Building a Functionalizable, Potent Chemiluminescent Agent: A Rational Design Study on 6,8-Substituted Luminol Derivatives

Article abstract of DOI:10.1021/acs.joc.1c00890

Luminol is a prominent chemiluminescent (CL) agent, finding applications across numerous fields, including forensics, immunoassays, and imaging. Different substitution patterns on the aromatic ring can enhance or decrease its CL efficiency. We herein report a systematic study on the synthesis and photophysics of all possible 6,8-disubstituted luminol derivatives bearing H, Ph, and/or Me substituents. Their CL responses are monitored at three pH values (8, 10, and 12), thus revealing the architecture with the optimum CL efficiency. The most efficient pattern is used for the synthesis of a strongly CL luminol derivative, bearing a functional group for further, straightforward derivatization. This adduct exhibits an unprecedented increase in chemiluminescence efficiency at pH = 12, pH = 10, and especially at pH = 8 (closer to the biologically relevant conditions) compared to luminol. Complementary work on the fluorescence of the emissive species as well as quantum chemistry computations are employed for the rationalization of the observed results.

Full text of DOI:10.1021/acs.joc.1c00890

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Article
Building a Functionalizable, Potent Chemiluminescent Agent: A
Rational Design Study on 6,8-Substituted Luminol Derivatives
Theodoros Mikroulis, M. Consuelo Cuquerella, Angelo Giussani, Anna Pantelia,
̃
Gemma M. Rodríguez-Muniz, Georgios Rotas, Daniel Roca-Sanjuán,* Miguel A. Miranda,*
and Georgios C. Vougioukalakis*
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ABSTRACT: Luminol is a prominent chemiluminescent (CL)
agent, finding applications across numerous fields, including
forensics, immunoassays, and imaging. Different substitution
patterns on the aromatic ring can enhance or decrease its CL
efficiency. We herein report a systematic study on the synthesis and
photophysics of all possible 6,8-disubstituted luminol derivatives
bearing H, Ph, and/or Me substituents. Their CL responses are
monitored at three pH values (8, 10, and 12), thus revealing the
architecture with the optimum CL efficiency. The most efficient
pattern is used for the synthesis of a strongly CL luminol derivative,
bearing a functional group for further, straightforward derivatization. This adduct exhibits an unprecedented increase in
chemiluminescence efficiency at pH = 12, pH = 10, and especially at pH = 8 (closer to the biologically relevant conditions)
compared to luminol. Complementary work on the fluorescence of the emissive species as well as quantum chemistry computations
are employed for the rationalization of the observed results.
INTRODUCTION
■
The demand for rapid detection and testing in diverse aspects
of human life has driven the invention of molecular sensors
with high sensitivity and specificity. Among them, chemilumi-
nescence (CL)-based sensors comprise an exceptional
category, due to their outstanding properties, including fast
response and a high signal-to-noise ratio. A wide range of
application fields, such as forensics,1 (immuno)assays2
(including even the detection of the lately emerged SARS-
CoV-2),3 bioimaging,4 and theranostics,5 benefit from their
supreme sensing ability. CL agents emit light upon chemical
stimuli, usually triggered by oxidation from reactive oxygen or
nitrogen species.6 The most widely used CL probe is 5-amino-
Figure 1. Phthalhydrazide derivatives and their reported relative CL
efficiency.
2,3-dihydro-1,4-phthalazinedione, commonly known as lumi-
nol, whose CL is triggered by oxidation. Luminol’s strong CL
has been known for almost a century,7 and since its discovery,
numerous works have studied its properties and applications.8
Lf-i, prepared using a similar strategy, have been shown to
The need for molecular probes with optimized CL
greatly enhance the CL up to 20-fold,9 an observation
performance has triggered the design of a variety of substituted
attributed to a “steric gearing” effect. A strong CL response
luminols. To this end, 6-Me and 7-Me9 derivatives (La,b,
Figure 1) exhibit a weaker CL than luminol. On the contrary,
8-alkyl substituted derivatives (prepared using a cycloaddition/
Received: April 17, 2021
aromatization strategy) have been shown to enhance the CL.
In fact, it seems that the less bulky the substituent, the higher
the CL quantum yield (ΦCL) (Lc-e). These findings suggest
that both steric and electronic effects impact luminol’s CL.10
Recently, the 6,8-symmetrical alkyl disubstituted derivatives
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has also been reported for L-012, a related, commercially
available pyridopyridazine-based hydrazide derivative.11 De-
spite these impressive results, the phthalhydrazide derivatives
ABMI and ABEI,12 which are currently used as chemilumi-
nescent probes with functional tips for the attachment on
target molecules, are based on the substituted isoluminol
isomer, rather than luminol itself. These derivatives show a
weaker CL, but are more easily accessible.
which were separated using column chromatography in 13 and
60% yields, respectively. Again, Suzuki methylation or
phenylation of 6a,b afforded the corresponding monosub-
stituted 3-aminophthalimide derivatives 5c−f. The 6-mono-
substituted derivatives 5e,f were further brominated toward
7a,b, followed by coupling with the appropriate boronic
compound toward the nonsymmetrically 4,6-disubstituted
aminophthalimide derivatives 5g,h.
With this knowledge in mind, we herein report the synthesis
of all possible 6,8-substituted luminol derivatives, bearing
methyl, phenyl, and/or hydrogen substituents, as well as their
photophysical characterization, regarding their CL properties.
A novel, modular, reproducible, and highly efficient synthesis
procedure, based on a Suzuki coupling reaction in its key step,
is developed, both for the phthalhydrazides and their
corresponding phthalic acid derivatives. The chemilumines-
cence properties of the former and the fluorescence properties
of the latter have shed light on the CL reaction mechanism of
luminol at pH 10, as well as under milder conditions (pH 8),
more closely resembling the environment of biological
applications. Quantum chemistry computations have also
been carried out, aiming to shed light on and rationalize the
observed experimental results. Equally important, our study
points toward the structural characteristics for the rational
design of a highly efficient chemiluminescent phthalhydrazide,
also bearing an easy-to-functionalize handle, thus paving the
way for the preparation of innovative, tailor-designed, luminol-
based, and strongly chemiluminescent probes with variable
uses.
Once phthalimides 5a−h were synthesized, the desired
luminol derivatives 8a−f were easily acquired via hydrazinol-
ysis15 in moderate to high yields (Scheme 3). Moreover, in
order to fully assess the corresponding CL results, the
fluorescence behavior of the phthalates 10, being the light-
emitting species of the corresponding phthalhydrazides 8 (see
below), had to be investigated. In this respect, complete
hydrolysis of phthalimides 5a−h in strongly alkaline conditions
yielded, upon acidic workup of the reaction mixtures, the
corresponding phthalic acid anhydrides 9a−h. These anhy-
drides were readily hydrolyzable towards the phthalates 10 in
aqueous alkaline media (see Figure S36) and, therefore, they
were used as their precursors in the fluorescence experiments.
Finally, luminol derivative 8i, bearing an easy-to-further-
functionalize hydroxyl moiety, was prepared from 7a according
to the above-described procedure (Scheme 4).
To assess the CL properties of the newly synthesized
luminol derivatives, these were dissolved in aqueous solutions
at pH = 10 or pH = 8, to a final concentration of 7.5 μM. Two
milliliters of each solution was placed in a quartz cuvette, and
the CL reaction was triggered by the addition of H2O2 and
K3[Fe(CN)6], under vigorous stirring. The process was
monitored using a fluorometer running in the time-based
mode (with its own lamp switched off, and 425 nm as the
monitoring wavelength). All of the synthesized luminol
derivatives were screened for chemiluminescence by registering
their CL kinetic traces at pH = 12, pH = 10, and pH = 8
(Figure 2); the absolute quantum yields (ΦCL) were obtained
by comparison with that of the parent luminol at pH = 12
(ΦCL = 0.012)16 (Table 1). The results clearly show that
substitution at positions 6 and/or 8 has a direct influence on
the overall process, irrespective of the pH.
RESULTS AND DISCUSSION
■
The synthesis of the desired ring-substituted luminol
derivatives was initially approached through a published
procedure.13 According to that route, a 3-component
condensation, followed by Pd-catalyzed aromatization with
concomitant de-benzylation, affords the corresponding amino-
phthalimide (Scheme 1). Hydrazinolysis of the latter yields the
a
Scheme 1. Synthesis of 2
At the three pH values, methyl substitution at position 8
(para to the amino group, 8e,a,g) increases the ΦCL
significantly, compared to luminol. An enhanced CL effect
(albeit much weaker) is also observed for the corresponding 8-
phenyl derivatives (8f,h,b). Substitution at position 6 seems to
have a smaller influence on the ΦCL.
