Abolishing Dopamine D2long/D3Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2Receptor Agonists

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Abolishing Dopamine D_2long/D₃Receptor Aﬃnity of Subtype-

Selective Carbamoylguanidine-Type Histamine H₂Receptor

Agonists

Katharina Tropmann, Merlin Bresinsky, Lisa Forster, Denise M ö nnich, Armin Buschauer,

Hans-Joachim Wittmann, Harald Hu

̈

bner, Peter Gmeiner, Ste ﬀ en Pockes,* and Andrea Strasser*

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ABSTRACT: 3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted

carbamoylguanidines are potent, subtype-selective histamine H₂

receptor (H₂R) agonists, but their applicability as pharmacological

tools to elucidate the largely unknown H₂R functions in the central

nervous system (CNS) is compromised by their concomitant high

aﬃnity toward dopamine D₂-like receptors (especially to the D₃R).

To improve the selectivity, a series of novel carbamoylguanidine-

type ligands containing various heterocycles, spacers, and side

residues were rationally designed, synthesized, and tested in

binding and/or functional assays at H₁₋₄and D_2long/3receptors.

This study revealed a couple of selective candidates (among others

31 and 47), and the most promising ones were screened at several

oﬀ-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42,

and 7.35) are responsible for the diﬀerent aﬃnities at the H₂- and D_2long/3-receptors. These results provide a solid base for the

exploration of the H₂R functions in the brain in further studies.

Starting from the H₂R agonists of the arpromidine (3, Figure

1A) series, several highly potent (up to 3000 times the potency

of histamine) monomeric and dimeric H₂R agonists with the

acylguanidine or carbamoylguanidine partial structure were

developed (4−6, Figure 1A).¹⁹⁻²⁴In contrast to acylguanidines,

the carbamoylguanidines are chemically stable and possess an

excellent selectivity over the other three histamine receptors

(H_1,3,4) if a 2-aminothiazole ring is used for the bioisosteric

replacement of the imidazole ring (5, 6, Figure 1A).^22,23,25Based

on the existing knowledge about the physicochemical and/or

pharmacokinetic properties of acyl- and carbamoylguanidines,

we assume that carbamoylguanidines are also able to overcome

the blood−brain barrier (BBB).^19,20,23,26This advantage over

previously reported H₂R agonists (cf. guanidines, e.g., 3, Figure

1A) should enable access to the H₂R in the CNS.^19,20On the

other hand, the direct injection of the ligand into the brain is also

possible as a means of application in the case of insuﬃcient

bioavailability and/or BBB penetration.²⁷

INTRODUCTION

■

The histamine H₂receptor (H₂R) has been the subject of many

research studies due to its versatile physiological properties.^1,2

The H₂R belongs to the class A G protein-coupled receptors

(GPCRs) and is expressed throughout the whole human body,

most importantly in the stomach, heart, and central nervous

system (CNS).¹⁻⁴Activation of the H₂R by its endogenous

ligand histamine (1, Figure 1A) leads to adenylyl cyclase

activation by coupling to the Gs protein.²The central role of the

H₂R in the stimulation of gastric acid secretion^2,5is the basis for

the therapeutic use of H₂R antagonists to treat the gastro-

esophageal reﬂux disease and gastroduodenal ulcers.^1,6The

function of the H₂R in the CNS is largely unknown but includes,

e.g., modulation of cognitive processes and circadian rhythm.⁷

Furthermore, positive eﬀects of the H₂-antagonist famotidine (2,

Figure 1A) in schizophrenia and an improvement in ^L-3,4-

dihydroxyphenylalanine (^L-DOPA)-induced dyskinesia are

reported in the literature.⁷⁻¹⁵In addition, there are studies

reporting that the stimulation of the postsynaptic H₂R has

positive eﬀects on learning and memory.¹⁶So far, these eﬀects

have only been shown with dual-acting acetylcholinesterase

inhibitors and H₃R antagonists as these molecules initiate this

process through the inhibition of presynaptic H₃-autorecep-

tors.¹⁶⁻¹⁸Therefore, the use of CNS-penetrating H₂R agonists is

of great interest.

Received: April 16, 2021

Published: June 10, 2021

American Chemical Society

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Figure 1. (A) Structures of histamine (1), famotidine (2), arpromidine (3), and related prototypical acylguanidine-type (4) and carbamoylguanidine-

type (5, 6) H₂R agonists, as well as the D₂-like receptor agonist pramipexole (7). (B) Structural modiﬁcations of N^G-carbamoylated guanidines

resulting in the title compounds. Het: heterocycle.

However, the 2-aminothiazole structural motif is also part of

the dopamine receptor agonist pramipexole (7, Figure 1A),

which is used as a drug for the treatment of Parkinson’s

disease.^28,29Due to these similarities, we assumed that H₂R

agonists containing the 2-aminothiazole motif might also bind to

dopamine receptors. Indeed, we could prove this assumption

during previous studies with radioligand binding experiments

and found that such H₂R agonists revealed a considerable

aﬃnity to dopamine receptors of the D₂-like family, in particular

to the D₃receptor.^23,24

To enable the use of carbamoylguanidines as pharmacological

tools to elucidate the H₂R functions in the CNS, we addressed

the need to develop improved molecules that bind exclusively

selective to the H₂receptor. Thus, we herein report the synthesis

and pharmacological characterization of these novel, subtype-

selective H₂R ligands by variation of the carbamoylguanidine-

based scaﬀold with diﬀerent heterocycles, spacers, and side

residues (Figure 1B).

The synthesized compounds were investigated for their

functional activity at the H₂R and/or D_2long/3R in minimal G

(mini-G) protein- and/or β-arrestin2-recruitment assays as well

as on the isolated spontaneously beating guinea pig (gp) right

atrium in a more complex but well-established standard model

for the characterization of H₂R ligands.^1,30The selectivity for the

human (h) H₂R over hH_1/3/4R and hD_2long/3R was evaluated in

radioligand competition binding experiments. To support our

investigations in silico, molecular docking studies were

performed. We compared the binding of the selected (4-

methyl)thiazolyl- or thiadiazolyl-containing carbamoylguani-

dines to identify the amino acid (aa) residues that might be

responsible for the diﬀerent binding aﬃnities of these ligands at

the H₂- and D_2long/3-receptors. Finally, an aﬃnity screening of

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Figure 2. Structures of amines 8−17 used for the synthesis of monomeric (30−36, 38−57, and 59−66) and dimeric (67−70) carbamoylguanidines.

Het: heterocycle. For more details regarding 8−17, see the SI.

a

Scheme 1. Synthesis of Monomeric Carbamoylguanidines 30−66

a

Reagents and conditions: (a) NEt₃, HgCl₂, CH₂Cl₂, room temperature (rt), 4−48 h and (b) TFA, CH₂Cl₂, rt, 7−18 h, 4−64% over two steps.

b

Isolated yields over two steps are given in brackets. For more details regarding 8−27, see the SI. Modiﬁed synthetic procedure (see the

Experimental Section and Scheme S8). BB: building block and Het: heterocycle. Amines 8−17: all compounds were used as free bases; and

guanidinylating reagents 18−27: all compounds were used as mono-Boc-protected (or di-Boc-protected if applicable) intermediates. The target

compounds 30−52, 55, and 57−66 were obtained as TFA and 53, 54, and 56 as HCl salts.

the best compounds at common oﬀ-target GPCRs was

performed and the gastrointestinal (GI) absorption and BBB

penetration of these ligands were estimated by the SwissADME

online tool.³¹

RESULTS AND DISCUSSION

■

Chemistry. The amines 8−17^{19,20,32−34}(Figure 2) and the

guanidinylating reagents 18−29^22−24,26(Schemes 1 and 2) were

synthesized as reported in the SI or in the literature. We chose

several diﬀerent side residues for the guanidinylating reagents

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a

Scheme 2. Synthesis of the Dimeric N^G-Carbamoylated Guanidines 67−70

a

Reagents and conditions: (a) 11, 15, or 16, NEt₃, HgCl₂, CH₂Cl₂, rt, 8 h and (b) TFA, CH₂Cl₂, rt, 6−16 h, 7−23% over two steps. Isolated yields

over two steps are given in brackets. For more details regarding 11, 15, 16, 28, and 29, see the SI. Amines 11, 15, and 17: all compounds were used

as free bases; and guanidinylating reagents 28 and 29: all compounds were used as di-Boc-protected intermediates. The target compounds 67−70

were obtained as TFA salts.

18−29, which performed well in our recent studies about 2-

aminothiazoles.^19−24,26The monomeric (Scheme 1) or dimeric

(Scheme 2) carbamoylguanidine-type ligands were prepared by

reacting the amines 8−17 with the guanidinylating reagents 18−

29 in the presence of HgCl₂and triethylamine (NEt₃).³⁵Finally,

the protected carbamoylguanidine-type intermediates were

treated with triﬂuoroacetic acid (TFA), giving compounds

30−36, 38−57, and 59−70 (Schemes 1 and 2), which were

puriﬁed by preparative high-performance liquid chromatog-

raphy (HPLC) (acetonitrile (MeCN)/0.1% TFA in H₂O) or

column chromatography (CH₂Cl₂/7 N NH₃in MeOH) and

subsequent recrystallization into the corresponding HCl salts.

37 and 58 were synthesized using a modiﬁed synthetic

procedure (for details, see the Experimental Section and

Scheme S8 in the SI).

Chemical Stability of Carbamoylguanidines. The

chemical stability of the selected compounds (30−35, 37, 41,

and 57) was investigated in binding buﬀer²²(pH 7.4) at room

temperature (rt) over a time period of 2 weeks. Under these

conditions, the investigated N^G-carbamoylated guanidines

proved to be stable (for graphs, see Figures S123 −S131 in the

SI, for details, see the SI).

Pharmacology. H₂R Aﬃnity and Receptor Subtype

Selectivity. The pK_ivalues of all target compounds were

determined in competition binding studies on membrane

preparations of Sf9 cells expressing the hH₂R-G_sαSfusion

protein using the radioligand [³H]UR-DE257³⁶(Table 1). At

ﬁrst, we investigated the inﬂuence of the linker on the binding

aﬃnity. The conformationally restricted compounds (e.g., 33

(para, pK_i= 6.34), 34 (meta, pK_i= 6.72), and 35 (bicyclic, pK_i=

6.81), Table 1) bind well to the hH₂R albeit with lower aﬃnities

compared to their ﬂexible (propyl linker) counterparts (e.g., 6²³

(pK_i= 8.32²³), Table 1).

Next, we investigated the inﬂuence of the heterocycle. The

replacement of the sulfur atom in the 2-amino-4-methylthiazole

by an oxygen atom resulted in a decreased hH₂R aﬃnity (e.g.,

oxazole: 45 (pK_i= 6.41, Table 1) vs thiazole UR-CH22^23,24(pK_i

= 7.16,^23,24see Figure S135 and Table S1 in the SI)). The

omission of the methyl group in position 4 of the heterocyclic

ring did not cause a signiﬁcant change in the hH₂R aﬃnity (32,

48, and 59 vs 6²³(Table 1), UR-CH22^23,24and UR-SB257,^23,24

see Figure S135 and Table S1 in the SI). However, the

replacement of the amino(methyl)thiazole by a 2-amino-1,3,4-

thiadiazole was favorable: the K_i-values of compounds 31 (pK_i=

8.52), 36 (pK_i= 8.29), 47 (pK_i= 8.30), 56 (pK_i= 8.09), and 57

(pK_i= 8.19) were in the single-digit nanomolar range (cf. Table

1). Also, in the case of the diazoles, the substitution of the sulfur

atom by an oxygen atom resulted in a decreased hH₂R aﬃnity

(e.g., oxadiazole: 58 (pK_i= 6.17) vs thiadiazole 57 (pK_i= 8.19),

Table 1). The replacement of the free amine group in the 2-

amino-1,3,4-thiadiazole by a methyl group (30, 38, 42, and 46)

resulted in a dramatic decrease of the hH₂R aﬃnity (cf. Table 1),

indicating that the heteroaromatic amine group is extremely

important for high aﬃnity. This observation was also supported

by docking experiments (see the Molecular Docking Studies

section). Finally, using the reported 1H-1,2,4-triazole³⁴or a

more explorative 4-(dimethylamino)methyl-1,2,3-triazole in-

stead of the 2-amino-4-methylthiazole resulted in decreased

hH₂R aﬃnities (1,2,4-triazole 54 (pK_i= 7.27, Table 1) and 1,2,3-

triazole 55 (pK_i= 5.35, Table 1) vs thiazole UR-Po563²³(pK_i=

7.75,²³see Figure S135 and Table S1 in the SI)).

It is literature known that dimeric ligands possess a

signiﬁcantly increased H₂R aﬃnity (human or guinea pig).^22,25

Therefore, we also synthesized several dimeric compounds, e.g.,

the 2-amino-1,3,4-thiadiazole heterocycle containing ligands 69

(hexyl-spacer, pK_i= 8.28, Table 1) and 70 (octyl-spacer, pK_i=

8.32, Table 1). However, no further increase in aﬃnity could be

achieved compared to the monomeric compounds 31 (pentyl,

pK_i= 8.52, Table 1) and 36 (hexyl, pK_i= 8.29, Table 1).

The pK_ivalues of all synthesized compounds were also

determined at the hH₁, hH₃, and hH₄receptors on membranes

of Sf9 cells expressing the respective histamine receptor using

the radioligands [³H]mepyramine (hH₁R), [³H]N^α-methylhist-

amine, or [³H]UR-PI294³⁷(hH₃R) and [³H]1 (hH₄R, cf. Table

1). The imidazole-containing ligand 52 was synthesized as a

control compound to showcase that the subtype selectivity is

largely inﬂuenced by the heterocycle. Unsurprisingly, despite a

high aﬃnity at the H₂R, it bound similarly well or even better to

the H₃R and H₄R. In contrast, neither of the 2-amino-1,3,4-

thiadiazoles (31, 36, 43, 47, 53, 56, 57, 63, 69, and 70) displayed

remarkable aﬃnity to the hH₁, hH₃, or hH₄receptors leading to

at least 100-fold selectivity for the hH₂R (cf. Table 1). The only

exception among the thiadiazoles was observed for compound

39, which contains the 8-aminooctyl side chain. Within the

synthesized series, compound 31 showed the highest aﬃnity

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Table 1. Binding Data of the Compounds 30−70 on Human Histamine Receptor Subtypes

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Table 1. continued

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Table 1. continued

a

Radioligand competition binding assay using membrane preparations of Sf9 cells expressing the hH₁R + RGS4, hH₂R-G_sαS, hH₃R + G_αi2+ G_β1γ2

,

or hH₄R + G_αi2+ G_β1γ2. All compounds were tested as TFA salts unless otherwise noted. Data represent mean values standard error of the mean

(SEM) of N independent experiments, each performed in triplicate. The displacement curves of compounds 31, 32, 36, 43, 47, 53, 54, 56, 57, 59,

b

c

63, and 69 are presented in Figures S133 and 134 in the SI. Displacement of 5 nM [³H]mepyramine (K_d= 4.5 nM). Displacement of 20 nM

[³H]UR-DE257³⁶(K_d= 12.2 nM). Displacement of 8.6 nM [³H]N^α-methylhistamine (K_d= 3 nM). Displacement of 2 nM [³H]UR-PI294³⁷(K_d

d

e

f

g

h

= 3 nM). Displacement of 15 nM [³H]1 (K_d= 16 nM). Tested as the HCl salt instead of the TFA salt. n.d.: not determined.

(pK_i= 8.52, Table 1) and subtype selectivity (ratio of K_iH₁R/

H₃R/H₄R of 2138:>3311:>3311, Table 1).

In summary, although many ligands (31, 36, 47, 53, 54, and

57) showed a decent selectivity for the hH₂R over the hD_2long

and hD₃receptors (ratios of K_i> 100), 31 and 47 turned out to

be the most promising candidates due to their excellent

selectivity proﬁles.

D_2longR and D₃R Aﬃnities of N^G-Carbamoylated Guani-

dines. N^G-Carbamoylated guanidines with a pK_ivalue >7.0 at

the hH₂R were investigated for their aﬃnities to the hD_2longand

hD₃receptors in radioligand binding assays on homogenates of

HEK293T-CRE-Luc cells coexpressing the respective receptor

(Table 2). Ligands with pK_ivalues <7.0 were not tested, as they

are not suitable as pharmacological tools to study the H₂R

function in the brain due to their low aﬃnity. Compounds

containing the 2-aminothiazole heterocycle without a methyl

group in position 4 (32, 48, and 59) still showed high to

moderate aﬃnities to the hD_2long/3receptors, especially to the

hD₃R (cf. Table 2). The determined hD_2long/3receptor aﬃnities

were comparable to aﬃnities published for 2-amino(4-methyl)-

thiazoles.²³Fortunately, compounds containing the 2-amino-

1,3,4-thiadiazole or the 1H-1,2,4-triazole heterocycle displayed

only low aﬃnity to the hD_2longand hD₃receptors. We observed

that some of them (31, 36, 47, 53, 54, and 57) showed even

more than 100-fold selectivity for the hH₂R over the hD_2longand

hD₃receptors (cf. Table 2). This trend indicates that the

nitrogen in position 4 might be responsible for the lower aﬃnity

to the hD₃R (for more details, see docking results; Figure 3D).

In addition to the eﬀect of the heterocycle, the side residue

played an important role in the dopamine hD_2long/3receptor

aﬃnities. For example, thiadiazoles 43 (2-cyclohexylpropyl side

residue) and 63 (2-methyl-5-phenylpentyl side residue) still had

a moderate aﬃnity for the hD_2long/3receptors, which might

indicate an additional (hydrophobic) interaction in the binding

pocket of the D_2long/3receptors (not further investigated).

Finally, the dimeric ligand 69 also possessed high hD_2longand

hD₃receptor aﬃnities compared to the corresponding

monomeric ligands 31 and 36 (cf. Table 2). Therefore, 70,

also being a dimeric ligand, was not further investigated. 39 and

52 were, despite their high H₂R aﬃnity, also excluded from

additional experiments due to their low subtype selectivity (cf.

Table 1).

Functional Studies at the Human H₂R. To gain further

insights into the general structure−activity relationship of the

N^G-carbamoylated guanidines, all target compounds 30−70

were investigated for hH₂R agonism and antagonism in the β-

arrestin2- and mini-G protein recruitment assays using

genetically engineered HEK293T cells, respectively. Only the

functional data of the most interesting molecules (31, 32, 36, 43,

47, 48, 53, 54, 56, 57, 59, 63, and 69) are shown in Table 3 (for

functional data of the remaining and reference compounds, see

Table S2 in the SI). The responses in both assays were

normalized to the maximum eﬀect induced by 100 μM

histamine (1, E_max= 1.00) and buﬀer control (E_max= 0).

