Improvement of fluorescence characteristics of coumarins: Syntheses and fluorescence properties of 6-methoxycoumarin and benzocoumarin derivatives as novel fluorophores emitting in the longer wavelength region and their application to analytical reagents

Article abstract of DOI:10.1248/cpb.53.750

To improve the fluorescence characteristics, especially emission wavelength, of coumarins, various 3-substituted-6-methoxycoumarin derivatives were synthesized, and then benzocoumarin derivatives were also synthesized in expectation of the shift to the longer wavelength region by the extension of the conjugated system. Their fluorescence properties were investigated spectrophotometrically in acetonitrile and evaluated from the viewpoint of the intramolecular charge transfer (ICT) between push- and pull-substituents in the ground and the excited states. Among them, benzocoumarin derivatives especially fluoresced in the longer wavelength around 540 nm with remarkably large Stokes shifts beyond 10000 cm^-1. Using such fluorophores, some novel fluorescence derivatization reagents for carboxylic acids, alcohols, phenols, and amines were preliminarily prepared as an example, and their derivatized products were also found to fluoresce in the longer wavelength region with large Stokes shifts.

Full text of DOI:10.1248/cpb.53.750

750
Chem. Pharm. Bull. 53(7) 750—758 (2005)
Vol. 53, No. 7
Improvement of Fluorescence Characteristics of Coumarins: Syntheses
and Fluorescence Properties of 6-Methoxycoumarin and Benzocoumarin
Derivatives as Novel Fluorophores Emitting in the Longer Wavelength
Region and Their Application to Analytical Reagents
Chiyomi MURATA,a Toshinobu MASUDA,a Yasuko KAMOCHI,a Kenichiro TODOROKI,b
Hideyuki YOSHIDA, Hitoshi NOHTA, Masatoshi YAMAGUCHI, and Akira TAKADATE
b
b
b
,a
*
b
a Daiichi College of Pharmaceutical Sciences; 22–1 Tamagawa, Minami-ku, Fukuoka 815–8511, Japan: and Faculty of
Pharmaceutical Sciences, Fukuoka University; 8–19–1 Nanakuma, Jonan-ku, Fukuoka 814–0180, Japan.
Received November 22, 2004; accepted March 16, 2005
To improve the fluorescence characteristics, especially emission wavelength, of coumarins, various 3-substi-
tuted-6-methoxycoumarin derivatives were synthesized, and then benzocoumarin derivatives were also synthe-
sized in expectation of the shift to the longer wavelength region by the extension of the conjugated system. Their
fluorescence properties were investigated spectrophotometrically in acetonitrile and evaluated from the view-
point of the intramolecular charge transfer (ICT) between push- and pull-substituents in the ground and the ex-
cited states. Among them, benzocoumarin derivatives especially fluoresced in the longer wavelength around
540 nm with remarkably large Stokes shifts beyond 10000 cmꢀ1. Using such fluorophores, some novel fluores-
cence derivatization reagents for carboxylic acids, alcohols, phenols, and amines were preliminarily prepared as
an example, and their derivatized products were also found to fluoresce in the longer wavelength region with
large Stokes shifts.
Key words fluorescence characteristic; stokes shift; methoxycoumarin; benzocoumarin; intramolecular charge transfer; fluores-
cence derivatization
Fluorometric analysis is one of the most sensitive methods also another interesting fluorophore, since its fluorescence
for detecting organic and/or inorganic compounds and there- changes drastically with substituents and their introduced po-
fore it has been widely used in many scientific fields with im- sitions.19—21) In previous papers, we reported the fluorescence
proving analytical instruments such as high-performance liq- characteristics of methoxycoumarins and discussed the struc-
uid chromatography (HPLC), capillary electrophoresis (CE), tural features of strongly fluorescing methoxycoumarins
fluorescence confocal microscopy, etc. For these purposes, a from the viewpoint of intramolecular charge transfer (ICT)
great deal of effort has gone into the development of fluores- between push- and pull-substituents in the ground and the
cent reagents using various fluorophores, which can be uti- excited states.22,23) Based on the above findings, we suc-
lized for derivatizing biologically active small molecules, la- ceeded in developing novel fluorescent reagents with both
beling macromolecules such as proteins and DNAs, and high sensitivity and functions such as self-catalytic
probing ions such as biologically relevant metal cations and reactivity.24—26) In the course of those studies, we suspected
inorganic or organic anions.1—4) In the development of such that most coumarin derivatives showed large Stokes shifts in
fluorescent reagents, it should first be ensured that the fluo- their fluorescence spectra as compared with other fluo-
rophore used has a high fluorescence quantum yield because rophores. These large Stokes shifts appear to be advanta-
of the reliably high sensitivity in the detection. Fluorescein is geous for escaping autofluorescence from biological mole-
one of the most widely used fluorophore for biological exper- cules and reducing the self-absorption of chromophores. Fur-
iments, since it has a high fluorescence quantum yield of ther, if the emission bands of coumarins could be shifted to
0.90 or grater in aqueous solution and its excitation and longer wavelengths with large Stokes shifts, analytical detec-
emission wavelengths are in the visible region. Recently, tion limits would markedly increase because of the clearing
Nagano and coworkers have succeeded in developing fluores- of interferences off and higher signal-to–noise (S/N) ratio.
cent reagents for the specific detection of nitric oxide With this in mind, we began to design novel coumarin fluo-
(NO),5—8) singlet oxygen (1O2),9,10) and others11,12) using fluo- rophores that can fluoresce in the longer wavelength region
rescein as the fluorophore. 4,4-Difluoro-4-bora-3a,4a-diaza- while considering the relationship between the chemical
s-indacenes (BODIPYs) are also well-known fluorophores structure of coumarins and their fluorescence properties.
that have very sharp and narrow fluorescence bandwidths in Here, we report the synthesis and the fluorescence properties
addition to their high fluorescence quantum yield in aqueous of 6-methoxycoumarin and benzocoumarin (naphtopyra-
solution.4,13,14) Therefore they have been used as mother fluo- none) derivatives as candidate novel fluorophores emitting in
rophores for many analytical purposes.15—18) However, the the longer wavelength region, and the preliminary investiga-
difference between the excitation and emission wavelengths tion of their application to fluorescence derivatization
of these derivatives are very small, less than approximately reagents using synthesized fluorophores as an example.
30 nm for fluoresceins and 15 nm for BODIPYs, and it is
necessary to correct their spectra from interferences such as Results and Discussion
Rayleigh or Raman scattering light.