These results point toward a more or less independent
substituent effect. A rough estimation shows that 8-Me yields a
marked ΦCL enhancement,17 followed by 8-Ph, 6-Ph, and 6-
Me. The CL quantum yields decrease sharply with the pH
decrease, an anticipated trend, as luminol’s CL is known to
occur in strongly alkaline conditions.8,18 At pH=8, although
ΦCL values are by far lower than those measured at pH = 12
and 10, the CL of the functional derivative 8i is most
impressive, showing a massive ΦCL increase at all pH values
(12, 10, and 8), relative to luminol. In addition, the ΦCL value
obtained for 8a at pH = 12 (0.17) is in good agreement with
that reported in the literature for the same compound at the
same pH (0.18).9 This validates the uniqueness of 8i as a
superior luminol-based chemiluminescent agent. It is quite
remarkable that the CL of 8i at pH = 8 is even higher than that
of parent luminol at pH=12 (ΦCL = 0.017 vs 0.012). This is
highly relevant, given that the use of luminol’s CL is often
a
Reagents and conditions: (i) pTsOH, Ac2O, NMP, 120 °C, 24 h.
(ii) Pd/C, triglyme, 140 °C, 2 days.
desired phthalhydrazide. However, in our hands, phthalimide’s
2 synthesis proved quite troublesome. Even though the first
step proceeded smoothly and afforded precursor 1 in good
yields, the aromatization step exhibited low reproducibility.
We thus opted for an alternative, modular, and reproducible
synthetic strategy. 3-Aminophthalimide 3, prepared easily from
3-aminophthalic acid,14 was used as the starting material.
Bromination, using excess of bromine or NBS, afforded the
4,6-dibrominated derivative 4 in high yield (Scheme 2). Then,
Suzuki coupling with trimethylboroxine or phenylboronic acid
yielded the corresponding symmetrically 4,6-disubstituted 3-
aminophthalimide derivatives 5a,b. Monobromination of 3
afforded a mixture of the 4- and 6-bromophthalimides 6a,b,
B
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a
Scheme 2. Synthesis of the 3-Aminophthalimide Derivatives
a
Reagents and conditions: (i) (a) Ac2O, Δ, (b) sec-BuNH2, AcOH, Δ, (c) H2, Pd/C, MeOH, (ii) Br2, CH3COOH/CH3COONa, r.t. 18 h, (iii)
NBS, DCM, r.t. 30 min, (iv) trimethylboroxine/phenylboronic acid, Pd(PPh3)4.
Scheme 3. Synthesis of the Phthalhydrazide and Phthalic
a
Anhydride Derivatives
a
Reagents and conditions: (i) Hydrazine monohydrate, 110 °C, 24−
48 h. (ii) 15 N KOH, EtOH, Δ, 3 days.
Scheme 4. Synthesis of the Hydroxy Functionalized
a
Luminol Derivative 8i
Figure 2. Typical chemiluminescence decay kinetics displayed by the
luminol derivatives 8a−i in aqueous solutions at pH = 12 (A), pH =
10 (B), and pH = 8 (C). The insets show the traces for the less-
efficient compounds, with a magnified CL scale. The kinetics was
followed for up to 250 s, but it is only shown until 50 s, to facilitate
comparison between derivatives.
a
Reagents and conditions: (i) 4-(hydroxymethyl)phenylboronic acid,
Pd(PPh3)4, K2CO3, H2O, 1,4-dioxane, 105 °C, 18 h. (ii) Hydrazine
monohydrate, 110 °C, 24 h.
hampered, or at least limited, by its low CL quantum yield at
near-neutral conditions, e.g., in biological environments.
C
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Table 1. Chemiluminescence Quantum Yields (ΦCL) of Luminol Derivatives 8 Registered in Aqueous Solutions at pH = 12, 10,
c
and 8 and Fluorescence Quantum Yield (ΦF) of the Corresponding Phthalates 10 at pH = 10
a
b
c
ΦF (3AP)= 0.3.19 The parent anhydride did not hydrolyze cleanly. The ΦCL values are compared to those of luminol at pH = 12.
Mechanistically, it is well established that luminol’s CL
chemiluminescence (the mechanism is discussed below in
more detail). As a result, luminol’s chemiluminescence
depends on the efficiency of both phases. To gain a deeper
insight into the second phase, we measured the fluorescence
quantum yields of some of the phthalates corresponding to our
luminol derivatives (Scheme 5).
The fluorescence quantum yields of isoabsorptive samples of
the phthalates (Amax = 0.1) were measured and compared to
that of 3AP (ΦF (3AP) = 0.3,19 Figure S37); the results are
shown in Table 1. In general, the phthalates’ fluorescence
quantum yields are high and of the same order of magnitude as
that of 3AP. In this range, the 8b-phthalate exhibits a decreased
ΦF (0.16), while 8h nearly doubles the reference value. Clearly,
the high quantum yields of the phthalates reveal that the
oxidation phase of the chemiluminescence is the one limiting
the efficiency of the overall process. In other words, the
consists of several steps, grouped here in two phases: phase 1, a
multistep oxidation procedure of the luminol moiety, leading
to the formation of 3-aminophthalate in its excited singlet state
(13AP*), and phase 2, relaxation of this excited species to its
ground state, in part, emitting fluorescence with a maximum at
425 nm (Scheme 5), thus producing the observed
Scheme 5. Simplified Mechanism for the Luminol
Chemiluminescence Reaction
Figure 3. Scheme of the chemiexcitation of CP-2 in luminol illustrating theoretical quantities that are relevant for interpreting the ΦCL of luminol
derivatives 8.
D
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efficiency of the excited-state formation in the chemiexcitation
phase (rather than the fluorescence quantum yield of the
emitter) determines the CL efficiency.
Table 2. Computed Theoretical Quantities of Luminol
Derivatives 8 and Combined Quantum Yields of Oxidation
and Singlet Chemiexcitation (ΦOS)
To interpret our experimental data, quantum chemistry
computations were carried out focusing on the complex
molecular and electronic-structure transformations that are
involved in phases 1 and 2 of Scheme 5. In terms of chemical
mechanisms, phase 1 depends on the media. In aprotic
solvents, a strong base deprotonates the luminol to a dianion,
which is able to react directly with molecular oxygen. In protic
solvents, in which the details are less clear, the distinct
proposals imply a series of oxidation and deprotonation steps
and reaction with hydrogen peroxide or a superoxide radical.18
Most of the proposals involve the formation of a bicyclic
endoperoxide containing two rings with O−O and NN
bonds, which upon nitrogen extrusion gives rise to a
monocyclic peroxide (CP−2). In contrast to luminol, CP−2
can efficiently transfer the reactivity from the ground to the
excited state with low thermal energies (the so-called
chemiexcitation process).18b,20 This occurs upon O−O bond
breaking, which brings the molecule to the transition state
(TS) and nearby to a region of states crossing (conical
intersections, CI), allowing the production of the emissive
species, 3AP* (see Figure 3).
In aqueous solutions, such a structure is formed at high pH
values (pH > 8 according to the estimations by Yue and
Liu).18a At lower or equal values, double deprotonation is not
achieved and the corresponding cyclic peroxides, neutral (CP)
or monoanionic (CP−), are much less efficient for
chemiexcitation due to their dark path evolutions upon O−
O bond breaking. This correlates with the lower CL yields
measured in this work at pH = 8. We shall focus on the
theoretical analysis at pH = 10, where there are less doubts that
CP−2 is present and that it is the species responsible for the
chemiexcitation as in nonprotic solvents.18b Note also that the
chemiexcitation part is expected to be mostly affected by the
substitution pattern due to the large changes in the electronic
structure of the molecule happening in this step. Therefore, we
will theoretically analyze this part of the complex mechanism in
this work, to interpret the distinct chemiluminescence
efficiencies of the luminol derivatives. Future studies on the
previous oxidation steps shall be also interesting to find any
possible contribution from this part.
a
b
c
d
e
f
compound ΔE‡
ΔEπ→σ
Δρ
ΔE3AP*‑CP
f
ΦOS
luminol
8c
8d
8f
8h
8b
8e
8a
8g
0.14
0.25
0.25
0.15
0.56
0.58
0.42
0.37
0.36
0.38
0.02
0.04
0.13
−0.06
−0.06
0.05
−0.08
−0.1
0.1
−1.35
−1.4
−1.55
−1.19
−1.23
−1.4
−1.39
−1.46
−1.61
0.17
0.22
0.53
0.22
0.23
0.42
0.22
0.24
0.017
0.013
0.018
0.14
0.16
0.22
0.23
0.24
0.17
0.18
0.2
0.020
0.175
0.273
0.577
0.54
b
a
ΔE‡ (in eV): the energy barrier between CP−2 and TS. ΔEπ→σ* (in
eV): the energy gap between the ground and excited states at the TS.
c
Δρ: the charge difference of the carboxylate groups between the TS
d
and CP−2
.
