5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel non-nitrogenous ligand for 5-HT2B receptor

Article abstract of DOI:10.1016/j.bmcl.2014.02.029

Chromones are a class of natural products found in almost every known terrestrial plant with over 4000 naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-(2-phenylethyl) chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report our most recent characterization on this interesting natural product against a number of CNS receptors for the purpose to identify the potential molecular targets that may be related to its biological activity. Based on our studies, including radiobinding assays, calcium flux functional assays and molecular modeling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.

Full text of DOI:10.1016/j.bmcl.2014.02.029

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1489–1492
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel
non-nitrogenous ligand for 5-HT2B receptor
b
b,
Dwight A. Williams a, , Saheem A. Zaidi , Yan Zhang
⇑
⇑
a Department of Pharmacology and Toxicology, Virginia Commonwealth University, 410 North 12th Street, PO Box 980613, Richmond, VA 23298-0613, United States
b Department of Medicinal Chemistry, Virginia Commonwealth University, BioTech One, Suite 205, 800 E. Leigh St., PO Box 980540, Richmond, VA 23298-0540, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Chromones are a class of natural products found in almost every known terrestrial plant with over 4000
naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-
(2-phenylethyl)chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity
against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other
naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report
our most recent characterization on this interesting natural product against a number of CNS receptors
for the purpose to identify the potential molecular targets that may be related to its biological activity.
Based on our studies, including radiobinding assays, calcium flux functional assays and molecular mod-
eling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.
Published by Elsevier Ltd.
Received 23 October 2013
Revised 28 January 2014
Accepted 4 February 2014
Available online 19 February 2014
Keywords:
Chromone
Neuroprotection
5-HT2B receptor
Antagonist
Neurodegenerative diseases affecting the central nervous
system (CNS) are widely spread among the population.1 Conse-
quently, tremendous research effort has been directed towards
the development and application of small molecules to study the
proteins involved in these CNS disorders.2 The role of natural prod-
ucts in these efforts however has not been very significant.3 Chro-
mones (1) are a class of natural products found in almost every
known terrestrial plant with over 4000 naturally occurring deriva-
tives having been isolated and structurally elucidated.4 Perhaps the
most widely studied chromones are the flavonoids like Kaempferol
(2). Flavonoids bear a phenyl ring at the C2 position and are typi-
cally polyhydroxylated. The medicinal value of these chromones
has been known for centuries.5 They have demonstrated various
biological activity such as antioxidants, antivirals, antifungals,
antimicrobials, anti-inflammatories, neurotrophics and neuropro-
tectives.6–8 Such a broad range of activity makes chromone type
natural products a potential treasure trove for therapeutics. Thus
far, most of the research on the therapeutic value of chromones
as related to CNS disorders has been correlated to their antioxidant
properties.9 Until recently, little attention has been paid to the
ability of chromones to serve as small molecule modulators of en-
zymes and receptors within the CNS.10–12
potential tools to study CNS related disorders.13 Unlike the flavo-
noids, these chromones possess a phenylethyl substituent at C2
and to date less than 100 congeners of 2-(2-phenylethyl)chromone
have been isolated and characterized.14 In 2006 Yoon et al. isolated
5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC, 4) from Imperata
cylindrical. They showed that this natural product had neuropro-
tective activity against glutamate induced excitotoxicity in pri-
mary cultures of rat cortical cells.15 The neurotoxic effects of
glutamate have been shown to involve not only glutamate
receptors but also non-glutamate receptors, ion channels, and
transporters.16,17 Thus, elucidation of the potential molecular tar-
get(s) of 5-HPEC would be necessary in order to further study
the role of this type of natural products in their CNS neuroprotec-
tion mechanism.
In comparison to other naturally occurring neuroprotective chr-
omones, for example Kaempferol (2), 5-HPEC contains fewer hydro-
xyl groups.18 Therefore, we hypothesize that the neuroprotective
effects of 5-HPEC may be not only due to its antioxidant potential
but very possibly relate to its ability recognizing certain receptors
and/or ion channels in the CNS. We recently developed an efficient
method to synthesize 5-HPEC.19 Here we report our further charac-
terization on this interesting natural product against a number of
CNS receptors in order to identify the potential molecular targets
that may be related to its biological activity (Fig. 1).
Lately, 2-(2-phenylethyl)chromones (3), a relatively small and
under-explored class of chromones have shown promise as
The initial screening measured the inhibition by 5-HPEC (at
10 lM) to radioligand binding on a series of selected receptors
and ion channels. Inhibition of 50% or greater at this concentration
⇑
Corresponding authors. Tel.: +1 804 8280021 (Y.Z.).
E-mail addresses: williamsda2@vcu.edu (D.A. Williams), yzhang2@vcu.edu
was deemed meaningful. As shown in Figure 2, 5-HPEC only
(Y. Zhang).
http://dx.doi.org/10.1016/j.bmcl.2014.02.029
0960-894X/Published by Elsevier Ltd.

