Covalent inhibition of the histamine H3 receptor

Article

Covalent Inhibition of the Histamine H

3

Receptor

1,†

1

Gábor Wágner , Tamara A. M. Mocking , Albert J. Kooistra , Inna Slynko ,

2

1

Péter Ábrányi-Balogh , György M. Keserű , Maikel Wijtmans , Henry F. Vischer ,

1

1,

Iwan J. P. de Esch and Rob Leurs *

1

Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry,

Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108,

1081 HZ Amsterdam, The Netherlands; gaborwagner75@gmail.com (G.W.);

t.a.m.mocking@vu.nl (T.A.M.M.); kooistra.a.j@gmail.com (A.J.K.); innaslynko@gmail.com (I.S.);

m.wijtmans@vu.nl (M.W.); h.f.vischer@vu.nl (H.F.V.); i.de.esch@vu.nl (I.J.P.d.E.); r.leurs@vu.nl (R.L.)

Medicinal Chemistry Research Group, Research Centre for Natural Sciences, Hungarian Academy of

2

Sciences, Magyar tudósok krt 2, H-1117 Budapest, Hungary; abranyi-balogh.peter@ttk.mta.hu (P.Á.-B.);

keseru.gyorgy@ttk.mta.hu (G.M.K.)

* Correspondence: r.leurs@vu.nl; Tel.: +31-205987579

† These authors contributed equally to this work.

Received: 17 November 2019; Accepted: 8 December 2019; Published: 11 December 2019

Abstract: Covalent binding of G protein-coupled receptors by small molecules is a useful approach

for better understanding of the structure and function of these proteins. We designed, synthesized

and characterized a series of 6 potential covalent ligands for the histamine H

Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed

by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H

3

receptor (H

3

R).

3

R

ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water

and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with

nucleophilic residues. The covalent interaction of 44 with H

experiments and leads to inverse agonism on H R. Irreversible binder 44 (VUF15662) may serve as

a useful tool compound to stabilize the inactive H R conformation and to study the consequences

of prolonged inhibition of the H R.

3

R was validated with washout

3

Keywords: G protein-coupled receptor (GPCR); Histamine H

3

receptor; covalent binder;

isothiocyanate

1. Introduction

The histamine H

representative of the G protein-coupled receptor (GPCR) subfamily of histamine receptors (now

known to consist of H R, H R, H R, and H R). As an autoreceptor, H R presynaptically modulates the

3

receptor (H R) was discovered in 1983 by Arrang et al. [1] as the third

3

1

2

3

4

3

synaptic histamine level, while as a heteroreceptor it regulates the levels of other neurotransmitters

(e.g., acetylcholine, noradrenaline, serotonin, dopamine, glutamate). Due to the high expression in

brain areas such as the cerebral cortex, hypothalamus tuberomammillary nucleus, hippocampus, and

striatum, H

wake cycle, learning and memory processes, food intake, and susceptibility to seizures [2–4]. As such,

R attracts considerable interest as a therapeutic target from industry and academia alike. The

cloning of the human receptor in 1999 by Lovenberg et al. [5] and the discovery of several structurally

diverse drug-like ligands in the 2000s [6,7] gave a strong impetus to H R research. Although this

boom resulted in a large amount of novel H R ligands, several clinical trials ongoing in the early 2010s

[8,9] have so far resulted in only one approved H R ligand (Wakix, approved in 2016 by EMA and in

2019 by FDA) [10,11] suggesting that H R pharmacology is not yet fully understood and

3

R regulates several physiological processes of the central nervous system including sleep-

H

3

complementary approaches are needed to pave the way forward.

Molecules 2019, 24, 4541; doi:10.3390/molecules24244541

www.mdpi.com/journal/molecules

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One such approach is the use of covalent small-molecule binders, which can for example help in

the stabilization of H³R in the active or inactive conformation. This in turn could aid in crystallization

of a ligand-receptor complex (no crystal structure of H³R has been published to date), as well in the

exploration of the binding site, conformation, and signaling properties of the protein. Targeted

covalent inhibitors (TCIs) are ligands designed to bind irreversibly to a nucleophilic amino acid

residue when bound to the targeted protein. A typical strategy of TCI design is linking a

pharmacophore moiety (anchor) that leads the ligand into the binding pocket to a reactive moiety

(warhead) which reacts with a residue creating a covalent bond [12,13]. In the field of kinases the use

of TCIs has resulted in marketed drugs in past years [12], while the progress in covalent GPCR

ligands is still limited in comparison [13]. Nonetheless, aided by advances in GPCR crystallography,

covalent ligands targeting the orthosteric binding site of GPCRs have been disclosed [14–23]. The

targeted GPCR residue is mostly a cysteine with diverse warheads being used, such as a disulfide

[17,19,23], isothiocyanate [18], or Michael acceptor [15]. Other residues have been targeted as well

with various warheads, i.e., lysine with activated ester [16] or arylsulfonyl fluoride [20], tyrosine with

an arylsulfonyl fluoride [22], or asparagine with an aziridinium ion [17]. Indeed, recent extensions of

the diversity in warheads give an opportunity for the fine-tuning of the reactivity [24,25].

While no covalent ligand for H³R has been reported to date, the use of covalent strategies within

the histamine receptor family is not without precedent. In the 1940s, covalent histamine-protein

complexes were explored for pharmacological investigation using different warheads on histamine

[26,27]. More recently, mutated H¹R has been targeted by a disulfide-containing ligand [17].

Furthermore, our group has reported the discovery of a H⁴R covalent ligand VUF14480 (1, Figure 1)

which acts as a partial H⁴R agonist [15]. Its submicromolar affinity (pKi = 6.3) for human H4R is high

enough to ensure sufficient recognition by the receptor, whereupon the ethenyl moiety acts as a

3.36

Michael acceptor forming a covalent interaction with C98 [15].

In this study, we describe the design and synthesis of covalent H³R ligands, ultimately resulting

in the discovery of a covalent inverse agonist targeting the H³R.

2. Results

2.1. Design

Arguably, a H⁴R covalent ligand can be a suitable starting point for covalent H3R ligands given

the high homology of H⁴R and H3R (43% full sequence identity, 50% predicted transmembrane

bundle identity) [28]. Hence, the covalent interaction of Michael acceptor VUF14480 (1) with H⁴R [15]

was used as a template for the design of potential H³R covalent ligands (Figure 1). Similarly to the

3.36

H⁴R binding pocket, C is also accessible in the H3R binding pocket (C118 ) to potentially serve as

nucleophilic moiety [29] (throughout this manuscript, we use both the UniProt residue numbers and

the GPCR-specific Ballesteros–Weinstein residue numbering scheme [30]). Given the relatively high

3.36

similarity of the H³R and H4R orthosteric binding pockets [29], the C118 residue of H3R might be

targeted with a similar combination of anchor, central core and warhead moieties as 1 contains.