ΔE3AP*‑CP (in eV): the energy difference between CP−2
e
and 3AP*. f: the oscillator strength of the 3AP* for the electronic
f
transition from the excited to the ground state. ΦOS: combined
Φ
ΦF
quantum yield of oxidation and singlet chemiexcitation (ΦOS
=
CL ).
computed with high-level multiconfigurational quantum
chemistry for systems with smaller size (luminol, 8c,h,a).
Accurate CI characterizations for the larger-sized molecules are
prohibitive, due to the limitations of the active space.21 Energy
barriers to such CI points are also very similar (in the range
0.1−0.2 eV) and they are also unable to predict the correct
trends of the ΦCL. Note that a second CI structure is present in
the chemiexcitation region.18b,20b The higher ΦCL of 6,8-
methylated luminol (in comparison to luminol) was previously
attributed to the lower position of such CI,9 facilitated by the
steric gearing of the substituents. However, a more recent
study improving the determination of the CI and the path in
the excited state has shown that the second CI contributes to
the nonradiative decay rather than to the CL process.18b
Overall, these findings indicate that the decomposition
mechanism in the studied molecules is similar and the
computed magnitude analyzed above (energy barriers to the
TS and CI) and their associated errors (0.1−0.2 eV) might not
be enough to discriminate among the experimental yields. In
these conditions, analyses of other magnitudes might be
helpful to disentangle distinct electronic-structure features that
contribute to the trends for comparing all these rather similar
systems. Based on previous knowledge,18a the magnitudes
shown in Figure 3 were identified as relevant candidates for the
analyses of the CL process.
The energy gap between the excited and the ground state at
the TS (ΔEπ→σ*) might be linked to the accessibility of the
excited state, which initially carries the molecule toward 3AP*.
The difference between the charges of the carbonyl groups at
the TS and at the CP−2 (Δρ) shows any charge-transfer
contribution that could activate the O−O bond breaking
(resembling the CTIL or CIEEL mechanisms).20a The energy
difference between 3AP* and CP−2 (ΔE3AP*‑CP) represents the
energy released in such process. This magnitude has been
shown experimentally to correlate with ΦOS.22
Energy barriers between CP−2 and the transition state of the
O−O bond breaking reaction (TS), ΔE‡, were computed,
since this property has been previously shown to allow
distinction between highly efficient and less-efficient CL
systems, with catalyzed and noncatalyzed mechanisms,
respectively. A short range of values was obtained (0.14−
0.24 eV) and no correlation was found between the barriers
and the experimental ΦCL or the combined yield of oxidation
and singlet chemiexcitation (ΦOS), which can be defined as the
quotient between ΦCL and ΦF and represents the efficiency of
the chemical excitation process20a (see Table 2).
No significant differences were computed when changing
between distinct conditions (gas phase, DMSO solution, or
water solution; see Table S1). For a more precise character-
ization, the conical intersection (CI) at the region of the TS,
which mixes the ground electronic state and the excited state
that corresponds to an electron promotion from an occupied π
orbital delocalized over the conjugated system to the
antibonding σ* orbital of the O−O bond (π→σ*), and
opens the path towards the excited electronic state,18b was also
Note, at this point, that while in aprotic media the emissive
species is a structure produced in the excited state after proton
transfer from the amino to the adjacent carboxylate group, this
proton transfer does not happen in aqueous solutions.23
Finally, the oscillator strength for the electronic transition of
3AP* from the excited to the ground state (f) correlates with
E
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the strength of the radiative decay. Note, however, that ΦF
depends on both f and the efficiency of possible nonradiative
decay processes.
conical intersection related to the chemiexcitation step does
not allow further rationalization of the ΦCL trends. Based on
the experimental and theoretical analyses for 8a−g, the 6-aryl-
8-methyl luminol derivative 8i has been prepared, bearing a
hydroxyl function for further straightforward functionalization.
This derivative shows an absolute ΦCL value of 0.017 at pH =
8, which is even higher than that of luminol at pH = 12
(0.012). Hence, 8i appears as an ideal CL reagent for sensing,
tracking, and other possible biologically related applications.
Table 2 compiles the computed data for the above-
mentioned magnitudes together with the combined quantum
yield of oxidation and chemiexcitation (ΦOS). ΔEπ→σ* values
have similar trends as ΔE‡ and, analogously, their role in the
ΦCL is not evident. As can be seen, higher f values are obtained
with phenyl substitution at position 6 (8d,b,g). If compared
with the experimental ΦF, clear disagreements are found. For
example, 6-phenyl substitution is found to enhance f, whereas
this is not observed in the experiments. This suggests that
nonradiative decay paths are affecting each system differently.
Due to this disagreement of data, f values cannot be directly
compared with the ΦF.
EXPERIMENTAL SECTION
■
Materials, Instrumentation, and Methods. All chemicals were
obtained from commercial sources and were used without further
purification. Solvents were dried according to published procedures.25
The course of the reactions was monitored with thin-layer
chromatography (TLC), using aluminum sheets (0.2 mm) coated
with silica gel 60 with a fluorescence indicator (silica gel 60 F254).
Purification of the products was carried out by flash column
chromatography, using silica gel 60 (230−400 mesh). Nuclear
magnetic resonance (NMR) spectra were obtained with a Bruker
Avance 400 MHz or a Varian Mercury 200 MHz spectrometer.
Chemical shifts are reported in ppm. HRMS spectra were recorded in
a QTOF maXis impact (Bruker) spectrometer under electron spray
ionization conditions.
Regarding ΔE3AP*‑CP, it can be seen that adding a phenyl
moiety at position 6 significantly releases more energy in the
formation of the excited species 3AP* than when this position
is occupied by H (compare, for example, luminol and 8d),
while the opposite trend is obtained when the phenyl is added
at position 8 (compare, for example, luminol and 8f).
Methylation does not affect this property that much. Based
on the Δρ magnitude, Table 2 shows that 8-substitution helps
in activating the decomposition via a partial negative charge
donation to the peroxide region (Δρ becomes in general lower
when a substituent is added at this position). On the contrary,
6-substitution decreases the activation (Δρ in general
increases). To compensate for these properties, 6-phenyl and
8-methyl substitution (8g) arises as the optimal solution.
Finally, for further rationalization, we would like to remind
the possible contribution of the previous oxidation steps to the
chemiexcitation from the luminol starting molecule to CP−2
(mentioned above). We would like to point also to other
factors in the chemiexcitation part, such as dynamical aspects
and excited-state path evolution after chemiexcitation, which
are too challenging to study, due to the size and complexity of
the current set of molecules. In small-sized systems, in
particular, 1,2-dioxetane with and without methyl substitution,
dynamics simulations have shown that methylation enhances
the yield of chemiexcitation.24 Note, however, that 1,2-
dioxetane is based on a preferential triplet chemiexcitation,
and for luminol the dynamical effects might be different.
Fluorescence spectra were registered with a Photon Technology
International (PTI) spectrofluorometer (Photon Technology Interna-
tional, Inc., NJ), model LPS-220B, equipped with a 75 W Xe lamp as a
light source and with a monochromator, or with a FLS1000
spectrofluorometer (Edinburgh instruments), equipped with a N-
DMM double-emission monochromator and a N-G11 PMT-980
detector. Monitoring of the CL was performed using the same
spectrofluorometer with its own lamp switched off. The set was run in
the time-based mode with the detection dialed at 425 nm.26 Each
experiment was performed at least 5 times. To trigger the
chemiluminescence, luminols were dissolved in aqueous basic
solutions at pH 12 or 10, or in phosphate-buffered aqueous solutions
at pH 8, giving a final concentration of 7.5 μM. Then, 2 mL of each
sample was introduced in a quartz cuvette, and the CL was prompted
by addition of 2.5 μL of H2O2 (50% w/w) and 8 μL of 75 mM
K3[Fe(CN)6] while vigorously stirring. To do so, the total area under
the decay curve for each compound was measured 5 times, in separate
experiments, and the average value was used to determine the
absolute chemiluminescence quantum yields (ΦCL) by comparison
with luminol at pH = 12, whose absolute ΦCL is 0.012.16 The ΦCL
0
values were calculated as ΦCL= A/AL × ΦCL0, where A corresponds to
the average area of each compound and AL the value of the reference
compound luminol at pH = 12.
