Synthesis of two-photon active cinnamoyl coumarins for high-contrast imaging of cancer cells and their photophysical characterization

Article abstract of DOI:10.1016/j.jphotochem.2014.02.005

A series of two-photon (TP) active 4-dimethylaminocinnamoyl coumarins were synthesized. These compounds exhibit red shift in absorption and considerable Stokes shift in emission spectra in comparison to the parent coumarin. Large TP absorption cross-sections were measured for all the coumarins in dilute solutions. A correlation between the chemical structure and TP characteristics was established. TP confocal microscopy revealed that these coumarin derivatives can be internalized by cancer cells rendering them a potential candidate as a label in TP confocal imaging.

Full text of DOI:10.1016/j.jphotochem.2014.02.005

Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis of two-photon active cinnamoyl coumarins for
high-contrast imaging of cancer cells and their photophysical
characterization
Preeti Yadava,1, Soumitra Satapathib,1, Meena Kumaria, Akanksha Chaturvedic, Lian Lid,
Lynne A. Samuelsond, Jayant Kumarb,∗∗, Sunil K. Sharmaa,∗
a Department of Chemistry, University of Delhi, Delhi 110007, India
b Center for Advanced Materials, University of Massachusetts Lowell, Lowell, MA 01854, USA
c Indian Institute of Science Education and Research Pune, Pune, Maharashtra 411008, India
d US Army Natick Soldier Research, Development and Engineering Center, Natick, MA 01760, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of two-photon (TP) active 4-dimethylaminocinnamoyl coumarins were synthesized. These com-
pounds exhibit red shift in absorption and considerable Stokes shift in emission spectra in comparison to
the parent coumarin. Large TP absorption cross-sections were measured for all the coumarins in dilute
solutions. A correlation between the chemical structure and TP characteristics was established. TP con-
focal microscopy revealed that these coumarin derivatives can be internalized by cancer cells rendering
them a potential candidate as a label in TP confocal imaging.
Received 20 December 2013
Received in revised form 14 February 2014
Accepted 19 February 2014
Available online 1 March 2014
Keywords:
Two-photon active
© 2014 Elsevier B.V. All rights reserved.
4-Dimethylaminocinnamoyl coumarins
Stokes shift
Label
1. Introduction
the possibility of using novel coumarin derivatives as molecular
probes for confocal microscopy based bio-imaging.
Among all of the fluorescence microscopy techniques developed
and optimized during the last few decades [1], the two-photon
fluorescence (TPF) microscopy is of specific interest because of
its potential applications in clinical imaging [2], immunology [3]
and bio-engineering [4]. In TPF, a fluorescent molecule absorbs
two near IR (NIR) photons simultaneously and emits a photon
in the visible spectral region. Unlike conventional one-photon
confocal microscopy, the TPF emission is highly localized reducing
the photo damage and improving the spatial contrast in imaging.
The widespread applications of the TPF microscopy have led to
several design strategies and synthesis of organic molecules with
large TPA cross-sections (ı) [5,6]. The ı value for organic molecules
is usually small, thus it is desirable to search for newer organic
molecules with high absorption cross sections. We have explored
Coumarins, a member of benzopyrone family, are a group
of plant-derived polyphenolics and are well-known for their
antibacterial, anticoagulant, antioxidant, anticancer, and enzyme
inhibition properties [7–10]. These compounds are relatively non-
toxic as no adverse effects of coumarin have been reported [11].
Apart from being biologically active, coumarins also possess consid-
erable optical properties e.g. molar extinction coefficient, quantum
efficiency of fluorescence, and emission in the visible spectral
region [12]. These features render coumarin and its derivatives
potential candidates for high contrast in vivo imaging of tumor
cells. However, the application of coumarins for biological imaging
has not been well-established. This could be attributed to the fact
that the coumarins typically show small Stokes shift of the order of
10–30 nm [13–15]. However, Stokes shifts of 80 nm and larger are
desirable to minimize the background from the exciting radiation
and to reduce autofluorescence in cells [16]. In addition, molecules
with high quantum efficiency of fluorescence are considered as
prospective candidates for high contrast in vivo imaging. Therefore,
fluorophores with a higher Stokes shift and high quantum yield
(˚) are promising candidates for NIR fluorescence bioassays, which
can enhance the detection sensitivity to a large extent. With this
∗
Corresponding author. Tel.: +91 11 27666950; fax: +91 11 27666950.
Corresponding author.
