87 ± 0.04	3
131	5.39 ± 0.04	11	6.08 ± 0.12	0.86 ± 0.04	3
132	4.99 ± 0.03	14	5.87 ± 0.05	0.86 ± 0.08	3
133	n.d.g	0	4.82 ± 0.09	0.92 ± 0.03	3
134	5.35 ± 0.04	12	6.44 ± 0.11	0.90 ± 0.02	3
135	5.14 ± 0.03	19	6.62 ± 0.07	0.89 ± 0.05	3
136	6.10 ± 0.06	8	6.71 ± 0.10	1.13 ± 0.07	3
137	6.74 ± 0.05	12	6.53 ± 0.08	0.90 ± 0.03	3
138	6.71 ± 0.05	12	6.63 ± 0.07	0.91 ± 0.03	3
139	6.36 ± 0.06	6	6.57 ± 0.07	0.60 ± 0.02	3
140	6.43 ± 0.07	6	6.25 ± 0.07	0.70 ± 0.01	3
141	6.43 ± 0.06	6	n.a.	0	3
145	5.24 ± 0.04	9	5.82 ± 0.07	1.00 ± 0.03	3

Data represent mean values ± SEM from at least three independent experiments (N). Data were analyzed by nonlinear regression and were best-fitted to sigmoidal CRCs.

pA2: −log c(Ant) + log(r – 1); r = 10ΔpEC; ΔpEC50 was calculated from pEC50 of histamine and pEC50 of histamine in the presence of the respective antagonist.

pEC50: −log EC50.

pEC50 was calculated from the mean-corrected shift ΔpEC50 of the agonist curve relative to the histamine reference curve by equation pEC50 = 6.16 + ΔpEC50.

Emax: maximal response relative to the maximal increase in heart rate induced by histamine (Emax = 1.00).

n.a. = not active.

n.d. = not determined.

Figure 5

CRCs of histamine (reference) and 63 in the absence (A) and presence (B) of 30 μM cimetidine at the gpH2R (atrium). Displayed curves were calculated by endpoint determination (N = 1).

The maximum response of all H2R agonists at the right atrium could be completely antagonized by the addition of the H2R antagonist cimetidine (pA2 = 6.10[25,26]) (30 μM) after cumulative organ stimulation (63, cf. Figure A). Full CRCs in the presence of cimetidine (30 μM, 30 min preincubation) were obtained for compounds 5, 57, 62, 63 (Figure B), 127, 128, 132, 135, and 137. The presence of cimetidine led to a rightward shift of the CRCs in a way that is in accordance with the calculated values via Schild equation (Table S1, Supporting Information). These results proved that the positive chronotropic effect of the investigated (partial) agonists in the guinea pig right atrium assay was mediated via the H2R.

Computational Studies

To determine accurate binding modes of the most interesting bivalent compounds 5 and 58 at the hH2R, and of the monovalent ligand 129 at both the hH3R and hH4R, molecular docking studies were performed, coupled with molecular dynamics (MD) simulations and free-energy (molecular mechanics generalized born surface area (MM-GBSA)) calculations. Overall, five promising binding poses for 5 and 58 at the hH2R, two poses for 129 at the hH3R, and three poses for 129 at the hH4R were determined by docking studies. Visual analysis of ligand–receptor complexes refined by molecular dynamics simulations (30 ns) has shown that some poses primarily formed contacts within the orthosteric binding site, whereas other poses additionally interacted with residues of the allosteric site between extracellular loop (ECL)2 and ECL3 (Figures S29–S31 and Table S3). The binding free energies (MM-GBSA) of the respective refined lowest free-energy docking poses (pose 1) of 5 (hH2R), 58 (hH2R), 129 (hH3R), and 129 (hH4R) amounted to −49.0, −61.5, −30.4, and −46.0 kcal/mol (Figures , 7, and S28). Interestingly, compared to the other poses, these lowest free-energy conformations were among those with the strongest H-bond contacts between ligand atoms and acidic amino acids of the orthosteric binding site (D983.32, D1865.42 for 5 and 58 at hH2R, D1143.32 for 129 at hH3R, and D943.32, E1825.46 for 129 at hH4R) (cf. Figure S32). The probability for an interaction with these residues is supported by the fact that they were proven to act as key player in aminergic GPCR activation and ligand binding,[27−30] or to account for receptor selectivity (D1865.42 at hH2R).[31] Interestingly, both 5 and 58 showed high binding affinities at hH2R (Ki < 100 nM), low affinities at the hH1R (Ki > 1 μM), but varying affinities at the hH3R and hH4R (5: Ki < 100 nM; 58: Ki > 1 μM). Although, by contrast, the monomeric imidazole-type analogue 129 bound unexpectedly poor to the hH2R, and, comparable to the dimeric compounds, also to the hH1R (Ki > 1 μM), it bound to the hH3R and hH4R with high affinity (Ki < 100 nM). In this context, an amino(methyl)thiazole moiety present in 58, unlike an imidazole moiety present in 5 or 129, was shown to trigger hH2R selectivity also in the case of other ligands.[32,33] Aiming at an attempt to explain this behavior, different steric effects of amino acids enclosing the orthosteric binding pocket may come into play due to less voluminous residues at the hH2R (V993.33, V176ECL2.54-Q177ECL2.55) compared to hH1R (Y1083.33, F184ECL2.54-Y185ECL2.55), hH3R (Y1153.33, F192ECL2.54-F193ECL2.55), and hH4R (Y953.33, F168ECL2.54-F169ECL2.55). In addition, an absence (hH1R) or different locations (hH3R: E2065.46; hH4R: E1825.46) of fundamental acidic amino acids in TM5 compared to hH2R (D1865.42) may further contribute to hH2R selectivity of amino(methyl)thiazole-type compounds, but to preferential binding of imidazole-type ligands, such as 5 or 129, to the hH3R and hH4R. Although 5 is still capable of binding to hH2R, most probably due to its larger and more flexible chain, 129 merely binds to the hH3R and hH4R with high affinity in a well-defined binding mode. Noteworthy, the lowest free-energy pose (pose 1) of 129 bound to the hH4R showed H-bond contacts with E163ECL2 and T1785.42 in addition to contacts with D943.32 and E1825.46, compared to relatively few and weak H-bond interactions when bound to hH3R (Figures and S32). This may contribute to a lower 129 binding free energy when bound to the hH4R, compared to hH3R (Figure S28).

Figure 6

Most probable binding mode of 5 (A) and 58 (B) at the hH2R, showing the respective most predominant clustered structure with the lowest binding free energy of the docking poses investigated by 30 ns MD simulations. Fundamental amino acids involved in ligand binding are shown as light-blue sticks, 5 is colored in green and 58 in magenta. H-bond contacts are illustrated as dashed yellow lines. The allosteric binding site is located most likely between ECL2 and ECL3.