CONCLUSIONS
■
The fluorescence quantum yields (ΦF) were measured with the
same setup running in the spectral mode and by comparing the area of
the fluorescence of each sample with the one obtained for the
reference compound 3AP under the same conditions (ΦF (3AP) =
0.319). For that, isoabsorptive samples of 0.1 were prepared at their
UV absorption maximum (ca 300 nm) to ensure that all absorb the
same number of photons. The samples were prepared by dissolving
the corresponding phthalic anhydrides in pH 10 aqueous solutions
and allowing them to hydrolyze to give the final phthalate. This
reaction was monitored by UV spectroscopy as anhydrides’
absorption spectrum present a maximum around 400 nm that
disappears as the phthalate is formed. Moreover, phthalates display a
maximum at 300 nm (Figure S36).
We have synthesized the complete series of 6,8-disubstituted
luminol derivatives, having hydrogen, methyl, and/or phenyl
moieties. Their synthesis has been accomplished through a
new, modular, and reproducible, consecutive bromination/
Suzuki coupling protocol of the parent aminophthalimide,
followed by hydrazinolysis. The corresponding aminophtha-
lates have also been prepared, starting from their anhydrides.
The CL quantum yields of the full series of compounds ΦCL
were measured at pH = 12, 10, and 8, revealing a relatively
independent contribution of each substituent. 8-Me was found
to greatly enhance ΦCL, whereas Ph has a less marked effect.
This could be attributed to the fact that 8-substitution donates
negative charge to the cyclic peroxide moiety, thus assisting the
cleavage of the O−O bond. Substitution at position 6
contributes less, with the 6-phenyl yielding somewhat better
results, probably due to the higher release of energy required to
generate the emissive species from the peroxide intermediate.
Determination of energy barriers related to the O−O bond
breaking of this species or the relative energetic location of the
TD/DFT and CASPT221,27 methods were used herein as
implemented in the Gaussian 1628 and OpenMolcas29 programs,
respectively. All computations were carried out by imposing no
restrictions on the symmetry of the molecule (C1 symmetry). The
CAM-B3LYP functional30 was used with the 6-31G* basis set,
hereafter CAM-B3LYP/6-31G*, to determine minima and the TS.
The open-shell approach, as established in previous studies,31 was
used to correctly determine the geometries and energies. The
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CASPT2 method was used to optimize the CP−2 geometries and to
obtain the CI for selected molecules (see main text). Since no
analytical CASPT2 gradients are available at the moment in the
OpenMolcas code, all CASPT2 optimizations were performed
numerically. A basis set of atomic natural orbitals (ANO) of S-
type32 contracted to C,N,O[3s,2p,1d]/H[2s,1p], hereafter ANO-S-
VDZP, was used for all CASPT2 calculations. An active space of 12
electrons in 10 orbitals was employed. Within the CASPT2
calculations, an imaginary level-shift correction of 0.2 au was used
to minimize the effects of possible intruder states. The CASPT2
standard zeroth-order Hamiltonian was used as originally imple-
mented.33 The core orbitals were frozen in the CASPT2 calculations.
Such a CASPT2 approach has been validated in many different
studies on organic molecules.21,34 The nature of all minima and
transition states was confirmed by computing the corresponding
frequencies. All TD/DFT calculations were performed either in gas
phase or by simulating the solvent (either water or DMSO) using the
PCM model.35 All CASPT2 calculations were performed in gas phase.
ΔE‡ was calculated at the CAM-B3LYP/6-31G* by simulating the
solvent (water) with PCM. ΔEπ‑>σ* was obtained for the TS
computed at the CAM-B3LYP/6-31G* level and as the energy
difference between the ground and excited states computed at the
CASPT2(12,10) level of theory in the gas phase. ΔE3AP*‑CP was
computed at the CAM-B3LYP/6-31G*-PCM(water) level. The f
associated with the fluorescence emission at the 3AP* minimum was
evaluated by performing a TDDFT-B3LYP/6-31G*-PCM(water)
calculation at the 3AP* minimum. For Δρ, Mulliken charges
computed at the DFT-B3LYP/6-31G*-PCM(water) level were used.
Synthesis. Synthesis of Benzyl (2,5,7-trimethyl-1,3-dioxo-
2,3,3a,4,7,7a-hexahydro-1H-isoindol-4-yl)carbamate (1). Accord-
ing to a previously published procedure:13a In a pressure tube was
added benzyl carbamate (1 g, 6.6 mmol, 1.4 equiv) in NMP (1.65
mL) followed by N-methylmaleimide (535 mg, 4.8 mmol),
propionaldehyde (0.48 mL, 6.6 mmol, 1.4 equiv), acetic anhydride
(0.62 mL, 6.6 mmol, 1.4 equiv), and p-toluenesulfonic acid
monohydrate (catalytic, 19.2 mg, 0.1 mmol). The tube was sealed,
submerged in an oil bath, and the mixture was stirred at 120 °C for 48
h. The mixture was then quenched with H2O and extracted with ethyl
acetate. The combined organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo. Purification by column
chromatography, using a gradient of petroleum ether/EtOAc 70:30 to
60:40, afforded 1 as an orange oil (792 mg, 70%). 1H NMR (DMSO-
d6, 200 MHz): δ 7.46-7.28 (m, 5H, Ph), 6.93 (d, J = 9.2 Hz, 1H, NH),
5.34 (s, 1H, CH), 5.11 (s, 2H, CH2), 4.40-4.23 (m, 1H, NHCH),
3.28 (d, J = 6.1 Hz, 1H, NHCHCHCO), 3.08 (t, J = 7.5 Hz, 1H,
CH3CHCHCO), 2.75 (s, 3H, NCH3), 2.49-2.37 (m, 1H, CH3CH),
1.58 (s, 3H, CH3C), 1.25 (d, J = 7.1 Hz, 3H, CH3CH).
Procedure B. Phthalimide 3 (300 mg, 1.38 mmol) was added to a
solution of N-bromosuccinimide (510 mg, 2.89 mmol, 2.1 equiv) in
DMF (4 mL) and the whole was stirred in the dark o/n. Water (80
mL) was added, the mixture was stirred, left for 1 h, filtered, and
washed with water. The filter cake was suspended in methanol (30
mL) and reprecipitated with the addition of water (30 mL). The solid
was dried in vacuo, affording 4 as a yellow powder (430 mg, 83%). 1H
NMR (CDCl3, 200 MHz) δ = 7.79 (s, 1H, ArH), 5.76 (bs, 1H, NH),
4.34−4.06 (m, 1H, NCH), 2.19−1.61 (m, 2H, CH2), 1.44 (d, J = 6.9
Hz, 3H, CHCH3), 0.87 (t, J = 7.4 Hz, 3H, CH3). 13C{1H} NMR
(CDCl3, 50 MHz): δ 168.4, 166.0, 142.2, 141.0, 128.4, 115.6, 113.0,
103.9, 49.2, 26.7, 18.3, 11.3. Molecular ions could not be detected on
ES-MS nor ES-HRMS.
Monobromination of 3-Aminophthalimide 3. Phthalimide 3
(5.33 g, 24.43 mmol) was added to a solution of N-bromosuccinimide
(3.9 g, 21.91 mmol, 0.9 equiv) in dichloromethane (20 mL) and the
resulting mixture was stirred in the dark for 30 min. Then, the solvent
was evaporated and the residue was subjected to column
chromatography (5−10% EtOAc/PE), where the two mono-
brominated isomers 6a and 6b were successively eluted.
4-Amino-5-bromo-2-(sec-butyl)isoindoline-1,3-dione (6a). Yel-
1
low solid (846 mg, 13%). H NMR (CDCl3, 400 MHz): δ 7.64 (d,
J = 7.7 Hz, 1H, H-6), 6.97 (d, J = 7.6 Hz, 1H, H-7), 5.67 (bs, 2H,
NH2), 4.21−4.12 (m, 1H, CH), 2.05−1.68 (m, 2H, CH2), 1.42 (d, J =
6.9 Hz, 3H, CHCH3), 0.85 (t, J = 7.4 Hz, 3H, CH2CH3). 13C{1H}
NMR (CDCl3, 100 MHz): δ 170.0 (C-3), 168.2 (C-1), 142.8 (C-4),
137.8 (C-6), 131.9 (C-7a), 115.2 (C-3a), 112.8 (C-7), 112.2 (C-5),
49.0 (CH), 27.0 (CH2), 18.5 (CHCH3), 11.4 (CH2CH3). ESI-HRMS
m/z for C12H14BrN2O2 [M + H]+: calcd. 297.0233, found 297.0242.