Several design cycles were needed to tune this scaffold into a H3R covalent ligand suitable for our

purposes. In-house results (unpublished data) suggested that H⁴R/H3R selectivity of this scaffold

could be switched from H⁴R to H3R by suitable modification of the anchor and substituent pattern of

1

pyrimidine core. Thus, the aim of the first design cycle was the identification of a suitable anchor (R ).

Three different anchors were chosen based on publicly available and in-house data: (1) replacement

of the methyl substituent by a cyclobutyl group [31], (2) replacement of the piperazine moiety by a 3-

(methylamino)azetidine moiety, (3) replacement of the piperazine moiety by

a

3-

(dimethylamino)azetidine moiety to assist in synthetic feasibility (vide infra). Based on in-house

results, the phenyl group at Position 6 was changed to an isopropyl group to favor H³R selectivity.

Both the pyrimidine ring as central core and the vinyl group as warhead were unchanged. Instead,

2

the warhead (R ) was addressed in the second design cycle whilst the group at Position 6 was changed

to a methyl group to prevent the more lipophilic isopropyl moiety from targeting a lipophilic

subpocket next to the cysteine, thereby preventing the warhead from achieving its appropriate

Molecules 2019, 24, 4541

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orientation. The vinyl unit was changed to an ethynyl moiety in order to potentially improve the

orientation of the reactive carbon atom. Isothiocyanate and halomethylamido derivatives were also

designed. Notably, isothiocyanates are known to have high reactivity toward cysteines and reduced

albeit still substantial reactivity toward lysines [13,32,33] depending on the pH, while

chloroacetamides show reactivity only to cysteines [13]. Yet in our work, both warhead types caused

synthetic obstacles (vide infra) because of too high chemical reactivity that we attribute to the

electron-withdrawing pyrimidine nitrogen atoms. This reactivity issue combined with biological

inactivity of other compounds in this second cycle (vide infra) necessitated a third design cycle

comprising of scaffold hopping to the less electron-withdrawing phenyl ring as central core. This

switch in core was supported by a ChEMBL search on H³R ligands containing a phenyl group

connected with a 3-carbon linker to a basic amino moiety. Figure 2 illustrates the envisioned strategy

3.36

key to the third design cycle, with C118 forming a covalent bond with the isothiocyanate moiety.

2

The binding mode of a representative ligand from this design cycle (Figure 1 top right, R = -NCS)

was evaluated by covalent docking using GOLD (version 5.2.1). The binding site of the H³R protein

is characterized by two acidic residues that are known to interact with basic groups of the ligands,

3.32

5.46

3.32

i.e., D114 and E206 . Residue D114 is considered the anchoring point for the basic amines of

the endogenous ligands of aminergic GPCRs [34]. Residues in TM5 are also involved in ligand

5.46

binding and in the case of H³R it is residue E206 that is anticipated to interact with the imidazole

ring of the endogenous agonist histamine [35]. Interestingly, the predicted binding mode of the

isothiocyanate analog is flipped to allow covalent bond formation between the isothiocyanate and

3.36

5.46

C118 , resulting in a binding mode in which the basic piperazine moiety interacts with E206

through an ionic interaction. The nitrogen atom of the dithiocarbamate that is formed after covalent

3.32

bond formation is able to interact with D114 (Figure 2) through a hydrogen bond.

When studying ligands that form covalent bonds with protein targets, it is essential for

pharmacological characterization to have proper non-reactive reference compounds. In our previous

work on H⁴R ligand 1, the 2-methyl-analog 2 had served as non-covalent control [15]. Based on steric

and electronic considerations, in the current work several 2-methyl, 2-ethyl and 2-amino-derivatives

of the designed compound set were investigated as controls.

Figure 1. The three design cycles of the potential H³R covalent ligands and their non-covalent

reference compounds. Red compounds proved too unstable to be isolated in pure form.

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Figure 2. Proposed binding mode of a representative compound from the third design cycle (Figure

1 top right, R²= -NCS) covalently bound to C118^3.36. The ligand is shown in cyan sticks and the protein

is shown in light yellow sticks and ribbons. Only polar hydrogens are shown in white with protein-

ligand hydrogen bonding indicated in black dashed lines. For clarity purposes, parts of the protein

are not shown. Residue numbers are shown as UniProt and as GPCR-specific Ballesteros–Weinstein

numbers [30].

2.2. Synthesis

The compounds from Design Cycles 1 and 2 were synthesized as outlined in Schemes 1 and 2.

The core intermediates 5 and 6 were obtained from the appropriate pyrimidin-4(3H)-one derivatives

3 or 4 with POCl . The Boc-protected intermediates 7–9 were obtained via nucleophilic aromatic

3

substitution on 5 or 6 with aminoazetidine or piperazine moieties under microwave heating at 150

°C. Two 2-amino derivatives (10 and 11) were directly synthesized from N,N-dimethylazetidin-3-

amine or 1-cyclobutylpiperazine via an aromatic substitution on 5 or 6, while the other amino

derivative 12 was synthesized from the Boc-protected intermediate 9 via deprotection under acidic

conditions followed by a reductive amination. Pyrimidines 7–9 were transformed to 2-chloro-

pyrimidine intermediates 13–15 by nonaqueous diazotization with tBuONO or iPeONO and SbCl

[36]. Boc-protection was necessary for this step to prevent by-products. Formation of chlorides 13–15

was followed by deprotection under acidic condition and reductive amination with H CO or

3

2

cyclobutanone to afford the intermediates 16–18. A silyl-protected ethynyl group was incorporated

via a Sonogashira coupling on intermediate 18 to give 19, followed by removal of the protective group

to yield ligand 20. The vinyl derivatives 21–23 were obtained via Suzuki-Miyaura cross-coupling with

vinyl boronic acid N-methyliminodiacetic acid (MIDA) ester on the 2-chloro-pyrimidines 16–18 in a

4

microwave at 120 °C. The synthesis of the 3-(methylamino)azetidine analog of 21 (i.e., with R = H)

was undertaken (not shown), although we anticipated a modest stability because of the simultaneous

presence of nucleophilic and electrophilic moieties in the product. Therefore, we designed a protocol

4

in which deprotection on a precursor having R = Boc was carried out in a hot D

2

O/d6-DMSO mixture

[37] with the intent to use the solution as is for pharmacological purposes. However, even though

product was formed (typically ca. 70% conversion), overall conversion and impurity profiles were

not acceptable for further pursuit.

