Preparation and characterization of thermochemiluminescent acridine-containing 1,2-dioxetanes as promising ultrasensitive labels in bioanalysis

Article abstract of DOI:10.1021/jo401683r

Thermochemiluminescence is the luminescence process in which a thermodynamically unstable molecule decomposes with light emission when heated above a threshold temperature. We recently reported the thermochemiluminescence properties of an acridine-containing 1,2-dioxetane, which emits at relatively low temperatures (i.e., below 100 C). Herein, we explored the effect of the introduction of methyl substituents in the acridine system. The methyl group did not determine an excessive destabilization of 1,2-dioxetane ring nor significantly affect the general physical properties of the molecule. Monosubstituted methyl derivatives and a series of derivatives bearing several combinations of two, three, and four methyl groups were prepared. The rate of formation of 1,2-dioxetane derivatives 1b-k strongly depended on the methyl substitution pattern. All members of this library of mono-, di-, tri-, and tetramethyl-substituted derivatives were characterized in terms of photophysical and thermochemiluminescence properties. The introduction of methyl groups into the acridine ring caused a marked decrease in the activation energy of the thermochemiluminescent reaction. Tri- and tetramethyl-substituted acridones had the highest fluorescence quantum yields, in the range 0.48-0.52, and the corresponding 1,2-dioxetanes 1h and 1j showed in thermochemiluminescence imaging experiments limit of detection values more than ten times lower with respect to the unsubstituted derivative.

Full text of DOI:10.1021/jo401683r

Article
pubs.acs.org/joc
Preparation and Characterization of Thermochemiluminescent
Acridine-Containing 1,2-Dioxetanes as Promising Ultrasensitive
Labels in Bioanalysis
Massimo Di Fusco,*,† Arianna Quintavalla,*,‡ Claudio Trombini,‡ Marco Lombardo,‡ Aldo Roda,‡
Massimo Guardigli,‡ and Mara Mirasoli‡
†Advanced Applications in Mechanical Engineering and Materials Technology Interdepartmental Center for Industrial Research,
Alma Mater Studiorum, University of Bologna, Bologna, Italy
‡Department of Chemistry ‘‘G. Ciamician’’, Alma Mater Studiorum, University of Bologna, Bologna, Italy
S
* Supporting Information
ABSTRACT: Thermochemiluminescence is the luminescence process in which a thermodynamically unstable molecule
decomposes with light emission when heated above a threshold temperature. We recently reported the thermochemilumi-
nescence properties of an acridine-containing 1,2-dioxetane, which emits at relatively low temperatures (i.e., below 100 °C).
Herein, we explored the effect of the introduction of methyl substituents in the acridine system. The methyl group did not
determine an excessive destabilization of 1,2-dioxetane ring nor significantly affect the general physical properties of the molecule.
Monosubstituted methyl derivatives and a series of derivatives bearing several combinations of two, three, and four methyl groups
were prepared. The rate of formation of 1,2-dioxetane derivatives 1b−k strongly depended on the methyl substitution pattern.
All members of this library of mono-, di-, tri-, and tetramethyl-substituted derivatives were characterized in terms of
photophysical and thermochemiluminescence properties. The introduction of methyl groups into the acridine ring caused a
marked decrease in the activation energy of the thermochemiluminescent reaction. Tri- and tetramethyl-substituted acridones
had the highest fluorescence quantum yields, in the range 0.48−0.52, and the corresponding 1,2-dioxetanes 1h and 1j showed in
thermochemiluminescence imaging experiments limit of detection values more than ten times lower with respect to the
unsubstituted derivative.
emission intensities, which determined a poor sensitivity of
TCL-based bioanalytical applications. Nevertheless, TCL still
could offer interesting and largely unexplored analytical
opportunities. For instance, as in BL-CL and ECL, there is
no nonspecific signal due to the matrix components; this simply
triggered by heat, TCL would allow for highly sensitive reagent-
free detection.
Since the late 1960s, when 3,3,4-trimethyl-1,2-dioxetane was
synthesized,2 a number of 1,2-dioxetanes have been proposed
as CL and TCL substrates.3 1,2-Dioxetanes undergo thermally
induced decomposition into two carbonyl compounds, one of
which can be formed in an electronically excited state. The
excited product can thus emit light, either directly through its
radiative decay to the ground state (direct CL) or indirectly via
INTRODUCTION
■
Thermochemiluminescence (TCL) is the luminescence process
in which a thermodynamically unstable molecule decomposes
with light emission when heated above a threshold temperature.
This process is closely related to other luminescence
phenomena such as photoluminescence, biochemiluminescence
(BL-CL), and electrochemiluminescence (ECL), in which the
excited-state molecule responsible for light emission is
generated through different processes (i.e., light absorption
and chemical or electrochemical reactions) rather than heating
of a thermally unstable precursor. Even though TCL was
proposed in the late 1980s as a luminescence detection
technique for bioanalytical applications (e.g., immunoassays),1
it did not achieve the success of other luminescence detection
techniques. The main drawbacks that limited the use of TCL
were the high temperatures required to trigger the emission
(typically in the 200−250 °C range) and the relatively low
Received: August 1, 2013
© XXXX American Chemical Society
A
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a nonradiative energy transfer to a fluorescent acceptor
molecule (indirect CL). The latter case is exploited to amplify
the CL signal when the excited product is a poor fluorophore.
Adamantylidene adamantane 1,2-dioxetane was the first 1,2-
dioxetane proposed as a TCL label for bioanalytical
applications,1 and even now 1,2-dioxetanes represent the
main TCL species. We recently reported an efficient synthesis
of the thermochemiluminescent acridine-containing 1,2-dioxe-
tane 1a, which decomposes at relatively low temperature (<100
°C) into 2-adamantanone and ethyl 9-oxo-10(9H)-acridine
acetate. The latter compound, obtained in the singlet excited
state, is responsible for the TCL emission (Scheme 1).4 Two
molecular electron transfer from the lone electron pair of the
acridinic nitrogen to the 1,2-dioxetane ring, followed by
cleavage of the O−O bond and formation of a pair of radical
ions. Then, a back-electron transfer from the radical anion to
the radical cation produced the singlet excited-state N-
alkylacridone carbonyl fragment. Recently, the involvement of
an intramolecular electron transfer from the acridinium moiety
to the peroxide ring has been shown experimentally to occur.12
To improve the performance of 1a, we introduced suitable
structural modifications in order to (i) decrease the TCL
emission triggering temperature and (ii) increase the TCL
emission intensity by producing more efficient fluorophores as
excited state-products, thus optimizing detectability with no
need of an energy acceptor. A classic approach in optimization
of emission properties of aromatic molecules relies on the
introduction of substituents onto the aromatic scaffold.
Therefore, we explored the effect of methyl substituents in
the acridine system. The methyl group was selected because of
its relatively small electro-donating effect, which should not
determine an excessive destabilization of the 1,2-dioxetane ring.
Moreover, contrary to strong electron-withdrawing or -donat-
ing groups, it does not significantly affect the general physical
properties of the molecule, such as the solubility profile. In
more detail, the four different monosubstituted methyl
derivatives and a series of derivatives bearing several
combinations of two, three, and four methyl groups were
prepared. All members of this library of mono-, di-, tri-, and
tetramethyl-substituted derivatives were characterized in terms
of photophysical and TCL properties.
Scheme 1. Thermally Induced Fragmentation of 1a
structural properties of 1a deserve a comment: (i) the crucial
stabilizing role played, as in the majority of 1,2-dioxetane
substrates, by the spiro-bonded adamantylidene group, which
sterically protects the dioxetane ring from spontaneous
decomposition at room temperature,5 and (ii) the presence
of an ester group, which provides a binding site useful for
bioanalytical applications of 1a, e.g., for labeling analytes with
this TCL molecule.