E-mail addresses: jayant kumar@uml.edu (J. Kumar), sk.sharma90@gmail.com
∗∗
(S.K. Sharma).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jphotochem.2014.02.005
1010-6030/© 2014 Elsevier B.V. All rights reserved.

40
P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
O
O
HO
O
O
O
HO
O
OH
i
+
O
R1
R1
R1 = H/C2H5
1a/1b R1 = H/C2H5
Scheme 2. Reagents and conditions: (i) HClO4·SiO2, 130 ◦C, 30–90 min, solvent free.
Scheme 1. Reagents and conditions: (i) CH3COCHR1COOC2H5, conc. H2SO4, 30 ◦C;
(ii) (CH3CO)2O, DMAP, THF, 30 ◦C; (iii) AlCl3, 125–170 ◦C.
perspective we have designed and developed a series of fluorescent
coumarin derivatives for bio-imaging applications.
Wu et al. reported that 1,3-diarylprop-2-enones (chalcones)
exhibit promising nonlinear optical properties [17]. Therefore, it
is expected that the incorporation of a 3-aryl-2-propenoyl (cin-
namoyl) moiety on the coumarin skeleton might lead to new
materials that are fluorescent and also possess nonlinear optical
properties. A few such derivatives are reported in the litera-
ture; however, in almost all the cases the cinnamoyl moiety
was kept confined at the C-3 or C-4 position of the coumarin
skeleton (lactone ring) [18]. In addition, their capabilities for fluo-
rescence imaginghave notbeen fully explored [18]. Herein, wehave
synthesized C-6 and C-8 substituted 4-dimethylaminocinnamoyl
coumarins and studied their photophysical and TP bio-imaging
properties.
Scheme 3. Reagents and conditions: (i) DMB (4-dimethylaminobenzaldehyde),
ethanol, reflux; (ii) CH3I, dimethylformamide, K2CO3, 30 ◦C.
ortho-coupled protons H-5 and H-6 were observed at ı 7.60 and
6.82 (J = 8.8 Hz) respectively. The isomeric compounds 1b and 1d
were also characterized on a similar basis.
The C-6 and C-8 substituted 4-dimethylaminocinnamoyl
coumarins (2–5) were synthesized by piperidine catalyzed
Claisen–Schmidt condensation of 4-dimethylaminobenzaldehyde
(DMB) with 6/8-acetyl-7-hydroxy-3-alkyl-4-methylcoumarin.
Compounds 6, 7, and 8 in turn were synthesized by carrying
O-methylation of compounds 2, 3, and 4 respectively using methyl
iodide (Scheme 3). The use of a weak base piperidine was preferred
because chalcones having hydroxyl group at ortho to carbonyl
group in ring A may get cyclized to the corresponding flavanones
under stronger alkaline conditions. The compounds reported here
are novel except 2 [23] and 4 [24] and fully characterized.
2. Results and discussion
2.1. Chemistry
The reaction of ␤-resorcinol with corresponding ethylace-
toacetate under Pechmann condensation yielded 7-hydroxy-3-
alkyl-4-methyl-2H-benzopyran-2-one [19]. Acetylation of these
compounds with acetic anhydride under catalytic amount of
dimethylaminopyridine (DMAP) in THF resulted the correspond-
ing 7-acetoxy derivatives. The C-6 acetyl (1a, 1b) and C-8 acetyl
(1c, 1d) coumarin derivatives (Scheme 1) were then obtained
from 7-acetoxy-3-alkyl-4-methylcoumarin via Fries migration in
1:9 ratio, respectively [20,21]. As the yield of the 6-acetyl-7-
hydroxy-4-methyl-2H-chromen-2-one is low via Fries migration,
we have synthesized it by following another literature procedure
(Scheme 2) in about 60% yield [22]. The structural assignment of
1a and 1c was unambiguously carried on the basis of 1H NMR. For
compound 1a the protons H-5 and H-8 were observed as singlets
at ı 7.94 and 6.82 respectively, however for compound 1c the two
2.2. Photochemistry
The
optical
properties
of
all
of
the
six
4-
dimethylaminocinnamoyl coumarins (2, 4–8) were studied.
For spectroscopic measurement, the compounds were dissolved
in DMSO (1 mg/mL) and diluted further to get the required con-
centration for optical studies. A quartz cuvette of 1 cm path-length
was used to record both absorption and one-photon fluorescence
spectra. All of the compounds exhibited strong absorption maxima
8
7
6
5
4
2
1.0
0.8
0.6
0.4
0.2
0.0
400
500
600
700
800
Wavelength (nm)
Fig. 1. Normalized one-photon absorption (solid lines) and emission (dotted lines) spectra of cinnamoyl coumarins.

