4-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as High-Affinity Non-imidazole Histamine H3 Receptor Agonists with in Vivo Central Nervous System Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01462

Despite the high diversity of histamine H₃ receptor (H₃R) antagonist/inverse agonist structures, partial or full H₃R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded

Full text of DOI:10.1021/acs.jmedchem.9b01462

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Article
4-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as high-affinity non-imidazole
histamine H3 receptor agonists with in vivo central nervous system activity
Gábor Wágner, Tamara Mocking, Marta Arimont, Gustavo Provensi, Barbara Rani, Bruna
Silva-Marques, Gniewomir Latacz, Daniel Da Costa Pereira, Christina Karatzidou, Henry F.
Vischer, Maikel Wijtmans, Katarzyna Kiec-Kononowicz, Iwan J. P. De Esch, and Rob Leurs
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01462 • Publication Date (Web): 01 Nov 2019
Downloaded from pubs.acs.org on November 2, 2019
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-(3-Aminoazetidin-1-yl)pyrimidin-2-amines as
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high-affinity non-imidazole histamine H receptor
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agonists with in vivo central nervous system activity
a
a
a
b
Gábor Wágner, Tamara A. M. Mocking, Marta Arimont, Gustavo Provensi,
c
b,d
e
Barbara Rani, Bruna Silva-Marques, Gniewomir Latacz, Daniel Da Costa
a
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Pereira, Christina Karatzidou, Henry F. Vischer, Maikel Wijtmans, Katarzyna
e
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a*
Kieć-Kononowicz, Iwan J. P. de Esch and Rob Leurs
aAmsterdam 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.
b
Department of Neuroscience, Psychology, Drug Research and Child Health, Section of
Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, CAP
50139, Florence, Italy.
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Department of Health Sciences, University of Florence, Viale G. Pieraccini 6, CAP
0139, Florence, Italy.
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Department of Physiotherapy, Federal University of São Carlos, Washington Luís, km
35, SP-310, São Carlos, Brazil.
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Department of Technology and Biotechnology of Drugs, Medical College, Jagiellonian
University, Medyczna 9, PL 30-688 Cracow, Poland
KEYWORDS: GPCR, Histamine H receptor, agonism, non-imidazole, amino-pyrimidine,
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social recognition memory.
ABSTRACT: Despite the high diversity of histamine H receptor (H R) antagonist/inverse
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agonist structures, partial or full H R agonists have typically been imidazole derivatives.
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An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-
yl)pyrimidin-2-amine 11b as partial H R agonist. Here, the design, synthesis and
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structure-activity relationships of 11b analogues are described. This series yields several
non-imidazole full agonists with potencies varying with the alkyl substitution pattern on
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the basic amine following the in vitro evaluation of H R agonism using a CRE-luciferase
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reporter gene assay. Key compound VUF16839 (14d), combines nanomolar on-target
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activity (pK = 8.5, pEC = 9.5) with weak activity on CYP enzymes and good metabolic
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stability. The proposed H R binding mode of 14d indicates key interactions similar to
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those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice,
revealed an amnesic effect at 5 mg/kg i.p. The excellent in vitro and in vivo
pharmacological profile and the non-imidazole structure of 14d make it a promising tool
compound in H R research.
