Investigation of thiolysis of NBD amines for the development of H2S probes and evaluating the stability of NBD dyes

Article abstract of DOI:10.1039/c6ob02354a

In order to evaluate the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines for development of H₂S probes, herein we investigated the reactivity and selectivity of a series of NBD amines for the first time. The piperazinyl- and piperidyl-ba

Full text of DOI:10.1039/c6ob02354a

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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Investigation of Thiolysis of NBD Amines for the Development of
H S Probes and Evaluating the Stability of NBD dyes
2
Received 00th January 20xx,
Accepted 00th January 20xx
†a
†b
b
c
b
c
b
Fanbo Song, Zhifei Li, Jiayuan Li, Shuai Wu, Xianbo Qiu, * Zhen Xi and Long Yi *
DOI: 10.1039/x0xx00000x
In order to evaluate the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amines for development of H
investigated the reactivity and selectivity of a series of NBD amines for the first time. The piperazinyl- and piperidyl-based
NBD probes could react efficiently with micromolar H S in buffer (pH 7.4), while such NBD(S) (nitrobenzothiadiazole)
derivatives showed much slow thiolysis even in the presence of millimolar H S. Low reactivity was also observed for
thiolysis of these ethylamino-, ethanolamino- and anilino-based NBD probes. Therefore, almost NBD amines used in
bioimaging should be stable, in consideration of the presence of only micromolar endogenous H S in vivo. Moreover, the
2
S probes, herein we
www.rsc.org/
2
2
2
2 2
piperazinyl-NBD derivatives could be efficient in development of fluorescent H S probes and for directly visualizing H S by
paper-based detection.
2
6
27,28
29-43
and others,
precipitation, thiolysis of dinitrophenyl ether,
Introduction
have been successfully employed to develop fluorescent and/or
colorimetric probes for detection of biological H S. Among these
2
Hydrogen sulfide (H S) is an important endogenous signalling
2
29
30
1
-6
organic reactions, the thiolysis of NBD amine and NBD thioether
discovered by our group and Pluth group, respectively, were
molecule with multiple biological functions.
H S could be
2
enzymatically produced in vivo by three distinctive pathways
including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE)
and 3-mercaptopyruvate sulfurtransferase (3-MPST)/cysteine
3
1-36
successful for development of efficient H
2
S probes. We
further employed the thiolysis of NBD amines to develop
efficient fluorescent H S probes. The thiolysis of NBD ether was also
explored by us and others for biothiols‘ probes.
research indicated that the rate for thiolysis of NBD probes varied
and
3
7-39
others
7
-10
2
aminotransferase (CAT) in different organs and tissues.
In the
4
0-42
However, our
central nervous system, the biological concentration of H S is in the
2
range of 50-160 μM. The sulfide level in plasma blood is in the
-1
-1
31-36
from 1 to over 100 M s due to different chemical structures.
range of 10-100 μM. Studies have shown that the H S level in vivo is
2
In this study, we aim to investigate the reactivity and selectivity of
NBD amines for further understanding such thiolysis reaction.
correlated with numerous diseases, including the symptoms of
Alzheimer’s disease, Down syndrome, diabetes and liver
1
,11,12
On the other hand, though the success for thiolysis of NBD
cirrhosis.
Despite H S has been recognized to be linked to
2
31-42
probes in H
2
S detection,
the rising of issue whether NBD-based
numerous physiological and pathological processes, many of its
underlying molecular events in vivo remain largely unknown and
could be further explored. Therefore, it presents significant
research value to develop efficient chemical or advanced methods
dyes are stable in bioimaging should be addressed, because
endogenous H S exists in vivo and NBD-based dyes were widely
used in bioimaging.
and studied their thiolysis reactions in buffer solution. The reaction
2
4
4-48
Herein, we prepared a series of NBD amines
for selective and sensitive detection of biological H S.
2
-1 -1
1
3,14
2
rates of NBD amines towards H S varied from 43.2 to 0.05 M s or
H S-triggered chemical reactions,
including nucleophilic
2
1
5-19
20-25
smaller values dependent on the chemical structures. A sensitive
colorimetric probe was obtained for direct visualization of low
addition,
reduction of azide or nitro to amine,
copper
2 2
micromolar H S and for paper-based H S detection. Our studies also
a.
Tianjin Key Laboratory of Water Resources and Environment, Tianjin Normal
University, Tianjin 300387, China.
point out that almost NBD amines should be stable dyes for
bioimaging.
b.
c.
State Key Laboratory of Organic-Inorganic Composites and College of Information
Science and Technology, Beijing University of Chemical Technology (BUCT), 15
Beisanhuan East Road, Chaoyang District, Beijing 100029, China.
State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical
Biology, National Engineering Research Center of Pesticide (Tianjin), Collaborative
Innovation Center of Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, China.
Results and discussion
One major challenge in development of H S probes is
2
discovery of a chemical reaction to effectively separate the
†
These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any reactivity of biothiols and H S. To address this challenge, we
2
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
29
discovered such a reaction of thiolysis of NBD amine in 2013.
