Tetrahydroindazolone substituted 2-aminobenzamides as fluorescent probes: Switching metal ion selectivity from zinc to cadmium by interchanging the amino and carbamoyl groups on the fluorophore

Article abstract of DOI:10.1039/c2ob25852h

Three fluorescent probes CdABA′, CdABA and ZnABA′, which are structural isomers of ZnABA, have been designed with N,N-bis(2-pyridylmethyl) ethylenediamine (BPEA) as chelator and 2-aminobenzamide as fluorophore. These probes can be divided into two groups: CdABA, CdABA′ for Cd²⁺ and ZnABA, ZnABA′ for Zn²⁺. Although there is little difference in their chemical structures, the two groups of probes exhibit totally different fluorescence properties for preference of Zn²⁺ or Cd ²⁺. In the group of Zn²⁺ probes, ZnABA/ZnABA′ distinguish Zn²⁺ from Cd²⁺ with F_Zn ²⁺-F_Cd²⁺ = 1.87-2.00. Upon interchanging the BPEA and carbamoyl groups on the aromatic ring of the fluorophore, the structures of ZnABA/ZnABA′ are converted into CdABA/CdABA′. Interestingly, the metal ions selectivity of CdABA/CdABA′ was switched to discriminate Cd²⁺ from Zn²⁺ with F_Cd ²⁺-F_Zn²⁺ = 2.27-2.36, indicating that a small structural modification could lead to a remarkable change of the metal ion selectivity. ¹H NMR titration and ESI mass experiments demonstrated that these fluorescent probers exhibited different coordination modes for Zn²⁺ and Cd²⁺. With CdABA′ as an example, generally, upon addition of Cd²⁺, the fluorescence response possesses PET pathway to display no obvious shift of maximum λ_em in the absence or presence of Cd²⁺. However, an ICT pathway could be employed after adding Zn²⁺ into the CdABA′ solution, resulting in a distinct red-shift of maximal λ_em. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2ob25852h

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PAPER
Tetrahydroindazolone substituted 2-aminobenzamides as fluorescent probes:
switching metal ion selectivity from zinc to cadmium by interchanging the
amino and carbamoyl groups on the fluorophore†
Jia Jia, Qin-Chao Xu, Ri-chen Li, Xi Tang, Ying-Fang He, Meng-Yu Zhang, Yuan Zhang and Guo-Wen Xing*
Received 3rd May 2012, Accepted 11th June 2012
DOI: 10.1039/c2ob25852h
Three fluorescent probes CdABA′, CdABA and ZnABA′, which are structural isomers of ZnABA,
have been designed with N,N-bis(2-pyridylmethyl) ethylenediamine (BPEA) as chelator and
2-aminobenzamide as fluorophore. These probes can be divided into two groups: CdABA, CdABA′ for
Cd2+ and ZnABA, ZnABA′ for Zn2+. Although there is little difference in their chemical structures, the
two groups of probes exhibit totally different fluorescence properties for preference of Zn2+ or Cd2+. In
the group of Zn2+ probes, ZnABA/ZnABA′ distinguish Zn2+ from Cd2+ with FZn2+–FCd2+ = 1.87–2.00.
Upon interchanging the BPEA and carbamoyl groups on the aromatic ring of the fluorophore, the
structures of ZnABA/ZnABA′ are converted into CdABA/CdABA′. Interestingly, the metal ions
2+
selectivity of CdABA/CdABA′ was switched to discriminate Cd2+ from Zn2+ with FCd2+–FZn
=
2.27–2.36, indicating that a small structural modification could lead to a remarkable change of the metal
ion selectivity. 1H NMR titration and ESI mass experiments demonstrated that these fluorescent probers
exhibited different coordination modes for Zn2+ and Cd2+. With CdABA′ as an example, generally, upon
addition of Cd2+, the fluorescence response possesses PET pathway to display no obvious shift of
maximum λem in the absence or presence of Cd2+. However, an ICT pathway could be employed after
adding Zn2+ into the CdABA′ solution, resulting in a distinct red-shift of maximal λem
.
interference between Cd2+ and Zn2+ during the fluorometric
detection, because Cd and Zn are both group IIb elements,
which process very similar coordinating functions to the fluor-
escent sensors. For instance, pyridyl-containing ligands BPA (N,N-
bis(2-pyridylmethyl)diamine)3c,d,o,p,4 and BPEA (N,N-bis(2-pyr-
idylmethyl) ethylenediamine)3g,h,n,5 are widely used as chelators
for both Zn2+ and Cd2+ fluorescent sensors; however, it is
difficult to predict in which situation BPA/BPEA has more pre-
ference of Zn2+ to Cd2+, and vice versa. Therefore, to date, it
still a challenge to develop Cd2+-specific fluorescent probes,
especially with new fluorophores, that can distinguish Cd2+ from
Zn2+ in biological and environmental systems.
