Sydnone Reporters for Highly Fluorogenic Copper-Free Click Ligations

Article abstract of DOI:10.1021/acs.joc.7b03004

Bioorthogonal fluorescent turn-on reactions are attractive for the sensitive real-time detection of a variety of phenomena including bioconjugation, chemical reactivity, and material assembly. Herein we describe the use of 3,4-disubstituted sydnones, a singular class of mesoionic dipoles, for highly fluorescent turn-on copper-free click cycloadditions with the fluorogenic dibenzocyclooctyne Fl-DIBO. Coherent with time-dependent density functional theory calculations, the pyrazole cycloadducts were found to be highly fluorescent with compelling photophysical properties including excellent fluorescence enhancement (up to 240-fold), high quantum yields (over 45%), and large Stokes shift (over 100 nm). Furthermore, the good stability and reactivity of 4-chlorosydnones with Fl-DIBO allowed us to employ them as chemical reporters for the challenging detection of modified-proteins in complex cellular extracts, with exquisite specificity in no-wash conditions. This novel fluorogenic system significantly expands our chemical biology toolbox and should be beneficial in countless applications.

Full text of DOI:10.1021/acs.joc.7b03004

Article
pubs.acs.org/joc
Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX
Sydnone Reporters for Highly Fluorogenic Copper-Free Click
Ligations
Camille Favre,†,‡ Lucie de Cremoux,†,‡ Jerome Badaut,‡,§ and Frederic Friscourt*,†,‡
́
́
†Institut Europee
́
n de Chimie et Biologie, Universite
́
de Bordeaux, 2 rue Robert Escarpit, 33607 Pessac, France
‡Institut de Neurosciences Cognitives et Integ
́
ratives d’Aquitaine, CNRS UMR5287, Bordeaux, France
§Basic Science Department, Loma Linda University, Loma Linda, California 92350, United States
S
* Supporting Information
ABSTRACT: Bioorthogonal fluorescent turn-on reactions are attractive for the sensitive real-time detection of a variety of
phenomena including bioconjugation, chemical reactivity, and material assembly. Herein we describe the use of 3,4-disubstituted
sydnones, a singular class of mesoionic dipoles, for highly fluorescent turn-on copper-free click cycloadditions with the
fluorogenic dibenzocyclooctyne Fl-DIBO. Coherent with time-dependent density functional theory calculations, the pyrazole
cycloadducts were found to be highly fluorescent with compelling photophysical properties including excellent fluorescence
enhancement (up to 240-fold), high quantum yields (over 45%), and large Stokes shift (over 100 nm). Furthermore, the good
stability and reactivity of 4-chlorosydnones with Fl-DIBO allowed us to employ them as chemical reporters for the challenging
detection of modified-proteins in complex cellular extracts, with exquisite specificity in no-wash conditions. This novel
fluorogenic system significantly expands our chemical biology toolbox and should be beneficial in countless applications.
demand Diels−Alder reaction,10−13 and the strain-promoted
INTRODUCTION
■
alkyne−azide cycloaddition (SPAAC). Although numerous
fluorogenic azido-probes have been developed, notably for
copper-click chemistry,14,15 their utilization under metal-free
conditions is consequently limited by the necessity of
employing relatively bulky cyclooctynes as chemical reporters,16
that may not be well tolerated by the cell’s metabolic
machinery.
The recent advances in fluorescence microscopy, in coordina-
tion with the development of fluorescent probes, have
revolutionized our understanding of cell biology and physiol-
ogy.1 Biomolecules such as proteins, lipids, and complex
glycans can now be tracked effortlessly through the
incorporation of fluorescent reporters, in a spatial and temporal
manner, within their native environment.2−4 Of particular
importance, the bioorthogonal chemical reporter strategy has
emerged as a key technology for the challenging labeling of
post-translational modifications.5−7 In this two-step approach, a
uniquely reactive chemical functionality (the reporter) is first
introduced into the biomolecule of interest and is subsequently
utilized for the chemical ligation of a diverse set of probes (e.g.,
fluorescent tags), using strictly selective bioorthogonal
reactions. While this technology can ensure the visualization
of a specific target in complex biological settings, the fact that
fluorescent labels are always “on” precludes their direct
application for real-time bioimaging.
To address this major drawback, fluorogenic cyclooctynes
have recently been synthesized that can be activated upon
SPAAC with inconspicuous azido-reporters (Figure 1A).
Inspired by the coumarin quenching ability of terminal alkyne
through the stabilization of a nonemissive excited triplet3(n,π*)
state,17 Bertozzi and co-workers have developed a fluorogenic
copper-free click reagent CoumBARAC (1) by incorporating a
cyclooctyne ring into the coumarin dye.18 Although the
formation of the triazole moiety upon SPAAC with 2-
azidoethanol led to a 10-fold fluorescence enhancement (FE),
the requirement of relatively high energy of excitation (λexc
∼
300 nm) combined with a rather low fluorescence quantum
yield (ΦF = 0.04) made the reagent ill-suited for bioimaging.
Following a similar design, the Wong group recently reported
To overcome this limitation, fluorogenic molecules, that
increase in fluorescence after chemical reactions or target-
binding, have recently been developed for maximizing signal
over background, expanding significantly our chemical biology
toolbox.8 Popular fluorogenic metal-free bioorthogonal chem-
istries include the Staudinger ligation,9 the inverse electron-
Received: November 28, 2017
© XXXX American Chemical Society
A
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Article
that the fluorogenic probe Fl-DIBO (3) can indeed be
efficiently turned on upon reaction with sydnones and that
the generated cycloadducts exhibit compelling photophysical
properties including excellent fluorescence enhancement (up to
240-fold), high quantum yields (over 45%), and large Stokes
shift (over 100 nm). Time-dependent density functional theory
(TD-DFT) calculations indicate that the particular high
fluorescence quantum yields observed for the 1,5-disubstituted
pyrazoles are due to the large oscillator strengths of their S0↔S1
transitions. Ultimately, the optimal bioorthogonal fluorogenic
system was employed in a complex biological setting for the
selective visualization of sydnone-modified proteins in cell
lysate.
RESULTS AND DISCUSSION
■
Photophysical Properties of the Cycloadducts Gen-
erated from Fl-DIBO and Modified Sydnones. Previous
studies on the fluorogenic probe Fl-DIBO (3) revealed that the
nature of the heterocycle formed after cycloaddition is key for
the cycloadduct fluorescence emission or quenching.23 To
attest whether Fl-DIBO (3) can be activated upon reactions
with sydnones and investigate the fluorescence properties of the
resulting click products, we prepared an array of 3,4-
disubstituted sydnones (Scheme 1). Briefly, 3-modified
Figure 1. Fluorogenic strain-promoted reagents. A. Turn-on cyclo-
octynes. B. Fluorescence enhancement of Fl-DIBO (3) upon
bioorthogonal reactions with various chemical reporters.
the fluorogenic probe CoumOCT (2) that exhibited improved
photophysical properties (λexc = 330 nm, ΦF = 0.23, FE = 20)
and was successfully employed for the visualization of
metabolically labeled azido-glycoconjugates in living cells.19
While turning off known fluorescent dyes with bioorthogonal
functionalities clearly simplifies the design of novel fluorogenic
probes, potential degradation or unwanted side-reactions of the
quenching moiety20,21 may potentially activate the probe
leading to unspecific fluorescence. On the other hand,
fluorogenic probes that chemically assemble the fluorophore
during the bioorthogonal conjugation step are consequently
more reliable constructs for fluorescent bioimaging. In this
context, we recently developed a fluorogenic dibenzocyclooc-
tyne (Fl-DIBO (3)), which upon SPAAC with azido-
biomolecules generates ligated products with a 60-fold
fluorescence enhancement (Figure 1B). The fluorescent
triazoles exhibited good photostability and could be excited
above 350 nm, the typical cutoff wavelength of standard
fluorescence microscopes.22 The fluorogenic behavior of Fl-
DIBO was later shown to be highly dependent on the nature of
the 1,3-dipole used. For instance, while the reaction with nitrile
oxides, nitrones, and disubstituted diazo compounds gave
cycloadducts with low quantum yield, monosubstituted diazo
reagents produced 1H-pyrazole derivatives that displayed an
improved 160-fold fluorescence enhancement.23 However, the
high reactivity of diazo compounds toward metals and their
stability being strongly dependent on the electronic character of
their substituents24,25 make their synthesis and biological
utilization greatly challenging.