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Consequently, this reaction was widely applied for H
Journal Name
2
S probes
Because the biological H
2
S level normally stands in the range
3
1-39
by us and others.
fluorescence in buffer, while the thiolysis product 4-thiol-7- the fluorescence spectra of the reaction between NBD probes
nitrobenzofurzan (NBD-SH) is non-fluorescence but shows and 100 µM H S (using Na S as an equivalent) in PBS buffer (pH
The NBD amines show yellow of nanomolar to micromolar concentrations, we firstly tested
2
2
3
0
unique red color. Therefore, NBD-based probes could be 7.4, containing 20% DMSO) for 30 min. As shown in Figures 1
used to develop colorimetric and fluorescent dual-channel and S1-S2, all NBD probes exhibited a certain emission around
probes (Scheme 1). However, we observed that different NBD- 560 nm upon excitation at 470 nm. After reaction with H
2
S,
was observed, while
4-7) did not display any obviously
work, we prepared a series of NBD derivatives 1-9 (Scheme 1) fluorescence change. The fluorescence turn-off for probe is
3
1-42
based probes have various reaction rates toward H
2
S,
and large fluorescence change for
3b
1, 3a and 8
hope to further understand the thiolysis of NBD amines. In this other probes (
2
,
,
1
for comparative studies of their thiolysis reactions. The largest, and the final decrease factor was about 14-fold.
coupling reaction of NBD-Cl (or NBD(S)Cl) and commercial Furthermore, the fluorescent turn-off response of the probes
amines under basic conditions produced the target probes (see could be observed by naked eye under 365 nm UV lamp
experimental part). Such facile and economic synthesis is (Figure S3), and we found that high concentration H S (2 mM)
2
important for the wide use of the probes reported herein. The could also trigger fluorescent turn-off of probes including 5.
1
structural characterizations of all probes were confirmed by H These preliminary results encouraged us to further check the
1
NMR, C NMR, and high resolution mass spectra.
3
reactivity and selectivity of these probes.
To obtain the reaction kinetics, the time-dependent
fluorescence signal at 560 nm was recorded for data analysis
(Figures 2, S4-S7). The pseudo-first-order rate, kobs was
determined by fitting the fluorescence intensity data with
single exponential function. The linear fitting between kobs and
H S concentrations gives the reaction rate (k ). The thiolysis
2
2
-1
rate (Table 1) for
1
s , respectively, which showed near 1000-fold difference for
thiolysis of NBD amines. Probes , and have similar slow
1,
3a,
5
was found to be 43.26, 1.10, 0.05 M
-
2,
4
6
thiolysis rate with that of
H2S. The thiolysis rate of
5
even in the presence of millimolar
7
in millimolar H S is too slow to be
2
-
1 -1
observed. The k for probe
2
8
is 21.77 M s , which is slight
slower than
1. The thiolysis reactions in 1% DMSO-containing
buffer gave similar kinetic data as that in 20% DMSO-
containing buffer (Figure S2). These results clearly indicated
that piperazinyl- and piperidyl-based probes could react
Scheme 1. Fluorescent and colorimetric H
2
2
S probes based on efficiently with micromolar H S under physiological conditions,
thiolysis of NBD amine (a) and chemical structures of NBD while other NBD amines could react with millimolar H S slowly.
amines in this work (b).
2
Previous studies indicated that H S executes physiological
2
effects at a wide range of concentrations between 10 and 300
4
µM. Therefore, NBD amines including
2,
4-7 should be enough
stable in the presence of biological H S.
2
Pluth et al. have reported that H S and biothiols could
2
deactivate the NBD-based thiol labeling reagents (thiolysis of
4
NBD thioether bond), implying that NBD thioethers could not
9
4
0,50,51
be used in in vivo experiments. In our previous work,
we
have indicated that micromolar biothiols could efficiently react
with several NBD ethers which contained electron-
withdrawing group neighboring the NBD ether bond. Herein
we hope to state that since many NBD dyes are analogs of
ethylamino-NBD, such dyes could be safety used for biological
studies even in the presence of micromolar endogenous H
However, the piperazinyl- and piperidyl-based NBD amines
could react efficiently with micromolar H S under physiological
2
S.
2
conditions. The NBD(S) amines should be good alternatives for
the piperazinyl- and piperidyl-based amines, because
compounds
2
2 and 3b are stable toward micromolar H S and
Figure 1. The emission spectra (excitation = 470 nm) of probes
biothiols. We hope that this work could help scientists to
choose a suitable linker for labelling with NBD in biological
studies.
1
-4 (1 µM) before (black line) and after (red line) reaction with
H
2
S (100 µM) for 30 min in PBS buffer (pH 7.4).
2
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Figure 3. (a) The emission spectra (excitation = 470 nm) of
µM) before (black line) and after (red line) reaction with H
100 µM) for 30 min in SDS-containing PBS buffer (pH 7.4,
containing 40 mM SDS). (b) Time-dependent emission intensity
9 (1
2
S
(
at 550 nm of
buffer.
