Synthesis, biological evaluation and radiochemical labeling of a dansylhydrazone derivative as a potential imaging agent for apoptosis

Article abstract of DOI:10.1016/j.bmcl.2008.05.002

To develop a small molecule-based tracer for in vivo apoptosis imaging, dansylhydrazone (DFNSH) was synthesized in 93% yield in less than 30 min. The biological evaluation showed that DFNSH selectively binds to paclitaxel-induced apoptotic cancer cells. T

Full text of DOI:10.1016/j.bmcl.2008.05.002

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3573–3577
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and radiochemical labeling of a
dansylhydrazone derivative as a potential imaging agent for apoptosis
Wenbin Zeng a, Min-liang Yao b, David Townsend a, George Kabalka b, Jonathan Wall a, Michael Le Puil c,
John Biggerstaff c, Weimin Miao a,
*
a Molecular Imaging and Translational Research Program, University of Tennessee, Graduate School of Medicine, Box 93, 1924 Alcoa Highway, Knoxville, TN 37920, USA
b Department of Radiology, University of Tennessee, Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37920, USA
c Center for Environmental Biotechnology, 676 Dabney Hall, University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To develop a small molecule-based tracer for in vivo apoptosis imaging, dansylhydrazone (DFNSH) was
synthesized in 93% yield in less than 30 min. The biological evaluation showed that DFNSH selectively
binds to paclitaxel-induced apoptotic cancer cells. The high magnification fluorescent images demon-
strate that DFNSH is localized within the cytoplasm of cells that bound AlexaÒ 488 labeled annexin V
on the plasma membrane. [18F]-DFNSH ([18F]-3) was synthesized and isolated in 50–60% radiochemical
Received 3 March 2008
Revised 25 April 2008
Accepted 1 May 2008
Available online 6 May 2008
yields, based on [K/K222
fluorine-18 labeled 3 makes it a promising tracer for PET imaging of apoptosis.
]
18F, with a synthesis time of 50 min (EOB). The straightforward preparation of
Keywords:
Dansylhydrazone
Apoptosis detection
Annexin V
Ó 2008 Elsevier Ltd. All rights reserved.
Fluorescent imaging
Fluorine-18 labelling
Potential PET tracer
Apoptosis is an important process involved in the etiology,
pathogenesis, and response to therapy of a variety of diseases.
Imaging apoptosis in vivo is a potentially powerful tool for the
early diagnoses of strokes,1–3 myocardial infarctions,4 and a range
of neurodegenerative disorders.5,6 In addition, imaging apoptosis
in patients could also be valuable for the early evaluation of organ
transplant rejection7,8 and cancer treatment response.9,10 Applica-
tions of annexin V, a 35.8-kDa protein, to imaging apoptosis have a
long history.11–15 These studies generally involve the incorporation
of a probe, either a fluorochrome for fluorescence detection or a
radiolabeled linker for nuclear imaging studies. Annexin V binds
to externalized phosphatidylserine (PS) on the outer membrane
of apoptotic cells.16 However, this cell surface binding is not com-
pletely specific, as annexin V also binds to a small percentage of
normal cells.17 In addition, annexin V accumulates in human
healthy organs such as the kidney, bladder, liver, and spleen, which
limits its application in imaging studies focused on diseases in the
abdominal.18 The non-specific biodistribution profile, poor target/
background contrast ratio, slow clearance from the blood, along
with its cost, hinder the use of annexin V derivatives as imaging
agents for apoptosis in the clinic. Because of this, a number of small
molecules,19–23 peptides,24,25 and nanoparticles17 have been exam-
ined as alternatives to annexin V. Among the small molecules
investigated, compounds containing a fluorescent dansyl core21,22
have demonstrated great promise.
In vitro and ex vivo studies have demonstrated that dansyl
compounds bearing amino acid moieties [N0,N0-didansyl-
L-cystine
(DDC), 1 and 5-(dimethylamino)-1-naphthalene-sulfonyl-a-ethyl-
fluoroalanine (NST-732), 2] are selectively bound to apoptotic
cells.19–22 Confocal fluorescent imaging studies suggest that the
dansyl compounds accumulate within the cytoplasm of the apop-
totic cell. Generally speaking, intracellular uptake results in im-
proved target to background contrast ratios. The preparation of
fluorine-18 labeled NST-732 was recently reported26 for potential
use in positron emission tomographic (PET)27 studies. However,
the poor radiolabeling efficiency limits its clinical application.
Herein we report the preparation and preliminary evaluation of a
dansylhydrazone (DFNSH, 3) that demonstrates great selectivity
toward apoptotic cells. The straightforward, highly efficient syn-
thesis of the fluorine-18 analog of 3 makes it potentially valuable
for PET imaging studies when compared to NST-732 (Fig. 1).
Compound 3 was prepared by condensation of dansylhydrazine
with 4-fluorobenzaldehyde (Scheme 1). After a detailed evaluation
of reaction parameters (solvent and temperature), it was found
that 3 can be isolated in 93% yield in less than 30 min by carrying
out the reaction in refluxing methanol. These reaction conditions
are quite amenable to the preparation of the fluorine-18 labeled
analogue, [18F]-3.
* Corresponding author. Tel.: +1 865 544 6893; fax: +1 865 544 8694.
E-mail address: wmiao@utmck.edu (W. Miao).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.002