It should be pointed out that the reaction mechanism of
acridine-containing 1,2-dioxetanes is different from that of
“classical” TCL 1,2-dioxetanes such as the adamantylidene
adamantane 1,2-dioxetane. Indeed, alkyl- and aryl-substituted
1,2-dioxetanes decompose upon heating at relatively high
temperatures with low singlet excited state formation
efficiency.3a,6 Different reaction pathways have been proposed,
including a synchronous, concerted mechanism7 and the
formation of a biradical intermediate.8 Further studies
suggested that neither extreme is in accordance with the
reaction mechanism and an intermediate asynchronous
concerted mechanism has been described.9 N-Substituted
acridine-containing 1,2-dioxetanes are generally less stable
than alkyl- and aryl-substituted 1,2-ones and show higher
singlet excited-state formation efficiency.10 Their proposed
thermal decomposition mechanism5,10,11 postulated an intra-
RESULTS AND DISCUSSION
■
We report here the synthesis of a family of methyl-substituted
1,2-dioxetane derivatives with formulas 1b−k (Figure 1) and
the photophysical and TCL properties of these temperature-
sensitive molecules with the aim to correlate TCL properties,
i.e., triggering temperatures and TCL emission intensity, to
structural features.
The general synthetic pathway followed to obtain the
methyl-substituted acridine-containing 1,2-dioxetanes 1b−k is
reported in Scheme 2. An Ullmann-type coupling13 of properly
substituted anilines and 2-bromo- or 2-iodobenzoic acids
provided 2-aminoarylbenzoic acids 2b−k successively treated
with POCl3 to give the corresponding substituted acridones
3b−k.14 After N-alkylation with ethyl bromoacetate,15 the
resulting ethyl 9-oxo-10(9H)-acridineacetates 4b−k were
reductively coupled with 2-adamantanone under McMurry
conditions16 to give 5b−k, which were photooxygenated using
Figure 1. Structures of the methyl-substituted 1,2-dioxetane derivatives. Each member of this family is identified on the basis of the methyl
substitution pattern on the acridine ring system.
B
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a
Scheme 2
Table 1. Photophysical Properties of Acridone Derivatives
4b−k
a
a
a
a,b
compd
λmax (nm)
ε
(L mol−1 cm−1
)
λem (nm)
ϕF
4b
255
390
252
397
255
387
254
390
256
403
255
388
253
399
254
395
254
404
261
399
51900
10400
54300
11400
55500
12000
23100
3000
406
411
401
422
418
424
416
422
411
418
0.02
0.30
0.21
0.09
0.42
0.04
0.52
0.10
0.48
0.08
4c
4d
4e
4f
77000
12300
55100
8500
4g
4h
4i
50600
10500
48400
8900
a
Conditions: (i) Cu/K2CO3, 1-pentanol, reflux; (ii) POCl3, CH3CN/
water, reflux; (iii) NaH, BrCH2CO2Et, Bu4NI, DMF, rt; (iv) 2-
adamantanone, TiCl3/LiAlH4, Et3N, THF, reflux; (v) polymer-bound
Bengal Rose or Methylene Blue, hν, O2, CH2Cl2, 0 °C.
4j
63700
12400
59500
4k
7800
Bengal Rose or Methylene Blue as sensitizers to yield the 1,2-
dioxetane derivatives 1b−k.3j,17
a
b
Determined in acetonitrile solution. Determined using quinine
sulfate as standard (ϕF = 0.53 in H2SO4 0.05 mol L−1).
The critical step of the whole synthetic pathway is the
photooxygenation of the tetrasubstituted double bond of
compounds 5. The reaction is very sensitive to the π-electron
density of the double bond; thus, electron-rich olefins undergo
easier [2 + 2] cycloaddition reactions with singlet oxygen.18
The rate of formation of 1,2-dioxetane derivatives 1b−k
strongly depended on the methyl substitution pattern. While
the olefinic precursor of the unsubstituted 1,2-dioxetane 1a
required about 12 h to undergo quantitative photooxygenation,
under the same experimental conditions the monomethyl
olefins 5c−e required 7−10 h, the disubstituted olefin 5f 2 h,
and the tri- and tetrasubstituted olefins 5h and 5j only 1 h. On
the other hand, as demonstrated by HPLC−MS analysis, only
trace amounts of 1,2-dioxetane derivatives were observed after
photooxygenation of olefins 5b, 5g, 5i, and 5k, even after 20 h
of irradiation using polymer-bound Bengal Rose as sensitizer.
Furthermore, no products were observed in the same
conditions using Methylene Blue as sensitizer. Since these
four substrates share a methyl substituent on position 1 of the
acridine system, thus in proximity to the carbon−carbon double
bond, it can be concluded that in these compounds the [2 + 2]
cycloaddition reaction is inhibited for steric reasons. For the
other substrates, the reaction rate increases with the number of
methyl groups onto the acridine moiety, indicating that the
higher electron density on the double bond promotes a faster
[2 + 2] cycloaddition of the singlet oxygen.
We already demonstrated that the TCL emission of 1a was
due to the formation of singlet excited-state acridone.4
According to this finding, to investigate the TCL properties
of the 1,2-dioxetanes 1b−k we first studied the photophysical
properties of substituted acridone derivatives 4b−k, i.e., the
expected emitting species of the TCL reactions. Absorption and
fluorescence data (maximum absorption wavelengths, molar
absorption coefficients, maximum emission wavelengths and
fluorescence quantum yields) obtained for the compounds 4b−
k are collected in Table 1. For comparison, the unsubstituted
acridone derivative 4a has absorption maxima at λ = 250 and
390 nm with molar absorption coefficients of 46300 and 11100
L mol−1 cm−1, respectively, λem = 403 nm and ϕF = 0.11. The
experimental results suggest that the introduction of methyl
groups onto the aromatic system of acridone affects the
fluorescence quantum yield through different mechanisms.
Compounds containing methyl groups onto positions 2, 3, 6,
and 7 (4c, 4d, 4f, 4h, 4j) show a significant increase of the
fluorescence quantum yields, which could be due to a decrease
of the intersystem crossing rate constant and thus to a lower
efficiency of nonradiative deactivation processes, as already
reported for aromatic compounds such as anthracene.19 Tri-
and tetramethyl-substituted acridones have the highest
fluorescence quantum yield values, in the range 0.48−0.52.
On the other hand, the acridone derivatives with methyl groups
in positions 4 (compound 4e) and 1 (compounds 4b, 4g, 4i,
4k) show quite low fluorescence quantum yields. This behavior
can be attributed to the steric hindrance of the methyl
substituent, which causes distortion from planarity of the
aromatic system.20
Upon heating of compounds 1c, 1d, 1e, 1f, 1h, and 1j, a
weak TCL emission was observed starting from relatively low
temperatures (40−60 °C). With increasing temperature, the
TCL reaction became faster and the emission intensity
increased. To confirm the nature of the emitting species, we
compared the TCL spectra of the 1,2-dioxetane derivatives with
the fluorescence spectra of the corresponding acridones 4c, 4d,
4e, 4f, 4h, and 4j. In all cases, the TCL spectra closely matched
the fluorescence spectra of the corresponding acridones, thus
demonstrating that the TCL emission was primarily due to the
singlet excited state of acridone (Figure 2). Singlet excited-state
2-adamantanone could also be produced in the TCL reaction.
However, the possible emission of excited 2-adamantanone
could not be detected due to the overlap with the fluorescence
emission of acridone derivatives and the weak intensity (for 2-
adamantanone λmax = 425 nm21 and ϕF = 0.01522).
In the temperature interval between 70 and 120 °C, the TCL
emission showed a first-order decay. The activation parameters
C
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cooled CCD sensor. Samples were deposited as acetonitrile
solutions on conventional microscope glass slides glued to a flat
heating element or on high-resistivity (70−100 Ω) indium−tin
oxide (ITO)-coated microscope glass slides. Then, the TCL
signal was imaged upon heating the support. According to the
calibration curves so obtained (Figure 3), the LOD values for
Figure 2. Comparison between the TCL emission spectrum of
compound 1j recorded in acetonitrile solution upon heating to 60 °C
and the fluorescence emission spectrum of compound 4j measured in
acetonitrile solution (λexc = 380 nm).
of the reaction were determined by a standard isothermal
kinetic method: the measurement of the emission decay
kinetics at different temperatures allowed us to obtain the
kinetic constants k of the TCL reaction, and then the
temperature dependence of the kinetic constants was analyzed
according to a standard Arrhenius equation to evaluate the
activation energies (Ea) and the pre-exponential coefficients
(A) of the TCL reactions (Table 2).