Introduction
Cadmium is considered as a toxic and inessential element for
life, and generally exists in the form of compounds with low
quality in nature. However, Cd can be accumulated in organisms
and get into the human body by the food chain, causing severe
lesions and cancers in the brain, bone, kidney, liver and gastro-
intestinal tract etc.1 Therefore, considerable efforts have been
expended on developing highly sensitive and selective detecting
methods for Cd2+, among which fluorescent probes have
attracted great attention due to their simplicity, high degree of
specificity and low detection limit.2
In the previous study, ZnABA6 was found to be a Zn2+-
specific fluorescent probe (Fig. 1) with 2-aminobenzamide as
fluorophore and BPEA as chelator. As a part of our efforts to
further synthesize various structural isomers of ZnABA and
investigate their fluorescent properties, Herein, we reported
the new discovery of fluorescent probes for Cd2+ based on
2-aminobenzamide.
In the past decade, many fluorescent sensor molecules have
been reported to measure Cd2+ in vivo or in vitro,3 and all of
them employed traditional dyes as fluorophores, including fluor-
escein,3q rhodamine,3k cyanine,3b anthracene/phenanthrene,3l,s,t
quinoline/naphthyridine,3a,e–h,j,m,r,u
coumarin,3n
naphthali-
mide,3d,p BODIPY3i,o etc. More importantly, there is always
Department of Chemistry, Beijing Normal University, Beijing 100875,
China. E-mail: gwxing@bnu.edu.cn
†Electronic supplementary information (ESI) available: Fig. S1–S6 and
copies of NMR spectra for all the new compounds. CCDC 875185 for
CdABA′ and 875205 for 8b. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/c2ob25852h
Results and discussion
There are three functional groups on the benzene ring of
ZnABA: carbamoyl, BPEA and tetrahydroindazolone. To further
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firstly, 2-bromo-5-fluorobenzonitrile (2) was coupled with tetra-
hydro-4H-indazol-4-one 1 in the presence of K2CO3, to afford
the desired tetrahydroindazolone 1H-tautomer substituted benzo-
nitrile 3a as the major product (50% yield, Table 1, entry 1), and
2H-tautomer compound 3b as the minor product (<5% yield).
To increase the product yield of isomer 3b, Cs2CO3 instead of
K2CO3 was selected for the reaction, and the yield of 1H-tauto-
mer compound 3a declined to 38% with increasing the yield of
2H-tautomer compound 3b to 19% (Table 1, entry 2), indicating
that the stronger base Cs2CO3 with greater solubility leads to a
much higher ratio of 3b to 3a in the aromatic nucleophilic substi-
tution. Next, the Buchwald–Hartwig coupling8 was carried out
under the conditions of catalytic PdCl2, 1,1′-bis(diphenylphos-
phanyl)ferrocene (DPPF), and NaOtBu in toluene at 100 °C, and
then BPEA moiety was incorporated into the aromatic ring of
3a/3b to give compound 5/6 in 54–66% yield. Finally,
hydration9 of the yielding benzonitrile 5/6 with KOH catalyzed
by H2O2 in a mixture of EtOH/DMSO afforded the new probe
CdABA/CdABA′ in high yield of 82–90%. Additionally, the
crystal structure of CdABA′ was obtained by single-crystal X-ray
diffraction, which is demonstrated in Fig. 2.10
Fig. 1 Structures of ZnABA and its structural isomers. ZnABA has
been previously reported.6 The others are newly designed.
explore the possibilities of developing new fluorescent probes,
we interchanged the BPEA and carbamoyl groups on the fluoro-
phore of ZnABA to produce a new compound CdABA, whose
chemical structure is shown in Fig. 1.
3,6,6-Trimethyl-1(2),5,6,7-tetrahydro-4H-indazol-4-one
(1)
has a equilibrium of two tautomers (Scheme 1).7 Previously, the
1H-tautomer substituted derivative ZnABA has been syn-
thesized.6 To deeply evaluate the influence of different tautomers
of 1 on the fluorescent probes, the 2H-tautomer substituted
derivatives ZnABA′ and CdABA′ (Fig. 1) were then designed
and synthesized in this study.
The synthetic procedures of CdABA, CdABA′ and ZnABA′
are depicted in Scheme 2. For the synthesis of CdABA/CdABA′,
Scheme 1 The equilibrium of two tautomers of tetrahydro-4H-indazol-
4-one 1.
Scheme 2 Synthesis of tetrahydroindazolone substituted 2-aminobenzamides. Reagents and conditions: (a) K2CO3, or Cs2CO3, DMF; (b) PdCl2,
DPPF, NaOtBu, toluene, 100 °C; (c) KOH, H2O2, EtOH/DMSO.
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Table 1 Effect of base on the coupling of 1 with bromobenzonitrile
Table 2 Absorption and fluorescence properties of all probes used in
this study
Entry
Substrate
Base
Products (yield)
Compound
εa (M−1 cm−1
)
λemb (nm)
Φc
1
2
3
4
2
2
7
7
K2CO3
Cs2CO3
K2CO3
Cs2CO3
3a (50%)/3b (<5%)
3a (38%)/3b (19%)
8a (82%)/8b (<5%)
8a (57%)/8b (28%)
CdABAd
11 700
12 600
26 820
25 880
28 835
21 900
14 700
14 700
456
456
454
454
434
446
436
448
0.20
0.53
0.06
0.23
0.0096
0.077
0.015
0.059
CdABA + Cd2+ e
CdABA′d
CdABA′ + Cd2+ e
ZnABA d,f
ZnABA + Zn2+ e,f
ZnABA′ d
ZnABA′ + Zn2+ e
a Data were evaluated at the maximum λabs of 260 nm. b The excitation
wavelength was 260 nm. c The fluorescence quantum yields were
obtained by using quinine sulfate (in 0.05 M H2SO4, Φ = 0.55) as the
standard. d Data were determined in the absence of Zn2+ or Cd2+
.
e Data
were determined in the presence of 1.0 equiv. Zn2+ or Cd2+
.
f See ref. 6.