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D. A. Williams et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1489–1492
Table 1
Determined pKi values for 5-HPEC at 5-HT1E, 5-HT2B, and 5-HT3
Receptor
pKia
5-HT1E
5-HT2B
5-HT3
<5
5.61 0.1
5.60 0.08
a
pKi were determined by competitive inhibition of [3H]5-HT, [3H]LSD,
[3H]GR65630 at 5-HT1E
,
5-HT2B and 5-HT3, respectively. Concentration of the
,
radioligand was equal to the Kd of the radioligand which was determined by finding
the mean for 3 previously conducted saturation binding assays.
Next the functional activity of 5-HPEC at 5-HT2B was studied.
Several reasons directed our research focus toward the 5-HT2B
receptor at this stage: (1) the selectivity shown by 5-HEPC at 5-
HT2B over 5-HT2A and 5-HT2C; (2) the recently available crystal
structure of the 5-HT2B receptor as an advantage to further facili-
tate our research; (3) the accessibility of functional activity screen-
ing methods for the 5-HT2B receptor; and (4) some toxicity
concern, that is, should 5-HPEC show any significant agonist activ-
ity at the 5-HT2B receptor, enthusiasm for further investigation
would be diminished as 5-HT2B agonists are known to play a crit-
ical role in valvular heart disease.25 A preliminary calcium mobili-
Figure 1. Several natural products bearing the chromone core.
demonstrated notable inhibitory response at several serotonin
(5-HT) receptors, namely 5-HT1E, 5-HT2B, and 5-HT3, with inhibi-
tory activities of 55%, 62%, and 61%, respectively. Interestingly, 5-
HPEC showed very low inhibitory effects on other receptors from
the 5-HT2 subfamily, for example 5-HT2A (14.2%) or 5-HT2C
(4.8%). This suggested that 5-HPEC may act as a selective ligand
for 5-HT2B over other receptors in this subfamily.
Following this initial investigation, the binding affinity of 5-
HPEC at these identified receptors was determined (Table 1). The
affinity of 5-HPEC at the 5-HT1e receptor seemed to be insignifi-
cant. On the other hand, 5-HPEC showed almost equal affinity on
both 5-HT3 and 5-HT2B receptors with pKi values of 5.60 and
5.61, respectively. These results were very encouraging since sero-
tonergic pathways have been implicated to play an important role
in CNS neurorprotection.20–22 More specifically, both 5-HT2B and 5-
HT3 antagonists have demonstrated certain neuroprotective
properties.23,24
zation assay in Flp-In HEK cells using 5-HPEC at 10 lM showed
minimal agonist activity (0.6 0.2% of the maximal response)
when compared to 5-HT. Conversely, when challenged with an
EC50 dose (1.6 nM) of 5-HT, 5-HPEC demonstrated modest antago-
nists activity (6.9 1.9% inhibition). This suggested that 5-HPEC
did behave as an antagonist, not an agonist to the 5-HT2B receptor.
Following these, a concentration response curve in the presence of
an EC50 concentration of 5-HT gave a pIC50 value of 5.05 0.05 for
5-HPEC at the 5-HT2B receptor.
Figure 3 showed 5-HT (5) and three other well-known 5-HT2B
antagonists. It has long been assumed that ligands targeting the
5-HT receptors and transporters would contain an amino moiety
Figure 2. Representative data for the inhibitory responses produced by 5-HPEC at various CNS receptors.