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Scheme 1. Synthesis of pyrimidine-based covalent ligands. a) POCl3, reflux, 3 h, 26–33%; b) N,N-

dimethylazetidin-3-amine or 1-cyclobutylpiperazine, N,N-Diisopropylethylamine (DIPEA), dioxane,

150 °C, 30 min, μW, 59–65%; c) t-butyl azetidin-3-yl(methyl)carbamate or t-butyl piperazine-1-

carbamate, DIPEA, dioxane, 150 °C, 30 min, μW, 50–85%, 8: used without full purification; d) 1. HCl,

dioxane, rt, 2 h, 2. cyclobutanone, NaBH(OAc)³, dichloromethane (DCM), rt, overnight, 33% (two

steps); e) tBuONO or iPeONO, SbCl³, DCM, rt, 3 h, 23–40%; f) 1. HCl, dioxane, rt, 2 h-overnight, 2.

H²CO or cyclobutanone, NaBH(OAc)3, DCM, rt, overnight, 56–73% (two steps); g)

ethynyltrimethylsilane, CuI, Pd(dppf)Cl², TEA, DME, 100 °C, 1 h, μW, 54%; h) K2CO3, MeOH, rt, 1 h,

72%; i) vinyl boronic acid MIDA ester, Pd(PPh³)4, Na2CO3, DME, H2O, 120 °C, 1 h, μW, 21–62%.

Non-covalent 2-alkyl-pyrimidines were synthesized with similar synthetic steps, as outlined in

Scheme 2. Condensation of the appropriate amidines 24 or 25 and 4-methyl-3-oxopentanoate to 26

and 27 was followed by conversion to chloro-analog 28 and 29 with POCl³. A nucleophilic aromatic

substitution on 28 or 29 with t-butyl azetidin-3-yl(methyl)carbamate was performed in a microwave

at 150 °C and the obtained Boc-protected intermediate 30 and 31 were deprotected resulting in the

monomethyl intermediates 32 and 33. These in turn were subjected to a reductive amination with

H²CO to afford 34 and 35. Likewise, cyclobutyl-piperazine products 36 and 37 were obtained from

28 or 29 and 1-cyclobutylpiperazine.

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Scheme 2. Synthesis of pyrimidine-based reference ligands. a) methyl 4-methyl-3-oxopentanoate,

NaOMe, MeOH, rt, overnight, 91–97%; b) POCl³, reflux, 3 h, 86–87%; c) t-butyl azetidin-3-

yl(methyl)carbamate, DIPEA, dioxane, 150 °C, 30 min, μW, 34–38%; d) HCl, dioxane, rt, 3 h-overnight,

77–90%; e) H²CO, NaBH(OAc)3, DCM, rt, overnight, 82%; f) 1-cyclobutylpiperazine, DIPEA, dioxane,

150 °C, 30 min, μW, 36–51%.

In Design Cycle 2, we also attempted to prepare the designed isothiocyanate and

chloroacetamide derivatives from compound 12 via reaction with CSCl²or 2-chloroacetylchloride,

but all attempts were met by complex product profiles. We attribute this to the too high chemical

reactivity of the warhead (vide supra), leading to decomposition during workup and purification.

All derivatives with phenyl as core ring (i.e., those from the third design cycle) were synthesized as

outlined in Scheme 3. Compound 39 was built via a Buchwald–Hartwig amination of bromide 38

with t-butyl piperazine-1-carboxylate. Deprotection to 40 and reductive amination with

cyclobutanone yielded 41. The nitro group of 41 was reduced with HCOONH⁴and Pd/C to afford

key intermediate 42, which was used for incorporating reactive warheads under basic conditions.

Chloroacetamide 43 was formed with 2-chloroacetyl chloride, while isothiocyanate 44 was formed

with CSCl². Synthesis of the bromo analog of 43 was also attempted under similar conditions, but the

product decomposed during purification. All potential covalent ligands (20–23, 43, 44) were stored

as solids in the freezer (−20 °C). Under these conditions, key compound 44 was stable for >2 years (as

judged by NMR and LC-MS analysis).

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Scheme 3. Synthetic of phenylpiperazine derivatives. a) t-butyl piperazine-1-carboxylate, NaOtBu,

Pd²(dba)3, Xantphos, dioxane, 80 °C, 1 h, μW, 57%; b) HCl, dioxane, rt, 1 h, 78%; c) cyclobutanone,

NaBH(OAc)³, DCM, rt, overnight, 76%; d) HCOONH4, Pd/C, MeOH, H2O, rt, overnight, 25%; e) 2-

chloroacetyl chloride, TEA, DCM, rt, 1 h, 68%; f) CSCl², NaHCO3, DCM, H2O, rt, 1 h, 64%.

2.3. Structure-Activity Relationship

3

Compounds were evaluated for H³R binding in a [ H]N-α-methylhistamine ([ H]NAMH)

displacement assay on H³R-expressing HEK293T cell homogenates. The first step of a covalent ligand

binding is the reversible binding to the binding site, which is followed by the formation of the

covalent bond in case of appropriate orientation of the reactive warhead [13]. Consequently, binding

equilibrium cannot be established and therefore an ‘apparent’ affinity (pKⁱ) is reported for potential

covalent ligands 1, 20–23, 43 and 44 (Table 1). To identify potential irreversible binders, 100 μM ligand

was pre-incubated with human H³R (hH3R)-expressing cell homogenates for 1 h at 25 °C prior to

3

vigorous washout of the compounds. The washed membranes were then subjected to a [ H]NAMH

3

displacement assay to determine the amount of receptors that are still available for [ H]NAMH

binding, which is taken as a measure of potential covalent H³R labeling (Table 1).

We expected that H⁴R covalent binder 1 [15] can also covalently bind H3R due to the high

3.36

sequence similarity with H⁴R and the conservation of the anchor residue C [29]. Although ligands

1 and 2 showed both comparable micromolar affinity for hH⁴R (pKi 6.3 and 6.4, respectively) [15],

ligand 2 showed a remarkable increase in H³R binding affinity (pKi: 7.1), while 1 showed only weak

3

H³R affinity (pKi < 5). Despite its weak apparent affinity, binding of [ H]NAMH to H3R-expressing

cell homogenates pre-incubated with 1 after washout resulted in only 41% occupancy of the receptor

3

population by [ H]NAMH, suggesting that 1 irreversibly binds to hH3R since binding of its non-

covalent analog 2 is reversible (Table 1).