Fig. 2 X-ray crystallographic structure of CdABA′.
Similarly, ZnABA′ was also obtained through above synthetic
strategy. 8b was prepared by the substitution of 2-bromo-4-fluoro-
benzonitrile (7) with indazolone 1. Notably, Cs2CO3 was still a
better base for the reaction, since the expected 2H-tautomer com-
pound 8b was obtained in higher yield (28%, Table 1, entry 4).
In contrast, the yield of 8b was lower than 5% with K2CO3 as
base (Table 1, entry 3), and the 1H-tautomer compound 8a was
almost the major product. The crystal structure of 8b was also
confirmed by X-ray diffraction (Fig. S1†).10
With all of the newly designed probes CdABA, CdABA′ and
ZnABA′ in hand, we screened their fluorescence response to
various metal ions. Interestingly, CdABA/CdABA′ was demon-
strated to be a Cd2+-specific fluorescent probe, while ZnABA′
was a Zn2+ fluorescent probe.
The photophysical properties of all the 2-aminobenzamides
used in this study, including previously reported Zn2+ probe
ZnABA, are summarized in Table 2. Among these probes,
CdABA′ and ZnABA have greater ε values (2.68–2.88 ×
104 M−1 cm−1), whereas CdABA and ZnABA′ show much
smaller ε values (1.17–1.26 × 104 M−1 cm−1). CdABA′ has a
maximal absorption peak at 260 nm with a long tail to 400 nm
before titration (Fig. 3a). Upon addition of Cd2+ (0–2.0 equiv.),
the absorbance at 260 nm decreased and one isosbestic point at
283 nm appeared, suggesting that the CdABA′–Cd2+ complex
could be formed. Meanwhile, the maximum fluorescence emis-
sion of CdABA′ was at 454 nm with a very low quantum yield
of 0.06 in the absence of Cd2+ (Table 2). After Cd2+ (0–2.0
equiv.) was added into the probe solution, the maximum fluor-
escence emission peak didn’t change, and the fluorescence inten-
sity showed a 5.2-fold enhancement with remarkably improved
quantum yield of 0.23 (Fig. 4a). Considering above results on
UV and fluorescent studies, it is suggested that the observed flu-
orescence enhancement of CdABA′ should be attributed to the
fact that the lone pair of the tertiary nitrogen of BPEA chelates
to Cd2+, and then the photo-induced electron transfer (PET)
process is prevented to bring out an efficient off-on fluorescence
response.
Fig. 3 UV-vis spectra of (a) CdABA′ and (b) ZnABA′ (10 μM; in
HEPES buffer, 25 mM, 0.1 M NaClO4, pH = 7.4, I = 0.1) upon addition
of Cd2+ and Zn2+ (0–20 μM).
The only difference in structure between CdABA and
CdABA′ is the tetrahydroindazolone moiety with 1H-tautomer
and 2H-tautomer respectively. Upon addition of Cd2+
(1.0 equiv.), the fluorescence emission intensity of CdABA was
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Fig. 5 Selectivity of (a) CdABA′ and (b) ZnABA′ towards various
metal ions. Experimental conditions: CdABA′/ZnABA′ (10 μM, 25 mM
HEPES buffer, 0.1 M NaClO4, pH = 7.4, I = 0.1), 10 μM Ba2+, Ca2+
,
Co2+, Cr3+, Cu2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Cd2+ and Zn2+, λex
260 nm, λem = 454 nm for CdABA′ and λem = 448 nm for ZnABA′.
=
1-carbamoyl group goes up, which leads to the two obvious iso-
sbestic points in its UV spectra and the red-shift of its emission
peak. In general, ZnABA′ displayed similar fluorescence proper-
ties to ZnABA, which has been reported before.6
Fig. 4 Fluorescence emission spectra (λex = 260 nm) of (a) CdABA′
and (b) ZnABA′ (10 μM, 25 mM HEPES buffer, 0.1 M NaClO4, pH =
7.4, I = 0.1) upon addition of Cd2+ or Zn2+ [added as Cd(ClO4)2 or
Zn(ClO4)2, 0–20 μM].
The selectivity of CdABA′ towards various common metal
ions was examined and the results are shown in Fig. 5a. No
change could be observed upon addition of the metal cations
such as Ba2+, Ca2+, Cr3+, Fe3+, Mg2+, Mn2+, Ni2+ and Pb2+,
whereas the transition-metal ions Co2+ and Cu2+ quenched the
fluorescence to some extent. Besides, both Zn2+ and Cd2+
induced the fluorescence enhancement. The fluorescence
enhancement (5.2-fold) of CdABA′ towards Cd2+ is higher than
that towards Zn2+ (2.2-fold), suggesting that CdABA′ has a
enhanced 3.4-fold (Fig. S2†), which is slightly lower than that of
CdABA′. On the whole, CdABA has very similar photophysical
properties to CdABA′, indicating that 1H-tautomer and 2H-tau-
tomer of tetrahydroindazolone have no distinguishable influence
on the probe properties.
