Fluorescent push-pull pH-responsive probes for ratiometric detection of intracellular pH

Article abstract of DOI:10.1039/c4ob00147h

A family of fluorescent push-pull pH-responsive probes based on 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran as a strong electron acceptor group is described. Small structural variations allow obtaining pK_a ranging from 4.8 to 8.6, underlining the role of the substituent in modulating the acidic properties. Remarkable changes in the optical properties (in particular the fluorescence intensity ratios) were observed as a function of pH. The most interesting probes with pK_a close to neutrality were used for ratiometric imaging of intracellular pH. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00147h

Organic &
Biomolecular Chemistry
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Fluorescent push–pull pH-responsive probes
for ratiometric detection of intracellular pH†
Cite this: Org. Biomol. Chem., 2014,
12, 3641
Martin Ipuy,a Cyrielle Billon,b Guillaume Micouin,a Jacques Samarut,b
Chantal Andrauda and Yann Bretonnière*a
A family of fluorescent push–pull pH-responsive probes based on 2-dicyanomethylidene-3-cyano-4,5,5-
trimethyl-2,5-dihydrofuran as a strong electron acceptor group is described. Small structural variations
allow obtaining pKa ranging from 4.8 to 8.6, underlining the role of the substituent in modulating the
acidic properties. Remarkable changes in the optical properties (in particular the fluorescence intensity
ratios) were observed as a function of pH. The most interesting probes with pKa close to neutrality were
used for ratiometric imaging of intracellular pH.
Received 20th January 2014,
Accepted 31st March 2014
DOI: 10.1039/c4ob00147h
www.rsc.org/obc
Introduction
Phenolic compounds are amongst the most important classes
of dyes characterized by acidity of the hydroxy group. The pKa
of phenol is 9.98, but when electron-withdrawing groups that
can stabilize the phenoxide ion are present, a substantial
change in acidity of the OH group can be noted.1 For example,
the ionization constants of o- and p-nitrophenol are several
hundred times greater than those of phenol with pKa of 7.23
and 7.15 respectively. Moreover, the hydroxy group OH is a
weak electron donating group whereas the deprotonated O− is
a much stronger donating group with a negative charge that
can delocalize along a conjugated path. As a consequence, the
phenol and the corresponding phenolate ion display very
different optical properties in absorption and emission, with
the latter being usually much more emissive. This unique
feature has been intensively exploited in the design of fluo-
rescent indicators for intracellular pH.2 Most of the pH
indicators developed so far (Fig. 1), such as 1,4-DHPN (1,4-
dihydroxyphthalonitrile),3 HPTS (1-hydroxypyrene-3,6,8-tri-
sulfonate),4 BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6-)carboxy-
fluorescein),5 or the long-wavelength fluorescent SNAFL
(seminaphthofluoresceins),6 SNAFR (seminaphthofluorones),7
and SNARF (seminaphthorhodafluors),8 are phenol derivatives
spanning the physiological pH from different organelles
Fig. 1 Examples of commonly used phenol-based pH sensitive
fluorophores.
(4.50–6.00 range) to the cytosol (6.80–7.40 range) and covering
a wide range of absorption and emission wavelengths. These
indicators are often used as ratiometric (or dual) excitation or
emission pH indicators because the absorption or emission
profile of the fluorophore changes significantly with the pH.
Given the importance of modulating the pKa of phenol deriva-
tives, finding new simple fluorescent structures containing a
phenol group is of prime interest.
aLaboratoire de Chimie de l’ENS de Lyon, CNRS UMR 5182, Ecole Normale
Supérieure de Lyon, Université Lyon I, 46 allée d’Italie, 69364 Lyon cedex 07, France.