Design of Coumarin Fluorophores Although coumarin
On the other hand, coumarin (2H-benzopyran-2-one), is by itself is nonfluorescent, its derivatives with both electron-
∗ To whom correspondence should be addressed. e-mail: takadate@daiichi-cps.ac.jp
© 2005 Pharmaceutical Society of Japan

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Fig. 1. Absorption Spectra of 3-Substituted-6-methoxycumarins 1b, 1d, and 1f in Acetonitrile at 25 °C
Concentration of 3-substituted-6-methoxycumarinsꢁ2.0ꢂ10ꢀ5 M.
donating groups at the 6- and 7-positions and an electron- tive methylene compounds such as ethyl acetoacetate, diethyl
withdrawing group at the 3-position develop intense fluores- malonate, ethyl cyanoacetate, and ethyl nitroacetate by
cence,19—21) as shown in 3-acetyl-6,7-dimethoxycoumarin means of Knoevenagel condensation32) using piperidine as a
with a quantum yield of 0.52 and a large Stokes shift of catalyst in dry ethanol, and with phosphoranes such as car-
5200 cmꢀ1 in methanol.22) In previous studied, we examined bethoxymethyl triphenylphosphorane and carbethoxyethylli-
in detail the fluorescence characteristics of coumarins from dene triphenylphosphorane in diethylaniline, respectively.
the viewpoint of the substituent effect in connection with two The benzocoumarin derivatives 4a and b, 5a and b, and 6a—
ICT in the coumarin skeleton and postulated that one ICT d and f were also obtained from the appropriate hydroxy-
from an electron-donating group at the 6-position to an elec- naphthaldehyde in a similar manner to that for 6-methoxycou-
tron-withdrawing group at the 3-position contributed to the marin derivatives. 3-Formyl-6,7-benzocoumarin 6e was ob-
fluorescence wavelength and the other ICT from an electron- tained by the oxidation of 6d with selenium dioxide in
donating group at the 7-position to an electron-withdrawing toluene.33)
lactone carbonyl group at the 2-position contributed to the
Effects of Substituents on Spectroscopic Properties of
fluorescence intensity.22,23) Based upon the above hypotheses, 6-Methoxycoumarins The typical absorption spectra of 3-
only the former ICT may be allowed to shift the emission substituted-6-methoxycoumarins (1b, 1d, 1f) in acetonitrile
wavelength of coumarin to the longer wavelength region. are shown in Fig. 1. Their spectroscopic properties, absorp-
Thus 6-methoxycoumarin derivatives with various sub- tion maxima (lmax), molar absorptivities (e), fluorescence ex-
stituents at the 3-position were synthesized and their funda- citation maxima (lEx), fluorescence emission maxima (lEm),
mental fluorescence behaviors were investigated spectropho- Stokes shifts (Dn), and relative fluorescence intensities
tometrically.
(RFIs) are listed in Table 1, together with the Hammett sub-
It is well known that seminaphthofluoresceins (SNAFLs) stituent constants (sp-values).34,35) The RFIs are represented
and naphthofluoresceins, which are recognized as annellated as values against the fluorescence intensity (100) of the stan-
derivatives of fluorescein by one or two aromatic ring, have dard compound 1a. Although the Hammett substituent con-
longer emission wavelengths at 623 nm and 663 nm, respec- stants are commonly used for the estimation of reactivity and
tively, compared with fluorescein (516 nm).4,27—29) We there is no theoretical connection between sp constants and
thought that the introduction of such conjugated systems into fluorescence, these parameters appeared to be suitable and
the coumarin skeleton would make it possible to emit fluo- easily available to represent the total electronic effects in the
rescence in the longer wavelength region. However, it should ground state because of the fluorescence of coumarins based
be noted that the direction of annellation possibly affects the upon the typical ICT between push- and pull-subtituents in a
fluorescence properties of coumarin, as observed in studies molecule. Gottlieb and coworkers reported that the 13C-
of SNAFLs and naphthofluoresceins. Therefore a series chemical shifts of 6- and 7-substituted coumarins correlated
of benzo-annellated coumarins, that is, 5,6-benzocoumarin to the Hammett constants.36) Furthermore, Sherman and
(3H-naphto[2,1-b]pyran-3-one), 6,7-benzocoumarin (2H- Robins pointed out the close correlation of the fluorescence
naphto[2,3-b]pyran-2-one), and 7,8-benzocoumarin (2H- of substituted 7-hydroxycoumarins with the Hammett con-
naphto[1,2-b]pyran-2-one) derivatives were first synthesized, stants.37) Recently, Imai and coworkers have also succeeded
and their fundamental fluorescence properties were investi- in predicting the fluorescence characteristics of benzofurazan
gated for comparison.
compounds using the Hammett substituent constants.38)
Syntheses 6-Methoxycoumarin 1a was synthesized ac- Therefore it can be expected that the ICT character of
cording to the method described in the literature.30) Other de- coumarin derivatives in the ground state primarily reflects
rivatives 1b—f, with various substituents at the 3-position, their emissive ICT in the excited state. As shown in Fig. 1,
were synthesized from 5-methoxysalicylaldehyde31) with ac- the absorption spectra of 6-methoxycoumarins were influ-

752
Vol. 53, No. 7
Table 1. Spectral Properties of 3-Substituted-6-methoxycoumarins in Acetonitrile
Absorption
Fluorescence
Compound No.
R (sp)
l
max/nm
e
l
Ex/nm
l
Em/nm
Dn/cmꢀ1
RFI
1a
1b
1c
1d
1e
1f
H (0)
344
334
366
367
374
393
4000
8000
5200
5200
4800
4900
344
335
367
375
374
393
441
437
478
501
485
553
6400
7000
6300
6700
6100
7400
100
233
1871
1573
1067
223
CH3 (ꢀ0.17)
COOC2H5 (0.45)
COCH3 (0.50)
CN (0.66)
NO2 (0.78)
enced by a substituent at the 3-position. The lmax values of 3- the fluorescence properties of the compounds derivatized by
substituted compounds (1a—f) shifted to longer wavelengths such reagents.
