Triazole ligands reveal distinct molecular features that induce histaine H4 receptor affinity and subtly govern H4/H3 subtype selectivity

Article abstract of DOI:10.1021/jm1013488

The histamine H₃ (H₃R) and H₄ (H ₄R) receptors attract considerable interest from the medicinal chemistry community. Given their relatively high homology yet widely differing therapeutic promises, ligand selectivity for the two receptors is crucial. We interrogated H₄R/H₃R selectivities using ligands with a [1,2,3]triazole core. Cu(I)-assisted "click chemistry" was used to assemble diverse [1,2,3]triazole compounds (6a-w and 7a-f), many containing a peripheral imidazole group. The imidazole ring posed some problems in the click chemistry putatively due to Cu(II) coordination, but Boc protection of the imidazole and removal of oxygen from the reaction mixture provided effective strategies. Pharmacological studies revealed two monosubstituted imidazoles (6h,p) with <10 nM H₄R affinities and >10-fold H ₄R/H₃R selectivity. Both compounds possess a cycloalkylmethyl group and appear to target a lipophilic pocket in H ₄R with high steric precision. The use of the [1,2,3]triazole scaffold is further demonstrated by the notion that simple changes in spacer length or peripheral groups can reverse the selectivity toward H₃R. Computational evidence is provided to account for two key selectivity switches and to pinpoint a lipophilic pocket as an important handle for H₄R over H₃R selectivity.

Full text of DOI:10.1021/jm1013488

ARTICLE
pubs.acs.org/jmc
Triazole Ligands Reveal Distinct Molecular Features That Induce
Histamine H4 Receptor Affinity and Subtly Govern H4/H3
Subtype Selectivity
Maikel Wijtmans,* Chris de Graaf, Gerdien de Kloe, Enade P. Istyastono, Judith Smit, Herman Lim,
Ratchanok Boonnak, Saskia Nijmeijer, Rogier A. Smits, Aldo Jongejan, Obbe Zuiderveld, Iwan J. P. de Esch,
and Rob Leurs
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Exact Sciences, VU University Amsterdam,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
S Supporting Information
b
ABSTRACT:
The histamine H3 (H3R) and H4 (H4R) receptors attract considerable interest from the medicinal chemistry community. Given
their relatively high homology yet widely differing therapeutic promises, ligand selectivity for the two receptors is crucial. We
interrogated H4R/H3R selectivities using ligands with a [1,2,3]triazole core. Cu(I)-assisted “click chemistry” was used to assemble
diverse [1,2,3]triazole compounds (6a-w and 7a-f), many containing a peripheral imidazole group. The imidazole ring posed
some problems in the click chemistry putatively due to Cu(II) coordination, but Boc protection of the imidazole and removal of
oxygen from the reaction mixture provided effective strategies. Pharmacological studies revealed two monosubstituted imidazoles
(6h,p) with <10 nM H4R affinities and >10-fold H4R/H3R selectivity. Both compounds possess a cycloalkylmethyl group and
appear to target a lipophilic pocket in H4R with high steric precision. The use of the [1,2,3]triazole scaffold is further demonstrated
by the notion that simple changes in spacer length or peripheral groups can reverse the selectivity toward H3R. Computational
evidence is provided to account for two key selectivity switches and to pinpoint a lipophilic pocket as an important handle for H4R
over H3R selectivity.
’ INTRODUCTION
disease, allergic asthma, and pruritis.9,12 More recently, possible
roles in pain modulation and (breast) cancer have been
revealed.13,14 The high interest of medicinal chemists for H4R
has manifested itself in an increasing development of small
molecules able to modulate H4R. A few selective small-molecule
agonists have been disclosed15-17 and are useful tools in H4R
research.18,19 From a therapeutic point of view though, most of
the focus has been on H4R antagonists, and several different
classes of H4R antagonists have been disclosed to date.14,20-23
H4R antagonists have proven very useful in the confirmation of
postulated roles of the H4R.14,21,24-26
The neurotransmitter histamine exerts its biological action
through four histamine receptors, which all belong to the super-
family of G protein-coupled receptors (GPCRs).1 Whereas the
histamine H1 and H2 receptors are proven targets for blockbuster
drugs, the H3R and H4R both bear promise to reach that stage in
the near future. The H3R was discovered in 19832 and cloned in
1999.3 Ever since, intensive research has taken place to discover
H3R ligands. Consequently, the past decade has seen the
emergence of many different classes of H3R antagonists.4-7
The newest addition to the histamine receptor family is the
H4R. Since its discovery in 2000, it has attracted much interest
from academia and industry alike.8,9 The H4R is widely expressed
on hematopoetic and immune cells, where it mediates chemo-
taxis of eosinophils and mast cells.10,11 Therefore, it is believed to
play an important role in inflammation and immune responses
with possible applications in diseases such as inflammatory bowel
A reoccurring issue in the development of small H4R ligands is
the selectivity between H4R and H3R. These two receptors share
the highest homology within the histamine receptor family (31%
overall, 54% in the transmembrane region).8 This implies that
Received: October 18, 2010
Published: February 24, 2011
r
2011 American Chemical Society
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Figure 2. Initial design of the ligands.
