Rational design, green synthesis, and initial evaluation of a series of full-color tunable fluorescent dyes enabled by the copper-catalyzed N-arylation of 6-phenyl pyridazinones and their application in cell imaging

Article abstract of DOI:10.1002/chem.201302495

There is widespread interest in the application, optimization, and evolution of the transition-metal-catalyzed arylation of N-heteroarenes to discover full-color tunable fluorescent core frameworks. Inspired by the versatile roles of pyridazinone in organic synthesis and medicinal chemistry, herein, we report a simple and efficient copper-catalyzed cross-coupling reaction for the N-functionalization of pyridazinones in neat water. To achieve the efficient conversion of pyridazinones and organic halides in aqueous phase, a series of copper-salen complexes composed of different Schiff base ligands were investigated by rational design. A final choice of fine-tuned hydrophilicity balanced with lipophilicity among the candidates was confirmed, which affords excellent activity towards the reaction of a wide range of pyridazinones and organic halides. More importantly, the products as N-substituted pyridazinones were synthesized rationally by this methodology as full-color tunable fluorescent agents (426-612 nm). The N2 position of pyridazinones was modified by different aryl group such as benzothiazole, N,N-dimethylaniline, 3-quinoline, 4-isoquinoline and 2-thiophene, resulting in a series of full-color tunable fluorescent reagents. Meanwhile, the effects of electron-donating and electron-withdrawing groups of the 6-substituted phenyl ring have also been investigated to optimize the fluorescent properties. These fluorescent core frameworks were studied in several cell lines as fluorescent dyes. Different colors from blue to red were observed by using fluorescence microscopy and confocal microscopy. Colorful cores: A simple and efficient copper-catalyzed cross-coupling reaction for the N-functionalization of pyridazinones in neat water is reported (see figure). The N2 position of pyridazinones was modified by different aryl groups (R¹), such as benzothiazole, N,N-dimethylaniline, 3-quinoline, 4-isoquinoline, and 2-thiophene, resulting in a series of full-color tunable fluorescent reagents. Copyright

Full text of DOI:10.1002/chem.201302495

FULL PAPER
DOI: 10.1002/chem.201302495
Rational Design, Green Synthesis, and Initial Evaluation of a Series of Full-
Color Tunable Fluorescent Dyes Enabled by the Copper-Catalyzed N-
Arylation of 6-Phenyl Pyridazinones and Their Application in Cell Imaging
Lei Liang,[a] Wei Wang,[a] Jun Wu,[b] Fengrong Xu,[a] Yan Niu,[a] Bo Xu,*[b] and
Ping Xu*[a]
Abstract: There is widespread interest
in the application, optimization, and
evolution of the transition-metal-cata-
lyzed arylation of N-heteroarenes to
discover full-color tunable fluorescent
core frameworks. Inspired by the versa-
tile roles of pyridazinone in organic
synthesis and medicinal chemistry,
herein, we report a simple and efficient
copper-catalyzed cross-coupling reac-
tion for the N-functionalization of pyri-
dazinones in neat water. To achieve the
efficient conversion of pyridazinones
and organic halides in aqueous phase, a
series of copper-salen complexes com-
posed of different Schiff base ligands
were investigated by rational design. A
final choice of fine-tuned hydrophilicity
balanced with lipophilicity among the
candidates was confirmed, which af-
fords excellent activity towards the re-
action of a wide range of pyridazinones
and organic halides. More importantly,
the products as N-substituted pyridazi-
nones were synthesized rationally by
this methodology as full-color tunable
fluorescent agents (426–612 nm). The
N2 position of pyridazinones was modi-
fied by different aryl group such as
benzothiazole, N,N-dimethylaniline, 3-
quinoline, 4-isoquinoline and 2-thio-
phene, resulting in a series of full-color
tunable fluorescent reagents. Mean-
while, the effects of electron-donating
and electron-withdrawing groups of the
6-substituted phenyl ring have also
been investigated to optimize the fluo-
rescent properties. These fluorescent
core frameworks were studied in sever-
al cell lines as fluorescent dyes. Differ-
ent colors from blue to red were ob-
served by using fluorescence microsco-
py and confocal microscopy.
Keywords: arylation · cell imaging ·
copper · cross-coupling · fluorescent
dyes
Introduction
structure must be significantly changed to tune the emission
wavelength, which increases the difficulty of synthesis. An-
other group of fluorescent materials with tunable and pre-
dictable emission wavelengths is quantum dots (QDs),
which are generally composed of heavy metals such as CdSe
and CdTe.[3] However, QDs are still not completely suitable
for biological applications due to their innate toxicity.[4] Re-
cently, Park and co-workers developed a full-color tunable
Fluorescent organic materials have attracted considerable
attention due to their potential uses in biological science,
clinical diagnosis, and drug discovery.[1] Despite the rapid
development of organic fluorophores, the core skeletons
possessing tunable emission wavelength is quite limited, and
the rational design of new fluorescent reagents with desira-
ble photophysical properties is far from the requirement
fluorescent core skeleton, 1,2-dihydropyrroloACHTUNTRGNEU[GN 3,4-b]indoli-
of
scientific
research.
4-Bora-3a,4a-diaza-s-indacene
zin-3-one, with a tunable emission wavelength ranging from
420–613 nm. After incorporating a maleimide group for bio-
conjugation to thiol moieties, two selected compounds
proved their applicability as labeling agents for immunocy-
tochemistry.[5]
(BODIPY) and cyanine dyes are the well-known organic
fluorophores with correlation between structures and their
photophysical properties.[2] Nevertheless, their molecular
[a] Dr. L. Liang, W. Wang, F. Xu, Dr. Y. Niu, Dr. P. Xu
Department of Medicinal Chemistry
School of Pharmaceutical Sciences
Peking University Health Science Center
Beijing 100191 (P.R. China)
It is well-known that chemical modification of the p con-
jugation or variations in substituents and substitution posi-
tions can influence the energy levels and/or dipole moments
in the ground and excited states of the core fluorophores,
which gives rise to different optical and photochemical
properties of fluorescent materials.[6] Thus, to design an or-
ganic chromophore with desirable photochemical and pho-
tophysical properties, an understanding of the relationship
between its molecular structure and its function is required.