Figure 3. Left: TCL imaging calibration curves obtained for the 1,2-
dioxetane derivatives 1c−f, 1h, and 1j. Right: TCL image of an ITO-
coated glass with spots containing different amounts of the 1,2-
dioxetane 1j (four replicates for each amount of compound). The
TCL image is shown in pseudocolors, each color corresponding to a
different TCL emission intensity.
Table 2. Activation Parameters for the Thermal
Decomposition of 1,2-Dioxetane Derivatives 1c−f, 1h, and
1j
the 1,2-dioxetane derivatives 1c−f, 1h, and 1j, defined as the
amount of compound giving a signal higher than the
background signal plus three times its standard deviation,
were calculated (Table 3). For comparison, for the unsub-
stituted 1,2-dioxetane 1a the LOD value obtained under similar
experimental conditions using an ITO-coated glass slide was 3.5
× 10−13 mol spot−1.
a
a
compd
Ea (kcal mol−1
)
ln A (s−1
)
1c
1d
1e
1f
25.7 1.6
24.5 0.3
23.0 0.4
19.3 0.5
18.4 0.2
16.7 1.6
30.9 1.9
28.1 0.1
26.2 0.6
21.4 0.2
20.9 0.8
18.6 2.3
Table 3. LOD Values for 1,2-Dioxetane Derivatives 1c−f, 1h,
and 1j
1h
1j
a
a
LOD (mol spot−1
)
Mean SD of three independent measurements.
compd
support
1c
glass
(6.0 0.5) × 10−14
(5.4 0.4) × 10−14
(5.6 0.2) × 10−14
(7.6 0.8) × 10−14
(1.1 0.3) × 10−13
(4.7 0.7) × 10−13
(5.1 1.0) × 10−14
(4.1 0.4) × 10−14
(3.2 0.6) × 10−14
(2.7 0.4) × 10−14
(1.9 0.3) × 10−14
(2.1 0.3) × 10−14
ITO-coated glass
glass
For comparison, the parent unsubstituted 1,2-dioxetane 1a
1d
1e
1f
has Ea = 29.7 kcal mol−1 and ln A = 38.5 s−1, and the activation
energies reported for other 1,2-dioxetane derivatives ranged
between 12.2 and 32.5 kcal mol−1.3d,j,12a,23 These results clearly
show that the introduction of methyl groups into the acridine
ring causes a marked decrease in the activation energy of the
TCL reaction, which determined a lower triggering temperature
of the TCL emission. Nevertheless the methyl-substituted
compounds remained stable enough for their handling and
storage. Indeed, as shown in Table 2, the decrease in the
activation energy due to the presence of methyl groups is
paralleled by a reduction in the pre-exponential coefficient. As a
result, despite the quite different activation energies, at a given
temperature the kinetic rate constants of the TCL reactions of
the investigated compounds differ by no more than 1 order of
magnitude.
ITO-coated glass
glass
ITO-coated glass
glass
ITO-coated glass
glass
1h
1j
ITO-coated glass
glass
ITO-coated glass
a
Mean SD of four measurements.
The highest detectability was obtained for compound 1j,
whose LOD value is 17 times lower than that of the
unsubstituted 1,2-dioxetane 1a. The values reported in Table
3 show a good correlation between the LODs of 1,2-dioxetane
derivatives and the fluorescence quantum yields of the
corresponding acridone derivatives (i.e., the greater is the
fluorescence quantum yield the smaller is the LOD). This
suggests that, as expected, the fluorescence quantum yield of
the emitting carbonyl fragment is an important factor in
To preliminarily evaluate the suitability of the synthesized
1,2-dioxetane derivatives as labels for biospecific probes in
bioassays, we measured their limit of detection (LOD) by TCL
imaging. Measurements were performed by imaging arrays of
spots containing different amounts of compound using a low-
light luminograph equipped by an ultrasensitive, double Peltier-
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were then analyzed to measure the TCL signals using the image
analysis software provided with the instrument. The kinetic and
thermodynamic parameters of the TCL process were obtained by
measuring the TCL emission decay kinetics in the temperature range
between 70 and 120 °C. For each temperature, the kinetic constant k
of the TCL process (i.e., the inverse of the decay time in seconds) was
calculated by fitting the decay profile of the TCL emission with the
first-order decay equation
determining the overall TCL emission intensity of 1,2-
dioxetane derivatives.
CONCLUSIONS
■
With the aim to design new TCL labels potentially useful in
bioanalysis, a family of dispirodioxetanes containing an acridine
moiety 1b−k has been synthesized. The acridine ring system
was decorated with one or more methyl groups in different
positions to investigate the effect of weak electron donating
groups on the photophysical and TCL properties. The behavior
of the [2 + 2] cycloaddition reaction and the TCL properties of
the 1,2-dioxetanes markedly depend on the substitution pattern
of the acridine ring. Indeed, a methyl group in position 1
disfavors both 1,2-dioxetane formation and the cycloreversion
responsible for the TCL emission, as expected for a concerted
mechanism. On the other hand, an increasing number of
methyl groups located in positions 2, 3, 6, and 7 facilitates the
[2 + 2] cycloaddition and causes a decrease of the activation
energy of the TCL reaction. Moreover, the LOD value of the
tetrasubstituted derivative 1j was 17 times lower than that of
the unsubstituted 1,2-dioxetane 1a. With these compounds in
hands, we are investigating their application in ultrasensitive
bionalytical applications by binding the TCL molecules to
biospecific probes through the properly installed acetic acid
appendage. Results will be published in due time.
ITCL = (ITCL)0e−kt
(1)
in which ITCL is the TCL signal at time t and (ITCL)0 is the TCL signal
at zero time. Then, the activation energy Ea and the pre-exponential
factor A of the TCL process were calculated from the kinetic constants
k measured at different temperatures using the logarithmic form of the
Arrhenius equation
ln k = ln A − (Ea/RT)
(2)
in which R is the universal gas constant and T is the temperature.
Synthesis of 2-Iodo-4,5-dimethylbenzoic Acid.25 A solution of
NaClO2 (80% purity, by iodometric titration, 500 mg, 4.4 mmol) in
4.5 mL of water was added dropwise in 2 h to a stirred mixture of 2-
iodo-4,5-dimethylbenzaldehyde (820 mg, 3.1 mmol) in 3.5 mL of
acetonitrile, NaH2PO4 (110 mg) in 1.2 mL of water, and 30% H2O2
(370 μL, 3.6 mmol), keeping the temperature at 10 °C with water
cooling. After 1 h, a small amount of Na2SO3 (∼30 mg) was added to
destroy the unreacted HOCl and H2O2. Acidification with 10%
aqueous HCl afforded 2-iodo-4,5-dimethylbenzoic acid (850 mg, 98%
1
yield) as a crystalline solid: H NMR (400 MHz, CDCl3) δ 2.27 (s,
6H), 7.84 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 19.3, 19.4, 91.4,
130.0, 133.3, 136.9, 142.8, 143.7, 170.7; MS (ESI) [M + H]+ m/z
277.0. Anal. Calcd for C9H9IO2 (275.96): C, 39.16; H, 3.29. Found: C,
39.26; H, 3.31.
EXPERIMENTAL SECTION
Materials. All of the chemicals were used as received.
■
Characterization of Compounds. 1H and 13C NMR spectra
were recorded on a 200 or 400 NMR instrument with a 5 mm probe.
All chemical shifts have been quoted relative to deuterated solvent
signals, chemical shifts (δ) are reported in ppm, and coupling
constants (J) are reported in hertz. HPLC−MS analysis was performed
using an HPLC system coupled with a single-quadrupole mass
spectrometer. A ZOBRAX-Eclipse XDB-C8 column was employed for
the chromatographic separation; mobile phase: H2O/CH3CN,
gradient from 30% to 80% of CH3CN in 8 min, 80% of CH3CN
until 25 min, 0.4 mL min−1. Mass spectrometric detection was
performed in full-scan mode from m/z 50 to m/z 2600, scan time 0.1 s
in positive ion mode, ESI spray voltage 4500 V, nitrogen gas 35 psi,
drying gas flow 11.5 mL min−1, fragmentor voltage 20 V. Flash-
chromatography was carried out using Merck silica gel 60 (230−400
mesh particle size). Thin-layer chromatography was performed on
Merck 60 F254.