2
9 (1 µM) upon reaction with H S (5 mM) in PBS
We also checked the UV-Vis absorbance spectrum of NBD
probes upon treatment with H S. As shown in Figures 4, S6 and
S7, all probe solutions exhibited noticeable absorbance at 460-
10 nm, due to the NBD absorbance. After treatment with H S,
all probe solutions except exhibited time-dependent increase
2
5
2
7
absorbance at 530-550 nm with various rates, which could be
attributed to the produce of NBD-SH. Pluth group firstly
indentified the production of coloric NBD-SH from thiolysis of
3
0
NBD probes. For probe 1, the isosbestic point was at 510 nm,
Figure 2. Kinetic studies for thiolysis of NBD amines. (a) Time-
indicating a clear transformation. We also observed the
concentration-dependent absorbance spectrum changes
through treatment of the probe solution with different
dependent fluorescence intensity at 560 nm (excitation = 470
nm) of probe
concentrations of H
relationship of kobs versus H
kinetics constant k for (b) or 3a (c) or
1
(1 µM) upon reaction with different
S in PBS buffer (pH 7.4). The linear
S concentrations gives reaction
(d) and H S.
2
concentrations H S in PBS buffer (data not shown). The
2
2
reaction kinetics (Figure S7) based on absorbance spectra were
2
1
5
2
consistent with that from fluorescent tests (Table 1), namely, 1,
3
6
a
and
8 are fast-response toward micromolar H S, while 2, 4-
2
are slow-response toward even millimolar H S. NBD probes
2
-
1
-1 -1
2
k (M s )
λ
max (nm) Em. (nm)
k
obs (s ) ([H
2
S])
are also water-soluble based on absorbance spectra (Figure S8).
-
3
1
2
3
3
4
5
6
7
8
9
492
471
505
498
480
476
496
475
480
490
550
466
565
562
550
551
554
558
550
552
4.4*10 (75 μM)
43.26
ND
-
4
3.8*10 (1 mM)
-
4
a
b
2.6*10 (250 μM) 1.10
ND
2.8*10 (7 mM)
ND
ND
0.05
ND
ND
-
4
-
4
3.2*10 (3 mM)
-
4
1.3*10 (7 mM)
ND
-
3
2.0*10 (200 μM) 21.77
ND ND
Figure 4. The absorbance spectra of probes
the presence of 100 µM (for , a) or 2 mM H
buffer (50 mM, pH 7.4, containing 20% DMSO).
1
or 2 (10 µM) in
1
2
S (for 2, b) in PBS
Table 1. Spectra characterizations and kinetic data of 1-9. ND,
not determine.
Considering that NBD probes have been widely used in
member biology due to the environmental sensitivity of this
Inspiring with the above results, we further checked the
color change of NBD probes for directly visualizing different
fluorophore, we also prepared a lipid-containing NBD dye
thiolysis studies. As shown in Figure 3, the fluorescence change
of was much small in the presence of 100 μM H S. Time-
dependent emission of in millimolar H S (5 mM) only
triggered slight decrease. Therefore, lipid-containing NBD
9 for
concentrations of H
probes upon H S activation is compound
light brown to red through reaction with H
further used for visualization of various concentrations of
S (10-100 μM) at room temperature. The results indicated
that low concentration of H S (10 μM) have triggered obvious
color change even for 5 min reaction; and 1 h incubation led to
clearer color change for low micromolar H S (Figure 5a).
Therefore, we can clearly see physiological level of micromolar
S based on . Moreover, could be further employed for
paper-based detection of H S (Figure 5b). To our delight, the
labelling paper could response with H S over 10 to 1000 µM
range. The unassisted visual detection of low concentration of
S based on probe and paper-based method is an appealing
2
S. The obviously colorimetric change of
, which turned from
S (Figure 5). We
2
8
9
2
9
2
2
8
H
2
2
probes should be stable in the presence of endogenous H S in
vivo and could be used for biological studies in vivo.
2
2
H
2
8
8
2
8-
2
H
2
8
method. This no instrumentation requirement provides access
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to simple detection methods for applications in which
instrumentation or laboratory costs are unavailable.
Considering the facile synthesis of
8
and its colorimetric
response with low concentration H
has various future applications.
2
S, we believe the probe 8
Figure 6. Concentration-dependent fluorescence spectra of
probe 1 (1 µM) upon reaction with H S (5-40 µM) for 20 min in
2
PBS buffer (pH 7.4). Inset: emission intensity at 560 nm versus
H2S concentrations.
Figure 5. NBD probe
detection of H S. (a) Photographs of probe
presence of different concentrations of H S for 5 min or 1 h. (b)
Paper-based tests of probe (1 mM) in the presence of
different concentrations of H S for 30 min.
8
for colorimetric and paper-based
2
8
(10 µM) in the
2
8
2
Figure 7. Fluorescence responses at 560 nm of the probe
µM, a) or (1 µM, b) to various biologically relevant species in
PBS (pH 7.4, containing 1% DMSO) for 30 min. Lanes 1, 3, 5, 7,
1 (1
8
Because
further evaluated its usage as H
checked the fluorescence signal change of probe
various concentrations of H
1
had the fastest response rate toward H
2
S, we
with
2
S fluorescence probe. We
9
, probe or probe to 1 mM Cys, 1 mM GSH, 1 mM Hcy, or 100
2
1
-
3
µM SO , respectively. Lanes 2, 4, 6, 8, 10, probe to 100 µM
2
S (Figure 6). As expected,
2
-
2 2 3
H S or to both H S and Cys, GSH, Hcy, or SO , respectively.
fluorescence decrease at 560 nm could be detected when the
reaction mixture was excited at 470 nm. Higher concentration
of H
the range of H
determined to be 0.57 µM. These demonstrated that probe
could be used to fluorescent detection of low micromolar H S.