Pyridazinones, as a vital type of heterocycles, have been
used as a pharmacophore with versatile biological functions
in the past decades (Figure 1). Recently, N-arylated pyrida-
E-mail: pingxu@bjmu.edu.cn
[b] J. Wu, Dr. B. Xu
State Key Laboratory of Natural and Biomimetic Drugs
School of Pharmaceutical Sciences
Peking University Health Science Center
Beijing 100191 (P.R. China)
E-mail: xubo@bjmu.edu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201302495.
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
1
&
ÞÞ
These are not the final page numbers!

the catalyst.[16] As we know, rational screening of the dia-
mine moiety has not been achieved, thus the depth of infor-
mation of this type of catalyst is still ambiguous.
In spite of an increasing number of relevant reports, the
use of transition-metal-catalyzed N-arylation of heteroar-
enes to discover new full-color tunable fluorescent core
frameworks still remains less explored. Therefore, to achieve
À
the construction of C N bond of pyridazinones in aqueous
environment and also to overcome the disadvantages of tra-
ditional methods, herein we report a copper-catalyzed cross-
coupling reaction of pyridazin-3(2H)-ones with organic hal-
ides, based on our rational screening of the copper–salen
complexes as catalyst. The N-arylated products with diverse
colors were further applied as fluorescent dyes in cell imag-
ing.
Figure 1. Bioactive compounds containing a pyridazinone scaffold.
zinones have been developed as G-protein-coupled-receptor
(GPCR) histamine 3 receptor (H3R) antagonists associated
with several central nervous system (CNS) diseases.[7] Our
original attempt for the discovery of a new core skeleton
was based on extension of the conjugation system of pyrida-
zinone, by using a transition-metal-catalyzed cross-coupling
reaction. Nevertheless, the traditional synthetic method of
these pyridazinones suffered from several drawbacks, such
as a tedious procedure, hazardous solvents, and narrow tol-
erance of the substrates.[8] Furthermore, an efficient, eco-
nomical, and environmentally friendly procedure for the N-
functionalization of pyridazinones in aqueous phase has not
been fully developed. Given that both pyridazinone and or-
ganic halide substrates are insoluble in water, resulting in
poor reactivity, the application of an aqueous strategy for
Results and Discussion
Recently, we reported a relay catalysis by copper catalysis in
a tandem dehydrogenation/dechlorination sequence, which
was successfully applied in the synthesis and medicinal study
of pyridazinones.[17] We found that the electronic property of
substituents at the N2 position of pyridazinones was impor-
tant for the photophysical properties. To facilitate the aque-
ous synthesis of these compounds, a series of copper–salen
complexes Cu1–Cu14 were designed and the structures are
shown in Figure 2.
À
the C N cross-coupling reaction has long been lacking in
Screening, optimization, and evolution of the copper–salen
catalysts in the cross-coupling reaction: Initially, we chose
the cross-coupling reaction of 3’-chloro-6-phenylpyridazin-
3(2H)-one with bromobenzene for screening the catalysts.
The structure of the copper–salen complexes are shown in
Figure 2 and the results are summarized in Figure 3. We in-
vestigated different modifications of either the diamine or
salicylaldehyde moiety of the original copper–salen complex
Cu1, which exhibited negligible reactivity in water. First, we
equipped the diamine with carboxylic or phenoxyacetic acid
group(s); the yields of the desired cross-coupling product
were evidently increased (Figure 3; Cu2 and Cu3). Then, we
studied the modification of the salicylaldehydes by hydroxyl
or sulfonate groups, which resulted in quite different yields
(Figure 3; Cu4 and Cu5). As expected, when the salicylalde-
hyde moieties were substituted by hydrophilic group such as
sulfonate, the solubility of the copper complex was remarka-
bly improved and its performance in the cross-coupling reac-
tion was promoted considerably (Figure 3; Cu5). Additional-
ly, we also proved that hydroxyl group could improve the
solubility and then enhanced the reactivity slightly
(Figure 3; Cu4). With confirmation of the sulfonate-modi-
fied salicylaldehyde as one half part of the ligand, we con-
tinued to study the other half part of the Schiff base ligand
using 1,2-diaminecyclohexane and ethylenediamine. As
shown in Figure 3, both catalysts gave considerably im-
proved results (Cu6 and Cu8). To confirm the importance of
the N,N,O,O tetra-coordinated ligand, a half-condensed
development. In the past decades, DMF and some other or-
ganic solvents represent the most widely used solvents,
which is commonly adopted in catalysis of CuI with Cs2CO3
as a base.[9] If this reaction could be transferred into water,
it would provide us abundant opportunities in the combina-
torial synthesis of a diversity-oriented fluorescence library
without tedious work-ups and hazardous residual.
On the other hand, an increasing interest in developing
aqueous catalysis has emerged as important alternative
strategies for the traditional procedures carried out in or-
ganic solvents.[10] According to the major principles of green
chemistry, water is the ideal solvent due to the fact that it is
abundant, nontoxic, and nonflammable.[11] In
a recent
survey, most pharmaceutical companies are considering
water as a potential solvent.[12] So far, tremendous efforts
have been made to switch organic reactions to water, among
those cases, copper-catalyzed cross-coupling reaction is an
excellent example; this is one of the most powerful tools for
[13]
À
the construction of C N bonds.
In terms of the copper
catalysts, water-soluble copper–salen complexes were widely
used in aqueous catalysis such as Ullmann reactions,[14] and
azide–alkyne cycloadditions (CuAAC).[15] However, most at-
tention has been paid to the salicylaldehyde moieties that
are easily modified by hydrophilic groups, making the entire
complex soluble in water. On the contrary, the diamine
moiety has long been neglected despite subtle variations of
this component may influence the property and reactivity of
&
2
&
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Full-Color Tunable Fluorescent Dyes
FULL PAPER
Schiff base ligand was synthe-
sized and employed in the
cross-coupling reaction. As ex-
pected, the reactivity of Cu7
decreased dramatically, proba-
bly due to its poor solubility
and stability in water.