4-Amino-7-bromo-2-(sec-butyl)isoindoline-1,3-dione (6b). Yel-
1
low solid (3.9 g, 60%). H NMR (CDCl3, 200 MHz,): δ 7.42 (d, J
= 8.7 Hz, 1H, H-6), 6.73 (d, J = 8.7 Hz, 1H, H-5), 5.36 (s, 2H, NH2),
4.29−4.08 (m, 1H, CH), 2.14−1.62 (m, 2H, CH2), 1.42 (d, J = 6.9
Hz, 3H, CHCH3), 0.85 (t, J = 7.3 Hz, 3H, CH2CH3). 13C{1H} NMR
(CDCl3, 50 MHz): δ 169.2, 166.8, 144.7, 139.5, 129.3, 122.5, 112.9,
104.6, 49.1, 26.9, 18.5, 11.4. ESI-HRMS m/z for C12H14BrN2O2 [M +
H]+: calcd. 297.0233, found 297.0233.
General Procedure for the Bromination of the 7-Substituted
Aminophthalimides 5e and 5f. The phthalimide (2.8 mmol, 1
equiv) was added in a solution of N-bromosuccinimide (500 mg, 2.8
mmol, 1 equiv) in dichloromethane (6 mL) and the resulting mixture
was stirred in the dark for 2 h. Then, the solvent was evaporated and
the residue was subjected to column chromatography yielding the
corresponding phthalimide.
4-Amino-5-bromo-2-(sec-butyl)-7-methylisoindoline-1,3-dione
(7a). From phthalimide 5e (650 mg). Purification by column
chromatography (0-10% EtOAc, PE). Yellow solid (592 mg, 68%).
1H NMR (CDCl3, 200 MHz): δ 7.47 (s, 1H, H-6), 5.56 (s, 2H, NH2),
4.27−4.09 (m, 1H, CH), 2.50 (s, 3H, ArCH3), 2.14−1.64 (m, 2H,
CH2), 1.44 (d, J = 7.0 Hz, 3H, CHCH3), 0.87 (t, J = 7.4 Hz, 3H,
CH2CH3). 13C{1H} NMR (CDCl3, 100 MHz,): δ 170.0, 168.9, 141.2,
140.2, 128.0, 127.3, 115.3, 112.2, 48.9, 27.1, 18.6, 16.4, 11.5. GC-MS
(EI): 312 (M++2, 33), 310 (M+, 36), 283 (98), 281 (100), 266 (22),
264 (23%). Molecular ions could not be detected on ESI-MS or
HRMS.
4-Amino-5-bromo-2-(sec-butyl)-7-phenylisoindoline-1,3-dione
(7b). From phthalimide 5f (824 mg). Purification by column
chromatography (0-10% EtOAc, PE). Yellow solid (909 mg, 87%).
1H NMR (CDCl3, 200 MHz): δ 7.66 (s, 1H, H-6), 7.58−7.33 (m,
5H, PhH), 5.82 (bs, 2H, NH2), 4.33−4.03 (m, 1H, CH), 2.19−1.60
(m, 2H, CH2), 1.45 (d, J = 7.0 Hz, 3H, CHCH3), 0.88 (t, J = 7.3 Hz,
3H, CH2CH3). 13C{1H} NMR (CDCl3, 50 MHz): δ 169.7, 167.5,
142.1, 139.6, 135.6, 130.6, 129.3, 128.2, 128.0, 126.7, 115.2, 112.3,
48.9, 26.8, 18.4, 11.4. ESI-HRMS m/z for C18H18BrN2O2 [M + H]+:
calcd. 373.0546, found 373.0541.
General Procedure for the Suzuki−Miyaura Coupling. A
mixture of the brominated phthalimide (4 mmol, 1 equiv), the
boronic acid derivative (4.5 mmol, 1.1 equiv for mono- or 9 mmol, 2.3
equiv for dibrominated phthalimides), potassium carbonate (2.5 g, 18
mmol, 4.5 equiv), water (6 mL), and 1,4-dioxane (6 mL) was bubbled
Synthesis of 4-Amino-2,5,7-trimethylisoindoline-1,3-dione (2).
According to a modified published procedure:13a A round-bottom
flask was charged with carbamate 1 (200 mg, 0.58 mmol) in triglyme
(2.5 mL) and then 10% Pd/C was added (10 mol %). A CaCl2 tube
was attached, and the mixture was submerged in an oil bath and
stirred at 140 °C for 24 h. Palladium was removed via filtration
through a Celite pad with EtOAc, and the filtrate was washed with
H2O and brine. The organic phase was dried over anhydrous Na2SO4,
filtered, and concentrated in vacuo, and the residue was subjected to
column chromatography. The product was eluted with P.E./EtOAc
90:10 and was acquired as a yellow solid after condensation (62 mg,
1
52% max.large yield variations). H NMR (CDCl3, 200 MHz): δ
7.01 (s, 1H, ArH), 5.11 (bs, 2H, NH2), 3.06 (s, 1H, NCH3), 2.46 (s,
3H, CH3), 2.17 (s, 3H, CH3).
Synthesis of 4-Amino-5,7-dibromo-2-(sec-butyl)isoindoline-1,3-
dione (4). Procedure A. A mixture of phthalimide 3 (1.5 g, 6.88
mmol), sodium acetate (1.13 g, 13.76 mmol), and acetic acid (17
mL) was stirred for 30 min. Then, a solution of bromine (0.71 mL,
13.76 mmol, 2 equiv) in acetic acid (6 mL) was added dropwise and
the resulting solution was stirred for 18 h. The mixture was poured
into ice/water mixture (100 mL) and the precipitate was filtered,
washed with water, and dried in vacuo, leaving 4 as yellow powder
(2.39 g, 92%).
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with argon for 30 min. Tetrakis(triphenylphosphine)-palladium(0)
(69 mg, 0.06 mmol, 0.015 equiv) was added and bubbling continued
for another 15 min. Then, the mixture was stirred under an inert
atmosphere, in an oil bath at 105 °C for 18 h. After cooling, the
solvent was partially evaporated, water (60 mL) and EtOAc (150 mL)
were added, the phases were separated, the organic phase was washed
with 1N HCl (40 mL) and brine (40 mL), dried (MgSO4), and the
solvent was evaporated. The residue was further treated as indicated
in each case, affording the corresponding substituted derivative.
4-Amino-2-(sec-butyl)-5,7-dimethylisoindoline-1,3-dione (5a).
From dibromophthalimide 4 (1.5 g) and trimethylboroxine (3.5 M
in THF, 2.6 mL). Purification with column chromatography (10-30%
CH2), 1.47 (d, J = 6.9 Hz, 3H, CHCH3), 0.91 (t, J = 7.4 Hz, 3H,
CH2CH3). 13C{1H} NMR (CDCl3 50 MHz): δ 170.3, 168.2, 144.7,
137.4, 136.8, 129.9, 129.2, 127.9, 127,7, 127.2, 121.3, 111.1, 48.5,
26.8, 18.4, 11.3. ESI-HRMS m/z for C18H19N2O2 [M + H]+: calcd.
295.1441, found 295.1442.
4-Amino-2-(sec-butyl)-7-methyl-5-phenylisoindoline-1,3-dione
(5g). From bromophthalimide 7a (1.2 g) and phenylboronic acid
(553 mg). Purification with column chromatography (10% EtOAc/
1
P.E.). Yellow solid (679 mg, 55%). H NMR (CDCl3, 400 MHz): δ
7.50−7.35 (m, 5H, PhH), 7.12 (s, 1H, H-6), 5.30 (bs, 2H, NH2),
4.31−4.13 (m, 1H, CH), 2.54 (s, 3H, ArCH3), 2.06 (ddq, J = 14.7,
9.4, 7.4 Hz, 1H, CH2), 1.84−1.69 (m, 1H, CH2), 1.45 (d, J = 7.0 Hz,
3H, CHCH3), 0.90 (t, J = 7.4 Hz, 3H, CH2CH3). 13C{1H} NMR
(CDCl3, 100 MHz): δ 170.7, 169.3, 141.3, 138.5, 137.0, 134.0, 129.2,
128.7, 128.3, 127.7, 126.4, 111.6, 48.5, 27.0, 18.6, 16.5, 11.4. ESI-
HRMS m/z for C19H21N2O2 [M + H]+: calcd. 309.1598, found
309.1599.
1
EtOAc/P.E.). Yellow solid (552 mg, 56%). H NMR (CDCl3, 200
MHz): δ 7.01 (s, 1H, H-6), 5.15 (bs, 2H, NH2), 4.26−3.94 (m, 1H,
CH), 2.46 (s, 3H, 7-CH3), 2.17 (s, 3H, 5-CH3), 2.10−1.61 (m, 2H,
CH2) 1.41 (d, J = 7.0 Hz, 3H, CH2CH3), 0.85 (t, J = 7.4 Hz, 3H,
CHCH3). 13C{1H} NMR (CDCl3, 50 MHz): δ 170.9, 169.5, 142.3,
138.0, 129.5, 126.4, 126.3, 111.0, 48.3, 27.0, 18.6, 16.8, 16.4, 11.4.
