Synthesis and characterization of environment-sensitive fluorescent ligands for human 5-HT1A receptors with 1-arylpiperazine structure

Article abstract of DOI:10.1021/jm900706d

A series of "long-chain" 1-(2-methoxyphenyl)piperazine derivatives containing an environment-sensitive fluorescent moiety (4-amino-1,8-naphthalimide, 4-dimethylaminophthalimide, dansyl) was synthesized. The compounds displayed very high to moderate 5-HT

Full text of DOI:10.1021/jm900706d

7892 J. Med. Chem. 2009, 52, 7892–7896
DOI: 10.1021/jm900706d
Synthesis and Characterization of Environment-Sensitive Fluorescent Ligands for Human 5-HT1A
Receptors with 1-Arylpiperazine Structure†
Enza Lacivita,‡ Marcello Leopoldo,*,‡ Anna Carmela Masotti,‡ Carmela Inglese,‡ Francesco Berardi,‡ Roberto Perrone,‡
Sourav Ganguly,§ Md Jafurulla,§ and Amitabha Chattopadhyay§
§
‡Universitaꢀdegli Studi di Bari, Dipartimento Farmaco-Chimico, via Orabona 4, 70125 Bari, Italy, and Centre for Cellular and Molecular Biology,
Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India
Received May 25, 2009
A series of “long-chain” 1-(2-methoxyphenyl)piperazine derivatives containing an environment-sensi-
tive fluorescent moiety (4-amino-1,8-naphthalimide, 4-dimethylaminophthalimide, dansyl) was synthe-
sized. The compounds displayed very high to moderate 5-HT1A receptor affinity and good fluorescence
properties. 6-Amino-2-[5-[4-(2-methoxyphenyl)-1-piperazinyl]pentyl]-1H-benz[de]isoquinoline-1,3(2H)-
dione (4) combined very high 5-HT1A receptor affinity (Ki =0.67 nM), high fluorescence emission in
CHCl3, and undetectable fluorescence emission in aqueous solution. It was evaluated for its ability to
visualize 5-HT1A receptors overexpressed in CHO cells by fluorescence microscopy.
Introduction
for the orchestration of drug action within a given cell type
and the potential for cross-talk with other signaling path-
ways.4 The study of receptor pharmacology at single cell level
may benefit from use of fluorescent ligands. They offer a
multiplicity of information such as the mechanism of ligand
binding, the movement and internalization of receptors in
living cells, the distances between ligands and fluorescently
labeled aminoacids, the physicalnatureof the binding pocket,
and the visualization of labeled receptors.5
The serotonin1A (5-HT1A) receptor is an important mem-
ber of the large family of serotonin receptors and is perhaps
the most extensively studied for several reasons. One is the
availability of the selective agonist 8-OH-DPATa, which has
allowed extensive biochemical, physiological, and pharmaco-
logical characterization of the receptor. The 5-HT1A receptor
was the first serotonin receptor to be cloned and sequenced.
Human, rat, and mouse 5-HT1A receptors have been cloned
and their amino acid sequences deduced. More importantly,
the receptor has been stably expressed in a number of neural
and non-neural cell lines.1 It is generally accepted that 5-HT1A
receptor is involved in anxiety and depression. Recently, it has
been suggested that 5-HT1A receptor agonists have neuropro-
tective properties, whereas 5-HT1A receptor antagonists could
be useful in the treatment of cognitive disorders related to
Alzheimer’s disease.2 Transfection of the cDNA of this re-
ceptor into cells has resulted in the acquisition of large
amounts of information regarding signal transduction path-
ways linked to the receptor (adenylyl cyclase inhibition,
coupling to phospholipase, regulation of ion channels) and
pharmacological properties of the receptor. The realization
that the 5-HT1A receptor couples to multiple signaling path-
ways in cells and tissues in which it is normally expressed
reflects the promiscuity of GPCR signaling pathways and the
fact that single receptor subtypes might be linked to various
second messengers in a single cell system.3 It is likely that the
pharmacology of GPCRs differs markedly depending on their
membrane localization and the signaling proteins present
within that locality. This might have important implications
Thus, the identification of fluorescent ligands for 5-HT1A
receptor would be of interest. Fluorescent ligands are usually
designed starting from the selection of a pharmacophoric
moiety that is conjugated with a fluorescent tag through an
appropriate linker. For GPCRs activated by biogenic amines,
the ligand binding sites are buried within the transmembrane
regions of the receptor and interact with small molecules.