To further optimize the struc-
ture of the catalyst and improve
the cross-coupling reaction, we
hypothesized that suitable en-
hancement of the lipophilicity
of the diamine moiety might in-
crease the affinity between the
catalyst and lipophilic sub-
strates. We delicately tuned the
balance between the hydropho-
bic and hydrophilic property of
the catalyst by using a variety
of diamines including 1,2-di-
phenyl ethylenediamine, 1,1’-bi-
naphthyldiamine 4,5-dichloro-
benzene-1,2-diamine, 2,2’-((4,5-
diamino-1,2-phenylene)bis-
AHCTUNGTRENGN(UN oxy))diacetic acid, and 3,4-dia-
minobenzoic acid to modify the
upper-half of the ligand. As ex-
pected, Cu11 bearing dichloro
groups showed the highest reac-
tivity
(Figure 3).
However,
bulky groups on the diamines
such as 1,2-diphenyl and bi-
naphthyl showed
a negative
effect (Figure 3; Cu9 and
Cu10), as did hydrophilic
groups such as phenoxyacetic
acid and carboxylic acid (Cu12
and Cu13). Compared with
Cu13, if the sulfonate groups of
salicylaldehyde were replaced
by a hydrophobic group such as
Figure 2. Copper–salen complexes used as catalysts.
chloro, the reactivity was evi-
dently decreased (Figure 3;
Cu14). By design, the upper-half moiety of Cu11is lipophilic,
and affinitive to the organic substrates, whereas the lower-
half moiety is fully soluble in water, which facilitates the re-
action compatibility in water. Therefore, the potent reactivi-
ty of Cu11 is attributed to its oil/water amphoteric property
derived from the two halves of the ligand.
Screening of the optimized reaction conditions for Cu11 as
the catalyst in the cross-coupling reaction: After the screen-
ing of catalyst, the inorganic and organic bases including
Na2CO3, Cs2CO3, K2CO3, K3PO4, NaOH, KOH, and triethyl-
amine were investigated, in which Na2CO3 was found to be
the optimal choice (Table 1, entries 1–7). Moreover, the cat-
alytic system showed low reactivity in organic solvent such
Figure 3. Yields obtained by various copper complexes.
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
3
&
ÞÞ
These are not the final page numbers!

B. Xu, P. Xu et al.
Table 1. Selected representative screening of the reaction.[a]
yields (64 and 60%, respectively). Furthermore, disubstitut-
À
À
À
ed aryl bromides bearing CF3, Cl, and CH3 groups also
gave good yields. In addition, other aryl halides including 2-
bromonaphthalene, 6-bromo-1,4-benzodioxane, and 1-bro-
mopentafluorobenzene gave good to modest yields (85, 56,
and 67%, respectively). Alkyl bromides including cyclopen-
tyl bromide and cyclohexyl bromide were also employed in
this reaction and modest yields were obtained (65, 72%, re-
spectively). It is noteworthy that most of the solid products
could be separated from the aqueous mixture with high
purity through filtration and washing, which simplifies the
traditional procedures.
Entry
Catalyst
Base
Yield
[%][b]
1
2
3
4
5
6
7
8
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Cu11
Na2CO3
K2CO3
Cs2CO3
K3PO4
NaOH
KOH
86
77
79
75
64
50
trace
26[c]
30[d]
82[e]
52[f]
84[g]
0
To expand the scope of pyridazinones, we applied this cat-
alytic system to a variety of pyridazinone substrates. To our
delight, most of the substrates afforded the desired products
with good to excellent yields. As shown in Table 3, 6-phenyl-
Et3N
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
9
10
11
12
13
14
pyridazin-3ACTHNUGTRNEUNG(2H)-one was used with bromobenzene and 1,4-
dibromobenzene in the cross-coupling reaction to give the
products in excellent yields (95 and 86%, respectively).
Mono-chloro and dichloro-substituted substrates also exhib-
ited good yields. For example, both 4’-chloro-6-phenylpyri-
Cu11
0
[a] 3’-Chloro-6-phenylpyridazinone
(1.0 mmol),
bromobenzene
(1.1 mmol), Cu11 (5 mol%), base (2.0 mmol), in H2O at 808C for 12 h.
[b] Yield of the isolated product. [c] DMF as solvent. [d] O2 atmosphere.
[e] N2 atmosphere. [f] T=608C. [g] T=1008C.
dazin-3ACHTNUTRGENNUG(2H)-one and 2’-chloro-6-phenylpyridazin-3HCATUNGTRENN(UGN 2H)-one
afforded the N-arylated products in high yields above 85%.
2’,4’-dichloro-6-phenylpyridazinone was also used to the re-
action with bromobenzene and 89% yield was obtained. We
further investigated the reaction of 2’,4’-dichloro-6-phenyl-
pyridazinone and 1,4-dibromobenzene with a molar ratio
2.1:1.0 and the dimer product was obtained with 82% yield.
Other pyridazinones without 6-phenyl group were also ex-
amined. For instance, when the 6-phenyl group was replaced
as DMF (26%, Table 1, entry 8). Under different gas atmos-
phere, lower yields were detected especially in the oxygen
atmosphere (Table 1, entries 9 and 10). As we expected, a
lower temperature resulted in a lower yield (52%, Table 1,
entry 11). Increasing the temperature to 1008C also gave a
slight decreased yield (Table 1, entry 12).
À
by Cl, the cross-coupling reaction also proceeded with high
yield (83%). 4-bromophthalazin-1(2H)-one was also used to
Application of the catalytic system to the cross-coupling re-
action between various pyridazinones and organic halides:
With the optimized conditions in hand, we studied the gen-
erality of the reactions between different aryl/alkyl halides
give the product in 78% yield.