Scheme 1. Chemical Synthesis of Modified Sydnones 4a−n
sydnones 4a−d, bearing aryl groups with various electron
density, were synthesized in an efficient two-step protocol by
nitrosation of N-substituted glycines 5a−d with tert-butyl nitrite
followed by trifluoroacetic anhydride-induced cyclization
(Scheme 1A). Further C-4 chemical elaboration of the
mesoionic rings could easily be achieved by either halogenation,
Heck-coupling, or lithiation reactions generating the desired
3,4-disubstituted sydnones 4e−k in good yields (Scheme
1A,B). Finally, amide coupling protocols on the benzoic acid
derivatives allowed for additional modifications leading to
sydnones 4l−n (Scheme 1C).
Next we reacted 3-phenylsydnone 4a with Fl-DIBO (3), and
to our delight, the resulting 1-phenyl-5H-pyrazole 6a was found
to be strongly fluorescent when excited at 363 nm with a
maximum emission at 468 nm and a quantum yield of 0.35
(Table 1). Motivated by this result, we investigated the
influence of the substitution pattern of modified sydnones on
the photophysical properties of the click adducts (Table 1,
Figure 2, and Supporting Information, Figures S1−S13). While
switching substituents at the N-position of the pyrazole ring
had very little effect on the excitation wavelengths of the new
Sydnones are stable mesoionic 1,3-dipoles that undergo
thermal [3 + 2] cycloadditions in the presence of various
dipolarophiles.26 For instance, 1,4-pyrazoles can be generated
under mild conditions via regioselective copper(I)-catalyzed
sydnone cycloadditions with terminal alkynes.27 Similar to
azides, sydnones can also undergo metal-free strain-promoted
cycloadditions with various cyclooctyne-reporters.28−30 There-
fore, we envisaged that sydnones, which also generate 1H-
pyrazoles upon [3 + 2] cycloadditions with alkynes, could be
viable replacements of diazo reagents for highly fluorogenic
copper-free click ligations with Fl-DIBO (3). Herein we report
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Table 1. Photophysical Properties of Pyrazoles 6a−c, 6e−h, and 6j−n in Methanol
a
b
c
d
pyrazole
Z
X
λexc [nm]
λem [nm]
ΦF
FE
Pyrazoles Obtained from 3-Modified Sydnones
6a
6b
6c
6l
Ph
H
H
H
H
363
361
361
360
468
480
460
468
0.35
0.15
0.45
0.31
175
75
MeO-Ph
F3C-Ph
225
155
i-PrNHCOPh
Pyrazoles Obtained from 3,4-Modified Sydnones
6e
6f
Ph
Cl
Br
I
354
355
359
353
361
354
352
353
451
450
455
449
456
456
452
452
0.47
0.12
0.014
0.41
0.48
0.40
0.40
235
60
Ph
6g
6h
6j
Ph
7
F3C-Ph
Cl
Ph
SMe
Cl
205
240
200
200
235
Ph
6k
6n
Ph
e
TEGNHCOPh
f
f
f
f
0.47
a
b
c
Determined at λem = 460 nm. Determined at λexc = 360 nm. Fluorescence quantum yield, quinine sulfate in aqueous 1.0 N H2SO4 as standard.
d
e
Fluorescence enhancement compared to Fl-DIBO (sydnones having negligible fluorescence emission at λexc = 360 nm, Figure S13 in SI). TEGN =
f
triethylene glycol monoamine. Determined in 40% MeOH in PBS (pH 7.4).
structures at two different positions (3,4-substitutions of the
mesoionic ring). Consequently, we prepared 1,5-substituted
pyrazoles by reacting Fl-DIBO (3) with 3,4-modified sydnones
4e−h, 4j−k, and 4n and evaluated the effect of these
modifications on their fluorescence. While introducing a
substituent on C-5 of the pyrazole moiety significantly affected
the fluorescence quantum yield of the chromophores, only a
marginal impact on their emission maxima (λexc/em ≈ 355/455
nm) was observed (Table 1). For instance, C5-modification of
the pyrazole ring with a phenyl (6j) or an S-methyl (6k)
functionality noticeably enhanced the fluorescence quantum
yield to 0.48 and 0.40, respectively. Following a similar pattern,
4-chlorosydnones 4e, 4h, and 4n also efficiently increased the
quantum yield of their respective click products 6e, 6h, and 6n,
with a remarkable 235-fold fluorescence enhancement. On the
other hand, substitution of the chlorine atom with other
halogens such as bromine or iodine suppressed the fluorescence
emission, probably through the heavy atom effect via
intersystem crossing (S1 → T1).31 Because the polarity of
solvent is known to affect the photophysical properties of
chromophores, we measured the fluorescence emission of 5-
chloropyrazole 6n in pure methanol and in 40% methanol in
phosphate-buffered saline (PBS, pH 7.4) and were pleased to
observe similar spectral identification and quantum yield in
both conditions (ΦF/MeOH = 0.40 vs ΦF/40%MeOH‑PBS = 0.47), a
valuable result for bioimaging. Altogether, fluorogenic bio-
orthogonal strain-promoted cycloadditions between 3,4-modi-
fied sydnones and Fl-DIBO generate fluorescent pyrazoles with
emissions ranging from 450 to 480 nm, large Stokes shift
(∼100 nm), and considerable high fluorescence enhancement
(up to 240-fold).
Figure 2. Absorption (solid traces; 30 μM solutions) and fluorescence
emission (dashed traces; λexc = 360 nm; OD360 = 0.15) spectra of
pyrazoles 6a, 6c, and 6e in MeOH. Inset: Visual comparison of the
fluorescence intensity of cycloadducts in MeOH with excitation at 365
nm.
fluorophores, both the emission maxima and fluorescence
quantum yields were clearly impacted by the electron density of
the modifications. For example, pyrazole 6b, having an
electron-donating group, found its fluorescence emission to
be red-shifted (λem = 480 nm), accompanied by a decrease in
quantum yield (ΦF = 0.15). Conversely, pyrazole 6c, containing
an electron-withdrawing substituent, exhibited a clear hyp-
sochromic shift (λem = 460 nm) with an exciting increase in
fluorescence quantum yield (ΦF = 0.45). In general, 3-modified
sydnones, having substituents of moderate electron density,
generate pyrazoles (6a and 6l) with similar spectral identity
(λexc/em ≈ 360/470 nm) and good fluorescence quantum yield
(ΦF = 0.30).
