1-[(2,3-Dihydro-1-benzofuran-2-yl) methyl]piperazines as novel anti-inflammatory compounds: Synthesis and evaluation on H3R/H4R

Article abstract of DOI:10.1111/cbdd.12947

The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H₃R and H₄R have been explored as targets for drug discovery, including in the searc

Full text of DOI:10.1111/cbdd.12947

DR. JOÃO PAULO DOS-SANTOS FERNANDES (Orcid ID : 0000-0002-9089-273X)
Received Date : 22-Oct-2016
Revised Date : 10-Jan-2017
Accepted Date : 14-Jan-2017
Article type : Research Letter
1‐[(2,3‐Dihydro‐1‐benzofuran‐2‐yl) methyl]piperazines as Novel Anti-
inflammatory Compounds: Synthesis and Evaluation on H3R/H4R
Michelle Fidelis CORRÊAa, Marina Themoteo VARELAa, Aleksandro Martins
BALBINOa, Ana Claudia TORRECILHASa, Richardt Gama LANDGRAFa, Lanfranco
Ranieri Paolo TRONCONEb and João Paulo dos Santos FERNANDESa,∗
aDepartamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Rua São
Nicolau 210, Diadema-SP 09913-030, Brazil
bInstituto Butantan, Av. Vital Brazil 1500, São Paulo-SP 05503-900, Brazil
Keywords: H3R ligand, H4R ligand, histamine receptor, anti-inflammatory agent,
asthma.
Abstract
The histamine receptors (HRs) are members of G-protein coupled receptor (GPCR)
superfamily and traditional targets of huge therapeutic interests. Recently, H3R and
H4R have been explored as targets for drug discovery, including in the search for
dual-acting H3R/H4R ligands. The H4R, the most recent histamine receptor, is a
promising target for novel anti-inflammatory agents in several conditions such as
∗Corresponding author. Tel.: +55-11-3385-4137; e-mail: joao.fernandes@unifesp.br
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12947
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asthma and other chronic inflammatory diseases. Due to similarity with previously
reported
ligands
of
HRs,
a
set
of
1‐[(2,3‐dihydro‐1‐benzofuran‐2‐yl)methyl]piperazines
were
synthesized
and
evaluated in competitive binding assays as H3R/H4R ligands herein. The results
showed the compounds presented affinity (Ki) for H3R/H4R in micromolar range, and
are more selective to H3R. All the compounds showed no important cytotoxicity to
mammalian cells. The phenyl-substituted compound LINS01005 has shown the
higher affinity of the set for H4R but no considerable selectivity towards this receptor
over H3R. LINS01005 showed interesting anti-inflammatory activity in murine asthma
model, reducing the eosinophil counts in bronchoalveolar lavage fluid , as well as the
COX-2 expression. The presented compounds are valuable prototypes for further
improvements to achieve better anti-inflammatory agents.
Histamine is one of the most important chemical transmitters involved in several
biological processes. It has been widely studied since its discovery due to its role in
inflammatory and allergic reactions, but is also involved in gastric acid secretion,
sleep, mood and food intake regulations.[1] These processes are triggered by the
interaction of histamine with its receptors (HRs), namely from H1R to H4R. The HRs
are members of G-protein coupled receptor (GPCR) superfamily, and considering
they have shown biased behavior after the interaction with some ligands, they may
be also called as 7 transmembrane (7TM) receptors.[2,3]
H3R is found in CNS, and is involved in inhibition of the release of histamine, as well
as other neurotransmitters, including norepinephrine, acetylcholine and serotonin.[4]
Therefore, histamine H3R modulates neurotransmitter release acting as presynaptic
receptor (auto- and hetero-receptor). It was shown that signal transduction by H3R is
done by Gi/o proteins, leading to decrease in intracellular cAMP concentration and
decreased Ca2+ influx in neurons.[5] As result of its actions, H3R could be involved in
several neurological disorders, such as cognitive, convulsive and sleep-wake
disorders, as well as obesity.[6] So, H3R ligands could be promising drugs to treat
these conditions. Ligands of H3R have been widely explored, clobenpropit, ciproxifan
and ABT-239 (Figure 1) being examples of well-studied compounds.