Figure 7

Most probable binding modes of 129 at both the hH3R (A) and hH4R (B), showing the respective most predominant clustered structure with the lowest binding free energy of the docking poses investigated by 30 ns MD simulations. Fundamental amino acids involved in ligand binding are shown as light-blue sticks, 129 is colored in cyan when bound to the hH3R (A) and in orange when bound to the hH4R (B). H-bond contacts are illustrated as dashed yellow lines. The allosteric binding site is located most likely between ECL2 and ECL3 in both cases.

Summary and Conclusions

Homo- (5 and 53–60) and heterodimeric (61–63) as well as monomeric (127–141 and 145) hetarylpropylguanidine-type HR ligands were obtained in excellent yield by a six-step synthesis. The replacement of the imidazolyl by an aminothiazolyl moiety led, in accordance with previous reports on acylguanidine- and carbamoylguanidine-type HR ligands,[9,10,22] to highly selective and potent H2R agonists. The variation of the spacer length revealed best results for compounds containing a C8-, C10-, or C12-spacer (5, 56–59, and 61–63). The heterodimeric compounds showed potencies in a one-digit nanomolar range (up to 250 times the potency of histamine) as full agonists in the gpH2R atrium assay. In comparison to the monomeric ligands, the dualsteric structures showed up notably higher hH2R selectivity, higher affinities in the hH2R binding assay, and higher potencies at the functional H2R assays (e.g., guinea pig right atrium). The dimeric ligands displayed a slightly higher sensitivity for gpH2R and rH2R compared to the hH2R (maximum deviation less than 1 log unit). Monomeric imidazole-type compounds with small side chains, like 129, showing high hH4R affinity, could be of interest for the development of selective hH4R ligands to play an important role in different inflammatory, allergic, and immunological processes.[34,35] Molecular modeling studies, including the bivalent ligands 5 and 58, suggested an interaction of the guanidine groups with the acidic residues D983.32 and D1865.42 as key contributions to H2R binding. As the bisalkylguanidines turned out to be very stable chemical entities, the synthesized H2R selective aminothiazole derivatives represent promising lead structures for the development of pharmacological tools for the H2R, such as 58, 59, and 63. Moreover, the presented data could be of interest for the development of CNS penetrating H2R agonists—in consideration of changing the basicity of the strongly basic alkylguanidine structure, using bioisosteric approaches, and the lipophilicity—and H2R ligands useful for the treatment of acute myeloid leukemia.

Experimental Section

General Conditions

Commercially available chemicals (9, 10, 11–16, 64–69, and 82–87) and solvents were purchased from Acros Organics (Geel, Belgium), Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany), Iris Biotech GmbH (Marktredwitz, Germany), Merck KGaA (Darmstadt, Germany), Sigma-Aldrich Chemie GmbH (München, Germany), or TCI Europe (Zwijndrecht, Belgium) and were used as received. Deuterated solvents for nuclear magnetic resonance (1H NMR and 13C NMR) spectra were purchased from Deutero GmbH (Kastellaun, Germany). Compounds 6–8 were prepared as previously described.[22,32,36,37] The synthesis steps described in Section were carried out according to reported procedures.[8,17−19] All reactions involving dry solvents were accomplished in dry flasks under nitrogen or argon atmosphere. Millipore water was used for the preparation of buffers, HPLC eluents, and stock solutions. Column chromatography was carried out using Merck silica gel Geduran 60 (0.063–0.200 mm) or Merck silica gel 60 (0.040–0.063 mm) (flash column chromatography). The reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel 60 F254 aluminum sheets, and spots were visualized under UV light at 254 nm, by iodine vapor, ninhydrin, or fast blue B staining.

Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were recorded on a Bruker (Karlsruhe, Germany) Avance 300 (1H: 300 MHz, 13C: 75 MHz) or Avance 400 (1H: 400 MHz, 13C: 101 MHz) spectrometer using perdeuterated solvents. The chemical shift δ is given in parts per million (ppm). Multiplicities were specified with the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), and br (broad signal) as well as combinations thereof. 13C NMR peaks were determined by distortionless enhancement by polarization transfer (DEPT) 135 and DEPT 90: “+” primary and tertiary carbon atom (positive DEPT 135 signal), “–” secondary carbon atom (negative DEPT 135 signal), and “quat” quaternary carbon atom. NMR spectra were processed with MestReNova 11.0 (Mestrelab Research, Compostela, Spain). High-resolution mass spectrometry (HRMS) was performed on an Agilent 6540 UHD Accurate-Mass quadrupole time-of-flight liquid chromatography/mass spectrometry (MS) system (Agilent Technologies, Santa Clara, CA) using an electrospray ionization (ESI) source. Elemental analyses (EA) were performed on a Heraeus Elementar Vario EL III and are within ±0.4% unless otherwise noted. Melting points (mp) were measured on a Büchi (Essen, Germany) B-545 apparatus using an open capillary and are uncorrected. Preparative HPLC was performed with a system from Knauer (Berlin, Germany) consisting of two K-1800 pumps and a K-2001 detector. A Eurospher-100 C18 (250 × 32 mm2, 5 μm) (Knauer, Berlin, Germany) or a Kinetex XB-C18 (250 × 21.2 mm2, 5 μm) (Phenomenex, Aschaffenburg, Germany) served as stationary phase. As mobile phase, 0.1% TFA in millipore water and acetonitrile (MeCN) were used. The temperature was 25 °C, the flow rate was 15 mL/min, and UV detection was performed at 220 nm. Analytical HPLC was performed on system from Merck-Hitachi (Darmstadt/Düsseldorf, Germany) composed of an L-6200-A pump, an AS 2000A autosampler, an L-4000A UV detector, and a D-6000 interface. Stationary phase was a Eurosphere-110 C18 (250 × 4 mm2, 5 μm) (Knauer, Berlin, Germany) or a Kinetex XB-C18 (250 × 4.6 mm2, 5 μm) (Phenomenex, Aschaffenburg, Germany). As mobile phase, mixtures of MeCN and aqueous TFA were used (linear gradient: MeCN/TFA (0.1%) (v/v) 0 min: 5:95, 25 min: 50:50, 26–35 min: 95:5 (method A); 0 min: 10:90, 25 min: 50:50, 26–35 min: 95:5 (method B); flow rate = 0.80 mL/min, t0 = 3.32 min). Capacity factors were calculated according to k = (tR – t0)/t0. Detection was performed at 220 nm. All compounds were analyzed using method A, except for 139–141 (method B). Furthermore, filtration of the stock solutions with poly(tetrafluoroethylene) filters (25 mm, 0.2 μm, Phenomenex Ltd., Aschaffenburg, Germany) was accomplished before testing. Compound purities determined by HPLC were calculated as the peak area of the analyzed compound in % relative to the total peak area (UV detection at 220 nm). The HPLC purities (see Supporting Information) of the final compounds were ≥95% except for compound 58 (94.3%). The tested compounds were screened for pan-assay interference compounds (PAINS) and aggregation by publicly available filters (http://zinc15.docking.org/patterns/home, http://advisor.docking.org).[38,39] None of the screened structures have been previously reported as PAINS or an aggregator. Since Devine et al. described 2-aminothiazoles as a promiscuous frequent hitting scaffold at different enzymes,[40] full dose–response curves for all experiments and compounds, not only for the 2-aminothiazoles, were measured. None of the curves showed abnormalities, e.g., high Hill slopes, which could be a hint for PAINS.[39]

Chemical Synthesis and Analytical Data

General Procedure for the Synthesis of the Dibenzoylthioureas 17–22

To an ice-cold solution of the pertinent diamine (11–16, 1 equiv) in dichloromethane (DCM), benzoyl isothiocyanate (2 equiv) in DCM was added dropwise. The reaction was allowed to stir at room temperature (rt) overnight, and the organic solvent was concentrated under vacuum. The residue was suspended in 80 mL of methanol (MeOH) for 1 h and filtered to give the pure title compound.

