Design, synthesis, and biological evaluation of a dansyled amino acid derivative as an imaging agent for apoptosis

Article abstract of DOI:10.1016/j.tetlet.2008.08.091

To develop a small molecule-based biomarker for in vivo apoptosis imaging, a dansyled amino acid derivative (BNSBA) was designed and synthesized in good yield. The biological evaluation demonstrated that BNSBA selectively binds to apoptotic cancer cells and is localized within the cytoplasm of cells that bound annexin V on the plasma membrane.

Full text of DOI:10.1016/j.tetlet.2008.08.091

Tetrahedron Letters 49 (2008) 6429–6432
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design, synthesis, and biological evaluation of a dansyled amino
acid derivative as an imaging agent for apoptosis
b
c
c
a
Wenbin Zeng a, Weimin Miao a, , George Kabalka , Michael Le Puil , John Biggerstaff , David Townsend
*
a Department of Medicine, The University of Tennessee Graduate School of Medicine, 1924 Alcoa Hwy, Knoxville, TN 37920, United States
b Department of Radiology, The University of Tennessee Graduate School of Medicine, 1924 Alcoa Hwy, Knoxville, TN 37920, United States
c Center for Environmental Biotechnology, 676 Dabney Hall, University of Tennessee, Knoxville, TN 37996, United States
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 biomarker for in vivo apoptosis imaging, a dansyled amino acid deriv-
ative (BNSBA) was designed and synthesized in good yield. The biological evaluation demonstrated that
BNSBA selectively binds to apoptotic cancer cells and is localized within the cytoplasm of cells that bound
annexin V on the plasma membrane.
Received 1 August 2008
Revised 23 August 2008
Accepted 25 August 2008
Available online 29 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Amino acid
Imaging
Biomarker
Apoptosis
Apoptosis, or programmed cell death, is an essential biological
process for all living organisms. It is an active process of cellular
self-destruction that plays an important role in a large number of
disorders, including organ rejection after transplantation, strokes,1
myocardial infarct,2 and neurodegenerative diseases.3 Apoptosis
also plays a major role in cancer therapy.4 For this reason, consid-
erable efforts have been made in developing and validating meth-
ods to image apoptosis.
early marker of apoptosis.5 However, the non-specific biodistribu-
tion profile, poor target/background contrast ratio, slow clearance
from the blood, along with the cost, inhibit the use of annexin V
derivatives as imaging agents for apoptosis in the clinic. Therefore,
a number of small molecules,6 nanoparticles,7 and peptides8 have
been examined as alternatives to annexin V. Among the small
molecules investigated, N,N0-didansyl- -cystine (DCC, 1)9 and
L
5-(dimethylamino)-1-naphthalene-sulfonyl-a-ethylfluoroalanine
(NST-732, 2)10 have demonstrated great promise (Scheme 1).
Though NST-732 has potential use in positron emission
An often used agent for imaging apoptosis involves the protein
annexin V, which binds to extracellular phosphatidylserine, an
N
N
N
H
O
S
O
N
N
N
Br
S
HN
O
O
O
S
O S O
O
O
HN
HN
S
F
F
S
S
HN
OH
N
O
O
OH
HO
O
O
O
OH
N,N'-didansyl-L-cystine, 1
NST-732, 2
DFNSH, 3
BNSBA, 4
Scheme 1. Dansyl derivatives for imaging apoptosis.
* Corresponding author. Tel.: +1 865 305 6893; fax: +1 865 305 8694.
E-mail address: wmiao@utmck.edu (W. Miao).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.08.091