with an increase in electron-withdrawing ability (see sp) of
Fluorescence Derivatization by 6-Methoxycoumarin
substituents in the order of 1fꢃ1eꢃ1dꢃ1cꢃ1aꢃ1b, Reagents In general, the fluorescence derivatization
whereas their molar absorptivities (e) were similar except for reagents consist of two parts, a mother fluorophore and a
that of 1b (Table 1). In contrast to the absorption spectra, group reactive to target molecules. We have already reported
their fluorescence spectra differed markedly. In the RFI, a the effects of the reactive groups of 6,7-dimethoxycoumarin
drastic increase was observed for 1c—e substituted with reagents, such as the bromoacetyl group for carboxylic acids,
electron-withdrawing groups, i.e., ethoxycarbonyl (ester) to hydrazinocarbonyl group for ketones or aldehydes, and
cyano groups, as compared with that of 1a and b. The RFI chlorocarbonyl group for hydroxyl and amino groups, on the
value of 1f, with the strongest electron-withdrawing nitro fluorescence characteristics of derivatized compounds and
group, was comparable with that of 1b, with an electron-do- found that the compounds derivatized by both bromoacetyl-
nating methyl group. This prominent decrease in 1f may be and chlorocarbonyl-reactive groups showed excellent fluores-
attributed to the nonemissive twisted ICT (TICT) excited cence quantum yields in acetonitrile and aqueous acetoni-
state,39—43) where push-pull p-electron systems between a trile.45) In this study, we also chose bromoacetyl, carboxylic,
methoxyl group at the 6-position and a nitro group at the 3- and chlorocarbonyl groups as reactive groups of derivatizing
position in a molecule must be fully separated in polar sol- reagents and introduced them into the 3-position of the 6-
vent such as acetonitrile. Rettig and Klock reported that the methoxycoumarin skeleton. The synthetic route for the above
very weak fluorescence of 6-aminocoumarin with a stronger derivatization reagents and their derivatization reactions are
electron-donating amino group than the methoxyl group was shown in Chart 1. One reagent, 2a with a bromoacetyl-reac-
attributed to this TICT between the amino group and tive group, was synthesized from 1d as previously re-
coumarin ring.44) In spite of 6-methoxycoumarins 1a and ported,46,47) and the derivatization reaction of carboxylic
1c—f having similar molar absorptivities (e), the reason for acids with 2a was carried out using acetic acid, lauric acid,
the distinct differences in their fluorescence intensities is not and benzoic acid as model carboxylic acids in the presence
yet obvious. It is likely that the ICT characters such as dipole of catalysts such as KHCO3 and 18-crown-6 to yield the de-
moments of 6-methoxycoumarins in the excited state are rivatized compounds 2b—d, respectively. Another reagent,
more amplified than those in the ground state. In future, the 3a with a carboxylic group as a reactive group, was synthe-
study of computer calculations of substituent effects in the sized by the acid hydrolysis of 1c, and the derivatized com-
excited state may enable the fluorescence characteristics to pounds esters 3b and c and amides 3d—f were obtained
be predicted more precisely. On the other hand, the lEm val- using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
ues of these compounds were also shifted to longer wave- drochloride (EDC HCl) and 4-N,N-dimethylaminopyridine
length regions, with an increase in the electron-withdrawing (DMAP) as usual catalysts in dichloromethane. The fluores-
ability of substituents at the 3-position. Furthermore, the cence properties of these derivatized compounds in methanol
wavelengths were also about 20 nm longer than that of 6,7- and acetonitrile are listed in Table 2. The RFIs in this table
dimethoxycoumarins and resulted in the Stokes shifts (Dn) are represented as the relative values against the fluorescence
beyond 6000 cmꢀ1. The results were in accordance with our intensity (100) of reference compound 1d. These results
hypothesis that an ICT from a methoxyl group at the 6-posi- showed that the difference of excitation and emission wave-
tion to a substituent such as ethoxycarbonyl and acetyl lengths (Dl) and Stokes shifts (Dn) of all derivatized com-
groups at the 3-position greatly contributes to the fluores- pounds were greater than 100 nm and 6000 cmꢀ1, respec-
cence wavelength. Although the fluorescence quantum yields tively, except for 3f. In particular, those of 2b—d obtained
of 6-methoxycoumarins were lower than those of fluores- using reagent 2a reached more than 120 nm and 6900 cmꢀ1,
ceins and BODIPYs, as shown in 1d with a quantum yield of respectively. These large shifts in the fluorescence spectra in
0.02 in acetonitrile,22) their large Dn values appear to be use- the fluorescence spectra may be due to the strong electron-
ful for fluorometric analysis. To confirm this hypothesis, we withdrawing ability of a newly introduced functional group,
tried to synthesize novel fluorescence derivatizing reagents which has two carbonyl groups at the 3-position of the
based on the 6-methoxycoumarin skeleton and investigated coumarin skeleton due to the derivatization reaction. Al-

July 2005
753
Table 2. Fluorescence Spectral Data of Derivatized Products with 6-Methoxycoumarin Reagents in Acetonitrile and Methanol
Acetonitrile
Methanol
Compound
No.
l
Ex/nm
l
Em/nm
(Dla)/nm)
Dn/cmꢀ1
RFI
l
Ex/nm
l
Em/nm
(Dla)/nm)
Dn/cmꢀ1
RFI
1d
2b
2c
2d
3b
3c
3d
3e
3f
367
380
380
384
368
372
363
350
358
478
506
506
485
479
482
467
464
n.d.b)
(111)
(126)
(126)
(101)
(111)
(110)
(104)
(114)
—
6300
6600
6600
5400
6300
6100
6100
7000
—
76
114
126
186
90
137
51
19
369
380
380
383
370
373
367
352
358
480
519
514
515
480
484
471
469
n.d.b)
(111)
(139)
(134)
(132)
(110)
(111)
(104)
(117)
—
6300
7000
6900
6700
6200
6100
6000
7100
—
100
134
149
200
119
153
76
36
—
—
a) DlꢁlEmꢀlEx, b) not detected.
Chart 1
though a fluorescence quantum yield for 2c in methanol has small shoulder band at around 370—450 nm, whereas angu-
already been reported to be 0.25 (RFIꢁ149 in Table 2),22) lated compounds 4b and 5b definitely had two splitting ab-
those of 2b and 3b and c appear to be high as well. On the sorption bands at 337 and 382 nm for 4b and 329 and 390 nm
other hand, the RFIs of 3d—f obtained using reagent 3a were for 5b, respectively, as shown in Fig. 2. Although such annel-
slightly low as compared with the above compounds, because lation effects on the absorption spectra were frequently ob-
thir electron-withdrawing ability at the 3-position were re- served in condensed aromatic compounds, for example, a lin-
duced by an amide group. However, these Stokes shifts were ear compound such as anthracene generally has a lmax as-
1
still greater than 6000 cmꢀ1. This trend in fluorescence inten- cribed to Platt’s Lb or Clar’s p bands in the longer wave-
sity was also observed in the study of 6,7-dimethoxy- length region than that of an angulated phenanthrene. The
coumarin reagents.22,23) Therefore these fluorescence deriva- lmax values of benzocoumarin derivatives shifted to longer
tization reagents based on the 6-methoxycoumarin skeleton wavelengths in the order of 5bꢃ4bꢃ6b with decreasing ab-
are expected to have fluorescence properties superior to that sorbance. The same tendency was also observed in the 3-car-
of the 6,7-dimethoxycoumarin skeleton from the viewpoint boethoxy compounds (5aꢃ4aꢃ6a), as shown in Table 3.
of the highly sensitive fluorescence detection of analytes in These differences in the absorption spectra apparently were
the longer wavelength region.