4-Amino-2-(sec-butyl)-5-methyl-7-phenylisoindoline-1,3-dione
(5h). From bromophthalimide 7b (1.5 g) and trimethylboroxine (3.5
M in THF, 1.3 mL). Purification with column chromatography (10%
+
ESI-HRMS m/z for C14H19N2O2 [M + H]+: calcd. 247.1447, found
247.1429.
1
EtOAc/P.E.). Yellow solid (629 mg, 51%). H NMR (CDCl3, 200
4-Amino-2-(sec-butyl)-5,7-diphenylisoindoline-1,3-dione (5b).
From dibromophthalimide 4 (1.5 g) and phenylboronic acid (1.1
g). No further treatment needed. Yellow solid (1.4 g, 95%). 1H NMR
(CDCl3, 200 MHz): δ 7.65−7.31 (m, 11H, ArH), 5.59 (bs, 2H,
NH2), 4.37−4.10 (m, 1H, CH), 2.24−1.65 (m, 2H, CH2), 1.48 (d, J =
7.0 Hz, 3H, CHCH3) 0.92 (t, J = 7.4 Hz, 3H, CH2CH3). 13C{1H}
NMR (CDCl3, 50 MHz): δ 170.7, 168.1, 142.4, 138.1, 136.8, 136.7,
134.0, 130.1, 129.4, 129.4, 128.8, 128.6, 128.1, 127.9, 126.6, 111.9,
48.8, 26.9, 18.6, 11.5. ESI-HRMS m/z for C24H22NaN2O2+ [M+Na]+:
calcd. 393.1573, found 393.1572.
MHz): δ 7.58−7.34 (m, 5H, PhH), 7.26 (s, 1H, H-6), 5.27 (bs, 2H,
NH2), 4.33−3.99 (m, 1H, CH), 2.26 (s, 3H, ArCH3), 2.17−1.62 (m,
2H, CH2), 1.44 (d, J = 7.0 Hz, 3H, CHCH3), 0.88 (t, J = 7.4 Hz, 3H,
CH2CH3). 13C{1H} NMR (CDCl3, 50 MHz): δ 170.9, 168.2, 143.4,
137.7, 137.0, 130.1, 129.6, 129.4, 128.0, 127.8, 125.4, 111.4, 48.6,
26.9, 18.6, 17.0, 11.5. ESI-HRMS m/z for C19H20N2NaO2 [M + Na]+:
calcd. 331.1417, found 331.1417.
4-Amino-2-(sec-butyl)-5-(4-(hydroxymethyl)phenyl)-7-methyli-
soindoline-1,3-dione (5i). From bromophthalimide 7a (1.2 g) and 4-
(hydroxymethyl)phenylboronic acid (684 mg). Purification with
column chromatography (10% EtOAc/P.E.). Yellow solid (1.06 g,
4-Amino-2-(sec-butyl)-5-methylisoindoline-1,3-dione (5c). From
bromophthalimide 6a (1.2 g) and trimethylboroxine (3.5 M in THF,
1.3 mL). Purification with column chromatography (20% EtOAc/
1
78%). H NMR (CDCl3, 400 MHz): δ 7.50 (app d, J = 8.3 Hz, 2H,
1
H-3′), 7.43 (app d, J = 8.3 Hz, 2H, H-2′), 7.12 (s, 1H, H-6), 5.30 (bs,
2H, NH2), 4.77 (s, 2H, OCH2), 4.27−4.16 (m, 1H, CH), 2.55 (s, 3H,
ArCH3), 2.14−1.99 (m, 1H, CHCH2), 1.84−1.69 (m, 2H, OH,
CHCH2), 1.46 (d, J = 7.0 Hz, 3H, CHCH3), 0.90 (t, J = 7.4 Hz, 3H,
CH2CH3). 13C{1H} NMR (CDCl3, 50 MHz,) δ 170.8, 169.4, 141.3,
141.2, 138.5, 136.3, 133.8, 129.0, 127.8, 127.7, 126.5, 111.6, 64.9,
48.6, 27.1, 18.7, 16.6, 11.5. ESI-HRMS m/z for C20H22N2NaO3 [M +
Na]+: calcd. 361.1522, found 361.1520.
P.E.). Yellow solid (297 mg, 32%). H NMR (CDCl3, 200 MHz): δ
7.27 (d, J = 7.4 Hz, 1H H-7), 7.06 (d, J = 7.3 Hz, 1H, H-5), 5.23 (bs,
2H, NH2), 4.37−3.97 (m, 1H, CH), 2.23 (s, 3H, ArCH3), 2.15−1.66
(m, 2H, -CH2), 1.44 (d, J = 6.9 Hz, 3H, CHCH3) 0.88 (t, J = 7.4 Hz,
3H, CH2CH3). 13C{1H} NMR (CDCl3, 50 MHz): δ 171.1, 169.0,
143.9, 135.2, 130.6, 129.3, 112.4, 111.1, 48.6, 27.0, 18.6, 17.1, 11.4.
+
ESI-HRMS m/z for C13H16NaN2O2 [M+Na]+: calcd. 255.1104,
found 255.1104.
General Procedure for the Synthesis of Phthalhydrazides
(8). A suspension of phthalimide (0.4 mmol) in a large excess of
hydrazine monohydrate (5 mL) was heated in an oil bath at 110 °C
under Ar for 18 h. After cooling, the pH was adjusted to 2 using
concentrated hydrochloric acid and the precipitate was filtered and
washed with water, affording the corresponding phthalhydrazide.
5-Amino-6,8-dimethyl-2,3-dihydrophthalazine-1,4-dione (8a).
From phthalimide 5a (99 mg). Yellow solid (66 mg, 80%).13a 1H
NMR (DMSO-d6, 200 MHz): δ 11.08 (bs, 2H, NHNH), 7.21 (s, 1H,
H-7), 6.89 (bs, 2H, NH2), 2.48 (s, 3H, 8-CH3), 2.12 (s, 3H, 6-CH3).
5-Amino-6,8-diphenyl-2,3-dihydrophthalazine-1,4-dione (8b).
From phthalimide 5b (148 mg). Yellow solid (112 mg, 85%).13a 1H
NMR (DMSO-d6, 400 MHz): δ 9.67 (bs, 2H, NHNH), 8.11−6.64
(m, 13H, PhH, H-7, NH2). 13C{1H} NMR (DMSO-d6, 100 MHz): δ
162.0, 151.3, 147.1, 142.7, 138.4, 137.5, 129.3, 129.1, 128.8, 127.8,
127.2, 126.8, 125.9, 125.8, 122.5, 111.6. ESI-HRMS m/z for
C20H16N3O2 [M + H]+: calcd. 330.1237, found 330.1239.
5-Amino-6-methyl-2,3-dihydrophthalazine-1,4-dione (8c). From
phthalimide 5c (93 mg). Pale-yellow solid (70 mg, 92%).13b 1H NMR
(DMSO-d6, 200 MHz): δ 11.25 (br 2H, NHNH), 7.41 (d, J = 7.9 Hz,
1H, H-8), 6.95 (d, J = 7.7 Hz, 1H, H-7), 4.00 (bs, 2H, NH2), 2.16 (s,
3H, CH3). 13C{1H} NMR (DMSO-d6, 50 MHz) δ = 161.8, 151.7,
148.6, 134.9, 124.5, 124.2, 110.1, 109.5, 17.7. ESI-HRMS, m/z for
C9H10N3O2 [M + H]+: calcd. 192.0768, found 192.0756.
4-Amino-2-(sec-butyl)-5-phenylisoindoline-1,3-dione (5d). From
bromophthalimide 6a (1.2 g) and phenylboronic acid (553 mg). No
further treatment needed. Yellow solid (965 mg, 82%). 1H NMR
(CDCl3, 200 MHz): δ 7.55−7.37 (m, 5H, PhH), 7.34 (d, J = 7.3 Hz,
1H, H-6), 7.16 (d, J = 7.3 Hz, 1H, H-7), 5.42 (bs, 2H, NH2), 4.34−
4.02 (m, 1H, CH), 2.20−1.59 (m, 2H, CH2), 1.45 (d, J = 7.0 Hz, 3H,
CHCH3), 0.89 (t, J = 7.5 Hz, 3H, CH2CH3). 13C{1H} NMR (CDCl3,
50 MHz): δ 170.9, 168.7, 142.9, 136.9, 135.7, 133.7, 131.8, 129.3,
128.7, 128.4, 112.4, 111.5, 48.7, 27.0, 18.6, 11.4. ESI-HRMS m/z for
+
C18H18N2NaO2 [M+Na]+: calcd. 317.1260, found 317.1259.