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INTRODUCTION
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The four histamine receptors (H R, H R, H R, H R) belong to class A of the G protein-
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coupled receptor (GPCR) family. The histamine H receptor (H R) was discovered in
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983 by Arrang et al. and regulates the release of several neurotransmitters such as
histamine (1), acetylcholine, serotonin, noradrenaline and dopamine, both as auto- and
1
heteroreceptor. Due to its expression in cortex, striatum and hippocampus, H R
3
regulates several physiological processes such as sleep-wake regulation, cognition and
food intake.1,3
During the early years of discovery of H R ligands, the natural ligand histamine served
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as an initial structure for drug design, leading to a plethora of imidazole-containing
ligands. However, imidazole-containing ligands are associated with drug-drug
interactions due to the propensity for cytochrome P450 (CYP) inhibition and with poor
brain penetration.4,5 Therefore, research towards therapeutically relevant H R
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antagonists focused on drug-like non-imidazole structures6,7 and these efforts have led to
numerous clinical candidates for different indications connected to central nervous
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system diseases, e.g., Alzheimer`s disease, Attention Deficit Hyperactivity Disorders,
sleep disorders, schizophrenia, obesity, epilepsy and neuropathic pain and narcolepsy.8,9
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®
For the latter condition, the H R ligand pitolisant (Wakix ) was approved by the EMA in
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016, and most recently, by the FDA in 2019.
In sharp contrast, developing non-imidazole H R agonists has not been very successful.
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The best agonists contain an imidazole ring12,13,22,23,14–21 and, compared to the
endogenous ligand histamine (1), these derivatives show similar (e.g. imbutamine (2)) or
significantly higher affinity (pK) and/or functional activity (pEC ) on H R (e.g. imetit (3),
i
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3
methimepip (4) and AEIC (5)) (Fig. 1A). Imidazole-containing agonists have shown some
potential application in different therapeutic areas, such as mechanical nociception,23
obesity and diabetes mellitus (diet-induced obesity mice test)24 and stress (rodent-
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intruder mice test). Some data also support the hypothesis of cardioprotective effect of
H R receptor activation.25,26 However, it is fair to say that imidazole-containing agonists
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may suffer from the same imidazole-related drawbacks that were associated with the first
generation of imidazole-containing H R antagonists (vide supra). Future studies on the
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pharmacological and therapeutic role of H R agonists can therefore be helped by having
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non-imidazole H R agonists available.
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A very limited number of non-imidazole agonists has been published to date (Fig. 1B).
VUF8430 (6) was designed as an H R agonist based on the H R agonist dimaprit, and
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shows micromolar affinity and full H R agonism as well. The histamine analogues
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amthamine (7a) and amselamine (7b) were identified as H R agonists, but both show
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28,29
weak H R agonist activity in an electrically stimulated guinea-pig jejunum model.
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Three pentacyclic spiroindolinone derivatives were isolated from Penicillium waksmanii,
of which PF1270A (8) shows the best affinity for H R and moderate functional H R activity
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in a GTPS accumulation assay. ZEL-H16 (9) has been reported to have nanomolar
binding affinity to the H R, partial H R agonism in forskolin-stimulated cAMP accumulation
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and ERK1/2 signaling assays, and full H R agonism in guinea-pig ileum contraction
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assay. Finally, a compound set with 94 examples in 4 compound families with either a
-lactam or pyrrolidinone central core without basic amino moiety was published recently
and surprisingly included compounds with nanomolar functional H R agonist activities
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(e.g. compound 10). The fungal isolates and the multi-component reaction product 10
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are large and complex molecules, which are difficult to align with known H R
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pharmacophores or H R binding modes for agonists and antagonists. We therefore
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started a search for novel high-affinity non-imidazole H R full agonists with simpler
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structures to generate fundamental knowledge on ligand recognition and signaling of the
H R.
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Figure 1. A) Representative imidazole H R agonists. Activities are extracted from Igel et
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al. , Govoni et al. and Kazuta et al.12 B) Published non-imidazole H R (partial)
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agonists.27–32 Unless mentioned otherwise, compounds were tested on the human
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receptor. : intrinsic activity compared to histamine. -gal: CRE--galactosidase reporter
gene assay; cAMP: forskolin-stimulated cAMP accumulation assay.