N,N′-{[Octane-1,8-diylbis(azanediyl)]bis[carbonothioyl]}dibenzamide (20)

The title compound was prepared from octane-1,8-diamine (14, 1.08 g, 7.50 mmol) and 10 (2.02 mL, 15.00 mmol) in DCM (30 mL) according to the general procedure (Rf = 0.40 in ethyl acetate (EtOAc)/Hex 1:3). The product was obtained as a yellow solid (3.30 g, 93%), mp 146.8 °C. 1H NMR (300 MHz, CDCl3) δ (ppm) 10.74 (brs, 2H), 9.00 (brs, 2H), 7.88–7.78 (m, 4H), 7.68–7.57 (m, 2H), 7.56–7.45 (m, 4H), 3.71 (q, J = 7.2 Hz, 4H), 1.72 (quint, J = 6.7 Hz, 4H), 1.54–1.29 (m, 8H). 13C NMR (75 MHz, CDCl3) δ (ppm) 179.7, 166.9, 133.6, 131.8, 129.2, 127.4, 45.9, 29.1, 28.2, 26.8. HRMS (ESI-MS): m/z [M + H+] calculated for C24H31N4O2S2+: 471.1883, found 471.1884, C24H30N4O2S2 (470.65).

General Procedure for the Synthesis of the Bisthioureas 23–28

The corresponding dibenzoylthiourea (17–22, 1 equiv) was stirred in a solution of K2CO3 (4.1 equiv) in MeOH/H2O (7/3 v/v) for 3–5 h at rt. The proportion of MeOH was evaporated, and the resulting suspension was stirred for 1 h. The pure product was filtered with a Büchner funnel.

Because of the thione–thiol tautomerism, a splitting of the NH–C–(CH2)–C–NH signal could be observed in the following NMR spectra. Two broad singlets could be noted right next to each other. In each case, the integration value was exactly 4. For all of the other symmetric CH2 peaks, this peak splitting was not shown.

1,1′-(Octane-1,8-diyl)bis(thiourea) (26)[41]

The title compound was prepared from 20 (3.30 g, 7.01 mmol) and K2CO3 (3.88 g, 28.75 mmol) in MeOH/H2O (7/3 v/v, 100 mL) according to the general procedure (Rf = 0.43 in DCM/MeOH/NH3 90:10:0.1), yielding a colorless solid (1.80 g, 98%), mp 187.2 °C. 1H NMR (300 MHz, dimethyl sulfoxide (DMSO)-d6) δ (ppm) 7.60 (brs, 2H), 6.88 (brs, 4H), 3.32 + 3.00 (2 brs, 2.4H + 1.6H (thione–thiol tautomerism)), 1.43 (quint, J = 6.9 Hz, 4H), 1.31–1.20 (m, 8H). 13C NMR (75 MHz, DMSO-d6) δ (ppm) 182.9, 41.3, 32.7, 28.7, 26.2. HRMS (ESI-MS): m/z [M + H+] calculated for C10H23N4S2+: 263.1359, found 263.1359; C10H22N4S2 (262.44).

General Procedure for the Synthesis of the Bis-S-methylisothioureas (29–34)

The appropriate bisthiourea (23–28, 1 equiv) was dissolved in 50 mL of acetonitrile (MeCN) and treated with methyl iodide (2.1 equiv). The reaction mixture was stirred for 1 h under refluxing and the solvent was evaporated under vacuum. The resulting product (di-HI salt) was washed three times with 20 mL of diethylether (Et2O) and dried under vacuum.

1,1′-(Octane-1,8-diyl)bis(S-methylisothiourea) (32)

The compound 26 (1.80 g, 6.86 mmol) was dissolved in MeCN (50 mL) and treated with methyl iodide (0.90 mL, 14.40 mmol) according to the general procedure (Rf = 0.16 in DCM/MeOH/NH3 90:10:0.1). The resulting product was obtained as a yellow oil (32·2HI, 3.70 g, 99%). 1H NMR (300 MHz, CD3OD, hydrogen iodide) δ (ppm) 3.38 (t, J = 7.2 Hz, 4H), 2.65 (s, 6H), 1.66 (quint, J = 7.2 Hz, 4H), 1.44–1.36 (m, 8H). 13C NMR (75 MHz, CD3OD, hydrogen iodide) δ (ppm) 170.0, 45.5, 30.2, 29.0, 27.7, 14.4. HRMS (ESI-MS): m/z [M + H+] calculated for C12H27N4S2+: 291.1672, found 291.1674; C12H26N4S2·2HI (546.32).

General Procedure for the Synthesis of the Bis-N′-boc-S-methylisothioureas (35–40)

To a solution of the pertinent isothiourea (29–34) and 2 equiv of triethylamine (NEt3) in 50 mL of DCM, a solution of Boc2O (2 equiv) in 20 mL of DCM was added dropwise at rt. The reaction mixture was stirred overnight (rt) and washed with H2O and a saturated solution of NaCl. The organic layer was dried over Na2SO4, and the crude product was purified by column chromatography (EtOAc/petroleum ether (PE) 1/4–1/2 v/v).

1,1′-(Octane-1,8-diyl)bis(N′-tert-butoxycarbonyl-S-methylisothiourea) (38)

The reaction was carried out with 32 (3.70 g, 6.77 mmol), NEt3 (1.88 mL, 13.55 mmol), and Boc2O (2.96 g, 13.55 mmol) according to the general procedure (Rf = 0.62 in EtOAc/Hex 1:2), yielding a colorless oil (3.30 g, 99%). 1H NMR (300 MHz, CDCl3) δ (ppm) 9.56 (brs, 2H), 3.28 (q, J = 7.0 Hz, 4H), 2.45 (s, 6H), 1.65–1.56 (m, 4H), 1.49 (s, 18H), 1.38–1.30 (m, 8H). 13C NMR (75 MHz, CDCl3) δ (ppm) 173.5, 162.3, 79.2, 43.8, 29.3, 29.0, 28.3, 26.7, 13.6. HRMS (ESI-MS): m/z [M + H+] calculated for C22H43N4O4S2+: 491.2720, found 491.2721; C22H42N4O4S2 (490.72).