2
S induced weaker fluorescence of the reaction solution in
To test the biological applicability of probe
whether it can be used to detect exogenous H
HeLa cells were treated with probe and then washed with
PBS to remove excess . The -loaded cells were incubated
with and subsequently imaged using confocal
fluorescence microscopy (Figure 8). The addition of both probe
and H S resulted in nearly no fluorescence while the cells
treated with only probe showed obvious yellow fluorescence.
These preliminary studies suggested that NBD probe could be
used for visualization of H S in cells selectively.
1
, we examined
2
S (5-40 µM). The detection limit was
S in living cells.
2
1
1
2
1
1
To investigate the selectivity of NBD probes, various reactive
sulfur species were incubated with probe in PBS buffer and
their fluorescence responses were tested (Figures 7 and S9).
Small-molecules thiols such as glutathione (GSH) and cysteine
H
2
S
a
1
2
1
(
Cys) at 1 mM triggered no obvious fluorescence change for
1
2
-
probes
1
and
8
. SO
3
showed a certain fluorescence decrease,
S. While for probes and , no
obvious fluorescence decrease was observed for the tested
species. The pK of H
S is around 6.9, while the typical free Conclusions
thiols have higher pK values about 8.5. Thus, it should be
possible to selectively detect H S over biological thiols based
on the thiolysis of NBD-based amines. Our studies further
2
which is far below that for H
2
4
5
a
2
a
In summary, ten NBD amines were synthesized and
characterized for study of thiolysis reaction in PBS buffer. The
NBD-based piperazinyl- and piperidyl-probes could react
2
indicated piperazinyl-NBD derivatives are selective toward H
over biothiols.
2
S
efficiently with micromolar H S in buffer, while the NBD(S)
2
4
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(
nitrobenzothiadiazole) probes showed much slow thiolysis Agilent 6540 UHD Accurate-Mass Q-TOFLC/MS or Varian 7.0 T
even in the presence of millimolar H S in buffer. Low reactivity FTICR-MS. The UV-visible spectra were recorded on a UV-3600
was also observed for ethylamino-, ethanolamino- and anilino- UV-VIS-NIR spectrophotometer (SHIMADZU, Japan).
based probes. The acetylpiperazinyl NBD was found to be Fluorescence study was carried out using F-280
efficient in directly visualizing H S by naked eye and paper- spectrophotometer (Tianjin Gangdong Sci Tech.,
based H S detection. The piperazinyl-NBD could be used for Development. Co., Ltd).
highly selective detection and bioimaging of H S. This study
2
2
&
2
2
General Procedure for Spectroscopic Studies
also points out that many NBD amines used in bioimaging
should be stable dyes, in consideration of the presence of only
All spectroscopic measurements were performed in
phosphate-buffered saline buffer (PBS, 50 mM, pH 7.4,
containing 20% DMSO or 1% DMSO). Compounds were
dissolved into DMSO to prepare the stock solutions with a
concentration of 10.0 or 1.0 mM. 1-1000 mM Stock solutions
of Na S in degassed (by bubbling N for 30 min) PBS buffer
2
micromolar H S in vivo.
2
2
were used as H S source. Probes were diluted in PBS buffer
2
(pH = 7.4, 50 mM) to afford the final concentration of 1-10 µM.
For the selectivity experiment, different biologically relevant
molecules (100 mM) were prepared as stock solutions in PBS
buffer. Appropriate amount of biologically relevant species
were added to separate portions of the probe solution and
mixed thoroughly. All measurements were performed in a 3 ml
corvette with 2 ml solution. The reaction mixture was shaken
uniformly before emission spectra were measured. If it is not
stated specially, the excitation wavelength is 470 nm and the
emission at 560 nm was recorded. For compound 9, 40 mM
sodium dodecyl sulfate (SDS) was added in the test buffer.
Colorimetric and Paper-Based Assay for H2S
Probe
8 (10 μM) in 1 mL PBS buffer (50 mM, pH 7.4,
containing 20% DMSO) was treated with Na2S (final
concentration 10, 20, 30, 50 or 100 μM). The resulted solution
was incubated at 25 ºC for 5 min and 1 h and photos of the
solution were recorded. Filter papers (7 mm*7 mm) were
Figure 8. Confocal microscopy images of exogenous H
living cells using probe . HeLa cells were incubated with (a)
(5 µM) for 30 min, washed by PBS
S (100 µM) for 30 min. The merge images
2
S in
1
1
(
5 µM) for 30 min, (b) 1
immersed in probe
8 solution (1 mM, CH CN:DMSO = 9:1) for
3
buffer, and then Na
2
1
0 min and then dried on glass plate for 2 h. The dye-loaded
between fluorescent and bright-field images are right. Scale
bar, 50 µm.
papers were soaked into various concentrations of H2S
solution (10 μM to 1 mM) and took out immediately for
reaction at 25 ºC in a sealed container. After 30 min
incubation, the photos of papers were recorded.