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RESULTS
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Design
During an in-house compound screen aimed at identifying agonist activities in a set of
ligands using a H R-driven reporter gene assay in HEK293T cells, diaminopyrimidine 11b
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emerged as a H R partial agonist hit ( = 0.7), while its close derivative 11i showed only
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weak agonist activity ( = 0.4) (Fig. 2A). Interestingly, a set of four diaminopyrimidine
compounds has been tested before by others on H R en route to imbutamine (2)
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analogues, but the majority of these diaminopyrimidines were rather inactive and, where
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applicable, all were shown to be antagonists/inverse agonists. Intrigued by the agonist
activity of the diaminopyrimidine 11b and recognizing its core as a thoroughly explored
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heterocycle in the H R area, we decided to perform an in-depth patent search on this
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scaffold in an effort to capture the full array of industrial contributions. This resulted in the
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identification of 14a (Fig. 2B) as closest derivative with data associated to H R (Fig. 2B).
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Remarkably, 14a was claimed as an H R agonist by Abbott, although its actual synthesis
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was not included and only the potential synthetic route was described. In the same
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patent 25 related examples have been prepared and partial agonism at the human H R
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is reported.36
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Based on 11b and 14a, we designed a focused series of compounds to explore the H R
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affinity and activity in the chemical space between 14a and 11b (Fig. 2B). The design
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strategy targets four series with iPr, Et, Me or H as R group at position 6 (11-14). These
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R groups were combined with different substituents on the basic amino groups (R and
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R ). Beyond the evident H (a) and Me (b) substituents, dimethyl derivatives (i) were
synthesized due to the potential functional switch that appears to reside in the case of
11b and 11i. Based on the initial results of the designed compound set (vide infra), the
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R =H series was extended with elongated (d, f), branched (e, g, h) and disubstituted (j, k)
amino derivatives. This second design iteration was also inspired by previous work from
our labs on the imidazole-containing agonist imbutamine (2), which harbors a functional
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switch on the basic amine. Beyond 14a, some exact compounds from this designed set
are known but none in a context of H R. That is 11a, 11b, 12b, have been claimed as H R
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ligands,37,38 while 12i, 13i, 14i, 14j and 14k were offered in chemical catalogues (April
2019) without any synthesis description, analytical or pharmacological data.
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Figure 2. A) Initial functional data of compound 11b and 11i compared to histamine, as
obtained by ligand-induced activation of hH R expressed on HEK293T cells measured by
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CRE-luc reporter gene assay. Shown is a representative graph of at least 3 experiments
performed in triplicate. Data are mean ± S.D. B) Structures of 11b and 11i, the closest
relevant structure (14a) resulting from a subsequent extensive patent search and the
compound set designed for the current study.
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Chemistry
The designed compound set shown in Fig. 2B was synthesized as outlined in Scheme
. The key step of the synthetic route was a nucleophilic aromatic substitution on the
0
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0
1
appropriate aromatic cores with aminoazetidine moieties (step ii). The 4-chloro-2-
aminopyrimidines were commercially available (19, 20) or were synthesized (17, 18) from
2
the appropriate pyrimidin-4(3H)-one derivatives (15, 16) with POCl . The R =H or Me
3
derivatives of the Boc-protected 3-aminoazetidines (28a, b) were commercially available,
2
while the R =Et, nPr, nBu derivatives were synthesized. These intermediates were built
up from benzhydryl-protected 3-aminoazetidine (25) with Boc protection of the primary
amino group to give 26, followed by alkylation with the corresponding iodoalkyl reagents
resulting in the orthogonally protected intermediates (27c, d, f) and the removal of the
benzhydryl group with hydrogenation. The resulting mixtures of unprotected azetidine
intermediate and diphenylmethane were used directly for the ensuing nucleophilic
substitution with 17-20. The key nucleophilic aromatic substitution of the appropriate Boc-
protected intermediates was performed in a microwave at 120 - 150 °C to give 21-24.
