Discovery of naphthalimide conjugates as fluorescent probes for α1-adrenoceptors

Article abstract of DOI:10.1016/j.cclet.2015.12.002

α₁-Adrenoceptors (α₁-ARs), including at least three subtypes, α_1A, α_1B and α_1D, which play essential roles in G protein-coupled receptors (GPCRs), can convey multiple pivotal extracellular signals in varied tissues and organs. In this research, a series of napthalimide-based small-molecule fluorescent probes (1a–1f) for α₁-ARs, including two parts, a pharmacophore (quinazoline and phenylpiperazine) for α₁-AR recognition and a fluorophore (naphthalimide) for visualization, were designed and synthesized successfully. These compounds display excellent fluorescence property and high affinity to receptors, which were used successfully for in vitro visualization of α₁-adrenoceptors.

Full text of DOI:10.1016/j.cclet.2015.12.002

Chinese Chemical Letters 27 (2016) 185–189
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Discovery of naphthalimide conjugates as fluorescent probes for
1-adrenoceptors
a
*
Wei Zhang, Xin-Yang Zhou, Qin-Ying Yu, Lu-Pei Du, Min-Yong Li
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
1-Adrenoceptors (a1-ARs), including at least three subtypes, a1A, a1B and a1D, which play essential roles
in G protein-coupled receptors (GPCRs), can convey multiple pivotal extracellular signals in varied
tissues and organs. In this research, a series of napthalimide-based small-molecule fluorescent probes
Article history:
a
Received 25 July 2015
Received in revised form 30 September 2015
Accepted 20 October 2015
Available online 13 December 2015
(1a–1f) for a1-ARs, including two parts, a pharmacophore (quinazoline and phenylpiperazine) for a1-AR
recognition and a fluorophore (naphthalimide) for visualization, were designed and synthesized
successfully. These compounds display excellent fluorescence property and high affinity to receptors,
Keywords:
which were used successfully for in vitro visualization of
a1-adrenoceptors.
a1-Adrenergic receptors
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Fluorescent probes
High affinity
Published by Elsevier B.V. All rights reserved.
Cell imaging
1. Introduction
which could bind to the targets through the receptor–ligand
interaction, and the fluorophore which is used to trace the targets
As one of the essential members of G protein-coupled receptors
(GPCRs), 1-adrenoceptors ( 1-ARs), distributing in various cells,
by emitting fluorescence signals.
a
a
According to our previous work [11–15], a varied of fluorescent
tissues and organs, can convey multiple pivotal extracellular
signals. These receptors are categorized into at least three subtypes
probes for a1-ARs based on naphthalimide were well designed and
synthesized (Fig. 1). In this instance, the quinazoline and
phenylpiperazine moiety are chosen as the pharmacophore for
(
a1A, a1B, and a1D) based on their diversities on the biological
structure, pharmacological properties, tissue distributions, and
signaling pathways [1–3].
their high affinity to
fluorophore. With the help of the biological evaluation, we find
that our probes showed off the high affinities to 1-ARs and
a1-ARs, and naphthalimide is selected as the
It has been confirmed that
a
1-ARs are bound up with
a
hypertension, benign prostatic hyperplasia (BPH), and other
diseases [4–6]. In order to prevent and treat diseases connected
acceptable cell fluorescence imaging potential. It can be expected
that these probes could be utilized as useful tools for nowadays
high throughput screening of fluorescent competitive substrates in
with
a1-ARs anomalously expressed, numerous a1-ARs antago-
nists have been discovered, such as quinazoline or phenylpiper-
azine derivatives [7]. Nevertheless, we still face many challenges,
which become the stumbling obstacle to studying the biological
a1-ARs.
and pharmacological characteristics of
a
1-ARs, due to the lack of
2. Experimental
the three-dimensional crystal structures and tissue-selective
antagonists.
2.1. Materials and instruments
Fortunately, with the speedy growth of fluorescence technolo-
gy, small-molecule fluorescent probes have many merits such as
high sensitivity and selectivity for the detection of proteins,
enzymes, etc. [8–10]. Small-molecule fluorescent probes are
normally constitutive of two portions, the pharmacophore moiety
All materials were purchased from commercial companies
(Aladdin and J&K Scientific) and used without further purification.
Twice-distilled water was used throughout all experiments. Mass
spectra were performed by the analytical and the mass spectrom-
etry facilities in Drug Analysis Center at Shandong University on
Agilent Technologies 1100 infinity HPLC, Applied Biosystems
API4000. 1H NMR and 13C NMR were recorded on a Bruker
300 MHz NMR spectrometer.
*
Corresponding author.
E-mail address: mli@sdu.edu.cn (M.-Y. Li).
http://dx.doi.org/10.1016/j.cclet.2015.12.002
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