E-mail: yann.bretonniere@ens-lyon.fr; Fax: +33-4-7272-8860; Tel: +33-4-7272-8399
bInstitut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1,
CNRS, INRA, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon cedex
07, France
2-Dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydro-
furan (TCF) is a strong electron-withdrawing group that has
been extensively used in the design of highly active nonlinear
†Electronic supplementary information (ESI) available: Includes additional
figures, full spectroscopic properties of compounds 2a–2j and complete NMR
data for compounds 2a–2j. See DOI: 10.1039/c4ob00147h
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optical materials.9 In comparison, only very few examples
described its use in the design of red fluorescent probes for
bioimaging, only in combination with N,N-dialkylanilino
groups as electron-donating groups.10 Strong absorption, rela-
tively high fluorescence quantum yield, and sensitivity of their
fluorescence to the local environment make them interesting
for single molecule imaging,10a,e pH,11 thiol10d or K+ sen-
sors.10f TCF dyes containing other electron-donating groups
such as methoxy have seldom been described. And yet, besides
their highly environment sensitive optical properties, they have
been shown to be more photostable than their N,N-dialkyl-
anilino equivalents.12 In this article we report the design, syn-
thesis and study of the physical and optical properties of a
new series of simple push–pull dipolar phenol derivatives 2a–
2j (Scheme 1) containing the 2-dicyanomethylidene-3-cyano-
4,5,5-trimethyl-2,5-dihydrofuran (TCF) ring. These pH respon-
sive dyes are fluorescent in both the acidic (neutral) and the
basic (anionic) form and behave as selective fluorescent pH
receptors in aqueous solutions.
Results and discussion
The synthesis of 2a–g (Scheme 1) involved a simple Knoevena- Scheme 1 Synthesis of compounds 2a–2j.
gel condensation between TCF and the corresponding alde-
hyde. This reaction was performed by controlled microwave
irradiation in ethanol and in the presence of piperidine as a
catalyst. The synthesis of TCF derivatives by microwave heating tion with respect to that of the phenol induced a hypsochro-
is well established and has been shown to give a better yield mic shift in the absorption maxima (up to 34 nm for the nitro
than traditional heating.13 Using this simple procedure group in 2d). However, elongation of the conjugated path
allowed recovering the compounds by simple filtration from (2i–2j) or introduction of the electron-donating group in the
the reaction mixture in average to good yields (39 to 87%).
ortho position (2g–2h) was characterized by a bathochromic
Table 1 contains the spectroscopic data for compounds 2a– shift in the absorption maximum wavelength (up to 64 nm for
2i. The absorption spectra of 2a–2j in DMSO–water mixtures compound 2i).
(1/5 v/v) at low pH where only the phenol forms are present
The dramatic changes in the absorption spectra (Fig. 2a
display a broad band characteristic of a charge transfer tran- and 3a) observed when the pH is increased accounted for the
sition. Absorption maxima ranged from 414 nm for 2d to formation of the phenoxide at higher pH (Scheme 2). The
512 nm for 2i. When compared to the simplest compound 2a, band centred around 460 nm gradually decreased and a
introducing an electron-withdrawing group in the ortho posi- new narrow absorption peak appeared whose maximum was
Table 1 Spectroscopic properties of compounds 2a–2j
Neutral form
Phenoxide
λabs (ε)a nm (L mol−1 cm−1
)
λema nm
Δλc nm
Φd (εΦ)
λabs (ε)b nm (L mol−1 cm−1
)
λem
Δλc nm
Φe (εΦ)
b
2a
2b
2c
2d
2e
2f
2g
2h
2i
448 (48 500)
436 (39 400)
438 (44 000)
414 (60 000)
428 (43 200)
423 (31 600)
465 (56 600)
462 (60 800)
512 (52 300)
465 (51 500)
563
565
560
542
550
—
595
598
634
648
115
129
122
128
122
—
130
136
122
183
0.011 (530)
0.017 (670)
0.015 (660)
0.015 (900)
0.005 (215)
—
0.01 (570)
0.05 (3040)
0.035 (1830)
0.099 (5100)
588 (181 000)
586 (238 000)
584 (234 000)
537 (203 000)
570 (201 000)
574 (254 000)
605 (165 000)
610 (134 000)
632 (101 000)
—
614
618
619
614
611
626
650
644
655
—
26
32
35
77
41
52
45
34
23
—
0.002 (360)
0.004 (950)
0.007 (1640)
0.053 (10 760)
0.012 (2410)
0.012 (3050)
<0.005 (<825)
<0.002 (<270)
0.003 (300)
—
2j
a In water at pH = 2. b In water at pH = 10. λexc was set at the wavelength of the isosbestic point of the absorption-based pH titration. c Stokes shift.
d Measured in DMSO using Coumarin 153 in methanol (Φ = 0.54) as the reference. e Measured in DMSO using cresyl violet in methanol (Φ = 0.57)
as the reference.