Experimental
Cell Culture and Bioimaging
Materials and Methods
HeLa cells were cultured at 37 ºC, 5% CO in DMEM/HIGH
2
All chemicals and solvents used for synthesis were
purchased from commercial suppliers and applied directly in
the experiments without further purification. The progress of
the reaction was monitored by TLC on pre-coated silica plates
GLUCOSE (GIBICO) supplemented with 10% fetal bovine serum
(FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, and 4 mM
L-glutamine. The cells were maintained in exponential growth,
and then seeded in glass-bottom 35 mm plate at the density
about 2 × 104/well. Cells were passaged every 2-3 days and
used between passages 3 and 10. Cells were imaged on a
confocal microscope (Olympus FV1000 UPLSAPO40X) with a 40
(
Merck 60F-254, 250 µm in thickness), and spots were
visualized by basic KMnO , UV light or iodine. Merck silica gel
(100-200 mesh) was used for general column
4
60
1
13
chromatography purification. H NMR and C NMR spectra
were recorded on a Bruker 400 spectrometer. Chemical shifts
are reported in parts per million relative to internal standard
×
objective lens. All images were analyzed with Olympus
FV1000-ASW. Emission was collected at green channel (500-
00 nm) with 488 nm excitation.
Synthesis
-Nitro-7-(piperazin-1-yl)benzo[c][1,2,5]oxadiazole (1).
multiplicity is indicated as the following: s (singlet), d mixture of mono-tBoc-piperazine (223 mg, 1.2 mmol) and
doublet), t (triplet), dd (doublet of doublets), m (multiple). NBD-Cl (200 mg, 1.0 mmol) was dissolved in 20.0 mL
High-resolution mass spectra (HRMS) were obtained on a dichloromethane. N,N-Diisopropylethylamine (DIPEA, 0.25 mL,
6
tetramethylsilane (Si(CH
peaks (CDCl = 7.26 ppm; DMSO-d
coupling constants (J) are reported in Hertz (Hz), and
3 4
) = 0.00 ppm) or residual solvent
1
3
6
= 2.5 ppm). H NMR
4
A
(
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.5 mmol) was added to the reaction mixture. After stirring Diisopropylethylamine (DIPEA, 50
Journal Name
3
3
1
µ
L, 0.3 mmol) was added to
at room temperature for 4 h, the resulting solution was the reaction mixture. After stirring at room temperature for 4
evaporated by distillation under reduced pressure. The residue h, the resulting solution was evaporated by distillation under
was purified by flash column chromatography eluting with reduced pressure. The residue was purified by flash column
methanol/dichloromethane = 1.5/100 to give the protected chromatography
product. Then the product was dissolved in 10.0 mL ether/dichloromethane = 1/1 to give a red solid product 3b
eluting
with
petroleum
1
dichloromethane, 10.0 mL CF COOH was added. After stirring (59.7 mg, 68%). H NMR (400 MHz, CDCl ) δ 8.60-8.50 (m, 1 H),
3
3
at room temperature for 3 h, the resulting solution was 6.61-6.55 (m, 1 H), 4.05 (d, J = 5.6 Hz, 4 H), 1.88-1.76 (m, 6 H);
1
3
evaporated by distillation under reduced pressure to give a red
solid product (188 mg, 75%). 0.7 105.3, 51.3, 26.2, 24.5; HRMS (ESI): m/z [M+H] calcd. for
methanol/dichloromethane = 1/10); H NMR (400 MHz, C H N O S: 265.0754, found: 265.0752.
C NMR (100 MHz, CDCl ) δ 149.9, 148.7, 147.6, 132.4, 128.8,
3
+
1
R
f
=
1
(
1
1 13 4 2
DMSO-d
6
) δ 9.29 (brs, 1 H), 8.56 (d, J = 9.2 Hz, 1 H), 6.75 (d, J = 4-Nitro-7-(ethylamino)benzo[c][1,2,5]oxadiazole
(4).
mixture of ethylamine (0.38 mL, 2 M THF solution, 0.75 mmol)
) δ 144.9, 144.7, 144.5, 136.2, 122.6, and NBD-Cl (100 mg, 0.5 mmol) was dissolved in 10.0 mL dry
A
1
3
9
.2 Hz, 1 H), 4.30 (t, J = 5.2 Hz, 4 H), 3.39 (t, J = 5.2 Hz, 4 H);
NMR (100 MHz, DMSO-d
04.5, 46.1, 42.2; HRMS (ESI): m/z [M+H] calcd. for THF. N,N-Diisopropylethylamine (DIPEA, 0.25 mL, 1.5 mmol)
C
6
+
1
C
10
H
12
N
5
O
3
: 250.0935, found: 250.0936.
was added to the reaction mixture. After stirring at room
temperature for 1 h, the resulting solution was evaporated by
4-Nitro-7-(piperazin-1-yl)benzo[c][1,2,5]thiadiazole (2).