Thus, 1 is deﬁned as a full, unbiased agonist in either readout. 1

exhibits a signiﬁcantly lower potency in the β-arrestin2

recruitment assay compared to the mini-G protein recruitment

assay (pEC₅₀(β-arrestin2) = 5.42;⁴¹pEC₅₀(mGs (minimal G_αs

protein)) = 6.94;⁴²cf. Table 3). Similarly, the potencies of the

investigated N^G-carbamoylated guanidines were also lower in

the β-arrestin2 recruitment assay (cf. Table 3). A possible

explanation for this trend could be the use of the mGs protein

since it is known that mG proteins stabilize active states of

GPCRs, which favors the binding of agonists.⁴²⁻⁴⁵

The compounds shown in Table 3 proved to be strong partial

agonists (E_max= 0.83−0.95, cf. Table 3) in the mini-G protein

recruitment assay with pEC₅₀values >7.0. The determined

pEC₅₀values in most cases agree very well with the pK_ivalues

from the radioligand binding assay. Compound 47, containing

the benzyl side residue, showed the highest hH₂R potency with a

pEC₅₀of 8.48, and also 31 (pentyl side residue, pEC₅₀= 8.22)

showed an excellent potency in the single-digit nanomolar range

(cf. Table 3). The incorporation of a ring system (cf. Scheme 1,

BB9−11) in the spacer resulted in either antagonists or partial

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a

Table 2. Binding Data of the Selected N^G-Carbamoylated Guanidines on Human Dopamine D_2longand D₃Receptors

a

Data represent mean values SEM from N independent experiments, each performed in triplicate. All compounds were tested as TFA salts unless

b

otherwise noted. The radioligand competition binding assay with [³H]N-methylspiperone (hD_2longR: K_d= 0.0149 nM, c = 0.05 nM) using

homogenates of HEK293T-CRE-Luc-hD_2longR cells.⁴⁰The radioligand competition binding assay with [³H]N-methylspiperone (hD₃R: K_d=

c

0.0258 nM, c = 0.05 nM) using homogenates of HEK293T-CRE-Luc-hD₃R cells.⁴⁰Tested as the HCl salt instead of the TFA salt. The

d

e

displacement curves are presented in Figures S136 and S137 in the SI. Calculated using the high pK_ivalue.

agonists depending on the side residue (for details, see Table S2

in the SI). Surprisingly, some of the tested compounds revealed

a completely diﬀerent functional proﬁle in the β-arrestin2

recruitment assay (cf. Tables 3 and S2). Almost all tested 2-

aminothiazoles 6,²³UR-CH20,^23,24UR-CH22,^23,24UR-

Po563,²³UR-MB-69,²³UR-SB257,^23,24UR-KAT527,²³32,

48, and 59 and the thiadiazoles 57 and 63, containing a propyl

spacer, as well as triazole 54, exhibited a certain degree of eﬃcacy

bias toward G protein activation. The compounds acted as

strong partial agonists (E_max= 0.73−0.94, cf. Tables 3 and S2) in

the mini-G protein recruitment assay but were only partial

agonists in the β-arrestin2 recruitment assay (E_max= 0.10−0.73,

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Figure 3. (A) Active-state model of the hH₂R with 31 in the binding pocket. The most important amino acids interacting with 31 are highlighted. (B)

Diﬀerences between the interaction sites of the aminothiadiazole moiety of 31 and the hH₂R or the hD₂R, respectively. (C) The most important amino

acids of hH₂R (left), hD₂R-E^E2.49V-I^E2.51S (center), and hD₂R (right) interacting with the carbamoylguanidine moiety of 31. (D) The inﬂuence of the

isoleucine at position E2.52 of the E2 loop onto the interaction with the heterocycle of 31 (left), 32 (center), and 6 (right). The active-state β₂-

adrenergic receptor−Gs protein complex crystal structure (PDB ID: 3SN6⁵⁰) was used as a starting point for the homology model of the hH₂R. In the

case of the hD₂R, the PDB code is 6VMS.⁵¹

cf. Tables 3 and S2). The eﬃcacy bias was conﬁrmed by the

determination of eﬃcacy bias factors (eBF, for details, see

Section S10 in the SI). By contrast, since most of the thiadiazoles

(including the most promising candidates 31 and 47) exhibited

no signiﬁcant bias, they behave in a way similar to the

endogenous ligand histamine (1). This advantage should enable

an authentic examination of the function of the H₂R in the CNS

with these ligands. Finally, the dimeric ligands (e.g., thiazole:

5,²²thiadiazole: 69) exhibited similar characteristics as their

monomeric counterparts and all compounds containing a

rigidized spacer (cf. Scheme 1, BB9−11) acted as antagonists

in the β-arrestin2 recruitment assay (see Table S2 in the SI).

Functional Studies at the Guinea Pig H₂R. Furthermore, a

selection of compounds (with a pK_i> 7.0 at the hH₂R and a

selectivity over the hD_2long/3receptors) was investigated on the

isolated spontaneously beating guinea pig right atrium as a more

complex, well-established standard model for the character-

ization of H₂R ligands (Table 4).^1,30All compounds turned out

to be full agonists in this assay (E_max= 0.98−1.15, cf. Table 4).

The obtained data are generally comparable with the results

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Table 3. Potencies and Eﬃcacies of the Selected N^G-Carbamoylated Guanidines in the β-Arrestin2 and Mini-G Protein

a

Recruitment Assays at the hH₂R

a

Data represent mean values SEM from N independent experiments, each performed in triplicate. All compounds were tested as TFA salts unless

otherwise noted. The β-arrestin2 recruitment assay was performed using HEK293T-ARRB2-H₂R cells.^41,46The mini-G protein recruitment assay

b

c

was performed using HEK293T NlucN-mGs/hH₂R-NlucC cells.⁴²The response in both assays was normalized to the maximal eﬀect induced by

d

100 μM 1 (E_max= 1.00) and buﬀer control (E_max= 0.00). The concentration−response curves are presented in Figures S138 and S140 in the SI.

e

f

Tested as the HCl salt instead of the TFA salt. Selected compounds were investigated for functional activity in the [³⁵S]GTPγS binding assay at

the hH₂R-G_sαSfusion protein:²²31: pEC₅₀= 7.59 0.11, E_max= 0.84 0.04 (N = 3); 43: pEC₅₀= 7.88 0.09, E_max= 0.78 0.07 (N = 3); 57:

pEC₅₀= 7.89 0.11, E_max= 0.88 0.06 (N = 4); and 69: pEC₅₀= 7.46 0.09, E_max= 0.71 0.06 (N = 3). The obtained results were in good

agreement with the results from the mini-G protein recruitment assay.

from the gpH₂R mini-G protein recruitment assay in terms of

potency and eﬃcacy (Table 4). Noteworthily, 53, 56, and 57

showed the highest discrepancies regarding the potency in both

assays. However, 53 and 56 showed higher potencies by about

one logarithmic unit on the guinea pig right atrium, and 57

behaved exactly the opposite (Table 4). The thiadiazole 56

(pEC₅₀= 9.04) showed the highest potency on the guinea pig

right atrium, whereas 47 was the most potent compound in the

mini-G protein recruitment assay (pEC₅₀= 8.66). In general, a

comparison of the mini-G protein recruitment assay data at the

guinea pig and human H₂Rs showed that the potencies at the

gpH₂R were slightly better for all substances tested, while the

eﬃcacies were pretty much the same. A similar observation was

already published for the [³⁵S]GTPγS assay and the steady-state

GTPase assay.^20,25,47,48

Functional Studies at the Human D_2long/3Receptors.

Although the relevant N^G-carbamoylated guanidines (31, 32,

36, 47, 53, 54, 56, and 57) bind to the hD_2long/3receptors only

with low aﬃnity (pK_i< 6.5 (only 32 has a pK_i> 6.5 at the D₃R),

see Table 2), we decided to characterize these ligands in the β-

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Table 4. Potencies and Eﬃcacies of the Tested N^G-Carbamoylated Guanidines Determined in the Mini-G Protein Recruitment

a

Assay at the gpH₂R or by Organ Bath Studies at the Spontaneously Beating Guinea Pig Right Atrium

a

Data represent mean values SEM from N independent experiments, each performed in triplicate. All compounds were tested as TFA salts unless

otherwise noted. The mini-G protein recruitment assay was performed using HEK293T NlucN-mGs/gpH₂R-NlucC cells.²⁶The response was

b

c

normalized to the maximal eﬀect induced by 100 μM 1 (E_max= 1.00) and buﬀer control (E_max= 0.00). The concentration−response curves are

d

e

presented in Figure S139 in the SI. Organ bath studies using the isolated, spontaneously beating guinea pig right atrium.³⁸The pEC₅₀value was

calculated from the mean corrected shift ΔEC₅₀of the agonist curve relative to the histamine reference curve by the following equation: pEC₅₀

=

f

g

6.16 + ΔpEC₅₀. E_max: maximal response relative to the maximal increase in the heart rate induced by 30 μM 1 (E_max= 1.00). Tested as the HCl

h

salt instead of the TFA salt. n.d.: not determined.

arrestin2 assay, which is already established in our lab.⁴⁰In

addition, the data of 5,²²43, 48, 59, 63, and 69 were collected for

a broader comparison of the compounds. The measured

potencies and eﬃcacies are presented in Table 5. All tested

compounds showed agonistic activities in the β-arrestin2

recruitment assay at the hD₃R. In the β-arrestin2 recruitment

assay at the hD_2longR, 47 and 54 were inactive (up to a tested

concentration of 10 μM, cf. Table 5). The remaining

compounds (5,²²31, 32, 36, 43, 48, 53, 56, 59, and 69) acted

as agonists with the exception of 57 and 63, which were

antagonists. Some compounds (31, 36, 43, 53, and 56 at the

hD_2longR and 54 and 57 at the hD₃R) showed only very weak

partial agonism at the highest tested concentration of 10 μM,

which could not be ﬁtted. In general, thiadiazoles showed lower

potencies and eﬃcacies at the hD_2long/3receptors than their

thiazole counterparts (cf. Table 5).

Molecular Docking Studies. To shed light on the binding

modes of the amino(methyl)thiazole- and the aminothiadiazole-

containing carbamoylguanidines and to gain insight into the

speciﬁc molecular interactions leading to the diﬀerences in the

hH₂R, hD₂R, and hD₃R aﬃnities, we performed molecular

docking studies (Figure 3). We chose to investigate compounds

6 (2-amino-4-methylthiazole), 31 (2-aminothiadiazole) and, 32

(2-aminothiazole) on the active-state receptor models of the

hH₂R (homology model based on the β₂-adrenergic receptor−

Gs protein complex crystal structure 3SN6;⁵⁰sequence identity

of about 37%¹⁹) and the hD₂R (based on the D₂R-G protein

complex crystal structure 6VMS⁵¹). Since 6, 31, and 32 act as

agonists at the hD₃-receptor (β-arrestin2 assay, Table 5), they

should be docked into its active-state receptor model. However,

to the best of our knowledge, an active-state model of the D₃R

has not been reported yet. To investigate the binding mode at

the hD₃R despite this drawback, we decided to create mutants of

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Table 5. Potencies and Eﬃcacies of Selected N^G-Carbamoylated Guanidines Determined in the β-Arrestin2 Recruitment Assay at

a

the hD_2longR or hD₃R

a

Data represent mean values SEM from N independent experiments, each performed in triplicate. All compounds were tested as TFA salts unless

b

c

otherwise noted. The β-Arrestin2 recruitment assay was performed using HEK293T ElucN-βarr2 hD_2longR-ElucC cells. The β-Arrestin2

d

recruitment assay was performed using HEK293T ElucN-βarr2 hD₃R-ElucC cells. pK_b= −log K_b. K_bvalues were calculated according to the

Cheng−Prusoﬀ equation.⁴⁹The IC₅₀values of antagonists were determined in the antagonist mode vs quinpirole (50 nM, D_2longR). The response

e

f

in both assays was normalized to the maximal eﬀect induced by 10 μM quinpirole (E_max= 1.00) and buﬀer control (E_max= 0.00). Tested as the

HCl salt instead of the TFA salt.⁴⁰

E_maxat c = 10 μM. Silent antagonist. n.a. = not active. The concentration−response curves are presented in

g

h

Figures S141 −S143 in the SI.

the active-state hD₂R model, containing amino acid(s) (aa(s))

of the hD₃R.

First of all, we studied and analyzed literature data regarding

mutagenesis studies at aminergic GPCRs (primarily histamine

and dopamine receptors), focusing on the diﬀerent amino acids

of the orthosteric binding pocket at the hH₂R, hD₂R, and hD₃R.

The results are summarized in Table 6.

bind in a conformation, with the sulfur being located near C^3.36

and T^3.37. Additionally, 31 and 32 are stabilized by an

electrostatic interaction between the aspartate of the D^5.42

-

T^5.46motif and the NH₂-group of the heterocycle (Figure 3A,

shown for 31). The carbamoylguanidine moiety of both 31 and

32 forms an electrostatic interaction network with the amino

acids Y^3.28, D^3.32, and E^7.35(Figure 3A, shown for 31). 31 and 32

could also be docked into the analogue binding pocket of the

hD₂R. In contrast to the hH₂R, there is a serine instead of an

aspartate at position 5.42, which results in a reduced electrostatic

interaction of the hD₂R and the NH₂moiety of the heterocycle

(BB4 or BB6, respectively) (Figure 3B, shown for 31). As

The docking studies of 31 and 32 suggest that the heterocyclic

ﬁve-membered ring (BB4 or BB6, respectively), particularly if

surrounded by the amino acids C^3.36, T^3.37, D^5.42, T^5.46, and F^6.55

,

ﬁts well into the orthosteric binding pocket of the hH₂R (Figure

3A, shown for 31). However, both heterocycles are suggested to

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Table 6. Overview of the Literature Known Mutagenesis Studies at Aminergic GPCRs (Primarily Histamine and Dopamine

a

Receptors), Focusing on Diﬀerent Amino Acids of the Orthosteric Binding Pocket

position

2.61

receptor

mutation

most important new ﬁnding

^•approx. 50-fold decrease in K_iof clozapine, leading to a comparable aﬃnity 52

references

hD₂R

hH₁R

V^2.61

F

like at the wt D₄R

^•aa in position 2.61 is part of microdomain (including the amino acids at 3.28,

3.29, 7.35), which is partially accountable for the selectivity between the

hD₂R and the hD₄R

^•no or only a small inﬂuence on the binding aﬃnities of small molecules (e.g., 53 and 54

N^2.61S (hH₁R → gpH₁R)

mepyramine, cetirizine, histamine)

^•pK_iof more voluminous partial agonists (e.g., suprahistaprodifen and dimeric

histaprodifen) increased, resulting in an aﬃnity like at the gpH₁R

^•N^2.61is not the only responsible aa for the observed species diﬀerence between

the hH₁R and the gpH₁R

E2.49, E2.51

and E2.52 of

E2 loop

hD₂R/hD₃R

exemplarily: hD₃R-I183^E2.52F, ^•amino acids in these positions have inﬂuence on the binding aﬃnity

55−58

hD₂R-I184^E2.52A, hD₂R-

E181^E2.49

^5.42A

V

5.42

several GPCRs, e.g.,

hD₁R, mD₂R, hD₂R,

hD₃R

several GPCRs, e.g.,

hD₁R, gpH₁R, hH₄R

S

^•highly important for ligand binding (≥10-fold alteration of aﬃnity of diﬀerent 59−62

ligands)

5.46

hD₁R-S^5.46A, gpH₁R-N^5.46A,

^•≥10-fold alteration of aﬃnity for selected ligands

59 and

63−69

hH₄R-E^5.46D, hH₄R-E^5.46

Q

^•in the case of the hD₁R, aa in position 5.46 is suggested to inﬂuence the

subtype selectivity

rα₁AR

single mutations: S^5.42A and

^•no signiﬁcant reduction of the binding aﬃnity for e.g., (−)-epinephrine

^•approx. 100-fold reduced aﬃnity compared to the wt receptor

^•may aﬀect the aﬃnity up to 1000-fold

70

S^5.46

A

double mutation S^5.42A/

S^5.46

A

6.51

6.53

hD₂R

hH₁R

F^6.51A, F^6.51L, F^6.51

I^6.53

Y

71

V

^•K_dof [³H]mepyramine increased by approx. 10-fold; however, in another

53 and 72

study slightly decreased

6.55

gpH₁R, rD₂R, hD₃R

gpH₁R-F^6.55A, rD₂R-H^6.55L,

hD₃R-H^6.55L, rD₂R-H^6.55

^•up to 25-fold alteration of the binding aﬃnity

73−76

N

^•may aﬀect the binding aﬃnity not only by a direct interaction with the ligand

but also by changing the interaction network within the receptor

^•described to aﬀect the ligand aﬃnity

7.35

hM₁R

W^7.35A, W^7.35

F

77

a

m: mouse; r: rat; hM₁R: human muscarinic receptor M₁; and α₁AR: α₁adrenergic receptor.

roughly estimated by calculating the docking energy between

the hH₂R or the hH₂R-D^5.42S mutant and 31 or 32, respectively,

the interaction energy is considerably reduced for the hH₂R-

D^5.42S mutant. Thus, this missing interaction is probably one

reason for the reduced aﬃnity of 31 or 32 at the hD₂R and the

hD₃R, compared to the hH₂R. For the hD₂R, no compensating

interaction between the heterocycle (BB4 or BB6, respectively)

and the receptor could be identiﬁed.

probably another reason for the reduced aﬃnity of 31 or 32 at

the hD₂R or hD₃R compared to the hH₂R. An advanced

comparison of the amino acid sequence between the hD₂R and

the hD₃R revealed another two diﬀerences in the E2 loop in the

neighborhood to the highly conserved cysteine (hD₂R:

E^E2.49C^{E2.50 E2.51}; hD₃R: V^E2.49C^E2.50S^E2.51) (for amino acid

I

alignment, see Figure S146). In the case of the hD₂R, the

E^E2.49is too far from the carbamoylguanidine moiety of the

ligand and is not able to establish an electrostatic interaction

(Figure 3C, right). In contrast, at the hD₃R, the S^E2.51is able to

form a hydrogen bond with the carbamoylguanidine (cf. hD₂R-

E^2.49V-I^E2.51S, Figure 3C, center). Thus, the reduced interaction

between the receptor and the carbamoylguanidine moiety in the

series hH₂R → hD₃R → hD₂R will explain the reduced aﬃnity,

obtained by competition binding studies, within the same

sequence. However, this eﬀect could only be observed for the

double mutant hD₂R-E^E2.49V-I^E2.51S, not for the single mutant

hD₂R-I^E2.51S. As suggested by the modeling studies, in the case of

the single mutant, the S^E2.51interacts with E^E2.49and not with the

carbamoylguanidine moiety of the ligand. Thus, the double

mutation is suggested to be essential for the reduced aﬃnity of

the compounds at the hD₂R compared to the hD₃R.