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W. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3573–3577
N
O
H
N
O
H
N
O
O S O
HN
F
O
O
HO
S
O
S
N
S
HOOC
O
N
N
N
S
S
F
HO
N
O
H
N,N'-didansyl-L-cystine, 1
NST-732, 2
DFNSH, 3
Figure 1. Structure of N0,N0-didansyl-
L
-cystine (DDC), NST-732, and 3.
H
O
H
O
MeOH
O
N
N
H2N
S
N
S
+
H
N
N
O
O
F
F
4
5
3
Scheme 1. Synthesis of compound DFNSH (3).
A series of experiments were performed to evaluate the effi-
cacy of compound 3 in detecting apoptosis in breast cancer MCF-
7 cells by comparing 3 to AlexaÒ 488 labeled annexin V. Images
were obtained using Applied Spectral Imaging (ASI, Migdal Ha’E-
mek, Israel) microscopy. The apoptosis of MCF-7 cells was in-
duced by exposing the cells to 50 nM paclitaxel at 37 °C for
16 h. The paclitaxel-treated and untreated MCF-7 cells were du-
ally stained with AlexaÒ 488 labeled annexin V and compound 3
(50 lM in HEPES buffer, 2.5 mM CaCl2, pH 7.4) using a standard
protocol.22 Actively growing (approximately 80% confluent)
MCF7 cells were not stained by compound 3 and AlexaÒ 488 la-
beled annexin V (Fig. 2). Paclitaxel treatment (Fig. 3A), induced
approximately 30–40% of the cells to adopt a rounded morphol-
ogy, which was indicative of the induction of apoptosis [arrows
a and b identify apoptotic cells, and c and d identify actively
growing (spreading and dendritic morphology) cells]. The apop-
totic MCF-7 cells, which were dually stained with AlexaÒ 488 la-
beled annexin V and compound 3, were observed using Applied
Spectral Imaging microscope. The corresponding filters with
excitation at 300–400 nm and emission at 445–480 nm were
employed for the detection of compound 3 (Fig. 3B), where the
filters with excitation at 480–505 nm and emission at 510–
550 nm were used for AlexaÒ 488 detection (Fig. 3C). Figure 3B
illustrates that compound 3 was taken up and accumulated in
the cytoplasm of the apoptotic cells. By comparison with Figure
3C obtained using AlexaÒ 488 labeled annexin V, it was con-
firmed that the cells detected by compound 3 were apoptotic
cancer cells. The minimal nonspecific-binding of annexin V can
be seen in Figure 3C, where a few normal cells were also stained
(arrow d). Colocalization,of the two probes, performed using
AutoDeblurÒ (Silver Spring, MD), occurred on the periphery of
the apoptotic cells (Fig. 3D). Staining of apoptotic cells by com-
pound 3 was stable and was not reduced by additional washing,
suggesting that the uptake is irreversible.
The high magnification images demonstrate that compound 3
wastakenupby thecytoplasm(Fig. 4B andD)whileannexinV bound
to the outside membrane of the apoptotic cells (Fig. 4C and D). The
threedimensionalimaginganalysis(Fig. 5) confirmedtheintracellu-
lar uptake of compound 3. Dansylhydrazone analogues 7–10 (Fig. 6)
were also synthesized and subjected to evaluation. It was found that
these dansylhydrazone derivatives were also taken up by the cyto-
plasm in apoptotic cells. There is an argument that annexin V is
not specific for apoptosis since it images PS externalized on cells
undergoing necrosis, autophagy, and apoptosis,18 suggesting that
dansyl derivatives may also detect these modes of cell death. Fur-
thermore, these dansylhydrazones can also detect imatinib-induced
cell death in K562 leukemia cells and HT-29 colon carcinomas
(unpublished data). The ability to detect multiple pathways leading
to cell death may be advantageous in the evaluation of cancer treat-
ment response since necrosis, autophagy and apoptosis are all indic-
ative of a positive therapeutic outcome.18
Results from the biological evaluation encouraged us to prepare
the F-18 labeled analogue of 3 ([18F]-3) for the potential use in PET
imaging. The radiosynthetic approach to [18F]-3 is outlined in
Scheme 2. Fluorine-18 labeled fluorobenzaldehyde ([18F]-5) was
prepared from 4-formyl-N,N,N-trimethylanilinium triflate as previ-
Figure 2. Images in the same field of view of untreated MCF-7 cells dually stained with annexin V labeled with AlexaÒ 488, and DFNSH. (A) DIC image; (B) DFNSH in green;
(C) AlexaÒ 488 labeled annexin V in red. Total magnification (100ꢀ).