attributed to the substituent effect at the 3-position of the
Effects of Annellation on the Spectroscopic Properties benzocoumarin skeleton. The changes in transition moment
of Benzocoumarins The absorption and fluorescence spec- in a molecule may be reflected in their absorption spectra as
tra of three different benzocoumarins, 3-acetyl-5,6-benzo- the critical difference between linear compound and angu-
coumarin 4b, 3-acetyl-7,8-benzocoumarin 5b, and 3-acetyl- lated compounds. That is, this may be due to the increase in
6,7-benzocoumarin 6b, in acetonitrile are shown in Figs. 2 the CT character between the benzocoumarin skeleton and a
and 3, respectively, and the results are summarized in Table substituent at the 3-position. However, the fluorescence in-
3. As predicted, the direction of annellation affected both the tensity increased in the order of 5bꢃꢃ4bꢃ6b, as shown in
absorption and fluorescence properties of benzocoumarin de- Fig. 3. Konishi and coworkers also observed the same behav-
rivatives. The shape of the absorption band of 6b from iors in the fluorescence intensity of angulated and linear ben-
around 300 to 450 nm is markedly different from that of 4b zocoumarins in ethanol and benzene.48) Among these benzo-
and 5b. Linear compound 6b had a lmax at 337 nm with a coumarins, 4a of the ester-type and 5b of the acetyl-type

754
Vol. 53, No. 7
Fig. 2. Absorption Spectra of Benzocoumarins 4b, 5b, and 6b in Acetonitrile at 25 °C
Concentration of benzocoumarinsꢁ2.0ꢂ10ꢀ5 M.
substituents at the 3-position. In contrast to these behaviors
in the fluorescence intensity, the lEm values of the benzo-
coumarins shifted to the longer wavelength region in the
order of 6ꢃ5ꢃ4. In particular, that of linear compound 6b
reached the longest wavelength of 549 nm, in spite of lEx in
shorter wavelength (341 nm) and lower RFI. As a result, the
linear compounds 6a and b showed remarkably large Stokes
shifts (Dn) of greater than 11000 cmꢀ1, values two-fold
greater than those of the angulated compounds. Although
such fluorescence characteristics of the linear compounds 6a
and b are not clear, their emissive ICT characters in the ex-
cited state may be stabilized by the solvation of polar sol-
vents such as acetonitrile, resulting in shifts of their lEm val-
ues to the longer wavelength region and the lowering of their
fluorescence intensities.43,49) Thus the 6,7-benzocoumarin
skeleton was chosen as a novel fluorophore emitting in the
longer wavelength region, and its derivatives with various
substituents at the 3-position were synthesized to understand
the detailed fluorescence properties. Table 4 shows the fluo-
rescence spectral data of 3-substituted 6,7-benzocoumarins
6a—f. All the benzocoumarin compounds with electron-
withdrawing groups such as ester (6a), acetyl (6b), and
formyl (6e) groups at the 3-position developed intense fluo-
rescence. Langmuir and coworkers reported that the fluores-
cence quantum yields of linear benzocoumarin compounds
were very weak compared with those of angulated com-
pounds.49) For example, a quantum yield of methyl 7-
methoxy-2H-naphto[2,3,b]pyran-2-oxo-3-calboxylate, which
is a family compound of 6a, was found to be 0.13 in ethanol,
but its Dl value was 167 nm, resulting in a large Dn value
(8500 cmꢀ1). It was observed in this study that the lEm values
of the compounds with electron-withdrawing groups shifted
to the longer wavelength region with larger Dn values than
those of nonsubstituted derivative 6f and derivative 6d with
an electron-donating methyl group at the 3-position. In con-
trast, the RFI of 6c with a strong electron-withdrawing cyano
group apparently decreased. The reduction in the fluores-
cence intensity of 6c may be also ascribed to the nonemissive
TICT excited state due to the strong electron-withdrawing
ability of the cyano group, as well as in the case of 6-
methoxy-3-nitrocoumarin 1f. In this investigation, we con-
Fig. 3. Fluorescence Spectra of Benzocoumarins 4b, 5b, and 6b in Ace-
tonitrile at 25 °C
Concentration of benzocoumarinsꢁ1.0ꢂ10ꢀ6 M.
Table 3. Spectral Properties of Benzocoumarins in Acetonitrile
Absorption
Fluorescence
Compound
No.
l
max/nm
e
l
Ex/nm
l
Em/nm Dn/cmꢀ1
RFI
4a
4b
5a
5b
6a
6b
371
382
379
390
331
337
14200
15100
7900
8600
24200
22500
378
384
380
389
336
341
445
465
470
491
535
549
4000
4500
5000
5300
11100
11100
4050
90
1570
770
100
40
compounds showed the highest fluorescence intensity (Table
3). These results apparently indicate that the fluorescence in-
tensity of the benzocoumarins is influenced not only by the
direction of annellation, but also by the electronic effect of

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Table 4. Spectral Properties of 3-Substituted-6,7-benzocoumarins in Acetonitrile
Absorption
Fluorescence
Compound No.
R (sp)
l
max/nm
e
l
Ex/nm
l
Em/nm
Dn/cmꢀ1
RFI
6a
6b
6c
6d
6e
6f
COOC2H5 (0.45)
COCH3 (0.50)
CN (0.66)
CH3 (ꢀ0.17)
CHO (0.44)
H (0)
331
337
335
322
345
321
24200
22500
24000
19400
25100
19000
331
340
329
322
321
320
534
540
533
465
547
429
11500
10900
11600
9600
12900
7900
345
375
67
82
126
100
Chart 2
firmed that the linear benzocoumarin compounds with elec- the derivatives 6h—j and 6m—q obtained with these
tron-withdrawing groups at the 3-position had remarkably reagents in acetonitrile and methanol, which are frequently
large Stokes shifts of greater than 10000 cmꢀ1 in spite of low used as solvents in HPLC and CE, are summarized in Table
fluorescence intensity, and therefore that the introduction of 5, with reference compound 6b. The lEm values of these de-
such conjugated systems into the coumarin skeleton was ef- rivatives were shifted to the longer wavelength region with an
fective for the development of longer wavelength emissive increase in the electron-donating ability of the new func-
fluorophores. Next, we tried to synthesize novel fluorescence tional group (6h—jꢃ6m, nꢃ6o, p) introduced by the deriva-
derivatizing reagents based on the 6,7-benzocoumarin skele- tization reaction, and these lEm values reached more than
ton and investigated fluorescence properties of the deriva- 520 nm. As a result, their Dn values were surprisingly large,
tives prepared by such reagents.