Scheme 1. Synthesis of Imidazole-Containing Azidesa
Figure 1. Exemplary imidazole-containing H4R ligands with selectivity
over H3R.
potential selectivity issues are to be expected. Indeed, several
classical H3R tool compounds, such as thioperamide and clo-
benpropit, also display considerable H4R affinities.16
a Reagents and conditions: (a) TfN3, CuSO4, K2CO3, H2O, DCM,
MeOH, rt, 1 day. Yields: 10a, 92%; 10b, 88%; 10c, 47%. (b) Boc2O,
4-dimethylaminopyridine (DMAP), MeCN, H2O, dioxane, Et3N, rt,
1 day. Yields: 11a, 54%; 11b, 66%; 11c, 87%. (c) NH4CO2H, Pd/C,
MeOH, reflux, 6 h, 99%. (d) H2SO4, EtOH, reflux, 1 d, quant. (e)
LiAlH4, THF, rt, overnight, 50%. (f) Aqueous HBr (48%), reflux, 1 d,
quant. (g) NaN3, H2O, EtOH, reflux, 1 day. (h) Boc2O, DMAP, MeCN,
H2O, dioxane, Et3N, rt, 2 h, 40% over two steps (i.e., from 16).
In the area of non-imidazole ligands, H4R selectivities have
been proven to be readily accessible.24 A fundamentally inter-
esting and intrinsically challenging task is to achieve selectivity
with imidazole-containing ligands, as both receptors are
equipped with imidazole-binding pockets. H3R over H4R selec-
tivity for imidazoles has been widely documented,16,17,27,28 while
imidazole-containing molecules with H4R over H3R selectivity
appear more scarce. In two reports, 4-methylhistamine was identi-
fied as an agonist with high H4R selectivity (>100-fold),16,17
while others showed that varying degrees of H4R selectivity can
also be obtained with ligands having the “usual” monosubstituted
imidazole ring (1-4, Figure 1).27,29-32 Our lab recently disclosed
a study aimed at deciphering the factors for H4R and H3R
affinities in a series of clobenpropit analogues.33 From these
studies, isothiourea 5 emerged as a H4R agonist with high H4R
affinity (pKi = 8.8) and a 4-fold selectivity over H3R.
This modest list illustrates the subtle and poorly understood
nature of H4R/H3R selectivity with monosubstituted imidazoles.
In this report, we aim to pinpoint some important molecular
determinants capable of inducing selectivity in H4R/H3R affi-
nities and, as an expansion, of H3R/H4R selectivity. To this end, a
[1,2,3]triazole scaffold (compounds 6a-w and 7a-f) proved to
be a very suitable platform for pharmacological and computa-
tional investigations into the subtle factors that influence the
affinities of monosubstituted imidazole ligands for H3R and
especially for H4R.
groups in a very similar fashion. It is emphasized that the
[1,2,3]triazole ring is not a moiety with physiologically relevant
basicity (pKHBþ e 1),36 and hence, many of the envisioned com-
pounds are considered monobasic. It is noted that a recent report
shows how click chemistry was applied for non-imidazole
H3R ligands.37
Synthesis. During our structure-activity relationship (SAR)
efforts, the design protocol called for the synthesis of several
alkyne- and azide-building blocks. The synthesis of these will be
discussed in the following sections.
Synthesis of Imidazole-Containing Azides. The synthesis of
the required imidazole-containing azides is shown in Scheme 1.
Histamine (9a) or its homologues 9b,c were conveniently
converted to the corresponding azides 10a-c in one step using
a diazo-transfer step (47-92%).38 For reasons explained later,
the imidazole ring was Boc-protected, which after column
chromatography afforded 11a-c. Large amounts of azide 10b,
and hence 11b, could also be obtained by an alternative route.
Here, inexpensive urocanic acid (12) is converted in three steps
to alcohol 15.39 This is brominated to salt 16 and subjected to a
substitution reaction with NaN3 to give crude 10b with a minor
byproduct likely resulting from intramolecular attack by the
imidazole ring. Installation of the Boc group and purification
afforded 11b (40% from 16).
Synthesis of Non-Imidazole Azides. The synthesis of non-
imidazole azides proceeded along conventional synthetic trans-
formations (Scheme 2). 3-Chloro-1-bromopropane was reacted
with NaN3 to give azide 17, which was not isolated but kept as a
solution in ether because of the risk of explosion associated with
concentrated small organic azides. Reaction of 17 with piperidine
or N-methylpiperazine yielded azide 18a or 18b, respectively.
Amines 19c,d, which are commercially available, were directly
’ RESULTS AND DISCUSSION
Design. At the onset of our studies, we decided to explore
“click” chemistry as the key chemical transformation because of
its appealing ease and chemoselectivity.34,35 More specifically,
the Cu(I)-catalyzed coupling of azide and alkyne building blocks
was to be our key step affording an anti-[1,2,3]triazole element.