186
W. Zhang et al. / Chinese Chemical Letters 27 (2016) 185–189
Fig. 1. Designed fluorescent probes based on quinazoline and phenylpiperazine for
a1-ARs.
2.2. Synthesis of the probes
those compounds are measured, the consequences validated that
most of the probes owned reasonable optical properties (Table 1).
The two synthetic routes of these probes wereshown in Schemes
1 and 2. Thesynthesis of keyintermediatesc2and d2began withthe
CBZ protection of 3-bromopropan-1-amine, and then the nucleo-
philic substitution and deprotection reactions were conducted
(Scheme 1). The other key intermediates a2, e2, b2 and f2 were
synthesized through the Gabriel reactions (Scheme 2). All final
compounds (1a to 1f) were obtained from the acylation reactions of
the key intermediates with a 1,8-naphthalic anhydride.
The optical properties were performed on a Thermo-Fisher
Varioskan microplate reader by dissolving the probes in
50 mmol/L PBS, pH 7.4. As we can see in Table 1, the maximum
absorption wavelength, the excitation wavelength and the
fluorescence emission wavelength of all target compounds (1a–
1f) are approximately 355 nm, 355 nm and 470 nm, respectively.
3.2. Affinity to
a1-ARs
Another pivotal characteristic for fluorescent probes is the
affinity to the targets ( 1-ARs) besides the optical properties. For
3. Results and discussion
a
this reason, the radioligand binding assay for evaluating the
affinity of these probes to three different adrenergic receptor
3.1. Optical property
subtypes (
a1A-, a1B- and a1D-AR) was carried out, in which
It is important that the rational fluorescent probes should
possess the ideal optical property. After the optical properties of
phentolamine serves as the positive control and atropine served as
Scheme 1. Reagents and conditions: (a) Cbz-Cl, 3 mol/L NaOH, CHCl3, overnight; (b) K2CO3, CH3CN, 80 8C, 5 h, 92%; (c) H2, Pd/C, 30 8C, overnight, 95%; (d) 4-acetamino-1,8-
naphthalic anhydride, CH3CH2OH, 85 8C, 3 h, 39%–87%.