A
mixture of mono-tBoc-piperazine (115 mg, 0.62 mmol) and distillation under reduced pressure. The residue was purified
NBD(S)-Cl (110 mg, 0.5 mmol) was dissolved in 20.0 mL by flash column chromatography eluting with petroleum
dichloromethane. N,N-Diisopropylethylamine (DIPEA, 0.13 mL, ether/dichloromethane = 1/1 to give a red solid product
4 (70
0
.75 mmol) was added to the reaction mixture. After stirring at mg, 67%). R = 0.6 (petroleum ether/dichloromethane = 1/2);
f
1
room temperature for 2 h, the resulting solution was
H NMR (400 MHz, CDCl ) δ 8.50 (d, J = 8.4 Hz, 1 H), 6.17 (d, J =
3
1
3
evaporated by distillation under reduced pressure. The residue 8.4 Hz, 1 H), 3.59-3.53 (m, 2 H), 1.47 (t, J = 7.2 Hz, 3 H);
C
was purified by flash column chromatography eluting with NMR (100 MHz, DMSO-d ) δ 144.7 144.2, 143.9, 137.7, 120.4,
6
+
methanol/dichloromethane = 2/100 to give the protected 98.7, 64.9, 13.1; HRMS (ESI): m/z [M+H] calcd. for C H N O :
8
9
4
3
product. Then the product was dissolved in 15.0 mL 209.0669, found: 209.0647.
dichloromethane, 15.0 mL CF COOH was added. After stirring 4-Nitro-7-(2-ethanolamino)benzo[c][1,2,5]oxadiazole (5).
3
A
at room temperature for 2 h, the resulting solution was mixture of 2-ethanolamine (61 mg, 1.0 mmol) and NBD-Cl (300
evaporated by distillation under reduced pressure to give a red mg, 1.5 mmol) was dissolved in 10.0 mL dry THF. N,N-
solid product
2
(106
mg,
80%).
1
R
f
=
0.7 Diisopropylethylamine (DIPEA, 0.5 mL, 3.0 mmol) was added to
(methanol/dichloromethane = 1/10); H NMR (400 MHz, the reaction mixture. After stirring at room temperature for 1
6
DMSO-d ) δ 8.58 (d, J = 8.8 Hz, 1 H), 6.93 (d, J = 8.8 Hz, 1 H), h, the resulting solution was evaporated by distillation under
1
3
4
.20-4.10 (m, 4 H), 3.23-3.16 (m, 4 H), 1.23 (brs, 1 H); C NMR reduced pressure. The residue was purified by flash column
100 MHz, DMSO-d ) δ 148.7, 147.6, 146.9, 131.8, 128.6, chromatography eluting with petroleum ether/ethyl acetate =
06.4, 47.8, 43.7; HRMS (ESI): m/z [M+H] calcd. for 1/1 to give a red solid product
C H N O S: 266.0712, found: 266.0703.
(petroleum ether/ethyl acetate = 1/1); H NMR (400 MHz,
-Nitro-7-(piperidyl)benzo[c][1,2,5]oxadiazole (3a). A mixture DMSO-d ) δ 9.41 (brs, 1 H), 8.51 (d, J = 8.8 Hz, 1 H), 6.46 (d, J =
(
6
+
1
f
5 (110 mg, 49%). R = 0.3
1
1
0
12
5
2
4
6
of piperidine (43 mg, 0.5 mmol) and NBD-Cl (150 mg, 0.75 8.8 Hz, 1 H), 4.94 (brs, 1 H), 3.71-3.67 (dd, J = 5.6 Hz, 11.2 Hz, 2
3
1
mmol) was dissolved in 10.0 mL dry THF. N,N- H), 3.62-3.48 (m, 2 H); C NMR (100 MHz, DMSO-d ) δ 145.5,
6
Diisopropylethylamine (DIPEA, 0.25 mL, 1.5 mmol) was added 144.3, 143.9, 137.8, 120.4, 99.3, 58.8, 46.0; HRMS (ESI): m/z
+
to the reaction mixture. After stirring at room temperature for [M+H] calcd. for C H N O : 225.0624, found: 225.0620.
8
9 4 4
1
h, the resulting solution was evaporated by distillation under 4-Nitro-7-(diethanolamino)benzo[c][1,2,5]oxadiazole (6).
reduced pressure. The residue was purified by flash column mixture of diethanolamine (53 mg, 0.5 mmol) and NBD-Cl (150
chromatography eluting with petroleum mg, 0.75 mmol) was dissolved in 10.0 mL dry DMF. N,N-
ether/dichloromethane = 1/2 to give a red solid product 3a Diisopropylethylamine (DIPEA, 0.25 mL, 1.5 mmol) was added
110 mg, 88%). R = 0.3 (petroleum ether/dichloromethane = to the reaction mixture. After stirring at room temperature for
/2); H NMR (400 MHz, CDCl ) δ 8.41 (d, J = 8.8 Hz, 1 H), 6.27 1 h, the resulting solution was evaporated by distillation under
d, J = 8.8 Hz, 1 H), 4.14-4.07 (m, 4 H), 1.86-1.80 (m, 6 H);
A
(
f
1
1
3
1
3
(
C
reduced pressure. The residue was purified by flash column
NMR (100 MHz, DMSO-d
6
) δ 145.1, 144.9, 144.6, 136.3, 120.0, chromatography eluting with ethyl acetate to give a red solid
+
1
1
03.0, 50.9, 25.8, 23.4; HRMS (ESI): m/z [M+H] calcd. for product
: 249.0988, found: 249.0979.