P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
41
20000
15000
10000
5000
0
25 mW
50 mW
a
b
75 mW
10000
1000
100
100 mW
125 mW
slope 2.06
10
100
500
600
700
Log (laser power)
Wavelength (nm)
Fig. 2. TPF spectra (a) and quadratic dependence of the fluorescence (b) for compound 7.
in the visible region from 399 to 463 nm due to extended conju-
gation. When excited at their corresponding absorption maxima,
they emitted strong green and yellow fluorescence with ꢀmax in
the range of 505–561 nm. Fig. 1 shows the normalized absorption
and fluorescence spectra of the cinnamoyl coumarins. Stokes shifts
of the order 98–149 nm were observed for these compounds.
In DMSO, the compound 6 exhibited the highest Stokes shift
(149 nm). These compounds display strong solvatochromism [25]
as even larger Stokes shifts of 175 and 195 nm were observed for
compounds 2 and 3, respectively in low polarity solvent chloro-
form (Supporting Information Table S1). The Stokes shifts of C-6
and C-8 cinnamoyl coumarins were compared and it was observed
that 6-cinnamoyl coumarins exhibit larger shifts and this may be
due to molecular geometry relaxation of the fluorophore upon
photoexcitation [26,27]. The quantum yields of the compounds
were determined using a commercially available dye, Coumarin
153, in ethyl acetate as a reference. The quantum efficiency of
Coumarin 153 in ethyl acetate is 88%. A moderate quantum yield
value was observed for these compounds as listed in Table 1.
Strong TPF was observed from each of these compounds in their
dilute solutions of DMSO when excited with NIR femtosecond laser
pulses. TPF microscopy confirmed the cellular uptake of these cin-
namoyl coumarins. The details of TPF experimental set-up have
been reported elsewhere [28]. Fig. 2 shows the intensity dependent
10000
1000
10000
1000
slope 2.00
100
slope 1.7
100
10
100
10
100
10000
1000
100
Log(laser power)
Log (laser power)
a
b
slope 1.8
10
100
10
100
10000
Log(lascer power)
10000
1000
100
10000
1000
100
1000
100
slope 2.0
slope 2.00
10
100
10
100
Log(laser power)
Log(laser power)
e
d
Fig. 3. Quadratic dependence of TPF for compounds 2 (a), 4 (b), 5 (c), 6 (d), and 8 (e).

42
P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
Fig. 4. One-photon fluorescence and TPF confocal images of WEHI-231 cells (4 ␮m × 4 ␮m) with compound 7. 1P, one-photon; 2P, two-photon; FL, coumarin fluorescence
image; DIC, differential interference contrast image; and MERGE, fluorescence imaged merged with DIC.
TPF spectra for compound 7. While comparing the absorption spec-
tra recorded before and after the TPF studies, no noticeable change
in the absorption profile was observed, thus suggesting that the
compounds have reasonable photostability.
The laser power dependent TPF for the cinnamoyl coumarin was
also measured. In each of the cases, As the intensity of the laser
beam increased, the TPF intensity also increased. The quadratic
behavior of the TPF was also confirmed by plotting log[TPF inten-
sity] vs log[laser power] as shown in Fig. 2. A slope of approximately
two was determined, indicating that the fluorescence from the
coumarin derivative is indeed a TP phenomenon. TPF studies were
also carried out for the other five compounds (Fig. 3).
correlation of quantum efficiency and TPA action cross-sections
with the chemical structures of the coumarins was observed. An
enhancement in quantum efficiency was observed for both C-6
and C-8 cinnamoyl coumarins, when the C-3 position is substituted
with an alkyl group (Table 1). The highest TPA action cross-section
was observed in 3-alkyl coumarin derivative 7 (16 GM). This obser-
vation could be useful for the future design and development of
coumarin based TP fluorophores.
2.3. Biology
To demonstrate the potential applications of cinnamoyl
coumarins as TP probes, we investigated their uptake in a murine
immature B lymphoma cell line WEHI 231. Both one-photon and TP
confocal microscopies (Laser Scanning Microscope 710, Zeiss cor-
poration) were performed. The cells were maintained in RPMI and
10% serum at 37 ◦C and were incubated with 10 ␮g/mL concentra-
tion of the cinnamoyl coumarins directly under the microscope and
uptake was monitored. Fig. 4 shows the one-photon fluorescence
and TPF confocal images of the uptake of the cinnamoyl coumarin
7 molecules in WEHI-231 cells. These images clearly indicated that
the cinnamoyl coumarin molecules were internalized in WEHI-231
cells. We did not observe any cell death at the coumarin concentra-
tion used for imaging. These results strongly suggested that the
cinnamoyl coumarins could be used as a potential fluorescence
label for TPF imaging.