Furthermore, the experimental studies show a decrease in

aﬃnity to the hD₃R in the series 6 → 32 → 31. Here, the docking

studies suggest that an isoleucine of the E2 loop (I^E2.52) is

responsible for that trend (Figure 3D). This isoleucine is in close

contact with the methyl group of the heterocycle of compound

6, establishing an additional van der Waals interaction between 6

Next, we performed further investigations to elucidate why

the compounds 31 and 32 have a higher aﬃnity to the hD₃R

than to the hD₂R. Concerning the 5.42−5.46 motif, the situation

at the hD₃R is identical compared to the hD₂R. Although a

comparison of the amino acid sequence between the hD₂R and

the hD₃R revealed two diﬀerences at the positions 4.53 and 4.56

(hD₂R: S^4.53, I^4.56; hD₃R: A^4.53, V^4.56), which are in close

proximity to the 5.42−5.46 motif, subsequent docking studies at

the hD₂R-S^4.53A-I^4.56V mutant suggested that these amino acids

are not responsible for the subtype selectivity between the hD₂R

and the hD₃R. Therefore, we performed an analysis of the

interaction between the carbamoylguanidine moiety and the

hD₂R or hD₃R. We observed that at position 7.35, the glutamate

(hH₂R) is exchanged into a tyrosine, which is not able to

establish an as strong electrostatic interaction as the glutamate.

Furthermore, the Y^3.28, which also interacts at the hH₂R with the

carbamoylguanidine by an electrostatic interaction, is a

phenylalanine at the hD₂R and hD₃R, resulting in a deﬁcit in

the electrostatic interaction. These two reduced interactions

between the hD₂R and the carbamoylguanidine constitute

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Table 7. Receptor Selectivity Studies of 6, 31, 47, and 54

pK_i

receptor

6

N

31

N

47

N

54

N

a

dopamine D₁

6.59 0.05

6.96 0.01

5.40 0.04

<5

3

5.61 0.05

5.65 0.04

4.46 0.05

<5

3

5.86 0.12

6.17 0.10

4.95 0.07

<5

5.88 0.03

<5

3

4

5.66 0.05

5.18 0.03

4.62 0.05

<5

3

b

dopamine D_4.4

c

dopamine D₅

d

e

muscarinic acetylcholine M₁

muscarinic acetylcholine M₂

muscarinic acetylcholine M₃

muscarinic acetylcholine M₄

muscarinic acetylcholine M₅

f

g

h

<5

<5.5

<5

i

α_1Aadrenergic

5.69 0.08

6.05 0.05

4.66 0.04

4.93 0.04

5.99 0.01

5.18 0.05

5.45 0.11

5.31 0.02

<4

4.20 0.06

5.47 0.03

4.49 0.07

6.07 0.02

5.90 0.12

<4

4.40 0.02

5.40 0.02

4.56 0.05

5.72 0.07

6.07 0.11

4.31 0.05

4.45 0.03

4.76 0.03

4.29 0.08

j

α_2Aadrenergic

k

β₁adrenergic

l

β₂adrenergic

m

μ-opioid

n

serotonin 5-HT_1A

a

Determined by competition binding with [³H]SCH23390 (K_d/applied conc: D₁, 0.23/0.4 nM) using homogenates from HEK293T-CRE-Luc-

hD₁R cells.⁴⁰Determined by competition binding with [³H]N-methylspiperone (K_d/applied conc: D_4.4, 0.078/0.1 nM) using homogenates from

b

HEK293T ElucN-βarr2 hD_4.4R-ELuc cells.⁴⁰Determined by competition binding with [³H]SCH23390 (K_d/applied conc: D₅, 0.24/0.4 nM) using

c

homogenates from HEK293T-CRE-Luc-hD₅R cells.⁴⁰Determined by competition binding with [³H]N-methylscopolamine (K_d/applied conc: M₁,

d

0.17/0.2 nM) using the whole CHO-hM₁R cells.⁷⁸Determined by competition binding with [³H]N-methylscopolamine (K_d/applied conc: M₂,

e

0.10/0.2 nM) using the whole CHO-hM₂R cells.⁷⁸Determined by competition binding with [³H]N-methylscopolamine (K_d/applied conc: M₃,

f

0.12/0.2 nM) using the whole CHO-hM₃R cells.⁷⁸Determined by competition binding with [³H]N-methylscopolamine (K_d/applied conc: M₄,

g

0.052/0.1 nM) using the whole CHO-hM₄R cells.⁷⁸Determined by competition binding with [³H]N-methylscopolamine (K_d/applied conc: M₅,

h

0.20/0.3 nM) using the whole CHO-hM₅R cells.⁷⁸Determined by competition binding with [³H]prazosin (K_d/applied conc: α_1A, 0.25/0.4 nM)

i

using membranes from HEK293T cells transiently transfected with cDNA of human adrenoceptors α_1A.^79,80Determined by competition binding

j

with [³H]RX821002 (α_2A, 0.6/0.5 nM) using membranes from HEK293T cells transiently transfected with cDNA of human adrenoceptors

α_2A.^79,80Determined by competition binding with [³H]CGP12177 (β₁, 0.075/0.2 nM) using membranes from HEK293T cells transiently

k

transfected with cDNA of human adrenoceptors β₁.^79,80Determined by competition binding with [³H]CGP12177 (β₂, 0.070/0.2 nM) using

l

membranes from HEK293T cells transiently transfected with cDNA of human adrenoceptors β₂.^79,80Determined by competition binding with

m

[³H]diprenorphine (μOR, 0.090/0.3 nM) using membranes from HEK293T cells transiently transfected with cDNA of human μ opioid receptor

(μOR).^79,80Determined by competition binding with [³H]WAY600135 (5-HT_1A, 0.080/0.2 nM) using membranes from HEK293T cells

n

transiently transfected with cDNA of human serotonin 1A receptor (5-HT_1A).^79,80

Figure 4. Selectivity over representative aminergic GPCRs, including histaminergic (gray), dopaminergic (blue), muscarinic (green), adrenergic (red),

opioid (magenta), and serotonergic (orange) receptors, were determined by radioligand competition binding experiments and compared to the aﬃnity

at the H₂R of (A) 6, (B) 31, (C) 47, and (D) 54. The dashed line indicates an at least 100-fold selectivity for the H₂R of the respective compound. Bars

represent the mean SEM of three to four individual experiments each performed in triplicate.

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and the receptor (Figure 3D, right). For 32, this contact and,

therefore, the van der Waals interaction are reduced due to the

replacement of the methyl group with a proton at the

heterocycle (Figure 3D, center). For 31, this interaction is

completely missing due to the presence of an additional nitrogen

atom in the ring (Figure 3D, left). In summary, the docking

studies at the active-state models of the hH₂R and the hD₂R

suggest that the amino acids at the positions 3.28, 3.32, E2.49,

E2.51, 5.42, and 7.35 are responsible for diﬀerent aﬃnities of 6,

31, and 32 at the hH₂R, hD₂R, and hD₃R. However, this

participation has to be veriﬁed in detail by the corresponding

mutagenesis experiments in future studies.

hD_2longR (>1000-fold) and the highest selectivity for the hH₂R

over the hD₃R (>520-fold). Moreover, 31 and 47 were shown to

be selective H₂R agonists (>100-fold) relative to 14 additional

peripheral and central GPCRs (including dopaminergic,

muscarinic, adrenergic, serotonergic, and opioid receptors).

These key characteristics render 31 and 47 the most aﬃne and

selective monomeric carbamoylguanidine-type agonists known

so far. Therefore, we plan to employ them as pharmacological

tools for further investigations on the physiological and

pathophysiological role of the H₂R and hope that these studies

can contribute to clarify the largely unknown function of H₂

receptors in the CNS.

Oﬀ-Target Studies. To be able to diﬀerentiate between the

on-target and oﬀ-target eﬀects, the ligands should selectively

bind to the H₂R. Therefore, the selected compounds 6, 31, 47,

and 54 were additionally investigated in radioligand competition

binding studies for their ability to bind to other peripheral and

central aminergic GPCRs (oﬀ-target studies). Some of these

receptors are frequently found antitarget receptors (e.g., α_1A

adrenergic, M₁₋₅muscarinic). The results are summarized in

Table 7 and indicate that the most promising thiadiazoles 31 and

47 have a more than 100-fold higher aﬃnity at the H₂R

compared to the other tested GPCRs (cf. selectivity proﬁles B

and C in Figure 4). The thiazole 6, tested as a control, also

showed a moderate aﬃnity to D₁and D_4.4receptors (D₁R: pK_i=

6.59; D_4.4R: pK_i= 6.96, cf. Table 7) in addition to the already

published high aﬃnity for the D₃R (pK_i= 7.80, cf. Table 2).

Although triazole 54 showed low aﬃnities (pK_i≤ 6.07) at the 14

additionally tested aminergic GPCRs, it did not achieve a

suﬃcient selectivity (factor >100, cf. selectivity proﬁle D in

Figure 4) due to the double-digit nanomolar aﬃnity for the H₂R

(pK_i= 7.27, cf. Table 1). Since the selectivity of a ligand in

tissue/in in vivo experiments is not only dependent on its aﬃnity

but also on the relative abundance of the respective receptor, we

also estimated the “total selectivity” of 6, 31, 47, and 54 in seven

diﬀerent brain regions (for details, see the SI).

EXPERIMENTAL SECTION

■

Chemistry: General Conditions. Unless otherwise stated,

chemicals and solvents were from commercial suppliers and were

used as received. All of the solvents were of analytical grade or were

distilled prior to use. For column chromatography, silica gel 60 (0.04−

0.063 mm, Merck, Darmstadt, Germany) was used. Flash chromatog-

raphy was performed on an Intelli Flash-310 workstation from Varian

Deutschland GmbH (Darmstadt, Germany) with SuperFlash (SF)

columns (Si50, 4−40 g) from Agilent Technologies (Santa Clara, CA).

Reactions were monitored by thin-layer chromatography (TLC) on

Merck silica gel 60 F254 aluminum sheets, and spots were visualized

with UV light at 254 nm or ninhydrin staining. NMR spectra were

recorded on a Bruker Avance 300 (¹H: 300 MHz, ¹³C: 76 MHz), a

Bruker Avance 400 (¹H: 400 MHz, ¹³C: 101 MHz), and a Bruker

Avance 600 (¹H: 600 MHz, ¹³C: 151 MHz) (Bruker, Karlsruhe,

Germany) NMR spectrometer with deuterated solvents from Deutero

(Kastellaun, Germany). All chemical shifts are reported in the δ-scale as

parts per million (ppm, multiplicity, coupling constant (J), number of

protons) relative to the solvent residual peaks as the internal

standard.^81,82The spectra were analyzed by the ﬁrst order, and

coupling constants are given in hertz (Hz). Abbreviations for the

multiplicities of the signals are singlet (s), doublet (d), triplet (t),

quartet (q), quintet (quint), multiplet (m), doublet of doublets (dd),

and broad singlet (br s). High-resolution mass spectrometry (HRMS)

was performed on a Q-TOF 6540 ultrahigh deﬁnition (UHD) LC/MS

system (Agilent Technologies) using an electrospray ionization (ESI)

source or on an AccuTOF GCX GC/MS system (Jeol, Peabody, MA)

using an electron ionization (EI) source. Preparative HPLC was

performed with a system from Knauer (Berlin, Germany) consisting of

two K-1800 pumps, a K-2001 detector, and the column was a

Phenomenex Kinetex (250 × 21 mm², 5 μm) (Phenomenex,

Aschaﬀenburg, Germany). As a mobile phase, mixtures of MeCN and

0.1% aqueous (aq) TFA were used. The UV detection was carried out at

220 nm. Prior to lyophilization (a ScanVac CoolSafe 4-15L freeze dryer

from Labogene (LMS, Brigachtal, Germany), equipped with a RZ 6

rotary vane vacuum pump (Vacuubrand, Wertheim, Germany)),

MeCN was removed under reduced pressure. Analytical HPLC

experiments were performed on a 1100 HPLC system from Agilent

Technologies equipped with an Instant Pilot controller, a G1312A

binary pump, a G1329A ALS autosampler, a G1379A vacuum degasser,

a G1316A column compartment, and a G1315B diode array detector

(DAD). The column was a Phenomenex Kinetex XB-C18 column (250

× 4.6 mm², 5 μm) (Phenomenex, Aschaﬀenburg, Germany), tempered

at 30 °C. As a mobile phase, mixtures of MeCN/aqueous TFA were

used. The following linear gradients were applied. Compounds 30−51

and 57−70: MeCN/TFA (0.05%) (v/v) 0 min: 10:90, 30 min: 90:10,

33 min: 95:5, 40 min: 95:5; ﬂow rate: 0.8 mL/min, t₀= 3.21 min.

Compounds 52−56: MeCN/TFA (0.05%) (v/v) 0 min: 10:90, 25 min:

95:5, 35 min: 95:5; ﬂow rate: 1.0 mL/min, t₀= 2.67 min. The injection

volume was 5−50 μL. Absorbance was detected at 220 nm. Compound

concentration was between 100 and 1000 μM.

SUMMARY AND CONCLUSIONS

■

In summary, we aimed for the development of novel, subtype-

selective H₂R ligands, which also have a selectivity over

dopamine D_2long/3receptors. To achieve this goal, we

synthesized and characterized a series of 40 compounds

containing a carbamoylguanidine as a key motif, as well as

varying heterocycles, spacers, and side residues. We observed

that the replacement of the thiazole by a thiadiazole ring in N^G-

carbamoylated thiazolylpropylguanidines resulted in potent

H₂R agonists with aﬃnities in the low one-digit nanomolar

range. Furthermore, ligands containing this modiﬁcation possess

a signiﬁcantly increased selectivity for the hH₂R over dopamine

hD_2long/3receptors. To identify the molecular interactions

leading to this selectivity toward the hD_2long/3receptors,

molecular docking studies with 6,²³31 (UR-KAT505), and 32

(UR-KAT583) on the active-state models of the hH₂R and the

hD₂R were performed. We found that 3.28, 3.32, E2.49, E2.51,

5.42, and 7.35 are most likely the responsible amino acids, which

will be conﬁrmed in future receptor mutagenesis experiments.

Within the synthesized thiadiazole-containing ligand series,

compounds 31 and 47 (UR-KAT533) turned out to be the most

promising candidates reaching up to 1000-fold selectivity over

the other three receptor subtypes (hH_1,3,4R). 31 showed the

highest selectivity for the hH₂R over the hD_2longR (>2000-fold)

and 260-fold selectivity for the hH₂R over the hD₃R. 47, on the

other hand, showed very good selectivity for the hH₂R over the

Compound Characterization. Target compounds (30−70) were

1

characterized by H NMR (for spectra, see the SI), ¹³C NMR (for

spectra, see the SI), and two-dimensional (2D) NMR (correlation

spectroscopy (COSY), heteronuclear single quantum correlation

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(HSQC), heteronuclear multiple bond correlation (HMBC)) spec-

troscopy, HRMS, and reversed-phase HPLC (RP-HPLC) analysis. The

purities (for chromatograms, see the SI) of the H₂R ligands used for

pharmacological investigation were ≥95%. For biological testing, the

target compounds 30−70 (TFA or HCl salts) were dissolved in

dimethyl sulfoxide (DMSO), DMSO/H₂O 1:1 (v/v), or DMSO/20

mM aq HCl 1:1 (v/v) to obtain a ﬁnal concentration of 10 mM.

Screening for Pan Assay Interference Compounds (PAINS).

Screening of all target compounds (30−70) for PAINS via the public

tool http://zinc15.docking.org/patterns/home⁸³gave no hits.

s, 1H), 8.52 (br s, 2H), 8.10−7.34 (m, 4H), 3.30 (q, J = 6.7 Hz, 2H),

3.09 (q, J = 6.6 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 1.89 (quint, J = 7.4 Hz,

2H), 1.44 (quint, J = 7.1 Hz, 2H), 1.31−1.22 (m, 4H), 0.86 (t, J = 7.0

Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 168.77, 158.93 (q, J =

34.3 Hz, TFA), 157.42, 153.86, 153.68, 116.45 (q, J = 297.7 Hz, TFA),

40.05, 39.21, 28.60, 28.39, 27.34, 26.52, 21.75, 13.86. HRMS (ESI-

MS): m/z [M + H⁺] calcd for C₁₂H₂₄N₇OS⁺: 314.1758; found:

314.1761. MF: C₁₂H₂₃N₇OS·C₄H₂F₆O₄. MW: (313.42 + 228.05).

1-(Amino{[3-(2-aminothiazol-5-yl)propyl]amino}methylene)-3-

(pentyl)urea Dihydrotriﬂuoroacetate (32). 32 was prepared from

amine 12 (30 mg, 0.12 mmol, 1 equiv), 18 (39 mg, 0.13 mmol, 1.1

equiv), NEt₃(41 μL, 0.29 mmol, 2.5 equiv), and HgCl₂(64 mg, 0.23

mmol, 2 equiv) according to the general procedure, yielding the

product as a white, foamlike, and hygroscopic solid (16.9 mg, 26%). R_f=

0.56 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 99% (t_R= 11.0 min, k = 2.43).

¹H NMR (600 MHz, DMSO-d₆) δ 10.45 (br s, 1H), 9.41−8.85 (m,

General Procedure for the Synthesis of the Carbamoylgua-

nidine-Type Ligands (30−36, 38−57, and 59−70). The reaction

was performed in analogy to the published procedure for bivalent

carbamoylguanidine-type ligands.²²In this general procedure, mercuric

chloride (HgCl₂) is used as a reagent, which is very toxic and potentially

carcinogenic. It should be used only in a well-ventilated fume hood after

reading the safety precautions and wearing proper lab safety equipment

(gloves, safety goggles, and lab coats). Future synthetic work should

consider replacements for HgCl₂. The guanidinylating reagents 18−29

(1−1.1 equiv) and 1−2 equiv of the respective amines 8−17 were

dissolved in CH₂Cl₂(3−20 mL). NEt₃(2.5−3 equiv) and HgCl₂(1.1−

2 equiv) were added to the mixture and stirring was continued for 4−48

h. The precipitate was removed by ﬁltration through Celite 545 or

centrifugation (4000g, 5 min). In the case of 52−56, the reaction was

quenched with 7 N NH₃(5 mL) in MeOH prior to ﬁltration. The

solvent was removed in a vacuum. The crude product was puriﬁed by

ﬂash or column chromatography on silica gel (gradient: 0−20 min:

petroleum ether/ethyl acetate (PE/EtOAc) 100:0−50:50, SF 8−12 g,

gradient: CH₂Cl₂/MeOH 90:10 to CH₂Cl₂/MeOH/25% NH₃in H₂O

50:50:1, or isocratic: CH₂Cl₂/7 N NH₃in MeOH 99:1) and dried in a

vacuum. The isolated Boc-/trityl (Trt)-protected intermediates were

characterized by LC-MS (data are shown in Table S7 in the SI).

Subsequently, the deprotection was performed by stirring the respective

compound with 30−70% TFA in CH₂Cl₂(5−14 mL) for 7−18 h. The

obtained carbamoylguanidines (cf. 30−36, 38−52, 55, 57, and 59−70)

were puriﬁed by preparative HPLC. In the case of 53, 54, and 56, the

HCl salts were synthesized according to the following procedure. After

deprotection with TFA, the ligands were puriﬁed by column

chromatography (isocratic: CH₂Cl₂/7 N NH₃in MeOH 90:10),

yielding the free base. The free base was dissolved in 1,4-dioxane (10

mL), and 1−2 N HCl (5 mL) in diethyl ether (Et₂O) was added

dropwise so that the HCl salt precipitated. The suspension was

concentrated in a vacuum, and the solid was washed with Et₂O (3 × 15

mL). After removing the solvent in a vacuum, compounds 53, 54, and

56 were obtained as HCl salts.