In the first design cycle, two H³R aminopyrimidine anchor-moieties (R = B or C) were combined

with an ethenyl-warhead at Position 2 of pyrimidine core (R ) and an iPr moiety as R (Table 1). Good

1

2

3

2

binding affinity was obtained for the amino-derivative 10 while substitution at the R position by an

alkyl (34, 35), as well as ethenyl moiety (21), significantly reduced the binding affinity (pKⁱ< 5).

3

Washout of 10, 34, 35 and 21 all resulted in high occupancy of the H³R with [ H]NAMH, indicating

that receptors are not covalently labeled by 21 nor, as expected, by its non-covalent analogs 10, 34

3

2

and 35. To improve H³R binding affinity, the same compound set (R = iPr; R = NH2, Me, Et, ethenyl)

1

was synthesized with a cyclobutyl-piperazine moiety as anchor (R = C). Although the affinity of the

amino-analog 11 was reduced compared to 10, the affinities of non-covalent alkyl derivatives 36 and

37 as well as of ethenyl-derivative 22 increased to the micromolar range with respect to their

counterparts in series B (Table 1). Washout experiments on non-covalent analogs 11, 36 and 37 show

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3

reversibility, while 22 decreased the amount of receptors available for [ H]NAMH binding, indicating

that a covalent interaction had been formed between 22 and H³R. To further improve both affinity

3

2

and covalent labeling, optimization of core substitution (R ) and warhead (R ) were undertaken in

3

the second design cycle. Reducing the iPr moiety to a smaller Me substituent (R = Me) resulted in

similar binding affinities for both the non-covalent (compare 12 to 11) as well as covalent ethenyl

(compare 23 to 22) analog. Similar to 22 and 11, compound 23 led to covalent labeling whereas 12 did

not. In contrast, the ethynyl analog 20 displayed poor affinity and is incapable of inhibiting

3

[ H]NAMH binding after washout, suggesting lack of irreversible binding presumably due to

inappropriate orientation of the reactive electrophilic carbon. We explored more reactive warheads

in order to improve upon the covalent H3R labeling by 22 and 23.

Table 1. (Apparent) binding affinity (pKi) of ligands for hH3R expressed on HEK293T cell

homogenates, and the amount of unlabeled receptors remaining after 1 h preincubation of hH³R

expressing membranes with 100 μM ligand and subsequent washout as determined by radioligand

binding. Ligands are considered potential covalent binders when % unlabeled receptors remaining

after 1 h pre-incubation with ligand was <50%. Data are mean ± S.E.M of 3 experiments performed in

triplicate with the exception of 1 for which mean ± SD is reported for two experiments performed in

triplicate.

Cmpd.

Central

Ring

R¹

R²

R³

pKi

% Unlabeled

Receptors

2

Pyrimidine

Phenyl

A

B

Me

CH=CH2

NH²

phenyl

iPr

Me

-

7.1 ± 0.1

< 5

84 ± 16

41 ± 6 (SD)

100 ± 0.0

87 ± 13

93 ± 7

1

10

34

35

21

11

36

37

22

12

23

20

42

43

44

7.3 ± 0.1

< 5

B

Me

B

Et

< 5

B

CH=CH²

NH²

< 5

85 ± 15

96 ± 4

C

6.7 ± 0.1

5.9 ± 0.1

5.5 ± 0.1

5.5 ± 0.0

6.5 ± 0.1

5.4 ± 0.1

< 5

Me

96 ± 3

Et

89 ± 11

41 ± 9

CH=CH²

NH²

91 ± 5

CH=CH²

C≡CH

NH²

40 ± 8

80 ± 2

5.9 ± 0.3

6.2 ± 0.1

6.5 ± 0.3

72 ± 8

Phenyl

NH-CO-CH²-Cl

N=C=S

-

86 ± 5

Phenyl

-

15 ± 2

Synthesis of analogs of 22 and 23 with isothiocyanate- and chloromethylketone warheads was

attempted, but combining these warheads with the electron-withdrawing pyrimidine core was

synthetically challenging. Therefore, in the third and final design cycle, the pyrimidine core was

changed to a phenyl group. While use of the phenyl core still resulted in micromolar affinities for

non-covalent amino analog 42, the apparent affinity of potential covalent derivatives 43 and 44 was

improved with respect to 22 and 23 (Table 1). Non-covalent control 42 and chloroacetamide 43

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showed similarly low labeling in washout experiments, for the latter suggesting inappropriate

orientation of the electrophilic carbon or low reactivity. Gratifyingly, though, after washout of 44 only

3

15% of the receptor population was available for [ H]NAMH binding, suggesting that an efficient

formation of a covalent interaction with the H³R was formed.

2.4. Covalent Binding to Glutathione and Nonapeptide

Given the suggested covalent binding to the H³R (Table 1), key isothiocyanate 44 was tested for

its chemical stability and reactivity with glutathione (GSH) under the conditions of the

pharmacological experiments. Compound 44 (0.40 mM) was dissolved in Tris buffer (50 mM Tris-

HCl, pH 7.4) in absence or presence of GSH (1.2 mM). The mixtures were incubated for 10–100 min

at room temperature and analysed by LC-MS (Figure 3). Interestingly, the stability experiments

showed a very slow reaction with Tris. That is, while 44 remained intact after 10 min incubation

(Figure 3A), a very minor adduct of 44 and Tris (45) was visible (1.9%) after 100 min incubation

(Figure 3B). Indeed, phenyl isothiocyanates are known to display reactivity towards amines and

thiols albeit at different rates (vide supra) [12,32], but exert low reactivity towards alcohols [38].

Therefore, we postulate that the isothiocyanate moiety of 44 binds the amino group of Tris to afford

adduct 45 (Figure 3D). Yet, the still high integrity of 44 (95%) at the pharmacologically relevant

incubation time (100 min) indicates an appropriate stability of 44 in buffer for our pharmacology

assays. Co-incubation with GSH in a 1:3 (44:GSH) molar ratio shows high reactivity, with the GSH

adduct 46 visible at high conversion (91.5%) after 10 min incubation (Figure 3C). The reaction of 44

with GSH is postulated to proceed via the thiol group (Figure 3D).