Consequently, appropriate positioning of the fluorophore
within the final conjugate is critical to retain both receptor
binding affinity and efficacy. Thus, this structural modifica-
tion could significantly affect the pharmacological properties
of the parent ligands.6 As an alternative strategy, we have
recently designed fluorescent probes for 5-HT1A receptor by
incorporating the fluorophore moieties into the pharmaco-
phoric part of the ligand. In other words, the fluorescent
moiety itself had structural characteristics that could be well-
tolerated in the interaction with the target receptor. In this
way, a series of fluorescent ligands was generated, with the
possibility of optimizing the fluorescence properties and
receptor affinity at the same time. In particular, we described
a series of fluorescent N-[ω-[4-(2-methoxyphenyl)-1-pipera-
zinyl]alkyl]-2-quinolinamines (Chart 1, structure I) character-
ized by nanomolar affinity at 5-HT1A receptor, along with
acceptable fluorescence properties.7 However, the ligands
showed excitation wavelengths in the near UV region of the
spectrum (340-380 nm) and this could limit their use to
visualizing 5-HT1A receptors overexpressed in cell lines by
conventional confocal laser scanning microscopy due to cell
†Contribution to celebrate the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Phone: þ39 080
5442798. Fax: þ39 080 5442231. E-mail: leopoldo@farmchim.uniba.it.
a Abbreviations: 8-OH-DPAT, 8-hydroxy-N,N-dipropylaminotetra-
line; GPCR, G-protein coupled receptor.
r
pubs.acs.org/jmc
Published on Web 08/25/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7893
Scheme 1a
Chart 1. Structures of 5-HT1A Receptor Agents
autofluorescence. To overcome this, we have replaced the
2-quinolinamine with three fluorescent labels possessing
different fluorescent properties. The fluorophores were 4-
amino-1,8-naphthalimide (6-amino-1,3-dioxo-1H,3H-benzo-
[de]isoquinolinyl), 4-dimethylaminophthalimide (5-dimethy-
lamino-1H-isoindole-1,3(2H)-dione), and dansyl(5-dimethyl-
aminonaphthalene-1-sulfonyl), which are known to have
different excitation wavelengths (315 nm for the dansyl
moiety, and 450-470 nm for the others). Moreover they
possess the additional feature of being environment-sensitive.
These fluorophores exhibit a low quantum yield in aqueous
solution but become highly fluorescent in nonpolar solvents
or when bound to a hydrophobic site in proteins or mem-
branes. Environment-sensitive molecules may be useful in
fluorescence microscopy studies, where fluorescence will be
higher for ligand-receptor interaction and lower for the
unbound ligand in aqueous environment.
Here we report the design, synthesis, and binding properties
for the human 5-HT1A receptor of the new ligands 1-12. The
fluorescent properties of the ligands were determined in
CHCl3, PBS buffer solution, and in the presence of cell
membranes overexpressing the receptor. We also report some
results of visualization of fluorescent ligand-labeled cells using
fluorescence microscopy.
Chemistry. The synthesis of the final compounds 1-12
(Scheme 1) required the key amines 13a-d, which were
prepared according to the literature as detailed in Supporting
Information (SI). For target compounds 1-4, key amines
13a-d were condensed with 4-nitro-1,8-naphthalic anhy-
dride to give nitro derivatives 14a-d, which were subse-
quently reduced by catalytic hydrogenation with hydrazine
hydrate in the presence of 10% Pd/C. The other target
compounds were prepared from the condensation of
13a-d with 4-(dimethylamino)phthalic acid in the presence
of 1,10-carbonyldiimidazole (5-8) or with dansyl chloride
(9-12).
a Reagents: (A) hydrazine hydrate, 10% Pd/C.