In late 2000 and early 2001, several research groups described a new isoform of HR
expressed in immune cells such as eosinophils, basophils, mast cells, NK cells, DC,
monocytes and T cells, which is involved in modulation of chemotaxis as well as
other functions.[7,8] This receptor was namely H4R, and has considerable homology
to H3R (~39% total, 54% in TM domains, similarity 68%).[9] Since these cells are
involved in immune and inflammatory processes, H4R is considered a promising
target for future anti-inflammatory and immunological drugs. Even as H3R, the H4R is
also coupled to Gi/o proteins.[3] H4R ligands have continuously been designed and
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evaluated as drug candidates (Figure 1), the indolecarboxamides (JNJ-7777120)
and 2-aminopyrimidines (A-943931) being the most explored chemotypes with this
regard.
Due to the similarity between H3R and H4R, it is obvious that several compounds
which bind to one of them can present considerable affinity to the other. Hence,
some pharmacophore models to H3R ligands can be also applied to H4R ligands.[10]
In fact, the search for selective ligands for those receptors are always reported in
literature, and several medicinal chemists which are involved in HR ligands are
focused in this goal. However, dual-acting H3R/H4R ligands may present therapeutic
potential in some pathological conditions, such as neuropathic pain,[11] cancer,[12]
and more recently in Parkinson’s disease.[13,14] This potential was already reported
for imidazole-containing H3R/H4R ligands, such as imetit, immepip, clobenpropit and
thioperamide.[3,10]
To our knowledge, dihydrobenzofuran-containing molecules were not evaluated yet
as H3R and/or H4R ligands. Considering the chemical similarities observed in several
H3R and H4R ligands (such as a resonance center, and a polar basic group, Figure
1), we have designed a small set of novel 1‐[(2,3‐dihydro‐1‐benzofuran‐2‐yl)
methyl]piperazines (LINS01 series – 1-4) to evaluate their potential as ligands of
these receptors. The design was based on the presence of an aryl or heteroaryl
group linked to a polar moiety in H3R and H4R ligands. The presence of an extra
lipophilic group attached to the polar moiety was also evaluated. The designed
compounds can also help to evaluate the importance of the carbonyl and pyrrole NH
groups to H4R affinity.
The compounds were synthesized (see Supporting Information) according to the
Figure 2. 2-Allylphenol was used as starting material, which was then converted into
the (2-iodomethyl)-2,3-dihydrobenzofuran as previously described by our group.[15,16]
By using this methodology, the 2-allylphenol was then converted into the (2-
iodomethyl)-2,3-dihydrobenzofuran (5) giving excellent yield (90%). This procedure
can satisfy the demands of environmentally benign green chemistry,[17] since water
was used as solvent instead of environmentally undesired dichloromethane.
Moreover, the previous method employed NaHCO3, which did not show significant
contribution to the yields, and so it was discharged from the procedure presented
herein.
Subsequently, the halogenated compound (5) was reacted with the piperazine
derivatives to give the final compounds (1-4). The N-methyl (2) and N-phenyl (4)
derivatives were prepared from the commercially available reagents (1-
methylpiperazine and 1-phenylpiperazine, respectively) and using the methodology
adapted from Ohtaka et al.[18] using polar solvent and in the presence of base.
However, the use of ethanol as solvent and stirring at room temperature led to poor
yields. By using reflux and an aprotic polar solvent (tetrahydrofuran), the yields were
much higher, and then this procedure was employed in the preparation of non-
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alkylated (1) and N-allyl (3) compounds. The yields to the final products are
presented in table 1.
To obtain the molecule 1, the iodinated compound 5 was then reacted with
considerable excess of piperazine to avoid polyalkylation of the nitrogens.
Preliminary runs using different synthetic approaches have not shown adequate
results to prepare this compound. The protection of one of the nitrogen atoms of
piperazine through neutralization with HCl (i.e. piperazine monohydrochloride) as
reported by Cunico et al.[19] led to poor yield of the product 1 and thus was not
considered adequate. Moreover, solvents such as ethanol or acetone led to lower
yields, and THF showed again to be the best option to achieve high yields.
Using protecting groups (such as tert-butyloxycarbonyl) is another option to avoid the
formation of disubstituted products.[20] Although this strategy provides easy
purification of the product, it increases the synthetic steps and thus decreases the
overall yield.[21] Thus, the use of piperazine in excess was considered more viable,
giving good yields even for compound 1 (83%). For comparison, Meng et al.[22]
described the synthesis of compound 1 using 5 equivalents of piperazine in 2-hours
reaction. In addition to the lower yield obtained (54%), column chromatography was
used (methanol:dichloromethane as eluent) for purification. The present
methodology does not require chromatographic purification since the product can be
crystallized after the acid-base extraction procedure, and thus greater synthetic
viability is evident.