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W. Zeng et al. / Tetrahedron Letters 49 (2008) 6429–6432
tomographic (PET)11 studies, poor radiolabeling efficiency limits
the clinical application. We have been exploring small molecule-
based biomarkers for use as imaging agents for apoptosis. Recently,
we reported the preparation and preliminary evaluation of a dans-
ylhydrazone (DFNSH, 3) which demonstrates great selectivity for
apoptotic cells.12 The straightforward, highly efficient synthesis
of the fluorine-18 analog of 3 makes it potentially valuable for
PET imaging studies when compared to NST-732. Bromine-76 is
also useful as a radiolabel for PET imaging.13 In previous work,14
we developed a rapid and high yield synthesis of high specific
activity bromine-76 labeled molecules from the corresponding
organotrifluoroborates. In the light of the interest in developing a
biomarker with better in vivo features such as in vivo affinity,
metabolic stability, and pharmacokinetics, we investigated the
synthesis of a brominated dansyl derivative (4-bromophenyl)-
4-(5-(dimethylamino)naphthalene-1-sulfonamido)butanoic acid
(BNSBA, 4). In this Letter, we report both the facile preparation of
BNSBA and the in vitro evaluation as an imaging agent for
apoptosis.
The synthesis of dansyled c-amino acid 4 is outlined in Scheme
2. Methyl 4-bromocinnamate, prepared in 92% yield by the reac-
tion of 4-bromocinnamic acid (5) with methanol and thionyl chlo-
ride, was allowed to react overnight with a 10-fold excess of
nitromethane, using K2CO3/toluene and benzyltriethylammonium,
instead of 1,1,3,3-tetramethylguanidine/THF for four days, to pro-
duce nitro ester 7 in 75% yield. Ester 7 was reduced with nickel
O2N
O
O
O
NiCl2, NaBH4
SOCl2
MeOH
MeNO2, K2CO3
OH
O
O
Toluene
Br
Br
Br
5
6
7
O
O
ClH3N
NH
S
O
N
Dansyl Chloride
K2CO3, CH3CN
HN
5N HCl
O
OH
Br
O
Br
Br
OH
8
9
4
Scheme 2. Synthesis of DNSBA (4).
Figure 1. Images in the same field of view of MCF-7 cells dually stained with annexin V labeled with AlexaÒ488 and 4. (A) DIC image; (B) 4 in green; (C) AlexaÒ488 in red; (D)
composite image of A, B, and C. 100ꢀ total magnification.

W. Zeng et al. / Tetrahedron Letters 49 (2008) 6429–6432
6431
Figure 2. Images in the same field of view of MCF-7 cell dually stained with annexin V labeled with AlexaÒ488 and 4. (A) DIC image; (B) 4 in green; (C) AlexaÒ488 in red; (D)
composite image of A, B, and C. 600ꢀ total magnification.
boride15 to give the lactam 8 in 85% yield. Hydrochloride salt 9 was
obtained by hydrolysis of lactam 8 with aqueous 5 N HCl. The reac-
tion of 9 with dansyl chloride was carried out in acetonitrile to give
the desired biomarker 4 in 84% yield.
ployed to compare the degree of overlap between two images.
The result showed that co-localization of the two dyes occurred
on the periphery of the apoptotic cells (Fig. 1D). In addition, the
non-specific binding of annexin V can be seen in Figure 1C, where
the normal cells were also stained (arrow c), while compound 4
specifically binds to the apoptotic cancer cells. Moreover, it was
found that the staining of apoptotic cells by compound 4 was sta-
ble and was not reduced by additional washing, suggesting that the
uptake is irreversible.
The high magnification images demonstrate that compound 4
was taken up by the cytoplasm (Fig. 2B and D), while annexin V
bound to the outside membrane of the apoptotic cells (Fig. 2C
and D).
In summary, k-amino acid 4 was synthesized in good yield and
found to selectively bind to paclitaxel-induced apoptotic cancer
cells. The in vitro image analysis demonstrates that compound 4
exhibits intracellular uptake and accumulation in apoptotic cells.
The radiochemical production of [76Br]-4 and PET imaging studies
of tumor apoptosis in rodent models are currently underway.
A series of experiments were performed to evaluate the efficacy
of compound 4 in detecting apoptosis in MCF-7 breast cancer cells
by comparing 4 to AlexaÒ488 labeled annexin V. Images were ob-
tained using spectral imaging microscopy. The apoptosis of MCF-7
cells was induced by exposing the cells to 50 nM paclitaxel at 37 °C
for 16 h. The apoptotic MCF-7 cells were dually stained with
AlexaÒ488 labeled annexin V and compound 4 (50
lM in HEPES
buffer, 2.5 mM CaCl2, pH 7.4) using a standard protocol.10 Excita-
tion at 300–400 nm and emission at 445–480 nm were employed
for the detection of compound 4, whereas excitation at 480–
505 nm and emission at 510–550 nm were used for AlexaÒ488
detection. Actively growing (approximately 80% confluent) MCF7
cells were not stained by 4 and AlexaÒ488 labeled annexin V
(Fig. 1A). Approximately 30–40% of the cells were rounded, which
was indicative of the induction of apoptosis [arrows a and b iden-
tify apoptotic cells, and c and d identify actively growing (spread-
ing and dentritic morphology) cells]. The data in Figure 1B
illustrate that compound 4 undergoes exclusive uptake and accu-
mulation in the cytoplasm of the apoptotic cells. By comparison
with Figure 2C obtained using AlexaÒ488 labeled annexin V, it
was confirmed that the cells detected by compound 4 were apop-
totic cancer cells. A co-localization technique (Fig. 1D) was em-
Acknowledgments
We wish to recognize the contribution of Dr. Meixiang Yu in the
early stages of this study. This work was supported by a grant from
Physician Medical Education and Research Foundation. We also
wish to thank the Robert H. Cole Foundation for support.