MHz, DMSO-d ) δ 8.45 (d, J = 9.2 Hz, 1 H), 6.55 (d, J = 9.2 Hz, 1
-Nitro-7-(piperidyl)benzo[c][1,2,5]thiadiazole (3b). A mixture H), 5.00 (brs, 2 H), 4.40-4.00 (m, 4 H), 3.76 (t, J = 5.6 Hz, 4H);
6 (120 mg, 89%). R = 0.4 (ethyl acetate); H NMR (400
f
11 13 4 3
C H N O
6
4
1
3
of piperidine (27 mg, 0.3 mmol) and NBD(S)-Cl (60 mg, 0.28
C NMR (100 MHz, DMSO-d ) δ 146.0, 144.7, 143.4, 136.0,
6
mmol) was dissolved in 15.0 mL dry CH
2
Cl
2
.
N,N-
6
| J. Name., 2012, 00, 1-3
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Organic & B molecula hemistry
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DOI: 10.1039/C6OB02354A
Journal Name ARTICLE
+
1
19.7, 102.6, 58.2, 56.3; HRMS (ESI): m/z [M+H] calcd. for
: 269.0886, found: 269.0881.
-Nitro-7-(anilino)benzo[c][1,2,5]oxadiazole (7). A mixture of
Notes and references
10 13 4 5
C H N O
1
2
C. Szabó, Nat. Rev. Drug Discov., 2007, 6, 917.
L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol.,
2011, 51, 169.
4
aniline (93 mg, 1.0 mmol) and NBD-Cl (300 mg, 1.5 mmol) was
dissolved in 10.0 mL dry THF. N,N-Diisopropylethylamine
3
4
B. L. Predmore, D. J. Lefer and G. Gojon, Antioxid. Redox
Signaling, 2012, 17, 119.
G. K. Kolluru, X. G. Shen, S. C. Bir and C. G. Kevil, Nitric Oxide,
(DIPEA, 0.5 mL, 3.0 mmol) was added to the reaction mixture.
After stirring at room temperature for 1 h, the resulting
solution was evaporated by distillation under reduced
pressure. The residue was purified by flash column
chromatography eluting with petroleum ether/ethyl acetate =
2
H. Kimura, Exp. Physiol., 2011, 96, 833.
G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A. K.
Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang, Science,
013, 35, 5.
5
6
2008, 322, 587.
H. Kimura, Amino Acids, 2011, 41, 113.
6
/1 to give a red solid product
petroleum ether/ethyl acetate = 6/1); H NMR (400 MHz,
CDCl ) δ 8.46 (d, J = 8.4 Hz, 1 H), 7.74 (brs, 1 H), 7.55-7.51 (m, 2
H), 7.42-7.35 (m, 3 H), 6.73 (d, J = 8.4 Hz, 1 H); C NMR (100
MHz, DMSO-d ) δ 144.9, 144.1, 142.3, 137.7, 137.6, 129.6,
f
7 (150 mg, 58%). R = 0.4
7
8
1
(
L. Wei, Z. T. Zhu, Y. Y. Li, L. Yi and Z. Xi, Chem. Commun.,
015, 51, 10463.
2
3
9
N. Shibuya, S. Koike, M. Tanaka, M. Ishigami-Yuasa, Y.
Kimura, Y. Ogasawara, K. Fukui, N. Nagahara and H. Kimura,
Nat. Commun., 2013, 4, 1366.
1
3
6
+
1
26.4, 123.8, 123.0, 101.5; HRMS (ESI): m/z [M+H] calcd. for 10 L. Wei, L. Yi, F. B. Song, C. Wei, B. F. Wang and Z. Xi, Sci. Rep.,
2
014, 4, 4521.
C H N O : 257.0669, found: 257.0664.
12 9 4 3
1
1 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G.
Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005,
42, 539.
4-Nitro-7-(4-acetylpiperazin-1-yl)benzo[c][1,2,5]oxadiazole
(8). A mixture of compound 1 (250 mg, 1.0 mmol) and acetic
1
1
2 S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E.-H. Joe, B. R. Cho and
H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915.
3 V. S. Lin, W. Chen, M. Xian and C. J. Chang, Chem. Soc. Rev.,
anhydride (0.22 mL, 1.8 mmol) was dissolved in 20.0 mL
dichloromethane. After stirring at room temperature for 3 h,
the resulting solution was evaporated by distillation under
2
015, 44, 4596.
reduced pressure. The residue was purified by flash column 14 F. B. Yu, X. Y. Han and L. X. Chen, Chem. Commun., 2014, 50
2234.