This was followed by the deprotection under acidic condition to afford the majority of
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2
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monosubstituted (R =H or linear alkyl, R =H) products 11-14. Although the Boc-protection
was necessary at the precursor stage to avoid overalkylation in case of linear alkyl
derivatives, overalkylation was not a problem in case of the branched-alkyl derivatives as
a result of steric hindrance. Therefore, the branched-alkyl derivatives 14e, g, h as well as
the dialkylated derivatives 12i, 13i, 14i and 14j were synthesized from 14a with a reductive
amination. Last, 11i was synthesized directly from 17 and N,N-dimethylazetidin-3-amine
with a nucleophilic substitution, while the pyrrolidine ring of 14k was obtained from 1,4-
diiodobutane and 14a.
1
1
1
1
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Scheme 1. Reagents and conditions: i) POCl , 110 °C, 3h, 26-52 %; ii) DIPEA, dioxane
3
or NMP, W, 120 - 150 °C, 0.5 - 2 h, 27-50 % (a and b) or 35-71 % (c, d and f, two steps
0
1
2
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0
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9
0
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9
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9
0
1
2
3
4
5
6
7
8
9
0
from benzhydryl deprotection); iii) HCl, DCM, MeOH, rt - 50 °C, 3 h - overnight, 10 % -
quant.; iv) aldehyde/ketone, AcOH, NaHB(OAc) , DCM, MeOH, rt, 3 h - overnight, 16-44
3
%
; or 1,4-diiodobutane, K CO , MeCN, reflux, 16 h, 9 %; v) N,N-dimethylazetidin-3-amine
2
3
dihydrochloride, DIPEA, dioxane, W, 150 °C, 30 min, 65 %; vi) di-tert-butyl dicarbonate,
2
TEA, THF, rt, overnight, 63 %; vii) NaH, R I, THF, 0 °C - rt, overnight, 28-58 %; viii) H ,
2
Pd/C, MeOH, EtOH, rt - 60 °C, 1 h - overnight, not purified and used crude.
Pharmacological evaluation
The synthesized compound set was tested for their activity at the human H R transiently
3
3
expressed in HEK293T cells. Binding affinity (K) was evaluated using a [ H]NAMH
i
displacement assay, while potency (EC ) and intrinsic activity () were determined as
50
the H R-mediated inhibition of forskolin-induced CRE-driven luciferase reporter gene
3
activity with histamine as control (Table 1, Fig. 3). During the first iteration, the isopropyl
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group of 11b was gradually decreased in size to give 11-14, which were all combined with
2
3
small-size R /R amino substituents (a, b, c, i). The affinities of the unsubstituted
derivatives (14) stand out especially within the monomethylated (b) and monoethylated
series (c), with both 14b and 14c exceeding the affinity of histamine. Although
1
1
1
1
1
1
1
1
1
1
2
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2
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0
1
monomethylated (b) derivatives generally show the highest affinities in each R subseries
11-14, the most notable variation was observed in case of the monoethylated series c,
with affinities of Me/Et/iPr derivatives 11c, 12c and 13c being considerably reduced
compared to 14c (Fig. 3A). A more indicative trend was observed in case of the functional
results of this c series. Compound 14c has a higher potency than 14b and produces the
same intrinsic activity () as histamine but with higher potency (EC ), while 12c and 13c
50
show more than >1 log unit weaker EC albeit with maintained full agonism ().
50
1
Interestingly, isopropyl substitution on the R position (11c) turns this full agonism to
partial agonism () (Fig. 3B). A similar trend was observed in the other three series,
1
with the pyrimidine derivatives bearing R =H (14a-c, i) reaching or exceeding the affinity
1
(
K) and potency (EC ) of histamine, while any alkyl substituent at position 6 (R ) on the
i
50
pyrimidine leads to inferior results. The intrinsic activity () indicates full or almost full
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agonism ( ≥ 0.8) in the methyl (12a, b, i) and ethyl (13a, b, i) series, while it drops to
partial agonism in the isopropyl series (11a, b, i) (Table 1).