1-(Amino{[3-(5-methyl-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(pentyl)urea Hydrotriﬂuoroacetate (30). 30 was

prepared from amine 10 (29 mg, 0.19 mmol, 1.1 equiv), 18 (51 mg,

0.17 mmol, 1 equiv), NEt₃(58 μL, 0.42 mmol, 2.5 equiv), and HgCl₂

(91 mg, 0.34 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (25 mg,

34%). R_f= 0.01 (PE/EtOAc 3:7). RP-HPLC: 98% (t_R= 13.5 min, k =

3.21). ¹H NMR (600 MHz, DMSO-d₆): δ 10.46 (br s, 1H), 9.06 (br s,

1H), 8.54 (br s, 2H), 7.51 (br s, 1H), 3.37−3.29 (m, 2H), 3.12−3.04

(m, 4H), 2.68 (s, 3H), 1.96 (quint, J = 7.4 Hz, 2H), 1.44 (quint, J = 7.2

Hz, 2H), 1.32−1.20 (m, 4H), 0.86 (t, J = 7.1 Hz, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 169.15, 165.01, 159.81 (q, J = 31.9 Hz, TFA),

153.89, 153.69, 117.36 (q, J = 299.5 Hz, TFA), 40.02, 19.11, 28.61,

28.39, 27.94, 26.31, 21.75, 15.10, 13.85. HRMS (ESI-MS): m/z [M +

H⁺] calcd for C₁₃H₂₅NOS⁺: 313.1805; found: 313.1827. Molecular

formula (MF): C₁₃H₂₄N₆OS·C₂HF₃O₂. Molecular weight (MW):

(312.44 + 114.02).

3H), 8.50 (br s, 2H), 7.48 (br s, 1H), 7.07 (s, 1H), 3.26 (q, J = 6.6 Hz,

2H), 3.08 (q, J = 6.6 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 1.77 (quint, J =

7.3 Hz, 2H), 1.42 (quint, J = 7.2 Hz, 2H), 1.32−1.18 (m, 4H), 0.85 (t, J

= 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.37, 159.06 (q, J

= 33.2 Hz, TFA), 153.82, 153.68, 123.95, 123.48, 116.62 (q, J = 296.7

Hz, TFA), 39.87, 39.10, 28.60, 28.46, 28.38, 23.29, 21.75, 13.86. HRMS

(ESI-MS): m/z [M + H⁺] calcd for C₁₃H₂₅N₆OS⁺: 313.1805; found:

313.1807. MF: C₁₃H₂₄N₆OS·C₄H₂F₆O₄. MW: (312.44 + 228.05).

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(pentyl)urea Dihydrotriﬂuoroacetate (33). 33 was

prepared from amine 15 (60 mg, 0.20 mmol, 1 equiv), 18 (66 mg,

0.22 mmol, 1.1 equiv), NEt₃(68 μL, 0.49 mmol, 2.5 equiv), and HgCl₂

(107 mg, 0.39 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (55 mg,

45%). R_f= 0.45 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100% (t_R= 11.8

1

min, k = 2.68). H NMR (600 MHz, DMSO-d₆) δ 10.72 (br s, 1H),

10.13 (br s, 1H), 9.33−7.91 (m, 4H), 7.59 (t, J = 5.6 Hz, 1H), 7.51−

7.45 (m, 2H), 7.41−7.35 (m, 2H), 3.11 (t, J = 7.0 Hz, 2H), 2.25 (s, 3H),

1.44 (quint, J = 7.1 Hz, 2H), 1.33−1.19 (m, 4H), 0.86 (t, J = 7.1 Hz,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 166.94, 159.14 (q, J = 33.1

Hz, TFA), 153.48, 153.36, 138.18, 137.69, 132.72, 130.37, 129.35,

126.20, 116.55 (q, J = 298.0 Hz, TFA), 115.92, 39.20, 28.53, 28.39,

21.75, 14.43, 13.87. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₇H₂₅N₆OS⁺: 361.1805; found: 361.1806. MF: C₁₇H₂₄N₆OS·

C₄H₂F₆O₄. MW: (380.48 + 228.05).

1-(Amino{[3-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(pentyl)urea Dihydrotriﬂuoroacetate (34). 34 was

prepared from amine 16 (30 mg, 0.10 mmol, 1 equiv), 18 (33 mg,

0.11 mmol, 1.1 equiv), NEt₃(33 μL, 0.25 mmol, 2.5 equiv), and HgCl₂

(53 mg, 0.20 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (28 mg,

48%). R_f= 0.55 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100% (t_R= 12.0

1

min, k = 2.74). H NMR (600 MHz, DMSO-d₆) δ 10.64 (br s, 1H),

10.00 (br s, 1H), 9.30−7.78 (m, 4H), 7.57 (t, J = 5.6 Hz, 1H), 7.52 (t, J

= 7.9 Hz, 1H), 7.39−7.35 (m, 1H), 7.34 (t, J = 2.0 Hz, 1H), 7.29−7.24

(m, 1H), 3.11 (t, J = 7.0 Hz, 2H), 2.25 (s, 3H), 1.44 (quint, J = 7.1 Hz,

2H), 1.34−1.19 (m, 4H), 0.86 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz,

DMSO-d₆) δ 166.86, 159.00 (q, J = 32.8 Hz, TFA), 153.52, 153.34,

134.31, 133.13, 130.40, 127.21, 125.21, 124.28, 116.66 (q, J = 296.3 Hz,

TFA), 115.89, 39.17, 28.54, 28.38, 21.74, 14.82, 13.88. HRMS (ESI-

MS): m/z [M + H⁺] calcd for C₁₇H₂₅N₆OS⁺: 361.1805; found:

361.1811. MF: C₁₇H₂₄N₆OS·C₄H₂F₆O₄. MW: (360.48 + 228.05).

1-(Amino{[(2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-

methyl]amino}methylene)-3-(pentyl)urea Dihydrotriﬂuoroacetate

(35). 35 was prepared from amine 17 (77 mg, 0.18 mmol, 1.1 equiv),

18 (50 mg, 0.17 mmol, 1 equiv), NEt₃(57 μL, 0.41 mmol, 2.5 equiv),

and HgCl₂(90 mg, 0.33 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (21 mg, 22%). R_f= 0.59 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100%

(t_R= 10.6 min, k = 2.30). ¹H NMR (600 MHz, DMSO-d₆): δ 10.56 (br

s, 1H), 9.28−8.81 (m, 3H), 8.56 (br s, 2H), 7.51 (br s, 1H), 3.36−3.22

(m, 2H), 3.08 (q, J = 6.6 Hz, 2H), 2.67−2.58 (m, 1H), 2.56−2.50 (m,

1H), 2.47−2.36 (m, 1H), 2.23−2.16 (m, 1H), 2.10−2.00 (m, 1H),

1.92−1.83 (m, 1H), 1.50−1.38 (m, 3H), 1.31−1.17 (m, 4H), 0.85 (t, J

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(pentyl)urea Dihydrotriﬂuoroacetate (31). 31 was

prepared from amine 11 (53 mg, 0.18 mmol, 1 equiv), 18 (50 mg,

0.19 mmol, 1.1 equiv), NEt₃(61 μL, 0.44 mmol, 2.5 equiv), and HgCl₂

(96 mg, 0.35 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (19 mg,

19%). R_f= 0.49 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 99% (t_R= 11.4 min,

k = 2.55). ¹H NMR (600 MHz, DMSO-d₆): δ 10.37 (br s, 1H), 9.04 (br

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= 7.1 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 168.63, 159.46 (q, J

= 32.8 Hz, TFA), 154.08, 153.83, 134.60, 116.85 (q, J = 296.5 Hz,

TFA), 113.20, 44.62, 40.05, 39.16, 33.22, 28.63, 28.42, 25.52, 24.53,

22.00, 21.78, 13.88. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₅H₂₇N₆OS⁺: 339.1962; found: 339.1964. MF: C₁₅H₂₆N₆OS·

C₄H₂F₆O₄. MW: (338.47 + 228.05).

MS): m/z [M + H⁺] calcd for C₁₈H₂₅N₆OS⁺: 373.1805; found:

373.1804. MF: C₁₈H₂₄N₆OS·C₄H₂F₆O₄. MW: (372.49 + 228.05).

1-(Amino{[3-(2-amino-4-methyloxazol-5-yl)propyl]amino}-

methylene)-3-(2-cyclohexylpropyl)urea Dihydrotriﬂuoroacetate

(41). 41 was prepared from amine 9 (35 mg, 0.1 mmol, 1 equiv), 22

(36 mg, 0.1. mmol, 1 equiv), NEt₃(35 μL, 0.25 mmol, 2.5 equiv), and

HgCl₂(54 mg, 0.2 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (14.1

mg, 24%). R_f= 0.52 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 97% (t_R= 15.0

min, k = 3.67). ¹H NMR (600 MHz, DMSO-d₆) δ 10.38 (br s, 1H), 9.00

(br s, 3H), 8.51 (br s, 2H), 7.48 (br s, 1H), 3.28 (q, J = 6.7 Hz, 2H),

3.19−3.12 (m, 1H), 2.99−2.92 (m, 1H), 2.59 (t, J = 7.3 Hz, 2H), 2.01

(s, 3H), 1.80−1.69 (m, 4H), 1.66−1.56 (m, 4H), 1.51−1.44 (m, 1H),

1.26−0.91 (m, 7H), 0.81 (d, J = 6.9 Hz, 3H). ¹³C NMR (151 MHz,

DMSO-d₆) δ 158.83 (q, J = 32.5 Hz, TFA), 157.40, 153.84, 153.74,

139.03, 120.74, 116.81 (q, J = 295.7 Hz, TFA), 42.87, 40.06, 39.24,

37.79, 30.29, 27.92, 26.24, 26.17, 26.07, 25.93, 20.21, 14.04, 7.95.

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(hexyl)urea Dihydrotriﬂuoroacetate (36). 36 was

prepared from amine 11 (22 mg, 0.086 mmol, 1 equiv), 19 (30 mg,

0.095 mmol, 1.1 equiv), NEt₃(30 μL, 0.215 mmol, 2.5 equiv), and

HgCl₂(47 mg, 0.172 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

1

solid (8.44 mg, 18%). RP-HPLC: 96% (t_R= 12.9 min, k = 3.02). H

NMR (600 MHz, DMSO-d₆) δ 10.15 (br s, 1H), 9.01 (br s, 1H), 8.50

(br s, 2H), 8.03−7.24 (m, 3H), 3.30 (q, J = 6.7 Hz, 2H), 3.09 (q, J = 6.6

Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H), 1.89 (quint, J = 7.4 Hz, 2H), 1.43

(quint, J = 6.9 Hz, 2H), 1.31−1.21 (m, 6H), 0.89−0.83 (m, 2H). ¹³C

NMR (151 MHz, DMSO-d₆) δ 168.77, 157.42, 158.59 (q, J = 34.2 Hz,

TFA), 153.79, 153.60, 116.18 (q, J = 293.6 Hz, TFA), 39.97, 39.14,

30.87, 28.87, 27.29, 26.52, 25.86, 22.01, 13.87. HRMS (ESI-MS): m/z

[M + H⁺] calcd for C₁₃H₂₆N₇OS⁺: 328.1914; found: 328.1917. MF:

C₁₃H₂₅N₇OS·C₄H₂F₆O₄. MW: (327.45 + 228.05).

1-(Amino{[3-(5-methyl-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(8-aminooctyl)urea Dihydrotriﬂuoroacetate (38). 38

was prepared from amine 10 (17 mg, 0.11 mmol, 1.1 equiv), 20 (46 mg,

0.1 mmol, 1 equiv), NEt₃(35 μL, 0.25 mmol, 2.5 equiv), and HgCl₂(54

mg, 0.2 mmol, 2 equiv) according to the general procedure, yielding the

product as a white, foamlike, and hygroscopic solid (22 mg, 37%). R_f=

0.48 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 97% (t_R= 9.1 min, k = 1.83).

¹H NMR (600 MHz, DMSO-d₆): δ 10.54 (br s, 1H), 9.05 (br s, 1H),

8.53 (br s, 2H), 7.77 (br s, 3H), 7.52 (br s, 1H), 3.40−3.29 (m, 4H),

3.11−3.05 (m, 4H), 2.76 (t, J = 6.7 Hz, 2H), 2.67 (s, 3H), 1.96 (quint, J

= 7.3 Hz, 2H), 1.51 (quint, J = 7.3 Hz, 2H), 1.46−1.39 (m, 2H), 1.32−

1.21 (m, 8H). ¹³C NMR (151 MHz, DMSO-d₆): δ 169.62, 165.49,

159.38 (q, J = 31.3 Hz, TFA), 154.36, 154.18, 117.44 (q, J = 297.3 Hz,

TFA), 40.48, 40.52, 39.26, 29.37, 28.91, 28.89, 28.40, 27.42, 26.77,

26.55, 26.19, 15.57. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₆H₃₂N₇OS⁺: 370.2384; found: 370.2388 MF: C₁₆H₃₁N₇OS·

C₄H₂F₆O₄. MW: (369.53 + 228.05).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(8-aminooctyl)urea Trihydrotriﬂuoroacetate (39). 39

was prepared from amine 11 (15 mg, 0.054 mmol, 1 equiv), 20 (25 mg,

0.05 mmol, 1.1 equiv), NEt₃(19 μL, 0.14 mmol, 2.5 equiv), and HgCl₂

(29 mg, 0.11 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (10 mg,

26%). RP-HPLC: 96% (t_R= 7.9 min, k = 1.46). ¹H NMR (600 MHz,

DMSO-d₆): δ 10.39 (br s, 1H), 9.05 (br s, 1H), 8.54 (br s, 2H), 7.88−

7.29 (m, 6H), 3.32 (q, J = 6.7 Hz, 2H), 3.11 (q, J = 6.6 Hz, 2H), 2.87 (t, J

= 7.5 Hz, 2H), 2.82−2.74 (m, 2H), 1.91 (quint, J = 7.4 Hz, 2H), 1.53

(quint, J = 7.4 Hz, 2H), 1.45 (q, J = 6.7 Hz, 2H), 1.28 (s, 8H). ¹³C NMR

(151 MHz, DMSO-d₆): δ 168.65, 158.58 (q, J = 33.5 Hz, TFA), 157.35,

153.84, 153.67, 116.53 (q, J = 296.1 Hz, TFA), 39.94, 39.16, 38.80,

28.90, 28.44, 27.38, 26.96, 26.49, 26.10, 25.72. HRMS (ESI-MS): m/z

[M + H⁺] calcd for C₁₅H₃₁N₈OS⁺: 370.2384; found: 370.2388. MF:

C₁₅H₃₀N₈OS·C₆H₃F₉O₆. MW: (369.53 + 342.07).

+

HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₈H₃₃N₆O₂: 365.2660;

found: 365.2660. MF: C₁₈H₃₂N₆O₂·C₄H₂F₆O₄. MW: (364.26 +

228.05).

1-(Amino{[3-(5-methyl-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(2-cyclohexylpropyl)urea Hydrotriﬂuoroacetate (42).

42 was prepared from amine 10 (22 mg, 0.14 mmol, 1.1 equiv), 22 (46

mg, 0.13 mmol, 1 equiv), NEt₃(43 μL, 0.32 mmol, 2.5 equiv), and

HgCl₂(70 mg, 0.26 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (12 mg,

19%). R_f= 0.54 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 97% (t_R= 17.9 min,

k = 4.58). ¹H NMR (600 MHz, DMSO-d₆): δ 10.19 (br s, 1H), 9.02 (br

s, 1H), 8.51 (br s, 2H), 7.47 (br s, 1H), 3.35−3.31 (m, 2H), 3.17−3.04

(m, 3H), 2.97−2.89 (m, 1H), 2.68 (s, 3H), 1.97 (quint, J = 7.4 Hz, 2H),

1.75−1.66 (m, 2H), 1.64−1.53 (m, 3H), 1.51−1.42 (m, 1H), 1.25−

0.90 (m, 6H), 0.80 (d, J = 6.9 Hz, 3H). ¹³C NMR (151 MHz, DMSO-

d₆): δ 169.62, 165.49, 159.43 (q, J = 32.2 Hz, TFA), 154.23, 154.15,

117.50 (q, J = 296.6 Hz, TFA), 43.35, 40.51, 38.25, 30.76, 28.38, 26.78,

26.71, 26.64, 26.54, 15.58, 14.52. HRMS (ESI-MS): m/z [M + H⁺]

calcd for C₁₇H₃₁N₆OS⁺: 367.2275; found: 367.2301. MF:

C₁₇H₃₀N₆OS·C₂HF₃O₂. MW: (366.53 + 114.02).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(2-cyclohexylpropyl)urea Dihydrotriﬂuoroacetate

(43). 43 was prepared from amine 11 (20 mg, 0.08 mmol, 1 equiv),

22 (31 mg, 0.09 mmol, 1.1 equiv), NEt₃(27 μL, 0.20 mmol, 2.5 equiv),

and HgCl₂(43 mg, 0.16 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (8 mg, 17%). RP-HPLC: 99% (t_R= 15.5 min, k = 3.83). ¹H NMR

(600 MHz, DMSO-d₆): δ 10.01 (br s, 1H), 8.98 (br s, 1H), 8.48 (br s,

2H), 7.46 (br s, 3H), 3.29 (q, J = 6.7 Hz, 2H), 3.16−3.09 (m, 1H),

2.96−2.89 (m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 1.88 (quint, J = 7.3 Hz,

2H), 1.72−1.66 (m, 2H), 1.63−1.53 (m, 3H), 1.50−1.41 (m, 1H),

1.25−0.88 (m, 6H), 0.79 (d, J = 6.9 Hz, 3H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 168.60, 158.58 (q, J = 33.5 Hz, TFA), 157.33, 153.90,

153.68, 116.46 (q, J = 296.2 Hz, TFA), 42.88, 40.01, 39.45, 37.77,

30.28, 27.91, 27.37, 26.49, 26.24, 26.17, 26.07, 14.06. HRMS (ESI-

MS): m/z [M + H⁺] calcd for C₁₆H₃₀N₇OS⁺: 368.2227; found:

368.2230. MF: C₁₆H₂₉N₇OS·C₄H₂F₆O₄. MW: (367.52 + 228.05).

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(2-cyclohexylpropyl)urea Dihydrotriﬂuoroacetate

(44). 44 was prepared from amine 15 (32 mg, 0.11 mmol, 1.1 equiv),

22 (34 mg, 0.10 mmol, 1 equiv), NEt₃(33 μL, 0.24 mmol, 2.5 equiv),

and HgCl₂(52 mg, 0.19 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (41 mg, 64%). R_f= 0.50 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100%