almost greater than 10000 cmꢀ1. The actual fluorescence of
Fluorescence Derivatization by Benzocoumarin these products in methanol appeared greenish yellow with
Reagents The synthetic routes of novel fluorescence de- excitation at 365 nm on the transilluminator apparatus. A fur-
rivatization reagents based on the 6,7-benzocoumarin skele- ther examination of Table 5 shows that the tendency of these
ton and their derivatization reactions are shown in Chart 2. A derivatives in terms of fluorescence intensity was apparently
bromoacetyl group was also selected as a reactive group for different from those of reagents 2a and 3a based on the 6-
carboxylic acids, and the derivatization reaction with reagent methoxycoumarin skeleton. Among the derivatives, the fluo-
6g was carried out in a similar manner to that with 2a. On rescence intensities of 6h—j obtained by a reagent 6g were
the other hand, a more reactive chlorocarbonyl group45) than lower and those of 6o—q obtained with reagent 6l were
the carboxylic group in 6-methoxycoumarin reagent 3a was higher, except for 6q in methanol. Interestingly, this tendency
introduced into the benzocoumarin skeleton to give reagent was reversed in acetonitrile. A strong electron-withdrawing
6l, and the derivatization reactions of alcohols, phenols, and group with two carbonyl groups is present in derivatives
amines with reagent 6l proceeded smoothly at room tempera- 6h—j, whereas a weak electron-withdrawing amide group is
ture as compared with 3a. The fluorescence spectral data of present in derivatives 6o, p. Therefore the difference in fluo-

756
Vol. 53, No. 7
Table 5. Fluorescence Spectral Data of Derivatized Products with Benzocoumarin Reagents in Acetonitrile and Methanol
Acetonitrile
Methanol
Compound
No.
l
Ex/nm
l
Em/nm
(Dla)/nm)
Dn/cmꢀ1
RFI
l
Ex/nm
l
Em/nm
(Dla)/nm)
Dn/cmꢀ1
RFI
6h
6i
6j
6b
6m
6n
6o
6p
6q
345
345
346
331
332
337
334
326
343
544
544
544
534
534
539
515
510
n.d.b)
(199)
(199)
(198)
(203)
(202)
(202)
(181)
(184)
—
10700
10600
10500
11500
11400
11100
10500
11100
—
131
125
136
52
346
346
347
336
336
339
336
328
345
550
550
550
535
536
539
525
521
n.d.b)
(204)
(204)
(203)
(199)
(200)
(200)
(189)
(193)
—
10700
10700
10600
11100
11100
10900
10700
11300
—
55
58
64
100
114
138
471
124
—
60
115
270
31
—
a) DlꢁlEmꢀlEx, b) not detected.
Preparation of 1b This compound was prepared from 5-methoxysalicyl-
aldehyde31) and calbethoxyethylidene triphenylphosphorane in diethylaniline
according to the method described in a literature.30) Crude product was sub-
jected to silica gel chromatography using chloroform as the eluent and re-
crystallized from hexane to give 1b as pale yellow needles. 1H-NMR
rescence intensity could be attributed to the difference in
electron-donating ability between the introduced benzene
ring and methoxyl group. It is likely that the ICT characters
involving solvation of these benzocoumarins in the excited
state are different from those of 6-methoxycoumarin deriva- (CDCl3, ppm) d: 2.22 (3H, s, C3-CH3), 3.84 (3H, s, C6-OCH3), 6.86 (1H, s,
C5-H), 7.04 (1H, d, C7-H), 7.25 (1H, d, C8-H), and C8-H), 7.48 (1H, s, C4-
tives. In any case, these findings indicate that such derivatiza-
tion reagents based on the benzocoumarin skeleton would be
useful for selective and sensitive detection of specific mole-
H). HR-MS (EIꢄ) m/z: 190.0628 (Calcd for C11H10O3: 190.0630).
General Synthetic Method of 1c—f A mixture of 5-methoxysalicyl-
aldehyde (10 mmol) and the appropriate ester (10 mmol) in 25 ml of absolute
cules in complex biological systems, because of their large
Stokes shifts of more than 10000 cmꢀ1. In the future, we will
attempt to confirm the above results in further, more precise
spectroscopic experiments and elucidate the theoretical rela-
tionships between spectroscopic behaviors such as absorp-
tion and fluorescence and chemical structures involving the
substituent effects of coumarin derivatives.
In conclusion, we have found two candidates to be novel
fluorophores, 6-methoxycoumarin and 6,7-benzocoumarin,
which can fluoresce in the longer wavelength region with
large Stokes shifts. The former was designed based on the
emission mechanism of coumarins, while the latter was
based on the introduction of conjugated systems due to an-
nellation.
ethanol was refluxed in the presence of a few drops of piperidine for 30 min.
After cooling, the resulting precipitates were recrystallized from ethanol to
give 6-methoxycoumarins 1c—f as pale yellow needles.
1
Ethyl 6-Methoxycoumarin-3-carboxylate (1c): H-NMR (CDCl3, ppm) d:
1.41 (3H, t, –CH3), 3.87 (3H, s, C6-OCH3), 4.42 (2H, q, –CH2–), 7.01 (1H, s,
C5-H), 7.22—7.31 (2H, m, C7-, C8-H), 8.48 (1H, s, C4-H). HR-MS (EIꢄ)
m/z: 248.0685 (Calcd for C13H12O5: 248.0685).
1
3-Acetyl-6-methoxycoumarin (1d): H-NMR (CDCl3, ppm) d: 2.73 (3H,
t, C3-COCH3), 3.87 (3H, s, C6-OCH3), 7.04 (1H, s, C5-H), 7.24 (1H, d, C7-
H), 7.31 (1H, d, C8-H), 8.47 (1H, s, C4-H). HR-MS (EIꢄ) m/z: 218.0579
(Calcd for C12H10O4: 218.0579).
1
3-Cyano-6-methoxycoumarin (1e): H-NMR (CDCl3, ppm) d: 3.88 (3H,
s, C6-OCH3), 6.98 (1H, s, C5-H), 7.26—7.35 (2H, m, C7-, C8-H), 8.21 (1H,
s, C4-H). HR-MS (EIꢄ) m/z: 201.0423 (Calcd for C11H7O3: 201.0426).
1
3-Nitro-6-methoxycoumarin (1f): H-NMR (CDCl3, ppm) d: 3.90 (3H, s,
C6-OCH3), 7.09 (1H, s, C5-H), 7.36—7.39 (2H, m, C7-, C8-H), 8.70 (1H, s,
C4-H). HR-MS (EIꢄ) m/z: 221.0326 (Calcd for C10H7O5: 221.0324).