The selectivity of 44 toward nucleophilic residues was further investigated with an oligopeptide

KGDYHFPIC. This nonapeptide (NP) is designed to harbor an array of nucleophilic residues, i.e.,

cysteine, lysine, tyrosine, aspartate and histidine [25]. Analysis by LC-MS/MS can identify the

binding to a particular residue from fragmentation patterns. NP and 44 were incubated in phosphate-

buffered saline pH 7.4 with 10% MeCN at rt for 16 h. LC-MS analysis indicates a reaction with the

NP as evidenced by a NP+44+H+ ion at m/z 1351.5 as well as its doubly-charged ion at m/z 676.5 in

the peak at 4.3 min (Figure S1A). The MS/MS spectrum of the two-fold charged NP modified by 44

(m/z 676.5) shows a fragmentation pattern indicative of the cysteine monoadduct of 44 (Figure 4),

suggesting selective binding of the isothiocyanate moiety of 44 to the cysteine residue. The non-

covalent analog 42 does not show any reaction with NP (Figure S1B).

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Figure 3. LC-MS analysis of 44 (0.40 mM) in 50 mM Tris-HCl pH 7.4 without (A, B) and with (C) GSH

(1.2 mM). The mixtures were incubated for 10 min (A, C) or 100 min (B) at rt. The incubated mixtures

were analysed by LC-MS. A signal from the DMSO used for the stock solution of 44 is visible at 1.2

min. (D) Postulated identified products.

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Figure 4. MS/MS spectrum of NP modified by 44 (m/z 676.5, see Figure S1A). Two-fold charged

species were examined.

2.5. Covalent Labeling of the H R by 44

3

To further validate that 44 binds irreversibly to the H

washout of H

washout of three reference non-covalent H

3

R, recovery of [ H]NAMH binding after

3

R-expressing cell homogenates preincubated with 10 μM 44 or 42 was compared to

R ligands (namely thioperamide, pitolisant, and

3

histamine) with relatively long to short residence times [39]. Indeed, 10 μM 44 could significantly (p

3

< 0.05, one-way ANOVA with Fisher’s LSD test) inhibit recovery of [ H]NAMH binding after

washout, while washout of H

pitolisant, or thioperamide recovered [ H]NAMH occupancy to vehicle levels (Figure 5A).

3

R expressing cell homogenates preincubated with 42, histamine,

3

Figure 5. (A) Binding of [³H]NAMH to hH

incubated with vehicle, 10 μM 44, 10 μM 42 or 10 μM of non-covalent reference H

3

R-expressing HEK293T cell homogenates that were pre-

R ligands

3

thioperamide, pitolisant and histamine for 1 h at 25 °C, followed by washout. (B, C) Radioligand

displacement curves of [³H]NAMH with histamine on cell homogenates preincubated for 1 h at 25 °C

with increasing concentrations of (B) 44 or (C) 42. After thorough washing pretreated cell

homogenates were subjected to displacement of [³H]NAMH with increasing histamine

concentrations. Data are normalized to specific binding of vehicle treated cell homogenates. Data

shown are pooled data mean ± S.E.M. of at least three experiments performed in triplicate. * p < 0.05

(one-way ANOVA with Fisher’s LSD test).

Molecules 2019, 24, 4541

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Moreover, after washout of cell homogenates pretreated with 0.1–10 μM 44 a concentration-

3

dependent decrease in [ H]NAMH occupancy of the H

3

R up to 20% is observed, while the affinity of

histamine was unaffected (Figure 5B). In contrast, pretreatment with non-covalent analog 42 showed

3

no significant decrease in [ H]NAMH binding up to 1 μM (Figure 5C). However, at 10 μM 42 only

3

56% [ H]NAMH labeling could be observed despite three vigorous wash steps to remove the ligand.

Due to high homology between H

NanoBRET-based fluorescent ligand displacement assay on the Nanoluc-H

incapable of displacing the fluorescent tracer clobenpropit-BODIPY from the Nanoluc-H

3

R and H

4

R binding of 42 and 44 was evaluated in a

R. Both 42 and 44 were

R up to a

4

concentration of 10 μM (Figure S2), indicating that binding of 44 and 42 is selective to H R.

3

2.6. Functional Characterization

35

Functional characterization of 44 in a [ S]GTPyS assay showed that 44 inhibited basal H

3

R

signaling with a pEC⁵⁰value of 7.1 ± 0.3 (Figure 6A). Moreover, 44 could inhibit H

by immepip (31.6 nM) to levels below basal signaling with a pIC⁵⁰value of 6.1 ± 0.1, suggesting that

it might act as inverse agonist (Figure 6B). In comparison, the marketed H R inverse agonist pitolisant

more potently inhibited immepip-induced H R activity (pIC50 8.1 ± 0.1), however, inhibition of basal

R signaling was more pronounced for 44.

3

R activity induced

3

H

3

Figure 6. Functional characterization of 44 as measured by [³⁵S]GTPγS binding to hH

HEK293T cell homogenates at 25 °C for 1 h. (A) Inhibition of basal hH R activity by 44. (B) Inhibition

of 31.6 nM immepip-induced hH R activity by 44 or pitolisant.

3

R expressing

3

3. Discussion

Covalent small-molecule ligands can be useful tools for the better understanding of GPCR

structure and function [14,17]. Despite intense investigations on the histamine receptor GPCR family

[2], histamine receptors have only been targeted with covalent ligands sporadically [15,17]. The aim

of the current study was the identification of a covalent H

3

R ligand. The high homology of H

3

R and

H

4

R [28] suggested that the published covalent partial H

4

R agonist VUF14480 [15] was a suitable

starting point. The first design cycle resulted in a cyclobutyl-piperazine anchor moiety, which was

combined with a change in warhead in the second design cycle, resulting in Michael acceptor vinyl-

ligands 22 and 23 with micromolar apparent affinity for H

3

R. In a subsequent scaffold hopping, the

pyrimidine core was changed to phenyl in the third design cycle, providing the phenyl-

isothiocyanate 44 with subnanomolar apparent affinity for H

3

R and no measurable interaction with

−5

H

4

R up to least 10 M.

The alkyl-isothiocyanate moiety as reactive warhead has been regularly used for covalent

targeting, e.g., for labeling of cannabinoid receptors [18,40,41], and were found to predominantly

interact with cysteine residues. Under physiological conditions alkyl-isothiocyanate rapidly form

adducts with cysteines, however, in the presence of lysines, a slow but irreversible reaction could

also form lysine adducts [32] as was observed with the some reactive phenyl isothiocyanates [33].