(t_R= 15.6 min, k = 3.86). ¹H NMR (600 MHz, DMSO-d₆): δ 10.86 (br

s, 1H), 10.42 (br s, 1H), 9.48−8.14 (m, 4H), 7.61 (t, J = 5.8 Hz, 1H),

7.54−7.47 (m, 2H), 7.43−7.37 (m, 2H), 3.21−3.12 (m, 1H), 3.01−

2.92 (m, 1H), 2.27 (s, 3H), 1.75−1.66 (m, 2H), 1.65−1.55 (m, 3H),

1.52−1.44 (m, 1H), 1.28−0.90 (m, 6H), 0.81 (d, J = 6.9 Hz, 3H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 167.22, 159.16 (q, J = 33.8 Hz, TFA),

153.50, 136.35, 133.09, 129.90, 129.43, 126.13, 116.56 (q, J = 297.3 Hz,

TFA), 115.82, 42.99, 39.71, 37.75, 30.29, 27.92, 26.23, 26.17, 26.06,

14.07, 13.99. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₂₁H₃₁N₆OS⁺:

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(cyclohexyl)urea Dihydrotriﬂuoroacetate (40). 40 was

prepared from amine 15 (50 mg, 0.16 mmol, 1 equiv), 21 (57 mg, 0.18

mmol, 1.1 equiv), NEt₃(57 μL, 0.41 mmol, 2.5 equiv), and HgCl₂(89

mg, 0.33 mmol, 2 equiv) according to the general procedure, yielding

the product as a white, foamlike, and hygroscopic solid (51 mg, 53%). R_f

= 0.54 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100% (t_R= 11.6 min, k =

2.61). ¹H NMR (600 MHz, DMSO-d₆): δ 10.87 (br s, 1H), 10.18 (br s,

1H), 9.39−8.20 (m, 4H), 7.64 (d, J = 7.6 Hz, 1H), 7.54−7.48 (m, 2H),

7.43−7.37 (m, 2H), 3.55−3.44 (m, 1H), 2.27 (s, 3H), 1.86−1.73 (m,

2H), 1.71−1.60 (m, 2H), 1.58−1.48 (m, 1H), 1.36−1.12 (m, 5H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 167.25, 159.13 (q, J = 33.7 Hz, TFA),

153.55, 152.60, 136.24, 133.04, 129.92, 129.45, 126.26, 116.57 (q, J =

297.3 Hz, TFA), 115.79, 48.40, 32.05, 24.96, 24.12, 13.95. HRMS (ESI-

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415.2275; found: 415.2275. MF: C₂₁H₃₀N₆OS·C₄H₂F₆O₄. MW:

(414.57 + 228.05).

yielding the product as a white, foamlike, and hygroscopic solid (58 mg,

48%). R_f= 0.46 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 99% (t_R= 10.9 min,

1

1-(Amino{[3-(2-amino-4-methyloxazol-5-yl)propyl]amino}-

methylene)-3-(benzyl)urea Dihydrotriﬂuoroacetate (45). 45 was

prepared from amine 9 (35 mg, 0.1 mmol, 1 equiv), 23 (32 mg, 0.1

mmol, 1 equiv), NEt₃(35 μL, 0.25 mmol, 2.5 equiv), and HgCl₂(54

mg, 0.2 mmol, 2 equiv) according to the general procedure, yielding the

product as a white, foamlike, and hygroscopic solid (14.9 mg, 27%). R_f=

0.52 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 96% (t_R= 10.4 min, k = 2.24).

¹H NMR (600 MHz, DMSO-d₆) δ 10.51 (br s, 1H), 9.14−8.85 (m,

k = 2.40). H NMR (600 MHz, DMSO-d₆) δ 11.69−10.16 (m, 2H),

9.42−8.58 (m, 4H), 8.15 (t, J = 5.9 Hz, 1H), 7.54−7.49 (m, 2H), 7.45−

7.39 (m, 2H), 7.38−7.25 (m, 5H), 4.35 (d, J = 5.8 Hz, 2H), 2.27 (s,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.46, 159.52 (q, J = 33.6

Hz, TFA), 153.62, 138.52, 135.20, 133.36, 129.52, 128.43, 127.33,

127.17, 126.19, 116.55 (q, J = 295.9 Hz, TFA), 115.73, 42.88, 13.63.

HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₉H₂₁N₆OS⁺: 381.1492;

found: 381.1491. MF: C₁₉H₂₀N₆OS·C₄H₂F₆O₄. MW: (380.47 +

228.05).

3H), 8.55 (br s, 2H), 8.00 (br s, 1H), 7.39−7.19 (m, 5H), 4.31 (d, J =

5.8 Hz, 2H), 3.27 (q, J = 6.7 Hz, 2H), 2.57 (t, J = 7.3 Hz, 2H), 1.99 (s,

3H), 1.75 (quint, J = 7.2 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ

158.79 (q, J = 32.4 Hz, TFA), 157.42, 153.82, 153.77, 139.01, 138.68,

128.40, 127.22, 127.11, 120.81, 116.87 (q, J = 298.7 Hz, TFA), 42.77,

40.06, 25.92, 20.22, 7.98. HRMS (ESI-MS): m/z [M + H⁺] calcd for

1-(Amino{[3-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(benzyl)urea Dihydrotriﬂuoroacetate (50). 50 was

prepared from amine 16 (30 mg, 0.10 mmol, 1 equiv), 23 (35 mg,

0.22 mmol, 1.1 equiv), NEt₃(33 μL, 0.25 mmol, 2.5 equiv), and HgCl₂

(53 mg, 0.20 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (27 mg,

44%). R_f= 0.52 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100% (t_R= 11.1

min, k = 2.46). ¹H NMR (600 MHz, DMSO-d₆): δ 10.90 (br s, 1H),

10.54 (br s, 1H), 8.42−9.20 (m, 4H), 8.11 (t, J = 5.9 Hz, 1H), 7.53 (t, J

= 8.0 Hz, 1H), 7.42−7.23 (m, 8H), 4.34 (d, J = 5.8 Hz, 2H), 2.26 (s,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.32, 159.14 (q, J = 33.5

Hz, TFA), 153.64, 153.60, 138.50, 136.81, 134.48, 132.36, 130.47,

128.42, 127.30, 127.16, 125.36, 124.75, 116.52 (q, J = 295.6 Hz, TFA),

115.65, 42.85, 14.04. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₉H₂₁N₆OS⁺: 381.1492; found: 381.1498. MF: C₁₉H₂₀N₆OS·

C₄H₂F₆O₄. MW: (380.47 + 228.05).

1-(Amino{[(2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-

methyl]amino}methylene)-3-(benzyl)urea Dihydrotriﬂuoroacetate

(51). 51 was prepared from amine 17 (85 mg, 0.20 mmol, 1 equiv),

23 (71 mg, 0.22 mmol, 1.1 equiv), NEt₃(76 μL, 0.55 mmol, 2.5 equiv),

and HgCl₂(119 mg, 0.44 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (45 mg, 38%). R_f= 0.48 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100%

(t_R= 9.8 min, k = 2.05). ¹H NMR (600 MHz, DMSO-d₆): δ 10.65 (br s,

1H), 9.27−8.84 (m, 3H), 8.61 (br s, 2H), 8.03 (br s, 1H), 7.35−7.30

(m, 2H), 7.30−7.23 (m, 3H), 4.31 (d, J = 5.8 Hz, 2H), 3.28 (q, J = 6.8

Hz, 2H), 2.63 (dd, J = 16.2, 5.0 Hz, 1H), 2.55−2.50 (m, 1H), 2.47−

2.36 (m, 1H), 2.23−2.15 (m, 1H), 2.10−2.01 (m, 1H), 1.91−1.83 (m,

1H), 1.50−1.40 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆): δ 168.60,

159.33 (q, J = 32.7 Hz, TFA), 154.04, 153.98, 138.73, 134.64, 128.44,

127.26, 127.14, 116.82 (q, J = 299.9 Hz, TFA), 113.21, 44.67, 42.80,

33.20, 25.53, 24.54, 22.01. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₇H₂₃N₆OS⁺: 359.1649; found: 359.1645. MF: C₁₇H₂₂N₆OS·

C₄H₂F₆O₄. MW: (358.46 + 228.05).

+

C₁₆H₂₃N₆O₂: 331.1877; found: 331.1882. MF: C₁₆H₂₂N₆O₂·

C₄H₂F₆O₄. MW: (330.39 + 228.05).

1-(Amino{[3-(5-methyl-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(benzyl)urea Hydrotriﬂuoroacetate (46). 46 was

prepared from amine 10 (25 mg, 0.16 mmol, 1.1 equiv), 23 (47 mg,

0.15 mmol, 1 equiv), NEt₃(50 μL, 0.36 mmol, 2.5 equiv), and HgCl₂

(79 mg, 0.29 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (13.5

mg, 20%). R_f= 0.54 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 98% (t_R= 12.6

min, k = 2.93). ¹H NMR (600 MHz, DMSO-d₆): δ 10.40−10.08 (m,

1H), 9.03 (br s, 1H), 8.54 (br s, 2H), 8.05−7.95 (m, 1H), 7.38−7.22

(m, 5H), 4.31 (d, J = 5.8 Hz, 2H), 3.39−3.28 (m, 2H), 3.07 (t, J = 7.6

Hz, 2H), 2.67 (s, 3H), 1.96 (quint, J = 7.2 Hz, 2H). ¹³C NMR (151

MHz, DMSO-d₆): δ 169.62, 165.50, 159.42 (q, J = 30.9 Hz, TFA),

154.24, 139.13, 128.88, 127.70, 127.58, 117.47 (q, J = 296.9 Hz, TFA),

43.24, 40.55, 28.35, 26.79, 15.58. HRMS (ESI-MS): m/z [M + H⁺]

calcd for C₁₅H₂₁N₆OS⁺: 333.1492; found: 333.1501. MF:

C₁₅H₂₀N₆OS·C₂HF₃O₂. MW: (332.43 + 114.02).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(benzyl)urea Dihydrotriﬂuoroacetate (47). 47 was

prepared from amine 11 (48 mg, 0.19 mmol, 1.2 equiv), 23 (50 mg,

0.16 mmol, 1 equiv), NEt₃(54 μL, 0.39 mmol, 2.5 equiv), and HgCl₂

(84 mg, 0.31 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (20.8

mg, 23%). RP-HPLC: 98% (t_R= 10.4 min, k = 2.24). ¹H NMR (600

MHz, DMSO-d₆) δ 10.46 (br s, 1H), 9.06 (br s, 1H), 8.56 (br s, 2H),

8.35−7.51 (m, 2H), 7.36−7.22 (m, 5H), 4.31 (d, J = 5.8 Hz, 2H), 3.30

(q, J = 6.7 Hz, 2H), 2.86 (t, J = 7.6 Hz, 2H), 1.89 (quint, J = 7.3 Hz, 2H).

¹³C NMR (151 MHz, DMSO-d₆) δ 169.07, 158.95 (q, J = 34.7 Hz,

TFA), 157.58, 153.84 (2C), 138.70, 128.41 (2C), 127.24 (2C), 127.10,

116.14 (q, J = 295.1 Hz, TFA) 42.78, 40.00, 27.16, 26.59. HRMS (ESI-

MS): m/z [M + H⁺] calcd for C₁₄H₂₀N₇OS⁺: 334.1445; found:

334.1447. MF: C₁₄H₁₉N₇OS·C₄H₂F₆O₄. MW: (333.41 + 228.05).

1-(Amino{[3-(2-aminothiazol-5-yl)propyl]amino}methylene)-3-

(benzyl)urea Dihydrotriﬂuoroacetate (48). 48 was prepared from

amine 12 (30 mg, 0.12 mmol, 1 equiv), 23 (41 mg, 0.13 mmol, 1.1

equiv), NEt₃(41 μL, 0.29 mmol, 2.5 equiv), and HgCl₂(64 mg, 0.23

mmol, 2 equiv) according to the general procedure, yielding the

product as a white, foamlike, and hygroscopic solid (20.4 mg, 30%). R_f=

0.56 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100% (t_R= 10.2 min, k = 2.18).

¹H NMR (600 MHz, DMSO-d₆) δ 10.68 (br s, 1H), 9.10 (br s, 3H),

8.57 (br s, 2H), 8.01 (br s, 1H), 7.43−7.20 (m, 5H), 7.06 (s, 1H), 4.32

(d, J = 5.9 Hz, 2H), 3.27 (q, J = 6.6 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H),

1.79 (quint, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ

169.35, 159.24 (q, J = 32.7 Hz, TFA), 153.88, 153.83, 138.72, 128.40

(2C), 127.22 (2C), 127.09, 123.96, 123.88, 116.7 (q, J = 298.4 Hz,

TFA), 42.76, 39.94, 28.49, 23.30. HRMS (ESI-MS): m/z [M + H⁺]

calcd for C₁₅H₂₁N₆OS⁺: 333.1492; found: 333.1495. MF:

C₁₅H₂₀N₆OS·C₄H₂F₆O₄. MW: (332.43 + 228.05).

1-(Amino{[3-(1H-imidazol-4-yl)propyl]amino}methylene)-3-((R)-

(1-phenylethyl))urea Dihydrotriﬂuoroacetate (52). 52 was prepared

from 8 (327 mg, 0.89 mmol, 1 equiv), 24 (300 mg, 0.89 mmol, 1 equiv),

HgCl₂(265 mg, 0.98 mmol, 1.1 equiv), and NEt₃(372 μL, 2.67 mmol, 3

equiv) according to the general procedure, yielding a 429.1 mg (73%)

of Trt-/Boc-protected intermediate. Thereof, 306 mg was deprotected

in the next step, yielding 52 as a white, foamlike, and hygroscopic solid

after puriﬁcation by preparative HPLC (129.7 mg, 51%). RP-HPLC:

100% (t_R= 8.3 min, k = 2.11). ¹H NMR (300 MHz, MeOD) δ 8.75 (d, J

= 1.4 Hz, 1H), 7.35−7.18 (m, 6H), 4.94−4.85 (m, 1H), 3.38−3.29 (m,

2H), 2.81 (t, J = 7.3 Hz, 2H), 2.00 (quint, J = 7.4 Hz, 2H), 1.47 (d, J =

7.0 Hz, 3H). ¹³C NMR (75 MHz, MeOD) δ 161.87 (q, J = 34.4 Hz,

TFA), 154.56, 153.29, 143.49, 133.46, 132.86, 128.72 (2C), 126.89,

125.56 (2C), 116.7 (q, J = 288.2 Hz, TFA), 114.97, 49.71, 39.99, 26.73,

21.29, 21.10. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₆H₂₃N₆O⁺:

315.1928, found: 315.1932. MF: C₁₆H₂₂N₆O·C₄H₂F₆O₄. MW: (314.39

+ 228.05).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-((R)-(1-phenylethyl))urea Dihydrochloride (53). 53

was prepared from 11 (407 mg, 1.47 mmol, 1 equiv), 24 (494 mg,

1.47 mmol, 1 equiv), HgCl₂(438 mg, 1.61 mmol, 1.1 equiv), and NEt₃

(613 μL, 4.40 mmol, 3 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (50 mg,

8%). RP-HPLC: 100% (t_R= 9.3 min, k = 2.48). ¹H NMR (300 MHz,

MeOD) δ 7.37−7.20 (m, 5H), 4.89−4.85 (m, 1H), 3.49−3.35 (m,

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(benzyl)urea Dihydrotriﬂuoroacetate (49). 49 was

prepared from amine 15 (60 mg, 0.20 mmol, 1 equiv), 23 (70 mg,

0.22 mmol, 1.1 equiv), NEt₃(68 μL, 0.49 mmol, 2.5 equiv), and HgCl₂

(107 mg, 0.39 mmol, 2 equiv) according to the general procedure,

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2H), 3.02 (t, J = 7.2 Hz, 2H), 2.09 (quint, J = 6.8 Hz, 2H), 1.47 (d, J =

6.9 Hz, 3H). ¹³C NMR (75 MHz, MeOD) δ 143.33, 128.26 (2C),

126.97, 125.65 (2C), 49.80, 40.08, 26.80, 26.63, 21.44, 4 C-signals are

missing due to the low concentration of the sample. HRMS (ESI-MS):

m/z [M + H⁺] calcd for C₁₅H₂₂N₇OS⁺: 348.1601, found: 348.1605.

MF: C₁₅H₂₁N₇OS·H₂Cl₂. MW: (347.44 + 72.92).

H⁺] calcd for C₁₈H₂₈N₇OS⁺: 390.2071; found: 390.2077. MF:

C₁₈H₂₇N₇OS·C₄H₂F₆O₄. MW: (389.52 + 228.05).

1-(Amino{[3-(2-aminothiazol-5-yl)propyl]amino}methylene)-3-

(2-methyl-3-(p-tolyl)propyl)urea Dihydrotriﬂuoroacetate (59). 59

was prepared from amine 12 (37 mg, 0.15 mmol, 1.1 equiv), 26 (50 mg,

0.13 mmol, 1 equiv), NEt₃(46 μL, 0.33 mmol, 2.5 equiv), and HgCl₂

(72 mg, 0.26 mmol, 2 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (18.2

mg, 23%). R_f= 0.55 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 99% (t_R= 14.5

1-(Amino{[3-(1H-1,2,4-triazol-5-yl)propyl]amino}methylene)-3-

((R)-(1-phenylethyl))urea Dihydrochloride (54). 54 was prepared

from 13 (420 mg, 1.14 mmol, 1 equiv), 24 (423 mg, 1.25 mmol, 1.1

equiv), HgCl₂(340 mg, 1.25 mmol, 1.1 equiv), and NEt₃(474 μL, 3.42

mmol, 3 equiv) according to the general procedure, yielding 560 mg

(75%) of Trt-/Boc-protected intermediate. Thereof, 540 mg was

deprotected in the next step, yielding 54 as a white, foamlike, and

hygroscopic solid after puriﬁcation by preparative HPLC (200 mg,

63%). RP-HPLC: 100% (t_R= 9.1 min, k = 2.41). ¹H NMR (300 MHz,

MeOD) δ 9.27 (s, 1H), 7.41−7.19 (m, 5H), 4.88 (q, J = 6.8 Hz, 1H),

3.56−3.37 (m, 2H), 3.21−3.01 (m, 2H), 2.15 (quint, J = 7.3 Hz, 2H),

1.46 (d, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz, MeOD) δ 155.98, 155.71,

154.19, 144.64, 143.46, 129.56 (2C), 128.27, 126.95 (2C), 51.11,

41.42, 26.36, 22.97, 22.84. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₅H₂₂N₇O⁺: 316,1880, found: 316.1883. MF: C₁₅H₂₁N₇O·H₂Cl₂.

MW: (315.38 + 72.92).