General Procedure for the Synthesis of Compounds 2b−k. A
solution of substituted aniline (6.5 mmol), substituted 2-bromo- or 2-
iodobenzoic acid (4.7 mmol), anhydrous K2CO3 (0.9 g, 6.5 mmol),
and copper (57 mg, 0.9 mmol) in anhydrous 1-pentanol (5.0 mL) was
heated under reflux for 3 h. The solvent was evaporated under reduced
pressure, and the residue was dissolved in hot H2O (100 mL) and then
filtered through Celite. The Celite was washed with H2O (300 mL),
and the filtrate was acidified with concentrated HCl to pH 6. The
precipitate was isolated by filtration and washed with H2O. The solid
was recrystallized from CHCl3 to give compounds 2b−k.
2-(m-Tolylamino)benzoic acid (2b):26 yield 825 mg (77%).
5-Methyl-2-(phenylamino)benzoic acid (2c):27 yield 761 mg
(71%).
4-Methyl-2-(phenylamino)benzoic acid (2d): yield 825 mg
1
(77%); H NMR (400 MHz, CDCl3) δ 2.28 (s, 3H), 6.59 (d, J = 8.0,
Spectrophotometric Measurements. Absorption spectra were
recorded using a UV−vis spectrophotometer. Fluorescence emission
spectra and TCL emission spectra were recorded using a
spectrofluorimeter. Fluorescent quantum yields of compounds 4a−k
were measured using quinine sulfate as standard (ϕF = 0.53 in H2SO4
0.05 mol L−1).24
1H), 7.05 (s, 1H), 7.15 (t, J = 7.6, 1H), 7.28 (d, J = 8.4, 2H), 7.39 (t, J
= 7.6, 2H), 7.94 (d, J = 8.4, 1H), 9.30 (br, 1H); 13C NMR (100 MHz,
CDCl3) δ 22.1, 107.9, 114.0, 118.6, 123.2, 123.9, 129.4, 132.5, 140.4,
146.3, 148.9, 173.3; MS (ESI) [M + H]+ m/z 228.1. Anal. Calcd for
C14H13NO2 (227.09): C, 73.99; H, 5.77; N, 6.16. Found: C, 73.94; H,
5.76; N, 6.16.
TCL Measurements. TCL imaging experiments were performed
with a low-light luminograph. Microscope glass slides glued to a flat
heating element or high-resistivity (70−100 Ω) ITO-coated micro-
scope glass slides were used as solid supports. In the latter case,
heating was provided by a suitable electrical current flowing through
the conductive ITO coating. The temperature of the support was
controlled by varying the applied current and monitored by a copper/
constantan thermocouple. Samples were deposited on the supports as
acetonitrile solutions either with a micropipet or using a manual
microarrayer. The manual microarrayer deposited arrays of spots of
about 10 nL with diameters in the range of 500−800 μm depending
on the nature of the surface. The spots were allowed to dry before the
TCL measurement. TCL images were acquired upon heating the
support to the desired temperature (90−100 °C) using integration
times varying from 5 s (for evaluation of the TCL decay kinetics) to 5
min (for assessment of the detectability by TCL imaging). Images
2-(o-Tolylamino)benzoic acid (2e):26a yield 793 mg (74%).
5-Methyl-2-(p-tolylamino)benzoic acid (2f):28 yield 967 mg
(85%).
6-Methyl-2-(o-tolylamino)benzoic acid (2g): yield 762 (67%);
1H NMR (400 MHz, CDCl3) δ 2.27 (s, 3H), 2.61 (s, 3H), 6.64 (d, J =
7.6, 1H), 6.76 (d, J = 8.4, 1H), 7.06−7.22 (m, 3H), 7.28 (m, 2H); 13
C
NMR (50 MHz, CDCl3) δ 18.0, 23.8, 112.8, 121.2, 123.8, 124.4, 126.7,
131.1, 132.5, 133.0, 139.5, 142.1, 148.8, 175.0; MS (ESI) [M + H]+ m/
z 242.1. Anal. Calcd for C15H15NO2 (241.11): C, 74.67; H, 6.27; N,
5.81. Found: C, 74.35; H, 6.29; N, 5.87.
2-((3,4-Dimethylphenyl)amino)-5-methylbenzoic acid (2h):
1
yield 951 mg (79%); H NMR (400 MHz, CDCl3) δ 2.27 (s, 9H),
7.02 (m, 2H), 7.12 (m, 2H), 7.18 (d, J = 8.8, 1H), 7.84 (s, 1H); 13C
NMR (100 MHz, CDCl3) δ 19.1, 19.8, 20.2, 110.0, 114.3, 120.4, 124.5,
125.8, 130.3, 132.0, 132.1, 136.2, 137.6, 138.4, 147.3, 173.6; MS (ESI)
E
dx.doi.org/10.1021/jo401683r | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
[M + H]+ m/z 256.1. Anal. Calcd for C16H17NO2 (255.13): C, 75.27;
H, 6.71; N, 5.49. Found: C, 74.90; H, 6.72; N, 5.44.
3H), 2.49 (s, 3H), 4.30 (q, J = 7.0, 2H), 5.02 (s, 2H), 7.05 (s, 1H),
7.11 (d, J = 8.4, 1H), 7.27 (m, 2H), 7.67 (m, 1H), 8.42 (d, J = 8.0,
1H), 8.53 (dd, J1 = 8.0, J2 = 8.0, 1H); 13C NMR (100 MHz, CDCl3) δ
14.2, 22.5, 48.4, 62.1, 114.0, 114.1, 120.7, 121.7, 122.7, 123.5, 127.9,
129.7, 133.8, 142.3, 142.5, 145.1, 168.4, 177.9; MS (ESI) [M + H]+ m/
z 296.1. Anal. Calcd for C18H17NO3 (295.12): C, 73.20; H, 5.80; N,
4.74. Found: C, 73.39; H, 5.81; N, 4.74.
2-((3,4-Dimethylphenyl)amino)-4,5-dimethylbenzoic acid
1
(2j): yield 1.05 g (83%); H NMR (400 MHz, CDCl3) δ 2.19 (s,
6H), 2.28 (s, 6H), 7.02 (m, 3H), 7.13 (d, J = 8.4, 1H), 7.78 (s, 1H);
13C NMR (100 MHz, CDCl3) δ 18.6, 19.1, 19.8, 20.5, 115.0, 120.5,
124.6, 125.3, 130.3, 132.0, 132.5, 137.6, 138.4, 145.2, 147.6, 173.7; MS
(ESI) [M + H]+ m/z 270.0. Anal. Calcd for C17H19NO2 (269.14): C,
75.81; H, 7.11; N, 5.20. Found: C, 75.60; H, 7.07; N, 5.18.
General Procedure for the Synthesis of Compounds 3b−k.
Compounds 2b−k (4.0 mmol) were dissolved in CH3CN (9 mL) and
heated to reflux. Phosphorus(V) oxychloride (1.35 g, 8.8 mmol) was
added over 1 h. The solution was refluxed for further 2 h and then
cooled to 10−15 °C. H2O (5 mL) was added, and the mixture was
heated to reflux for 2.5 h. The suspension was cooled to 10 °C and
filtered. The solid was washed with H2O and CH3CN and then dried
under vacuum to obtain 3b−k.29
4-Methyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester (4e):
1
yield 51 mg (19%); H NMR (400 MHz, CDCl3) δ 1.19 (t, J = 7.0,
3H), 2.62 (s, 3H), 4.22 (q, J = 7.0, 2H), 4.89 (s, 2H), 7.29 (m, 3H),
7.53 (d, J = 6.8, 1H), 7.67 (m, 1H), 8.36 (d, J = 7.6, 1H), 8.43 (dd, J1 =
7.6, J2 = 8.0, 1H); 13C NMR (50 MHz, CDCl3) δ 14.0, 21.8, 55.8, 61.8,
116.5, 122.3, 122.8, 123.9, 125.5, 125.9, 126.2, 127.3, 133.9, 137.7,
145.4, 146.4, 169.9, 179.2; MS (ESI) [M + H]+ m/z 296.1. Anal. Calcd
for C18H17NO3 (295.12): C, 73.20; H, 5.80; N, 4.74. Found: C, 73.13;
H, 5.78; N, 4.73.