The TPA cross-section and TPA action cross-section (defined
as ı·˚) were determined using fluorescein in water as a refer-
ence. The TPA action cross-section offers a quantitative measure of
a chromophore for fluorescence microscopy application. The TPA
cross-section was calculated using the following formula:
ꢀ
ꢁꢀ ꢁꢀ
ꢁ
Fc
Fr
˚
Cr
Cc
r
ıc = ır
˚
c
where F is the integrated TPF intensity; C is the concentration;
subscripts c and r stand for the cinnamoyl coumarin and the ref-
erence molecule, respectively [29]. Using the TPA cross-section of
fluorescein of 36 GM at 800 nm [29], the calculated TPA cross-
sections of the cinnamoyl compounds (2, 4–8) (Table 1) were
found to be comparable to the dyes used as fluorescent labels. A
3. Conclusion
Table 1
Optical properties of the cinnamoyl coumarins in DMSO.
We have synthesized a series of fluorescent TP active biocom-
patible cinnamoyl coumarins. Strong TPF was observed for each
of the compounds. These coumarins exhibited considerable Stokes
shifts and moderate fluorescence quantum efficiencies. A correla-
tion of the chemical structure and TPF has been established. This
opens up a new design strategy of synthesizing organic molecules
with high ı value. The TPF images confirmed the rapid uptake of
the coumarins by the murine immature B lymphoma cell line WEHI
Compd. ꢀAb. (nm) ꢀEm. (nm) Stokes shift ˚ (%) ı (GM)
ı˚ (GM)
(nm)
2
4
5
6
7
8
463
399
406
408
409
408
561
512
510
557
553
505
98
113
104
149
144
97
5.2
8.6
12.0
10.0
18.0
1.0
53
29
63
38
93
2.75
2.50
8.00
3.80
16.00
1.75
175

P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
43
231. These coumarins have the potential to be used in the develop-
ment of a biological imaging probe.
round bottom flask fitted with a reflux condenser. The temper-
ature of the reaction mixture was raised quickly to 125 ◦C and
then slowly over a period of 2 h to 170 ◦C. Then, crushed ice was
added to the reaction mixture followed by the acidification using
dilute hydrochloric acid. The crude product was filtered, washed
with water followed by ether and then subjected to column
chromatography (petroleum ether/ethyl acetate).
4. Experimental
4.1. Materials and methods
4.1.1. Materials
4.2.1.1. 6-Acetyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1a).
All of the chemicals and reagents were procured from Spec-
trochem Pvt. Ltd., India, and Sigma–Aldrich Chemicals Pvt. Ltd.,
USA. The organic solvents were dried and distilled prior to their
use. Reactions were monitored by precoated TLC plates (Merck sil-
ica gel 60F254); the spots were visualized either by UV light, or by
spraying with 5% alcoholic FeCl3 solution. Silica gel (100–200 mesh)
was used for column chromatography.
Compound 1a was obtained as light yellow solid in 60% yield
by column chromatography using ethyl acetate/petroleum ether
(1:10) as an eluent. Melting point: 210–211 ◦C; UV (acetonitrile)
ꢀmax: 257, 297 and 342 nm; IR (KBr) vmax: 3438.76 (OH), 2357.39,
1738.11 (CO), 1614.66, 1369.57, 1308.01, 1233.59, 1059.21, 876.65,
657.39 cm−1 1H NMR (CDCl3, 400 MHz): ı 2.42 (s, 3H, C-4 CH3),
;
2.68 (s, 3H, –COCH3), 6.15 (s, 1H, H-3), 6.82 (s, 1H, H-8), 7.94 (s, 1H,
H-5), 12.61 (brs, 1H, OH); 13C NMR (CDCl3, 100.5 MHz): ı 18.57,
26.63, 105.39, 112.86, 112.99, 117.07, 128.12, 151.62, 158.71,
159.88, 165.31, 203.30; ESI MS: Calculated for C12H10O4 [M+H]+
219, found 219.