arylpiperazines). The general formula of these compounds
presents a 1-arylpiperazine linked through an alkyl chain to a
terminal fragment, belonging to one of the following four
structural classes: (i) imides, (ii) amides, (iii) alkyl, arylalkyl,
or heteroarylalkyl derivatives, and (iv) tetralins.8 In particu-
lar, we focused our study on 1-(2-methoxyphenyl)piperazine
derivatives because they frequently display high affinity for
the 5-HT1A receptor as for the antagonists15 (WAY-100635)9
and 16 (NAN-190)10 (Chart 1). As outlined above, several
examples from the literature indicate that various terminal
fragments are well-tolerated. Thus, the replacement of
2-quinolinamine nucleus in structure I (Chart 1) with the
fluorescent moieties 4-amino-1,8-naphthalimide, 4-dimethy-
laminophthalimide, and dansyl should be tolerated with
respect to the 5-HT1A affinity. In doing this, we were encour-
aged by the structural similarity between the terminal frag-
ment of the 5-HT1A antagonist 16 and the 4-dimethyla-
minophthalimide fluorophore. Finally, optimization of the
intermediate alkyl chain length was accomplished because it is
well-documented that each combination arylpiperazine/term-
inal fragment requires a spacer of appropriate length.8 Over-
all, our initial considerations were confirmed by Ki values
of the target compounds, which ranged between 0.47 and
151 nM. In particular, considering the 4-amino-1,8-naphtha-
limide derivatives 1-4, we note that length of the spacer
greatly influence the affinity. The compound 4 with a five
methylene spacer displayed subnanomolar affinity (Ki=0.67
nM). When the linker was shortened, a considerable loss in
affinity was observed (>65-fold), especially for compounds 1
(n=2) and 2 (n=3), whereas 3 was only 10-fold less potent
than 4. A different trend was found in the group of 4-
dimethylaminophthalimide derivatives 5-8. The compounds
6 (n=3) and 7 (n=4) displayed subnanomolar affinity (Ki=
0.47 and 0.50 nM, respectively), whereas 5 (n=2) and 8 (n=5)
were 30- and 15-fold less potent. Compound 7 is formally
derived from 16 by introduction of a dimethylamino substi-
tuent in the 4-position of the phthalimide moiety. This
modification had no influence on affinity: in fact, both
compounds displayed Ki values in the nanomolar range
(0.50 and 0.60 nM, respectively).10 The use of dansyl as
terminal fragment was also tolerated, although none of the
derivatives showed subnanomolar affinity at 5-HT1A recep-
tor. Ki values of compounds 9-12 were within a narrow range
Results and Discussion
The affinity values for 5-HT1A receptor and fluorescent
properties of the target compounds 1-12 are displayed in
Table 1.
To develop the new series of fluorescent probes for 5-HT1A
receptors, we took advantage of existing structure-
activity relationship studies on 5-HT1A receptor ligands
with N1-substituted N4-arylpiperazines (the “long-chain”

7894 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lacivita et al.
(29-151 nM). It is likely that the different spatial orientation
of dansyl group with respect to that of phthalimido or
naphthalimido is less favorable for interaction with the 5-
HT1A receptor. Overall, affinity data showed that various
terminal fragments are well-tolerated with respect to the
5-HT1A receptor affinity and that the optimization of the
intermediate alkyl chain length is a key step when studying
“long-chain” arylpiperazines. Considering the fluorescent
properties in ethanol of the target compounds 1-12, we note
that the structural modification of the fluorescent core has
little or no influence on either excitation or emission wave-
lengths.11,12 The excitation wavelengths of the dansyl deriva-
tives 9-12 are more shifted toward the UV region of the
spectrum as compared to the 4-dimethylaminophthalimide
derivatives 5-8 (400-420 nm) and the 4-amino-1,8-naphtha-
limides 1-4 (440-441 nm). All the target compounds showed
high difference of excitation to emission maximal wavelengths
(Stokes shift). To probe the environment affecting the sensi-
tivity of the fluorescent ligand, the quantum yield (Φ) in
PBS buffer and apolar solvent CHCl3 were determined
(Table 1). 1-12 exhibited very low fluorescence in PBS
buffer but became fluorescent in CHCl3. In particular, com-
pounds 1, 5, and 9, characterized by a two methylene spacer,
showed the lower Φ value within each series. Compounds 2, 4,
and 7 displayed the highest Φ values in CHCl3 (Φ=0.67, 0.59,
0.86, respectively). Taken together, 5-HT1A receptor affinity
data and fluorescence properties showed that the pursued
optimization strategy was successful, especially for derivatives 4
and 7, which combined very high 5-HT1A receptor affinity (Ki=
0.67 and 0.50 nM, respectively), high Φ values in CHCl3, and
excitation and emission wavelengths in a suitable range for
visualization experiments by confocal laser scanning micro-
scopy. On this basis, compounds 4 and 7 were incubated with
cell membranes overexpressing human 5-HT1A receptor to
evaluate how the interaction of the fluorescent ligand with the
Figure 1. Spectrofluorimetric determination of the fluorescence of
4 (A) or 7 (B) in the presence of cell membranes overexpressing 5-
HT1A receptors (c) in comparison with cell autofluorescence (b) and
PBS buffer alone (a).