The modular nature of click chemistry should thus pave the way
for rapid and efficient exploration of H4R and H3R affinities. We
envisioned an initial design based on a “scaffold hopping”
approach, and toward this end, we selected burimamide ana-
logues (8) reported by us to have good H4R affinity (Figure 2).16
That is, our scaffold contains the [1,2,3]triazole core instead of
the thiourea unit but can otherwise be decorated with peripheral
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Scheme 2. Synthesis of Non-Imidazole Azidesa
Scheme 3. Synthesis of Imidazole-Containing Alkynesa
a Reagents and conditions: (a) Ph3CCl, Et3N, DMF, rt, 2 h. (b) [1]
Oxalyl chloride, DMSO, DCM, -78 °C, 10 min; [2] alcohol, -78 °C,
10 min; [3] Et3N, 39% from 15. (c) Phosphonate 24, K2CO3, MeOH, rt,
4 h, 90%. (d) Ph3CCl, Et3N, DMF, rt, 4 h. (e) 2-(1,3-Dioxolan-2-yl)-
ethyltriphenylphosphonium bromide, n-BuLi, THF, rt, 18 h, 51%
over two steps. (f) H2, Pd/C, MeOH, EtOH, rt, 72 h. (g) 2 N HCl,
acetone, rt, 1 day, quantitative yield from 25. (h) Phosphonate 24,
K2CO3, MeOH, rt, 4 h, 79%. ( i) Concentrated aq HCl, MeOH, H2O,
reflux, 1 h. (j) Boc2O, MeCN, H2O, dioxane, Et3N, rt, 1 day, 60%
from 27.
a Reagents and conditions: (a) NaN3, DMSO, rt, 72 h, >90% conversion,
kept in ether solution. (b) Amine, NaI, Na2CO3, MeCN, reflux, 24 h.
Yields: 18a, 27%; 18b, 32%. (c) TfN3, CuSO4, K2CO3, H2O, DCM,
MeOH, rt, 1 d. Yields: 18c, 76%; 18d, 85%. (d) Concentrated aq
HBr, concentrated H2SO4, reflux, 24 h, 92%. (e) NaN3, EtOH or
MeOH, reflux, 24 h. Yields: 21a, 89%; 21b, 78%; 21c, 11%; 21d, 99%;
21e, 82%.
byproducts (e.g., allenes) did not bode well for purification, and we
opted to continue with the click reaction using impure 30i,j.
Fusion of Alkynes and Azides to Final [1,2,3]Triazoles. The
syntheses of the final receptor ligands were completed by a Cu-
catalyzed click reaction between the azides and alkynes followed,
where applicable, by a deprotection (Scheme 5). While the click
reaction is an extremely attractive and widely applicable tool and
has without doubt also lived up to this reputation in our research,
it is of interest to note that we encountered two unusual
problems in our early attempts using alkyne 30a and azide 10a.
The click reaction between 30a and 10a under standard condi-
tions (cat. Cu(II), sodium ascorbate, air, t-BuOH/H2O)34
proceeded very sluggishly and with formation of several unde-
sired products. Qualitative TLC experiments with model re-
agents shed light on possible reasons for this. First, subjecting
30a to the click conditions with but also without model azide 21a
gave several products more polar than 30a. This led to the
hypothesis that 30a is oxidized by Cu(II). In line with this
hypothesis we found that keeping the amount of Cu(II) minimal
with respect to Cu(I), either by using strictly deoxygenated
conditions or by using large amounts of reducing factor (sodium
ascorbate), led to a successful click reaction between 30a and
21a. Even so, application of these improved conditions to 30a
and azide 10a led to a clean but still unacceptably slow reaction. A
similar effect was observed for the reaction of 30a and model 21a
in the presence of stoichiometric amounts of non-azidoimidazole
15. It was thought that the imidazole group complexes Cu(II)
and thereby reduces the amount of active catalyst. We took this
hurdle by increasing the amount of Cu catalyst while simulta-
neously reducing the complexing potential of the imidazole ring
by use of the electron-withdrawing protecting group Boc.45 This
had the added bonus of easier purification of any protected click
intermediates.
converted to the corresponding azides 18c,d by the azide-transfer
protocol (76-85%).38 A range of halides, either commercially
available (20a-d) or prepared from the alcohol precursor (20e),
were converted to the corresponding azides 21a-e by reaction
with NaN3.
Synthesis of Imidazole-Containing Alkynes. Alkyne 23 has
been reported before.40 Its preparation starts with the synthesis
of aldehyde 22, itself obtained by tritylation of alcohol 15 follo-
wed by a Swern reaction (Scheme 3). However, the reported40
Corey-Fuchs protocol on 22 (i.e., CBr4, PPh3 followed by n-
BuLi) in our hands failed when attempted on a large scale. Hence,
we resorted to a convenient one-step protocol using dimethyl (1-
diazo-2-oxopropyl)phosphonate (24).41 This afforded alkyne 23
in high yield (90%). For the longer-chain alkyne homologue of
(un)protected 23, we sought to apply a similar sequence as for
23. The required aldehyde 26 was prepared through modifica-
tion of a reported procedure.42 It involves a Wittig reaction to
give 25 followed by hydrogenation and aldehyde deprotection.
Gratifyingly, aldehyde 26 smoothly underwent the one-pot
alkynation procedure with phosphonate 24 to give a 79% yield
of 27. A required switch of protecting groups (vide infra)
afforded building block 28.
Synthesis of Non-Imidazole Alkynes. Aminoalkynes 29a,b were
obtained by alkylation with propargyl bromide (Scheme 4). Al-
kynes 30a-h are commercially available, whereas 30i,j require
prior synthesis. Methyl-substituted alkyne 30i was obtained in
racemic form following an adapted literature procedure.43 This
involves the LiCuI2-induced reaction between a Grignard reagent
and the propargylic mesylate 31 reportedly through the interme-
diacy of a propargylic iodide.43 For 3-cycloheptylpropyne (30j), we
used a methoxyallene-based synthetic strategy described for 30d44
and successfully obtained 30j by switching to c-HepMgBr. The
volatility and very low polarities of both 30i,j and associated
For practical reasons, all discussed remedies were successfully
combined into one general protocol consisting of the use of
1-20 mol % CuSO4, 10-200 mol % sodium ascorbate, strict N2
atmosphere, and Boc-protected imidazole rings (Scheme 5).