2+
selectivity to prefer Cd2+ to Zn2+ (FCd2+–FZn = 2.36). CdABA
For ZnABA′, its UV titration spectra show two absorption
bands centered at 260 and 354 nm. On the addition of 0–2.0
equiv. Zn2+, the intensities of both bands decreased (Fig. 3b).
Two isosbestic points at 272 and 321 nm were observed due to
the formation of the ZnABA′-Zn2+ complex. The quantum
yields of ZnABA′ in the absence and presence of 1.0 equiv.
Zn2+ were 0.015 and 0.059 respectively, and its fluorescence
intensity was enhanced by 10.2-fold (Fig. 4b). Moreover, the
maximal emission peak of ZnABA′ showed a 12 nm red-shift,
which is totally different from the case of CdABA′ with no
change of the emission peak. The results can be explained by the
intermolecular charge transfer (ICT) effect of the aromatic plane
with BPEA as the conjugated electron donor and tetrahydroinda-
zolone as the receptor. Upon Zn2+ binding to ZnABA′, the elec-
tron-donating ability of the 2-amino group in the BPEA moiety
drops down, and the electron-withdrawing ability of the
also exhibited the preference for Cd2+ compared to Zn2+. Upon
addition of Cd2+ and Zn2+ (1.0 equiv.), the fluorescence intensity
of CdABA increased 3.4-fold and 1.5-fold, respectively
2+
(Fig. S3†), and the value of FCd2+–FZn is 2.27. Based on the
results, CdABA has a comparable selectivity to CdABA′ to dis-
criminate Cd2+ from Zn2+ in aqueous solutions.
As for the Zn2+ probes, the value of FZn2+–FCd is 1.87 for
2+
ZnABA′ (Fig. 5b), and 2.00 for ZnABA,6 which provides a
approximate selectivity to distinguish Zn2+ from Cd2+.
All the fluorescent probes used in this work have a 1 : 1
binding ratio with Zn2+ or Cd2+, which is determined by the Job
plot (Fig. S4†). The dissociation constants of CdABA′/CdABA
for Cd2+ and ZnABA′/ZnABA for Zn2+ were determined by flu-
orescence spectroscopy in metal–ligand-buffered solutions with
different Cd2+/Zn2+ concentrations. CdABA′ and CdABA
responded to the picomolar concentration of free Cd2+ (Kd
=
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Fig. 6 1H NMR spectra of CdABA′, CdABA′–Cd2+ complex and CdABA′–Zn2+ complex in DMSO-d6.
1.66–1.86 pM), indicating the high binding affinity of CdABA′/
Conclusion
CdABA for Cd2+. In contrast, the competitive binding experi-
ments afforded the estimated Kd of 1.06–2.06 nM for Zn2+-
ZnABA′ or Zn2+-ZnABA complex. Therefore, CdABA′/CdABA
has more sensitivity to detect Cd2+ than ZnABA′/ZnABA to
detect Zn2+.
Three novel fluorescent probes, CdABA′, CdABA and ZnABA′
have been designed with BPEA as chelator and the 2-aminoben-
zamide as fluorophore. All the probes used in this study are the
structural isomers of ZnABA, which has been previously
reported as Zn2+ probe. For the preparation of the probes, three
reactions including the aromatic nucleophilic substitution, the
Buchwald–Hartwig coupling and the subsequent hydration were
employed to successfully afford the desired fluorescent mol-
ecules. It should be noted that the utilization of Cs2CO3 as base
is crucial to the synthesis of 2H indazolone tautomer substituted
benzonitriles.
Notably, above results and Fig. S5† show the maximum λem
of the probes used in this work always exhibits a red-shift after
the addition of Zn2+, while there is no obvious changes of λem
upon addition of Cd2+, which indicates that there could be differ-
ent mechanisms of coordination for Zn2+ and Cd2+. To further
1
understand the mechanisms, the H NMR titration experiments
of CdABA′ with Zn2+ and Cd2+ in DMSO-d6 have been taken to
provide further evidence. As shown in Fig. 6, addition of 1.0
equiv. of Zn2+ or Cd2+ promoted downfield shifts of the H(5), H(6),
H(8,8′) and H(9,9′) protons and upfield shifts of the H(7,7′)
protons, indicating that the chemical circumstances of these
protons have been changed through the interaction between
Zn2+/Cd2+ and CdABA′. Moreover, the H(2) and H(3) protons
of amide experienced approximate 0.8–1.0 ppm downfield shifts
with Zn2+/Cd2+ adding into the CdABA′ solution, which demon-
strates the amide group directly participated the metal ion coordi-
nation. In addition, a huge upfield shift of the H(1) proton from
8.63 to 6.48 was observed as a result of the aniline nitrogen
atom coordinating with Cd2+ to form [CdABA′ + Cd]2+.