1-(Amino{[3-(4-((dimethylamino)methyl)-1H-1,2,3-triazol-1-yl)-

propyl]amino}methylene)-3-((R)-(1-phenylethyl))urea Trihydrotri-

ﬂuoroacetate (55). 55 was prepared from 14 (298 mg, 1.63 mmol, 1

equiv), 24 (549 mg, 1.63 mmol, 1 equiv), HgCl₂(486 mg, 1.79 mmol,

1.1 equiv), and NEt₃(680 μL, 4.88 mmol, 3 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (50 mg, 5%). RP-HPLC: 98% (t_R= 8.3 min, k = 2.11).

¹H NMR (300 MHz, MeOD) δ 8.22 (s, 1H), 7.36−7.20 (m, 5H),

1

min, k = 3.52). H NMR (600 MHz, DMSO-d₆) δ 10.51 (br s, 1H),

9.46−8.69 (m, 3H), 8.51 (br s, 2H), 7.56 (br s, 1H), 7.13−6.97 (m,

5H), 3.27 (q, J = 6.6 Hz, 2H), 3.13−3.03 (m, 1H), 2.98−2.87 (m, 1H),

2.64 (t, J = 7.5 Hz, 2H), 2.60−2.55 (m, 1H), 2.35−2.28 (m, 1H), 2.25

(s, 3H), 1.92−1.82 (m, 1H), 1.78 (quint, J = 7.3 Hz, 2H), 0.79 (d, J =

6.7 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.30, 159.18 (q, J =

32.9 Hz, TFA) 153.81, 153.77, 136.97, 134.72, 128.81 (2C), 128.77

(2C), 123.96 (2C), 116.69 (q, J = 297.1 Hz, TFA), 44.64, 40.06, 39.90,

34.87, 28.52, 23.30, 20.61, 17.07. HRMS (ESI-MS): m/z [M + H⁺]

calcd for C₁₉H₂₉N₆OS⁺: 389.2118; found: 389.2122. MF:

C₁₉H₂₈N₆OS·C₄H₂F₆O₄. MW: (388.53 + 228.05).

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(2-methyl-3-(p-tolyl)propyl)urea Dihydrotriﬂuoroa-

cetate (60). 60 was prepared from amine 15 (50 mg, 0.16 mmol, 1

equiv), 26 (68 mg, 0.18 mmol, 1.1 equiv), NEt₃(57 μL, 0.41 mmol, 2.5

equiv), and HgCl₂(89 mg, 0.33 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (58 mg, 55%). R_f= 0.64 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100%

(t_R= 15.2 min, k = 3.74). ¹H NMR (600 MHz, DMSO-d₆): δ 10.88 (br

s, 1H), 10.47 (br s, 1H), 9.29−8.29 (m, 4H), 7.70 (t, J = 5.8 Hz, 1H),

7.55−7.48 (m, 2H), 7.44−7.38 (m, 2H), 7.12−7.02 (m, 4H), 3.15−

3.08 (m, 1H), 3.00−2.93 (m, 1H), 2.60 (dd, J = 13.5, 6.1 Hz, 1H), 2.32

(dd, J = 13.5, 8.2 Hz, 1H), 2.29−2.24 (m, 6H), 1.93−1.83 (m, 1H),

0.81 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.31,

159.20 (q, J = 33.5 Hz, TFA), 153.55, 153.50, 136.94, 134.75, 133.23,

129.70, 129.47, 128.81, 128.79, 126.11, 116.59 (q, J = 295.9 Hz, TFA),

115.77, 44.74, 39.60, 34.83, 20.62, 17.09, 13.82. HRMS (ESI-MS): m/z

[M + H⁺] calcd for C₂₃H₂₉N₆OS⁺: 437.2118; found: 437.2118. MF:

C₂₃H₂₈N₆OS·C₄H₂F₆O₄. MW: (436.58 + 228.05).

4.90−4.85 (m, 1H), 4.55 (t, J = 6.7 Hz, 2H), 4.42 (s, 2H), 3.34 (t, J = 6.7

Hz, 2H), 2.88 (s, 6H), 2.25 (p, J = 6.9 Hz, 2H), 1.47 (d, J = 7.0 Hz, 3H).

¹³C NMR (75 MHz, MeOD) δ 156.03, 144.86, 138.09, 129.63 (2C),

128.33, 128.05, 126.97 (2C), 52.42, 51.09, 48.65, 42.92 (2C), 39.51,

29.69, 22.70, the 1C signal is missing due to the low concentration of

the sample. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₈H₂₉N₈O⁺:

373.2459, found: 373.2463. MF: C₁₈H₂₈N₈O·C₆H₃F₉O₆. MW: (372.48

+ 342.07).

1-(Amino{[3-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(2-methyl-3-(p-tolyl)propyl)urea Dihydrotriﬂuoroa-

cetate (61). 61 was prepared from amine 16 (20 mg, 0.07 mmol, 1

equiv), 26 (28 mg, 0.073 mmol, 1.1 equiv), NEt₃(23 μL, 0.17 mmol,

2.5 equiv), and HgCl₂(36 mg, 0.13 mmol, 2 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (16 mg, 34%). R_f= 0.58 (CH₂Cl₂/MeOH 9:1). RP-

HPLC: 100% (t_R= 15.4 min, k = 3.80). ¹H NMR (600 MHz, DMSO-

d₆): δ 10.81 (br s, 1H), 10.37 (br s, 1H), 9.30−8.53 (m, 4H), 7.68 (t, J =

5.8 Hz, 1H), 7.59−7.53 (m, 1H), 7.43−7.38 (m, 2H), 7.35−7.30 (m,

1H), 3.15−3.06 (m, 1H), 3.00−2.91 (m, 1H), 2.63−2.55 (m, 1H),

2.35−2.20 (m, 7H), 1.93−1.82 (m, 1H), 0.80 (d, J = 6.7 Hz, 3H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 167.56, 158.84 (q, J = 34.5 Hz, TFA),

153.56, 136.92, 134.76, 134.56, 131.80, 130.57, 128.81, 128.79, 127.35,

125.47, 125.10, 116.83 (q, J = 295.7 Hz, TFA), 115.54, 44.71, 40.06,

34.85, 20.61, 17.07, 13.50. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₂₃H₂₉N₆OS⁺: 437.2118; found: 437.2116. MF: C₂₃H₂₈N₆OS·

C₄H₂F₆O₄. MW: (436.58 + 228.05).

1-(Amino{[(2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-

methyl]amino}methylene)-3-(2-methyl-3-(p-tolyl)propyl)urea Dihy-

drotriﬂuoroacetate (62). 62 was prepared from amine 17 (82 mg, 0.19

mmol, 1.1 equiv), 26 (80 mg, 0.21 mmol, 1 equiv), NEt₃(67 μL, 0.48

mmol, 2.5 equiv), and HgCl₂(104 mg, 0.38 mmol, 2 equiv) according

to the general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (22 mg, 16%). R_f= 0.49 (CH₂Cl₂/MeOH 9:1). RP-

HPLC: 99% (t_R= 14.1 min, k = 3.39). ¹H NMR (600 MHz, DMSO-d₆):

δ 10.36 (br s, 1H), 9.10 (br s, 1H), 8.90−8.32 (m, 4H), 7.58 (br s, 1H),

7.12−7.02 (m, 4H), 3.33−3.26 (m, 2H), 3.14−3.03 (m, 1H), 2.97−

2.86 (m, 1H), 2.67−2.56 (m, 2H), 2.48−2.36 (m, 2H), 2.34−2.28 (m,

1H), 2.26 (s, 3H), 2.24−2.15 (m, 1H), 2.10−2.01 (m, 1H), 1.93−1.82

(m, 2H), 1.51−1.41 (m, 1H), 0.79 (d, J = 6.6 Hz, 3H). ¹³C NMR (151

MHz, DMSO-d₆) δ 168.13, 158.75 (q, J = 31.4 Hz, TFA), 153.94,

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(1-(3-ﬂuorophenyl)ethyl)urea Dihydrochloride (56).

56 was prepared from 11 (130 mg, 0.50 mmol, 1 equiv), 25 (179 mg,

0.50 mmol, 1 equiv), HgCl₂(150 mg, 0.55 mmol, 1.1 equiv), and NEt₃

(211 μL, 1.51 mmol, 3 equiv) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (70 mg,

32%). RP-HPLC: 100% (t_R= 9.8 min, k = 2.67). ¹H NMR (300 MHz,

MeOD) δ 7.40−7.29 (m, 1H), 7.20−7.06 (m, 2H), 7.02−6.92 (m,

1H), 4.91−4.86 (m, 1H), 3.46−3.38 (m, 2H), 3.03 (t, J = 7.4 Hz, 2H),

2.08 (p, J = 7.1 Hz, 2H), 1.47 (d, J = 7.0 Hz, 3H). ¹³C NMR (75 MHz,

MeOD) δ 172.03, 164.29 (d, J = 244.2 Hz), 159.72, 155.61, 154.34,

147.72 (d, J = 7.0 Hz), 131.41 (d, J = 8.2 Hz), 122.88 (d, J = 2.7 Hz),

114.92 (d, J = 21.3 Hz), 113.81 (d, J = 22.2 Hz), 50.76, 41.44, 28.22,

27.50, 22.63. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₁₅H₂₁FN₇OS⁺: 366.1507, found: 366.1509. MF: C₁₅H₂₀FN₇OS·

H₂Cl₂. MW: (365.43 + 72.92).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(2-methyl-3-(p-tolyl)propyl)urea Dihydrotriﬂuoroa-

cetate (57). 57 was prepared from amine 11 (66 mg, 0.26 mmol, 1.1

equiv), 26 (85 mg, 0.22 mmol, 1 equiv), NEt₃(78 μL, 0.56 mmol, 2.5

equiv), and HgCl₂(121 mg, 0.45 mmol, 2 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (24 mg, 18%). RP-HPLC: 98% (t_R= 15.0 min, k =

3.67). ¹H NMR (600 MHz, DMSO-d₆): δ 10.28 (br s, 1H), 9.01 (br s,

1H), 8.50 (br s, 2H), 7.40−8.03 (s, 3H), 7.11−6.99 (m, 4H), 3.29 (q, J

= 6.7 Hz, 2H), 3.12−3.02 (m, 1H), 2.96−2.89 (m, 1H), 2.85 (t, J = 7.5

Hz, 2H), 2.62−2.54 (m, 1H), 2.36−2.27 (m, 1H), 2.25 (s, 3H), 1.93−

1.82 (m, 3H), 0.78 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, DMSO-

d₆) δ 168.81, 158.87 (q, J = 34.7 Hz, TFA), 157.45, 153.77, 136.97,

134.72, 128.81, 128.78, 116.33 (q, J = 294.0 Hz, TFA), 44.05, 40.06,

39.98, 34.86, 27.30, 26.53, 20.61, 17.08. HRMS (ESI-MS): m/z [M +

8702

https://doi.org/10.1021/acs.jmedchem.1c00692

J. Med. Chem. 2021, 64, 8684−8709

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

153.71, 136.95, 134.73, 128.81, 128.77, 117.06 (q, J = 297.8 Hz, TFA),

113.15, 44.66, 40.06, 34.86, 33.27, 25.59, 24.70, 22.44, 20.61, 17.08.

HRMS (ESI-MS): m/z [M + H⁺] calcd for C₂₁H₃₁N₆OS⁺: 415.2275;

found: 415.2278. MF: C₂₁H₃₀N₆OS·C₄H₂F₆O₄. MW: (414.57 +

228.05).

3H), 3.31−3.25 (m, 2H), 3.08−3.00 (m, 1H), 2.95−2.87 (m, 1H),

2.57−2.50 (m, 2H), 2.47−2.37 (m, 1H), 2.24−2.14 (m, 1H), 2.10−

2.01 (m, 1H), 1.92−1.83 (m, 1H), 1.65−1.40 (m, 4H), 1.36−1.26 (m,

1H), 1.14−1.05 (m, 1H), 0.83 (d, J = 6.7 Hz, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 168.85, 159.44 (q, J = 30.5 Hz, TFA), 154.38,

142.61, 135.59, 128.70, 128.66, 126.08, 117.35 (q, J = 297.5 Hz, TFA),

113.63, 45.39, 45.10, 40.50, 35.79, 33.67, 33.08, 28.79, 26.00, 25.04,

22.62, 17.87. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₂₂H₃₃N₆OS⁺:

429.2431; found: 429.2433. MF: C₂₂H₃₂N₆OS·C₄H₂F₆O₄. MW:

(428.60 + 228.05).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-(2-methyl-5-phenylpentyl)urea Dihydrotriﬂuoroace-

tate (63). 63 was prepared from amine 11 (20 mg, 0.077 mmol, 1

equiv), 27 (33.5 mg, 0.085 mmol, 1.1 equiv), NEt₃(27 μL, 0.19 mmol,

2.5 equiv), and HgCl₂(42 mg, 0.15 mmol, 2 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (5.94 mg, 12%). RP-HPLC: 97% (t_R= 16.4 min, k =

4.11). ¹H NMR (600 MHz, DMSO-d₆) δ 9.92 (br s, 1H), 8.98 (br s,

1H), 8.49 (br s, 2H), 7.62−7.29 (m, 3H), 7.27−7.22 (m, 2H), 7.16 (d, J

= 25.0 Hz, 3H), 3.29 (q, J = 6.7 Hz, 2H), 3.07−3.00 (m, 1H), 2.95−

2.87 (m, 1H), 2.84 (t, J = 7.5 Hz, 2H), 2.58−2.52 (m, 2H), 1.88 (quint,

J = 7.4 Hz, 2H), 1.66−1.48 (m, 3H), 1.36−1.27 (m, 1H), 1.13−1.05

(m, 1H), 0.83 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ

168.58, 158.46 (q, J = 33.9 Hz, TFA), 157.32, 153.69 (2C), 142.12,

128.23 (2C), 128.20 (2C), 125.62, 44.92, 40.00, 35.31, 33.21, 32.61,

28.31, 27.38, 26.48, 17.41, second TFA quartet at approx. 116 ppm was

not visible. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₉H₃₀N₇OS⁺:

404.2227; found: 404.2232. MF: C₁₉H₂₉N₇OS·C₄H₂F₆O₄. MW:

(403.55 + 228.05).

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(2-methyl-5-phenylpentyl)urea Dihydrotriﬂuoroace-

tate (64). 64 was prepared from amine 15 (50 mg, 0.16 mmol, 1

equiv), 27 (71 mg, 0.18 mmol, 1.1 equiv), NEt₃(57 μL, 0.41 mmol, 2.5

equiv), and HgCl₂(89 mg, 0.33 mmol, 2 equiv) according to the general

procedure, yielding the product as a white, foamlike, and hygroscopic

solid (45 mg, 41%). R_f= 0.64 (CH₂Cl₂/MeOH 9:1). RP-HPLC: 100%

(t_R= 16.2 min, k = 4.05). ¹H NMR (600 MHz, DMSO-d₆): δ 10.84 (br

s, 1H), 10.33 (br s, 1H), 9.15−8.40 (m, 2H), 7.64 (t, J = 5.8 Hz, 1H),

7.55−7.47 (m, 2H), 7.42−7.38 (m, 2H), 7.30−7.23 (m, 2H), 7.21−

7.14 (m, 3H), 3.11−3.04 (m, 1H), 2.99−2.91 (m, 1H), 2.60−2.52 (m,

2H), 2.27 (s, 3H), 1.69−1.50 (m, 3H), 1.38−1.29 (m, 1H), 1.17−1.06

(m, 1H), 0.85 (d, J = 6.7 Hz, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ

167.14, 158.99 (q, J = 34.7 Hz, TFA), 153.54, 153.49, 142.13, 132.98,

130.03, 129.41, 128.23, 126.16, 125.63, 116.47 (q, J = 295.5 Hz, TFA),

115.83, 45.00, 35.32, 33.22, 32.58, 28.33, 17.41, 14.10. HRMS (ESI-

MS): m/z [M + H⁺] calcd for C₂₄H₃₁N₆OS⁺: 451.2275; found:

451.2274. MF: C₂₄H₃₀N₆OS·C₄H₂F₆O₄. MW: (450.61 + 228.05).

1-(Amino{[3-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-(2-methyl-5-phenylpentyl)urea Dihydrotriﬂuoroace-

tate (65). 65 was prepared from amine 16 (20 mg, 0.07 mmol, 1

equiv), 27 (29 mg, 0.073 mmol, 1.1 equiv), NEt₃(23 μL, 0.17 mmol,

2.5 equiv), and HgCl₂(36 mg, 0.13 mmol, 2 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (18 mg, 38%). R_f= 0.58 (CH₂Cl₂/MeOH 9:1). RP-

HPLC: 99% (t_R= 16.3 min, k = 4.08). ¹H NMR (600 MHz, DMSO-d₆):

δ 10.80 (br s, 1H), 10.29 (br s, 1H), 9.17−8.26 (m, 2H), 7.63 (t, J = 5.8

Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 7.42−7.36 (m, 2H), 7.32−7.24 (m,

3H), 7.21−7.14 (m, 3H), 3.12−3.04 (m, 1H), 2.99−2.92 (m, 1H),

2.62−2.51 (m, 2H), 2.27 (s, 3H), 1.68−1.50 (m, 3H), 1.37−1.29 (m,

1H), 1.16−1.08 (m, 1H), 0.85 (d, J = 6.7 Hz, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 167.20, 158.53 (q, J = 32.7 Hz, TFA), 153.55,

142.13, 134.43, 132.54, 130.47, 128.23, 127.25, 125.63, 125.30, 124.61,

116.53 (q, J = 297.6 Hz, TFA), 115.73, 44.98, 35.33, 33.21, 32.59,

28.33, 17.40, 14.22. HRMS (ESI-MS): m/z [M + H⁺] calcd for

C₂₄H₃₁N₆OS⁺: 451.2275; found: 451.2283. MF: C₂₄H₃₀N₆OS·

C₄H₂F₆O₄. MW: (450.61 + 228.05).

1-(Amino{[4-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-{6-[3-(amino{[4-(2-amino-4-methylthiazol-5-yl)-

phenyl]amino}methylene)ureido]hexyl}urea tetrahydrotriﬂuoroa-

cetate (67). 67 was prepared from amine 15 (22 mg, 0.073 mmol, 2

equiv), 28 (20 mg, 0.036 mmol, 1 equiv), NEt₃(25 μL, 0.18 mmol, 5

equiv), and HgCl₂(40 mg, 0.144 mmol, 4 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (9.3 mg, 23%). R_f= 0.21 (CH₂Cl₂/MeOH 9:1). RP-

HPLC: 98% (t_R= 10.3 min, k = 2.21). ¹H NMR (600 MHz, DMSO-d₆):

δ 11.70−10.09 (m, 4H), 9.54−8.44 (m, 8H), 7.65 (t, J = 5.7 Hz, 2H),

7.53−7.45 (m, 4H), 7.42−7.33 (m, 4H), 3.12 (q, J = 6.6 Hz, 4H), 2.26

(s, 6H), 1.46 (t, J = 7.0 Hz, 4H), 1.34−1.25 (m, 4H). ¹³C NMR (151

MHz, DMSO-d₆): δ 167.93 (2C), 160.05 (q, J = 33.2 Hz, TFA), 154.07

(2C), 153.96 (2C), 135.55 (2C), 133.83 (2C), 129.97 (4C), 129.92

(2C), 126.56 (4C), 117.01 (q, J = 294.0 Hz, TFA), 116.17 (2C), 39.65

(2C), 29.26 (2C), 26.31 (2C), 14.03 (2C). HRMS (ESI-MS): m/z [M

+

+ H⁺] calcd for C₃₀H₃₉N₁₂O₂S₂: 663.2755; found: 663.2752. MF:

C₃₀H₃₈N₁₂O₂S₂·C₈H₄F₁₂O₈. MW: (662.84 + 456.09).