4.1.2. Instruments
Melting points were measured on a Buchi M-560 instrument and
are uncorrected. Infrared spectra were recorded on a Perkin-Elmer
FT-IR model 9 spectrometer. The 1H NMR and 13C NMR spectra were
recorded on a Jeol-400 (400 MHz, 100.5 MHz) NMR spectrometer
and Avance-300 (300 MHz, 75.5 MHz) spectrometer using tetram-
ethylsilane as the internal standard. The chemical shift values are
on a ı scale and the coupling constant values (J) are in hertz. The
HRMS data were recorded on Agilent-6210 ES-TOF, JEOL JMX-SX-
102A and Waters LCT Micromass-KC455. UV–Visible absorption
spectra were recorded using a Perkin-Elmer Lambda-9 spectropho-
tometer. Photoluminescence spectra were recorded using a Varian
Cary Eclipse Fluorescence spectrophotometer (Cary 300). A quartz
cuvette of 1 cm path-length was used to record absorption and
fluorescence spectra.
4.2.1.2. 6-Acetyl-3-ethyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1b). Compound 1b was obtained as light yellow solid in 60% yield
by column chromatography using ethyl acetate/petroleum ether
(1:20) as an eluent. Melting point: 144–146 ◦C; UV (acetonitrile)
ꢀmax: 210, 230, 256, 305 and 341 nm; IR (KBr) vmax: 3413.02
(OH), 3057.27, 2973.31, 1721.02 (CO), 1646.41, 1386.40, 1254.88,
1167.11, 1058.49, 930.50, 805.41 cm−1 1H NMR (CDCl3, 400 MHz):
;
ı 1.10 (t, 3H, J = 7.3 Hz, –CH2CH3), 2.38 (s, 3H, C-4 CH3), 2.61 (q, 2H,
J = 7.3 Hz, –CH2CH3), 2.67 (s, 3H, –COCH3), 6.74 (s, 1H, H-8), 7.91 (s,
1H, H-5), 12.50 (brs, 1H, OH); 13C NMR (CDCl3, 100.5 MHz): ı 13.03,
14.77, 20.86, 26.57, 104.72, 113.52, 116.92, 125.99, 127.88, 144.75,
157.27, 160.65, 164.16, 203.38; HRMS: Calculated for C14H14O4
[M+Na]+ 269.0790, found 269.0784.
4.1.3. Quantum efficiency measurement
For quantum efficiency measurement, the compounds were dis-
solved in DMSO (1 mg/mL) and diluted further to get the required
concentration for optical studies. Coumarin 153 in ethyl acetate
was used as reference. For quantum efficiency measurement iden-
tical optical density solution was prepared. The relative quantum
efficiency was measured using the following equation:
4.2.1.3. 8-Acetyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1c).
Compound 1c was obtained as light yellow solid in 70% yield by
column chromatography using ethyl acetate–petroleum ether
(1:20) as an eluent; Melting point: 162–163 ◦C; UV (acetonitrile)
ꢀmax: 211, 308 and 350 nm; IR (Nujol) vmax: 2920.95, 2853.95,
1738.43 (COCH3), 1732.03 (CO), 1610.85, 1463.28, 1371.81,
ꢀ
ꢁ
ꢀ
ꢁ
2
Fc
Fr
nc
nr
˚
c
= ˚r ×
×
1234.08, 1087.60, 1059.53, 878.03, 658.62 cm−1 1H NMR (CDCl3,
;
400 MHz): ı 2.36 (s, 3H, C-4 CH3), 2.86 (s, 3H, –COCH3), 6.08 (s, 1H,
H-3), 6.82 (d, 1H, J = 8.8 Hz, H-6), 7.60 (d, 1H, J = 8.8 Hz, H-5), 13.48
(brs, 1H, OH); 13C NMR (CDCl3, 100.5 MHz): ı 19.08, 33.79, 109.09,
110.89, 111.73, 114.96, 131.19, 152.95, 154.98, 159.19, 166.47,
204.21; ESI MS: Calculated for C12H10O4 [M]+• 218, found 218.
where ˚c = quantum efficiency of the cinnamoyl coumarin;
˚r = quantum efficiency of the reference molecule; Fc = integrated
fluorescence intensity of the cinnamoyl coumarin; Fr = integrated
fluorescence intensity of the reference molecule; nc = refractive
index of the cinnamoyl coumarin; and nr = refractive index of the
reference molecule.