W. Zhang et al. / Chinese Chemical Letters 27 (2016) 185–189
187
Scheme 2. Reagents and conditions: (a) K2CO3, DMF, 30 8C, overnight; (b) triethylamine, CH3CN, 85 8C, 6 h, 60%–88%; (c) (i) hydrazine hydrate, EtOH, 85 8C, 3 h, (ii) HCl/EtOH;
(d) 4-acetamino-1,8-naphathalic anhydride, EtOH, 85 8C, 3 h, 19%–38%.
Table 1
Table 2
Optical properties of compounds 1a–1f.
The affinity of probes to a1-ARs.
Compound
UV lmax (nm)
lex (nm)
lem (nm)
Compound
Kia (nmol/L)
IC50 (nmol/L)
1a
1b
1c
1d
1e
1f
355
355
355
355
355
350
355
355
355
355
355
350
470
470
470
470
470
470
a1A
a1B
a1D
a1A
a1B
a1D
Phentolamine
0.8
7.7
11.1
NAb
0.34
81.4
4.3
1.4
19.2
NAb
1.97
64.0
40.8
38.4
52.1
153
18.0
NAb
0.75
183
9.8
Atropine
NAb
0.09
8.1
NAb
0.33
10.7
6.8
NAb
0.2
1a
1b
1c
1d
1e
1f
17.5
1.4
0.65
4.3
6.4
32.5
0.97
148
9.4
73.1
2.2
0.2
8.7
0.5
39.1
25.5
85.2
333
the negative control. According to the data in Table 2, all probes to
1-ARs had a high affinity at the nanomolar level or even lower the
affinity of phenylpiperazine derivatives to the 1-ARs is much
a
Ki was calculated from IC50 using the Cheng–Prusoff equation.
b
a
Not available.
a
higher than that of quinazoline derivatives. However, some recent
study indicated that phenylpiperazine derivatives may have high
shorter linker shows the higher affinity for the targets
ꢀ
ꢁ
affinity to some subtypes of 5-HT besides
a
1-ARs [16]. When the
Ki < Ki < Ki , but for quinazoline pharmacophore, compound
1a
1c
1e
pharmacophore is either phenylpiperazine, the compound with a
1d with the linker of three carbons shows highest affinity, which is
Fig. 2. The fluorescence images of probe 1a–1f to HEK293A-a1A-AR cells (left image is the bright field, right image is the fluorescence image), the concentration of each probe
was 50 nmol/L.

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W. Zhang et al. / Chinese Chemical Letters 27 (2016) 185–189
Fig. 3. The fluorescence images of probe 1a–1f to HEK293A-a1D-AR cells (Left image is the bright field, right image is the fluorescence image), the concentration of each probe
was 50 nmol/L.
approximately 2-fold more potent than 1b, and the affinity of
compund 1f with longer linker is lowest. Phenylpiperazine
a1-adrenergic receptor, accelerating the development of a1-
adrenergic receptor research. In addition, these probes can be
synthesized easily from inexpensive starting materials. It is
expected that these probes could be utilized as useful tools for
nowadays high throughput screening as fluorescent competitive
derivatives show higher affinity to
derivatives show higher affinity to both
Additionally, lengthening the linker may increase the affinity to
1D-AR of phenylpiperazine derivatives.
a1A-AR, while quinazoline
a
1A-AR and 1B-AR.
a
a
substrates in a1-ARs.
3.3. Cell fluorescence imaging
Acknowledgments
These compounds showed no difference in fluorescence
The present work was supported by grants from the Fok Ying
Tong Education Foundation (No. 122036), the Program of New
Century Excellent Talents in University (No. NCET-11-0306), the
Shandong Natural Science Foundation (No. JQ201019) and the
Independent Innovation Foundation of Shandong University,
IIFSDU (No. 2010JQ005). Our cell imaging work was performed
at the Microscopy Characterization Facility, Shandong University.
We also thank Professor You-Yi Zhang from Peking University for
intensity when incubation with
no ‘‘off-on’’ process happened. We incubated HEK293A cells
transfected stably with 1A-AR and 1D-AR with the probes to gain
a1-AR proteins, which indicated
a
a
the cell fluorescence imaging. Cells were incubated in DMEM
medium complemented with 10% (v/v) fetal bovine serum under
the condition of 5% CO2 at 37 8C.
The cell lineages were reared in 35 mm glass bottom culture
dishes (Mat Tek) at 37 8C for 24 h. Then cells were washed with
DMEM medium and cultured in DMEM medium including the
probes for 10 min at 37 8C. Fluorescence imaging was displayed on
a Zeiss Axio Observer A1.
her generous gift, the
cells.
a1A-AR- and a1D-AR-transfected HEK293A
Appendix A. Supplementary data
As a result, in Figs. 2 and 3, these cells highly expressing
and 1D-AR could be highlighted by fluorescent probes at the
nanomolar concentration, which would be an axis for exploiting
a1A-AR
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.cclet.2015.
12.002.
a
new longer wavelength probes for
a1-adrenergic receptors.
4. Conclusion
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-
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