Time-Dependent Density Functional Theory Calcu-
lations. To elucidate the observed influence of the C5-
substitution of the pyrazole framework on their fluorescence
quantum yield and emission wavelength, we decided to
One of the main advantages of sydnones, compared to other
1,3-dipoles such as azides, is the possibility to modulate their
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investigate the electronic properties of three representative
model pyrazoles: 1-Me-5-H-pyrazole 6o, 1-Me-5-Me-pyrazole
6p, and 1-Me-5-Cl-pyrazole 6q, by TD-DFT quantum chemical
calculations at the B3LYP/6-31+G(d) level (Figure 3). We
High reactivity and biological stability are critical features of
bioorthogonal reagents. Therefore, we examined the effect of
sydnone substitutions on their reaction kinetics with Fl-DIBO
(3) (Figure 4). The second-order rate constants of cyclo-
Figure 4. Kinetics data of cycloaddition reactions between Fl-DIBO
(3) (6.6 mM in a mixture of CDCl3 and MeOD (4:1) at 25 °C) in the
presence of various sydnones (4a, 4c, 4e, 4g, and 4h (6.6 mM)). The
lines shown were drawn using parameters obtained by linear fitting
with k representing the second-order rate constant of the reaction.
addition between Fl-DIBO (3) and sydnones 4a, 4c, 4e−h, and
4j−k were determined by monitoring product formation by 1H
NMR spectroscopy in a mixture of CDCl3 and CD3OD
(Supporting Information, Figures S14−S17). While 3-phenyl
sydnone 4a reacted with Fl-DIBO (3) at a rate of 6.0 × 10−4
M−1 s−1, adding an electron-withdrawing substituent was clearly
beneficial with p-CF3-phenyl sydnone 4c having a second-order
rate constant of 1.8 × 10−3 M−1 s−1. Regarding 3,4-disubstituted
sydnones, introduction of bulky substituents, such as phenyl, S-
methyl, and iodine, on C-4 of the mesoionic ring drastically
decreased their reactivity with kinetics below 4.0 × 10−4 M−1
s−1, probably due to steric hindrance. On the other hand, 4-
substitution of the sydnone scaffold with the smaller chlorine
atom significantly enhanced the 1,3-dipole reactivity with
second-order rate constants up to 4.7 × 10−3 M−1 s−1, a rate
twice as fast as the previously reported reaction between Fl-
DIBO and ethyl diazoacetate23 and 30-fold faster than the
Staudinger ligation, a bioconjugation reaction often used for the
in vitro and in vivo labeling of biological macromolecules.32
Next we investigated the stability of sydnones in aqueous
solution. 4-Chloro-3-benzoic acid sydnone 4i was incubated in
a mixture of DMSO-d6 and deuterated water (1:1), and its
Figure 3. A. Energy diagrams and contour plots of the HOMO and
LUMO of pyrazoles 6o−q with their transitions oscillator strength
values (f). B. Chemical structures of pyrazoles 6o−q.
have previously shown that the absorption and emission data of
pyrazole structures, generated from Fl-DIBO, were well
predicted with this method when the long-range corrected
hybrid functional CAM-B3LYP and a polarized continuum
model (PCM) for solvent were used.22 As depicted in Figure 3,
the calculated first excited singlet state S1 of all pyrazoles (6o−
q) correspond to a HOMO−LUMO transition having a π,π*
character, with coefficients mainly located on the dibenzocy-
clooctene moiety and only to a small extent on the pyrazole
ring, explaining for the overall very similar experimental
absorption and emission profiles of these molecules. Never-
theless, introduction of either a methyl group or a chlorine
atom on C5 of the pyrazole ring generated a slight but
consistent increase in energy values of their S0↔S1 transitions
with the chloro-substitution having the most pronounced
influence (over 0.1 eV difference), reflecting well the
experimental hypsochromic shift observed for the chloropyr-
azole 6e, 6h, and 6n absorption and emission wavelengths
(Table 1). More remarkably, the calculated emission oscillator
strength (f), which is a good representation of the radiative rate
constant of chromophores,31 significantly increased with the
C5-substitution pattern, going from 0.24 for 1-Me-5-H pyrazole
6o to 0.33 for 1-Me-5-Me pyrazole 4p and 0.43 for 1-Me-5-Cl
pyrazole 4q (Figure 3). Taken together, these TD-DFT
computed results suggest that the significantly high fluores-
cence quantum yields observed for the 1,5-disubstituted
pyrazoles 6e, 6h−k, and 6n is due to the large oscillator
strengths of their S0↔S1 transitions compared to their
monosubstituted analogues 6a and 6l.
1
stability was monitored by H NMR spectroscopy over time
(Supporting Information, Figure S18). The 4-chlorosydnone
was found highly stable, as no decomposition was observed for
over 25 h. Overall, 4-chlorosydnones are stable 1,3-dipoles in
aqueous solution that react fast with the Fl-DIBO probe to give
highly fluorescent pyrazoles.
Finally, to evaluate the performance of 4-chlorosydnones as
reporters for the fluorogenic labeling of biomolecules, we
functionalized the exposed lysines of bovine serum albumin
(BSA), as a model protein, with the mesoionic dipoles.
Accordingly, NHS-activated benzoic ester chlorosydnone 4m
was reacted with BSA in PBS (pH 7.4) (Figure 5A). The
modified protein (BSA-PhSydCl) was then incubated with Fl-
DIBO (3) in PBS (containing 4% of DMF) at 37 °C and
visualized by in-gel fluorescence imaging. As expected, while no
Kinetic and Stability Studies of Sydnones and Their
Application for the Fluorogenic Labeling of Proteins.
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making them suitable for biochemical applications. Accordingly,
these new reporters were appended to protein surface and the
modified protein could be detected by Fl-DIBO in complex
cellular extracts with exquisite specificity via in-gel fluorescence
imaging. Interestingly, Taran and co-workers recently reported
the synthesis of 4-fluorosydnones that was found to react
exceptionally fast toward alkynes,33 highlighting the promising
future of this new class of reagents. However, the challenging
isolation of the fluorosydnones preclude their current use as
bioorthogonal chemical reporters. While highly fluorogenic
reactions between sydnone reporters and Fl-DIBO clearly offer
advantages in cell biology, where background labeling is often
an issue, we look forward to using this exciting system for other
applications including the screening of chemical reactivity, the
sensitive detection of material assembly, and the sensing of
various analytes.
EXPERIMENTAL SECTION
■
Materials and Methods. All reactions were carried out under a
dry argon environment. All solvents were of reagent grade and used as
received. Dichloromethane and tetrahydrofuran were dried over
alumina columns under nitrogen through a solvent purification
system. Chemicals and reagents were used as commercially supplied
without any further purification unless otherwise stated. Room
temperature refers to ambient temperature (20−22 °C). Column
chromatography was carried out using silica gel G60 (Fluka analytical,
230−400 mesh, 40−63 μm particle size, 60 Å) as the stationary phase.