Interestingly, in retrospect the O2 removal and reduction of
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Scheme 4. Synthesis of Non-Imidazole Alkynesa
a Reagents and conditions: (a) propargyl bromide, Cs2CO3, acetone, 20 h, rt. Yields: 29a, 26%; 29b, nd. (b) MeSO2Cl, Et3N, CH2Cl2, 3 h, 0 °C, 95%. (c)
[1] LiI, CuI, THF; [2] mesylate 31, 2 h, rt; [3] c-HexMgCl, -70 °C, <1 min; [4] sat. aq NH4Cl, -70 °C, yield: nd. (d) [1] Mg, THF, rt f 40 °C, 3.5 h;
[2] CuI, methoxyallene, 10 °C, 40 min; [3] rt, 12 h, yield: nd. nd = not determined.
Scheme 5. Synthesis of the Final [1,2,3]Triazolesa
found in the Supporting Information, including 2D NMR
evidence for selected compounds concerning the correct regio-
chemistry of the [1,2,3]triazole.
Pharmacology. As can be seen in Figure 2, our final com-
pounds can be divided into four parts: an imidazole, a spacer, the
[1,2,3]triazole, and the peripheral R group. We started by
inspecting the spacer length with the aid of benzyl-substituted
derivatives 6a-c. H3R and H4R affinities were measured by
radioligand displacement assays using [3H]NR-methylhistamine
and [3H]histamine as radioligand, respectively.16,21 Histamine
and thioperamide were used as controls. The results (Table 1)
reveal that the n = 3 spacer length is the most attractive for H4R
affinity (6b, pKi = 6.74), albeit that the same held true for H3R
a Reagents and conditions: (a) CuSO4 5H2O (1-20 mol %), sodium
affinity. Using this finding, we next scanned a selected set of
aliphatic and polar peripheral R groups that included the known
preferred H4R element 4-methylpiperazine (6d-g).24 This led
to the conclusion that H4R affinity benefits best from an aliphatic
group R and that, interestingly, 6f and 6g provided a first glimpse
into H4R/H3R selectivity. The aliphatic group therefore became
our point of focus.
Remarkably, H4R affinity and selectivity got a large boost
when a cyclohexylmethyl group was installed (6h, pKi(H4R) =
8.08, pKi(H3R) = 6.90), prompting us to interrogate this specific
group in a detailed SAR study (Table 2). The optimal spacer
length of n = 3 for H4R affinity was reinforced (6h-j), and spacer
lengths of n = 1 or n > 4 were therefore not pursued. A striking
reversal in H4R/H3R selectivity upon simple spacer shortening
(compare 6h to 6i) was observed. Additional SAR studies on the
cyclohexylmethyl moiety showed that methylene removal (6k)
or insertion (6l) and ring-opening (6m) all led to compromised
H4R affinity. A sterically more subtle exercise, i.e., methylene
substitution to rac-6n, still afforded good affinity (H4R pKi =
7.67), but the H4R/H3R selectivity was reduced by a factor ∼5.
Ring enlargement to a cycloheptyl (6o) also led to a substantial
drop in H4R affinity and H4/H3 selectivity. Ring contraction, on
the other hand, gave a compound with H4R affinity and selectivity
3
ascorbate (10-200 mol %), strict N2 atmosphere, t-BuOH/H2O, rt,
4 - 72 h. (b) TFA, DCM, rt, 2-4 h. Except for 7d: aq HCl, MeOH,
reflux, 1 h.
electron density in the imidazole ring by Boc installation may
have very well prevented a recently disclosed problem, i.e., the
aerobic oxidation of imidazole rings under click conditions
lacking stabilizing ligands.46 The synthesis of “inverted” triazoles
7a-c,e,f generally proceeded more smoothly compared to that
of the imidazole-containing members of the 6 series. In such
cases, amounts of Cu and ascorbate could be reduced. Note-
worthy, however, is the click reaction leading to 7d (i.e., with 23)
which proceeded very sluggishly. This being the only instance
where a trityl-protecting group was used instead of a Boc, it
appears to reconfirm the importance of reducing the electron
density on the imidazole ring and is the reason why a protecting
group switch was applied (from 27 to 28) when we embarked on
the longer-chain homologues 7a-c,e,f.
Any Boc group was deprotected using trifluoroacetic acid
(TFA) to give the final compounds. The exception was com-
pound 7d, for which the trityl group was removed with refluxing
HCl. Details for all click reactions and deprotections can be
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Table 1. H3R and H4R Affinities of Compounds with Varying
Triazole Substituents
Table 2. H3R and H4R Affinities of Compounds with
Aliphatic Triazole Substituentsd
a Homogenates of human embryonic kidney (HEK) 293T cells, stably
expressing either the human H3R or the human H4R, were used for
determining ligand affinities for H3R and H4R with [3H]NR-methylhis-
tamine and [3H]histamine as radioligand, respectively. Histamine and
thioperamide are reference compounds. Measurements shown are the
mean of at least three experiments.
matching those of 6h (6p, pKi(H4R) =8.10, pKi(H3R) =
7.04). Last, as a final confirmation of the n = 3 spacer length,
the best compounds from this mini-SAR (i.e., rac-6n and 6p)
were subjected to spacer-shortening, affording almost a 100-fold
drop in affinity (rac-6r, 6q).
a Homogenates of human embryonic kidney (HEK) 293T cells, stably
expressing either the human H3R or the human H4R, were used for
determining ligand affinities for H3R and H4R with [3H]NR-methylhis-
tamine and [3H]histamine as radioligand, respectively. Measurements
shown are the mean of at least three experiments. b Measured as racemic
mixture. c Defined as the Ki for H4R divided by the Ki for H3R. d Results
of reference compounds are given in Table 1.