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1
Due to the better results of the pyrimidines lacking a R -substituent (14), in a
2
subsequent iteration the amine NHR substituent was replaced with longer linear groups
(14d, f), branched groups (14e, g, h), or dialkylamino (14j, k) moieties. Representative
curves illustrate the SAR (Fig. 3C) and SFR (Fig. 3D) of this series. The nPr derivative
1
4d shows the highest affinity (pK=8.5) from all linear monoalkyl substituents. Although
i
2
both shorter and longer R moieties resulted in lower binding affinities (e.g. 14c: pK=8.0
i
or 14f: pK=7.8), all derivatives remained in the same affinity range (Fig. 3C). The
i
branched alkyl moieties as well as the dialkylated derivatives display loss of affinity
(
compare e.g. 14d vs 14e, or 14b vs 14i). The potencies (EC ) show almost the same
50
trends as observed for the affinities (Fig. 3D). Highly noteworthy, the potency of 14d
(
pEC =9.5) is almost a log unit higher than that of histamine (pEC =8.6), while chain
50
50
shortening (e.g. 14b, c), chain lengthening (14f), chain branching (e.g. 14e), or
dialkylation (e.g. 14i) results in lower potencies. In contrast to the observed differences in
affinity and potency, the intrinsic activities indicate that all derivatives of 14 remain full
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agonists ( ≥ 1.0) (Table 1). A combination of highest affinity (pK=8.5), highest potency
i
(
pEC =9.5) and full agonism resides in 14d. The potential aggregation of GPCR ligands
50
1
1
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1
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5
5
5
5
5
5
5
5
6
0
1
2
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6
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8
9
0
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0
39
might cause nonspecific effect on the receptor activity, but nephelometry revealed no
microprecipitation of 14d up to 100 mM concentration (Fig. S1) and underscores the high
aqueous solubility of 14d (soluble up to at least 100 mM in 50 mM Tris-HCl, pH 7.4). All
this led to identification of 14d as key compound (VUF16839) in this study.
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Table 1. Pharmacological evaluation of designed compound set. Affinity values (pK) were
i
3
determined by [ H]NAMH displacement assay on hH R expressed on HEK293T cell
3
0
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homogenates. Potency (pEC ) and intrinsic activity ( were determined by ligand
50
induced activation of hH R expressed on HEK293T cells as measured by a CRE-
3
luciferase reporter gene assay. Data are mean ± S.E.M. of at least 3 experiments
performed in triplicate.
R2
N
R3
R1
N
N
N
NH2
Compound
R1
-
R2
-
R3
-
pKi
pEC50

Histamine
7.9 ± 0.2
6.7 ± 0.0
7.0 ± 0.0
7.0 ± 0.1
8.6 ± 0.0
7.1 ± 0.1
7.9 ± 0.4
6.8 ± 0.3
1.0 ± 0.0
11a
11b
11c
iPr
iPr
iPr
H
H
H
H
0.7 ± 0.0
0.7 ± 0.1
0.3 ± 0.1
Me
Et
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11i
iPr
Et
Me
H
Me
H
6.9 ± 0.0
7.1 ± 0.0
7.3 ± 0.1
6.9 ± 0.1
6.6 ± 0.2
7.3 ± 0.1
7.5 ± 0.1
7.1 ± 0.1
6.5 ± 0.2
7.8 ± 0.1
8.2 ± 0.1
7.0 ± 0.2
7.5 ± 0.0
7.6 ± 0.0
7.5 ± 0.1
7.2 ± 0.1
7.8 ± 0.0
8.0 ± 0.0
7.9 ± 0.0
7.4 ± 0.0
8.3 ± 0.0
8.9 ± 0.1
0.4 ± 0.0
0.9 ± 0.0
0.8 ± 0.0
0.9 ± 0.0
0.8 ± 0.0
0.9 ± 0.0
0.8 ± 0.1
1.0 ± 0.0
0.9 ± 0.1
1.1 ± 0.0
1.1 ± 0.1
12a
1
1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
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7
8
9
0
12b
Et
Me
Et
H
12c
Et
H
12i
Et
Me
H
Me
H
13a
Me
Me
Me
Me
H
1
3bb
Me
Et
H
13c
H
13i
Me
H
Me
H
1
4aa
4ba
1
H
Me
H
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14ca
H
H
H
H
H
H
H
H
H
Et
nPr
H
H
8.0 ± 0.1
8.5 ± 0.1
7.4 ± 0.1
7.8 ± 0.1
7.9 ± 0.2
7.4 ± 0.2
7.3 ± 0.1
7.2 ± 0.1
9.2 ± 0.0
9.5 ± 0.1
8.5 ± 0.1
9.1 ± 0.3
8.7 ± 0.1
8.3 ± 0.0
8.4 ± 0.1
8.1 ± 0.1
8.1 ± 0.0
1.0 ± 0.1
1.2 ± 0.1