1H, 13C, and 19F NMR spectra were recorded on a Bruker 300 MHz
̈
Figure 5. Fluorogenic labeling of chlorosydnone-modifed BSA
conjugate (BSA-PhSydCl) with Fl-DIBO (3). A. Incorporation of 4-
chloro-3-arylsydnones 4m onto BSA using NHS-chemistry and
subsequent reaction with Fl-DIBO in PBS at 37 °C. B. In-gel
visualization of BSA-PhSydCl or native BSA incubated with Fl-DIBO
(25−500 μM) or no reagent (−) for 3 h. C. In-gel visualization of
BSA-PhSydCl or native BSA incubated with Fl-DIBO (100 μM) or no
reagent (−) for 60−180 min. D. In-gel visualization of BSA-PhSydCl
or native BSA incubated with Fl-DIBO (100 μM) or no reagent (−)
for 3 h in U2OS cell extracts.
spectrometer. Chemical shifts are reported in δ units, parts per million
(ppm) downfield from TMS. Coupling constants (J) are reported in
hertz (Hz) without adjustments. Splitting patterns are designed as
follows: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; dt,
doublet of triplets; td, triplet of doublets; ddd, doublet of doublet of
doublets; tt, triplet of triplets; m, multiplet; br, broad. All NMR signals
were assigned on the basis of 1H NMR, COSY, HSQC, and 13C
experiments. High-resolution mass spectra were obtained with an
electrospray ionization Thermo Exactive orbitrap mass spectrometer.
General Procedure for the Formation of 3-Modified
Sydnones 4a−d. tert-Butyl nitrite (0.27 mL, 2.2 mmol) was added
dropwise to a solution of N-aryl glycine (2 mmol) in THF (5 mL).
The reaction mixture was stirred at room temperature for 30 min.
Trifluoroacetic anhydride (0.31 mL, 2.2 mmol) was then added to the
previous solution, and the reaction mixture was stirred for an
additional 1 h. The mixture was then quenched with a saturated
aqueous solution of NaHCO3 (10 mL). The organic layer was
extracted with ethyl acetate (3 × 15 mL), dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (20 g) using a mixture of 1% of
methanol in dichloromethane to afford the respective pure 3-
substituted sydnone 4a−d.
labeling was detected when native BSA was used, BSA
displaying chlorosydnone moieties showed a clear dose- and
time-dependent increase in fluorescence intensity (Figure
5B,C). Optimal fluorescent labeling was achieved at a
concentration of 100 μM of Fl-DIBO after 3 h of incubation.
Cyclooctynes have recently been reported to react with
biological entities such as thiols20 and sulfenic acids,21 which
could potentially generate background labeling. To challenge
the signal specificity of our fluorogenic system in a more
stringent biological environment, we performed the turn-on
protein ligation in cell lysate. Human bone osteosarcoma
(U2OS) cell lysate was spiked with 10% of BSA-PhSydCl, or
regular BSA for control, and incubated for 3 h at 37 °C in the
presence of Fl-DIBO (Figure 5D). Gratifyingly, only BSA-
PhSydCl conjugate displayed a robust fluorescent signal,
whereas all controls showed no detectable labeling.
3-Phenyl-1,2,3-oxadiazol-3-ium-5-olate 4a. White solid (289 mg,
89%): 1H NMR (300 MHz, DMSO-d6): δ 7.69−7.73 (m, 3H), 7.77 (s,
1H), 7.93 (dd, J = 8.0, 1.9 Hz, 2H); 13C NMR (75.5 MHz, DMSO-d6):
δ 95.0 (CH), 121.6 (2 × CH), 130.2 (2 × CH), 132.4 (CH), 134.6
(C), 168.5 (C−O−) in agreement with the literature data.28
3-(4′-Methoxyphenyl)-1,2,3-oxadiazol-3-ium-5-olate 4b. Brown
1
solid (227 mg, 59%): H NMR (300 MHz, CDCl3): δ 3.90 (s, 3H),
6.65 (s, 1H), 7.07 (d, J = 9.1 Hz, 2H), 7.64 (d, J = 9.1 Hz, 2H); 13C
NMR (75.5 MHz, CDCl3): δ 56.0 (CH3), 93.5 (CH), 115.4 (2 ×
CH), 122.8 (2 × CH), 134.5 (C), 162.6 (C), 169.2 (C−O−) in
agreement with the literature data.28
CONCLUSION
■
In summary, strain-promoted cycloaddition reactions between
3,4-disubstituted sydnones and Fl-DIBO generated highly
fluorescent pyrazoles, exhibiting emissions ranging from 450
to 480 nm, large Stokes shift (∼100 nm), and high fluorescence
quantum yields (up to 47%), leading to remarkable
fluorescence enhancements (up to 240-fold). In addition, 4-
chlorosydnones proved to be stable reporters in aqueous
solution that react noticeably fast with the Fl-DIBO probe,
3-[4′-(Trifluoromethyl)phenyl]-1,2,3-oxadiazol-3-ium-5-olate 4c.
1
White solid (298 mg, 65%): H NMR (300 MHz, CDCl3): δ 6.84 (s,
1H), 7.92 (s, 4H); 13C NMR (75.5 MHz, CDCl3): δ 94.0 (CH), 122.1
(2 × CH), 123.0 (q, J = 272.9 Hz, CF3), 127.8 (q, J = 3.7 Hz, 2 ×
CH), 134.7 (q, J = 33.7 Hz, C), 137.3 (C), 168.7 (C−O−); 19F NMR
(282 MHz, CDCl3) δ −63.08 (s) in agreement with the literature
data.28
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1
3-(4′-Carboxyphenyl)-1,2,3-oxadiazol-3-ium-5-olate 4d. Orange
solid (240 mg, 58%): 1H NMR (300 MHz, DMSO-d6): δ 7.88 (s, 1H),
8.07 (d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.8 Hz, 2H); 13C NMR (75.5
MHz, DMSO-d6): δ 95.3 (CH), 121.9 (2 × CH), 131.0 (2 × CH),
134.3 (C), 137.3 (C), 166.0 (CO), 168.4 (C−O−) in agreement
with the literature data.28
desired sydnone 4j as a brown solid (95 mg, 80%): H NMR (300
MHz, CDCl3): δ 7.27−7.29 (br s, 5H), 7.46−7.50 (m, 2H), 7.54−7.60
(m, 2H), 7.63−7.69 (m, 1H); 13C NMR (75.5 MHz, CDCl3): δ 108.0
(C), 124.5 (C), 124.9 (2 × CH), 127.5 (2 × CH), 128.8 (CH), 128.8
(2 × CH), 130.3 (2 × CH), 132.2 (CH), 134.8 (C), 167.3 (C−O−) in
agreement with literature data.34
General Procedure for the Formation of 4-Chlorosydnone
4e and 4h−i. N-Chlorosuccinimide (134 mg, 1.0 mmol) was added
to a solution of 3-aryl sydnone (0.5 mmol) in acetic acid (1.2 mL).
The reaction mixture was stirred at room temperature for 8 h and then
diluted with ethyl acetate, and all the volatiles were removed under
reduced pressure. The residue was then purified by flash
chromatography on silica gel (15 g) using an appropriate mixture of
solvents to afford pure 4-chloro-3-arylsydnone.