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considerably red-shifted compared to the neutral form. The
absorption maxima now ranged from 537 nm for 2d, 588 nm
for the simplest compound 2a up to 632 nm for 2i with a
naphthalene ring. The two bands interconverted with an iso-
sbestic point. The influence of the substituent on the absorp-
tion of the phenoxides was less pronounced than that for the
neutral forms. Based on the absorption of 2a only small hypso-
chromic shifts were observed. It has to be noted that for all
compounds the molar absorption coefficient of the deproto-
nated form was much higher (around 5 times) than the proto-
nated form (Table 1). The switch between the phenol and the
phenoxide (Scheme 2) was fully reversible. The absorption of
the phenol re-appeared upon re-acidification of the solution
and no noticeable changes in the absorption of 2a could be
1
observed after 10 cycles between pH = 4 and pH = 10. H NMR
experiments confirmed the reversibility of the proton exchange
(see ESI†).
Recording the absorption at various pH values enabled
determining the pKa values of all compounds by fitting by non-
linear regression the sigmoid response of absorption value vs.
pH according to eqn (1):
Fig. 2 (a) Absorption spectra of 2a at different pH values in HEPES
buffer. (b) Changes of the absorbance at 590 nm (A590 nm) over the pH
range 2–10 with non-linear least-square fit of the experimental data. (c)
Emission spectra of 2a at different pH in HEPES buffer (λexc = 500 nm).
(d) Emission intensity ratio changes (F554 nm/F614 nm) over the pH range
2–10.
Ka ꢀ AF þ 10ꢁpH ꢀ A0
A ¼
ð1Þ
Ka þ 10ꢁpH
where AF and A0 are respectively the final (high pH) and the
initial (low pH) absorption. The pKa values obtained (Table 2)
varied from 4.2 for 2f to 8.9 for 2j, spanning the intracellular
pH range. As expected, installation of the electro-withdrawing
group ortho to the hydroxy group induced a decrease of the
pKa value,14 the nitro substituted compound 2d being almost
1000 times more acidic than the parent 2a. Surprisingly, intro-
duction of the OR (OH and OCH3) electro-donating group
ortho to the hydroxy group also induced a small decrease in
the pKa probably due to the inductive withdrawing effect of the
O atom. Indeed a good correlation was found when plotting
the pKa value of all phenyl compounds (i.e. 2a–2h, Fig. 4) vs.
the Hammet σm value (Table 2) characteristic of the inductive
effect of the substituent.15 The deviation observed for com-
Fig. 3 (a) Absorption spectra of 2d at different pH in HEPES buffer. (b)
Changes of the absorbance at 540 nm (A540 nm) over the pH range 2–10
with non-linear least-square fit of the experimental data. (c) Emission
spectra of 2d at different pH in HEPES buffer (λexc = 455 nm). (d) Emis-
sion intensity ratio change (F533 nm/F617 nm) over the pH range 2–10.
Table 2 Hammett σm constant, pKa and the excited state pK*a of com-
pounds 2a–2j
a
b
Substituent group
σm
pKa
pK*ac
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
F
Cl
NO2
COOMe
Cl, Cl
OH
OCH3
—
0
7.6
6.4
6.0
4.8
7.2
4.2
6.9
7.1
5.3
8.9
0.5
−1.3
−1.8
−3.3
−0.8
—
0.2
0.3
0.9
—
0.34
0.37
0.71
0.37
0.74
0.12
0.12
—
2j
—
—
a Substituent inductive value taken from ref. 15. b Values obtained
from eqn (1) by non-linear least-square fitting of experimental data.
c Values calculated according to eqn (2) and the experimental data
from Table 1.
Scheme 2 pH modulated switching of 2a.
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obtained by fitting the absorption data. This is surprising
because estimation of the excited state pKa (pK*a, Table 2)
using the Förster cycle equation17 (2):
ꢀ
ꢁ
ArOH
Abs
ArOꢁ
Abs
ArOH
Em
ArOꢁ
Em
hc eν
ꢁ eν
þ eν
ꢁ eν
pKa ꢁ pKa* ¼
ð2Þ
2ð2:303kTÞ
where the terms ˜ν represent the energy of the electronic tran-
sitions for the species ArOH and ArO− respectively, obtained
from the maxima of the absorption and emission spectra at
low and high pH given in Table 1, clearly differentiated strong
photoacids (with pK*a < 0 for 2b–2e) and weaker photoacids
(pK*a > 0 for 2a, 2g–2i).