1.2 ± 0.1
1.2 ± 0.0
1.1 ± 0.0
1.1 ± 0.0
1.0 ± 0.1
1.2 ± 0.1
1.3 ± 0.2
1
4da
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
14ea
iPr
H
1
4fa
nBu
rac-sBu
iBu
H
1
4ga
4ha
H
1
H
1
4ia
Me
Me
Et
14ja
Et
4ka
NR R = Pyrrolidine 7.4 ± 0.1
2
3
1
a
b
Measured as fumarate salt.
Measured as dihydrochloride salt.
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0
1
2
3
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8
9
0
1
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3
4
5
6
7
8
9
0
Figure 3. Representative Structure-Affinity (A, C) and Structure-Function relationship (B,
1
2
3
D) effects selected from Table 1. (A, B) Different R substituents with R = Et and R = H
2
3
1
(
11c, 12c, 13c, 14c); (C, D) Different R and R substituents with R = H (14b, 14d, 14e,
14f, 14i). Shown is a representative graph of at least 3 experiments performed in triplicate.
Data are mean ± S.D.
Computational studies of 14d.
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A combination of molecular docking and molecular dynamics simulations was used in
order to evaluate the potential binding mode of the key compound 14d and to compare it
0
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1
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1
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9
0
to the binding mode of the endogenous ligand histamine. A homology model of H R based
3
on the available crystal structure of H R was used (see Experimental section). This model
1
was validated by its ability to retrospectively discriminate between known H R fragment-
3
like ligands and true inactives.40 Histamine was docked in the receptor model using
PLANTS 1.1 (Fig. 4A). The best-scored docking pose showing interactions with both
D1143.32 and E2065.461 (residues known to be involved in H R ligand binding40–45) was
3
selected. During 100 ns of molecular dynamics (MD) simulations, histamine is able to
maintain stable interactions with residues D1143.32 and E2065.461 as well as with Y3746.51
(
Fig. 4B and 4E). Using similar procedures, compound 14d was also docked into the
same homology model using PLANTS 1.1 (Fig. 4A) and the best scored docking results
show similar interactions of 14d with D1143.32 and E2065.461. The basic amine of 14d forms
an ionic interaction with the negatively-charged side chain of D1143.32, and the amino
group in the pyrimidine ring makes a hydrogen bond with E2065.461. Different docking
poses maintain these key interactions but show a different positioning of the linear nPr
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moiety at the R position: towards the extracellular surface of the receptor or towards the
intracellular side (Fig. S2A and Fig. S2B, respectively). MD simulations of the two
alternative models were performed (Supplementary Movies 1 and 2). The model in which
the nPr group of 14d is directed towards the intracellular half of the receptor was not
stable along 100 ns of MD simulations (Fig. S2D, Supplementary Movie 2), while the
model where the nPr group of 14d is pointing towards the extracellular vestibule remained
stable throughout the entire simulation time (Fig. S2C, Supplementary Movie 1). This
binding mode is shown in Fig. 4D and the interactions that remained stable during the
simulations are depicted in Fig. 4C and the interaction finger prints (IFP) in Fig. 4E. It can
be concluded that the non-imidazole H R ligand 14d exerts its unusual agonist H R
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
4
4
4
4
4
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4
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activity by showing a similar pharmacophore as the endogenous H R ligand. That is, it
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may achieve its agonist activity by forming similar interactions with the same residues as
histamine.