4-(Methylsulfanyl)-3-phenyl-1,2,3-oxadiazol-3-ium-5-olate 4k. A
solution of n-butyllithium in THF (1.6 M, 0.34 mL, 0.55 mmol) was
added dropwise to a cold (−78 °C) solution of 3-phenylsydnone 4a
(81 mg, 0.5 mmol) in THF (2 mL). After 1 h, dimethyl disulfide (0.04
mL, 0.5 mmol) was then added dropwise and the reaction mixture was
stirred for 1 h (the temperature did not rise above −50 °C). The
reaction was then quenched by addition of water (20 mL), and the
aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel (10 g) using a mixture of petroleum
ether and ethyl acetate (5:1) to afford the pure desired sydnone 4k as
a brown solid (49 mg, 73%): 1H NMR (300 MHz, CDCl3): δ 2.27 (s,
3H), 7.58−7.71 (m, 5H); 13C NMR (75.5 MHz, CDCl3): δ 18.0
(CH3), 125.0 (2 × CH), 129.0 (C), 129.9 (2 × CH), 131.1 (C), 132.5
(CH), 168.1 (C−O−) in agreement with literature data.35
3-{4′-[Isopropylamino)carbonyl]phenyl}-1,2,3-oxadiazol-3-ium-
5-olate 4l. Isopropylamine (0.05 mL, 0.55 mmol) was added to a
solution of PyBop (312 mg, 0.6 mmol), 3-carboxyphenylsydnone 4d
(103 mg, 0.5 mmol), and DIPEA (0.17 mL, 1 mmol) in DMF (5 mL).
The reaction mixture was stirred overnight and then diluted with ethyl
acetate. The organic layer was washed with a saturated aqueous
solution of NH4Cl (3 × 30 mL) and brine (30 mL), dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash chromatography on silica gel (10 g) using a mixture of
dichloromethane and acetone (95:5) to afford the pure sydnone 4l as
an off-white solid (50 mg, 40%): 1H NMR (300 MHz, CDCl3) δ 1.30
(d, J = 6.6 Hz, 6H), 4.31 (hept, J = 6.6 Hz, 1H), 6.05 (br d, J = 6.6 Hz,
1H), 6.76 (s, 1H), 7.80 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H);
13C NMR (75 MHz, CDCl3) δ 22.9 (2 × CH3), 42.6 (CH), 93.8
4-Chloro-3-phenyl-1,2,3-oxadiazol-3-ium-5-olate 4e. Purified
with a mixture of petroleum ether and ethyl acetate (7:3); green
1
solid (16 mg, 16%): H NMR (300 MHz, methanol-d4): δ 7.70−7.78
(m, 5H); 13C NMR (200 MHz, methanol-d4): δ 100.3 (C−Cl), 126.1
(2 × CH), 131.3 (2 × CH), 134.0 (CH), 134.5 (C), 166.1 (C−O−) in
agreement with literature data.28
4-Chloro-3-[4′-(trifluoromethyl)phenyl]-1,2,3-oxadiazol-3-ium-5-
olate 4h. Purified with a mixture of petroleum ether and ethyl acetate
1
(4:1); orange solid (127 mg, 96%): H NMR (300 MHz, CDCl3): δ
7.84 (d, J = 8.4 Hz, 2H), 7.97 (d, J = 8.4 Hz, 2H); δ 13C NMR (75.5
MHz, CDCl3): 123.0 (q, J = 273.1, CF3), 125.3 (2 × CH), 127.6 (q, J
= 3.6 Hz, 2 × CH), 135.0 (q, J = 33.7 Hz, C), 135.7 (C), 163.7 (C−
O−), missing C−Cl signal due to relaxation issue; 19F NMR (282
MHz, CDCl3): δ − 63.11 (s); HRMS (m/z): [M + H+] calcd for
C9H5ClF3N2O2, 264.9986; found, 264.9987.
3-(4′-Carboxyphenyl)-4-chloro-1,2,3-oxadiazol-3-ium-5-olate 4i.
On 2.4 mmol scale; purified with a mixture of 1% of methanol in
1
dichloromethane with 0.5% acetic acid; white solid (64 mg, 20%): H
NMR (300 MHz, methanol-d4): δ 7.90 (d, J = 8.8 Hz, 2H), 8.30 (d, J
= 8.8 Hz, 2H); 13C NMR (75 MHz, methanol-d4): δ 126.4 (2 × CH),
132.4 (2 × CH), 136.3 (C), 137.5 (C), 166.0 (C−O−), 167.6 (CO),
missing C−Cl signal due to relaxation issue in agreement with
literature data.28
(CH), 121.6 (2 × CH), 129.1 (2 × CH), 136.7 (C), 139.0 (C), 164.5
(CO), 168.8 (C−O−); HRMS (m/z): [M + H+] calcd for
C12H14N3O3, 248.1030; found, 248.1031.
4-Bromo-3-phenyl-1,2,3-oxadiazol-3-ium-5-olate 4f. N-Bromo-
succinimide (178 mg, 1.6 mmol) was added to a solution of 3-
phenylsydnone 4a (172 mg, 1.06 mmol) in acetic acid (4 mL). The
reaction mixture was stirred at room temperature for 2 h. Water (30
mL) was added, and the resulting precipitate was washed with cold
ethanol (2 mL) and dried under reduced pressure to afford the pure
NHS-Activated 3-(4′-Carboxyphenyl)-4-chloro-1,2,3-oxadiazol-3-
ium-5-olate 4m. N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide
hydrochloride (39 mg, 0.2 mmol) was added to a solution of 3-(4′-
carboxyphenyl)-4-chlorosydnone 4i (43 mg, 0.17 mmol) and N-
hydroxysuccinimide (23 mg, 0.2 mmol) in DMF (3 mL). The reaction
mixture was stirred at room temperature overnight. The reaction was
then quenched with a saturated aqueous solution of NH4Cl. The
aqueous layer was extracted with ethyl acetate (2 × 20 mL). The
combined organic layers were washed with brine (2 × 30 mL), dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel (5 g) using a
mixture of dichloromethane and methanol (99:1) to afford the pure
1
brominated sydnone 4f as an orange solid (174 mg, 68%): H NMR
(300 MHz, methanol-d4): δ 7.67−7.79 (m, 5H); 13C NMR (75.5
MHz, methanol-d4): δ 86.3 (C−Br), 126.3 (2 × CH), 131.2 (2 ×
CH), 133.9 (CH), 135.4 (C), 168.0 (C−O−) in agreement with
literature data.28
4-Iodo-3-phenyl-1,2,3-oxadiazol-3-ium-5-olate 4g. N-Iodosucci-
nimide (169 mg, 0.75 mmol) was added to a solution of 3-
phenylsydnone 4a (81 mg, 0.5 mmol) in acetic acid (1.3 mL). The
reaction mixture was stirred at room temperature for 1 h 30 min.
Water (20 mL) was added, and the resulting precipitate was washed
with cold ethanol (3 mL) and dried under reduced pressure to afford
1
compound as an off-white solid (37 mg, 65%): H NMR (300 MHz,
CDCl3): δ 2.95 (s, 4H), 7.87 (d, J = 8.6 Hz, 2H), 8.45 (d, J = 8.6 Hz,
2H); 13C NMR (75 MHz, CDCl3): δ 25.8 (2 × CH2), 125.2 (2 ×
CH), 129.6 (C), 132.5 (2 × CH), 137.6 (C), 160.3 (C), 168.8 (3 ×
C), missing C−Cl signal due to relaxation issue; HRMS (m/z): [M +
H+] calcd for C13H9ClN3O6, 338.0174; found, 338.0175.
1
the iodinated sydnone 4g as a light orange solid (95 mg, 66%): H
NMR (300 MHz, methanol-d4): δ 7.58−7.73 (m, 5H); 13C NMR
(75.5 MHz, methanol-d4): δ 50.6 (C−I), 125.3 (2 × CH), 130.2 (2 ×
CH), 132.7 (CH), 135.3 (C) 168.9 (C−O−) in agreement with
literature data.28
4-Chloro-3-{4-[({2-[2-(2-hydroxyethoxy)ethoxy]ethyl}amino)-
carbonyl]phenyl}-1,2,3-oxadiazol-3-ium-5-olate 4n. A solution of
NHS-chlorosydnone 4m (0.24 mmol, 81 mg) in DMF (2 mL) was
added to a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanol (0.2
mmol, 30 mg) and triethylamine (0.8 mmol, 0.1 mL) in DMF (2 mL).