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According to our reactivity/stability assays, phenyl-isothiocyanate 44 forms an adduct with cysteine

in a rapid reaction without significant aqueous decomposition. A more in-depth nonapeptide assay

shows that the reaction is selective for cysteine in the presence of other nucleophilic residues, and

formation of lysine adducts did not significantly occur during an incubation period of 16 h.

Determination of washout resistance by radioligand binding is a frequently used method for the

validation of a postulated irreversible interaction between a potential covalent ligand and the

targeted receptor [15–23]. In this experimental design, the bound ligand 44 was washed out after the

preincubation with hH³R expressing cell homogenate and showed a concentration-dependent

labeling of the receptor, while the non-covalent control 42 and reference ligands recovered

radioligand binding to vehicle levels. This result indicates the irreversible nature of 44 binding to the

binding pocket.

The unraveling of the crystal structure of H¹R [42] has significantly impacted research on the

histamine receptor family. However, the moderate similarity between H¹R and H3R causes

limitations in obtaining H³R models based on the structure of H1R [29]. Covalent ligands have been

shown to facilitate GPCR crystallization [14,43]. We therefore believe that the identified covalent

inverse agonist 44 could aid in stabilizing the H³R in the inactive conformation and as such function

as a tool compound in H³R crystallization. The covalent binder 44 might also serve as a useful

extension of the diverse H³R compound set for studying H3R signaling [39,44].

In conclusion, we designed and synthesized potential covalent ligands for H³R. Three successive

design cycles led to identification of 44 (VUF15662) as an irreversible H³R binder. Tool compound 44

can be used to stabilize the inactive conformation of hH³R and to evaluate the effects of long-term

blockage of H³R signaling.

4. Materials and Methods

4.1. Pharmacology

4.1.1. Materials

3

35

[ H]NAMH (specific activity: 79.7 Ci/mmol) and [ S]GTPyS (specific activity 2200 Ci/mmol)

were purchased from Perkin Elmer (Boston, MA, USA). All other chemicals were obtained from

commercial suppliers and were of analytical grade.

4.1.2. Cell Culture and Transfection

Human embryonic kidney 293T cells (HEK293T) (ATCC, Manassas, VA, USA) were cultured in

DMEM (Gicbo, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Bodinco,

Alkmaar, the Netherlands) and 1% pen/step (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

2

Two million cells per 10 cm dishes were plated 24 h prior to transfection. Cells were transfected using

the polyethyleneimine (PEI) method [44] with 2500 ng cDNA encoding the hH³R and 2500 ng empty

plasmid pcDEF3.

4.1.3. Preparation of Cell Homogenates

Cell homogenates expressing the hH³R were harvested 48 h after transfection as reported

previously [39].

4.1.4. Radioligand Displacement Assays

3

[ H]NAMH displacement assays were perform in binding buffer [50 mM Tris-HCl pH 7.4, 25

3

°C] by co-incubation of ~2 nM [ H]NAMH, increasing concentration of unlabeled ligand and cell

homogenates expressing the hH³R. Assay mixture was incubated for 2 h at 25 °C before rapid

filtration over a PEI-coated GF/C filter with a Perkin Elmer filtermate harvester. Filterplate was dried

and 300 min after 25 μL Microsint O was added filterbound radioactivity was measured with a

Microbeta scintillation counter (Perkin Elmer).

Molecules 2019, 24, 4541

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4.1.5. Receptor Recovery Assay

To assess covalent binding of unlabeled ligands, hH³R expressing cell homogenates were pre-

incubated for 1 h with either Tris buffer [50 mM Tris-HCl pH 7.4], 10 μM, 1 μM or 0.1 μM unlabeled

ligand, or 100 μM unlabeled ligand for screening, in a thermoshaker (1000 RPM, 25 °C). Subsequently,

cell homogenates were vigorously washed according to the procedure described in Nijmeijer et al.

(2013) [15] and pelleted cell homogenates were reconstituted in Tris buffer and subjected to

radioligand displacement assay (vida supra).

35

4.1.6. [ S]GTPγs Assay

35

In [ S]GTPyS assay, hH3R expressing cell homogenates (20 μg/well) were incubated in GTPγs

buffer [50 mM HEPES, 150 mM NaCl, 10 mM MgCl², 4 μM GDP, 0.2 μg saponin, pH 7.4] with ~0.5

35

−4

−12

nM [ S]GTPγS and increasing ligand concentrations (10 M to 10 M) for 1 h at 25 °C. Reaction was

stopped by rapid filtration of a GF/B filter with a Perkin Elmer filtermate harvester. Filterplate was

dried and 25 μL Microsint O was added to measure filterbound radioactivity with a Microbeta

scintillation counter (Perkin Elmer) at 500 min.

4.1.7. Chemical Stability and Reactivity of 44

The reactivity of 44 (400 μM) was tested in the mixture of binding buffer (96% 50 mM Tris-HCl

pH 7.4 and 4% DMSO) with or without GSH (1.2 mM) at rt. At various timepoints, the mixtures were

directly injected and analysed by LC-MS, using the acidic mode elution programme described in the

General information of the chemistry experimental section.

4.1.8. Nonapeptide Assay

Analysis of NP (KGDYHFPIC) incubated (16 h, rt) with 44 or 42 was performed according to

Ábrányi-Balogh et al. [25].

4.1.9. Data Analysis:

All data was analysed and statistical analysis performed using Graphpad prism 7.02 (Graphpad

software inc, San Diego, USA). Competition binding curves were fitted using a one-site binding

model and affinity (pKⁱ) values were calculated using Cheng–Prusoff equation [45].

4.2. Modelling

The homology model of H³R was constructed according to Hauwert et al. [46].

The covalent compound docking (Figure 2) was performed using GOLD version 5.2.1 using the

Piecewise Linear Potential (PLP) fitness function. A flood fill of the orthosteric binding site was used

3.36

as reference for the docking site. The sulfur atom of C118 was assigned as covalent anchor point

3.28

3.32

3.33

5.46

6.48

7.43

and the pocket residues W110 , D114 , Y115 , E206 , W371 , and W402 were treated as

semi-flexible by sampling different rotamers from the GOLD rotamer library.

4.3. Chemistry

4.3.1. General Information

Chemicals and solvents were obtained from commercial suppliers and were used without

further purification. Dry DCM and dioxane were obtained from PureSolv solvent purification system

®

by Inert . All reactions were carried out under an inert N2 atmosphere. Microwave reactions were

performed with Biotage Initiator microwave system. TLC analyses were performed with Merck F254

alumina silica plates using UV visualization or staining. Column purifications were carried out

automatically using Biotage Isolera and Silicycle Ultra Pure silica gel. Melting point (Mp) for final

compounds was determined using a Büchi M-565 melting point apparatus with a rate of 1 °C/min.