2,7-Dimethyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester
(4f): yield 165 mg (59%); 1H NMR (400 MHz, CDCl3) δ 1.28 (t, J =
7.0, 3H), 2.48 (s, 6H), 4.30 (q, J = 7.0, 2H), 5.04 (s, 2H), 7.22 (d, J =
8.4, 2H), 7.53 (dd, J1 = 8.4, J2 = 8.4, 2H), 8.37 (s, 2H); 13C NMR (100
MHz, CDCl3) δ 14.1, 20.5, 48.3, 62.1, 114.0, 122.4, 127.3, 131.2,
135.3, 140.3, 168.4, 178.0; MS (ESI) [M + H]+ m/z 310.3. Anal. Calcd
for C19H19NO3 (309.14): C, 73.77; H, 6.19; N, 4.53. Found: C, 73.64;
H, 6.18; N, 4.52.
1,5-Dimethyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester
(4g): yield 223 mg (80%); 1H NMR (400 MHz, CDCl3) δ 1.10 (t, J =
7.0, 3H), 2.55 (s, 3H), 2.88 (s, 3H), 4.12 (q, J = 7.0, 2H), 4.81 (s, 2H),
7.01 (d, J = 7.2, 1H), 7.19 (m, 2H), 7.44 (m, 2H), 8.20 (d, J = 8.0,
1H); 13C NMR (100 MHz, CDCl3) δ 13.9, 21.3, 23.4, 56.3, 61.6,
114.9, 122.8, 125.2, 125.5, 126.2, 128.2, 132.6, 136.8, 141.8, 144.7,
148.1, 169.8, 181.4; MS (ESI) [M + H]+ m/z 310.1. Anal. Calcd for
C19H19NO3 (309.14): C, 73.77; H, 6.19; N, 4.53. Found: C, 73.86; H,
6.14; N, 4.75.
2,3,7-Trimethyl-9-oxo-10(9H)-acridineacetic acid, ethyl
ester (4h): yield 90 mg (31%); 1H NMR (400 MHz, CDCl3) δ
1.29 (t, J = 7.0, 3H), 2.37 (s, 3H), 2.42 (s, 3H), 2.46 (s, 3H), 4.30 (q, J
= 7.0, 2H), 5.03 (s, 2H), 7.05 (s, 1H), 7.19 (d, J = 8.8, 1H), 7.50 (dd,
J1 = 8.8, J2 = 8.8, 1H), 8.28 (s, 1H), 8.34 (s, 1H); 13C NMR (100
MHz, CDCl3) δ 14.2, 19.0, 20.5, 21.1, 48.2, 62.0, 114.0, 114.5, 120.7,
122.3, 127.2, 127.7, 130.7, 131.1, 135.0, 140.2, 140.6, 144.2, 168.5,
177.8; MS (ESI) [M + H]+ m/z 324.0. Anal. Calcd for C20H21NO3
(323.15): C, 74.28; H, 6.55; N, 4.33. Found: C, 74.19; H, 6.54; N,
4.32.
1-Methylacridin-9(10H)-one (3b):30 yield 301 mg (36%).
2-Methylacridin-9(10H)-one (3c):31 yield 829 mg (99%).
3-Methylacridin-9(10H)-one (3d):31 yield 578 mg (69%).
4-Methylacridin-9(10H)-one (3e):31 yield 720 mg (86%).
1
2,7-Dimethylacridin-9(10H)-one (3f): yield 887 mg (99%); H
NMR (400 MHz, DMSO-d6) δ 2.41 (s, 3H), 7.43 (d, J = 8.4, 2H),
7.54 (d, J = 8.4, 2H), 8.00 (s, 2H), 11.58 (s, 1H); 13C NMR (100
MHz, d6-DMSO) δ 21.3, 118.0, 122.2, 125.8, 130.6, 135.4, 139.7,
170.6; MS (ESI) [M + H]+ m/z 224.1. Anal. Calcd for C15H13NO
(223.10): C, 80.69; H, 5.87; N, 6.27. Found: C, 80.25; H, 5.93; N,
6.24.
1
1,5-Dimethylacridin-9(10H)-one (3g): yield 161 mg (18%); H
NMR (400 MHz, DMSO-d6) δ 2.56 (s, 3H), 2.86 (s, 3H), 6.96 (d, J =
6.8, 1H), 7.11 (t, J = 7.6, 1H), 7.52 (m, 2H), 7.75 (d, J = 8.4, 1H), 8.06
(d, J = 7.6, 1H), 10.32 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ
17.6, 23.7, 116.3, 118.9, 120.4, 122.0, 123.8, 124.1, 124.7, 132.2, 133.7,
138.8, 140.1, 142.7, 179.1; MS (ESI) [M + H]+ m/z 224.1. Anal. Calcd
for C15H13NO (223.10): C, 80.69; H, 5.87; N, 6.27. Found: C, 81.07;
H, 5.88; N, 6.22.
2,3,7-Trimethylacridin-9(10H)-one (3h) and 1,2,7-trimethy-
lacridin-9(10H)-one (3i): yield 494 mg (52%) (mixture of 3h and
3i).
2,3,6,7-Tetramethylacridin-9(10H)-one (3j) and 1,2,6,7-tetra-
methylacridin-9(10H)-one (3k): yield 653 mg (65%) (mixture of 3j
and 3k).
General Procedure for the Synthesis of Compounds 4b−k. A
solution of 3b−k (0.9 mmol) in DMF (4 mL) was added to a
suspension of NaH (29 mg, 1.2 mmol) in dry DMF (2 mL). The
mixture was stirred for 30 min at room temperature, and then, after the
mixture was cooled to 0 °C, ethyl 2-bromoacetate (234 mg, 1.4 mmol)
and tetrabutylammonium iodide (4 mg, 0.01 mmol) were added. The
solution was stirred for further 24 h at room temperature and then
poured into cold water. The precipitate was collected by filtration,
dried under vacuum, and purified by flash chromatography on silica gel
using 9:1 (v/v) dichloromethane/ethyl acetate as the eluent to obtain
4b−k.
1,2,7-Trimethyl-9-oxo-10(9H)-acridineacetic acid, ethyl
ester (4i): yield 140 mg (48%); 1H NMR (400 MHz, CDCl3) δ
1.30 (t, J = 7.0, 3H), 2.39 (s, 3H), 2.46 (s, 3H), 2.92 (s, 3H), 4.31 (q, J
= 7.0, 2H), 4.96 (s, 2H), 7.01 (d, J = 8.8, 1H), 7.13 (d, J = 8.4, 1H),
7.45 (m, 2H), 8.27 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.2,
18.6, 20.3, 20.6, 49.2, 62.0, 111.1, 113.6, 121.5, 124.1, 127.3, 130.5,
130.9, 134.6, 135.1, 139.7, 140.5, 142.3, 168.7, 180.5; MS (ESI) [M +
H]+ m/z 324.0. Anal. Calcd for C20H21NO3 (323.15): C, 74.28; H,
6.55; N, 4.33. Found: C, 74.62; H, 6.53; N, 4.34.
2,3,6,7-Tetramethyl-9-oxo-10(9H)-acridineacetic acid, ethyl
1-Methyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester (4b):
1
1
ester (4j): yield 100 mg (33%); H NMR (400 MHz, CDCl3) δ 1.31
yield 173 mg (65%); H NMR (400 MHz, CDCl3) δ 1.31 (t, J = 7.0,
(t, J = 7.0, 3H), 2.37 (s, 6H), 2.42 (s, 6H), 4.32 (q, J = 7.0, 2H), 5.03
(s, 2H), 7.04 (s, 2H), 8.29 (s, 2H); 13C NMR (100 MHz, CDCl3) δ
14.2, 19.0, 21.1, 48.2, 62.0, 114.5, 120.8, 127.7, 130.6, 140.6, 143.9,
168.6, 177.5; MS (ESI) [M + H]+ m/z 338.1. Anal. Calcd for
C21H23NO3 (337.17): C, 74.75; H, 6.87; N, 4.15. Found: C, 74.60; H,
6.88; N, 4.17.