General Procedure for the Guanidinylation Reaction of 41–49

To a suspension of the corresponding amine 6, 7, 8, or 9 (2 equiv), the pertinent bis-N′-boc-S-methylisothiourea 35–40 (1 equiv), and HgCl2 (2 equiv) in DCM, NEt3 (6 equiv) was added. The mixture was stirred overnight at rt. A possible excess of HgCl2 was quenched with 7 N NH3 in MeOH (3–5 mL). The resulting suspension was filtered over Celite, and the crude product was purified by column chromatography (DCM/MeOH/7 N NH3 in MeOH 98/1/1–95/3/2 v/v/v).

1,1′-(Octane-1,8-diyl)bis{N″-tert-butoxycarbonyl-N′-[3-(1-trityl-1H-imidazol-4-yl)propyl]guanidine} (44)

The title compound was synthesized from 6 (500 mg, 1.36 mmol), 38 (334 mg, 0.68 mmol), HgCl2 (369 mg, 1.36 mmol), and NEt3 (0.57 mL, 4.08 mmol) in DCM (20 mL) according to the general procedure (Rf = 0.27 in DCM/MeOH/NH3 98:2:0.1). The product was obtained as a yellow oil (460 mg, 60%). 1H NMR (300 MHz, CDCl3) δ (ppm) 9.05 (brs, 2H), 7.38–7.19 (m, 20H), 7.15–7.01 (m, 12H), 6.53 (d, J = 1.4 Hz, 2H), 3.46–3.02 (m, 8H), 2.55 (t, J = 6.4 Hz, 4H), 1.84 (quint, J = 6.8 Hz, 4H), 1.44 (m + s, 4 + 18H), 1.22–1.12 (m, 8H). 13C NMR (75 MHz, CDCl3) δ (ppm) 164.4, 160.5, 142.3, 140.6, 138.0, 129.7, 128.1, 128.1, 118.3, 77.5, 75.2, 41.4, 40.7, 29.2, 28.8, 28.7, 28.6, 26.9. HRMS (ESI-MS): m/z [M + H+] calculated for C70H85N10O4+: 1129.6750, found 1129.6737; C70H84N10O4 (1129.51).

1,1′-(Octane-1,8-diyl)bis{N″-tert-butoxycarbonyl-N′-[(2-tert-butoxycarbonyl-amino-4-methylthiazol-5-yl)propyl]guanidine} (47)

The title compound was synthesized from 7 (554 mg, 2.04 mmol), 38 (500 mg, 1.02 mmol), HgCl2 (554 mg, 2.04 mmol), and NEt3 (0.85 mL, 6.12 mmol) in DCM (20 mL) according to the general procedure (Rf = 0.28 in DCM/MeOH/NH3 98:2:0.1). The product was obtained as a yellow foamlike solid (520 mg, 54%). 1H NMR (300 MHz, CDCl3) δ (ppm) 11.28 (brs, 2H), 3.24–3.06 (m, 8H), 2.67 (t, J = 6.5 Hz, 4H), 2.17 (s, 6H), 1.82 (quint, J = 7.3 Hz, 4H), 1.44 (s, 18H), 1.39 (m + s, 4 + 18H), 1.23–1.16 (m, 8H). 13C NMR (75 MHz, CDCl3) δ (ppm) 164.2, 160.0, 158.3, 152.9, 142.1, 122.9, 82.2, 77.9, 41.1, 40.3, 30.7, 29.1, 29.0, 28.4, 28.3, 26.7, 23.3, 14.5. HRMS (ESI-MS): m/z [M + H+] calculated for C44H77N10O8S2+: 937.5362, found 937.5367; C44H76N10O8S2 (937.27).

General Procedure for the Guanidinylation Reaction of 50–52

To a suspension of the corresponding amine 6, 7, or 8 (two out of them, each 1 equiv), 38 (1 equiv), and HgCl2 (2 equiv) in DCM, NEt3 (6 equiv) was added. The mixture was stirred overnight at rt. A possible excess of HgCl2 was quenched with 7 N NH3 in MeOH (3–5 mL). The resulting suspension was filtered over Celite, and the crude product was used in the next step without further purification.

2-tert-Butoxycarbonyl-1-[3-(2-tert-butoxycarbonyl-amino-4-methylthiazol-5-yl)propyl]-3-{8-[2-tert-butoxycarbonyl-3-(3-(2-tert-butoxycarbonylaminothiazol-5-yl)propyl)guanidino]octyl}guanidine (52)

The title compound was synthesized from 7 (111 mg, 0.41 mmol), 8 (105 mg, 0.41 mmol), 38 (200 mg, 0.41 mmol), HgCl2 (222 mg, 0.82 mmol), and NEt3 (0.17 mL, 1.22 mmol) in DCM (20 mL) according to the general procedure. The crude product was used in the next step without further purification. HRMS (ESI-MS): m/z [M + H+] calculated for C43H75N10O8S2+: 923.5204, found 923.5208; C43H74N10O8S2 (923.25).

General Procedure for the Synthesis of the Bivalent Ligands (5 and 53–63)

TFA (4.0 mL) was added to a solution of the protected precursors 41–52 in DCM (16.0 mL, 20% TFA in DCM), and the mixture was refluxed overnight until the protecting groups were removed (TLC control). Subsequently, the solvent was evaporated in vacuo and the residue was washed three times with Et2O (each 20 mL). The crude product was purified by preparative RP-HPLC (MeCN/0.1% TFA (aq): 5/95–40/60). All compounds were dried by lyophilization and obtained as tetra-trifluoroacetates.

1,1′-(Octane-1,8-diyl)bis{3-[3-(1H-imidazol-4-yl)propyl]guanidine} (5)[13]

Prepared from 44 (280 mg, 0.25 mmol) in DCM (16.0 mL) and TFA (4.0 mL) according to the general procedure, 5 was yielded as a yellow oil (150 mg, 67%): RP-HPLC: 98%, (tR = 13.97, k = 3.21). 1H NMR (300 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 8.83 (d, J = 1.2 Hz, 2H), 7.39 (s, 2H), 3.28 (t, J = 7.5 Hz, 4H), 3.20 (t, J = 7.0 Hz, 4H), 2.83 (t, J = 7.8 Hz, 4H), 1.98 (quint, J = 7.4 Hz, 4H), 1.59 (quint, J = 6.9 Hz, 4H), 1.42–1.35 (m, 8H). 13C NMR (75 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 163.1 (q, J = 35.2 Hz), 157.6, 154.3, 134.9, 134.5, 117.1, 42.7, 41.7, 30.3, 30.0, 28.9, 27.7, 22.6. HRMS (ESI-MS): m/z [M + H+] calculated for C22H41N10+: 445.3510, found 445.3506; C22H40N10·4TFA (900.72).