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Figure 4. Predicted binding mode of 14d. A) Overview of the H R homology model based
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on the H R crystal structure (PDB ID: 3RZE). The experimentally validated binding
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mode of histamine (magenta) is shown in more detail in (B) and the predicted binding
mode of 14d (green) is schematically represented in (C) and shown in more detail in (D).
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Interaction fingerprint representations of histamine and compound 14d are shown in (E),
where a one represents the presence of an interaction according to the color coding: blue
for apolar, dark green for face-to-face aromatic, green for edge-to-face aromatic, red for
protein hydrogen bond donor, purple for ligand hydrogen bond donor, grey for ligand
negative charge, and orange for protein negative charge.
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Pharmacological and pharmacokinetic characterization of 14d.
Functional characterization of key compound 14d in a direct G protein activation assay,
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i.e. [ S]-GTPS accumulation assay on hH R expressing cell homogenates (Fig. 5A),
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results in potent, but partial agonism (pEC = 8.4 ± 0.3,  = 0.5 ± 0.05) compared to
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histamine (pEC = 7.2 ± 0.3,  = 1.0 ± 0.0).
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Figure 5. (A) Dose-dependent Gα activation by 14d and histamine as measured by
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[35
S]GTPS accumulation on HEK293T cell homogenates expressing the hH R. (B, C)
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Dose-response curves of 14d and histamine for ligand-induced activation of mH R (B)
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and mH R (C) expressed on HEK293T cells as measured by CRE-luciferase reporter
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gene assay. Representative graphs of at least three experiments performed in triplicate
are shown. Data are mean ± S.D.
Due to the high homology of the hH R with hH R (43 % full sequence identity, 58 %
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predicted transmembrane regions identity), several, mainly imidazole-containing, hH R
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ligands are known to possess high affinity for hH R as well,
although there are
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examples of imidazole-containing H R agonists with high H R/H R selectivity as well
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(
e.g., 4 and 5).12,33 Therefore, 14d was tested for its H R/H R selectivity. The pyrimidine
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shows only marginal selectivity with respect to binding hH R or hH R (hH R pK = 8.5 ±
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.1 vs hH R pK = 8.1 ± 0.0), but encouragingly a 10-fold selectivity in potency is observed
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in favour of the hH R (hH R pEC = 9.5 ± 0.1 vs hH R pEC = 8.5 ± 0.2) with full agonism
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on hH R ( = 1.1 ± 0.1) in a CRE-luc reporter gene assay. Moreover, 14d does not
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activate the H R and H R up to 10 µM (Fig. S3).
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Equally encouragingly, the binding affinity of 14d is increased for mH R (pK = 9.0 ± 0.1)
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compared to hH R, while for mH R the pK value is decreased to 7.8 ± 0.0, thus yielding
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a substantial H R/H R binding selectivity for mouse receptors. Compound 14d was also
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functionally evaluated as agonist on the mH R and mH R using the CRE-luciferase
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reporter gene assay. In these experiments 14d displays a 10-fold selectivity in potency
(mH R pEC = 10.0 ± 0.1 versus mH R pEC = 9.0 ± 0.1), while acting as a full agonist
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on both murine receptors (mH R  = 1.2 ± 0.1 and mH R  = 1.1 ± 0.1) (Fig. 5B, C).
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The metabolic stability of 14d was determined in vitro by incubation with rodent liver
microsomes (Table 2). The pharmacokinetic properties for mouse (t1/2 = 130.8 min; Clint
−
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−1
= 20.7 mL·min ·kg ) indicate more than 2 times slower elimination compared to the
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reference control verapamil. For rat, this difference between 14d and verapamil is even
more pronounced.
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