For all compounds, with the notable exception of 2d, the
fluorescence quantum yield of the neutral form was higher
than that of the anionic form. The fluorescence quantum
yields are rather low (the maximum is for 2j with 9.9%), but
the overall brilliance εΦ of these objects reaches 500 to 4000 L
mol−1 cm−1. Moreover, the study of the variation of fluo-
rescence or fluorescence intensity ratios as a function of pH
showed a good response around physiological pH, especially
for probes 2a, 2g and 2h. With an excitation at the wavelength
of the isosbestic point, large ratio increases of the fluorescence
intensity ratios R (RpH=2/RpH=8 = 70 for the best compound 2a)
were determined between the two extreme pH values. As
shown in Fig. 2d and 3d, the highest variation occurs around
pH = 7, indicating that these dyes are well suited for imaging
of intracellular pH with a ratiometric response in emission.
To determine whether these new dyes can be used in intra-
cellular pH imaging, preliminary experiments were carried
out with probes 2a and 2g using laser confocal scanning
microscopy. These probes were excited at 488 nm or 514 nm
respectively with an argon ion laser and showed well separated
emissions from the protonated and deprotonated forms of the
fluorophores for ratiometric emission experiments. Specifi-
cally, HeLa cells were incubated with 1 µM compound 2a
(resp. 2g) for 5 minutes. Good quality images were obtained
upon excitation at 488 nm (resp. 514 nm) revealing that the
two probes enter the cells immediately after loading (Fig. 5).
Bright-field transmission measurements after dye incubation
confirmed that the cells were viable. At physiological pH (pH =
7.4), the fluorescence could be detected in the green (500/
550 nm for 2a and 535/590 nm for 2g) and the red (600/
650 nm for 2a and 650/710 nm for 2g resp.) emission chan-
Fig. 4 Plot of pKa as a function of σm for phenyl compounds 2a–2h
showing good correlation between the pKa value and the inductive
effect of the substituent. The solid line is the linear fit (r = 0.971).
pound 2e bearing an ester group can be explained by the pres-
ence of a five member hydrogen bonding between the OH
group and the CvO bond of the ester that stabilizes the
phenol and decreases its acidity.
The fluorescence properties of all compounds were also
studied in water: first at low pH, where only the neutral form
was present, and then the pH was increased. Significant differ-
ences were observed, depending on the nature of the substitu-
ent group. For the four compounds having the highest pKa
(2a, 2g, 2h and 2j), as well as 2i, low pH excitation at the wave-
length of the isosbestic point of the absorption-based pH titra-
tion gave rise to a single emission (Fig. 2c, and SI-5 to SI-8†)
attributed to the phenol. This emission was characterized by a
large band with a red shift in the maxima and a large Stokes’
shift increasing from 115 nm for 2a up to 183 nm for 2j with
the presence of an ortho-electron-donating group. When the
pH was increased, the emission of the phenoxide appeared as
a narrow band with a small Stokes’ shift. The only exception
was 2j for which the phenoxide was hardly fluorescent. The
introduction of an electron donating group ortho to the phenol
induced a considerable red shift in the emission of the
phenoxide (up to 40 nm for 2g).
For the other compounds with lower pKa (i.e. 2b–2f), exci-
tation of the neutral form at low pH resulted in dual emission
of phenol and phenoxide (Fig. 3c, and SI-1 to SI-4†). The emis-
sion of the phenol was also characterized by a large band and
a large Stokes’ shift, whereas the emission of the phenoxide
ion was narrow and displayed a smaller Stokes’ shift. The pres-
ence of an electron-withdrawing group ortho to the phenol
induced a blue shift in the emission maxima of the phenol
compared to 2a, but a small increase in the Stokes’ shift.
Contrary to what was recently reported for other phenol
based fluorophores,16 for which two inflexion points corres-
ponding to the ground state pKa and the excited state pKa
(pK*a) were observed, for all compounds 2a–2j a plot of the
maximum intensities vs. pH displayed only one inflexion point
(data not shown). Fitting gave pKa values close to what was
Fig. 5 Confocal images of HeLa cells incubated at pH = 7.4 and with
1 µM 2a for 5 minutes at 37 °C (λexc = 488 nm): (a) bright field; (b) green
emission channel (500–550 nm); (c) red emission channel
(600–650 nm); scale bar: 25 µm.
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a Merck 60 F254 precoated silica gel plate (0.2 mm thickness).