Evidently, the nature of the cycloaliphatic group must meet
very strict steric demands, as no manipulation on 6h improved on
H4R affinity. Bearing in mind that analogues of 6h with N-atoms
inserted (i.e., 6d and 6e, Table 1) led to dramatic loss of H4R
affinity, the collective data imply that a complementary lipophilic
pocket in H4R is targeted with high steric precision, most notably
in 6h and 6p. In contrast to this sensitivity associated with H4R
affinity, corresponding H3R affinities remain strikingly similar in
the SAR (Table 2) irrespective of the SAR manipulation involved
(spacer shortening or elongation, methylene substitution, ring-
contraction or -expansion or -opening). This puts forward
the peripheral cycloaliphatic group as an excellent handle to
modulate H4R/H3R selectivity. Indeed, 6h and 6p display
H4R/H3R selectivities that are noteworthy for monosubstituted
imidazoles.
Toward this end, we swapped the alkyne and azide functional
groups of selected combinations, giving the alternative [1,2,3]-
triazole fusion product shown in Scheme 5 (7a-f, Table 3).
Decoration with benzyl groups (7a,b) reduced the H4R affinity
compared to benzyl isomer 6b. Likewise, a ∼10-fold reduction of
H4R affinity was observed when the cyclohexylmethyl-group was
installed (compare 7c to 6h). Nevertheless, within a mini-SAR
on 7c involving spacer shortening, methylene insertion, and ring-
shift (7d-f), the cyclohexylmethyl group was still preferred for
H4R affinity. It stands to reason that the 7 series is targeting the
same H4R pocket as the 6 series does. Nonetheless, the consistent
decrease in affinity of 7a,c,e compared to 6b,h,l, respectively,
We were interested to see to what extent the central
[1,2,3]triazole unit contributes to the observed H4R affinities.
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Table 3. H4R Affinities of Compounds with an “Inverted”
replacement of the imidazole by alternative basic groups.4 To
confirm this hypothesis, we first used the best H4R binder 6h as a
case. Replacement of the C-linked imidazole in 6h by an N-linked
imidazole (6s) proved to be fruitless. However, within the
explored replacements by cyclic amines (6t-v), ligands 6u,v
pushed the pKi for H4R below 5.0 while H3R affinity could be
largely maintained with respect to 6h, especially so for piperidine
6v. With the latter finding at hand, we next returned to starting
point 6d and replaced its imidazole group by a piperidine
(affording 6w). Gratifyingly, 6w displayed a H3R affinity of
6.92 with a H3R/H4R selectivity >300. Indeed, the double-
piperidine motif is not uncommon among other H3R ligands
reported in the literature.47,48
Triazole Corec
In all, by replacing the peripheral imidazole and cyclohexyl
units in H4R-selective compound 6h by two piperidines (i.e.,
6w), we were able to maintain the exact same H3R affinity but
induced a concomitant drop in H4R affinity by almost a factor
5000. As a whole, this reinforces the notion that H3R is generally
forgiving toward decoration of our [1,2,3]triazole based scaffold
while H4R affinities are dramatically affected. Future studies
could address the question of whether these observations extend
from human H4R and H3R to those of other species.
Structure-Based Rationalization of Structure-Activity Re-
lationships. Molecular modeling studies based on three-dimen-
sional H3R and (previously validated49) H4R receptor models
and ligand-receptor interaction fingerprint analysis50,51 of dock-
ing simulations52 (described in the Supporting Information)
were used to explain two illustrative selectivity switches.
Known selective H4R agonists 4-methylhistamine16 and OUP-
16 (1)29 could be accommodated in the H4R model while
forming H-bonds with both essential negatively ionizable resi-
dues D3.32 and E5.4653,54 simultaneously (see Supporting
Information Figure S2). In the H3R model, on the other hand,
no binding modes of 4-methylhistamine and 1 could be gener-
ated, which satisfied both essential H-bond interactions55 (see
Supporting Information Figure S2), demonstrating the suitability
of the H3R and H4R models to rationalize H4R over H3R
selectivity.
a Homogenates of human embryonic kidney (HEK) 293T cells, stably
expressing the human H4R, were used for determining ligand affinities
for H4R with [3H]histamine as radioligand. Measurements shown are
the mean of at least three experiments. b Tested as the dihydro-
chloride salt. c Results of reference compounds are given in Table 1.