5 Y. Qian, J. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee,
J. Zhao and C. He, Nat. Commun., 2011, , 495.
,
1
chromatography eluting with methanol/dichloromethane =
.8/100 to give a red solid product (151 mg, 52%). R = 0.2
methanol/dichloromethane = 2/100); H NMR (400 MHz,
DMSO-d ) δ 8.52 (d, J = 8.8 Hz, 1 H), 6.62 (d, J = 8.8 Hz, 1 H),
.27-4.10 (m, 4 H), 3.80-3.70 (m, 4 H), 2.06 (s, 3 H); C NMR
100 MHz, DMSO-d ) δ 168.7, 145.4, 144.7, 144.7, 136.2,
21.1, 103.1, 48.8, 44.2, 21.1; HRMS (ESI): m/z [M+H] calcd.
: 292.1046, found: 292.1047.
-Nitro-7-(didodecylamino)benzo[c][1,2,5]oxadiazole (9).
1
1
0
8
f
2
1
(
6 L. W. He, W. Y. Lin, Q. Y. Xu and H. P. Wei, Chem. Commun.,
2015, 51, 1510.
17 Y. C. Chen, C. C. Zhu, Z. H. Yang, J. J. Chen, Y. F. He, Y. Jiao, W.
6
1
3
4
J. He, L. Qiu, J. J. Cen and Z. J. Guo, Angew. Chem. Int. Ed.,
(
6
2
013, 52, 1688.
8 Y. Qian, L. Zhang, S. T. Ding, X. Deng, C. He, X. E. Zheng, H. L.
Zhu and J. Zhao, Chem. Sci., 2012, , 2920.
9 X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem.
Sci., 2013, , 2551.
+
1
1
1
2
for C12
H
14
N
5
O
4
3
4
A
4
mixture of dodecylamine (88 mg, 0.25 mmol) and NBD-Cl (75
mg, 0.375 mmol) was dissolved in 10.0 mL dry THF. N,N-
0 A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc.,
2011, 133, 10078.
Diisopropylethylamine (DIPEA, 0.13 mL, 0.75 mmol) was added 21 H. J. Peng, Y. F. Cheng, C. F. Dai, A. L. King, B. L. Predmore, D.
J. Lefer and B. H. Wang, Angew. Chem. Int. Ed., 2011, 50
672.
,
to the reaction mixture. After stirring at room temperature for
h, the resulting solution was evaporated by distillation under
reduced pressure. The residue was purified by flash column
chromatography eluting with petroleum
ether/dichloromethane = 3/1 to give a red solid product (96
mg, 74%). R = 0.3 (petroleum ether/dichloromethane = 3/1);
9
1
2
2
2
2 Q. Q. Wan, Y. C. Song, Z. Li, X. H. Gao and H. M. Ma, Chem.
Commun., 2013, 49, 502.
3 L. Zhang, S. Li, M. Hong, Y. Xu, S. Wang, Y. Liu, Y. Qian and J.
Zhao, Org. Biomol. Chem., 2014, 12, 5115.
4 L. Zhang, W. Q. Meng, L. Lu, Y. S. Xue, C. Li, F. Zou, Y. Liu and
9
f
J. Zhao, Sci. Rep., 2014,
H NMR (400 MHz, CDCl ) δ 8.43 (d, J = 9.2 Hz, 1 H), 6.07 (d, J = 25 H. A. Henthorn and M. D. Pluth, J. Am. Chem. Soc., 2015, 137
4, 5870.
1
3
,
1
5330.
9
.2 Hz, 1 H), 4.00-3.70 (m, 4 H), 1.79-1.70 (m, 4 H), 1.38-1.27
1
m, 36 H), 0.88 (t, J = 6.8 Hz, 6 H); C NMR (100 MHz, DMSO-
3
26 K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T.
Komatsu, T. Ueno, T. Terai, H. Kimura and T. Nagano, J. Am.
Chem. Soc., 2011, 133, 18003.
27 X. W. Cao, W. Y. Lin, K. B. Zheng and L. W. He, Chem.
Commun., 2012, 48, 10529.
8 Z. J. Huang, S. S. Ding, D. H. Yu, F. H. Huang and G. Q. Feng,
Chem. Commun., 2014, 50, 9185.
9 C. Wei, L. Wei, Z. Xi and L. Yi, Tetrahedron Lett., 2013, 54
6937.
0 L. A. Montoya, T. F. Pearce, R. J. Hansen, L. N. Zakharov and
M. D. Pluth, J. Org. Chem., 2013, 78, 6550.
1 C. Y. Zhang, L. Wei, J. Zhang, R. Y. Wang, Z. Xi and L. Yi. Chem.
Commun., 2015, 51, 7505.
(
d6) δ 144.6, 144.6, 144.0, 135.6, 119.8, 101.6, 64.7, 31.1, 28.8,
+
28.8, 28.8, 28.7, 28.5, 21.9, 13.7; HRMS (ESI): m/z [M+H]
53 4 3
calcd. for C30H N O : 517.4118, found: 517.4104.
2
2
3
3
,
Acknowledgements
This work was supported by NSFC (81371711, 21402007,
21332004) and the Doctor Foundation of Tianjin Normal
University (52XB1210).
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ARTICLE
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| J. Name., 2012, 00, 1-3
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