Visualization was performed using a UV lamp. Microwave
syntheses were conducted in a 20 mL sealed tube on a Biotage
Initiator 2.5 single-mode reactor using external IR temperature
control. TCF (2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-
2,5-dihydrofuran) was synthesized according to a literature
procedure.18 Melting points were recorded on a differential
scanning calorimeter operating between 30 and 400 °C. NMR
spectra were recorded at ambient temperature on a standard
spectrometer operating at 500 MHz for 1H and 125 MHz for
13C. Chemical shifts are given in parts per million (δ/ppm) and
are reported relative to tetramethylsilane (1H, 13C) using the
1
residual solvent peaks as the internal standard. H NMR split-
ting patterns are designated as singlet (s), doublet (d), triplet
(t), quartet (q), dd (doublet of doublets) or m (multiplets).
Infrared spectra (IR) were recorded on a FT-IR spectro-
photometer and are reported as wavelength numbers (ν/cm−1).
Fig. 6 (a) Confocal images of HeLa cells incubated at different pH and
with 1 µM 2a for 5 minutes at 37 °C (λexc = 488 nm). From left to right:
bright field, green emission channel (500–550 nm), red emission
channel (600–650 nm); scale bar: 25 µm. (b) Evolution of the emission Low resolution mass spectra were taken on a LC-MS instru-
ratio R = Fgreen/Fred as a function of pH. The black squares represent the
emission ratio F554 nm/F614 nm obtained from the titration (Fig. 1d).
ment and high resolution mass spectra (HRMS) were recorded
on an IF-TOF spectrometer.
Spectroscopy
nels, proving that the two forms exist in agreement with the
measured pKa. We then applied 2a and 2g to HeLa cells grown
at different pH values (6.8 to 7.4) to observe the changes in
fluorescence on the two channels. Bright-field transmission
images confirmed the viability of cells after incubation even
at lower pH (Fig. 6). Different areas of interest (ROI) were
defined. The fluorescence intensity in the two channels in
Absorption spectra (UV-Vis) were recorded on a dual beam
Jasco 670 spectrometer. Data are reported as absorption
maximum wavelength (λmax/nm) and molar extinction coeffi-
cient at the absorption maximum wavelength (ε/L mol−1
cm−1). Fluorescence spectra were performed on a Horiba
Jobin-Yvon Fluorolog-3® spectrofluorimeter equipped with a
red-sensitive Hamamatsu R928 photomultiplier tube. Spectra
were reference corrected for both the excitation source light
intensity variation (lamp and grating) and the emission
spectral response (detector and grating).
Fluorescence quantum yields Φ were measured in diluted
water solutions. Coumarin-153 in methanol (Φ = 0.54)19 or
Cresyl Violet in methanol (Φ = 0.57)19 were used as the refer-
ence for the emission range 500–700 nm and 575–750 nm
respectively. The sample and the reference are excited at the
same wavelength (λexc). The quantum yield relative to the refer-
ence is given by eqn (3):
those ROI was measured to give the values of Fgreen and Fred
.
Similarly, fluorescence intensity ratios R = Fgreen/Fred of 4 for 2a
and 2.7 for 2g were obtained between pH = 6.8 and pH = 7.4
(Fig. 6b and SI-9†) closely matching the values obtained in
solution (Fig. 2 and SI-5†). The data showed that these new
probes strongly depend on pH and could distinguish near-
neutral minor pH fluctuations in cells.
Conclusions
In conclusion, we developed a family of fluorescent push–pull
pH-responsive probes based on TCF as a strong electron accep-
tor group. Small structural variations allowed fine tuning of
the pKa between 4.8 and 8.9. In aqueous solution, the fluo-
rescence showed remarkable changes in ratiometric response
to small pH variations around neutrality with large emission
shifts. The simple structure of these fluorophores allows
further chemical functionalization for the targeting of specific
subcellular compartments. Compound 2h, in particular,
having a pKa of 7.1 and a methoxy group that can easily be
modified, looks rather interesting in that view.
ꢂ
ꢃ
2
SS
SRef
ndS
nRdef
ΦS ¼ ΦRef
ꢀ
ð3Þ
where S is the slope obtained by plotting the integrated area
under the fluorescence emission spectrum vs. the absorbance
at λexc and nd is the refractive index of the solvents. Super-
scripts Ref and S correspond to the reference and the sample
respectively. For each experiment 5 points were recorded, all
corresponding to an absorbance at λexc below 0.1.