Table 1. 5-HT1A Receptor Affinities and Fluorescence Properties of the Final Compounds 1-12a
a The values are the mean ( SEM from three independent experiments in triplicate.

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7895
receptor reflects on the fluorescence emission. Figure 1 shows
typical fluorescence curves representing the fluorescence emis-
sion of the ligands in PBS, the autofluorescence of cell
membrane alone, and the total observed fluorescence due to
the membranes incubated with 4 and 7, respectively. The
incubation of the fluorescent ligands with cell membranes
led to a marked increase in fluorescence emission intensity
when compared to PBS solution for both compounds. The
naphthalimide derivative 4 determined a 3-fold increase of
fluorescence emission when compared to cell membrane
autofluorescence, whereas 7 determined only a slight increase
in fluorescence emission. Therefore, compound 4 was selected
for labeling 5-HT1A receptor in CHO cells. The experiments
were performed by conventional confocal laser scanning
microscopy at a λexc =458 nm for single photon excitation
and at 900 nm for multiphoton excitation. In the first attempt
to visualize 5-HT1A receptors, stably transfected CHO cells
(TCHO) were incubated with 4 at different concentrations
(0.125, 0.5, and 1 μM) at ∼23 °C. In parallel, the same
experiments were performed on wild-type CHO cells to
evaluate if the binding of the fluorescent ligand was specific.
Compound 4 exhibited reasonably bright fluorescence when
imaged under these experimental conditions. Representative
fluorescence micrographs under various conditions are shown
in Figure 2. Unfortunately, 4 appeared to undergo almost
instantaneous nonspecific aggregation as well as internaliza-
tion upon addition to cells. The patterns of labeling exhibited
are similar in both TCHO and CHO cells, suggesting pre-
dominantly nonspecific interactions. To inhibit endocytosis,
labeling was also performed at 4 °C without appreciable
improvement in the labeling pattern. Moreover, competition
with 5-HT did not induce any perceivable change in the
labeling pattern. Therefore, 4 appeared to be unsuitable for
visualizing 5-HT1A receptors specifically in CHO cells. An
explanation for the observed high nonspecific binding is
difficult to address. It has been proposed that high lipophili-
city is a factor contributing to the nonspecific binding of some
fluorescent probes.13-15 However, literature data do not
suggest anoptimal lipophilicityrange thatmight be associated
with low nonspecific binding for fluorescent ligands. Also,
specific interaction of compound 4 with molecular targets
other than 5-HT1A receptors present in CHO cells cannot be
ruled out. For instance, it has been reported that 1,8-naphtha-
limide derivatives can strongly interact with DNA.16 Clearly,
these possibilities cannot be properly evaluated during the
design process.
Conclusions
We have identified a series of environment-sensitive fluor-
escent ligands for 5-HT1A receptors by inserting an environ-
ment-sensitive moiety in the N-[4-(2-methoxyphenyl)-1-
piperazinyl)alkyl framework. Several of the newly prepared
ligands displayed nanomolar affinity at 5-HT1A receptor and
fluorescent properties suitable for use in fluorescence micro-
scopy. In particular, the fluorescent ligand 4 showed a favor-
able combination of 5-HT1A receptor affinity (Ki=0.67 nM),
Stokes shift (excitation wavelength=400 nm, emission wave-
length=496 nm), and quantum yield in CHCl3 (Φ= 0.59). In
spite its favorable profile, compound 4 was unable to speci-
fically label the human 5-HT1A receptor overexpressed in
CHO cell lines.
Experimental Section
General Procedure for Preparation of Nitro Derivatives
14a-d. A mixture of 4-nitro-1,8-naphthalic anhydride (0.48 g,
2.0 mmol) and the appropriate amine 13a-d (2.2 mmol) in
absolute EtOH (20 mL) was refluxed for 3 h. After cooling, the
solvent was evaporated to dryness and the crude residue was
chromatographed (CHCl3/AcOEt, 1:1, as eluent) to afford pure
compound 14a-d as a brown semisolid.