3-Bromoacetyl-6-methoxycoumarin (2a): This compound was prepared
Using the fluorophores, we obtained fluorescence derivati-
zation reagents 2a, 3a, 6g, and 6l for target molecules. Since by the bromination of acetyl compound 1c using tetrabuthylammonium tri-
bromide (TBABr3) as previously reported46,47) and recrystallized from
the derivatized compounds can emit fluorescence in the
longer wavelength region with large Stokes shifts, they
ethanol to give yellow needles of 2a. 1H-NMR (CDCl3, ppm) d: 3.38 (3H, s,
C6-OCH3), 4.77 (2H, s, COCH2Br), 7.07 (1H, s, C5-H), 7.28 (1H, d, C7-H),
should be useful for selective and sensitive detection of vari-
ous biological molecules in analytical procedures such as
HPLC and CE. In addition, the application of these reagents
to multicolored imaging, which can distinguish many target
molecules simultaneously owing to the varying bright fluo-
rescence from white blue to greenish yellow even if excited
at the same wavelength, would be possible.
7.34 (1H, d, C8-H), 8.60 (1H, s, C4-H). HR-MS (EIꢄ) m/z: 295.9691 (Calcd
for C12H9O4Br: 295.9684).
General Synthetic Method of Derivatized Products 2b—d46,47)
A
mixture of 2a (1.1 mmol), the appropriate carboxylic acid (1.2 mmol), 18-
crown-6 (1.0 mmol), and KHCO3 (800 mg) in acetone (200 ml) was stirred at
room temperature for 30 min. The solvent was evaporated to dryness under
reduced pressure. The residue was purified by column chromatography on
silica gel eluted with chloroform–ethyl acetate (9 : 1). Recrystallization from
ethanol gave 2b—d as pale yellow needles in 60—80% yields.
Experimental
6-Methoxycoumarin-3-carbonylmethyl Acetate (2b): 1H-NMR (CDCl3,
ppm) d: 2.22 (3H, s, –CH3), 3.88 (3H, s, C6-OCH3), 5.39 (2H, s, –COCH2–),
7.06 (1H, s, C5-H), 7.29—7.35 (2H, m, C7-, C8-H), 8.60 (1H, s, C4-H). HR-
1H-NMR spectra were obtained with a JEOL LNM-GSX 500FT-NMR
spectrometer. Chemical shifts are expressed in d ppm downfield from an in-
ternal tetramethylsilane (TMS) signal. The following abbreviations are used: MS (EIꢄ) m/z: 276.0630 (Calcd for C14H12O6: 276.0634).
s, singlet; d, doublet; t, triplet; and m, multiplet. The mass spectra (MS) were
6-Methoxycoumarin-3-carbonylmethyl Laurate (2c): 1H-NMR (CDCl3,
recorded with a JEOL JMS-DX303 spectrometer using the electron impact ppm) d: 0.88 (3H, t, –CH3), 1.27—1.38 (16H, m, –(CH2)8–), 1.70 (2H, m,
ionization (EI) mode at 70 eV. The fluorescence spectra were recorded with a –CH2–), 2.47 (2H, t, –CH2–), 3.87 (3H, s, C6-OCH3), 5.38 (2H, s,
Hitachi F-4500 fluorescence spectrophotometer. The slit width was 5.0 nm
for both excitation and emission. RFI values were obtained by comparing
the area under the recorded spectrum of coumarin derivatives with the stan-
dard one. All chemicals were of reagent grade and were used without further
purification. The solvents (Luminasol) used for the fluorescence measure-
–COCH2–), 7.05 (1H, s, C5-H), 7.27—7.34 (2H, m, C7-, C8-H), 8.59 (1H, s,
C4-H). HR-MS (EIꢄ) m/z: 416.2216 (Calcd for C24H32O6: 416.2199).
1
6-Methoxycoumarin-3-carbonylmethyl Benzoate (2d): H-NMR (CDCl3,
ppm) d: 3.88 (3H, s, C6-OCH3), 5.64 (2H, s, –COCH2–), 7.07 (1H, s, C5-H),
7.29 (1H, d, C7-H), 7.36 (1H, d, C8-H), 7.48—8.14 (5H, m, Ar-H), 8.62 (1H,
ment were purchased from Dojindo Laboratories (Kumamoto, Japan). Silica s, C4-H). HR-MS (EIꢄ) m/z: 338.0781 (Calcd for C19H14O6: 338.0790).
gel chromatography was performed using Silica gel 60 (Merck).
6-Methoxycoumarin-3-carboxylic Acid (3a): A suspension of 1c

July 2005
757
cording to the method of Ishii et al.51)
(5.0 mmol) in concentrated hydrochloric acid (10 ml) containing acetic acid
(5 ml) was refluxed for 2 h. The mixture was poured into ice-water (100 ml),
and then the resulting precipitate was collected, washed with water, and re-
crystallized from ethanol to give 3a as yellow needles. 1H-NMR (DMSO-d6,
ppm) d: 3.82 (3H, s, C6-OCH3), 7.33 (1H, d), 7.40 (1H, d), 7.48 (1H, s),
8.70 (1H, s), 13.25 (1H, broads, C3-COOH). HR-MS (EIꢄ) m/z: 220.0365
(Calcd for C11H8O5: 220.0372).
3-Formyl-6,7-benzocoumarin (6e): A solution of 6d (1.5 mmol) dissolved
in toluene was refluxed with selenium dioxide (2.0 mmol) for 2 h. The mix-
ture was filtered hot to remove black selenium, and the filtrate was concen-
trated under reduced pressure. The residue was purified by column chro-
matography on silica gel eluted with chloroform–ethyl acetate (7 : 3), and the
1
desired fractions were concentrated to give 6e as yellow crystals. H-NMR
General Synthetic Method of Derivatized Products 3b—f To an ice-
cold suspension of 3a (1.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDC HCl) (1.1 mmol), N,N-dimethylaminopyri-
dine (DMAP) (1.1 mmol) in dichloromethane (20 ml) was added dropwise to
the appropriate alcohols, phenols, or amines with stirring. The reaction mix-
ture was stirred for a further 1 h at room temperature, and then the mixture
was washed with saturated aqueous NaHCO3 and water. The solution was
dried over anhydrous Na2SO4 and the solvent was evaporated. The residue
was purified by column chromatography on silica gel eluted with ethyl ac-
etate. Recrystallization from ethanol gave 3b—f as pale yellow needles in
62—92% yield.
(CDCl3, ppm) d: 7.56 (1H, t), 7.67 (1H, t), 7.77 (1H, s), 7.91 (1H, d), 7.98
(1H, d), 8.26 (1H, d), 8.56 (1H, s), 10.36 (1H, s, C3-CHO). HR-MS (EIꢄ)
m/z: 224.0475 (Calcd for C14H8O3: 224.0474).
6,7-Benzocoumarin (6f): This compound was synthesized in a similar
manner to that described for 1b and purified by recrystallization from
ethanol–chloroform to give the product as yellow needles.