However, the disappearance of H(1) signal in CdABA′ − Zn2+
proton NMR spectrum implied that the [CdABA′ + Zn − H]+
could be formed in the solution, which is consistent to the
reports that zinc ion usually cause deprotonation during the
coordination with probes.3h,3p The ESI mass spectra also pro-
vided the supplemental evidence for the existence of the singly
charged [CdABA′ + Zn − H]+ complex (Fig. S6†).
These probes can be divided into two groups, CdABA,
CdABA′ for Cd2+ and ZnABA, ZnABA′ for Zn2+. ZnABA and
CdABA, ZnABA′ and CdABA′ are positional isomers on the
benzene ring; while the couples of ZnABA and ZnABA′,
CdABA and CdABA′ are 1H or 2H indazolone tautomer substi-
tuted functional isomers. Although there is little difference in the
chemical structures of these 2-aminobenzamide probes, these
two groups of probes exhibit totally different fluorescence prop-
erties for the preference of Zn2+ or Cd2+. In the group of probes
2+
for Zn2+, ZnABA/ZnABA′ prefers Zn2+ to Cd2+ with FZn
–
2+
FCd = 1.87–2.00. It is noted that the structure of ZnABA/
ZnABA′ is converted into CdABA/CdABA′ by interchanging
the BPEA and carbamoyl groups on the 2-aminobenzamide
fluorophore. Interestingly, the metal ions selectivity of CdABA/
CdABA′ was switched to prefer Cd2+ to Zn2+ with FCd2+–FZn
2+
= 2.27–2.36, indicating that a small structural modification could
lead to a remarkable change in the metal ion selectivity.
1H NMR titration and ESI mass experiments showed that
these fluorescent probes exhibited different coordination modes
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for Zn2+ and Cd2+. With CdABA′ as an example, generally,
upon addition of Cd2+, the fluorescence response possesses PET
pathway to display no obvious shift of maximum λem in the
absence or presence of Cd2+. However, an ICT pathway could be
employed when adding Zn2+ into the CdABA′ solution, as a
consequence a distinct red-shift of maximal λem is observed.
The current study should be helpful for the design and syn-
thesis of other metal fluorescent probes with 2-aminobenzamide
as the fluorophore. Also, the deep investigations of these probes
concerning biological activities and applications are under way
and will be reported in due course.
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-5-(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)benzonitrile (5). A
Schlenk flask was charged with compound 3a (96.3 mg,
0.275 mmol, 1.00 equiv.), BPEA11 (66.4 mg, 0.274 mmol, 1.00
equiv.), sodium tert-butoxide (96 mg, 1.00 mmol, 2.60 equiv.),
palladium chloride (10 mg, 0.06 mmol, 0.20 equiv.), 1,1′-bis-
(diphenylphosphanyl)ferrocene (DPPF; 48 mg, 0.086 mmol,
0.31 equiv.), and toluene (8 mL) under argon. The flask was
immersed in an oil bath at 100 °C with stirring until the starting
material had completely disappeared as judged by TLC analysis.
The solution was then allowed to cool to room temperature,
diluted with DCM (100 mL), filtered through Celite, and concen-
trated. The crude product was purified by column chromato-
graphy (CH2Cl2–MeOH, 40 : 1) on silica gel to give 5 (93 mg,
0.18 mmol, 66%) as a dark-red viscous oil. 1H NMR (400 MHz,
CDCl3): δ 1.10 (s, 6 H), 2.38 (s, 2 H), 2.52 (s, 3H), 2.67 (s, 2
H), 2.96 (t, J = 5.60 Hz, 2 H), 3.28 (s, 2H), 3.93 (s, 4 H), 6.11
(bs, 1 H), 6.62 (d, J = 9.00 Hz, 1 H), 7.18 (dd, J = 6.70, 5.50 Hz,
1 H), 7.42 (dd, J = 9.00, 2.50 Hz, 1 H), 7.59 (d, J = 7.80 Hz,
2 H), 7.71 (td, J = 7.70, 1.50 Hz, 2 H), 8.58 (d, J = 4.30 Hz, 2
H). 13C-Apt (100 MHz, CDCl3): δ 13.25, 28.38, 28.42, 29.66,
35.75, 36.82, 40.40, 51.58, 52.35, 60.15, 95.55, 111.32, 116.51,
117.02, 122.28, 122.32, 123.27, 123.29, 127.41, 130.15, 130.19,
131.25, 136.86 (2 C), 148.84, 149.10, 149.12, 149.62, 149.92,
158.69, 193.20 ppm. HRMS (ESI) calcd for C31H34N7O:
520.2825 [M + H]+; Found 520.2834.
Experimental
General
All chemicals were purchased as reagent grade and used without
further purification. Buchwald–Hartwig cross-coupling reactions
were performed in flame-dried glassware under argon. Toluene
was distilled from calcium hydride. The reactions were moni-
tored by analytical thin-layer chromatography (TLC) on silica
gel F254 glass plates and visualized under UV light (254 and
365 nm) and/or by staining with ninhydrin. Flash column chrom-
atography was performed on silica gel (200–300 mesh). 1H
NMR spectra were recorded with a Bruker Avance III 400 MHz
NMR spectrometer at 20 °C. Chemical shifts (in ppm) were
determined relative to tetramethylsilane (δ = 0 ppm) in deu-
teriated solvents. Coupling constants in Hz were measured from
the one-dimensional spectra. 13C NMR or 13C attached-proton-
test (13C-Apt) spectra were recorded with the 400 MHz NMR
spectrometer (100 MHz) and calibrated with CDCl3 (δ =
77.23 ppm). High-resolution mass spectra were recorded with a
Waters LCT Premier XE mass spectrometer. UV absorption and
emission spectra were recorded with a GBC Cintra 10e UV/Vis
spectrophotometer and a Varian Cary Eclipse spectro-fluorimeter,
respectively, in a quartz cell with a 1 cm path length.
