Development of a chalcone-triazine fusion library: Combination of a fluorophore and biophore

Article abstract of DOI:10.1016/j.tetlet.2013.03.129

A chalcone-triazine fusion library has been developed. We designed fused chalcone-triazine structure by combining a fluorophore and a biophore for imaging and biological probe development. Through solid support chemistry, 80 compounds with amine building blocks were synthesized. Spectroscopic studies and cell image testing of the compounds showed their potential to be used as probes for bio-imaging.

Full text of DOI:10.1016/j.tetlet.2013.03.129

Tetrahedron Letters 54 (2013) 2976–2979
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Tetrahedron Letters
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Development of a chalcone–triazine fusion library: combination of a
fluorophore and biophore
Sung-Chan Lee a, Duanting Zhai b, Young-Tae Chang a,b,
⇑
a Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A⁄STAR), Singapore 138667, Singapore
b Department of Chemistry & Medicinal Chemistry Program of Life Sciences Institute, National University of Singapore, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
A chalcone–triazine fusion library has been developed. We designed fused chalcone–triazine structure by
combining a fluorophore and a biophore for imaging and biological probe development. Through solid
support chemistry, 80 compounds with amine building blocks were synthesized. Spectroscopic studies
and cell image testing of the compounds showed their potential to be used as probes for bio-imaging.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 24 January 2013
Revised 13 March 2013
Accepted 28 March 2013
Available online 8 April 2013
Keywords:
Chalcone
Triazine
Fluorophore
Biophore
The chalcone1 moiety is a well-known biologically active motif,
however, it has not been explored in detail as a fluorescent probe.
We previously reported the first construction of a fluorescent
chalcone library and its application in stem cell probe develop-
ment, which successfully demonstrated the potential of chalcone
as a bio-imaging probe.2 With a significant progress in the develop-
ment of this chalcone bio-imaging probe, we explored the design of
a chalcone library based on combination of a biophore and fluoro-
phore (Fig. 1).
1,3,5-Triazine (s-triazine) derivatives are applied widely as
biophores, due to their structural similarity with naturally
abundant heteroaromatic components (e.g., the bases in nucleo-
sides and nucleotides). Among interest devoted to triazine deriva-
tives, we have reported a variety of their biological activities,
including biofuel production, HSP90 interruption, insulin mimet-
ics, neuroblastoma inhibition, antimicrobial, tyrosinase inhibition,
and microtubule inhibition.3 The 1,3,5-triazine scaffold is also of
particular interest in combinatorial chemistry approaches because
of its synthetic accessibility;4 using 2,4,6-trichloro-1,3,5-triazine
(cyanuric chloride), three chlorides can be replaced by amine,
alcohol, sulfur, and Grignard reagents.5 The convenience of
manipulating the triazine scaffold and the diverse biological
properties inspired us to design a fusion library by combining a
fluorophore and biophore utilizing chalcone and triazines, respec-
tively (Fig. 1). To facilitate synthesis on a solid support, we
introduced a 2-hydroxyethyl(methyl)amino group on the chalcone
side, and two amino groups on each side of the chalcone scaffold
were retained for their strong fluorescence properties.2 This novel
fusion library was named CT (Chalcone Triazine), and we expect to
discover useful imaging and biological probes from it.
The overall synthetic procedure for the CT library is shown in
Scheme 1. The preparation of intermediate 4 was approached
through two different pathways, routes A and B. In route A, nitro
reduction and coupling of cyanuric chloride followed after loading
nitrochalcone compound 1 on to a solid support resin. In route B,6
two steps of the solid phase reaction were removed and the
triazine-coupled chalcone was directly loaded onto 2-chlorotrityl
resin. Both routes A and B were practical for the synthesis of the
CT library, but we elected to follow route A to save on the workload
involved in the extra work-up procedure. However, route B
remains an optional procedure for individual compound scale-up
synthesis.
The key step in the solid supported CT library generation is
amine substitution. Synthetic methods for 1,3,5-triazine substitu-
R1
NH
O
N
N
OH
R2R2'N
N
N
H
N
Biophore
Fluorophore
⇑
Corresponding author. Tel.: +65 6516 7794; fax: +65 6779 1691.
E-mail address: chmcyt@nus.edu.sg (Y.-T. Chang).
Figure 1. The design of the CT library.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.03.129