General procedure for the Knoevenagel reaction
The aldehyde 1 (1 mmol) and TCF (1.15 mmol, 230 mg) were
dissolved in 10 mL of anhydrous ethanol in a 20 mL micro-
wave vial. 2 drops of piperidine were added. The vial was
Experimental section
Commercially available materials were used as received. sealed with a pressure septum and the mixture was irradiated
Analytical thin-layer chromatography (TLC) was carried out on by focused microwaves at 100 °C for 15 minutes by controlling
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the temperature. After cooling, the precipitate was filtered off C 66.48, H 4.18, N 11.63; found: C 65.95, H 4.23, N 11.71. MS
and washed with ethanol and cyclohexane to give the pure (ES−): m/z 360.1 for [M − H]−. IR (KBr): ν/cm−1 3448, 3235,
desired product.
2231, 2215, 1685, 1575, 1529, 1319, 1216.
2a. Yield: 66% (200 mg), orange solid. m.p. >260 °C. 1H
2f. Yield: 60% (223 mg), orange solid. m.p. >260 °C.
NMR (500 MHz, DMSO-d6): δ/ppm 10.57 (s, 1 H, OH), 7.86 (d, 1H NMR (500 MHz, DMSO-d6): δ/ppm 8.08 (s, 2 H), 7.76 (d,
1 H, J = 16.4 Hz), 7.77 (d, 2 H, J = 8.4 Hz), 6.98 (d, 1 H, J = 1 H, J = 16.6 Hz), 7.21 (d, 1 H, J = 16.6 Hz), 1.77 (s, 6 H, –CH3).
16.4 Hz), 6.86 (d, 1 H, J = 8.4 Hz), 1.74 (s, 6 H, –CH3). 13C NMR 13C NMR (125 MHz, DMSO-d6): δ/ppm 177.7, 175.5, 145.5,
(125 MHz, DMSO-d6): δ/ppm 177.9, 176.5, 162.9, 148.9, 132.9 130.5 (2 C), 127.9, 123.4 (2 C), 115.4, 113.3, 112.8, 112.5, 111.3,
(2 C), 126.3, 117.0 (2 C), 113.5, 112.7, 112.3, 111.8, 99.6, 97.3, 101.9, 99.8, 54.8, 25.7 (2 C). Elemental analysis calcd for
53.8, 25.9 (2 C). Elemental analysis calcd for C18H13N3O2: C18H11Cl2N3O2: C 58.08, H 2.98, N 11.29; found: C 57.92;
C 71.28, H 4.32, N 13.85; found: C 71.92, H 4.46, N 13.88. MS H 3.45; N 11.22. MS (ES−): m/z 370.0 (100%), 371.0 (19%),
(ES−): m/z 302.0 for [M − H]−. IR (KBr): ν/cm−1 3366, 2224, 372.0 (67%) for [M − H]−. IR (KBr): ν/cm−1 3364, 2233, 2212,
1554, 1524, 1380, 1281, 1211, 1162.
1573, 1561, 1525, 1411, 1286.
2b. Yield: 39% (125 mg), brown solid. m.p. >260 °C.
2g. Yield: 59% (188 mg), dark red solid. m.p. >260 °C.
1H NMR (500 MHz, DMSO-d6): δ/ppm 11.02 (s, 1 H, –OH), 7.89 1H NMR (500 MHz, DMSO-d6): δ/ppm 10.26 (s, 1 H), 9.46 (s,
(dd, 1 H, J = 12.6 Hz, J = 1.5 Hz), 7.85 (d, 1 H, J = 16.3 Hz), 7.58 1 H), 7.83 (d, 1 H, J = 16.2 Hz), 7.31 (d, 1 H, J = 2.0 Hz), 7.28
(dd, 1 H, J = 8.4 Hz, J = 1.5 Hz), 7.08 (d, 1 H, J = 16.3 Hz), 7.05 (dd, 1 H, J = 8.0 Hz, J = 2.0 Hz), 6.90 (d, 1 H, J = 16.2 Hz), 6.86
(t, 1 H, J = 8.4 Hz), 1.77 (s, 6 H). 13C NMR (125 MHz, DMSO- (d, 1 H, J = 8.0 Hz), 1.77 (s, 6 H, –CH3). 13C NMR (125 MHz,
d6): δ/ppm 177.8, 175.9, 151.5 (d, 1 C, JC–F = 240 Hz), 150.5 (d, DMSO-d6): δ/ppm 177.8, 176.3, 152.1, 149.3, 146.7, 126.8,
1 C, JC–F = 15 Hz), 147.5, 128.8, 126.8 (d, 1 C, JC–F= 7 Hz), 118.8 125.4, 116.8, 115.6, 113.6, 112.7, 112.1, 111.9, 99.5, 97.0, 53.5,
(d, 1 C, JC–F = 5 Hz), 117.3 (d, 1 C, JC–F = 18 Hz), 113.7, 113.4, 25.9 (2 C). Elemental analysis calcd for C18H13N3O3: C 67.71,
112.6, 111.7, 99.7, 97.9, 54.2, 25.8 (2 C). Elemental analysis H 4.10, N 13.16; found: C 67.60, H 4.11, N 13.15. MS (ES−): m/z
calcd for C18H12FN3O2: C, 67.29; H, 3.76; N, 13.08; found: 318.0 [M − H]−. IR (KBr): ν/cm−1 3444, 3324, 2242, 2224, 1572,
C 67.28; H 4.10; N 13.00. MS (ES−): m/z 320.0 (100%), 321.0 1519, 1444, 1287, 1192, 1149.