NMR spectra were recorded on a Bruker 250 or 500 MHz spectrometer. Chemical shifts are reported

Molecules 2019, 24, 4541

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1

in ppm (δ), and the residual solvent was used as internal standard (δ H NMR: CDCl3 7.26; D2O 4.79;

13

C NMR: CDCl3 77.16). Data are reported as follows: chemical shift, multiplicity (s = singlet, d =

doublet, t = triplet, q = quartet, p = pentet, hept = heptet, br = broad signal, m = multiplet, app =

apparent), coupling constants (Hz) and integration. HRMS spectra were recorded on Bruker

microTOF mass spectrometer using ESI in positive ion mode. Analytical HPLC-MS analyses were

conducted using a Shimadzu LC-20AD liquid chromatograph pump system connected to a Shimadzu

SPDM20A diode array detector with MS detection using a Shimadzu HPLC-MS 2010EV mass

spectrometer. The column used is an Xbridge C18 5 mm column (50mm × 4.6 mm). Acidic mode:

Solvent B (MeCN/0.1% formic acid) and solvent A (water/0.1% formic acid), flow rate of 1.0 mL/min

with a run time of 8 min. For compounds which retention time (tR) was less than 1.5 min with acidic

solvent system, a basic solvent system was used. Basic mode: Solvent B (MeCN/10% buffer), Solvent

A (water/10% buffer). The buffer is a 0.4% (w/v) NH⁴HCO3 solution in water, adjusted to pH 8.0 with

NH⁴OH. The analysis was conducted using a flow rate of 1.0 mL/min with a total run time of 8 min.

Gradient settings (basic and acidic system): start 5% B, linear gradient to 90% B in 4.5 min, then isocratic

for 1.5 min at 90% B, then linear gradient to 5% B in 0.5 min, then isocratic for 1.5 min at 5% B. Unless

specified otherwise, all compounds have a purity of ≥95%, calculated as the percentage peak area of

the analysed compound by UV detection at 254 nm. Yields reported are not optimized. The

compounds described in Table 1 were checked for the presence of PAINS substructures as described

by Baell and Holloway [47], and no PAINS substructures were identified.

4.3.2. Synthesis

The syntheses of final compounds 10–12, 20–23, 34–37 and 43 are described in the

Supplementary Materials.

Tert-butyl 4-(3-nitrophenyl)piperazine-1-carboxylate (39). A solution of bromide 38 (808 mg, 4.00 mmol)

and NaOtBu (461 mg, 4.80 mmol) in dioxane (15 mL) was degassed with N²for 10 min. Next,

Pd²(dba)3 (183 mg, 0.20 mmol), Xantphos (347 mg, 0.60 mmol) and tert-butyl piperazine-1-carboxylate

(1.12 g, 6.00 mmol) were added. The reaction mixture was heated for 1 h at 80 °C under microwave

irradiation. The reaction mixture was diluted with water (40 mL) and extracted with DCM (3 × 30

mL). The combined organic layers were dried over Na²SO4, filtered and concentrated in vacuo.

Purification by flash chromatography (heptane:EtOAc 5:0 to 4:1) gave the title compound as an

1

orange solid (700 mg, 57%). H NMR (500 MHz, CDCl3) δ 7.72 (t, J. = 2.2 Hz, 1H), 7.71–7.67 (m, 1H),

7.39 (t, J. = 8.2 Hz, 1H), 7.20 (dd, J. = 8.3, 2.1 Hz, 1H), 3.61 (t, J. = 5.2 Hz, 4H), 3.24 (t, J. = 5.1 Hz, 4H),

+

1.49 (s, 9H). HPLC-MS (acidic mode): t^R= 5.3 min, purity: 98.6%, [M + H] : 308.

1-(3-Nitrophenyl)piperazine (40). To a solution of carbamate 39 (1.20 g, 3.90 mmol) in dioxane (20 mL)

was added HCl in dioxane (4N, 9.75 mL, 39.0 mmol). The reaction mixture was stirred for 1 h at rt.

The solvent was removed under reduced pressure. The residue was mixed with satd. aq. Na²CO3 (40

mL) and extracted with DCM (3 × 30 mL). The combined organic layers were dried over Na²SO4,

filtered and concentrated in vacuo. The title compound was obtained as a brown solid (633 mg, 78%).

1

H NMR (500 MHz, CDCl3) δ 7.71 (s, 1H), 7.66 (d, J. = 8.0 Hz, 1H), 7.37 (t, J. = 8.2 Hz, 1H), 7.19 (d, J. =

8.3 Hz, 1H), 3.28–3.21 (m, 4H), 3.10–3.02 (m, 4H). HPLC-MS (acidic mode): t^R= 2.6 min, purity: 97.3%,

+

[M + H] : 208.

1-Cyclobutyl-4-(3-nitrophenyl)piperazine (41). To a solution of amine 40 (630 mg, 3.04 mmol) in DCM

(20 mL) was added cyclobutanone (273 μL, 3.66 mmol). After 10 min of stirring at rt, NaBH(OAc)³

(966 mg, 4.56 mmol) was added and the resulting mixture was stirred at rt overnight. The reaction

mixture was quenched with satd. aq. Na2CO3 (30 mL) and extracted with DCM (3 × 15 mL). The

combined organic phases were dried over Na²SO4, filtered and concentrated in vacuo. Purification

by flash chromatography (DCM:MeOH 20:0 to 19:1) gave the title compound as a yellow oil (600 mg,

1

76%). H NMR (500 MHz, CDCl3) δ 7.71 (t, J. = 2.3 Hz, 1H), 7.65 (dd, J. = 8.1, 1.5 Hz, 1H), 7.37 (t, J. =

8.2 Hz, 1H), 7.18 (dd, J. = 8.3, 2.1 Hz, 1H), 3.38–3.23 (m, 4H), 2.80 (p, J. = 7.9 Hz, 1H), 2.51 (t, J. = 5.1

Molecules 2019, 24, 4541

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Hz, 4H), 2.13–2.04 (m, 2H), 2.00–1.87 (m, 2H), 1.81–1.57 (m, 2H, overlaps with residual water). HPLC-

+

MS (acidic mode): t^R= 2.8 min, purity: 97.8%, [M + H] : 262.