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W. Zeng et al. / Tetrahedron Letters 49 (2008) 6429–6432
7. Kim, K.; Lee, M.; Park, H.; Kim, J.; Kim, S.; Chung, H.; Choi, K.; Kim, I.; Seong, B.;
References and notes
Kwon, I. J. Am. Chem. Soc. 2006, 128, 3490.
8. (a) Bullok, K.; Piwnica-Worms, D. J. Med. Chem. 2005, 48, 5404; (b) Swairjo, M.;
Seaton, B. Annu. Rev. Biophys. Struct. 1994, 23, 193.
1. (a) Hayashi, T.; Abe, K. Neurol. Res. 2004, 26, 827; (b) Prunell, G. F.; Arboleda, V.
A.; Troy, C. M. Drug Targets CNS Neurol. Disord. 2005, 4, 51; (c) Gu, Z.; Cui, J.;
Brown, A.; Fridman, R.; Mobashery, S.; Strongin, A. Y.; Lipton, S. A. J. Neurosci.
2005, 25, 6401.
2. Abbate, A.; Bussani, R.; Biondi Zoccai, G. G.; Santini, D.; Petrolini, A.; de Giorgio,
F.; Vasaturo, F.; Scarpa, S.; Severino, A.; Liuzzo, G.; Leone, A. M.; Baldi, F.;
Sinagra, G.; Silvestri, F.; Vetrovec, G. W.; Crea, F.; Biasucci, L. M.; Baldi, A. Eur.
Heart J. 2005, 26, 2039.
3. (a) Hartmann, A.; Hunot, S.; Michel, Patrick P.; Muriel, M.; Vyas, S.; Faucheux,
B.; Mouatt, A.; Turmel, H.; Srinivasan, A.; Ruberg, M.; Evan, G.; Agid, Y.; Hirsch,
E. Proc. Natl. Acad. Sci. 2000, 97, 2875; (b) Marx, J. Science 2001, 293, 2192.
4. (a) Okada, H.; Mak, T. Nat. Rev. Cancer 2004, 4, 592; (b) Fischer, U.; Schulze-
Osthoff, K. Pharmacol. Rev. 2005, 57, 187.
9. Reshef, A.; Shirvan, A.; Grimberg, H.; Levin, G.; Cohen, A.; Mayk, A.; Kidron, D.;
Djadetti, R.; Melamed, E.; Ziv, I. Brain Res. 2007, 1144, 156.
10. Damianovich, M.; Ziv, I.; Heyman, S.; Rosen, S.; Shina, A.; Kidron, D.; Aloya, T.;
Grimberg, H.; Levin, G.; Reshef, A.; Bentolila, A.; Cohen, A.; Shirvan, A. Eur. J.
Nucl. Med. Mol. Imaging 2006, 33, 281.
11. Weissleder, R. Science 2006, 312, 1168.
12. Zeng, W.; Yao, M.; Townsend, D.; Kabalka, G.; Wall, J.; Le Puil, M.; Biggerstaff, J.;
Miao, W. Bioorg. Med. Chem. Lett. 2008, 18, 3573.
13. Loevqvist, A.; Sundin, A.; Ahlstroem, H.; Carlsson, J.; Lundqvist, H. J. Nucl. Med.
1997, 38, 395.
14. (a) Kabalka, G.; Mereddy, M.; Green, J. J. Label Compd. Radiopharm. 2006, 49, 11;
(b) Kabalka, G.; Tang, G.; Mereddy, M. J. Label Compd. Radiopharm. 2007, 50,
446; (c) Tang, G.; Zeng, W.; Yu, M.; Kabalka, G. J. Label Compd. Radiopharm.
2008, 51, 68.
5. Gottlieb, R.; Kitsis, R. Nat. Med. 2001, 7, 1277.
6. (a) Zhou, D.; Chu, W.; Rothfuss, J.; Zeng, C.; Xu, J.; Jones, L.; Welch, M.; Mach, R.
Bioorg. Med. Chem. Lett. 2006, 16, 5041; (b) Aloya, R.; Shirvan, A.; Grimberg, H.;
Reshef, A.; Levin, G.; Kidron, D.; Cohen, A.; Ziv, I. Apoptosis 2006, 11, 2089; (c)
Wang, W.; Kim, S.; El-Deiry, W. Proc. Natl. Acad. Sci. 2006, 103, 11003.
15. Corey, E. J.; Zhang, F.-Y. Org. Lett. 2000, 2, 4257.