(20.6%) for [M − H]−. IR (KBr): ν/cm−1 3420, 2222, 1563, 1531,
1516, 1300, 1282, 1180, 1165, 1101.
2h. Yield: 55% (183 mg), brown solid. m.p. >260 °C.
1H NMR (500 MHz, DMSO-d6): δ/ppm 10.30 (s, 1 H), 7.88 (d,
2c. Yield: 45% (150 mg), orange solid. m.p. >260 °C. 1 H, J = 16.2 Hz), 7.50 (s, 1 H), 7.42 (d, 1 H, J = 8.2 Hz), 7.04 (d,
1H NMR (500 MHz, DMSO-d6): δ/ppm 11.28 (s, 1 H, –OH), 8.03 1 H, J = 16.2 Hz), 6.90 (d, 1 H, J = 8.2 Hz), 3.87 (s, 1 H, –OCH3),
(d, 1 H, J = 1.5 Hz), 7.80 (d, 1 H, J = 16.0 Hz), 7.70 (dd, 1 H, J = 1.79 (s, 6 H, –CH3). 13C NMR (125 MHz, DMSO-d6): δ/ppm
8.4 Hz, J = 1.5 Hz), 7.05 (d, 1 H, J = 16.0 Hz), 7.03 (d, 1 H, J = 177.9, 176.4, 152.7, 149.4, 148.8, 126.8, 126.1, 116.7, 113.5,
8.4 Hz), 1.74 (s, 6 H, –CH3). 13C NMR (125 MHz, DMSO-d6): 113.3, 112.7, 112.5, 111.8, 99.6, 96.9, 56.4, 53.7, 25.9 (2 C).
δ/ppm 177.8, 176.1, 157.7, 147.2, 132.2, 130.8, 127.4, 121.5, Elemental analysis calcd for C19H15N3O3: C 68.46, H 4.54,
117.7, 113.8, 113.4, 112.6, 111.6, 99.8, 98.4, 54.4, 25.8 (2 C). N 12.61; found: C 68.81, H 4.66, N 12.65. MS (ES−): m/z 332.1
Elemental analysis calcd for C18H12ClN3O2: C 64.01, H 3.58, for [M − H]−. IR (KBr): ν/cm−1 3405, 2224, 1568, 1506, 1437,
N 12.44; found: C 64.18, H 3.69, N 12.11. MS (ES−): m/z 336.0 1301, 1287.
1
(100%), 337.0 (19%), 338.0 (30%) for [M − H]−. IR (KBr):
2i. Yield: 73% (258 mg), violet solid. m.p. >260 °C. H NMR
(500 MHz, DMSO-d6): δ/ppm 11.56 (s, 1 H, –OH), 8.84 (d, 1 H,
ν/cm−1 3307, 2237, 2225, 1556, 1513, 1414, 1385, 1298, 1278.