The reaction mixture was stirred at room temperature during 48 h.
The reaction was quenched with a saturated aqueous solution of
NH4Cl. The aqueous layer was extracted with dichloromethane (2 ×
20 mL), and the organic layer was successively washed with water (3 ×
30 mL) and brine (30 mL), dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel (5 g) using a mixture of dichloromethane
and methanol (95:5) to afford the pure chlorosydnone 4n (26 mg,
3,4-Diphenyl-1,2,3-oxadiazol-3-ium-5-olate 4j. Iodobenzene
(0.08 mL, 0.75 mmol) was added to a solution of 3-phenylsydnone
4a (81 mg, 0.5 mmol), palladium(II) acetate (5.6 mg, 0.025 mmol),
potassium carbonate (138 mg, 1 mmol), and triphenylphosphine (13
mg, 0.05 mmol) in wet DMF (2 mL). The reaction mixture was
refluxed for 12 h. After being cooled to room temperature, water was
added to the mixture and the aqueous layer was extracted with ethyl
acetate (3 × 15 mL). The combined organic layers were then dried
over MgSO4 and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel (10 g) using a
mixture of petroleum ether and ethyl acetate (5:1) to afford the pure
F
DOI: 10.1021/acs.joc.7b03004
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
1
35%) as an orange solid: H NMR (300 MHz, CDCl3): δ 3.62−3.65
151.8 (C), 152.3 (CO), 162.3 (C), 162.7 (C); HRMS (m/z): [M +
H+] calcd for C26H18ClN2O3, 441.1000; found, 441.1010.
(m, 2H), 3.66−3.75 (m, 11H), 7.23 (br s, 1H), 7.73 (d, J = 8.8 Hz,
2H), 8.15 (d, J = 8.7 Hz, 2H); 13C NMR (75.5 MHz, CDCl3): δ 40.2
(CH2), 61.8 (CH2), 69.9 (CH2), 70.4 (CH2), 70.5 (CH2), 72.6 (CH2),
124.7 (2 × CH), 129.3 (2 × CH), 135.0 (C), 138.8 (C), 161.9 (C−
O−), 165.4 (CO), missing C−Cl signal due to relaxation issue;
HRMS (m/z): [M+Na+] calcd for C15H18ClN3O6Na, 394.0776; found,
394.0775.
General Procedure for the Cycloaddition of Fl-DIBO with
Various Sydnones. A solution of Fl-DIBO (3) (14.4 mg, 0.05 mmol)
and the respective sydnone (0.1 mmol) in a mixture of dichloro-
methane and methanol (4:1, 5 mL, solvent system A) or a mixture of
1,2-dichloroethane and methanol (4:1, 5 mL, solvent system B) was
stirred at 50 °C (system A) or 70 °C (system B). All volatiles were
then removed under reduced pressure, and the residue was purified by
flash chromatography on silica gel (5 g) using a mixture of 1% of
methanol in dichloromethane, affording the desired pure cycloadducts,
respectively.
3-Bromo-5,11-dimethoxy-2-phenyldibenzo[3,4:7,8]cyclopropa-
[5,6]cycloocta[1,2-c]pyrazol-8(2H)-one 6f. Obtained with system A
overnight. Off-white solid (21 mg, 88%): 1H NMR (300 MHz,
CDCl3): δ 3.86 (s, 3H), 3.89 (s, 3H), 6.93 (dd, J = 8.5, 2.7 Hz, 1H),
6.95 (dd, J = 8.5, 2.7 Hz, 1H), 7.16 (d, J = 2.6 Hz, 1H), 7.31 (d, J = 2.6
Hz, 1H), 7.47−7.64 (m, 7H); 13C NMR (75.5 MHz, CDCl3): δ 55.7
(CH3), 55.8 (CH3), 113.5 (CH), 114.9 (CH), 116.8 (C−Br), 117.6
(CH), 118.0 (C), 119.2 (C), 119.9 (CH), 120.2 (C), 126.2 (2 × CH),
129.2 (2 × CH), 129.3 (CH), 132.8 (CH), 133.0 (CH), 135.3 (C),
138.1 (C), 139.0 (C), 149.3 (C), 151.4 (C), 152.1 (C), 152.2 (C
O), 162.2 (C), 162.6 (C); HRMS (m/z): [M + H+] calcd for
C26H18BrN2O3, 485.0495; found, 485.0494.
3-Iodo-5,11-dimethoxy-2-phenyldibenzo[3,4:7,8]cyclopropa[5,6]-
cycloocta[1,2-c]pyrazol-8(2H)-one 6g. Obtained with system A
during 24 h. Yellow solid (11 mg, 41%): 1H NMR (300 MHz,
CDCl3): δ 3.86 (s, 3H), 3.91 (s, 3H), 6.94 (dd, J = 8.5, 2.6 Hz, 1H),
6.95 (dd, J = 8.5, 2.7 Hz, 1H), 7.18 (d, J = 2.6 Hz, 1H), 7.30 (d, J = 2.6
Hz, 1H), 7.49−7.57 (m, 5H), 7.59 (d, J = 8.5 Hz, 1H), 7.61 (d, J = 8.5
Hz, 1H); 13C NMR (75.5 MHz, CDCl3): δ 55.7 (CH3), 55.8 (CH3),
91.1 (C−I), 113.7 (CH), 114.9 (CH), 117.5 (CH), 118.1 (C), 119.5
(C), 120.4 (CH), 125.5 (C), 127.0 (2 × CH), 129.2 (2 × CH), 129.5
(CH), 132.6 (CH), 133.0 (CH), 136.6 (C), 138.1 (C), 140.6 (C),
149.5 (C), 151.7 (C), 152.2 (C), 152.5 (CO), 162.0 (C), 162.6
(C); HRMS (m/z): [M + H+] calcd for C26H18IN2O3, 533.0357;
found, 533.0357.
5,11-Dimethoxy-2-phenyldibenzo[3,4:7,8]cyclopropa[5,6]-
cycloocta[1,2-c]pyrazol-8(2H)-one 6a. Obtained with system A
overnight. Light yellow solid (18 mg, 89%): H NMR (300 MHz,
1
CDCl3): δ 3.89 (s, 3H), 3.91 (s, 3H), 6.89 (dd, J = 8.5, 2.5 Hz, 1H),
6.95 (dd, J = 8.5, 2.7 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 7.34 (tt, J =
7.4, 1.5 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.48 (t, J = 7.4 Hz, 2H),
7.63 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.74 (dd, J = 7.4, 1.5
Hz, 2H), 7.86 (s, 1H); 13C NMR (75.5 MHz, CDCl3): δ 55.8 (2 ×
CH3), 112.6 (CH), 114.5 (CH), 117.4 (C), 117.7 (CH), 117.7 (C),
118.3 (CH), 119.1 (2 × CH), 122.3 (C), 127.4 (CH), 129.7 (2 ×
CH), 130.7 (CH), 133.7 (CH), 134.0 (CH), 137.0 (C), 138.3 (C),
139.3 (C), 148.4 (C), 149.8 (C), 150.2 (C), 152.8 (CO), 162.6
(C), 162.9 (C); HRMS (m/z): [M + H+] calcd for C26H19N2O3,
407.1390; found, 407.1393.