1-(Amino{[3-(2-amino-4-methylthiazol-5-yl)phenyl]amino}-

methylene)-3-{6-[3-(amino{[3-(2-amino-4-methylthiazol-5-yl)-

phenyl]amino}methylene)ureido]hexyl}urea Tetrahydrotriﬂuoroa-

cetate (68). 68 was prepared from amine 16 (22 mg, 0.073 mmol, 2

equiv), 28 (20 mg, 0.036 mmol, 1 equiv), NEt₃(25 μL, 0.18 mmol, 5

equiv), and HgCl₂(40 mg, 0.144 mmol, 4 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (5.8 mg, 14%). R_f= 0.23 (CH₂Cl₂/MeOH 9:1). RP-

HPLC: 98% (t_R= 10.2 min, k = 2.18). ¹H NMR (600 MHz, DMSO-d₆):

δ 10.72 (br s, 2H), 10.26 (br s, 2H), 9.08−8.53 (m, 1H), 8.10 (br s,

3H), 7.62 (t, J = 5.7 Hz, 2H), 7.52 (t, J = 7.9 Hz, 2H), 7.39−7.35 (m,

2H), 7.33 (t, J = 2.0 Hz, 2H), 7.28−7.23 (m, 2H), 3.11 (q, J = 6.1 Hz,

4H), 2.25 (s, 6H), 1.44 (quint, J = 6.9 Hz, 5H), 1.32−1.24 (m, 4H).

+

HRMS (ESI-MS): m/z [M + H⁺] calcd for C₃₀H₃₉N₁₂O₂S₂: 663.2755;

found: 663.2747. MF: C₃₀H₃₈N₁₂O₂S₂·C₈H₄F₁₂O₈. MW: (662.84 +

456.09).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-{6-[3-(amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)-

propyl]amino}methylene)ureido]hexyl}urea Tetrahydrotriﬂuoroa-

cetate (69). 69 was prepared from amine 11 (53.3 mg, 0.21 mmol, 2

equiv), 28 (57 mg, 0.10 mmol, 1 equiv), NEt₃(71 μL, 0.52 mmol, 5

equiv), and HgCl₂(112 mg, 0.41 mmol, 4 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (35 mg, 34%). RP-HPLC: 96% (t_R= 8.6 min, k =

1.68). ¹H NMR (600 MHz, DMSO-d₆) δ: 10.45 (br s, 2H), 9.05 (br s,

2H), 8.53 (br s, 4H), 8.06 (br s, 4H), 7.52 (br s, 2H), 3.30 (q, J = 6.7 Hz,

4H), 3.09 (q, J = 6.6 Hz, 4H), 2.87 (t, J = 7.5 Hz, 4H), 1.89 (quint, J =

7.4 Hz, 4H), 1.43 (quint, J = 6.7 Hz, 4H), 1.31−1.22 (m, 4H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 169.06, 159.16 (q, J = 34.4 Hz, TFA),

157.57, 153.90, 153.71, 39.60, 38.83, 28.88, 27.21, 26.58, 25.88. HRMS

+

(ESI-MS): m/z [M + H⁺] calcd for C₂₀H₃₇N₁₄O₂S₂: 569.2660; found:

569.2660. MF: C₂₀H₃₆N₁₄O₂S₂·C₈H₄F₁₂O₈. MW: (568.26 + 456.09).

1-(Amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)propyl]amino}-

methylene)-3-{8-[3-(amino{[3-(5-amino-1,3,4-thiadiazol-2-yl)-

propyl]amino}methylene)ureido]octyl}urea Tetrahydrotriﬂuoroa-

cetate (70). 70 was prepared from amine 11 (99 mg, 0.38 mmol, 2.2

equiv), 29 (100 mg, 0.17 mmol, 1 equiv), NEt₃(118 μL, 0.85 mmol, 5

equiv), and HgCl₂(185 mg, 0.68 mmol, 4 equiv) according to the

general procedure, yielding the product as a white, foamlike, and

hygroscopic solid (11.9 mg, 6.7%). RP-HPLC: 96% (t_R= 10.3 min, k =

2.21). ¹H NMR (600 MHz, DMSO-d₆) δ 10.26 (br s, 2H), 9.02 (br s,

2H), 8.50 (br s, 4H), 7.97 (br s, 3H), 7.49 (br s, 2H), 3.29 (q, J = 6.7 Hz,

4H), 3.07 (q, J = 6.6 Hz, 4H), 2.85 (t, J = 7.5 Hz, 4H), 1.88 (quint, J =

1-(Amino{[(2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-

methyl]amino}methylene)-3-(2-methyl-5-phenylpentyl)urea Dihy-

drotriﬂuoroacetate (66). 66 was prepared from amine 17 (81 mg,

0.19 mmol, 1 equiv), 27 (83 mg, 0.21 mmol, 1.1 equiv), NEt₃(66 μL,

0.48 mmol, 2.5 equiv), and HgCl₂(104 mg, 0.38 mmol, 2 equiv)

according to the general procedure, yielding the product as a white,

foamlike, and hygroscopic solid (18 mg, 14%). R_f= 0.62 (CH₂Cl₂/

MeOH 9:1). RP-HPLC: 100% (t_R= 15.2 min, k = 3.74). ¹H NMR (600

MHz, DMSO-d₆): δ 10.39 (br s, 1H), 9.12 (br s, 1H), 8.88 (br s, 2H),

8.54 (br s, 2H), 7.52 (br s, 1H), 7.29−7.21 (m, 2H), 7.20−7.11 (m,

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7.3 Hz, 4H), 1.46−1.37 (m, 4H), 1.28−1.22 (m, 8H). ¹³C NMR (151

MHz, DMSO-d₆) δ 168.97 (2C), 158.81 (q, J = 35.1 Hz, TFA), 157.53

(2C), 153.83 (2C), 153.64 (2C), 115.98 (q, J = 293.8 Hz, TFA), 40.06

(2C), 39.31 (2C), 28.92 (2C), 28.61 (2C), 27.20 (2C), 26.56 (2C),

26.18 (2C). HRMS (ESI-MS): m/z [M + H⁺] calcd for

binding experiments were performed as previously described in

detail^22,38with one minor modiﬁcation: PBS (8 g NaCl, 0.2 g KCl,

1.0 g Na₂HPO₄·2H₂O, 0.15 g NaH₂PO₄·H₂O, 0.1 g KH₂PO₄in 1 L

Millipore H₂O; pH 7.4; 4 °C) was used as washing buﬀer while

harvesting instead of the previously used binding buﬀer.^22,38[³H]-

Mepyramine (speciﬁc activity (spec. act.): 20.0−87 Ci/mmol) was

+

C₂₂H₄₁N₁₄O₂S₂: 597.2973; found: 597.2967. MF: C₂₂H₄₀N₁₄O₂S₂·

C₈H₄F₁₂O₈. MW: (596.78 + 456.09).

from Hartmann analytics (Braunschweig, Germany) or Novandi

3

̈

Chemistry AB (Sodertalje, Sweden), [ H]1 (speciﬁc activity: 25.0

Ci/mmol) and [³H]N^α-methylhistamine (speciﬁc activity: 85.3 Ci/

mmol) were from Hartmann analytics (Braunschweig, Germany).

[³H]UR-DE257³⁶(speciﬁc activity: 63.0 Ci/mmol) and [³H]UR-

PI294³⁷(speciﬁc activity: 41.8 Ci/mmol) were synthesized and

characterized in our laboratories. Histamine dihydrochloride and

diphenhydramine hydrochloride were from TCI Deutschland GmbH

(Eschborn, Germany). Famotidine was from Alfa Aesar (Karlsruhe,

Germany).

General Procedure for the Synthesis of Oxadiazole Deriva-

tives 37 and 58. The oxadiazole heterocycle was formed according to

a previously published procedure.⁸⁴Cyanogen bromide (CNBr) is used

as a reagent in this procedure, which is acutely toxic and potentially

carcinogenic. It should be used only in a well-ventilated fume hood after

reading the safety precautions and wearing proper lab safety equipment

(gloves, safety goggles, and lab coats). Future synthetic work should

consider replacements for CNBr. The respective acylhydrazine (1

equiv, for details regarding 108 and 109, see SI) was dissolved in a

mixture of H₂O/ethanol (EtOH, 1:1 or 2:3 (v/v), 1−2 mL), and

KHCO₃(3.2 equiv) was added. After addition of BrCN (3 M in

CH₂Cl₂, 1 equiv), the reaction mixture was heated at 60 °C for 2 h. The

reaction mixture was cooled to rt and stirred for an additional hour.

EtOH was removed in a vacuum, and the residue was puriﬁed by

preparative HPLC.

Dopamine D₁₋₅Receptors. The competition binding experiments

were performed on homogenates of HEK293T-CRE-Luc-hD_xR (x = 1,

2long, 3 or 5) or HEK293T ElucN-βarr2 hD_4.4R-ELuc cells using [³H]

N-methylspiperone (hD_2long/3/4.4R, speciﬁc activity: 77 Ci/mmol,

Novandi Chemistry AB) or [³H]SCH23390 (hD_1/5R, speciﬁc activity:

81 Ci/mmol, Novandi Chemistry AB) using the previously published

protocol.⁴⁰Generation and cell culture of HEK293T-CRE-Luc cells,

expressing the hD₁-, hD_2long-, hD₃-, or hD_4.4receptors as well as the

general procedure for the homogenate preparation have been described

in the same publication.⁴⁰

The HEK293T-CRE-Luc cell line stably expressing the hD₅R was

generated in an analogous manner as published for the hD₁R, hD_2longR,

hD₃R, or hD_4.4R.⁴⁰In brief, 2 μg of the pIRESneo3 SP-FLAG-hD₅R

vector (generated in an analogous manner as described for the hD₁R,

hD_2longR, hD₃R, and hD_4.4R⁴⁰) was used and selection was achieved in

the presence of 600 μg/mL of G418. The preparation of cell

homogenates was performed as previously described⁴⁰with the

following modiﬁcation: after centrifugation (6 °C, 50 000g, 15 min)

and resuspension of the remaining pellet in tris(hydroxymethyl)-

aminomethane (Tris)−MgSO₄buﬀer⁴⁰homogenization was per-

formed with a Potter homogenizer (10 times, ice-cooled) instead of a

syringe and needle.

1-(Amino{[3-(5-amino-1,3,4-oxadiazol-2-yl)propyl]amino}-

methylene)-3-(hexyl)urea Dihydrotriﬂuoroacetate (37). 37 was

prepared from 108 (82 mg, 0.16 mmol, 1 equiv), KHCO₃(51 mg,

0.51 mmol, 3.2 equiv), and BrCN (3 M in CH₂Cl₂, 53 μL, 0.16 mmol, 1

equiv) in H₂O/EtOH (1:1.5 mL) according to the general procedure,

yielding the product as a white, foamlike, and hygroscopic solid (5.25

mg, 6.1%). RP-HPLC: 99% (t_R= 13.5 min, k = 3.21). ¹H NMR (600

MHz, DMSO-d₆) δ 10.03 (br s, 1H), 9.00 (br s, 1H), 8.50 (br s, 2H),

7.49 (t, J = 5.6 Hz, 1H), 6.93 (br s, 2H), 3.31 (q, J = 6.7 Hz, 2H), 3.09

(p, J = 6.4 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 1.87 (p, J = 7.4 Hz, 2H),

1.43 (p, J = 7.2 Hz, 2H), 1.29−1.24 (m, 6H), 0.91−0.82 (m, 3H). ¹³C

NMR (151 MHz, DMSO) δ 163.52, 158.59 (q, J = 34.5 Hz, TFA),

158.48, 153.76, 153.55, 39.61, 38.85, 30.86, 28.86, 25.86, 24.71, 22.01,

+

21.73, 13.88. HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₃H₂₆N₇O₂:

312.2142; found: 312.2154. MF: C₁₃H₂₅N₇O₂·C₄H₂F₆O₄. MW:

(311.39 + 228.05).

Competition binding experiments with hD₁and hD₅receptors were

performed by incubating homogenates in binding buﬀer⁴⁰at a ﬁnal

concentration of 0.3 μg (hD₁R) or 0.4 μg (hD₅R) protein/well together

with [³H]SCH23390 (hD₁R (K_d= 0.23 nM) and hD₅R (K_d= 0.2 nM):

c = 0.4 nM) and increasing concentrations of the competing ligands (6,

31, 47, and 54) for 120 min at room temperature. Binding studies at the

hD_2longR, hD₃R, and hD_4.4R were performed in binding buﬀer⁴⁰at a

concentration of 0.3 μg (hD_2longR), 0.7 μg (hD₃R), or 0.5−1.0 μg

1-(Amino{[3-(5-amino-1,3,4-oxadiazol-2-yl)propyl]amino}-

methylene)-3-(2-methyl-3-(p-tolyl)propyl)urea Dihydrotriﬂuoroa-

cetate (58). 58 was prepared from 109 (22 mg, 0.038 mmol, 1

equiv), KHCO₃(12.2 mg, 0.122 mmol, 3.2 equiv), and BrCN (3 M in

CH₂Cl₂, 15.3 μL, 0.046 mmol, 1.2 equiv) in H₂O/EtOH (0.5 mL: 0.5

mL) according to the general procedure, yielding the product as a

white, foamlike, and hygroscopic solid (13.97 mg, 61%). RP-HPLC:

1

100% (t_R= 15.8 min, k = 3.92). H NMR (600 MHz, DMSO-d₆) δ

(hD_4.4R) protein/well together with [³H]N-methylspiperone (hD_2long

R

10.13 (br s, 1H), 9.02 (br s, 1H), 8.52 (br s, 2H), 7.57 (t, J = 5.8 Hz,

1H), 7.36−7.01 (m, 6H), 3.34 (q, J = 6.7 Hz, 2H), 3.14−2.90 (m, 2H),

2.71 (t, J = 7.4 Hz, 2H), 2.60 (dd, J = 13.5, 6.1 Hz, 1H), 2.36−2.30 (m,

1H), 2.28 (s, 3H), 1.95−1.83 (m, 3H), 0.81 (d, J = 6.7 Hz, 3H). ¹³C

NMR (151 MHz, DMSO) δ 163.17, 158.59 (q, J = 34.7 Hz, TFA),

158.52, 153.72, 153.68, 136.95, 134.72, 128.80, 128.77, 116.05 (q, J =

293.9 Hz, TFA), 44.64, 39.59, 39.39, 34.85, 24.65, 21.73, 20.60, 17.08.

(K_d= 0.0149 nM): c = 0.05 nM; hD₃R (K_d= 0.0258 nM): c = 0.05 nM;

hD_4.4R (K_d= 0.078 nM): c = 0.1 nM) and varying concentrations of the

competing ligands (6, 31, 32, 46, 43, 47, 48, 53, 54, 56, 57, 59, 63, and/

or 69) for 60 min in the case of the hD_2longR and hD₃R and 140 min in

the case of the hD_4.4R at room temperature. Nonspeciﬁc binding was

determined in the presence of 2 μM (+)-butaclamol (Sigma,

Taufkirchen, Germany, hD₁R, hD_2longR, hD₃R) or nemonapride

(Tocris Bioscience, Bristol, U.K., hD_4.4R).

Muscarinic Acetylcholine M₁₋₅Receptors. Binding studies at

human muscarinic receptors (stably expressed in CHO-hM_xR cells, x

= 1−5) were performed using previously described radioligand

competition binding assays.⁷⁸

Binding aﬃnities toward the human adrenoceptors α_1A, α _2A, β₁, and

β₂as well as to the human serotonin receptor 5-HT_1Aand the human

opioid receptor μOR were determined as previously described.^79,80In

brief, membranes were prepared from HEK293T cells each transiently

transfected with appropriate cDNAs (cDNA of α_1A, β₂, and 5-HT_1A

from the cDNA Resource Center, Bloomsburg, PA, for α_2A: a gift from

the D. Yang, Chinese University of Hong Kong, Shenzhen, China, for

μOR: a gift from the Ernest Gallo Clinic and Research Center, UCSF,

CA, for β₁: a gift from the R. Sunahara, UCSD, CA). Receptor density

(B_maxvalue) and speciﬁc binding aﬃnities (K_Dvalue) for α_1Awere

determined as 6500 fmol/mg and 0.25 nM, respectively ([³H]prazosin

+

HRMS (ESI-MS): m/z [M + H⁺] calcd for C₁₈H₂₈N₇O₂: 374.2299;

found: 374.2300. MF: C₁₈H₂₇N₇O₂·C₄H₂F₆O₄. MW: (373.22 +

228.05).

Pharmacological Protocols: Cell Culture. Cells were maintained

in 25 or 75 cm²ﬂasks (Sarstedt, Nu

̈

mbrecht, Germany) in a humidiﬁed

atmosphere (95% air, 5% CO₂) at 37 °C. HEK293T-CRE-Luc-hD_2long

R

cells,⁴⁰HEK293T NlucN-mGs/gpH₂R-NlucC cells,²⁶HEK293T-

ARRB2-H₂R cells,⁴¹HEK293T ElucN-βarr2 hD_2longR-ElucC cells³⁹,

and HEK293T ElucN-βarr2 hD₃R-ElucC cells⁴⁰were cultured as

described previously.

Radioligand Competition Binding. Histamine H₁₋₄Recep-

tors.^22,38Competition binding experiments were performed with

membrane preparations of Sf9 insect cells, expressing the hH₁R +

86

RGS4,⁸⁵hH₂R-G_sαSfusion protein,⁴⁸hH₃R + G_αi2+ G_β1γ2

,

or the

87

hH₄R + G_αi2+ G_β1γ2

.