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W. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3573–3577
Figure 3. Images in the same field of view of MCF-7 cells dually stained with annexin V labeled with AlexaÒ 488, and DFNSH. (A) DIC image; (B) DFNSH in green; (C) AlexaÒ
488 labeled Annexin V in red; (D) composite image of (A), (B), and (C). Total magnification (100ꢀ).
Figure 4. High magnification images in the same field of view of MCF-7 cell dually stained with annexin V labeled with AlexaÒ 488, and DFNSH. (A) DIC image showed a
round-shaped apoptotic cell (arrow b) beside a normal cell (arrow a); (B) the apoptotic cell was specifically stained by DFNSH in green; (C) the apoptotic cell was also stained
by AlexaÒ 488 labeled annexin V in red; (D) composite image of (A), (B), and (C). Total magnification (600ꢀ).

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W. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3573–3577
Figure 5. 3D representation of 4.8 mm thick MCF-7 cell dually stained with annexin V labeled with AlexaÒ 488, and DFNSH, comprising five optical slices captured at 1.2 mm
intervals. (A) Composite image of all slices; (B) bottom slice at 0 mm; (C) slice at 1.2 mm; (D) slice at 2.4 mm; (E) slice at 3.6 mm; (F) top slice at 4.8 mm. Total magnification
(600ꢀ).
ously reported.28 After purification using a semi preparative silica
I
O2N
Me2N
Sep-Pak, compound [18F]-5 was allowed to react with dansylhydr-
azine at 100 °C for 30 min. [18F]-DFNSH ([18F]-3) was isolated in
50–60% radiochemical yields, based on [K/K222]
18F, with a synthe-
N
N
N
N
NH
O
O
NH
O
NH
O
NH
O
sis time of 50 min (EOB). The [18F]-DFNSH was purified by HPLC in
a radiochemical purity greater than 99%. PET imaging studies of tu-
mor apoptosis in rodent models are currently underway.
In summary, we have found that dansylhydrazone derivatives
selectively bind to paclitaxel-induced apoptotic cancer cells. 3D
image analysis demonstrates that compound 3 exhibits intracellu-
lar uptake and accumulation in apoptotic cells. The straightforward
preparation of fluorine-18 labeled 3 makes it a promising tracer for
PET imaging of apoptosis. The evaluation of related dansyl deriva-
tives is currently underway.
S
O
O
S
S
S
O
N
N
N
N
8
9
7
10
Figure 6. Other dansylhydrazone derivatives synthesized for biological evaluation.
Acknowledgments
O
H
We thank Mr. Craig Wooliver for assistance with the fluores-
cence microscopy. We thank Dr. Timothy Panella and Dr. Jay
Wamalasena for insightful discussions. The work was partially
supported by a grant (to Dr. Weimin Miao) from the Physician’s
Medical Education and Research (PMERF). Support from the Robert
H. Cole foundation is gratefully acknowledged.
OTf
Me3N
6
18F−/K222
DMSO
H
O
H
O
O
MeOH
N
N
S
H2N
S
N
+
H
N
N
O
O
18F
18 F
Supplementary data
4
[
18F]-5
3
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.05.002.
Scheme 2. The synthesis of [18F]-3.

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W. Zeng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3573–3577
13. Schellenberger, E.; Bogdanov, A.; Hogemann, D.; Tait, J.; Weissleder, R.;
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