4-Amino-2-(sec-butyl)-7-methylisoindoline-1,3-dione (5e). From
bromophthalimide 6b (1.2 g) and trimethylboroxine (3.5 M in THF,
1.3 mL). Purification with column chromatography (10% EtOAc/
1
P.E.). Yellow solid (650 mg, 70%). H NMR (CDCl3, 400 MHz): δ
7.13 (dq, J = 8.4, 0.6 Hz, 1H, H-6), 6.73 (dd, J = 8.4, 0.3 Hz, 1H, H-
5), 5.13 (s, 2H, NH2), 4.22−4.13 (m, 1H, CH), 2.50 (s, 3H, ArCH3),
2.02 (ddq, J = 7.4, 9.3, 13.8 Hz, 1H, CH2), 1.74 (ddq, J = 6.1, 7.4,
13.5 Hz, 1H, CH2), 1.43 (d, J = 7.0 Hz, 3H, CHCH3), 0.87 (t, J = 7.4
Hz, 3H, CH2CH3). 13C{1H} NMR (CDCl3, 100 MHz): δ 170.6 (C-
3), 169.6 (C-1), 143.5 (C-4), 137.9 (C-6), 128.5 (C-7a), 126.8 (C-7),
121.2 (C-5), 111.4 (C-3a), 48.5 (C-2′), 27.03 (C-3′), 18.6 (C-1′),
+
16.6 (ArCH3), 11.5 (C-4′). ESI-HRMS m/z for C13H16N2NaO2 [M
+Na]+: calcd. 255.1104, found 255.1103.
4-Amino-2-(sec-butyl)-7-phenylisoindoline-1,3-dione (5f). From
bromophthalimide 6b (1.2 g) and phenylboronic acid (553 mg).
Purification with column chromatography (20% EtOAc/P.E.). Yellow
5-Amino-6-phenyl-2,3-dihydrophthalazine-1,4-dione (8d). From
1
phthalimide 5d (118 mg). Pale-yellow solid (93 mg, 92%). H NMR
1
solid (1.1 g, 92%). H NMR (CDCl3, 200 MHz): δ 7.61−7.38 (m,
(DMSO-d6, 400 MHz): δ 7.63−7.29 (m, 6H, H-6, PhH), 7.09 (d, J =
7.9 Hz, 1H, H-8), 7.30−6.98 (bs, 2H, NH2). 13C{1H} NMR (DMSO-
d6, 100 MHz): δ 161.7, 151.6, 147.3, 138.0, 135.1, 129.1, 128.8, 127.9,
5H, PhH), 7.34 (d, J = 8.6 Hz, 1H, H-6), 6.85 (d, J = 8.5 Hz, 1H, H-
5), 5.47 (bs, 2H, NH2), 4.37−4.06 (m, 1H, CH), 2.21−1.64 (m, 2H,
H
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Article
127.7, 126.0, 111.2, 110.0. ESI-HRMS, m/z for C14H12N3O2 [M +
H]+: calcd. 254.0924, found 254.0923.
5-Amino-8-methyl-2,3-dihydrophthalazine-1,4-dione (8e). From
phthalimide 5e (93 mg). Pale-yellow solid (57 mg, 74%).13b 1H NMR
(DMSO-d6, 400 MHz): δ 11.03 (bs, 2H, NHNH), 7.24 (d, J = 8.4
Hz, 1H, H-6), 6.80 (d, J = 8.4 Hz, 1H, H-7), 2.52 (s, 3H, CH3).
13C{1H} NMR (DMSO-d6, 100 MHz): δ 161.3, 153.3, 149.2, 137.5,
4-Amino-5-methylisobenzofuran-1,3-dione (9c). From phthali-
mide 5c (70 mg). Purification with column chromatography (10%
EtOAc/P.E.). Yellow solid (25 mg, 47%). H NMR (CDCl3, 200
MHz): δ 7.45 (d, J = 7.3 Hz, 1H, H-6), 7.19 (d, J = 7.3 Hz, 1H, H-7),
5.33 (bs, 2H, NH2), 2.29 (s, 3H, CH3). 13C{1H} NMR (CDCl3, 50
MHz): δ 165.1, 163.4, 145.6, 137.8, 130.7, 129.5, 114.7, 110.4, 17.2.
ESI-HRMS m/z for C9H8NO3 [M + H]+: calcd. 178.0499, found
178.0500.
1
124.7, 121.4, 116.9, 111.2, 22.0. ESI-HRMS, m/z for C9H10N3O2 [M
+ H]+: calcd. 192.0768, found 192.0758.
4-Amino-5-phenylisobenzofuran-1,3-dione (9d). From phthali-
mide 5d (88 mg). Purification with column chromatography (10%
5-Amino-8-phenyl-2,3-dihydrophthalazine-1,4-dione (8f). From
phthalimide 5f (118 mg). Yellow solid (96 mg, 95%). 1H NMR
(DMSO-d6, 400 MHz): δ 11.28 (bs, 2H, NHNH), 7.32−7.13 (m, 6H,
PhH, H-6), 6.95 (d, J = 8.6 Hz, 1H, H-7). 13C{1H} NMR (DMSO-d6,
100 MHz): δ 161.5, 151.4, 149.8, 143.0, 137.4, 129.3, 126.8, 126.2,
125.7, 123.1, 116.4, 111.1. ESI-HRMS, m/z for C14H10N3O2 [M-H]−:
calcd. 252.0778, found 252.0762
5-Amino-8-methyl-6-phenyl-2,3-dihydrophthalazine-1,4-dione
(8g). From phthalimide 5g (123 mg). Yellow solid (53 mg, 50%). 1H
NMR (DMSO-d6, 400 MHz): δ 11.25 (bs, 2H, NHNH), 7.52−7.39
(m, 5H, PhH), 7.22 (s, 1H, H-7), 7.02 (bs, 2H, NH2), 2.57 (s, 3H,
CH3). 13C{1H} NMR (DMSO-d6, 101 MHz): δ 162.2, 153.5, 146.3,
139.2, 138.3, 129.5, 129.3, 128.6, 128.2, 124.6, 122.2, 112.5, 22.5. ESI-
HRMS, m/z for C15H14N3O2 [M + H]+: calcd. 268.1081, found
268.1066.
5-Amino-6-methyl-8-phenyl-2,3-dihydrophthalazine-1,4-dione
(8h). From phthalimide 5h (123 mg). Yellow solid (84 mg, 79%). 1H
NMR (DMSO-d6, 400 MHz): δ 11.31 (bs, 2H, NHNH), 7.29−7.17
(m, 6H, PhH, H-7), 2.18 (s, 3H, CH3). 13C{1H} NMR (DMSO-d6,
101 MHz): δ 162.0, 151.5, 148.3, 143.1, 138.4, 129.3, 126.8, 125.7,
125.6, 123.7, 121.0. ESI-HRMS, m/z for C15H14N3O2 [M + H]+:
calcd. 268.1081, found 268.1080.
1
EtOAc/P.E.). Yellow solid (22 mg, 31%). H NMR (CDCl3, 400
MHz): δ 7.58−7.39 (m, 6H, 5-PhH, H-6), 7.31 (d, J = 7.3 Hz, 1H, H-
7), 5.50 (s, 2H, NH2). 13C{1H} NMR (CDCl3, 101 MHz): δ 165.0,
163.2, 144.8, 138.1, 135.9, 135.0, 130.7, 129.7, 129.1, 128.7, 114.7,
111.0. ESI-HRMS m/z for C15H12NO4 [M + MeOH − H]−: calcd.
270.0772, found 270.0778.
4-Amino-7-methylisobenzofuran-1,3-dione (9e). From phthali-
mide 5e (70 mg). Purification with column chromatography (20%
1
EtOAc/P.E.). Yellow solid (13 mg, 25%). H NMR (CDCl3, 400
MHz), δ = 7.34 (d, J = 8.4 Hz, 1H, H-6), 6.89 (d, J = 8.4 Hz, 1H, H-
5), 5.21 (s, 2H, NH2), 2.54 (s, 3H, CH3). 13C{1H} NMR (CDCl3,
101 MHz), δ = 164.8, 163.6, 145.3, 140.0, 129.1, 128.0, 122.3, 110.5,
16.7. ESI-HRMS m/z for C9H8NO3 [M + H]+: calcd. 178.0499,
found 178.0504.
4-Amino-7-phenylisobenzofuran-1,3-dione (9f). From phthali-
mide 5f (88 mg). Purification with column chromatography (20%
1
EtOAc/P.E.). Yellow solid (32 mg, 45%). H NMR (CDCl3, 400
MHz), δ = 7.57−7.06 (m, 6H, PhH, H-6), 7.04 (d, J = 8.5 Hz, 1H, H-
5), 5.43 (s, 2H, NH2). 13C{1H} NMR (CDCl3, 101 MHz), δ = 164.8,
162.5, 146.2, 139.6, 135.6, 132.5, 129.2, 128.7, 128.5, 126.7, 122.3,
111.0. ESI-HRMS m/z for C14H10NO3 [M + H]+: calcd. 240.0655,
found 240.0638.