General procedures for the generation of

recombinant baculoviruses, the culture of Sf9 cells, and membrane

preparations have been described elsewhere.⁸⁸The competition

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(speciﬁc activity: 84 Ci/mmol, PerkinElmer, Rodgau, Germany) at a

concentration of 0.4 nM), for α_2A: 1800 fmol/mg and 0.60 nM,

respectively ([³H]RX821002 (spec. act. 52 Ci/mmol, Novandi

Chemistry AB) at 0.5 nM), for β₁: 3500 fmol/mg and 0.075 nM,

respectively ([³H]CGP12177 (spec. act. 52 Ci/mmol, PerkinElmer) at

0.2 nM), for β₂: 2000 fmol/mg and 0.070 nM, respectively

([³H]CGP12177 at 0.2 nM), for 5-HT_1A: 1100 fmol/mg and 0.080

nM, respectively ([³H]WAY600135 (spec. act. 80 Ci/mmol, Biotrend,

Cologne, Germany) at 0.2 nM), and μOR: 1700 fmol/mg and 0.090

nM, respectively ([³H]diprenorphine (spec. act. 31 Ci/mmol,

PerkinElmer) at 0.3 nM). Competition binding experiments with α_1A

and 5-HT_1Awere performed by incubating membranes in buﬀer A (50

mM Tris, 5 mM MgCl₂, 0.1 mM ethylenediaminetetraacetic acid

(EDTA), 5 μg/mL bacitracin, and 5 μg/mL soybean trypsin inhibitor at

pH 7.4) at a ﬁnal protein concentration of 1 μg/well and 6 μg/well

together with the radioligand and varying concentrations of the

competing ligands for 60 min at 37 °C. Binding at α_2Aand μOR was

performed in buﬀer B (50 mM Tris at pH 7.4) at a protein

concentration of 10 and 6 μg/well, respectively, and for β₁and β₂in

buﬀer C (25 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic

acid (HEPES), 5 mM MgCl₂, 1 mM EDTA, and 0.01% bovine serum

albumin (BSA) at pH 7.4) at a protein concentration of 2 and 4 μg/well,

respectively. Nonspeciﬁc binding was determined in the presence of 10

μM of prazosin (α_1A), RX821002 (α_2A), CGP12177 (β₁and β₂),

WAY600135 (5-HT_1A), and naloxone (μOR). The protein concen-

tration was established using the method of Lowry.⁸⁹The resulting

competition curves were analyzed by nonlinear regression using the

algorithms implemented in PRISM 6.0 (GraphPad Software, San

Diego, CA) to provide an IC₅₀value, which was subsequently

transformed into the K_ivalue, employing the equation of Cheng and

Prusoﬀ.⁴⁹

ﬁrst 14 amino acids of the N-terminus of the hH₂R were added to the

ﬁrst amino acid present in the receptor-part (E30) of the 3SN6 crystal

structure with a random conformation using the tool “Build Protein” of

Sybyl. The amino acid sequences RNETSKGNHTTSK (part of the E2

Loop) and GDD (part of the E3-Loop) were modeled using the “Loop

Search” Module. In addition, the amino acids NHISSWKAA (part of

the I3-Loop) were added using the “Loop Search” Module to close the

gap, which was present in the 3SN6 template, between TM5 and TM6.

The C-Terminus of the hH₂R, starting with C³⁵⁴, was not included in

the homology model because the C-terminus was not present in the

3SN6 template. The resulting active-state model of the hH₂R was

minimized with the Amber 7 FF99 force ﬁeld.

Since the compounds, for which the interaction with the hD₂R

should be analyzed, were also identiﬁed as partial agonists at the hD₂R

by functional studies, an active-state model of the hD₂R was used. For

this purpose, the crystal structure of the dopamine D₂receptor−G

protein complex (protein databank code: 6VMS) was used as a

template.⁵¹The Gαβγ-subunits (chain A, B, C) and the scFv16 (chain

E) were deleted. This model was reﬁned by exchanging all amino acids

of the template, which is diﬀerent from the corresponding amino acid of

the hD₂R in the correct amino acid of the hD₂R (tool: “Mutate

Monomers” of Sybyl). The N-terminus (the ﬁrst 31 amino acids) of the

hD₂R was not added. The I3-loop (R²²⁷−R³⁶⁰), which was not present

in the template structure, was not modeled. However, the gap between

TM5 and TM6 was already closed in the template by connecting K²²⁶

with R³⁶¹. The resulting active-state hD₂R was minimized with the

Amber 7 FF99 force ﬁeld. Based on the model of the hD₂R, the receptor

mutant hD₂R-E^E2.49V-I^E2.51S was generated, using the “Mutate

Monomers” tool to exchange E^E2.49into V and E^E2.51into S followed

by minimization as described above.

The compounds 6, 31, and 32, provided with the Gasteiger−Huckel

̈

[³⁵S]GTPγS Binding Assay. The assay was performed on Sf9

membranes expressing the hH₂R-G_sαSfusion protein as previously

described^22,38,48with one minor modiﬁcation: PBS (8 g NaCl, 0.2 g

KCl, 1.0 g Na₂HPO₄·2H₂O, 0.15 g NaH₂PO₄·H₂O, 0.1 g KH₂PO₄in 1

L Millipore H₂O; pH 7.4; 4 °C) was used as washing buﬀer while

harvesting instead of the previously used binding buﬀer. [³⁵S]GTPγS

was from Hartmann Analytics (Braunschweig, Germany). GTPγS and

GDP were purchased from Roche (Mannheim, Germany).

charges, were docked manually into the orthosteric binding pocket of

the respective receptor, considering mutations studies, described in the

literature, diﬀerences in the amino acid alignment of the hH₂R, hD₂R,

and hD₃R (Figure S146 in the SI), and the pharmacological results of

the present study. The net charge for 6, 31, and 32 was 1, with the

positive charge being located on the carbamoylguanidine group. The

resulting ligand−receptor complexes were minimized with the Amber 7

FF99 force ﬁeld.

Data Processing. Compound purities were calculated as the

percentage peak area of the analyzed compound by UV detection at 220

nm. Retention (capacity) factors (k) were calculated from retention

times (t_R) according to k = (t_R− t₀)/t_0,t₀= dead time. Data from

Mini-G Protein Recruitment Assay. The mini-G protein

recruitment assay at the hH₂R or gpH₂R was performed using

HEK293T NlucN-mGs/hH₂R-NlucC⁴²or HEK293T NlucN-mGs/

gpH₂R-NlucC²⁶cells as previously described in detail. Furimazine was

from Promega (Mannheim, Germany).

β-Arrestin2 Recruitment Assay. Histamine H₂Receptor. The β-

arrestin2 recruitment assay at the hH₂R was performed using

HEK293T-ARRB2-H₂R⁴¹cells, as previously described in detail.⁴⁶

Dopamine D_2long/3Receptors. The β-arrestin2 recruitment assay at

the hD_2longR or hD₃R was performed using HEK293T ElucN-βarr2

hD_2longR-ElucC or HEK293T ElucN-βarr2 hD₃R-ElucC cells, as

previously described in detail.⁴⁰Quinpirole was from Tocris Bioscience

(Bristol, U.K.). Pierce ^D-luciferin was purchased as the potassium salt

from Fisher Scientiﬁc GmbH (Schwerte, Germany).

Docking. Models of the Active-State hH₂R and Active-State hD₂R.

For the receptor modeling, docking studies, and presentation of the

results, Sybyl 7.3 software (Tripos Inc., St. Louis, MO) was used. Since

the compounds, for which the interaction with the hH₂R should be

analyzed, were experimentally identiﬁed as partial agonists at the hH₂R,

an active-state model of the hH₂R was generated by homology

modeling, using the crystal structure of the β₂adrenergic receptor−Gs

protein complex (protein databank code: 3SN6) as a template.⁵⁰The

Gαβγ-subunits (chain A, B, G), the endolysin, and the camelid antibody

variable domain of the heavy chain of the heavy-chain antibody (VHH)

fragment (chain N) were deleted. According to an appropriate

sequence alignment between hβ₂R and hH₂R, the homology model

was generated by exchanging all amino acids of the template, being

diﬀerent to the corresponding amino acid of the hH₂R sequence using

the tool “Mutate Monomers” of Sybyl, into the correct amino acid of

the hH₂R. N-terminus and loops (tool: “Loop Search” of Sybyl) were

modeled, according to procedures, as described elsewhere.⁹⁰Brieﬂy, the

38

40

radioligand competition binding assays (hH₁₋₄and hD_2long/3

receptors), from the [³⁵S]GTPγS binding assay,³⁸from the mini-G

41,46

40

protein (hH₂R,⁴²gpH₂R²⁶), or β-arrestin2 (hH₂

and hD_2long/3

receptors) recruitment assays, and from the H₂R assay on isolated

guinea pig right atrium³⁸were processed, as reported previously. K_i

values for the calculation of relative aﬃnities (H₂R selectivity, Tables 1

and 2) were obtained by transforming the pK_imean value to K_i(K_i=

10^−pK).

i

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00692.

■

sı

Experimental details for the amines 8−17 and the

guanidinylating reagents 18−29; experimental details

for the acylhydrazines 108 and 109; NMR spectra for the

carbamoylguanidines 30−70; RP-HPLC chromatograms

of compounds 30−70; RP-HPLC chromatograms:

chemical stability of 30−35, 37, 41, and 57; determi-

nation of the pK_avalue; radioligand competition binding

data; concentration−response curves obtained using β-

arrestin2 or mini-G protein recruitment assays; inves-

tigation of β-arrestin2 and mGs recruitment at

HEK293T-hH₂R cells for cmpds UR-CH20, UR-SB291,

UR-CH22, UR-Po563, UR-MB-69, UR-SB257, UR-

8705

https://doi.org/10.1021/acs.jmedchem.1c00692

J. Med. Chem. 2021, 64, 8684−8709

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Harald Hu

Medicinal Chemistry, Friedrich-Alexander-University of

Erlangen-Nurnberg, 91058 Erlangen, Germany

̈

bner − Department of Chemistry and Pharmacy,

KAT527, 30, 33−35, 37−42, 44−46, 49−52, 55, 58,

60−62, 64−68, and 70; bias analysis, docking; synthesis

and characterization of zolantidine dihydrotriﬂuoroace-

tate; estimation of the selectivity with respect to the

relative receptor expression in diﬀerent CNS tissues;

computation prediction of the membrane permeability;

and MS (ESI) data of Boc-/Trt-protected intermediates

(PDF)

̈

Peter Gmeiner − Department of Chemistry and Pharmacy,

Medicinal Chemistry, Friedrich-Alexander-University of

Erlangen-Nurnberg, 91058 Erlangen, Germany; orcid.org/

̈

0000-0002-4127-197X

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00692

Molecular formula strings, hH₁₋₄R and hD_2long/3binding

data, h/gpH₂R, and hD_2long/3functional data binding data

(CSV)

Author Contributions

M.B. and L.F. contributed equally to this work. K.T. and M.B.

performed the synthesis and analytical characterization of

chemical compounds. K.T. performed the investigation of the

chemical stability. M.B. determined the pK_avalue for the 2-

aminothiadiazole. K.T. and M.B. performed radioligand

competition binding experiments at H_1‑4Rs and analyzed the

data. L.F. performed radioligand competition binding experi-

ments at D₁₋₅Rs and analyzed the data. D.M. generated the

HEK293T-CRE-Luc-hD₅R cell line and prepared the corre-

sponding homogenates. K.T. performed radioligand competi-

tion binding experiments at M₁₋₅R and analyzed the data. H.H.

and P.G. performed the radioligand binding studies at the

human adrenoceptors α_1A, α_2A, β₁, and β₂as well as to the human

serotonin receptor 5-HT_1Aand the human opioid receptor μOR

and analyzed the data. K.T. performed the functional studies at

H₂R and analyzed the data, with the exception of guinea right

atrium experiments, which were performed and analyzed by

M.B. L.F. and K.T. performed and analyzed functional studies at

D_2long/3Rs. H.-J.W. and A.S. performed molecular docking and

processed the data. A.B. and A.S. initiated and planned the

project. S.P. and A.S. supervised the research. K.T., S.P., and A.S.

wrote the manuscript. All authors have given approval to the

ﬁnal version of the manuscript.

Three-dimensional (3D) coordinates of the hD₂R in

complex with 6, obtained through molecular docking; 3D

coordinates of the hD₂R in complex with 31, obtained

through molecular docking; 3D coordinates of the hD₂R

in complex with 32, obtained through molecular docking;

3D coordinates of hD₂R-E249V-I251S in complex with 6,

obtained through molecular docking; 3D coordinates of

hD₂R-D249V-I251S in complex with 31, obtained

through molecular docking; 3D coordinates of hD₂R-

E249V-I251S in complex with 32, obtained through

molecular docking; 3D coordinates of the hH₂R in

complex with 6, obtained through molecular docking; 3D

coordinates of the hH₂R in complex with 31, obtained

through molecular docking; 3D coordinates of the hH₂R

in complex with 32, obtained through molecular docking;

3D coordinates of hH₂R-D542S in complex with 31,

obtained through molecular docking; and 3D coordinates

of hH₂R-D542S in complex with 32, obtained through

molecular docking (ZIP)

AUTHOR INFORMATION

■

Corresponding Authors

Steﬀen Pockes − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany; Department of

Neurology, University of Minnesota, Minneapolis, Minnesota

55455, United States; Department of Medicinal Chemistry,

Institute for Therapeutics Discovery and Development,

University of Minnesota, Minneapolis, Minnesota 55414,

United States; orcid.org/0000-0002-2211-9868;

Phone: (+49)941 943-4814/4825; Email: ste ﬀ en.pockes@

ur.de; Fax: (+49)941 943-4809

Andrea Strasser − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany; orcid.org/

0000-0002-3994-3079; Phone: (+49)941 943-4821/4694;

Email: andrea.strasser@ur.de; Fax: (+49)941 943-4820

Funding

This work was funded by the Graduate Training Program

(Graduiertenkolleg) GRK1910, “Medicinal chemistry of

selective GPCR ligands”, of the Deutsche Forschungsgemein-

schaft (K.T., A.B., H.H., P.G., A.S.).

Notes

The authors declare no competing ﬁnancial interest.

^⊥Deceased, July 18, 2017.

ACKNOWLEDGMENTS

■

The authors are grateful to Christine Gebhardt-Braun and

Kerstin Röhrl for carrying out guinea pig right atrium

experiments, Brigitte Wenzl for excellent technical assistance,

and Dr Max Keller for supporting the muscarinic receptor

binding studies. We thank Dr Anja Kraus for the synthesis of 3-

methyl-6-phenylhexanoic acid and 3-cyclohexylbutanoic acid.

We also thank Claudia Honisch, Drs Sabrina Biselli and Nicole

Plank for synthesis and providing the compounds UR-CH22,

UR-SB257, UR-SB291, and UR-NK22. We thank Dr Johannes

Felixberger for proving the HEK293T-ARRB2-H₂R cell line and

Carina Höring for providing the HEK293T NlucN-mGs/hH₂R-

NlucC and the HEK293T NlucN-mGs/gpH₂R-NlucC cell lines.

We thank Josef Kiermaier and Wolfgang Söllner for mass

spectrometry analysis. We thank Fritz Kastner for NMR

measurements.

Authors

Katharina Tropmann − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany

Merlin Bresinsky − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany

Lisa Forster − Institute of Pharmacy, University of Regensburg,

D-93053 Regensburg, Germany

Denise Mönnich − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany

^⊥Armin Buschauer − Institute of Pharmacy, University of

Regensburg, D-93053 Regensburg, Germany; orcid.org/

0000-0002-9709-1433

Hans-Joachim Wittmann − Institute of Pharmacy, University

of Regensburg, D-93053 Regensburg, Germany

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(3) Dove, S.; Elz, S.; Seifert, R.; Buschauer, A. Structure-activity

relationships of histamine H₂receptor ligands. Mini-Rev. Med. Chem.

2004, 4, 941−954.

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59, 290−299.

ABBREVIATIONS

■

5-HT_1A, serotonin 1A receptor; α_1A, adrenoreceptor α 1A; α_2A,

adrenoreceptor α 2A; β₁, adrenoceptor β₁, adrenoceptor β₂,

βarr2 or ARRB2, β-arrestin2 gene, and protein; δ, chemical shift

in ppm; μOR, μ opioid receptor; AU, absorption units; BB,

building block; c, molar concentration; CDCl₃, deuterated

chloroform; cf., confer/conferatur; CRE-Luc, cAMP-response

element driven transcriptional luciferase reporter; CH₂Cl₂,

dichloromethane; CHO, Chinese hamster ovary; CNS, central

nervous system; cmpd, compound; DAD, diode array detector;

DMSO-d₆, deuterated DMSO; D_xR, dopamine receptor subtype

x; E, extracellular loop; EI, electron ionization; Eluc, Emerald

luciferase; ElucC, C-terminal Eluc fragment; ElucN, N-terminal

Eluc fragment; E_max, maximal inducible receptor response

referenced to a standard compound; Et₂O, diethyl ether;

EtOAc, ethyl acetate; EtOH, ethanol; FLAG, polypeptide

protein tag with sequence motif DYKDDDDK; g, gram(s) or

number of times the gravitational force; G418, geneticin; G_αi2, α-

subunit of the G_i2protein that mediates the inhibition of

adenylyl cyclase; G_β1γ2, G protein β₁- and γ₂-subunit; gp, guinea

pig; Gs, adenylyl cyclase stimulatory G protein; G_sαS, α-subunit

(short splice variant) of the Gs protein that mediates stimulation

of adenylyl cyclase; GTPγS, guanosine 5′-O-[γ-thio]phosphate;

h, human; HEK293T, human embryonic kidney 293T cells; Het,

heterocycle; H_xR, histamine receptor subtype x; k, retention (or

capacity) factor (HPLC); K_a, acid dissociation constant; K_b,

dissociation constant obtained from a functional assay; K_d,

dissociation constant obtained from a saturation binding

experiment; K_i, dissociation constant obtained from a

competition binding experiment; LC, liquid chromatography;

MeOD, deuterated methanol; MeCN, acetonitrile; MeOH,

methanol; MF, molecular formula; mG or mini-G, engineered

minimal G protein; mGs, engineered guanosine triphosphate

hydrolase domain of Gα_ssubunit (long splice variant); L-DOPA,

levodopa; M_xR, muscarinic receptor subtype x; NEt₃, triethyl-

amine; Nluc, NanoLuc luciferase; NlucC, C-terminal Nluc

fragment; NlucC, N-terminal Nluc fragment; PE, petroleum

ether; pEC₅₀, negative logarithm of the half-maximum activity

concentration in M; pH, potential or power of hydrogen; pK_a,

negative logarithm of the K_ain M; pK_b, negative logarithm of the

K_bin M; pK_i, negative logarithm of the K_iin M; r, rat; R, residue;

RGS4, regulator of G protein signaling proteins 4; RP-HPLC,

reversed-phase HPLC; Q-TOF, quadrupole time of ﬂight;

scFv16, single-chain variable fragment derived from mAb16

antibody; SEM, standard error of the mean; SF, SuperFlash; Sf9,

Spodoptera frugiperda insect cell line; SI, Supporting Informa-

tion; SP, signal peptide: subunit A of the murine type 3 receptor

for 5-hydroxytryptamine; spec. act., speciﬁc activity; t₀, dead

time; t_R, retention time; Tris, tris(hydroxymethyl)-

aminomethane; Trt, trityl; UHD, ultrahigh deﬁnition; VHH,

variable domain of heavy chain of heavy-chain antibody

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https://doi.org/10.1021/acs.jmedchem.1c00692

J. Med. Chem. 2021, 64, 8684−8709

Article Doi

Abolishing Dopamine D2long/D3Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2Receptor Agonists

DOI: 10.1021/acs.jmedchem.1c00692

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Full text of DOI:10.1021/acs.jmedchem.1c00692

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