3-Bromoacetyl-6,7-benzocoumarin (6g): This compound was synthesized
from acetyl compound 6b in a similar manner to that described for 2a and
purified by recrystallization from ethanol–chloroform to give the product as
yellow needles. 1H-NMR (CDCl3, ppm) d: 4.80 (2H, s, C3-COCH2Br), 7.56
(1H, t), 7.67 (1H, t), 7.77 (1H, s), 7.92 (1H, d), 7.99 (1H, d), 8.27(1H, s),
8.78 (1H, s). HR-MS (EIꢄ) m/z: 315.9734 (Calcd for C15H9O3Br: 315.9735).
General Synthetic Method of Derivatized Products 6h—j These
compounds were synthesized from 6g in a similar manner to that described
for 2b—d and purified by column chromatography on silica gel eluted with
chloroform–ethyl acetate (9 : 1). Recrystallization from ethanol–chloroform
gave 6h—j as pale yellow needles in 60—92% yields.
1
Methyl 6-Methoxycoumarin-3-carboxylate (3b): H-NMR (CDCl3, ppm)
d: 3.87 (3H, s, C6-OCH3), 3.96 (3H, s, COOCH3), 7.03 (1H, s, C5-H), 7.24
(1H, d, C7-H), 7.31 (1H, d, C8-H), 8.53 (1H, s, C4-H). HR-MS (EIꢄ) m/z:
234.0521 (Calcd for C12H10O5: 234.0528).
1
Phenyl 6-Methoxycoumarin-3-carboxylate (3c): H-NMR (CDCl3, ppm)
d: 3.88 (3H, s, C6-OCH3), 7.06 (1H, s, C5-H), 7.24—7.30 (4H, m, C7-H, Ar-
H), 7.34 (1H, d, C8-H), 7.44 (2H, t, Ar-H), 8.69 (1H, s, C4-H). HR-MS (EIꢄ)
m/z: 296.0690 (Calcd for C17H12O5: 296.0685).
6,7-Benzocoumarin-3-carbonylmethyl acetate (6h): 1H-NMR (CDCl3,
ppm) d: 2.24 (3H, s, –CH3), 5.42 (2H, s, –COCH2–), 7.54—8.25 (6H, m),
8.77 (1H, s). HR-MS (EIꢄ) m/z: 296.0694 (Calcd for C17H12O5: 296.0685).
6,7-Benzocoumarin-3-carbonylmethyl Laurate (6i): 1H-NMR (CDCl3,
ppm) d: 0.89 (3H, t, –CH3), 1.25—1.39 (16H, m), 1.72 (2H, m), 2.49 (2H,
t), 5.42 (2H, s, –COCH2–), 7.56 (1H, t), 7.67 (2H, t), 7.77 (1H, s), 7.92 (1H,
d), 7.98 (1H, d), 8.25 (1H, s), 8.77 (1H, s). HR-MS (EIꢄ) m/z: 436.2242
(Calcd for C27H32O5: 436.2250).
6-Methoxycoumarin-3-N-methylcarboxamide (3d): 1H-NMR (CDCl3,
ppm) d: 3.03 (3H, d, NHCH3), 3.88 (3H, s, C6-OCH3), 7.08 (1H, s, C5-H),
7.23 (1H, d, C7-H), 7.34 (1H, d, C8-H), 8.88 (1H, s, C4-H). HR-MS (EIꢄ)
m/z: 233.0682 (Calcd for C12H11NO4: 233.0688).
6-Methoxycoumarin-3-N,N-dimethylcarboxamide (3e): 1H-NMR
(DMSO-d6, ppm) d: 3.02 (3H, s, N-CH3), 3.13 (3H, s, N-CH3), 3.86 (3H, s,
C6-OCH3), 6.94 (1H, s, C5-H), 7.13 (1H, d, C7-H), 7.30 (1H, d, C8-H), 7.86
(1H, s, C4-H). HR-MS (EIꢄ) m/z: 247.0843 (Calcd for C13H13NO4:
247.0845).
6,7-Benzocoumarin-3-carbonylmethyl Benzoate (6j): 1H-NMR (CDCl3,
ppm) d: 5.67 (2H, s, –COCH2–), 7.49 (2H, t), 7.55—7.69 (3H, m), 7.78 (1H,
s), 7.92 (1H, d), 7.98 (1H, d), 8.27 (1H, s), 8.15 (2H, d), 8.26 (1H, s), 8.79
(1H, s). HR-MS (EIꢄ) m/z: 358.0842 (Calcd for C22H14O5: 358.0841).
6-Methoxycoumarin-3-N-phenylcarboxamide (3f): 1H-NMR (DMSO-d6,
ppm) d: 3.90 (3H, s, C6-OCH3), 7.12 (1H, s, C5-H), 7.17 (1H, t, Ar-H), 7.28
(1H, m, C7-H), 7.37—7.40 (3H, m, C8-H, Ar-H), 7.75 (2H, d, Ar-H), 8.98
(1H, s, C4-H), 10.90 (1H, s, NH). HR-MS (EIꢄ) m/z: 295.0848 (Calcd for
C17H13NO4: 295.0845).
6,7-Benzocoumarin-3-carbonylchloride (6l):
A
suspension of 6a
(10 mmol) in concentrated hydrochloric acid (50 ml) containing acetic acid
(10 ml) was refluxed for 2 h. The mixture was poured into ice-water
(500 ml), and then the resulting precipitate was collected, washed with water,
and recrystallized from ethanol to give 6k as yellow needles. 1H-NMR
(DMSO-d6, ppm) d: 7.58 (1H, t), 7.69 (1H, t), 7.92 (1H, s), 8.04 (1H, d),
8.08 (1H, d), 8.56 (1H, s), 8.84 (1H, s), 13.32 (1H, broads, C3-COOH). HR-
MS (EIꢄ) m/z: 240.0432 (Calcd for C14H8O4: 240.0423). A suspension of 6k
(5.0 mmol) in thionyl chloride (15 ml) was refluxed for 12 h. After cooling,
excess thionyl chloride was evaporated to afford a crude product. This was
purified by recrystallization from benzene–petroleum ether to give pale yel-
low needles. 1H-NMR (CDCl3, ppm) d: 7.59 (1H, t), 7.71 (1H, t), 7.76 (1H,
s), 7.93 (1H, d), 8.01 (1H, d), 8.32 (1H, s), 9.00 (1H, s). HR-MS (EIꢄ) m/z:
258.0087 (Calcd for C14H7O3Cl: 258.0084).
General Synthetic Method of 4a and b, 5a and b, and 6a—c A mix-
ture of the hydroxynaphtaldehyde, 2-hydroxy-1-naphtaldehyde for 4a and b,
1-hydroxy-2-naphtaldehyde for 5a and b, and 2-hydroxy-3-naphtaldehyde50)
for 6a—c (10 mmol) and the appropriate ester (10 mmol) in 25 ml of ab-
solute ethanol was refluxed in the presence of a few drops of piperidine for
30 min. After cooling, the resulting precipitates were recrystallized from
ethanol to give benzocoumarins as yellow needles.