3-(4-Cyclobutylpiperazin-1-yl)aniline (42). To a solution of nitrocompound 41 (59 mg, 0.23 mmol) in

MeOH (2 mL) was added HCOONH⁴(71 mg, 1.13 mmol) as a solid followed by a suspension of Pd/C

(10%, 24 mg) in water (2 mL). The reaction mixture was stirred at rt overnight. The mixture was

filtered over Celite and the filtrate was concentrated in vacuo. The residue was diluted with aq.

Na²CO3 (1.0 M, 10 mL) and extracted with DCM (3 × 5 mL). The combined organic layers were dried

over Na²SO4, filtered and concentrated in vacuo. The crude product was purified by flash

chromatography (DCM:MeOH 20:0 to 19:1). The selected fractions were collected and the solvents

were evaporated. The residue was dissolved in aq. HCl (1.0 M, 10 mL), washed with EtOAc (2 × 5

mL) and c-hexane (3 × 10 mL). The pH of the aqueous layer was adjusted to 10 with satd. aq. Na²CO3

and extracted with EtOAc (3 × 10 mL). The combined organic phases were dried over Na²SO4, filtered

and concentrated in vacuo. The title compound was obtained as an off-white solid (13 mg, 25%). Mp:

1

92.5–92.7 °C. H NMR (500 MHz, CDCl3) δ 7.04 (t, J. = 8.0 Hz, 1H), 6.36 (dd, J. = 8.2, 2.3 Hz, 1H), 6.25

(t, J. = 2.3 Hz, 1H), 6.21 (dd, J. = 7.7, 2.0 Hz, 1H), 3.59 (br, 2H), 3.26–3.10 (m, 4H), 2.78 (p, J. = 7.9 Hz,

13

1H), 2.56–2.37 (m, 4H), 2.12–2.02 (m, 2H), 2.00–1.88 (m, 2H), 1.80–1.64 (m, 2H). C NMR (126 MHz,

CDCl³) δ 152.6, 147.4, 130.0, 107.1, 107.1, 103.0, 60.4, 49.6, 48.8, 27.1, 14.4. HPLC-MS (basic mode): tR =

+

4.1 min, purity: 98.4%, [M + H] : 232. HR-MS [M + H] calcd for C14H22N3 : 232.1808, found 232.1818.

1-Cyclobutyl-4-(3-isothiocyanatophenyl)piperazine (44). To an ice-cold mixture of aniline 42 (46 mg, 0.20

mmol) in DCM (2 mL) and aq. NaHCO³(1.0 M, 30 mL) was added dropwise a solution of CSCl2 (18

μL, 0.24 mmol) in DCM (1 mL). The reaction mixture was stirred for 1 h at rt. The reaction mixture

was diluted with water (10 mL) and extracted with DCM (3 × 5 mL). The combined organic layers

were dried over Na²SO4, filtered and concentrated in vacuo. Purification by flash chromatography

(DCM:MeOH 20:0 to 19:1) gave the title compound as a white solid (35 mg, 64%). The compound is

stable as a solid in the freezer (−20 °C) over a period of at least 2 years as judged by NMR and LC-MS

1

analysis. Mp: 79.7–80.1°C. H NMR (500 MHz, CDCl3) δ 7.18 (t, J. = 8.1 Hz, 1H), 6.81 (dd, J. = 8.5, 2.4

Hz, 1H), 6.72–6.65 (m, 2H), 3.23–3.15 (m, 4H), 2.77 (p, J. = 7.9 Hz, 1H), 2.50–2.42 (m, 4H), 2.11–2.02 (m,

13

2H), 1.96–1.86 (m, 2H), 1.79–1.64 (m, 2H). C NMR (126 MHz, CDCl3) δ 152.2, 134.2, 131.9, 130.0,

116.5, 114.9, 112.6, 60.3, 49.3, 48.3, 27.1, 14.4. HPLC-MS (acidic mode): t^R= 3.6 min, purity: 98.9%, [M

+

+ H] : 274. HR-MS [M + H] calcd for C15H20N3S : 274.1372, found 274.1374.

Supplementary Materials: The following are available online. LC-MS analysis of nonapeptide assays with 44

and 42; Nluc-H⁴R displacement of clobenpropit-BODIPY by 42, 44 and reference ligands; syntheses of final

compounds 10–12, 20–23, 34–37 and 43; LC-MS chromatogram and ¹H, ¹³C, HSQC and HMBC NMR spectra for

44 and 42.

Author Contributions: G.W. performed the synthesis and reactivity/stability studies. T.A.M.M. performed the

pharmacological experiments. A.J.K., I.S., G.W. and M.W. contributed to the design of the compound set. G.M.K.

and P.Á.-B. supervised the nonapeptide assay. G.W. and T.A.M.M. wrote the original draft with input from A.J.

K. and P.Á.-B. M.W., H.F.V., I.J.P.d.E. and R.L. conceptualized and supervised the whole research project.

G.M.K., P.Á.-B., M.W., H.F.V., I.J.P.d.E. and R.L. reviewed and edited the manuscript.

Funding: This research was funded by The Netherlands Organization for Scientific Research (NWO) TOPPUNT

[“7 ways to 7TMR modulation (7-to-7)”] [Grant 718.014.002]. P. Ábrányi-Balogh was supported by the

Hungarian Science Foundation OTKA (PD124598) grant.

Acknowledgments: We thank Hans Custers for HRMS measurements. Tímea Imre is acknowledged for

performing the nonapeptide assay. László Petri is acknowledged for his efforts with the nonapeptide

measurements.

Conflicts of Interest: The authors declare no conflicts of interest.

Molecules 2019, 24, 4541

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Abbreviations

[³H]NAMH

ANOVA

DCM

DIPEA

DME

DMSO

GPCR

GSH

GTPγS

LSD

MeCN

MIDA

Mp

[³H]N-α-methylhistamine

analysis of variance

Dichloromethane

N,N-Diisopropylethylamine

1,2-Dimethoxyethane

dimethyl sulfoxide

G protein-coupled receptor

glutathion

guanosine 5′-O-[gamma-thio]triphosphate

least significant difference

Acetonitrile

N-methyliminodiacetic acid

Melting point

NP

SAR

satd. aq.

SD

nonapeptide (KGDYHFPIC)

structure-activity relationship

saturated aqueous

standard deviation

SEM

rt

standard error of mean

room temperature

TCI

TEA

targeted covalent inhibitors

Triethylamine

Tris

μW

2-Amino-2-(hydroxymethyl)propane-1,3-diol

microwave reaction

wt

wild type

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Sample Availability: Samples of the compound 44 are available from the authors.

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Covalent inhibition of the histamine H3 receptor

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