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-5-(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)benzamide
(CdABA). KOH (70 mg, 1.24 mmol, 7.75 equiv.) and com-
pound 5 (84.6 mg, 0.16 mmol, 1.00 equiv.) were added to a sol-
ution of EtOH and DMSO (4 : 1, 3.50 mL). The reaction mixture
was stirred in a 50 °C oil bath, and H2O2 (30%, 0.4 mL) was
slowly added dropwise through a syringe over 0.5 h. Then the
resulting solution was stirred for another 3.5 h until the disap-
pearance of benzonitrile, as shown by TLC. After removal of the
solvent by rotary evaporation, the mixture was diluted with
DCM (150 mL), washed with saturated NaCl solution (3 ×
30 mL), dried with MgSO4, filtered, and concentrated. The
mixture was purified by column chromatography (CH2Cl2–
MeOH, 20 : 1) to give CdABA (73.4 mg, 0.136 mmol, 82%) as
a colorless solid. Mp. 235–236 °C. 1H NMR (400 MHz,
CDCl3): δ 1.08 (s, 6 H), 2.35 (s, 2 H), 2.52 (s, 3H), 2.65 (s, 2
H), 2.95 (t, J = 5.90 Hz, 2 H), 3.36 (t, J = 6.00 Hz, 2H), 3.97 (s,
4 H), 6.65 (d, J = 8.90 Hz, 1 H), 7.13–7.20 (m, 2 H), 7.23 (dd, J
= 8.90, 2.10 Hz, 1 H), 7.55 (d, J = 2.10 Hz, 1 H), 7.65–7.80 (m,
4 H), 8.53 (d, J = 4.50 Hz, 2 H). 13C-Apt (100 MHz, CDCl3): δ
13.31, 28.34 (2C), 35.66, 36.68, 40.41, 52.31, 52.55, 60.41
(2C), 111.77, 113.77, 116.15, 122.11 (2C), 123.81 (2C), 124.90,
125.66, 128.71, 136.66 (2C), 148.82 (2C), 149.08, 149.18,
149.46, 159.07 (2C), 171.06, 193.43. HRMS (ESI) calcd for
C31H35N7O2Na: 560.2750 [M + Na]+; Found 560.2740.
2-Bromo-5-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)-
benzonitrile (3a). Indazolone 17 (250 mg, 1.47 mmol, 1.47
equiv.) was dissolved in DMF, and 2-bromo-5-fluorobenzonitrile
(2) (200 mg, 1.00 mmol, 1.00 equiv.) and K2CO3 (350 mg,
2.54 mmol, 2.54 equiv.) were added. The reaction mixture was
stirred at room temperature for 30 min and then heated in an
80 °C oil bath until the starting material had been completely
consumed as detected by TLC. The solution was then allowed to
cool to room temperature, and the DMF was evaporated under
vacuum to leave a yellowish oil. The crude oil was then diluted
with DCM (150 mL), washed with saturated NaCl solution (3 ×
30 mL), and dried with MgSO4. After removal of the solvent,
the mixture was purified by column chromatography (hexanes–
EtOAc, 5 : 1) and then recrystallized (CH2Cl2–hexanes) to give
3a (175 mg, 0.50 mmol, 50%) as
a colorless solid.
1
Mp. 152–153 °C. H NMR (400 MHz, CDCl3): δ 1.13 (s, 6 H),
2.42 (s, 2 H), 2.53 (s, 3 H), 2.81 (s, 2 H), 7.65 (dd, J = 4.39,
1.28 Hz, 1 H), 7.80–7.83 (m, 2 H). 13C-Apt (100 MHz, CDCl3):
δ 13.28, 28.40 (2C), 35.94, 37.45, 52.19, 116.16, 116.95,
117.88, 123.85, 128.11, 128.14, 134.25, 138.35, 148.92, 150.99,
193.00. HRMS (ESI) calcd for C17H17N3OBr: 358.0555
[M + H]+; Found 358.0563.