1
Ethyl 5,6-Benzocoumarin-3-carboxylate (4a): H-NMR (CDCl3, ppm) d:
1.46 (3H, t, –CH3), 4.49 (2H, q, –CH2–), 7.49 (1H, d), 7.63 (1H, t), 7.77
(1H, t), 7.95 (1H, d), 8.11 (1H, d), 8.35 (1H, d), 9.35 (1H, s). HR-MS (EIꢄ)
m/z: 268.0739 (Calcd for C16H12O4: 268.0736).
General Synthetic Method of Derivatized Products 6m—q To an ice-
cold solution of the appropriate alcohol, phenol, or amine (1.0 mmol) and
triethylamine (TEA) (1.1 mmol) in dichloromethane (20 ml) was added
dropwise 6l in dichloromethane (5 ml) with stirring. The reaction mixture
was stirred for a further 1 h at room temperature, and then the mixture was
washed with saturated aqueous NaHCO3 and water. The solution was dried
over anhydrous Na2SO4 and the solvent was evaporated. The residue was pu-
rified by recrystallization from ethanol to give 6m—q as pale yellow needles
in 60—70% yield.
3-Acetyl-5,6-benzocoumarin (4b): 1H-NMR (CDCl3, ppm) d: 2.80 (3H, s,
C3-COCH3), 7.50 (1H, d), 7.63 (1H, t), 7.77 (1H, t), 7.94 (1H, d), 8.12 (1H,
d), 8.39 (1H, d), 9.35 (1H, s). HR-MS (EIꢄ) m/z: 238.0639 (Calcd for
C15H10O3: 238.0630).
1
Ethyl 7,8-Benzocoumarin-3-carboxylate (5a): H-NMR (CDCl3, ppm) d:
1.44 (3H, t, –CH3), 4.45 (2H, q, –CH2–), 7.54 (1H, d), 7.68—7.73 (3H, m),
7.77 (1H, t), 7.90 (1H, d), 8.60 (1H, d), 8.35 (1H, d), 8.68 (1H, s). HR-MS
(EIꢄ) m/z: 268.0724 (Calcd for C16H12O4: 268.0736).
3-Acetyl-7,8-benzocoumarin (5b): 1H-NMR (CDCl3, ppm) d: 2.79 (3H, s,
C3-COCH3), 7.58 (1H, d), 7.70—7.74 (3H, m), 7.91 (1H, d), 8.60 (1H, d),
8.67 (1H, s). HR-MS (EIꢄ) m/z: 238.0620 (Calcd for C15H10O3: 238.0630).
1
Methyl 6,7-Benzocoumarin-3-carboxylate (6m): H-NMR (CDCl3, ppm)
d: 3.99 (3H, s, –COOCH3), 7.54 (1H, t), 7.64 (1H, t), 7.73 (1H, s), 7.90 (1H,
d), 7.96 (1H, d), 8.17 (1H, s), 8.70 (1H, s). HR-MS (EIꢄ) m/z: 254.0589
(Calcd for C15H14O4: 254.0579).
1
Ethyl 6,7-Benzocoumarin-3-carboxylate (6a): H-NMR (CDCl3, ppm) d:
Phenyl 6,7-Benzocoumarin-3-carboxylate (6n): 1H-NMR (CDCl3, ppm)
d: 7.28—7.32 (3H, m), 7.45 (2H, t), 7.56 (1H, t), 7.67 (1H, t), 7.78 (1H, s),
7.93 (1H, d), 7.99 (1H, d), 8.24 (1H, s), 8.87 (1H, s). HR-MS (EIꢄ) m/z:
316.0733 (Calcd for C20H12O4: 316.0736).
1.44 (3H, t, –CH3), 4.44 (2H, q, –CH2–), 7.54 (1H, d), 7.64 (1H, t), 7.73
(1H, s), 7.90 (1H, d), 7.96 (1H, d), 8.17 (1H, s), 8.66 (1H, s). HR-MS (EIꢄ)
m/z: 268.0746 (Calcd for C16H12O4: 268.0736).
3-Acetyl-6,7-benzocoumarin (6b): 1H-NMR (CDCl3, ppm) d: 2.77 (3H, s,
C3-COCH3), 7.55 (1H, d), 7.65 (1H, t), 7.75 (1H, s), 7.90 (1H, d), 7.97 (1H,
d), 8.22 (1H, s), 8.65 (1H, s). HR-MS (EIꢄ) m/z: 238.0620 (Calcd for
C15H10O3: 238.0630).
6,7-Benzocoumarin-3-N-methylcarboxamide (6o): 1H-NMR (CDCl3,
ppm) d: 3.05 (3H, d, –NHCH3), 7.56 (1H, t), 7.65 (1H, t), 7.78 (1H, s), 7.92
(1H, d), 7.99 (1H, d), 8.25 (1H, s), 8.78 (1H, s, –NH–), 9.06 (1H, s). HR-MS
(EIꢄ) m/z: 253.0740 (Calcd for C11H13NO3: 253.0739).
3-Cyano-6,7-benzocoumarin (6c): 1H-NMR (CDCl3, ppm) d: 7.59 (1H, t),
7.70 (1H, s), 7.81 (1H, s), 7.93 (1H, d), 7.99 (1H, d), 8.16 (1H, s), 8.39 (1H,
s). HR-MS (EIꢄ) m/z: 221.0479 (Calcd for C14H7NO2: 221.0477).
3-Methyl-6,7-benzocoumarin (6d): This compound was synthesized ac-
1
6,7-Benzocoumarin-3-N,N-dimethylcarboxamide (6p): H-NMR (CDCl3,
ppm) d: 3.05 (3H, s, N–CH3), 3.15 (3H, s, N–CH3), 7.53 (1H, t), 7.62 (1H,
t), 7.75 (1H, s), 7.90 (1H, d), 7.95 (1H, d), 8.04 (1H, s), 8.07 (1H, s). HR-

758
Vol. 53, No. 7
MS (EIꢄ) m/z: 267.0899 (Calcd for C16H13NO3: 267.0896).
6,7-Benzocoumarin-3-N-phenylcarboxamide (6q): 1H-NMR (CDCl3,
ppm) d: 7.18 (1H, t), 7.40 (2H, t), 7.57 (1H, t), 7.67 (1H, t), 7.77 (2H, d),
7.83 (1H, s), 7.94 (1H, d), 8.02 (1H, d), 8.30 (1H, s), 9.16 (1H, s), 10.86
(1H, s, –NH–). HR-MS (EIꢄ) m/z: 315.0909 (Calcd for C20H13NO3:
315.0896).
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