3-Chloro-5,11-dimethoxy-2-[4-(trifluoromethyl)phenyl]dibenzo-
[3,4:7,8]cyclopropa[5,6]cycloocta[1,2-c]pyrazol-8(2H)-one 6h. Ob-
1
tained with system A overnight. Off-white solid (25 mg, 98%): H
NMR (300 MHz, CDCl3): δ 3.88 (s, 3H), 3.90 (s, 3H), 6.95 (dd, J =
8.7, 2.6 Hz, 1H), 6.98 (dd, J = 8.7, 2.6 Hz, 1H), 7.13 (d, J = 2.6 Hz,
1H), 7.31 (d, J = 2.6 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.66 (d, J = 8.6
Hz, 1H), 7.80 (s, 4H); 13C NMR (75.5 MHz, CDCl3): δ 55.7 (CH3),
55.8 (CH3), 113.5 (CH), 114.9 (CH), 117.8 (CH), 117.9 (C−Cl),
118.3 (C), 119.1 (C), 119.5 (CH), 123.7 (q, J = 271.7 Hz, C), 125.5
(2 × CH), 126.5 (q, J = 3.6 Hz, 2 × CH), 128.2 (C), 131.0 (q, J = 33.2
Hz, C), 133.0 (CH), 133.2 (CH), 134.1 (C), 137.7 (C), 140.8 (C),
149.8 (C), 151.3 (C), 151.9 (C), 152.1 (CO), 162.4 (C), 162.6
(C); 19F NMR (282 MHz, CDCl3): δ − 62.62 (s); HRMS (m/z): [M
+ H+] calcd for C27H17ClF3N2O3, 509.0874; found, 509.0876.
5,11-Dimethoxy-2,3-diphephenyldibenzo[3,4:7,8]cyclopropa-
[5,6]cycloocta[1,2-c]pyrazol-8(2H)-one 6j. Obtained with system B
during 120 h. Yellow solid (10 mg, 42%): 1H NMR (300 MHz,
CDCl3): δ 3.30 (s, 3H), 3.93 (s, 3H), 6.29 (d, J = 2.6 Hz, 1H), 6.75
(dd, J = 8.5, 2.6 Hz, 1H), 6.81−7.06 (m, 3H), 7.16−7.35 (m, 8H),
7.50 (d, J = 2.6 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 8.5 Hz,
1H); 13C NMR (75.5 MHz, CDCl3): δ 55.2 (CH3), 55.7 (CH3), 114.4
(CH), 114.6 (CH), 117.9 (CH), 118.0 (C), 119.1 (CH), 119.4 (C),
125.3 (2 × CH), 127.9 (CH), 128.7 (CH), 128.8 (2 × CH), 129.0 (2
× CH), 130.0 (2 × C), 130.9 (2 × CH), 132.5 (CH), 133.3 (CH),
136.6 (C), 138.7 (C), 139.5 (C), 144.5 (C), 148.7 (C), 151.4 (C),
151.9 (C), 152.6 (CO), 161.8 (C), 162.6 (C); HRMS (m/z): [M +
H+] calcd for C32H23N2O3, 483.1703; found, 483.1710.
5,11-Dimethoxy-2-(4-methoxyphenyl)dibenzo[3,4:7,8]-
cyclopropa[5,6]cycloocta[1,2-c]pyrazol-8(2H)-one 6b. Obtained
with system B during 48 h. Light yellow solid (14 mg, 65%): H
1
NMR (300 MHz, CDCl3): δ 3.85 (s, 3H), 3.88 (s, 3H), 3.90 (s, 3H),
6.87 (dd, J = 8.5, 2.6 Hz, 1H), 6.93 (dd, J = 8.5, 2.7 Hz, 1H), 6.06−
6.99 (m, 3H), 7.41 (d, J = 2.7 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.62
(d, J = 8.5 Hz, 1H), 7.63 (d, J = 9.1 Hz, 2H), 7.76 (s, 1H); 13C NMR
(75.5 MHz, CDCl3): δ 55.7 (3 × CH3), 112.5 (CH), 114.3 (CH),
114.7 (2 × CH), 117.4 (C), 117.6 (CH), 117.7 (C), 118.2 (CH),
120.8 (2 × CH), 121.8 (C), 130.8 (CH), 133.0 (C), 133.7 (CH),
133.9 (CH), 137.1 (C), 138.4 (C), 147.8 (C), 149.9 (C), 150.2 (C),
152.8 (CO), 158.9 (C), 162.6 (C), 162.8 (C); HRMS (m/z): [M +
H+] calcd for C27H21N2O4, 437.1496; found, 437.1500.
5,11-Dimethoxy-2-(4-trimethylfluorophenyl)dibenzo[3,4:7,8]-
cyclopropa[5,6]-cycloocta[1,2-c]pyrazol-8(2H)-one 6c. Obained with
system A overnight. Light yellow solid (23 mg, 97%): H NMR (300
1
MHz, CDCl3): δ 3.89 (s, 3H), 3.91 (s, 3H), 6.90 (dd, J = 8.5, 2.6 Hz,
1H), 6.97 (dd, J = 8.5, 2.7 Hz, 1H), 7.01 (d, J = 2.6 Hz, 1H), 7.41 (d, J
= 2.7 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.74
(d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.93 (s, 1H); 13C NMR
(75.5 MHz, CDCl3): δ 55.8 (CH3), 55.8 (CH3), 112.8 (CH), 114.5
(CH), 117.4 (C), 117.6 (C), 117.8 (CH), 118.4 (CH), 118.7 (2 ×
CH), 123.2 (C), 123.9 (q, J = 272.0 Hz, CF3), 127.0 (q, J = 3.7 Hz, 2
× CH), 129.2 (q, J = 33.0 Hz, C), 130.6 (CH), 133.8 (CH), 134.0
(CH), 136.5 (C), 137.8 (C), 141.6 (C), 149.4 (C), 149.7 (C), 150.1
(C), 152.7 (CO), 162.6 (C), 162.9 (C); 19F NMR (282 MHz,
CDCl3): δ − 62.35 (s); HRMS (m/z): [M + H+] calcd for
C27H18F3N2O3, 475.1264; found, 475.1269.