D. A. Williams et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1489–1492
1491
Table 2
The optimal docking scores of 5-HT (5) and 5-HPEC (4) in the 5-HT2B receptor crystal
structure
Compound
HINT
ChemPLP
5-HT
5-HPEC
2669
537
59.44
50.75
and Ser139 were involved in a potential hydrogen bonding net-
work with the chromone hydroxyl group. Meanwhile, 5-HEPC also
seemed to take advantage of a hydrophobic cavity around helix 7
and extracellular loop 2 lined with Leu362 and Leu209 which
may interact with the phenylethyl substituent of 5-HEPC. A quali-
tative comparison of the suggested binding mode of 5-HPEC indi-
cated somehow less hydrogen bonding potential than 5-HT,
which was reflected in its lower HINT score. This may explain
the lower affinity of 5-HPEC (pKi 5.60) compared to 5-HT (pKi
7.87).32 To be noticed, the binding mode comparison between
the endogenous ligand 5-HT (an agonist) and 5-HPEC (an antago-
nist) in an agonist bound crystal structure may not be ideal; how-
ever, recent reports demonstrated that only relatively subtle
structural changes within the ligand binding pocket were observed
between agonist and antagonist bound protein.33 Therefore we be-
lieve that such an approximation would be tolerable in order to
serve the purpose of preliminarily predicting the putative binding
mode of 5-HPEC in the 5-HT2B receptor.
Figure 3. The chemical structures of serotonin and selected 5-HT2B antagonists.
in order to facilitate ligand binding.26 However, this idea has been
challenged recently and a few non-nitrogenous compounds have
demonstrated high affinity for the 5-HT transporter.27 To our
knowledge, no such ligand has ever been reported for the 5-HT2B
receptor, and in this respect 5-HPEC may represent a structurally
unique non-nitrogenous 5-HT2B ligand. To ascertain the putative
binding mode of 5-HPEC on the 5-HT2B receptor, an automated
To gain some insight into what structural modifications might
be tolerated to maintain the binding affinity of 5-HPEC to the
receptor, six analogues of 5-HPEC were synthesized (Fig. 5) and
docking experiment was performed using the recently available
28
agonist bound X-ray crystal structure of 5-HT2B
.
A generic algo-
rithm docking program GOLDv51 was used to explore docking
poses for both 5-HT and 5-HPEC.29 The generated binding modes
were then re-scored using Hydropathic INTeraction (HINT) that
calculates free energy associated with non-bonded interactions
based on a natural force field generated by employing experimen-
tally determined partition co-efficients (Table 2).30 As shown in
Figure 4 the highest scored 5-HT binding mode in the receptor
indicated that the indole ring of 5-HT located in a hydrophobic
pocket between helices 3 and 6. The indole nitrogen atom and
the hydroxyl group seemed to be involved in plausible hydrogen
bonding interactions with Thr140 and Ser222, respectively. The
terminal amino group was shown to interact with Asp135 and
Ser139. The above binding mode for 5-HT was in general agree-
ment with previously reported site-directed mutagenesis
studies.31
their inhibitory activity (at 10 lM) was assessed preliminarily at
each 5-HT2 receptor (Table 3). The design of these analogues was
focused the effects of the alkyl chain length and the aromatic ‘C’
ring on the affinity. Interestingly none of the modifications gave
rise to any significant affinity change at the 5-HT2A or 5-HT2C
receptors. With respect to 5-HT2B these data suggested that the
nature of the alkyl chain may be important. For example, replacing
C12 with an oxygen atom (10) led to a decrease in activity, which
meant that a hydrocarbon chain would be preferred. Additionally,
the alkyl chain length seemed to be critical because extension of
the alkyl chain by only one methylene group (12) led to an increase
in activity while any other changes in chain length were detrimen-
tal. Finally, exchanging the phenyl ring with a thiophene (13)
showed improved activity over the parent compound.
In summary, the goal of this study was to identify potential target
protein(s) relevant to the biological activity of 5-hydroxy-2-(2-
phenylethyl)chromone (5-HPEC). Accordingly 5-HPEC was screened
In contrast, the best scored docking solution for 5-HPEC showed
that its chromone core recognized a similar binding pocket com-
pared to that of 5-HT, that is, between helices 3 and 6 Asp135
Figure 4. (a) Docking of serotonin (5-HT) in the 5-HT2B receptor. (b) Putative binding mode of 5-HPEC (4) in the 5-HT2B receptor. Small molecules were shown in balls and
sticks (cyan), residues in sticks (with carbons colored as green). Numbers ‘x.yy’ referred to Ballesteros–Weinstein numbering. Possible hydrogen bonds were shown with
yellow dashed lines.

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D. A. Williams et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1489–1492
Figure 5. Chemical synthesis route of 5-HPEC analogues.
Table 3
References and notes
Inhibitory activity of 5-HPEC analogues at the 5-HT2 family of receptors
Compound
R
% Inhibition
5-HT2A
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29. Molecular modeling procedure: the molecular structures of 5-HT and 5-HPEC
were sketched, and their Gasteiger–Hückel charges were assigned before
energy minimization (10,000 iterations) with the TAFF within SYBYL-X2.0. The
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Acknowledgments
This work was supported by the NIH/NIDA Training Grant
DA007027 (DAW) and PHS Research Funds DA024022 (Y.Z.). Ki
determinations, receptor binding profiles, agonist and/or antago-
nist functional data was generously provided by the National Insti-
tute of Mental Health’s Psychoactive Drug Screening Program,
Contract # HHSN-271-2008-00025-C (NIMH PDSP). The NIMH
PDSP is directed by Bryan L. Roth, MD, PhD at the University of
North Carolina at Chapel Hill and Project Officer Jamie Driscol at
NIMH, Bethesda MD, USA. For experimental details please refer
to the PDSP web site http://pdsp.med.unc.edu/ and click on ‘Bind-
ing Assay’ or ‘Functional Assay’ on the menu bar.
atoms within 10 Å of the c-carbon atom of Asp135. Based on the fitness scores
and the binding orientation of each ligand within the binding cavity, the best
GOLD-docked solution was selected and merged into the receptor. The
combined receptor–ligand structures were then energy-minimized to
optimize the interactions between ligand and receptor by removing
structural clashes and minimizing strain energy. These optimized models
were then subjected to hydropathic analysis with the HINT program. Pictures
depicting docking poses were generated using PyMOL Molecular Graphics
System, V1.5.0.4.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02
.029.