S.-C. Lee et al. / Tetrahedron Letters 54 (2013) 2976–2979
2977
Route B
Table 1
Route A
Spectroscopic summary of representative compounds
O
O
a
CT {R1, R2R2
}
MW
kabs (nm)
kem (nm)
e
(Mꢀ1 cmꢀ1
)
Ub
0
+
O
{335, 180}
{382, 180}
{211, 376}
{211, 421}
{374, 376}
{374, 421}
571.8
591.8
567.7
579.7
553.7
565.7
427
428
428
428
428
428
545
544
548
547
545
547
14.333
15.000
17.666
18.666
23.333
15.666
0.37
0.40
0.38
0.36
0.38
0.36
OH
OH
OH
N
N
O2N
N
H2N
1
a
g
O
O
O
MW: molecular weight, kabs: absorption maxima,
kem: fluorescence emission maxima,
e
: molar extinction coefficient,
H2N
O2N
N
6
2
U: quantum yield.
a
b
Excited at 430 nm.
h
b
Quantum yield was calculated using the known reference compound, dansyl
M in DMSO.
amide at 10
l
O
Cl
O
N
N
O
3
7
OH
H2N
N
Cl
N
N
N
in tetrahydrofuran (THF)/dichloromethane (CH2Cl2) co-solvent,
which is a slight modification from the reported conditions.3e
H
c
Incorporation of R1-NH2 and R R NH (1° or 2° amine) was con-
trolled by the temperature and solvent. Conjugation of the R1
amine successfully occurred at room temperature in THF, and even
with 3–5 equiv of the R1 amine, no further substitution of chloride
was observed. In total, 11 commercially available primary amines
were loaded as the R1 substituents. The second substitution was
carried out at a higher temperature (60 °C) with a more polar
2
2
0
a
Cl
N
O
N
N
O
Cl
N
N
4
H
d,e
solvent (DMF) and another nine different R2R2 amines were
0
R1
N
NH
O
incorporated.
N
The products were then cleaved from the resin under mild
acidic conditions. We used 1%, 2%, and 5% trifluoroacetic acid
(TFA) in CH2Cl2 and found that 5% TFA in CH2Cl2 afforded the prod-
uct free from the polymer resin within 10 min. The cleaved prod-
ucts were simply filtered through a short silica plug to give 99
library compounds. By removing 19 impure compounds, 80 rela-
tively pure compounds (see Supplementary data, Tables S1 and
S2) were collected as the final CT library (average purity was 85%
at 430 nm by LC/MS).
All the compounds showed similar absorption and emission
profiles (i.e., kabs: 424–429 nm; kem: 549–553 nm) with an average
quantum yield of 0.35. Representative compounds from the library
and their absorption and emission spectra are shown in Figure 2
and Table 1. The Stokes shifts of the CT compounds are around
125 nm, which are larger than those of typical fluorescent dyes,
such as BODIPY, rhodamine, and fluorescein, which have Stokes
shift ranging from 15 to 60 nm. The full width at half maximum
peak height absorptions and emissions of the CT compounds ran-
ged from 60 to 80 nm.
O
R2R2'N
N
N
N
H
5
f
R1
N
NH
O
N
OH
R2R2'N
N
N
H
N
80 compounds
R2R2'
R1
O
O
Cl
NH2
O
NH
NH2
NH2
NH2
211
335
381
421
364
376
262
374
NH2
NH2
HO
NH2
180
O
n-Bu
N
NH2
NH2
NH2
O
n-Bu
382
395
368
To gain preliminary proof of our concept of designing a fusion
library, we selected two CT compounds (CT {335, 180} and CT
{382, 180}) and tested them in normal and cancer liver cells (Chang
and HepG2). The compounds were incubated for one hour, and the
images were acquired using the fluorescein isothiocyanate (FITC)
filter set (ex: 450–490 nm, em: 515 nm).
NH2
N
NH2
NH2
NH2
NH2
442
401
277
H2N
NH2
NH2
N
F
N
O
NH2
348
272
575
386
375
The two CT compounds clearly stained cells in the cytosol with
low background, implying that they are cell permeable and have
the potential to be useful tools for bio-imaging studies (Fig. 3).
The unique fluorescent properties of the CTs would facilitate inves-
tigation of cellular mechanism and target identification.
In summary, we have designed and synthesized a novel CT fu-
sion library, utilizing the advantages of chalcone and 1,3,5-triazine
as a fluorophore and biophore, respectively. The CT library contains
80 compounds with structures of two-dimensional amine building
blocks, and their spectroscopic properties showed the potential for
study as bio-imaging probes. Furthermore, the CT compounds can
penetrate cell membranes and clearly stain cells in the cytosol,
indicating that they should be useful imaging tools for studying
cellular function. Their biological applications will be reported in
due course.
Scheme 1. Synthesis of the CT library. Reagents and conditions: (a) 2-ClTr chloride
resin, pyridine, CH2Cl2, 5 h, RT; (b) SnCl2, 6% H2O in DMF, rt, overnight; (c) 3 equiv
cyanuric chloride, DIEA, THF/CH2Cl2, 0 °C, 2 h; (d) 3 equiv R1-NH2 in THF at rt, 1 h;
(e) R2 R20NH in DMF at 60 °C, 3 h; (f) 5% TFA in CH2Cl2; (g) 4 M NaOH, EtOH, reflux,
overnight; and (h) 1.2 equiv cyanuric chloride, DIEA, THF 0 °C, 1 h. The R1, R2R2
0
amine numbers originate from in-house building block code numbers.
tion have been well studied, mostly based on derivatization of
cyanuric chloride.7 The decreasing reactivity of cyanuric chloride
with increasing numbers of substituents is an advantage for
controlling nucleophilic substitution of each chloride. The first
reactive chloride of cyanuric chloride was successfully substituted
by the aniline of resin-loaded chalcone (intermediate 3) at 0 °C

2978
S.-C. Lee et al. / Tetrahedron Letters 54 (2013) 2976–2979
NH
O
NH
O
OH
OH
N
N
N
N
N
H
N
N
H
N
N
H
N
N
H
N
CT{382, 180}
CT{335, 180}
ZDYHꢀOHQJWK
ZDYHꢀOHQJWK
Figure 2. Representative compound structures and absorption/emission spectra at 10
l
M in dimethylsulfoxide (DMSO). Fluorescence was measured by excitation at 430 nm.
Each of the numbers corresponds to the ID number of the R1, R2R2 amine building block.
0
Figure 3. Normal/cancer cell images of representative compounds. Upper panel: change cells; lower panel: HepG2. FITC/bright field images were taken, respectively. a = CT
{335, 180}, b = CT {382, 180}, at 5 lM final concentrations; scale bar; 10 lm.
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M. L. J. Med. Chem. 2001, 44, 4443–4452; (f) Epifano, F.; Genovese, S.; Menghini,
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Acknowledgments
This study was supported by intramural funding and grant 10/
1/21/19/656 from A⁄STAR (Agency for Science, Technology and Re-
search, Singapore) Biomedical Research Council.
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Commun. 2012, 6681–6683.
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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.tetlet.2013.
03.129.
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