1,1′-(Dodecane-1,12-diyl)bis{3-[3-(1H-imidazol-4-yl)propyl]guanidine} (57)

Prepared from 46 (240 mg, 0.20 mmol) in DCM (16.0 mL) and TFA (4.0 mL) according to the general procedure, 57 was yielded as a yellow oil (120 mg, 62%): RP-HPLC: 99%, (tR = 17.84, k = 4.37). 1H NMR (300 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 8.77 (d, J = 1.3 Hz, 2H), 7.32 (d, J = 0.9 Hz, 2H), 3.27 (t, J = 7.0 Hz, 4H), 3.17 (t, J = 7.1 Hz, 4H), 2.80 (t, J = 7.7 Hz, 4H), 1.95 (quint, J = 7.3 Hz, 4H), 1.56 (quint, J = 6.8 Hz, 4H), 1.34–1.26 (m, 16H). 13C NMR (75 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 163.1 (q, J = 34.8 Hz), 157.6, 157.6, 134.8, 134.6, 129.0 (q, J = 44.3 Hz), 117.0, 42.7, 41.6, 30.7, 30.7, 30.4, 30.0, 28.9, 27.8, 22.6. HRMS (ESI-MS): m/z [M + H+] calculated for C26H49N10+: 501.4136, found 501.4131; C26H48N10·4TFA (956.83).

1,1′-(Octane-1,8-diyl)bis{3-[3-(2-amino-4-methylthiazol-5-yl)propyl]guanidine} (58)

Prepared from 47 (520 mg, 0.55 mmol) in DCM (16.0 mL) and TFA (4.0 mL) according to the general procedure, 58 was yielded as a yellow oil (280 mg, 51%): RP-HPLC: 94%, (tR = 15.53, k = 3.68). 1H NMR (300 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 3.26–3.10 (m, 8H), 2.71 (t, J = 7.1 Hz, 4H), 2.20 (s, 6H), 1.86 (quint, J = 7.3 Hz, 4H), 1.61–1.52 (m, 4H), 1.37–1.30 (m, 8H). 13C NMR (75 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 170.4, 162.8 (q, J = 35.6 Hz), 157.6, 157.6, 132.4, 118.5, 42.6, 41.5, 30.6, 30.2, 29.9, 27.7, 23.5, 11.5. HRMS (ESI-MS): m/z [M + H+] calculated for C24H45N10S2+: 537.3265, found 537.3261; C24H44N10S2·4TFA (992.90).

1-[3-(2-Amino-4-methylthiazol-5-yl)propyl]-3-{8-[3-(3-(2-aminothiazol-5-yl)propyl)guanidino]octyl}guanidine (63)

Prepared from 53 (without purification) in DCM (16.0 mL) and TFA (4.0 mL) according to the general procedure, 63 was yielded as a yellow oil (28.3 mg, 7.1%): RP-HPLC: 95%, (tR = 15.87, k = 3.78). 1H NMR (300 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 7.02 (s, 1H), 3.29–3.05 (m, 8H), 2.85–2.55 (m, 4H), 2.19 (s, 3H), 2.00–1.73 (m, 4H), 1.69–1.50 (m, 4H), 1.46–1.30 (m, 8H). 13C NMR (75 MHz, CD3OD, tetra-trifluoroacetate) δ (ppm) 171.7, 170.3, 157.5, 132.4, 126.7, 123.7, 118.9, 43.1, 42.0, 41.9, 31.0, 30.8, 30.4, 30.2, 27.9, 25.3, 24.0, 12.4. HRMS (ESI-MS): m/z [M + H+] calculated for C23H43N10S2+: 523.3108, found 523.3106; C23H42N10S2·4TFA (978.87).

General Procedure for the Synthesis of the Benzoylthioureas 70–75

To an ice-cold solution of the pertinent amine (64–69, 1 equiv) in MeCN, benzoyl isothiocyanate (1 equiv) was added dropwise. The reaction was stirred at room temperature (rt) for 2 h, and the organic solvent was concentrated under vacuum. The residue was dissolved in DCM (50 mL) and washed three times with H2O and saturated solution of NaCl. The organic layer was dried over Na2SO4, and the crude product was purified by column chromatography (EtOAc/PE 1/9–1/6 v/v).

N-(Isobutylcarbamothioyl)benzamide (70)[42]

The title compound was prepared from isobutylamine (64, 1.00 mL, 10.00 mmol) and 10 (1.34 mL, 10.00 mmol) in MeCN (30 mL) according to the general procedure (Rf = 0.41 in EtOAc/Hex 1:7). The product was obtained as a beige-colored solid (2.10 g, 89%), mp 79.6 °C. 1H NMR (300 MHz, CDCl3) δ (ppm) 10.83 (brs, 1H), 8.98 (brs, 1H), 7.88–7.80 (m, 2H), 7.67–7.59 (m, 1H), 7.56–7.48 (m, 2H), 3.55 (td, J = 6.9, 5.4 Hz, 2H), 2.06 (m, 1H), 1.03 (d, J = 6.7 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 179.85, 166.90, 133.59, 131.81, 129.19, 127.41, 53.38, 27.61, 20.29. HRMS (ESI-MS): m/z [M + H+] calculated for C12H17N2OS+: 237.1056, found 237.1057; C12H16N2OS (236.33).

General Procedure for the Synthesis of the Thioureas 76–81

The corresponding benzoylthiourea (70–75, 1 equiv) was stirred in a solution of K2CO3 (2.1 equiv) in MeOH/H2O (7/3 v/v) for 3–5 h at rt. The proportion of MeOH was evaporated, and the resulting suspension was extracted with DCM and stirred for 1 h. The organic layer was dried over Na2SO4, and the crude product was purified by column chromatography (DCM/MeOH/7M NH3 in MeOH 95/3/2 v/v).

Because of the thione–thiol tautomerism, a splitting of the NH–C–R-signal could be observed in the following NMR spectra. Two broad singlets could be noted right next to each other. In each case, the integration value was exactly 2. For all of the other symmetric CH2 peaks, this peak splitting was not shown.

N-Isobutylthiourea (76)[42]

The title compound was prepared from 70 (2.00 g, 8.46 mmol) and K2CO3 (2.46 g, 17.77 mmol) in MeOH/H2O (7/3 v/v, 50 mL) according to the general procedure (Rf = 0.38 in DCM/MeOH/NH3 95:5:0.1), yielding a colorless oil (0.78 g, 70%). 1H NMR (300 MHz, CDCl3) δ (ppm) 6.96 (brs, 1H), 6.32 (brs, 2H), 3.34 + 2.93 (2 brs, 0.9H + 1.1H (thione–thiol tautomerism)), 1.88 (m, 1H), 0.94 (d, J = 6.6 Hz, 6H). 13C NMR (75 MHz, CDCl3) δ (ppm) 180.76, 51.82, 27.85, 20.23. HRMS (ESI-MS): m/z [M + H+] calculated for C5H13N2S+: 133.0794, found 133.0794; C5H12N2S (132.23).

General Procedure for the Synthesis of the S-Methylisothioureas (88–99)

The appropriate thiourea (76–87, 1 equiv) was dissolved in 30 mL of MeCN and treated with methyl iodide (1.1 equiv). The reaction mixture was stirred for 1 h under refluxing, and the solvent was evaporated under vacuum. The resulting product (HI salt) was washed three times with 20 mL of diethylether (Et2O) and dried under vacuum.