3H), 3.00 (s, 3H), 4.33 (q, J = 7.0, 2H), 5.00 (s, 2H), 7.07 (d, J = 7.2,
1H), 7.11 (d, J = 8.4, 1H), 7.25 (m, 2H), 7.53 (t, J = 8.0, 1H), 7.66 (t,
J = 7.2, 1H), 8.48 (dd, J1 = 7.6, J2 = 8.0, 1H); 13C NMR (100 MHz,
CDCl3) δ 14.2, 24.5, 49.4, 62.1, 112.3, 113.8, 121.4, 121.6, 124.1,
125.2, 127.8, 132.8, 133.5, 141.8, 143.0, 144.0, 168.5, 180.1; MS (ESI)
[M + H]+ m/z 296.1. Anal. Calcd for C18H17NO3 (295.12): C, 73.20;
H, 5.80; N, 4.74. Found: C, 72.90; H, 5.79; N, 4.75.
2-Methyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester
(4c):32 yield 184 mg (69%); 13C NMR (100 MHz, CDCl3) δ 14.2,
20.6, 48.4, 62.1, 114.0, 114.1, 121.5, 122.5, 127.3, 128.0, 131.5, 133.9,
135.4, 140.4, 142.2, 168.3, 178.1; MS (ESI) [M + H]+ m/z 296.1.
Anal. Calcd for C18H17NO3 (295.12): C, 73.20; H, 5.80; N, 4.74.
Found: C, 73.54; H, 5.81; N, 4.71.
1,2,6,7-Tetramethyl-9-oxo-10(9H)-acridineacetic acid, ethyl
1
ester (4k): yield 85 mg (28%); H NMR (400 MHz, CDCl3) δ 1.30
(t, J = 7.0, 3H), 2.38 (m, 9H), 2.92 (s, 3H), 4.32 (q, J = 7.0, 2H), 4.96
(s, 2H), 6.98 (m, 2H), 7.42 (d, J = 8.4, 1H), 8.20 (s, 1H); 13C NMR
(100 MHz, CDCl3) δ 14.2, 18.6, 19.0, 20.3, 21.0, 49.1, 61.9, 111.2,
114.1, 121.5, 122.5, 127.8, 130.4, 130.5, 134.9, 140.1, 140.5, 142.3,
143.4, 168.8, 180.3; MS (ESI) [M + H]+ m/z 338.1. Anal. Calcd for
C21H23NO3 (337.17): C, 74.75; H, 6.87; N, 4.15. Found: C, 74.74; H,
6.85; N, 4.14.
3-Methyl-9-oxo-10(9H)-acridineacetic acid, ethyl ester (4d):
1
yield 99 mg (37%); H NMR (400 MHz, CDCl3) δ 1.28 (t, J = 7.0,
F
dx.doi.org/10.1021/jo401683r | J. Org. Chem. XXXX, XXX, XXX−XXX

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General Procedure for the Synthesis of Compounds 5b−k.
Under a nitrogen atmosphere, LiAlH4 (41 mg, 1.08 mmol) was added
to a suspension of TiCl3·3THF (438 mg, 1.08 mmol) in anhydrous
THF (1.5 mL) at 0 °C, and the suspension was stirred for 10 min.
After addition of triethylamine (109 mg, 1.08 mmol) at room
temperature, the reaction mixture was refluxed for 1 h. A solution of
compounds 4b−k (0.36 mmol) and 2-adamantanone (54 mg, 0.36
mmol) in dry THF (3.0 mL) was added dropwise over a period of 30
min. Then, the reaction mixture was refluxed for 17 h, cooled to room
temperature, and filtered over silica gel. Silica was washed with
dichloromethane, and the filtrate was evaporated under vacuum. The
crude product was purified by flash chromatography on silica gel using
1:1 (v/v) dichloromethane/hexane as the eluent to obtain 5b−k.
1-Methyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-acridi-
neacetic acid, ethyl ester (5b): yield 72 mg (48%); 1H NMR (400
MHz, CDCl3) δ 1.23 (t, J = 7.2, 3H), 1.42 (m, 2H), 1.60 (m, 2H),
1.78 (s, 3H), 1.93−2.16 (m, 5H), 2.34 (s, 3H), 2.59 (s, 1H), 3.27 (s,
1H), 4.23 (q, J = 7.2, 2H), 4.62 (AB, 2H), 6.60 (d, J = 8.0, 1H), 6.78
(d, J = 8.0, 1H), 6.87 (d, J = 7.6, 1H), 6.98 (t, J = 7.6, 1H), 7.04 (t, J =
8.0, 1H), 7.13 (t, J = 7.2, 1H), 7.20 (d, J = 7.2, 1H); 13C NMR (50
MHz, CDCl3) δ 14.2, 20.3, 27.8, 27.9, 32.6, 33.4, 36.9, 37.3, 39.1, 39.7,
40.0, 48.8, 61.2, 109.2, 112.1, 119.8, 120.5, 123.1, 125.5, 125.6, 125.9,
127.1, 127.5, 134.7, 143.1, 143.7, 145.8, 169.8; MS (ESI) [M + H]+ m/
z 414.2. Anal. Calcd for C28H31NO2 (413.24): C, 81.32; H, 7.56; N,
3.39. Found: C, 81.11; H, 7.54; N, 3.40.
1H), 4.15 (m, 2H), 4.57 (AB, 2H), 6.90 (d, J = 7.2, 1H), 6.98 (m, 2H),
7.06 (m, 2H), 7.11 (d, J = 7.6, 1H); 13C NMR (100 MHz, CDCl3) δ
14.2, 19.9, 20.1, 27.8, 28.0, 33.0, 33.7, 37.0, 37.6, 39.0, 39.1, 39.3, 54.3,
60.7, 115.2, 120.9, 122.5, 124.2, 125.1, 125.5, 128.3, 129.2, 132.3,
134.4, 135.4, 143.9, 144.9, 146.7, 170.2; MS (ESI) [M + H]+ m/z
428.1. Anal. Calcd for C29H33NO2 (427.25): C, 81.46; H, 7.78; N,
3.28. Found: C, 81.45; H, 7.74; N, 3.31.
2,3,7-Trimethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-
acridineacetic acid, ethyl ester (5h): yield 97 mg (61%); 1H NMR
(400 MHz, CDCl3) δ 1.28 (t, J = 7.2, 3H), 1.50−2.20 (m, 12H), 2.22
(s, 3H), 2.24 (s, 3H), 2.31 (s, 3H), 3.45 (s, 2H), 4.27 (q, J = 7.2, 2H),
4.60 (s, 2H), 6.54 (s, 1H), 6.66 (d, J = 8.4, 1H), 6.94 (d, J = 8.4, 1H),
6.98 (s, 1H), 7.01 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.2, 19.1,
20.1, 20.8, 26.9, 32.1, 32.2, 37.2, 48.7, 61.1, 111.9, 113.5, 119.9, 123.7,
126.1, 126.5, 127.8, 128.0, 128.3, 129.1, 134.0, 141.2, 141.4, 143.3,
170.1; MS (ESI) [M + H]+ m/z 442.3. Anal. Calcd for C30H35NO2
(441.27): C, 81.59; H, 7.99; N, 3.17. Found: C, 81.76; H, 7.95; N,
3.17.
1,2,7-Trimethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-
1
acridineacetic acid, ethyl ester (5i): yield 81 mg (51%); H NMR
(200 MHz, CDCl3) δ 1.26 (t, J = 7.2, 3H), 1.44−2.11 (m, 12H), 2.23
(s, 3H), 2.26 (s, 3H), 2.33 (s, 3H), 2.56 (s, 1H), 3.30 (s, 1H), 4.24 (q,
J = 7.2, 2H), 4.60 (AB, 2H), 6.53 (d, J = 8.4, 1H), 6.68 (d, J = 8.0,
1H), 6.94 (m, 2H), 7.02 (s, 1H); 13C NMR (100 MHz, CDCl3) δ
14.2, 18.1, 19.7, 20.7, 27.8, 28.0, 29.7, 32.6, 33.4, 36.9, 37.2, 39.1, 39.8,
40.0, 48.6, 61.1, 108.7, 111.8, 120.4, 126.4, 126.7, 127.5, 129.3, 129.4,
133.1, 141.1, 141.9, 145.1, 170.0; MS (ESI) [M + H]+ m/z 442.3.