5-Amino-6-(4-(hydroxymethyl)phenyl)-8-methyl-2,3-dihydroph-
thalazine-1,4-dione (8i). From phthalimide 5i (135 mg). Yellow
solid (113 mg, 95%). 1H NMR (400 MHz, MeOD-d4): δ 7.77 (s, 1H,
H-7), 7.59 (app d, J = 8.5 Hz, 2H, H-3′), 7.52 (app d, J = 8.4 Hz, 2H,
4-Amino-7-methyl-5-phenylisobenzofuran-1,3-dione (9g). From
phthalimide 5g (93 mg). Purification with column chromatography
1
(20% EtOAc/P.E.). Yellow solid (22 mg, 29%). H NMR (CDCl3,
1
H-2′), 4.72 (s, 2H, OCH2), 2.89 (s, 3H, ArCH3). H NMR (200
400 MHz) δ = 7.53−7.37 (m, 6H, PhH, H-6), 5.43 (bs, 2H, NH2),
2.32 (s, 3H, CH3). 13C{1H} NMR (CDCl3, 101 MHz) δ = 165.3,
162.5, 145.0, 139.8, 135.7, 132.2, 129.2, 128.5, 128.4, 124.4, 110.7,
17.1. ESI-HRMS, m/z for C15H12NO4 [M+H2O-H]−: calcd.
270.0772, found 270.0770.
MHz, DMSO-d6) δ 7.43 (app s, 4H, H-2′,3′), 7.28 (s, 1H, H-7), 4.55
(s, 2H, OCH2), 2.59 (s, 3H, ArCH3). 13C{1H} NMR (50 MHz,
DMSO-d6): δ 161.7, 153.1, 148.5, 145.3, 142.1, 138.7, 136.0, 128.5,
127.1, 124.1, 122.2, 112.3, 62.6, 22.0. ESI-HRMS m/z for
C16H15N3NaO3 [M+Na]+: calcd. 320.1006, found 320.1003.
4-Amino-5-methyl-7-phenylisobenzofuran-1,3-dione (9h). From
phthalimide 5h (93 mg). Purification with column chromatography
General Procedure for the Synthesis of Phthalic Acid
Anhydrides (9). A large excess of aqueous potassium hydroxide
(15 N, 3 mL) was added to a solution of the phthalimide (0.3 mmol)
in ethanol (3 mL), and the mixture was heated in an oil bath to reflux
and stirred at that temperature for 3 days. Subsequently, ethanol was
removed under reduced pressure, water (10 mL) was added, and the
resulting solution was extracted with DCM (3 × 15 mL). The
aqueous phase was collected and acidified with 1 N hydrochloric acid
until pH=3. Immediately after acidification, the aqueous phase turned
fluorescent green, indicative of the anhydride formation. The solution
was extracted with ethyl acetate (3 × 20 mL) and the combined
organic phase was dried (MgSO4) and concentrated in vacuo. The
residue was further treated as indicated in each case below, affording
the corresponding anhydride.
1
(20% EtOAc/P.E.). Yellow solid (25 mg, 33%). H NMR (CDCl3,
400 MHz) δ = 7.54−7.41 (m, 5H, PhH), 7.30 (s, 1H, H-6), 5.37 (bs,
2H, NH2), 2.56 (s, 3H, CH3). 13C{1H} NMR (CDCl3, 101 MHz) δ =
165.0, 163.4, 143.2, 140.5, 136.0, 135.4, 129.6, 129.0, 128.7, 128.7,
127.0, 110.7, 16.6. ESI-HRMS, m/z for C15H12NO3 [M + H]+: calcd.
254.0812, found 254.0811.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c00890.
1H and 13C NMR spectra for all the synthesized
compounds, as well as UV spectra from the hydrolysis
of anhydrides 9a−h; fluorescence spectra of phthalates
10a−e, 10h and 3AP; comparative CL kinetics of
luminol, 8a, 8i at given pH; energy barrier calculations
between CP−2 and TS of 8a−h (PDF)
4-Amino-5,7-dimethylisobenzofuran-1,3-dione (9a). From
phthalimide 5a (74 mg). No further treatment needed. Yellow solid
(47 mg, 82%). 1H NMR (DMSO-d6, 200 MHz): δ 7.34 (s, 1H, H-6),
6.38 (s, 2H, NH2), 2.39 (s, 3H, 7-CH3), 2.21 (s, 3H, 5-CH3).
13C{1H} NMR (DMSO-d6, 50 MHz): δ 164.4, 163.5, 145.2, 139.8,
131.9, 126.4, 124.8, 107.7, 17.3, 15.9. ESI-HRMS, m/z for C10H8NO3
[M − H]−: calcd. 190.0509, found 190.0497.
4-Amino-5,6-diphenylisobenzofuran-1,3-dione (9b). From
phthalimide 5b (111 mg). Purification with column chromatography
AUTHOR INFORMATION
■
1
(20% EtOAc/P.E.). Yellow solid (36 mg, 38%). H NMR (CDCl3,
Corresponding Authors
400 MHz): δ 7.58−7.38 (m, 11H, PhH, H-6), 5.61 (s, 2H, NH2).
13C{1H} NMR (CDCl3, 101 MHz): δ 165.1, 162.3, 144.1, 140.1,
135.7, 135.5, 135.4, 132.2, 129.7, 129.2, 129.2, 128.7, 128.5, 125.6,
111.3. ESI-HRMS, m/z for C20H14NO3 [M + H]+: calcd. 316.0968,
found 316.0942.
Daniel Roca-Sanjuán − Instituto de Ciencia Molecular,
̀
̀
Universitat de Valencia, 46071 Valencia, Spain;
orcid.org/0000-0001-6495-2770; Email: daniel.roca@
uv.es
I
https://doi.org/10.1021/acs.joc.1c00890
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
Miguel A. Miranda − Instituto de Tecnología Química,
035123-I). D.R.-S. is also thankful to the MICINN for a
“Ramón y Cajal” grant (Ref RYC-2015-19234) and to the
BBVA Foundation for a 2019 Leonardo Grant for Researchers
and Cultural Creators.
̀
̀
UPV−CSIC, Universitat Politecnica de Valencia, 46022
̀
Valencia, Spain; orcid.org/0000-0002-7717-8750;
Email: mmiranda@qim.upv.es
Georgios C. Vougioukalakis − Laboratory of Organic
Chemistry, Department of Chemistry, National and
Kapodistrian University of Athens, 15771 Athens, Greece;
orcid.org/0000-0002-4620-5859; Email: vougiouk@
chem.uoa.gr
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Authors
Theodoros Mikroulis − Laboratory of Organic Chemistry,
Department of Chemistry, National and Kapodistrian
University of Athens, 15771 Athens, Greece
M. Consuelo Cuquerella − Instituto de Tecnología Química,
̀
̀
UPV−CSIC, Universitat Politecnica de Valencia, 46022
̀
Valencia, Spain; orcid.org/0000-0002-2640-1123
Angelo Giussani − Instituto de Ciencia Molecular, Universitat
de Valencia, 46071 Valencia, Spain; orcid.org/0000-
0002-9452-7641
Anna Pantelia − Laboratory of Organic Chemistry,
Department of Chemistry, National and Kapodistrian
University of Athens, 15771 Athens, Greece
̀
̀
Gemma M. Rodríguez-Muniz − Instituto de Tecnología
̃
̀
̀
Química, UPV−CSIC, Universitat Politecnica de Valencia,
̀
46022 Valencia, Spain; orcid.org/0000-0001-8989-2401
Georgios Rotas − Laboratory of Organic Chemistry,
Department of Chemistry, National and Kapodistrian
University of Athens, 15771 Athens, Greece
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c00890
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Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors would like to thank Ekaterini Bouga and
Christina-Ioanna Vrettou for assisting with certain synthesis
steps. This project was financially supported by the European
Union’s Horizon 2020 framework program for research and
innovation under Grant Agreement No. 712921. T.M. and A.P.
would like to thank the State Scholarships Foundation (IKY)
for financial support via Ph.D. fellowships through the
“Strengthening of Human Resources through Doctoral
Research” program of the Operational Program “Human
Resource Development, Education and Lifelong Learning”
2014−2020, co-financed by the European Union (European
Social Fund ESF) and Greek national funds. The authors
would also like to thank Prof. N. Thomaidis and Dr. Maria-
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