2d. Yield: 51% (178 mg), brown solid. m.p. >260 °C. J = 15.8 Hz), 8.34 (d, 1 H, J = 8.4 Hz), 8.26 (d, 1 H, J = 7.8 Hz),
1H NMR (500 MHz, DMSO-d6): δ/ppm 11.96 (s, 1 H, –OH), 8.50 8.22 (d, 1 H, J = 7.8 Hz), 7.72 (t, 1 H, J = 7.8 Hz), 7.58 (t, 1 H, J =
(d, 1 H, J = 1.8 Hz), 8.08 (dd, 1 H, J = 9.0 Hz, J = 1.8 Hz), 7.86 7.8 Hz), 7.22 (d, 1 H, J = 15.8 Hz), 7.07 (d, 1 H, J = 8.4 Hz), 1.80
(d, 1 H, J = 16.6 Hz), 7.16 (d, 1 H, J = 9.0 Hz), 7.12 (d, 1 H, J = (s, 6 H, –CH3). 13C NMR (125 MHz, DMSO-d6): δ/ppm 178.1,
16.6 Hz), 1.76 (s, 6 H, –CH3). 13C NMR (125 MHz, DMSO-d6): 176.3, 160.3, 144.0, 133.8, 130.5, 129.4, 126.2, 125.1, 123.8,
δ/ppm 177.6, 175.6, 155.9, 146.1, 138.2, 135.2, 128.3, 126.1, 122.8, 122.0, 113.7, 113.3, 112.9, 112.8, 110.2, 99.6, 95.3, 53.4,
120.6, 115.0, 113.3, 112.5, 111.3, 99.9, 99.6, 54.7, 25.8. Elemen- 25.7 (2 C). Elemental analysis calcd for C22H15N3O2: C 74.68,
tal analysis calcd for C18H12N4O4: C 62.07, H 3.47, N 16.09; H 4.28, N 11.89; found: C 73.62, H 4.41, N 11.97. MS (ES−): m/z
found: C 61.92, H 3.62, N 16.11. MS (ES−): m/z 347.0 for 352.1 for [M − H]−. IR (KBr): ν/cm−1 3280, 2228, 2209, 1540,
[M − H]−. IR (KBr): ν/cm−1 3257, 2233, 1579, 1544, 1531, 1311, 1510, 1377, 1354, 1262, 1218, 1189.
1168.
2j. Yield: 87% (307 mg), brown solid. m.p. >260 °C.
2e. Yield: 51% (184 mg), red solid. m.p. >260 °C. H NMR 1H NMR (500 MHz, DMSO-d6): δ/ppm 10.27 (s, 1 H, –OH), 8.27
(500 MHz, DMSO-d6): δ/ppm 11.12 (s, 1 H, –OH), 8.29 (d, 1 H, (s, 1 H), 8.05 (d, 1 H, J = 16.4 Hz), 7.92 (d, 1 H, J = 8.6 Hz), 7.86
J = 2.3 Hz), 8.15 (dd, 1 H, J = 8.4 Hz, J = 2.3 Hz), 7.94 (d, 1 H, (d, 1 H, J = 8.7 Hz), 7.75 (d, 1 H, J = 8.6 Hz), 7.21 (d, 1 H, J =
J = 16.5 Hz), 7.12 (d, 1 H, J = 16.5 Hz,), 7.11 (d, 1 H, J = 8.4 Hz), 16.4 Hz) 7.15 (d, 1 H, J = 2.5 Hz), 7.13 (dd, 1 H, J = 8.7 Hz, J =
3.92 (s, 3 H, –O–CH3), 1.80 (s, 6 H, –CH3). 13C NMR (125 MHz, 2.5 Hz), 1.78 (s, 6 H, –CH3). 13C NMR (125 MHz, DMSO-d6):
DMSO-d6): δ/ppm 177.7, 176.1, 168.4, 163.2, 147.2, 135.1, δ/ppm 177.8, 176.0, 158.8, 148.8, 137.4, 133.7, 131.8, 129.6,
134.5, 126.6, 119.4, 114.1, 115.6, 113.3, 112.5, 111.5, 99.9, 99.1, 127.9, 127.8, 124.6, 120.3, 114.4, 113.4, 112.6, 111.8, 109.9,
54.5, 53.2, 25.8 (2 C). Elemental analysis calcd for C20H15N3O4: 99.9, 98.3, 54.2, 25.7 (2 C). Elemental analysis calcd for
1
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C22H15N3O2: C 74.68, H 4.28, N 11.89; found: C 73.92, H 4.30,
N 11.73. MS (ES−): m/z 352.1 for [M − H]−. IR (KBr): ν/cm−1
3357, 2225, 1560, 1525, 1478, 1375, 1294, 1160.
Notes and references
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