2-Bromo-5-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-2-
yl)benzonitrile (3b). Compound 1 (170 mg, 1.00 mmol, 1.00
equiv.) was dissolved in DMF, and 2-bromo-5-fluorobenzonitrile
(2, 200 mg, 1.00 mmol, 1.00 equiv.) and Cs2CO3 (648 mg,
2.00 mmol, 2.00 equiv.) were added. The reaction mixture was
stirred at room temperature for 30 min and then heated in an
6284 | Org. Biomol. Chem., 2012, 10, 6279–6286
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80 °C oil bath until the starting material had been completely
consumed as detected by TLC. The solution was then allowed to
cool to room temperature, and the DMF was evaporated in
vacuum to leave a yellowish oil. The crude oil was then diluted
with DCM (150 mL), washed with saturated NaCl solution (3 ×
30 mL), and dried with MgSO4. After removal of the solvent,
the mixture was purified by column chromatography (hexanes–
EtOAc, 6 : 1) and then recrystallized (CH2Cl2–hexanes) to give
40 °C oil bath until the starting material had been completely
consumed as detected by TLC. The solution was then allowed to
cool to room temperature, and the DMF was evaporated under
vacuum to leave a yellowish oil. The crude oil was then diluted
with DCM (150 mL), washed with saturated NaCl solution (3 ×
30 mL), and dried with MgSO4. After removal of the solvent,
the mixture was purified by column chromatography (hexanes–
EtOAc, 5 : 1) and then recrystallized (CH2Cl2–hexanes) to give
3b (66.4 mg, 0.19 mmol, 19%) as
a
colorless solid.
8b (150 mg, 0.42 mmol, 28%) as a colorless solid.
1
1
Mp. 152–153 °C. H NMR (400 MHz, CDCl3): δ 1.13 (s, 6 H),
2.40 (s, 2 H), 2.66 (s, 3 H), 2.73 (s, 2 H), 7.60 (dd, J = 8.70,
2.50 Hz, 1 H), 7.83 (dd, J = 5.60, 3.00, 2 H). 13C-Apt
(100 MHz, CDCl3): δ 12.27, 28.44 (2C), 34.99, 36.87, 53.34,
116.04, 116.84, 117.01, 124.70, 129.64, 134.12, 138.26, 141.77,
156.94, 194.81. HRMS (ESI) calcd for C17H17N3OBr: 358.0555
[M + H]+; Found 358.0549.
Mp. 159–160 °C. H NMR (400 MHz, CDCl3): δ 1.13 (s, 6 H),
2.40 (s, 2 H), 2.70 (s, 3 H), 2.73 (s, 2 H), 7.57 (dd, J = 8.40,
2.00 Hz, 1 H), 7.78 (d, J = 8.40, 1 H), 7.93 (d, J = 1.90, 1 H).
13C-Apt (100 MHz, CDCl3): δ 12.50, 28.41 (2C), 34.91, 36.68,
53.36, 114.99, 116.40, 117.15, 123.07, 126.06, 128.85, 134.77,
142.07, 142.67, 157.13, 194.73. HRMS (ESI) calcd for
C17H17N3OBr: 358.0555 [M + H]+; Found 358.0556.
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-5-(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-2-yl)benzamide
(CdABA′). A Schlenk flask was charged with compound 3b
(78.0 mg, 0.22 mmol, 1.00 equiv.), BPEA (63.4 mg, 0.26 mmol,
1.17 equiv.), sodium tert-butoxide (96 mg, 1.00 mmol, 4.50
equiv.), palladium chloride (10 mg, 0.06 mmol, 0.27 equiv.),
DPPF (48 mg, 0.086 mmol, 0.39 equiv.), and toluene (8 mL)
under argon. The flask was immersed in an oil bath at 100 °C
with stirring until the starting material had completely dis-
appeared as judged by TLC analysis. The solution was then
allowed to cool to room temperature, was diluted with DCM
(100 mL), filtered through Celite, and concentrated. The crude
product was then purified further by column chromatography
(CH2Cl2–MeOH, 40 : 1) on silica gel to give 6 (61.6 mg,
0.12 mmol, 54%) as a viscous oil. 1H NMR (400 MHz, CDCl3):
δ 1.12 (s, 6 H), 2.37 (s, 2 H), 2.52 (s, 3H), 2.70 (s, 2 H), 2.96 (t,
J = 5.60 Hz, 2 H), 3.28 (s, 2H), 3.94 (s, 4 H), 6.12 (bs, 1 H),
6.61 (d, J = 9.00 Hz, 2 H), 7.18 (dd, J = 6.70, 5.50 Hz, 2 H),
7.37 (dd, J = 9.00, 2.50 Hz, 1 H), 7.46 (d, J = 2.50 Hz, 1 H)
7.60 (d, J = 7.80 Hz, 2 H), 7.71 (td, J = 7.70, 1.60 Hz, 2 H),
8.57 (d, J = 4.70 Hz, 2 H). Then, by using the same procedure
as that described for the preparation of CdABA, compound 6
was hydrated with KOH catalyzed by H2O2 to give CdABA′ in
90% yield as a colorless solid. Mp. 235–236.5 °C. 1H NMR
(400 MHz, CDCl3): δ 1.12 (s, 6 H), 2.37 (s, 2 H), 2.50 (s, 3H),
2.70 (s, 2 H), 2.93 (t, J = 5.70 Hz, 2 H), 3.33 (t, J = 5.80 Hz,
2H), 3.94 (s, 4 H), 5.87 (bs, 2 H), 6.63 (d, J = 8.90 Hz, 1 H),
7.14–7.18 (m, 2 H), 7.24 (dd, J = 8.90, 2.40 Hz, 1 H), 7.48 (d,
J = 2.40 Hz, 1 H), 7.68 (t, J = 7.60 Hz, 2 H), 7.76 (d, J = 7.80 Hz,
2 H), 8.52 (d, J = 4.70 Hz, 2 H). 13C-Apt (100 MHz, CDCl3):
δ 11.97, 28.49 (2C), 29.67, 35.08, 40.32, 52.51, 53.29, 60.35
(2C), 111.80, 113.52, 115.51, 122.21 (2C), 123.41 (2C), 125.50,
125.83, 130.05, 136.78 (2C), 141.76, 148.78 (2C), 149.70,
155.73, 158.83 (2C), 170.90, 194.90. HRMS (ESI) calcd for
C31H36N7O2: 538.2930 [M + H]+; Found 538.2929.