This procedure was also applied to prepare the non-commercially available 1-
allylpiperazine. Interestingly, the yields were quite variable through several runs, and
considerable amounts of 1,4-diallyl product was always detected. However, the N-
allyl derivative 3 also was obtained with success.
Binding assays were performed through the displacement of [3H]-histamine by the
tested compounds from human H3R and H4R membranes commercially available
from PerkinElmer (cat. no. ES-392-M400UA and ES-393-M400UA, respectively),
following its recommended protocol (see Supporting Information). Commercially
available compounds A-943931 and (R)-α-methylhistamine were used as reference
ligands of H4R and H3R, respectively. The binding affinities (Ki) were determined
from the IC50 values (concentration to displace 50% of the radioligand from the
receptors) using Cheng-Prusoff equation.[23] Determinations were done in three
independent triplicates. The results are shown in table 1.
In order to evaluate the cytotoxicity of the synthesized compounds, cell viability tests
were performed in HeLa (human cervix carcinoma, ATCC CCL-2) and J774A.1
(mouse macrophages, ATCC TIB-67) mammalian cells after 24, 48 and 72 h
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incubation with each compound. Cell viability was assessed using rezasurin as
indicator of cell activity, where each determination was compared to a compound-
free control (see Supporting Information).[24] The compounds were tested in
concentrations from 300 to 1 µM, and used to calculate the CC50 values
(concentration to reduce rezasurin conversion in 50%). The evaluations were done in
triplicate, and the results of cytotoxicity are also shown in table 1.
The synthesized compounds have shown poor affinity for both H3R and H4R,
exhibiting Ki values in micromolar range. However, they showed more affinity to H3R
than H4R. Regarding H3R affinity, the N-allyl derivative 3 was the most potent,
presenting Ki 7 µM, followed by N-phenyl (4) and N-methyl (2) derivatives. The non-
alkylated molecule 1 did not show appreciable affinity for H3R. The results suggest
the presence of an alkyl group linked to the polar moiety of the molecule increases
the affinity for H3R.
Several 2-aminoethylbenzofurans were already evaluated as H3R ligands, showing
good affinity.[25] These compounds can be viewed as rigid analogues of ciproxifan,
as ABT-239. Hence, the compounds here reported can also be considered rigid
ciproxifan derivatives. As observed in ABT-239 analogues, small alkyl substituents in
the polar moiety increase H3R affinity. This can be the explanation to the poor affinity
of 1 and higher affinity of 3 for H3R (Figure 1). Although the similarity between
ciproxifan and ABT-239 is evident, the later shows that imidazole nucleus (the
heteroaryl group in ciproxifan) is not essential to the binding in H3R, and possibly its
role in ciproxifan binding can be related to the polar group present in the structure.
Since ABT-239 and its analogues possess a 5-aryl substituted benzofuran, and also
much improved potency, further 5-substituted analogues 2,3-dihydrobenzofurans
should be evaluated in the future.
Regarding the affinity for H4R, almost all molecules showed no considerable affinity,
with exception of 4, which showed affinity Ki 28 µM. Considering the similarity of the
presented compounds with JNJ-7777120, possibly the absence of carbonyl group, in
addition to the change of indole nucleus, is much detrimental to the activity in H4R. In
fact, these changes in affinity could be expected, and it was already studied in
benzofuran compounds.[26] However, dihydrobenzofurans had not been evaluated
until this letter. Nevertheless, to the indolecarboxamide series the methyl substituent
in piperazine nitrogen leads to high affinity compounds, and bulkier groups disrupt
the binding activity in H4R.[27] This pattern was not observed in dihydrobenzofuran
chemotype, since compound 2 has not shown considerable affinity for H4R, and the
phenyl derivative 4 exhibited the higher affinity in the series. The role of phenyl
substituent in H4R binding, as well as the lack of affinity of the other compounds to
this receptor are being studied by our group using molecular modeling approaches,
however to date no reasonable docking poses were achieved.
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The results indicated the compounds have not shown considerable cytotoxicity at the
higher concentrations tested (300 µM) from 24 to 72 h after cell incubation with the
compounds. Although the compounds showed poor binding affinity for both
histamine receptors, the concentrations to affect the viability of the mammalian cells
are much higher than the IC50 values, allowing its application in further
pharmacological evaluations. Moreover, the compounds have not affected the cell
viability even after 72 hours in contact with the mammalian cells, indicating low
toxicity.