We used the same docking approach56 to propose binding
modes for the H4R selective agonist 6h in the H3R and H4R
receptor models (Figure 4A,B). The imidazole group of 6h forms
an H-bond to D3.32 in H3R (Figure 4A) and H4R (Figure 4B)
receptors. This binding mode is in line with earlier site-directed
mutagenesis (SDM) studies indicating the essential role of D3.32
in ligand binding in H3R55 and H4R.53,54 Mutation of E5.46,
another conserved negatively charged residue in H3R and H4R
binding pockets, diminishes histamine binding54,55 but does not
affect binding of iodoproxyfan (which does not bind to the D3.32
mutant in H3R).55 Altogether this suggests that the imidazole
headgroup of iodoproxyfan interacts with D3.32.57 In H3R, 6h
accepts an H-bond from T6.52 to the triazole moiety while
accommodating its lipophilic cyclohexyl ring into the hydro-
phobic pocket between TM helices 3 (A3.40), 5 (F5.47), and 6
(F6.44, W6.48) (Figure 4A). In H4R, the smaller T6.55 residue
(M6.55 H3R) allows the triazole group of 6h to approach TM5
and accept an H-bond from S5.42 (Figure 4B).
indicates that it does so in a less efficient manner as a result of
inverting the [1,2,3]triazole core. Hence, this scaffold was not
pursued further.
The functional activities of 6h and 6i on both H4R (Figure 3A)
and H3R (Figure 3B) were tested in a CRE (cAMP response
element) luciferase reporter gene assay with agonist histamine
and inverse agonist thioperamide as controls. Figure 3 clearly
shows that 6h and 6i display agonistic behavior on H4R, with the
corresponding pEC50 values being 9.0 ( 0.1 and 6.8 ( 0.1,
respectively (n = 3). Noteworthy in this respect is the absence of
a second basic site in these agonists (triazole pKHBþ e 1),36
which is frequently associated with H4R agonism.15,16,33 While 6i
is also an agonist on H3R (pEC50 = 6.6 ( 0.1), we found that 6h
is unable to evoke a response on H3R. These data point toward a
spacer-dependent agonism/antagonism switch for H3R in this
class of compounds.
The striking sensitivity with H4R affinities and contrasting
insensitivity with H3R affinities in the 6 series led us to investigate
whether the same versatile [1,2,3]triazole scaffold could be used
to make H3R-selective ligands (Table 4). The starting point was
compound 6d which displays dramatically reduced H4R affinity
(pKi = 5.7) compared to 6h with, however, an accompanying
smaller drop in H3R affinity (Table 1). It was hypothesized that
H3R affinity could be increased more than the H4R affinity by
Our receptor models can be used to rationalize the overall H4R
over H3R selectivity drop from 6h to 6w by a factor ∼5000
(Table 4) that can be achieved by our [1,2,3]triazole compounds.
Modification of the imidazole head to a piperidine or piperazine
group has a dramatic effect on the affinity for H4R because the
intramolecular H-bond interaction between D3.32 and Q7.42
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Figure 3. Functional activity of 6i and 6h on hH4R (A) and hH3R (B). Effect of histamine (O), thioperamide (b), 6i (0), and 6h (4) on 1 μM
forskoline-stimulated HEK293T cells transiently expressing CRE luciferase and hH4R or hH3R was measured. Agonist histamine (pEC50 = 7.6 ( 0.1
(H4R), 7.8 ( 0.1 (H3R)) and inverse agonist thioperamide (pEC50 = 7.1 ( 0.2 (H4R), 7.4 ( 0.2 (H3R)) were used as controls, and their values were set
at -100% and 100%, respectively. Graphs shown are pooled data from at least three independently performed experiments. Error bars indicate SEM
values.
Table 4. H3R and H4R Affinities of Compounds with Varying Peripheral Groupsd
a Homogenates of human embryonic kidney (HEK) 293T cells, stably expressing either the human H3R or the human H4R, were used for determining
ligand affinities for H3R and H4R with [3H]NR-methylhistamine and [3H]histamine as radioligand, respectively. Measurements shown are the mean of at
least three experiments. b Defined as the Ki for H4R divided by the Ki for H3R. c Tested as the fumarate salt. d Results of reference compounds are given in
Table 1.
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’ CONCLUSION
This paper describes compounds containing a [1,2,3]triazole
core, obtained by Cu(I)-catalyzed fusions of an alkyne and azide
(“click reaction”). Some synthetic hurdles were encountered
concerning imidazole moieties and an oxidizable alkyne. These
issues were successfully counteracted by applying several reme-
dies: O2 removal, increase in amount of ascorbate, decrease of
electron density on the imidazole, and an increase in amount of
catalysts. The general skeleton thus synthesized consists of an
imidazole, a spacer, the [1,2,3]triazole, and the peripheral R
group. In terms of decoration of the [1,2,3]triazole scaffold, H4R
affinities proved remarkably sensitive while H3R affinities were
strikingly insensitive, allowing a spectrum of H4R/H3R selectiv-
ities to be obtained. With subtle changes in aliphatic group, H4R-
agonist 6h was obtained which boosted high H4R affinity (pKi =
8.08) and a good H4R/H3R selectivity of 15, noteworthy for a
monosubstituted imidazole compound. In contrast, a re-
placement of both peripheral groups by piperidines led to
compound 6w with good H3R affinity (pKi = 6.92) and excellent
H3R/H4R selectivity (320). Molecular modeling studies were
used to address the role of the cycloalkylmethyl group, to inspect
receptor-ligand interactions when two peripheral piperidines
are installed, and to explain a key selectivity switch upon spacer
shortening. In all, the versatile [1,2,3]triazole core has proven to
be a useful tool scaffold to investigate the at times intriguingly
subtle differences between affinities for H4R and H3R.