S-Methyl-N-propylisothiourea (90)[43]

The compound 84 (1.18 g, 10.00 mmol) was dissolved in MeCN (30 mL) and treated with methyl iodide (0.69 mL, 11.00 mmol) according to the general procedure (Rf = 0.41 in DCM/MeOH/NH3 90:10:0.1). The resulting product was obtained as an orange oil (90·HI, 2.50 g, 96%). 1H NMR (300 MHz, CDCl3, hydrogen iodide) δ (ppm) 3.30 (t, J = 7.1 Hz, 2H), 2.79 (s, 3H), 1.74 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3, hydrogen iodide) δ (ppm) 170.49, 46.60, 21.93, 15.28, 11.45. HRMS (ESI-MS): m/z [M + H+] calculated for C5H12N2S+: 133.0794, found 133.0796; C5H12N2S·HI (260.14).

General Procedure for the Synthesis of the N′-Boc-S-methylisothioureas (100–111)

To a solution of the pertinent isothiourea (88–99) and 1 equiv of triethylamine (NEt3) in 50 mL of DCM, a solution of Boc2O (1 equiv) in 20 mL of DCM was added dropwise at rt. The reaction mixture was stirred overnight (rt) and washed with H2O and saturated solution of NaCl. The organic layer was dried over Na2SO4, and the crude product was purified by column chromatography (EtOAc/PE 1/9–1/5 v/v).

N′-tert-Butoxycarbonyl-S-methyl-N-propylisothiourea (102)

The reaction was carried out with 90 (2.46 g, 9.46 mmol), NEt3 (1.31 mL, 9.46 mmol), and Boc2O (2.06 g, 9.46 mmol) according to the general procedure (Rf = 0.50 in EtOAc/Hex 1:8), yielding a colorless oil (1.74 g, 79%). 1H NMR (300 MHz, CDCl3) δ (ppm) 9.81 (brs, 1H), 3.26 (q, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.64 (m, 2H), 1.49 (s, 9H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 173.44, 162.27, 79.17, 45.52, 28.25, 22.65, 13.56, 11.39. HRMS (ESI-MS): m/z [M + H+] calculated for C10H21N2O2S+: 233.1318, found 233.1321; C10H20N2O2S (232.34).

General Procedure for the Guanidinylation Reaction of 112–126 and 144

To a suspension of the corresponding amine 6, 7, 8, or 9 (1 equiv), the pertinent N′-boc-S-methylisothiourea 100–111 or 143 (1 equiv), and HgCl2 (2 equiv) in DCM, NEt3 (3 equiv) was added. The mixture was stirred overnight at rt. A possible excess of HgCl2 was quenched with 7 N NH3 in MeOH (3–5 mL). The resulting suspension was filtered over Celite, and the crude product was purified by column chromatography (DCM/MeOH/7 N NH3 in MeOH 98/1/1–95/3/2 v/v/v).

2-tert-Butoxycarbonyl-1-propyl-3-[3-(1-trityl-1H-imidazol-4-yl)propyl]guanidine (114)

The title compound was synthesized from 6 (808 mg, 2.20 mmol), 102 (510 mg, 2.20 mmol), HgCl2 (1.19 g, 4.40 mmol), and NEt3 (0.91 mL, 6.60 mmol) in DCM (20 mL) according to the general procedure (Rf = 0.24 in DCM/MeOH/NH3 98:2:0.1). The product was obtained as a yellow foamlike solid (620 mg, 51%). 1H NMR (300 MHz, CDCl3) δ (ppm) 9.05 (brs, 1H), 7.39–7.21 (m, 10H), 7.16–7.04 (m, 6H), 6.54 (s, 1H), 3.56–2.95 (m, 4H), 2.57 (t, J = 6.2 Hz, 2H), 1.86 (quint, J = 6.6 Hz, 2H), 1.51 (m, 2H), 1.47 (s, 9H), 0.83 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ (ppm) 164.49, 160.52, 142.38, 140.67, 138.07, 129.73, 128.34, 128.06, 118.25, 75.20, 53.49, 43.03, 40.67, 29.63, 28.69, 28.57, 22.52, 11.53. HRMS (ESI-MS): m/z [M + H+] calculated for C34H42N5O2+: 552.3333, found 552.3338; C34H41N5O2 (551.74).

General Procedure for the Synthesis of the Monomeric Ligands (127–141 and 145)

TFA (4.0 mL) was added to a solution of the protected precursors 112–126 or 144 in DCM (16.0 mL, 20% TFA in DCM), and the mixture was refluxed overnight until the protecting groups were removed (TLC control). Subsequently, the solvent was evaporated in vacuo and the residue was washed three times with Et2O (each 20 mL). The crude product was purified by preparative RP-HPLC (MeCN/0.1% TFA (aq): 5/95–40/60). All compounds were dried by lyophilization and obtained as di-trifluoroacetates.

1-[3-(1H-Imidazol-4-yl)propyl]-3-propylguanidine (129)[44]

Prepared from 114 (450 mg, 0.82 mmol) in DCM (16.0 mL) and TFA (4.0 mL) according to the general procedure, 129 was yielded as a beige-colored solid (310 mg, 87%), mp 126.7 °C. 1H NMR (300 MHz, CD3OD, di-trifluoroacetate) δ (ppm) 8.80 (d, J = 1.3 Hz, 1H), 7.34 (s, 1H), 3.26 (t, J = 7.0 Hz, 2H), 3.15 (t, J = 7.1 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 1.96 (quint, J = 7.3 Hz, 2H), 1.61 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CD3OD, di-trifluoroacetate) δ (ppm) 157.58, 134.92, 134.57, 116.97, 44.27, 41.58, 28.82, 23.25, 22.56, 11.41. HRMS (ESI-MS): m/z [M + H+] calculated for C10H20N5+: 210.1713, found 210.1714; C10H19N5·2TFA (437.34).

Synthesis of Di-tert-butyl-2-thioxodihydropyrimidin-1,3(2H,4H)-dicarboxylat (143)[45]

To a stirred solution of 142 (871 mg, 7.50 mmol) in THF (150 mL) under argon at 0 °C, hexane (20 mL) and sodium hydride (1.35 g, 33.8 mmol, 60% in mineral oil) were added. After 5 min, the ice bath was removed, and the reaction mixture was stirred at room temperature for 10 min. The mixture was cooled to 0 °C again, di-tert-butyl dicarbonate (3.60 g, 16.5 mmol) was added, and the ice bath was removed after 30 min of stirring at that temperature. The resulting slurry was stirred for another 2 h at room temperature. Then, the reaction was quenched with an aqueous solution of saturated NaHCO3 (10 mL). The reaction mixture was poured into water (250 mL) and extracted with EtOAc (3 × 70 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to give 143 as a yellow solid (1.88 g, 79%), mp 84.7 °C. 1H NMR (300 MHz, CDCl3) δ (ppm) 3.67 (t, J = 6.9 Hz, 4H), 2.15 (quint, J = 6.9 Hz, 2H), 1.53 (s, 18H). 13C NMR (75 MHz, CDCl3) δ (ppm) 182.68, 153.66, 84.09, 44.26, 27.77, 24.02. HRMS (ESI-MS): m/z [M + H+] calculated for C14H25N2O4S+: 317.1530, found 317.1532; C14H24N2O4S (316.42).