2-({2-[Bis(pyridin-2-ylmethyl)amino]ethyl}amino)-4-(3,6,6-
trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-2-yl)benzamide
(ZnABA′). A Schlenk flask was charged with compound 8b
(115.5 mg, 0.33 mmol, 1.20 equiv.), BPEA (66.7 mg,
0.275 mmol, 1.00 equiv.), sodium tert-butoxide (96 mg,
1.00 mmol, 3.60 equiv.), palladium chloride (10 mg, 0.06 mmol,
0.22 equiv.), DPPF (48 mg, 0.086 mmol, 0.31 equiv.), and
toluene (8 mL) under argon. The flask was immersed in an oil
bath at 100 °C with stirring until the starting material had com-
pletely disappeared as judged by TLC analysis. The solution was
then allowed to cool to room temperature, was diluted with
DCM (100 mL), filtered through Celite, and concentrated. The
crude product was then purified further by column chromato-
graphy (CH2Cl2–MeOH, 40 : 1) on silica gel to give 9 (73.9 mg,
0.14 mmol, 53%) as a viscous oil. 1H NMR (400 MHz, CDCl3):
δ 1.12 (s, 6 H), 2.38 (s, 2 H), 2.60 (s, 3H), 2.71 (s, 2 H), 2.93 (t,
J = 5.60 Hz, 2 H), 3.27 (d, J = 3.70 Hz, 2H), 3.91 (s, 4 H), 6.09
(bs, 1 H), 6.66–6.68 (m, 2 H), 7.16 (dd, J = 6.70, 5.60 Hz, 2 H),
7.49 (d, J = 8.20 Hz, 1 H), 7.58 (d, J = 7.80 Hz, 2 H), 7.69 (td,
J = 7.70, 1.5 Hz, 2 H), 8.55 (d, J = 4.80 Hz, 2 H). Then, by
using the same procedure as that described for the preparation of
CdABA, compound 9 was hydrated with KOH catalyzed by
H2O2 to give ZnABA′ in 90% yield as a colorless solid.
1
Mp. 198–199 °C. H NMR (400 MHz, CDCl3): δ 1.13 (s, 6 H),
2.38 (s, 2 H), 2.57 (s, 3H), 2.72 (s, 2 H), 2.90 (t, J = 6.00 Hz, 2
H), 3.30 (t, J = 6.00 Hz, 2H), 3.90 (s, 4 H), 5.93 (bs, 2 H), 6.60
(d, J = 8.3 Hz, 1 H), 7.08–7.18 (m, 2 H), 7.49 (d, J = 8.30 Hz, 1
H), 7.65 (t, J = 7.00 Hz, 2 H), 7.72 (d, J = 7.70 Hz, 2 H), 8.48
(d, J = 4.50 Hz, 2 H). 13C-Apt (100 MHz, CDCl3): δ 12.32,
28.48 (2C), 35.02, 36.99, 40.45, 52.57, 53.40, 60.37 (2C),
108.04, 110.68, 113.43, 116.19, 122.09 (2C), 123.29 (2C),
129.28, 136.64 (2C), 141.68, 142.61, 148.80 (2C), 150.51,
156.08, 159.04 (2C), 171.04, 194.93. HRMS (ESI) calcd for
C31H36N7O2: 538.2930 [M + H]+; Found 538.2923.
Spectroscopic materials and methods. Stock solutions (0.001
M) of zinc/cadmium perchlorate were prepared in HEPES buffer
(25 mM HEPES, 0.1 M NaClO4, pH = 7.4, I = 0.1). Stock solu-
tions (0.001 M) of ZnABA′, CdABA and CdABA′ were pre-
pared in EtOH. All the fluorescence spectra of ZnABA′, CdABA
and CdABA′ were also measured in HEPES buffer [25 mM
HEPES, 0.1 M NaClO4, pH = 7.4, I = 0.1, 1% (v/v) EtOH] and
2-Bromo-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-2-
yl)benzonitrile (8b). Compound 1 (250 mg, 1.47 mmol, 1.00
equiv.) was dissolved in DMF, and 2-bromo-4-fluorobenzonitrile
(7; 354 mg, 1.77 mmol, 1.20 equiv.) and Cs2CO3 (955 mg,
2.94 mmol, 2.00 equiv.) were added. The reaction mixture was
stirred at room temperature for 30 min and then heated in a
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 6279–6286 | 6285

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Acknowledgements
The project was financially supported by the National Natural
Science Foundation of China (20772013), Beijing National
Natural Science Foundation (2122031) and Beijing Municipal
Commission of Education.
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6286 | Org. Biomol. Chem., 2012, 10, 6279–6286
This journal is © The Royal Society of Chemistry 2012