Rational design and study of N-substituted 6-phenylpyrida-
zinones as fluorescent reagents: With the optimized reaction
conditions in hand, a series of pyridazinones and organic
halides were applied in this reaction and the results are
listed in Table 4. In general, all of the substrates afforded
the N-arylation products (PyL1–PyL8) with moderate to ex-
cellent yields ranging from 60 to 84%. With the compilation
of these fluorescent compounds, we evaluated the photo-
physical and photochemical properties of each individual
fluorophore. As shown in Figure 4a, the overlaid fluorescent
emission spectra highlight the accomplishment of full-color
tunable emissive fluorophores based on the 6-phenyl pyrida-
zinone skeleton. As expected, a dramatic change in the fluo-
rescence properties can be achieved successfully within a
single molecular framework simply by changing the substitu-
ents at only two variation points in the fluorophore. The
direct comparison of the electronic properties of the R1 sub-
stituents clearly demonstrates the tunability of this core
skeleton. The maximum emission wavelengths are increased
in the following order: R1 =phenyl (the model N-arylation
product), 4-isoquinoline (PyL7), 3-quinoline (PyL6), 2-ben-
zothiazole (PyL4), 4-methoxyphenyl (2ag), 2-thiophenyl
(PyL8), 4-dimethylamino phenyl (PyL1) on a single skele-
and 3’-chloro-6-phenylpyridazin-3ACTHNUTRGNEUNG(2H)-one. Various aryl
halides bearing either electron-donating or electron-with-
drawing groups converted to the desired products in modest
to excellent yields and the results are listed in Table 2. For
example, the para-substituted aryl bromide substrates bear-
À
À
À
À
À
ing NO2, CF3, CN, Cl, and F groups afforded excel-
lent yields. Electron-donating group (methoxyl and methyl)-
substituted substrates showed slightly lower yields (Table 2,
81 and 71%, respectively). The meta-substituted aryl bro-
mide substrates also exhibited good yields. Similarly, sub-
strates bearing electron-withdrawing groups showed higher
yields than those bearing electron-donating groups. In this
group, 3-bromobenzonitrile resulted in the highest yield
(88%). 3-bromonitrobenzene and 1-bromo-3-chlorobenzene
afforded high yields (80 and 86%, respectively). 3-bromoto-
luene and 3-bromoanisole afforded modest yields (74 and
66%, respectively). The ortho-substituted aryl bromides
showed remarkably lower yields, probably due to the steric
effect. 2-bromonitrobenzene and 1-bromo-2-chlorobenzene
afforded modest yields (70 and 71%, respectively). 1-
bromo-2-fluorobenzene and 2-bromotoluene gave lower
&
4
&
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Full-Color Tunable Fluorescent Dyes
FULL PAPER
Table 2. Reaction of various organic halides with 3’-chloro-6-phenylpyridazin-3(2H)-one under the optimized
zole was used as the organic
halide for the cross-coupling re-
action, which afforded modest
yields (PyL4 and PyL5). Com-
pared with PyL5, which has a
non-substituted 6-phenyl group,
the emission of PyL4 showed a
blueshift to 478 nm when the
meta-position was substituted
by chloro group (Figure 4c, i).
Meanwhile, the fluorescent in-
tensity of PyL4 was increased
to 0.25 compared with PyL5
(0.19). Moreover, quinoline
conditions.[a]
1
2
Yield [%]
1
2
Yield [%]
1
2
Yield [%]
R=
NO2
CF3
CN
Cl
F
CH3
OMe
R=
NO2
CN
Cl
CH3
OMe
R=
NO2
Cl
F
CH3
2aa
2ab
2ac
2ad
2ae
2af
89
90
92
90
87
81
71
2ah
2ai
2aj
2ak
2al
80
88
86
74
66
2am
2an
2ao
2ap
70
71
64
60
(PyL6)
and
isoquinoline
(PyL7) were also independently
investigated as donors to study
the photophysical properties.
As shown in Figure 4c, iii,
PyL7 displayed negligible fluo-
rescence compared with PyL6.
Thiophene, which was widely
used in optical materials and
devices, was also employed in
the cross-coupling reaction with
3’-chloro-6-phenylpyridazinone
and green fluorescence was ob-
tained (PyL8).
2ag
R=
CF3
Cl
2at
85
2au
56
2aq
2ar
2as
84
86
70
CH3
Application of the N-arylated
pyridazinones in cell imaging
by using fluorescent and confo-
cal microscopy: Molecular fluo-
rescent-imaging techniques are
helpful to understand biological
processes at the molecular level
2av
67
2aw
65
2ax
72
[a] 3’-Chloro-6-phenylpyridazinone (1.0 mmol), bromobenzene (1.1 mmol), Cu11 (5 mol%), Na2CO3
(2.0 mmol), in H2O at 808C for 12 h.
ton with fixed R2 substituent such as meta-Cl group. The
corresponding emission wavelengths of PyL7, PyL6, PyL4,
2ag, PyL8, and PyL1 are 426, 458, 478, 501, 517 and
612 nm, respectively. Fine tuning of the fluorescent property
of PyL compounds is feasible by changing the R2 group but
keeping a fixed R1 substituent. For instance, the electron-do-
nating group on the 6-phenyl ring blueshifts the emission
peak, which was achieved by our further modification of the
structures of PyLs. As shown in Table 4, PyL1 showed a dra-
matic increase of fluorescence with large bathochromic shift
to 612 nm. When the 6-phenyl group was substituted by
electron-donating ortho-ethoxy group, the fluorescent emis-
sion of PyL2 could be tuned slightly with a hypochromatic
shift to 597 nm. Further study of structure–activity relation-
ship (SAR) showed the 6-phenyl group is indispensable for
the whole conjugated system of fluorescent core framework.