Pharmacological Methods

Materials

Histamine dihydrochloride was purchased from Alfa Aesar GmbH & Co. KG (Karlsruhe, Germany). [3H]mepyramine (specific activity: 20.0 Ci/mmol), [3H]tiotidine (specific activity: 78.4 Ci/mmol), [3H]Nα-methylhistamine (specific activity: 85.3 Ci/mmol), and [3H]histamine (specific activity: 25.0 Ci/mmol) were obtained from Hartmann Analytic (Braunschweig, Germany). GTPγS was purchased from Roche (Mannheim, Germany), and [35S]GTPγS was purchased from PerkinElmer Life Science (Boston) or Hartmann Analytic (Braunschweig, Germany). [3H]UR-DE257 was synthesized in our laboratories. All stock solutions were dissolved in millipore water or in a mixture of Millipore water/DMSO. In all assays, the final DMSO content amounted to <0.5%.

Membrane Preparations[46]

Sf9 cells were seeded at 3.0 × 106 cells/mL and infected with a 1:100 dilution of high-titer baculovirus stocks encoding hH1R + RGS4, h/gp/r H2R + Gsαs, hH3R Giα2 + Gβ1γ2, or hH4R Giα2 + Gβ1γ2. The cells were cultured for 48 h before the production of the membrane preparation according to a previously reported protocol, using 1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/mL benzamidine, and 10 μg/mL leupeptin as protease inhibitors. Membranes were suspended in binding buffer (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) and stored at −80 °C until use.

Radioligand Binding Assay[14,47−49]

Sf9 membranes were thawed and sedimented by centrifugation at 13 000g at 4 °C for 10 min. The membranes (hH1R + RGS4, hH2R + Gsαs, hH3R Giα2 + Gβ1γ2, hH4R Giα2 + Gβ1γ2) were resuspended in a cold (4 °C) binding buffer (BB: 12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) at a final soluble protein concentration of 2–6 μg per 1 μL BB. Experiments were performed in 96-well plates (polypropylene (PP) microplates 96 well, Greiner Bio-One, Frickenhausen, Germany) using a total volume of 100 μL of BB, which contained 40–90 μg of membrane, 0.05% bovine serum albumin (BSA), the respective radioligand ([3H]mepyramine 5 nM, [3H]tiotidine 10 nM, [3H] UR-DE257 20 nM, [3H]Nα-methylhistamine 3 nM, or [3H]histamine 15 nM), and the investigated ligands at various concentrations. During the incubation period (60 min) at room temperature, the plates were shaken at 250 rpm with a Heidolph Titramax 101 (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Afterward, bound radioligand was separated from free radioligand by collection of the membranes on polyethylenimine-pretreated GF/C filters (Whatman, Maidstone, U.K.) using a 96-well Brandel harvester (Brandel Inc., Unterföhring, Germany). After two washing steps with binding buffer, filter pieces were punched and transferred into 1450-401 96-well sample plates (PerkinElmer, Rodgau, Germany). Scintillation cocktail (200 μL, Rotiszint Eco plus, Roth, Karlsruhe, Germany) was added, and the plates were sealed with Plateseal 1450-461 (PerkinElmer, Rodgau, Germany) and incubated in the dark for 12 h. Radioactivity (dpm) was measured with a MicroBeta2 1450 scintillation counter (PerkinElmer). Experimental data were analyzed by nonlinear regression and were best-fitted to sigmoidal concentration–response curves using Prism 5.0c software (GraphPad, San Diego, CA).[50]

[35S]GTPγS Binding Assay[14,49,51,52]

Membranes of Sf9 cells expressing the h/gp/rH2R + Gsαs, hH3R Giα2 + Gβ1γ2, or hH4R Giα2 + Gβ1γ2 were thawed and sedimented by centrifugation at 13 000g at 4 °C for 10 min. The membranes were resuspended in a cold (4 °C) binding buffer BB (12.5 mM MgCl2, 1 mM EDTA, and 75 mM Tris/HCl, pH 7.4) at a final soluble protein concentration of 1 μg per 1 μL BB. All H4R experiments contained additionally 100 mM NaCl. For investigations in the antagonist mode, histamine was added to the reaction mixture (hH2R: 1 μM, hH3,4R: 100 nM). Experiments were performed in 96-well plates (PP microplates 96 well, Greiner Bio-One, Frickenhausen, Germany) in a total volume of 100 μL, containing 10 μg of membrane, 1 μM GDP, 0.05% BSA, 20 nCi [35S]GTPγS, and the investigated ligands at various concentrations. During the incubation period (90 min) at room temperature, the plates were shaken at 250 rpm with a Heidolph Titramax 101 (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). Afterward, bound radioligand was separated from free radioligand by filtration through GF/C filters (Whatman, Maidstone, U.K.) using a 96-well Brandel harvester (Brandel Inc., Unterföhring, Germany). After two washing steps with binding buffer, the filter pieces were punched and transferred into 1450-401 96-well sample plates (PerkinElmer). Unspecific binding was determined in the presence of 10 μM GTPγS. Each well was supplemented with 200 μL of scintillation cocktail (Rotiszint Eco plus, Roth, Karlsruhe, Germany), and the plates were sealed with Plateseal 1450-461 (PerkinElmer, Rodgau, Germany) and incubated in the dark for 12 h. Radioactivity (ccpm) was measured with a MicroBeta2 1450 scintillation counter (PerkinElmer). Data were analyzed by nonlinear regression and were best-fitted to sigmoidal concentration–response curves with the Prism 5.0c software (GraphPad, San Diego, CA).

Histamine H1 Receptor Assay on Isolated Guinea Pig Ileum[15]

Guinea pigs of either sex (250–500 g) were stunned by a blow on the neck and exsanguinated. The ileum was rapidly removed, rinsed, and cut into segments of 1.5–2.0 cm length. The tissues were mounted isotonically (preload of 5 mN) in a jacketed 20 mL organ bath that was filled with Tyrode’s solution (composition [mM]: NaCl, 137; KCl, 2.7; CaCl2, 1.8; MgCl2, 1.0; NaH2PO4, 0.4; NaHCO3, 11.9; and glucose 5.0) supplemented with atropine, at a concentration not affecting H1 receptors (0.05 μM), to block cholinergic muscarinic receptors. The bath was aerated with 95% O2/5% CO2 and heated to 37 °C. During an equilibration period of 80 min, the tissues were stimulated three times with histamine (2 × 1 μM, 1 × 10 μM) followed by washout. Up to four cumulative CRCs were determined on each tissue: the first by addition of histamine (0.01–30 μM) and the second to fourth by addition of histamine in the presence of increasing concentrations of antagonist (preincubation: 10–15 min). pEC50 differences were not corrected because four successive curves for histamine were superimposable under these conditions (N > 10). Data were analyzed by nonlinear regression and were best-fitted to sigmoidal concentration–response curves using Prism 5.0c software (GraphPad, San Diego, CA).