Anal. Calcd for C30H35NO2 (441.27): C, 81.59; H, 7.99; N, 3.17.
Found: C, 81.30; H, 7.99; N, 3.16.
2,3,6,7-Tetramethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-
10(9H)-acridineacetic acid, ethyl ester (5j): yield 34 mg (21%);
1H NMR (400 MHz, CDCl3) δ 1.28 (t, J = 7.2, 3H), 1.44−2.10 (m,
12H), 2.22 (s, 6H), 2.24 (s, 6H), 3.45 (s, 2H), 4.28 (q, J = 7.2, 2H),
4.60 (s, 2H), 6.54 (s, 2H), 6.97 (s, 2H); 13C NMR (100 MHz, CDCl3)
δ 14.2, 19.1, 20.1, 32.2, 37.2, 48.7, 61.0, 113.6, 119.7, 123.9, 127.8,
128.3, 133.9, 141.4, 142.7, 170.2; MS (ESI) [M + H]+ m/z 456.2.
Anal. Calcd for C31H37NO2 (455.28): C, 81.72; H, 8.19; N, 3.07.
Found: C, 81.76; H, 8.17; N, 3.07.
2-Methyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-acridi-
1
neacetic acid, ethyl ester (5c): yield 54 mg (36%); H NMR (400
MHz, CDCl3) δ 1.29 (t, J = 7.2, 3H), 1.45−2.25 (m, 12H), 2.33 (s,
3H), 3.45 (ds, 2H), 4.28 (q, J = 7.2, 2H), 4.64 (s, 2H), 6.69 (d, J = 8.0,
1H), 6.77 (d, J = 8.0, 1H), 6.98 (d, J = 7.2, 2H), 7.04 (s, 1H), 7.16 (dt,
J1 = 7.2, J2 = 8.0, 1H), 7.25 (d, J = 7.2, 1H); 13C NMR (50 MHz,
CDCl3) δ 14.2, 20.8, 22.7, 27.5, 28.1, 28.7, 31.9, 32.2, 36.3, 37.2, 39.3,
39.7, 47.0, 48.7, 61.2, 112.1, 120.1, 120.2, 126.0, 126.1, 126.7, 127.4,
127.9, 129.6, 141.0, 143.4, 144.3, 169.9; MS (ESI) [M + H]+ m/z
414.2. Anal. Calcd for C28H31NO2 (413.24): C, 81.32; H, 7.56; N,
3.39. Found: C, 81.17; H, 7.55; N, 3.38.
3-Methyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-acridi-
neacetic acid, ethyl ester (5d): yield 106 mg (71%); 1H NMR (400
MHz, CDCl3) δ 1.29 (t, J = 7.2, 2H), 1.40−2.30 (m, 12H), 2.35 (s,
3H), 3.45 (s, 2H), 4.29 (q, J = 7.2, 2H), 4.65 (s, 2H), 6.59 (s, 1H),
6.78 (d, J = 8.0, 1H), 6.82 (d, J = 7.6, 1H), 6.99 (t, J = 7.6, 1H), 7.14
(m, 2H), 7.23 (d, J = 7.6, 1H); 13C NMR (50 MHz, CDCl3) δ 14.2,
21.7, 28.0, 32.2, 37.1, 39.5, 48.8, 61.2, 112.3, 113.1, 119.9, 120.3, 121.3,
123.5, 126.1, 127.2, 127.4, 135.8, 143.2, 144.0, 169.9; MS (ESI) [M +
H]+ m/z 414.2. Anal. Calcd for C28H31NO2 (413.24): C, 81.32; H,
7.56; N, 3.39. Found: C, 81.53; H, 7.59; N, 3.36.
1,2,6,7-Tetramethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-
10(9H)-acridineacetic acid, ethyl ester (5k): yield 62 mg (38%);
1H NMR (400 MHz, CDCl3) δ 1.26 (t, J = 7.2, 3H), 1.30−2.10 (m,
12H), 2.22 (s, 3H), 2.23 (s, 3H), 2.24 (s, 3H), 2.25 (s, 3H), 2.55 (s,
1H), 3.31 (s, 1H), 4.25 (m, 2H), 4.59 (AB, 2H), 6.52 (d, J = 8.4, 1H),
6.57 (s, 1H), 6.92 (d, J = 8.4, 1H), 6.98 (s, 1H); 13C NMR (100 MHz,
CDCl3) δ 14.2, 18.1, 19.1, 19.7, 20.1, 26.9, 27.8, 28.0, 32.6, 33.3, 36.9,
37.2, 39.1, 39.7, 40.0, 48.7, 61.0, 108.7, 113.4, 120.2, 125.3, 125.8,
126.6, 128.0, 128.1, 129.2, 133.0, 133.7, 141.3, 142.0, 144.5, 170.1; MS
(ESI) [M + H]+ m/z 456.2. Anal. Calcd for C31H37NO2 (455.28): C,
81.72; H, 8.19; N, 3.07. Found: C, 81.58; H, 8.20; N, 3.07.
General Procedure for the Synthesis of Compounds 1b−k.
Compounds 5b−k (0.07 mmol) were dissolved in CH2Cl2 (6.0 mL),
0.05 g of polymer-bound Bengal Rose or Methylene Blue were added,
and the suspension was cooled to 0 °C. The solution was bubbled with
oxygen and irradiated under stirring using a 500 W halogen lamp
equipped with an UV cutoff-filter (0.5% transmission at 400 nm). The
irradiation was continued until the starting material disappeared, and
the conversion was monitored by means of HPLC−MS. The reaction
mixture was treated with activated charcoal, filtered, and dried under
vacuum. The products 1c−f, 1h, and 1j were isolated pure or almost
pure (≥95%, on the basis of the NMR spectra); conversely, the
products 1b, 1i, and 1k were observed only in trace amounts by
HPLC−MS, and compound 1g was never observed.
4-Methyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-acridi-
1
neacetic acid, ethyl ester (5e): yield 84 mg (56%); H NMR (400
MHz, CDCl3) δ 1.17 (t, J = 7.2, 3H), 1.25−2.25 (m, 12H), 2.42 (s,
3H), 3.44 (s, 1H), 3.50 (s, 1H), 4.12 (q, J = 7.2, 2H), 4.58 (AB, 2H),
6.98−7.28 (m, 7H); 13C NMR (100 MHz, CDCl3) δ 14.1, 19.9, 27.6,
28.4, 32.5, 32.7, 32.9, 36.2, 37.1, 38.5, 38.7, 40.0, 54.6, 60.7, 118.8,
121.5, 121.9, 122.6, 125.4, 126.0, 127.1, 128.8, 129.3, 133.5, 134.6,
143.7, 144.2, 146.4, 170.2; MS (ESI) [M + H]+ m/z 414.2. Anal. Calcd
for C28H31NO2 (413.24): C, 81.32; H, 7.56; N, 3.39. Found: C, 81.26;
H, 7.61; N, 3.37.
2,7-Dimethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-ac-
1
ridineacetic acid, ethyl ester (5f): yield 71 mg (46%); H NMR
(400 MHz, CDCl3) δ 1.26 (t, J = 7.2, 3H), 1.43−2.20 (m, 12H), 2.31
(s, 6H), 3.44 (s, 2H), 4.26 (q, J = 7.2, 2H), 4.60 (s, 2H), 6.65 (d, J =
8.0, 2H), 6.95 (d, J = 8.0, 2H), 7.01 (s, 2H); 13C NMR (100 MHz,
CDCl3) δ 14.1, 20.5, 20.8, 21.8, 29.7, 31.9, 32.2, 33.7, 36.3, 37.2, 39.2,
46.9, 48.6, 61.1, 111.9, 114.0, 126.6, 127.3, 127.8, 129.3, 131.3, 134.8,
135.3, 140.3, 141.1, 144.0, 170.0; MS (ESI) [M + H]+ m/z 428.3.