Figure 4. Binding modes of 6h (green carbon atoms) and 6i (magenta
atoms) in H3R (A) and H4R (B) and 6w (orange atoms) in H3R (C) and
H4R (D) receptor models. The backbones of TM helices 5, 6, and 7 are
represented by yellow ribbons, and part of TM3 is shown as ribbon (the
top of the helix is not shown for clarity). Important binding residues
are depicted as ball-and-sticks with gray carbon atoms. The bottom of
the lipophilic binding pocket between TM helices 3, 5, and 6 is displayed
as a gray surface. Oxygen, nitrogen sulfur, and hydrogen atoms are
colored red, blue, yellow, and cyan, respectively. H-Bonds are depicted
by black dotted lines.
’ EXPERIMENTAL SECTION
General Remarks. Given the possibility of explosions, caution
should be exercised when working with organic azides, especially those
with low molecular weight. Unless reported otherwise, all chemicals
were from Aldrich. THF, toluene, and CH2Cl2 were freshly distilled
from CaH2. All other solvents were used as received. Unless indicated
otherwise, all reactions were carried out under an inert atmosphere. TLC
analyses were performed with Merck F254 alumina silica plates using
UV visualization or staining. Column purifications were carried out
manually using Silicycle Ultra Pure silica gel or automatically using the
Biotage equipment. All HRMS spectra were recorded on Bruker
micrOTOF mass spectrometer using ESI in positive ion mode. The
1H, 13C, and 2D NMR spectra were recorded on a Bruker 200, 250, 400,
or 500 MHz spectrometer. Depending on the exact conditions, 1H and/or
13C signals for the protons and carbons of unprotected imidazole rings
were not or only partially visible. On a few occasions, high-temperature
NMR was shown to restore the visibility of these signals (see data for
6h and 6p). Infrared spectra were recorded on a Galaxy series FT-IR
6030. Melting points were taken using the Stanford Research Systems
Optimelt apparatus, and values given are uncorrected. Elemental
analysis results were recorded at Mikroanalytisches Labor Pascher
(Remagen-Bandorf, Germany). Systematic names for molecules accord-
ing to IUPAC rules were generated using the Chemdraw AutoNom
program. Unless specified otherwise, all compounds have a purity of
g95%. This was determined using a Shimadzu HPLC/MS workstation
with a LC-20AD pump system, SPD-M20A diode array detection, and a
LCMS-2010 EV liquid chromatograph mass spectrometer. The buffer
mentioned is a 0.4% (w/v) NH4HCO3 solution in water, adjusted to pH
8.0 with NH4OH. The column used is an Xbridge C18 5 μm column
(100 mm ꢀ 4.6 mm). Compound purities were calculated as the
percentage peak area of the analyzed compound by UV detection at
230 nm. Solvents used in this paragraph were the following: solvent B =
90% MeCN-10% buffer; solvent A = 90% water-10% buffer. The
analysis was conducted using a flow rate of 1.0 mL/min, start 5% B,
linear gradient to 90% B in 8 min, then 0.5 min at 90% B, then 6.5 min at
(L7.42 in H3R) in H4R does not allow binding of a large moiety
between TM3 and TM7 as exemplified for compound
6w (Figure 4C). Reorientation of the ligand shifts the other
piperidine ring of 6w even further out of the apolar binding
pocket of H4R. Docking simulations of 6w in H3R suggest that
the two piperidine rings cannot be simultaneously accommo-
dated in the apolar binding pockets between D3.32 and L7.42
and between TM3, TM5, and TM6 (Figure 4D). This is,
however, compensated by favorable ionic interactions with
D3.32 and E5.46.
The most intriguing H4R/H3R selectivity switch observed in
our SAR, however, was the 151-fold change in receptor selectivity
from compound 6h (H4R over H3R selectivity, 15.1) to 6i (H4R
over H3R selectivity, 0.1) by decreasing the linker length with just
a single C-C bond (Table 2). Compounds 6h and 6i adopt
similar binding modes in the H3R receptor model (Figure 4A), in
line with the very similar binding affinities of 6h and 6i for H3R
(Table 2). In H4R, however, compound 6h can accommodate its
cyclohexyl ring significantly deeper in the hydrophobic pocket
between TM helices 3 (V3.40), 5 (F5.47), and 6 (F6.44, W6.48)
than 6i (Figure 4B), providing a plausible explanation for its
higher affinity for H4R (Table 2). Interestingly, mutation of
A3.40 into a valine residue (the corresponding residue in H4R at
this position) increases H3R affinity for neutral imidazole con-
taining ligands,58 supporting the important role of this hydro-
phobic pocket in H4R over H3R selectivity of 6h (Table 2), as
suggested by our modeling studies.
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Journal of Medicinal Chemistry
ARTICLE
5% B, total run time of 15 min. The occasional fumarate counterion is
also visible by UV. For compound 6w, purity is >95% as determined by
elemental analysis.
General Procedure for Synthesis of 6a-w and 7a-f. In a
round-bottom flask, the azide and alkyne were mixed with the indicated
volume of t-BuOH/H2O. Then the indicated amount of sodium
ascorbate was added as a solid and a septum was placed on the flask.
The mixture was degassed by bubbling N2 through the solution for 5 min
characterization of all compounds; representative 1D, 2D, and
high-temperature NMR spectra; and selected LC chromato-
grams. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ31-20-5987603. Fax:þ31-20-5987610. E-mail: m.wijtmans@
using a needle. The indicated amount of a solution of CuSO4 5H2O in
3
vu.nl.
water (0.3 M) was added with the aid of a syringe. The mixture was
briefly degassed again by bubbling N2 through for 2 min. The mixture
was stirred under a N2 atmosphere and at room temperature for the
indicated time. One of three workup/deprotection protocols (see
Supporting Information) was subsequently used to provide 6a-w and
7a-f.