Considering the low toxicity profile of the compounds, we decided to perform a
preliminary evaluation of the anti-inflammatory activity of compound 4 in a mouse
asthma model. Experimental murine asthma was induced by sensitization and
provocation with ovalbumine (OVA). Compound 4 was administered intraperitoneally
in a fixed dose of 5 mg/kg and the inflammatory cells were counted. As can be seen
in Figure 3, compound 4 decreased the total inflammatory cell count obtained from
bronchoalveolar lavage fluid in treated mice to close the control group. The reduction
was more important regarding eosinophils, which are the most important cells in
allergic asthma.
In bronchoalveolar lavage fluids with experimental allergic asthma, enhanced
numbers of cells are observed. This enhanced cellularity of the fluids is mainly due to
the occurrence of high eosinophillia, which are virtually absent in the controls.[28,29]
The involvement of H4R in experimental murine asthma in mice have been
demonstrated in several studies. Neumann et al.[30] demonstrated that JNJ-7777120
is capable of reducing the inflammatory infiltrations (including eosinophils) and other
asthma parameters in murine model. Thioperamide, a dual H3R/H4R antagonist, also
reduced the cell infiltration, but did not affect parameters such as anti-OVA IgE.
Since compound 4 (LINS01005) also reduced signifficantly the inflammatory
infiltration (especially eosinophils), we can attribute this effect to H4R binding,
although more specific studies must be done.
To test this hypothesis, we performed COX-2 analysis from lung tissue obtained from
the treated animals. The results (Figure 4) showed the expression of COX-2 was
significantly reduced, suggesting the anti-inflammatory effect was related to lowered
prostaglandin production. This effect was already observed with JNJ-777120 in
several studies,[31,32] being very similar to what we observed in our results. Thus, it
can be inferred that the anti-inflammatory effect of compound 4 is due to H4R
antagonism.
In conclusion, we presented quite simple compounds which showed preliminary
affinity to H3R and H4R in micromolar range, with relative selectivity to H3R, and no
significant cytotoxicity. Interesting anti-inflammatory activity was observed for
compound 4, possibly due to anti-H4R activity. The results presented here are
valuable as preliminary information to improve the anti-inflammatory properties of
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these novel chemotype, and further studies are under progress by our group to
achieve this goal.
Acknowledgements
The authors are grateful to FAPESP (2013/20479-9 and 2013/18897-7) and to CNPq
(455411/2014-0) for the grants provided to J.P.S.F. and to L.R.P.T., for the
scholarships provided to M.T.V. (2014/16564-3) by FAPESP and to M.F.C. by
CAPES.
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Figures captions
H3R ligands
H4R ligands
clobenpropit
AKOS011602151
JNJ-7777120
ciproxifan
ABT-239
A-943931
Designed compounds
LINS01001 ( )
LINS01005 ( )
4
1
LINS01003 ( )
LINS01004 ( )
3
2
Figure 1. Characteristics considered in the design of LINS01 compounds 1-4; green
– resonance center; orange – polar basic group; blue – additional lipophilic group.
(b)
(a)
(90%)
5
- (50-85%)
1 4
Figure 2. Synthesis of LINS01 compounds 1-4.
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Figure 3. Effects of compound 4 (LINS01005) in bronchoalveolar lavage from rat
asthma model. A: total cell count; B: mononuclear cells; C: eosinophils; D:
neutrophils. * p < 0.05.
Figure 4. A: Western blot analysis of COX-2 expression in the lung tissue from the
different groups. B: Densitometric values for the phosphorylated forms of COX-2
normalized to the values for β-actin. *p < 0.05.
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Tables
Table 1. Results of binding affinity assays and cytotoxic evaluation of the
compounds (n = 3; SD)
Compounds
Yield (%)
Ki H3R (µM)
>100
Ki H4R (µM)
>100
CC50 J774 (µM)
>300
CC50 HeLa (µM)
>300
1
83
85
66
50
2
25 7.0
7 1.5
>100
>300
>300
3
>100
>300
>300
4
17 4.9
28 3.8
>300
>300
(R)-α-Me-
histamine
A-943139
n.d.: not determined
-
-
0.031 0.01
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.012 0.01
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