3-Chloro-5,11-dimethoxy-2-phenyldibenzo[3,4:7,8]cyclopropa-
[5,6]cycloocta[1,2-c]pyrazol-8(2H)-one 6e. Obtained with system A
overnight. Yellowish solid (21 mg, 93%): 1H NMR (300 MHz,
CDCl3): δ 3.87 (s, 3H), 3.89 (s, 3H), 6.94 (dd, J = 8.6, 2.5 Hz, 1H),
6.96 (dd, J = 8.5, 2.6 Hz, 1H), 7.14 (d, J = 2.6 Hz, 1H), 7.33 (dd, J =
2.5 Hz, 1H), 7.49−7.56 (m, 3H), 7.60−7.66 (m, 4H); 13C NMR (75.5
MHz, CDCl3): δ 55.8 (CH3), 55.8 (CH3), 113.4 (CH), 114.9 (CH),
117.3 (C−Cl), 117.7 (CH), 117.8 (C), 119.1 (C), 119.4 (CH), 125.6
(2 × CH), 128.3 (C), 129.2 (CH), 129.3 (2 × CH), 133.0 (CH),
133.2 (CH), 134.6 (C), 138.1 (C), 138.2 (C), 148.8 (C), 151.2 (C),
5,11-Dimethoxy-3-(methylsulfanyl)-2-phenyldibenzo[3,4:7,8]-
cyclopropa[5,6]-cycloocta[1,2-c]pyrazol-8(2H)-one 6k. Obtained
1
with system B during 48 h. Yellow solid (21 mg, 91%): H NMR
(300 MHz, CDCl3): δ 1.79 (s, 3H), 3.85 (s, 3H), 3.90 (s, 3H), 6.94
(dd, J = 8.5 Hz, 2.6 Hz, 1H), 6.93 (dd, J = 8.5 Hz, 2.6 Hz, 1H), 7.26
(d, J = 2.6 Hz, 1H), 7.32 (d, J = 2.6 Hz, 1H), 7.45−7.54 (m, 3H),
7.57−7.64 (m, 4H); 13C NMR (75.5 MHz, CDCl3): δ 18.6 (CH3),
55.7 (CH3), 55.8 (CH3), 113.6 (CH), 114.7 (CH), 117.6 (CH), 117.9
(C), 119.2 (C), 119.8 (CH), 123.6 (C), 126.0 (2 × CH), 128.9 (CH),
129.1 (2 × CH), 132.5 (CH), 132.9 (CH), 136.2 (C), 137.7 (C),
138.5 (C), 139.5 (C), 148.7 (C), 151.6 (C), 152.3 (C), 152.4 (C
O), 162.2 (C), 162.6 (C); HRMS (m/z): [M + H+] calcd for
C27H21N2O3S, 453.1267; found, 453.1265.
5,11-Dimethoxy-2-(4-isopropylamidophenyl)dibenzo[3,4:7,8]-
cyclopropa[5,6]-cycloocta[1,2-c]pyrazol-8(2H)-one 6l. Obtained
with system B overnight. yellow solid (4 mg, 16%, low yield due to
G
DOI: 10.1021/acs.joc.7b03004
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The Journal of Organic Chemistry
Article
difficulties associated with the purification): 1H NMR (300 MHz,
CDCl3): δ 1.29 (d, J = 6.6 Hz, 6H), 3.89 (s, 3H), 3.92 (s, 3H), 4.31
(hept, J = 6.6 Hz, 1H), 5.97 (d, J = 7.8 Hz, 1H), 6.91 (dd, J = 8.5, 2.6
Hz, 1H), 6.97 (dd, J = 8.5, 2.7 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 7.43
(d, J = 2.6 Hz, 1H), 7.4 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H),
7.80 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.8 Hz, 2H), 7.91 (s, 1H); 13C
NMR (75.5 MHz, CDCl3): δ 23.0 (2 × CH3), 42.3 (CH), 55.8 (2 ×
CH3), 112.7 (CH), 114.5 (CH), 117.5 (C), 117.7 (C), 117.8 (CH),
118.4 (CH), 118.5 (2 × CH), 123.0 (C), 128.6 (2 × CH), 130.6
(CH), 133.5 (C), 133.8 (CH), 134.0 (CH), 136.6 (C), 137.9 (C),
141.2 (C), 149.1 (C), 149.9 (C), 150.2 (C), 152.7 (C), 162.6 (C),
162.9 (C), 165.7 (C); HRMS (m/z): [M + H+] calcd for C30H26N3O4,
492.1918; found, 492.1923.
bulk solvent effects, the gas-phase geometries were further geometry-
optimized with inclusion of the polarizable continuum model (PCM),
which creates a solute cavity based on a set of overlapping spheres.37
Coordinates for the final geometries are listed in Supporting
Information, Tables S1−S3. All structures were validated based on
vibrational frequency analysis to ensure a stationary point on the
ground-state potential surface. Vertical excitation energies were
computed based on the solvent-equilibrated ground-state geometries
(B3LYP/6-31G(d)) using TD-DFT with the CAM-B3LYP func-
tional38 and the 6-31+G(d) basis set with added diffuse functions.
ASSOCIATED CONTENT
* Supporting Information
■
S
4-(3-Chloro-5,11-dimethoxy-8-oxodibenzo[3,4:7,8]cyclopropa-
[5,6]cycloocta[1,2-c]pyrazol-2(8H)-yl)-N-{2-[2-(2-hydroxyethoxy)-
ethoxy]ethyl}benzamide 6n. Obtained with system A overnight;
Purified with a mixture of 5% of methanol in dichloromethane; Yellow
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.7b03004.
1H and 13C NMR spectra of all synthesized compounds,
absorbance and emission spectra of pyrazoles 6a−c, 6e−
h, and 6j−n, kinetics plots, 1H NMR data for the stability
study of sydnone 4e, and TD-DFT calculations (PDF)
1
solid (18 mg, 60%): H NMR (300 MHz, CDCl3): δ 3.59−3.62 (m,
2H), 3.66−3.73 (m, 11H), 3.87 (s, 3H), 3.88 (s, 3H), 6.94 (dd, J = 8.5,
2.6 Hz, 1H), 6.96 (dd, J = 8.5, 2.6 Hz, 1H), 7.12 (d, J = 2.5 Hz, 1H),
7.15 (br s, 1H), 7.31 (d, J = 2.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.64
(d, J = 8.6 Hz, 1H), 7.71 (d, J = 8.7 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H);
13C NMR (75.5 MHz, CDCl3): δ 40.0 (CH2), 55.8 (CH3), 55.8
(CH3), 61.8 (CH2), 70.1 (CH2), 70.4 (CH2), 70.5 (CH2), 72.7 (CH2),
113.5 (CH), 114.9 (CH), 117.7 (CH), 118.0 (C−Cl), 119.0 (C),
119.4 (CH), 125.2 (2 × CH), 128.2 (C), 128.3 (2 × CH), 133.0
(CH), 133.2 (CH), 134.3 (C), 134.9 (C), 137.8 (C), 140.3 (C), 149.4
(C), 151.1 (C), 151.7 (C), 152.2 (CO), 162.3 (C), 162.6 (C),
166.5 (CO); HRMS (m/z): [M + H+] calcd for C33H31ClN3O7,
616.1845; found, 616.1846.
AUTHOR INFORMATION
Corresponding Author
*E-mail: f.friscourt@iecb.u-bordeaux.fr.
■
ORCID
Frederic Friscourt: 0000-0002-0749-0861
́
́
Notes
Absorption and Fluorescence Measurements. UV−vis spectra
The authors declare no competing financial interest.
were recorded at 25
0.1 °C using a Varian Cary 300 UV−vis
spectrophotometer. Fluorescence spectra were recorded with a
SpectraMax M2E Multi-Mode Cuvette/Microplate Reader from
molecular devices using a cuvette with 1 cm path length. For spectra,
see Supporting Information Figures S1− S13.
Quantum Yield Determination. Quantum yields were deter-
mined from the slope of the integrated fluorescence emission between
380 and 700 nm (excitation at 360 nm) versus absorbance using
quinine sulfate in 1.0 N H2SO4 (ΦF = 0.54 0.03) as fluorescence
standard. For each compound, four data points were acquired with
absorbances ranging between 0.1 and 0.5 (l = 1 cm).
ACKNOWLEDGMENTS
■
This study has received financial support from the French State
in the frame of the “Investments for the Future” Programme
IdEx Bordeaux (ANR-10-IDEX-03-02 to F.F.) and from the
Laboratory of Excellence TRAIL (ANR-10-LABX-57 to F.F.).
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