For example, when the 6-phenyl group is replaced by a
chloro group, the fluorescent intensity of PyL3 decreased se-
verely (Figure 4c, ii). Besides the phenyl groups, we also in-
vestigated the effect of other aromatic groups on the N2 po-
sition of pyridazinones. For instance, 2-bromobenzo[d]thia-
(e.g., intracellular pH indicators,[18] sensors for reactive
oxygen and nitrogen species,[19] metal sensors,[20] diagnosis
imaging tools[21]) and mechanisms.[22] Thus, the development
of new fluorescent bioimaging probes still remains an attrac-
tive and promising goal.[23] To demonstrate the use of PyLs
as dye stains for cell imaging, their chemo- and photostabili-
ty, as well as the cytotoxicity or cell viability, were assessed
first. The pH-dependent absorption and emission spectra of
PyL2, PyL3, PyL6, and PyL8 in the solution of the mixed
buffers (NaOAc, Mes, Mops, and Tris) and DMSO (0.5%,
VDMSO/Vbuffer) were measured and shown in Figure S1 (see
the Supporting Information). Among our new fluorophores,
we chose PyL8 for direct comparison with the well-known
fluorescent molecule fluorescein.[24] As a result, PyL8 is
more resistant to photobleaching than fluorescein (see Fig-
ure S2 in the Supporting Information). Fluorescein is well-
known to have multiple ionization equilibria, which leads to
pH-dependent absorption and emission. Unlike fluorescein,
because of its new molecular framework, the fluorescence
intensity and maximum emission wavelength of PyL8 are
not influenced by pH value in the range 4–9 (physiological
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
5
&
ÞÞ
These are not the final page numbers!

B. Xu, P. Xu et al.
Table 3. Reaction of organic halides with various pyridazin-3(2H)-one
Table 4. Photophysical data of PyL compounds in DMSO.[a]
under the optimized conditions.[a]
[c]
[d]
Structure
PyLn
Yield[b]
[%]
emax
lex/lem
F[e]
PyL1
69
66
0.54
0.61
382, 612
375, 597
0.082
2ba 95%
2bd 85%
2bb 86%
2be 88%
2bc 92%
2bf 87%
PyL2
0.095
PyL3
PyL4
72
60
0.56
1.29
304, 603
363, 478
n.d[f]
2bg 89%
2bh 82%[b]
0.25
2bi 83%
2bj 78%
PyL5
PyL6
84
78
1.42
0.94
366, 489
304, 458
0.19
[a] Pyridazinone (1.0 mmol), bromobenzene (1.1 mmol), Cu11 (5 mol%),
Na2CO3 (2.0 mmol), in H2O at 808C for 12 h. [b] Molar ratio of pyridazi-
none/1,4-dibromobenzene=2.1:1.0.
0.064
range of in vivo system), which is an important element in
bioimaging and bioapplication.[25] Above all, it indicated
that these PyL compounds 1) were stable over the pH
ranges (4–9), and 2) are more resistant to photobleaching
than fluorescein.
PyL7
PyL8
70
68
0.60
1.02
304, 426
381, 517
n.d
Cell viability assays in four cell lines including MCF-7,
DU-145, Hela, and SW480 were conducted by using the 3-
(4,5-diethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide
(MTT>) assay. The cell viability data after treatment with
100 mm of the pyridazinone products and PyLs for 24 and
48 h are shown in Table S1 in the Supporting Information.
Although the growth inhibition rates of PyL7 were 67.4 and
75.0% in SW480 cells for treatment at 24 and 48 h, respec-
tively, other compounds are virtually nontoxic over a period
of 24 to 48 h (see Table S1 in the Supporting Information).
To further explore the practical application of these new
fluorescent molecules, the MCF-7 cells were incubated with
a series of PyLs. As shown in Figure 5, PyLs showed potent
cell permeability and successfully marked MCF-7 cells, and
preferentially accumulated in the cytoplasm, suggesting that
this class of membrane-permeable fluorophores (PyLs)
could be potentially useful reagents for biological imaging.
To further explore the application of PyLs in living cell
imaging, we observed the cell-imaging experiment by using
confocal microscopy. The confocal microscopy images
showed that PyL2 could be taken up efficiently by HeLa
cells and the optimum concentrations are 10 mm. As shown
0.077
[a] All data were measured in DMSO. [b] Yields of the isolated products.
[c] Extinction coefficient in 1ꢁ104 mÀ1 cmÀ1. [d] lmax values of the one-
photon absorption and emission spectra in nm of the absorption spec-
trum. [e] Fluorescence quantum yield, the uncertainty is Æ15%. [f] n.d=
not detected.
in Figure 6, we selected fluorescent images of HeLa cells in-
cubated with PyL2 (10 mm, 0.5 h) as a marker. In particular,
PyL2 displayed punctate luminescence in the cytoplasm.
Conclusion
A highly efficient and “green” protocol has been developed
for the synthesis of N-functionalized pyridazinones. The cat-
alytic system consists of a water-soluble copper–salen com-
plex, which is endowed with reasonable balance of hydro-
&
6
&
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Full-Color Tunable Fluorescent Dyes
FULL PAPER
Figure 5. Selected fluorescence images of MCF-7 cells incubated with
100 mm of a) PyL1, b) PyL2, c) PyL4, d) PyL8, e) PyL5.
Figure 6. a) and d) Bright-field images of the HeLa cells stained with
PyL2; b) and e) one-photon microscopy image of the same cells with ex-
citation at 488 nm; c) and f) merged image. Scale bar: 10 mm.
yields, pH-independent fluorescence, large Stokes shifts, and
drug-like lipophilicity for membrane permeability. The fluo-
rophores have successfully marked MCF-7 and Hela cells,
exhibiting their future potential as useful bioimaging fluo-
rescence probes. Further applications of these fluorophores
in biological imaging are currently being studied.
Figure 4. a) Synthesis and normalized emission spectra of PyL1, PyL2,
and PyL4–PyL8 (ꢀ2.0 mm) in DMSO. b) Selected fluorescence images of
PyLs in DMSO, irradiated at 365 nm. From left to right: PyL1, PyL3,
PyL2, PyL8, PyL4, and PyL7. c) Compared fluorescence of i) PyL4
versus PyL5, ii) PyL2 versus PyL3, and iii) PyL6 versus PyL7.