2-[5-[4-(2-Methoxyphenyl)-1-piperazinyl]pentyl]-6-nitro-1H-
benz[de]isoquinoline-1,3(2H)-dione (14d). Yield 40%. 1H NMR
(CDCl3) δ 1.41-1.51 (m, 2H), 1.63-1.72 (m, 2H), 1.76-1.85 (m,
2H), 2.47 (t, 2H, J=7.4 Hz), 2.69 (br s, 4H), 3.11 (br s, 4H), 3.83
(s, 3H), 4.25 (t, 2H, J = 7.4 Hz), 6.83-7.02 (m, 4H), 7.99 (t, 1H,
J = 8.2 Hz), 8.42 (d, 1H, J = 8.0 Hz), 8.69 (d, 1H, J=8.0 Hz),
8.75 (dd, 1H, J = 0.8, 7.4 Hz), 8.84 (dd, 1H, J = 0.8, 8.7 Hz).
ESIþ/MS m/z 503.0 (MHþ). ESIþ/MS/MS m/z 340 (72), 297
(82), 255 (100).
General Procedure for Preparation of Final Compounds 1-4.
Hydrazine hydrate (0.15 mL, 3 mmol) was added to a solution of
nitro derivatives 14a-d (1.0 mmol) in absolute EtOH (20 mL).
10% Pd/C (50 mg) was added portionwise to the warm solution,
and the mixture was refluxed for 1 h. After cooling, the catalyst
was removed by filtration through celite and the solvent was
evaporated to dryness. H2O (20 mL) was added to the residue,
and the aqueous phase was extracted with CHCl3 (2 ꢀ 30 mL).
The collected organic layers were dried over Na2SO4 and
evaporated under reduced pressure. The crude residue was
chromatographed (CHCl3/MeOH, 19:1, as eluent) to afford
pure compound 1-4 as yellow solid.
Figure 2. Representative fluorescent micrographs (color coded for
intensity scale) of CHO and TCHO cells labeled with 4 upon single
and multiphoton excitation: (A) CHO and (B) TCHO cells labeled
with 0.125 μM of 4 at 4 °C and excited at 458 nm (single photon
excitation), TCHO cells labeled with (C) 0.5 and (D) 1 μM of 4 at
∼23 °C and excited at 900 nm (multiphoton excitation), (E) TCHO
cells labeled with 0.125 μM of 4 at ∼23 °C and excited at 458 nm, and
(F) the same field of cells (as in (E)) excited at 900 nm. The scale bar
represents 10 μm in all cases.

7896 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lacivita et al.
6-Amino-2-[5-[4-(2-methoxyphenyl)-1-piperazinyl]pentyl]-1H-
benz[de]isoquinoline-1,3(2H)-dione (4). Yield 40%. 1H NMR
(CDCl3) δ 1.41-1.51 (m, 2H), 1.59-1.69 (m, 2H), 1.71-1.81 (m,
2H), 2.48 (app t, 2H), 2.69 (br s, 4H), 3.11 (br s, 4H), 3.85 (s, 3H),
4.16 (t, 2H, J=7.4Hz), 5.01(brs, 2H, D2O exchanged), 6.83-7.02
(m, 5H), 7.64 (d, 1H, J = 7.5 Hz), 8.11 (d, 1H, J = 8.5 Hz), 8.40 (d,
1H, J = 8.0 Hz), 8.58 (d, 1H, J = 7.4 Hz). ESIþ/MS m/z 473.0
(MHþ). ESIþ/MS/MS m/z 281 (28), 225 (100); mp 210-212 °C
(from CHCl3/n-hexane). Anal. (C28H32N4O3) C, H, N.
Fluorescence Spectroscopy. Emission and excitation spectra
of compounds 1-12 were recorded as detailed in SI. Fluore-
scence quantum yields were calculated in reference to that of
quinine sulfate in 0.5 M H2SO4 as a standard (excitation
wavelength 350 nm; Φ=0.546),17 as detailed in SI.
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Fluorescent Labeling of Cells. Fluorescence microscopy ob-
servation in CHO cells overexpressing human 5-HT1A receptors
were performed as detailed in SI.
Supporting Information Available: Spectral data of com-
pounds 1-3, 5-12, and 14a-c. Biological methods and statis-
tical analysis. Experimental procedure for fluorescence
spectroscopy. Fluorescent labeling of HEK-293 cell membranes
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