’ ACKNOWLEDGMENT
This work was supported by the Top Institute Pharma
(Project Number D1.105, the GPCR Forum), The Netherlands
Organization for Scientific Research (VENI Grant 700.59.408),
and COST Action BM0806. Hans Custers, Tarik Tagherbit, and
Kaamar Azijli are acknowledged for technical assistance.
1-(3-(1H-Imidazol-4-yl)propyl)-4-(cyclohexylmethyl)-1H-
1,2,3-triazole (6h). The general procedure was followed using azide
11b (100 mg, 0.4 mmol), alkyne 30d (64 μL, 0.44 mmol), water (2 mL),
t-BuOH (2 mL), sodium ascorbate (174 mg, 0.88 mmol), CuSO4
solution (0.3 M, 0.27 mL, 0.08 mmol), and a reaction time of 1 night.
Subsequently, protocol no. 2 (see Supporting Information) was used
including (a) column chromatography on the intermediate (6:2:1
hexane/DCM/TEA) and (b) deprotection using TFA (1 mL), DCM
(1 mL), and a reaction time of 2 h. This gave the product as a white solid
(60 mg, 55%). 1H NMR (CDCl3): δ = 9.33 (br, 1H), 7.61 (br, 1H), 7.28
(s, 1H), 6.84 (br, 1H), 4.33 (t, 2H, J = 6.9 Hz), 2.62 (t, 2H, J = 6.3 Hz),
2.54 (d, 2H, J = 6.8 Hz), 2.22 (p, 2H), 1.73-1.48 (m, 6H), 1.28-1.04
(m, 3H), 1.03-0.84 (m, 2H); imidazole protons and N-H are very
broad. 13C NMR (CDCl3): δ = 146.34, 120.98, 48.65, 37.44, 32.80,
32.32, 29.56, 26.00, 25.43, 23.18; imidazole carbons are difficult to
detect. Peaks were sharpened substantially in DMSO at higher tem-
perature: 1H NMR (DMSO-d6, 350 K): δ = 11.60 (v br, 1H), 7.75 (s,
1H), 7.49 (s, 1H), 6.77 (s, 1H), 4.33 (t, 2H, J = 7.1 Hz), 2.54-2.49 (m,
4H), 2.12 (p, 2H), 1.72-1.53 (m, 6H), 1.29-1.10 (m, 3H), 1.03-0.91
(m, 2H). HR-MS: [M þ H]þ C15H24N5 calcd, 274.2026; found,
274.2015. The Supporting Information contains graphical descriptions
on 2D NMR analysis and a LC-MS chromatogram.
1-(3-(1H-Imidazol-4-yl)propyl)-4-(cyclopentylmethyl)-1H-
1,2,3-triazole (6p). The general procedure was followed using azide
11b (100 mg, 0.4 mmol), alkyne 30h (52 mg, 0.48 mmol), water (2 mL),
t-BuOH (2 mL), sodium ascorbate (174 mg, 0.88 mmol), CuSO4
solution (0.3 M, 0.27 mL, 0.08 mmol), and a reaction time of 1 night.
Subsequently, protocol no. 2 (see Supporting Information) was used
including (a) column chromatography on the intermediate (6:2:1
hexane/DCM/TEA) and (b) deprotection using TFA (1 mL), DCM
(1 mL), and a reaction time of 2 h. This gave the product as a white oily
solid (52 mg, 50%). 1H NMR (CDCl3): δ = 8.41 (br, 1H), 7.57 (s, 1H),
7.30 (s, 1H), 6.80 (s, 1H), 4.33 (t, 2H, J = 7.0 Hz), 2.68 (d, 2H, J = 7.2
Hz), 2.60 (t, 2H, J = 7.1 Hz), 2.21 (p, 2H), 2.19-2.03 (m, 1H), 1.81-
1.64 (m, 2H), 1.65-1.42 (m, 4H), 1.27-1.09 (m, 2H). 13C NMR
(CDCl3): δ = 147.42, 135.37, 134.41, 119.83, 116.36, 48.65, 39.47,
32.06, 31.14, 29.58, 24.66, 23.06; imidazole carbons are visible but small.
Peaks were sharpened substantially in DMSO at higher temperature: 1H
NMR (DMSO-d6, 350 K): δ = 11.58 (br), 7.77 (s, 1H), 7.48 (s, 1H),
6.76 (s, 1H), 4.33 (t, 2H, J = 7.1 Hz), 2.63 (d, 2H, J = 7.3 Hz), 2.51 (t,
2H), 2.20-2.04 (m, 3H), 1.79-1.69 (m, 2H), 1.65-1.45 (m, 4H),
1.28-1.17 (m, 2H). HR-MS: [M þ H]þ C14H22N5 calcd, 260.1870;
found, 260.1862. The Supporting Information contains graphical de-
scriptions on 2D NMR analysis and a LC-MS chromatogram.
’ ABBREVIATIONS USED
H4R, histamine H4 receptor; H3R, histamine H3 receptor;
GPCR, G protein-coupled receptor; DMAP, 4-dimethylamino-
pyridine; TFA, trifluoroacetic acid; HEK, human embryonic kidney;
rac, racemic; CRE, cAMP response element; SAR, structure-
activity relationship; TM, transmembrane; SDM, site-directed
mutagenesis; rt, room temperature
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