Experimental Section
General: All reagents unless otherwise stated were purchased from com-
mercial suppliers and used without further purification. 1H, 13C and
19F NMR spectra were recorded on Bruker AVIII-400 spectrometers with
[D6]DMSO or CDCl3 as solvent. Chemical shifts are reported in ppm
using TMS as internal standard. Mass spectra (MS) and high-resolution
mass spectra (HRMS) were recorded with a Finnigan MAT 95 spectrom-
eter (EI) with an ionization potential of 70 eV. Infrared spectra (IR)
were recorded with a Bruker Tensor 27 spectrometer. Absorption bands
are reported in wave numbers (cmÀ1). For flash chromatography, silica
gel from Merck (200–300 mesh) was used. Merck silica gel GF254 plates
were used for TLC. The substances were detected with UV light
(254 nm) and iodine. Substances that have already been reported in the
literature were identified by comparison of the obtained 1H and
13C NMR spectra with those reported in the literature. New substances
were fully characterized by infrared (IR), 1H, 13C, 19F NMR (products
philic and hydrophobic properties. More importantly, this
reaction can be simply achieved in water and in an air at-
mosphere. Following this protocol, a full-color tunable fluo-
rescent core skeleton have been developed based on N-ary-
lated 6-phenylpyridazin-3(2H)-ones. This core skeleton can
accommodate various emission maxima simply by changing
substituents at the N2 and 6-phenyl positions, having differ-
ent electronic properties. These new fluorophores have ex-
cellent photophysical and photochemical properties, resist-
ance to photobleaching, moderate-to-considerable quantum
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
7
&
ÞÞ
These are not the final page numbers!

B. Xu, P. Xu et al.
consisting of fluoride) and mass spectrometry (MS). Additional charac-
terization data were obtained by high-resolution mass spectrometry
(HRMS).
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (21202003/21172012), the National Basic Research Program of
China (2012CB518000) and the Beijing Natural Science Foundation
(Design, synthesis and optimization of proteasome inhibitors as novel an-
titumor agents, 7112088).
All cells were incubated in complete medium (Dulbeccoꢂs modified
Eagleꢂs medium, DMEM, supplemented with 10% fetal bovine serum
(FBS), 1% penicillin-streptomycin) at 378C in an atmosphere containing
5% CO2. The steady-state absorption spectra were obtained with an Agi-
lent 8453 UV/Vis spectrophotometer in 10 mm path length quartz cuv-
ettes with a dye concentration of ꢀ1ꢁ10À5 m. Single-photon luminescence
spectra were recorded by using an Edinburgh Instrument FLS920 Com-
bined Fluorescence Lifetime and Steady-state spectrophotometer. All
measurements were performed at room temperature).
[1] a) Applied Fluorescence in Chemistry, Biology, and Medicine,
(Eds.:W. Rettig, B. Strehmel, S. Schrader, H. Seifert), Springer: New
York, 1999; b) J. J. Lavigne, E. V. Anslyn, Angew. Chem. 2001, 113,
3212–3225; Angew. Chem. Int. Ed. 2001, 40, 3118–3130; c) J. Rao,
A. Dragulescu-Andrasi, H. Yao, Curr. Opin. Biotechnol. 2007, 18,
17–25; d) J. Zhang, R. E. Campbell, A. Y. Ting, R. Y. Tsien, Nat.
Immunol. Nat. Rev. Mol. Cell. Biol. 2002, 3, 906–918.
[2] A. P. Demchenko, in Advanced Fluorescence Reporters in Chemistry
and Biology I, Springer-Verlag: Berlin, Heidelberg, 2010; Chapter
2.5, pp. 149–186.
[3] a) M. Bruchez Jr., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos,
Science 1998, 281, 2013–2016; b) C. B. Murray, D. J. Norris, M. G.
Bawendi, J. Am. Chem. Soc. 1993, 115, 8706–8715; c) Z. A. Peng,
X. Peng, J. Am. Chem. Soc. 2001, 123, 183–184; d) Z. A. Peng, X.
Peng, J. Am. Chem. Soc. 2002, 124, 3343–3353.
[4] R. Hardman, Environ. Health Perspect. 2006, 114, 165–172.
[5] a) E. Kim, M. Koh, J. Ryu, S. B. Park, J. Am. Chem. Soc. 2008, 130,
12206–12207; b) E. Kim, M. Koh, B. J. Lim, S. B. Park, J. Am.
Chem. Soc. 2011, 133, 6642–6649.
[6] a) C. L. Li, S. J. Shieh, S. C. Lin, R. S. Liu, Org. Lett. 2003, 5, 1131–
1134; b) Y. Yamaguchi, T. Ochi, T. Wakamiya, Y. Matsubara, Z.
Yoshida, Org. Lett. 2006, 8, 717–720; c) U. Nagata, U. Chujo, Mac-
romolecules 2008, 41, 2809–2813; d) G. Q. Zhang, S. E. Kooi, J. N.
Demas, C. L. Fraser, Adv. Mater. 2008, 20, 2099–2104; e) ##. Itami,
K. D. Yamazaki, J. Yoshida, J. Am. Chem. Soc. 2004, 126, 15396–
15397; f) C.-C. Chao, M.-K. Leung, Y. O. Su, K.-Y. Chiu, T.-H. Lin,
S.-J. Shieh, S.-C. Lin, J. Org. Chem. 2005, 70, 4323–4331; g) Q. Peng,
Y. P. Yi, Z. G. Shuai, J. S. Shao, J. Am. Chem. Soc. 2007, 129, 9333–
9339; h) R. Pohl, P. Anzenbacher, Org. Lett. 2003, 5, 2769–2772;
i) R. Pohl, V. A. Montes, J. Shinar, P. Anzenbacher, J. Org. Chem.
2004, 69, 1723–1725; j) Y. Qin, I. Kiburu, S. Shah, F. Jꢃkle, Org.
Lett. 2006, 8, 5227–5230; k) J. Cornil, D. Beljonne, J. P. Calbert, J. L.
Bredas, Adv. Mater. 2001, 13, 1053–1067; l) J. A. Marsden, J. J.