Anal. Calcd for C29H33NO2 (427.25): C, 81.46; H, 7.78; N, 3.28.
Found: C, 81.92; H, 7.79; N, 3.30.
Dispiro[2-methylacridine-9(10H),3′-[1,2]dioxetane-4′,2″-
tricyclo[3.3.1.13,7]decane]-10-acetic acid, ethyl ester (1c): yield
17 mg (56%); 1H NMR (400 MHz, CDCl3) δ 0.65 (m, 2H), 1.26 (t, J
= 7.2, 3H), 1.47−2.11 (m, 6H), 2.29 (s, 2H), 2.43 (s, 3H), 2.55 (s,
4H), 4.27 (q, J = 7.2, 2H), 4.63 (s, 2H), 6.73 (d, J = 8.0, 1H), 6.80 (d,
J = 8.4, 1H), 7.18 (m, 2H), 7.35 (m, 1H), 8.02 (s, 1H), 8.21 (d, J = 6.8,
1H); 13C NMR (50 MHz, CDCl3) δ 14.2, 20.8, 25.5, 25.7, 29.7, 31.8,
33.0, 36.2, 39.3, 47.0, 48.4, 61.5, 86.9, 97.8, 111.6, 111.7, 120.6, 121.5,
1,5-Dimethyl-9-(tricyclo[3.3.1.13,7]dec-2-ylidene)-10(9H)-ac-
1
ridineacetic acid, ethyl ester (5g): yield 89 mg (58%); H NMR
(400 MHz, CDCl3) δ 1.22 (t, J = 7.2, 3H), 1.83 (s, 4H), 1.96 (s, 3H),
1.99−2.18 (m, 5H), 2.32 (s, 3H), 2.40 (s, 3H), 2.62 (s, 1H), 3.37 (s,
G
dx.doi.org/10.1021/jo401683r | J. Org. Chem. XXXX, XXX, XXX−XXX

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Article
128.4, 128.5, 129.0, 129.8, 130.3, 137.2, 139.5, 169.4; MS (ESI) [M +
H]+ m/z 446.3. Anal. Calcd for C28H31NO4 (445.23): C, 75.48; H,
7.01; N, 3.14. Found: C, 75.46; H, 7.00; N, 3.13.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: massimo.difusco@unibo.it.
*E-mail: arianna.quintavalla@unibo.it.
■
Dispiro[3-methylacridine-9(10H),3′-[1,2]dioxetane-4′,2″-
tricyclo[3.3.1.13,7]decane]-10-acetic acid, ethyl ester (1d): yield
21 mg (66%); 1H NMR (400 MHz, CDCl3) δ 0.65 (m, 2H), 1.16 (m,
2H), 1.28 (t, J = 7.2, 3H), 1.38−2.11 (m, 6H), 2.29 (ds, 2H), 2.40 (s,
3H), 2.56 (s, 2H), 4.29 (q, J = 7.2, 2H), 4.64 (s, 2H), 6.62 (s, 1H),
6.82 (d, J = 8.0, 1H), 7.02 (d, J = 7.6, 1H), 7.19 (t, J = 7.2, 1H), 7.36
(m, 1H), 8.09 (d, J = 8.0, 1H), 8.22 (dd, J1 = 8.0, J2 = 8.0, 1H); 13C
NMR (100 MHz, CDCl3) δ 14.2, 21.8, 25.5, 25.7, 27.4, 31.7, 31.8,
32.9, 33.0, 36.2, 36.3, 39.2, 47.0, 48.5, 61.5, 86.8, 97.8, 111.8, 112.4,
118.9, 120.8, 121.8, 121.9, 128.3, 128.4, 129.0, 139.1, 139.2, 169.3; MS
(ESI) [M + H]+ m/z 446.1. Anal. Calcd for C28H31NO4 (445.23): C,
75.48; H, 7.01; N, 3.14. Found: C, 75.33; H, 7.02; N, 3.15.
Dispiro[4-methylacridine-9(10H),3′-[1,2]dioxetane-4′,2″-
tricyclo[3.3.1.13,7]decane]-10-acetic acid, ethyl ester (1e): yield
15 mg (46%); 1H NMR (400 MHz, CDCl3) δ 0.59 (m, 1H), 0.79 (m,
1H), 1.16−2.25 (m, 11H), 2.42 (s, 3H), 2.55 (s, 4H), 4.31−4.54 (m,
4H), 7.02 (d, J = 7.6, 1H), 7.10−7.36 (m, 5H), 8.13 (m, 1H); 13C
NMR (50 MHz, CDCl3) δ 14.4, 20.7, 25.5, 25.7, 27.6, 29.8, 31.8, 32.0,
32.8, 32.9, 33.0, 33.2, 36.4, 39.4, 47.0, 56.5, 61.7, 87.1, 97.1, 116.3,
119.0, 122.0, 122.4, 126.3, 126.5, 128.0, 129.5, 129.8, 133.3, 141.0,
143.4, 172.1; MS (ESI) [M + H]+ m/z 446.1. Anal. Calcd for
C28H31NO4 (445.23): C, 75.48; H, 7.01; N, 3.14. Found: C, 74.83; H,
6.95; N, 3.11.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support was provided by the Italian Ministry of
Instruction, University and Research (PRIN 2009 projects:
prot. 20099PKPHH_004 “Design and development of
innovative catalytic systems” and prot. 2009MB4AYL “Integra-
tion of biosensing and nanotechnology for medical diagnostics
with high analytical performance”), University of Bologna, and
Fondazione del Monte di Bologna e Ravenna.
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4′,2″-tricyclo[3.3.1.13,7]decane]-10-acetic acid, ethyl ester
1
(1f): yield 7 mg (23%); H NMR (400 MHz, CDCl3) δ 0.66 (m,
2H), 1.17 (m, 2H), 1.20−2.30 (m, 9H), 2.30 (s, 2H), 2.43 (s, 6H),
2.48 (s, 1H), 2.56 (s, 1H), 4.26 (q, J = 7.2, 2H), 4.60 (s, 2H), 6.70 (d, J
= 8.0, 2H), 7.16 (d, J = 8.0, 2H), 8.00 (s, 2H); 13C NMR (100 MHz,
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46.9, 48.3, 61.5, 87.0, 97.7, 111.5, 118.8, 121.3, 128.5, 129.7, 137.3,
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4′,2″-tricyclo[3.3.1.13,7]decane-10-acetic acid, ethyl ester (1h):
yield 24 mg (72%); 1H NMR (400 MHz, CDCl3) δ 0.67 (m, 2H), 1.16
(m, 2H), 1.27 (t, J = 7.2, 3H), 1.38−2.11 (m, 9H), 2.30 (s, 3H), 2.33
(s, 3H), 2.42 (s, 3H), 2.56 (s, 1H), 4.27 (q, J = 7.2, 2H), 4.60 (s, 2H),
6.57 (s, 1H), 6.69 (d, J = 8.4, 1H), 7.14 (d, J = 8.4, 1H), 7.92 (s, 1H),
8.00 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 14.2, 19.1, 20.3, 20.8,
25.5, 25.8, 27.4, 31.8, 31.9, 32.9, 33.0, 36.2, 39.2, 47.0, 48.3, 61.4, 86.9,
97.7, 111.5, 112.9, 118.9, 121.5, 128.4, 128.7, 129.0, 129.6, 129.7,
137.4, 137.5, 137.6, 169.6; MS (ESI) [M + H]+ m/z 474.2. Anal. Calcd
for C30H35NO4 (473.26): C, 76.08; H, 7.45; N, 2.96. Found: C, 76.50;
H, 7.46; N, 2.97.
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ethyl ester (1j): yield 29 mg (84%); H NMR (400 MHz, CDCl3)
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of new compounds, absorption and
fluorescence spectra of acridone derivatives, and TCL spectra of
1,2-dioxetane derivatives. This material is available free of
charge via the Internet at http://pubs.acs.org.
H
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