Histamine H2 Receptor Assay on the Isolated Guinea Pig Right Atrium (Spontaneously Beating)[2,16,53]

Hearts were rapidly removed from guinea pigs used for studies on the ileum (see above). The right atrium was quickly dissected and set up isometrically in the Krebs–Henseleit solution under a diastolic resting force of approximately 5 mN in a jacketed 20 mL organ bath at 32.5 °C. The bath fluid (composition [mM]: NaCl, 118.1; KCl, 4.7; CaCl2, 1.8; MgSO4, 1.64; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 5.0; sodium pyruvate, 2.0), supplemented with (RS)-propranolol (0.3 μM) to block β-adrenergic receptors, was equilibrated with 95% O2 and 5% CO2. Experiments were started after 30 min of continuous washing and an additional equilibration period of 15 min. Two successive histamine CRCs displayed a significant desensitization of 0.13 ± 0.02 (N = 16 control organs). This value was used to correct each individual experiment. For agonists, two successive concentration–frequency curves were generated: the first for histamine (0.1–30 μM) and the second for the agonist of interest either in the absence or in the presence of cimetidine (30 μM, 30 min preincubated). Additionally, the sensitivity to 30, 100, or 300 μM cimetidine was checked at the end of each H2R agonist CRC, and a significant reduction of frequency was observed. The relative potency of the agonist under study was calculated from the corrected pEC50 difference (ΔpEC50). pEC50 values are given relative to the long-term mean value for histamine (pEC50 = 6.16) determined in our laboratory (pEC50 = 6.16 + ΔpEC50). Data were analyzed by nonlinear regression and were best-fitted to sigmoidal concentration–response curves using Prism 5.0c software (GraphPad, San Diego, CA).

Computational Methods

Model Preparation

To examine possible binding modes of the bivalent ligands 5 and 58 at hH2R and of the monovalent ligand 129 at both hH3R and hH4R, homology models of these receptors were prepared as follows: For hH4R, the described hH4R model was used,[54] which was based on the inactive state crystal structure of the hH1R[55] (PDB ID: 3RZE), and the models comprising hH2R and hH3R were adapted from this model. Model preparation was essentially performed as described in Wifling et al.[54] using the modeling suite SYBYL-X 2.0 (Tripos Inc., St. Louis, MO).

Molecular Docking

The most interesting bivalent compounds 5 and 58 on the one hand and the monomeric ligand 129 on the other hand were geometry-optimized by means of Gaussian 09[56] at the HF/6-31(d,p) level, attributing the ligands a formal charge of +2 and +1, respectively. Upon file conversion by means of Open Babel[57] and assignment of physiological ionization states by means of ChemAxon (http://www.chemaxon.com) Marvin 16.3.28.0, 2016 Calculator Plugins, flexible docking was performed with the software package Autodock Vina.[58] The following hH2R amino acids were kept as flexible: K18, S75, Q79, Y94, T95, D98, V99, C102, R161, N168, H169, T170, T171, S172, K173, K175, V176, V178, N179, E180, G183, D186, G187, T190, W247, Y250, F251, F254, R257, R260, N266, E267, E270, L274, W275, G277, Y278. For hH3R, the amino acids T34, M41, Y91, Y94, V95, W110, L111, D114, Y115, C118, E185, H187, F192, F193, Y194, W196, L199, A202, S203, E206, W371, Y374, T375, M378, R381, H387, D391, D394, E395, D398, W399, L401, W402 were kept as flexible, and for hH4R the residues R15, M22, Y72, H75, T76, W90, L91, D94, Y95, C98, M150, K158, E160, S162, E163, F168, F169, S170, W172, L175, T178, S179, E182, W316, Y319, S320, T323, L326, S330, S331, T333, S337, Y340, R341, F344, W345, Q347, W348. The search box was set to a size of 28 Å × 32 Å × 32 Å, centered at the binding pocket. Up to 20 binding poses were exported for each ligand. Taking into account the results of the scoring function as well as experimental data, the respective best poses were selected for downstream molecular dynamics simulations.

Molecular Dynamics Simulations

To identify the respective most probable binding pose, 30 ns molecular dynamics simulations were performed in a water box at 310 K, and subsequently, free energy (MM-GBSA[59]) calculations were performed using the Amber 14[60] molecular dynamics package. To determine ligand force-field parameters, RESP[61] charges were determined by means of Gaussian 09[56] at the HF/6-31(d,p) level, and general amber force field[62] atom types as well as RESP[61] charges were assigned using antechamber.[60] parmchk2[60] and tleap[60] were used for input file generation. For ligand guanidine atoms and protein residues, the Amber ff99SB[63] force-field parameters were used. The simulation steps were carried out in an octahedral box comprising an 8.0 Å TIP3P[64] water layer and neutralizing chloride ions.[65] The mbondi2 parameter set was utilized.[66,67] The respective systems were minimized using the steepest descent (2500 steps) and conjugated gradient (7500 steps) methods without restraints, and were subsequently heated from 0 to 100 K over 50 ps in the NVT ensemble as well as from 100 to 310 K over 450 ps in the NPT ensemble. During the first ns of the 5 ns equilibration period, initial harmonic restraints of 5 kcal/mol Å2 were applied to all ligand and receptor atoms, and, beginning from 1 ns, harmonic restraints on receptor main chain atoms were reduced from 5.0 to 0.5 kcal/mol Å2 in a stepwise manner and maintained during the 30 ns simulation. Bonds involving hydrogen atoms were constrained using SHAKE[68] to enable a frame step size of 2 fs.[65,69] Nonbonded interactions were cut off at 8.0 Å, and long-range electrostatics were computed using the particle-mesh Ewald[70] method. The Langevin thermostat[71,72] with a collision frequency of 1.0 ps–1 and randomly assigned initial velocities was used to maintain a target temperature of 310 K. The Berendsen barostat[73] with isotropic position scaling and a pressure relaxation time of 1.0 ps was employed to keep the pressure constant at 1 bar. Data were collected every 10 ps. H-bond and cluster analysis were performed by means of CPPTRAJ[60] for the entire 30 ns trajectories. Moreover, the average linkage algorithm[74] was applied for cluster analysis, setting a cluster size of 5, and the programming language R[75−78] was used for the preparation of H-bond plots. Binding free-energy (MM-GBSA) calculations of the 30 ns trajectories were performed using MMPBSA.py[59] and a frame step size of 10 ps, and the corresponding plots were obtained using the Prism 5.01 software (GraphPad, San Diego, CA). All other figures were created with PyMOL Molecular Graphics system, version 1.8.2.1 (Schrödinger LLC, Portland, OR).