Miller, L. D. Shirtcliff, M. M. Haley, J. Am. Chem. Soc. 2005, 127,
2464–2476; m) B. Liu, Z. Wang, N. Wu, M. Li, J. You, J. Lan, Chem.
Eur. J. 2012, 18, 1599–1603.
[7] a) M. Tao, L. D. Aimone, Z. Huang, J. Mathiasen, R. Raddatz, J.
Lyons, R. Hudkins, J. Med. Chem. 2012, 55, 414–423; b) R. Hudkins,
R. Raddatz, M. Tao, J. Mathiasen, L. Aimone, N. Becknell, C.
Prouty, L. Knutsen, M. Yazdanian, G. Moachon, M. Ator, J. Malla-
mo, M. Marino, E. Bacon, M. Williams, J. Med. Chem. 2011, 54,
4781–4792.
[8] a) A. Coelho, E. Sotelo, E. RaviÇa, Tetrahedron 2003, 59, 2477–
2484; b) A. Monge, P. Parrado, M. Font, E. Alvarez, J. Med. Chem.
1987, 30, 1029–1035; c) I. Sircar, R. Weishaar, D. Kobylarz, W.
Moos, J. Bristol, J. Med. Chem. 1987, 30, 1955–1962; d) A. Akahane,
H. Katayama, T. Mitsunaga, J. Med. Chem. 1999, 42, 779–783; e) D.
Livermore, R. Bethell, N. Cammack, J. Med. Chem. 1993, 36, 3784–
3794; f) E. Sotelo, A. Coelho, E. Ravina, Tetrahedron Lett. 2003, 44,
4459–4462.
General procedure for the screening of the copper(II)–salen complexes
as catalysts: 3’-chloro-6-phenylpyridazinone (206 mg, 1.0 mmol), bromo-
benzene (173 mg, 1.1 mmol), and Na2CO3 (312 mg, 2.0 mmol) were
added to a stirred mixture of the copper–salen complex (0.05 mmol) in
water (2 mL) at room temperature. The tube was then sealed and the
temperature was increased to 808C in the air for 12 h. The reaction mix-
ture was filtered and the solid residue was purified by washing with distil-
led water (10 mLꢁ3) to remove the residual of catalyst and base; and n-
hexane (2 mLꢁ3) to remove the excessive organic residuals with spec-
trum purity. For further purification, the products could be purified by
column chromatography on silica gel using mixtures of n-hexane ether
and ethyl acetate (2:1) as the eluent.
General procedure for the reactions shown in Table 1: 3’-chloro-6-phenyl-
pyridazinone (206 mg, 1.0 mmol), bromobenzene (173 mg, 1.1 mmol),
and base (2.0 mmol) were added to a stirred mixture of Cu11 (32.5 mg,
0.05 mmol) in water (2 mL) at room temperature. The tube was then
sealed and the temperature was increased to 808C in the air. On comple-
tion of the reaction (as monitored by TLC) for 12 h, reaction mixture
was filtrated and the solid residue was purified by washing with distilled
water (10 mLꢁ3) to remove the residual of catalyst and base; and n-
hexane (2 mLꢁ3) to remove the excessive organic residuals with spec-
trum purity. For further purification, the products could be purified by
column chromatography on silica gel using mixtures of n-hexane ether
and ethyl acetate (2:1) as the eluent.
General procedure for the reactions shown in Table 2: 3’-chloro-6-phe-
nylpyridazinone (206 mg, 1.0 mmol), organic halide (1.1 mmol), and
Na2CO3 (312 mg, 2.0 mmol) were added to a stirred solution of Cu11
(32.5 mg, 0.05 mmol) in water (2 mL) at room temperature. The tube was
then sealed and the temperature was increased to 808C in the air. On
completion of the reaction for 12 h, reaction mixture was filtrated and
the solid residue was purified by washing with distilled water (10 mLꢁ3)
and n-hexane (2 mLꢁ3) with spectrum purity. For further purification,
the products were purified by column chromatography on silica gel using
mixtures of n-hexane and ethyl acetate (2:1) as the eluent.
General procedure for the reactions shown in Table 3: Pyridazinone
(1.0 mmol),
bromobenzene
(1.1 mmol)
or
1,4-dibromobenzene
(0.5 mmol), and Na2CO3 (312 mg, 2.0 mmol) were added to a stirred solu-
tion of Cu11 (32.5 mg, 0.05 mmol) in water (2 mL) at room temperature.
The tube was then sealed and the temperature was increased to 808C in
the air. On completion of the reaction for 12 h, reaction mixture was fil-
trated and the solid residue was purified by washing with distilled water
(10 mLꢁ3) and n-hexane (2 mLꢁ3) with spectrum purity. For further pu-
rification, the products were purified by column chromatography on
silica gel using mixtures of n-hexane and ethyl acetate (2:1) as the eluent.
General procedure for the synthesis of PyLs shown in Table 4: Pyridazi-
none (1.0 mmol), organic halide (1.1 mmol), and Na2CO3 (312 mg,
2.0 mmol) were added to a stirred solution of Cu11 (32.5 mg, 0.05 mmol)
in water (2 mL) at room temperature. The tube was then sealed and the
temperature was increased to 808C in the air. On completion of the reac-
tion for 12 h, reaction mixture was filtrated and the solid residue was pu-
rified by washing with distilled water (10 mLꢁ3) and n-hexane (2 mLꢁ3)
with spectrum purity. For further purification, the products were purified
by column chromatography on silica gel using mixtures of n-hexane and
ethyl acetate (2:1) as the eluent.
[9] I. P. Beletskaya, A. V. Cheprakov, Coord. Chem. Rev. 2004, 248,
2337–2364.
[10] a) D. Rowe, Science 2007, 317, 323–324; b) J. Liu, Science 2010, 328,
50; c) C.-J. Li, Acc. Chem. Res. 2002, 35, 533–538; d) C.-J. Li, Chem.
Rev. 2005, 105, 3095–3165.
[11] a) I. Beletskaya, A. Cheprakov, in Organic Synthesis in Water (Ed.:
P. Grieco), Blackie Academic & Professional, London, 1998, Ch. 5,
p. 141; b) C. I. Herrerꢄas, X. Yao, Z. Li, C.-J. Li, Chem. Rev. 2007,
107, 2546–2562; c) A. Modak, J. Mondal, M. Sasidharan, A. Bhau-
mik, Green Chem. 2011, 13, 1317–1331.
&
8
&
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Full-Color Tunable Fluorescent Dyes
FULL PAPER
[12] W. Watson, Green Chem. 2012, 14, 251–259.
[22] J. Wu, W. Liu, J. Ge, H. Zhang, P. Wang, Chem. Soc. Rev. 2011, 40,
3483–3495.
[23] a) B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien, Sci-
ence 2006, 312, 217–224; b) M. Vendrell, D. Zhai, J. Cheng Er, Y.-T.
[13] I. Beletskaya, A. Cheprakov, in Handbook of Organopalladium
Chemistry for Organic Synthesis, Vol. 2 (Eds.: E.-I. Negishi and A.
de Meijere), Wiley, New York, 2002, Ch. 10, p. 2957.
[14] a) Y. Wang, Z. Wu, L. Wang, Z. Li, X.-G. Zhou, Chem. Eur. J. 2009,
15, 8971–8974; b) L. Liang, Z. Li, X.-G. Zhou, Org. Lett. 2009, 11,
3294–3297.
[15] Y. Cai, L. Liang, J. Zhang, H. Sun, J.-L. Zhang, Dalton Trans. 2013,
42, 5390–5400.
[16] Y. Qu, F. Ke, L. Zhou, Z. Li, H. Xiang, D. Wu, X.-G. Zhou, Chem.
Commun. 2011, 47, 3912–3914.
[17] a) L. Liang, G. Yang, W. Wang, F. Xu, Y. Niu, Q. Sun, P. Xu, Adv.
Synth. Catal. 2013, 355, 1284–1290; b) L. Liang, G. Yang, W. Wang,
F. Xu, Y. Niu, Q. Sun, P. Xu, Org. Lett. 2013, 15, 2770–2773; c) L.
Liang, G. Yang, F. Xu, Y. Niu, Q. Sun, P. Xu, Eur. J. Org. Chem.
2013, DOI:10.1002/ejoc.201300640.
ˇ
Chang, Chem. Rev. 2012, 112, 4391–4420; c) G. Lukinavicius, K.
Umezawa, N. Olivier, A. Honigmann, G. Yang, T. Plass, V. Mueller,
L. Reymond, I. R. CorrÞa Jr, Z.-G. Luo, C. Schultz, E. A. Lemke, P.
Heppenstall, C. Eggeling, S. Manley, K. Johnsson, Nat. Chem. 2013,
5, 132–139; d) L. M. Wysockia, L. D. Lavis, Curr. Opin. Chem. Biol.
2011, 15, 752–759; e) E. Kim, S. B. Park, Chem. Asian J. 2009, 4,
1646–1658; f) Z. Chamas, E. Marchi, A. Modelli, Y. Fort, P. Ceroni,
V. Mamane, Eur. J. Org. Chem. 2013, 2316–2324.
[24] a) W.-B. Wu, M.-L. Wang, Y.-M. Sun, W. Huang, Y.-P. Cui, C.-X.
Xu, J. Phys. Chem. Solids 2008, 69, 76–82; b) M. Nakazono, S.
Nanbu, A. Uesaki, R. Kuwano, M. Kashiwabara, K. Zaitsu, Org.
Lett. 2007, 9, 3583–3586.
[18] J. Han, K. Burgess, Chem. Rev. 2010, 110, 2709–2728.
[19] a) E. W. Miller, C. J. Chang, Curr. Opin. Chem. Biol. 2007, 11, 620–
625; b) X. Chen, X. Tian, I. Shin, J. Yoon, Chem. Soc. Rev. 2011, 40,
4783–4804.
[20] a) Z. Xu, J. Yoon, D. R. Spring, Chem. Soc. Rev. 2010, 39, 1996–
2006; b) E. M. Nolan, S. J. Lippard, Chem. Rev. 2008, 108, 3443–
3480.
[25] a) D. Citterio, J. Takeda, M. Kosugi, H. Hisamoto, S.-I. Sasaki, H.
Komatsu, K. Suzuki, Anal. Chem. 2007, 79, 1237–1242; b) L. F. Mot-
tram, S. Boonyarattanakalin, R. E. Kovel, B. R. Peterson, Org. Lett.
2007, 9, 581–584; c) W.-C. Sun, K. R. Gee, D. H. Klaubert, R. P.
Haugland, J. Org. Chem. 1997, 62, 6469–6475.
[21] a) H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke, Y. Urano,
Chem. Rev. 2010, 110, 2620–2640; b) H. Kobayashi, P. L. Choyke,
Acc. Chem. Res. 2011, 44, 83–90.
Received: June 27, 2013
Published online: && &&, 2013
Chem. Eur. J. 2013, 00, 0 – 0
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
9
&
ÞÞ
These are not the final page numbers!

B. Xu, P. Xu et al.
Fluorescence
L. Liang, W. Wang, J. Wu, F. Xu,
Y. Niu, B. Xu,* P. Xu* ... ... &&&& — &&&&
Rational Design, Green Synthesis, and
Initial Evaluation of a Series of Full-
Color Tunable Fluorescent Dyes
Enabled by the Copper-Catalyzed N-
Arylation of 6-Phenyl Pyridazinones
and Their Application in Cell Imaging
Colorful cores: A simple and efficient
copper-catalyzed cross-coupling reac-
tion for the N-functionalization of pyr-
idazinones in neat water is reported
(see figure). The N2 position of pyrida-
zinones was modified by different aryl
groups (R1), such as benzothiazole,
N,N-dimethylaniline, 3-quinoline, 4-
isoquinoline, and 2-thiophene, result-
ing in a series of full-color tunable flu-
orescent reagents.
&
10
&
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!