4.2.1.4. 8-Acetyl-3-ethyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1d). Compound 1d was obtained as light yellow solid in 70%
yield by column chromatography using ethyl acetate/petroleum
ether (1:10) as an eluent. Melting point: 171–172 ◦C; UV (ace-
tonitrile) ꢀmax: 240, 273, 305 and 350 nm; IR (KBr) vmax: 2970.32,
4.1.4. Two-photon-fluorescence measurement
A Quantronix mode-locked Ti:sapphire laser at 800 nm with
100 fs pulse width and 1 kHz repetition rate was used as the exci-
tation source for TPF measurements. The 800 nm laser pulse was
passed through the sample cuvette and the fluorescence signal was
collected at right angle using a CCD array detector in conjunction
with a monochromator. A short-pass filter at 750 nm was placed
in front of the monochromator to remove the excitation. A 4.5 cm
focal length lens was used to collect the fluorescence.
2935.77, 1717.83 (CO), 1614.92, 1370.28, 1215.39, 1092.50, 834.73,
−1
647.36 cm
;
1H NMR (CDCl3, 400 MHz): ı 1.09 (t, 3H, J = 7.3 Hz,
–CH2CH3), 2.33 (s, 3H, C-4 CH3), 2.59 (q, 2H, J = 7.3 Hz, –CH2CH3),
2.86 (s, 3H, –COCH3), 6.78 (d, 1H, J = 8.7 Hz, H-6), 7.60 (d, 1H,
J = 9.5 Hz, H-5), 13.34 (brs, 1H, OH); 13C NMR (CDCl3, 100.5 MHz): ı
12.90, 14.77, 20.57, 33.82, 108.88, 112.46, 114.62, 124.14, 131.23,
146.03, 153.32, 160.07, 165.34, 204.24; HRMS: Calculated for
4.2. Chemistry
C
14H14O4 [M + H]+ 247.0926, found 247.0965.
4.2.1. General procedure for synthesis of
6/8-acetyl-7-hydroxy-4-methyl-2H-chromen-2-ones (1a–1d)
4.2.2. General procedure for the synthesis of C-6 and C-8
substituted cinnamoyl coumarins (2–5)
7-Acetoxy-3-alkyl-4-methyl-2H-chromen-2-one
and anhydrous aluminium chloride (34 mmol) were taken in a
(9.2 mmol)
A mixture of 8-acetyl-3-alkyl-7-hydroxy-4-methylcoumarin or
6-acetyl-7-hydroxy-4-methylcoumarin (0.01 mol) and an aromatic

44
P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
aldehyde (0.012 mol) in absolute ethanol (50–55 mL) was refluxed
with piperidine (1–2 drops) for 6–8 h (Scheme 3). On evaporating
the solvent, cinnamoyl coumarin separated out which was filtered
and purified by either column chromatography or crystallized from
methanol-methylene chloride.
by crystallization in methylene chloride–methanol. Melt-
ing point:164–166 ◦C; IR (KBr) vmax
3432.47 (OH), 2926.89,
1707.40 (OCO), 1596.08 (CO), 1517.55, 1374.88, 1168.14, 1092.78,
809.93 cm−1 1H NMR (CDCl3, 300 MHz): ı 1.18 (t, 3H, J = 7.5 Hz,
CH3 of ethyl), 2.41 (s, 3H, C-4 CH3), 2.70 (q, 2H, J = 7.5 Hz, CH2 of
ethyl), 3.06 (s, 6H, 2× NCH3), 6.71 (d, 2H, J = 9.0 Hz, H-3ꢀꢀ and H-5ꢀꢀ),
6.91 (d, 1H, J = 9.0 Hz, H-6), 7.64–7.68 (m, 3H, H-2ꢀꢀ, H-6ꢀꢀ and H-5),
8.01 (d, 1H, J = 15.3 Hz, H-2ꢀ), 8.19 (d, 1H, J = 15.0 Hz, H-3ꢀ), 14.32
(s, 1H, OH); 13C NMR (CDCl3, 75.5 MHz): ı 13.06, 14.92, 20.70,
40.04, 109.63, 111.86, 112.58, 114.92, 120.59, 122.79, 123.86,
130.35, 131.34, 146.43, 147.39, 152.42, 153.02, 160.52, 166.44,
192.53; HRMS: Calculated for C23H23NO4 [M−H]+ 376.1627, found
376.2679.
:
;
4.2.2.1. (E)-6-[3-(4-Dimethylaminophenyl)acryloyl]-7-hydroxy-
4-methyl-2H-chromen-2-one (2). The title compound (2) was
obtained from the reaction of 6-acetyl-7-hydroxy-4-methyl-
2H-chromen-2-one (1a) with 4-dimethylaminobenzaldehyde
as orange solid in 81% yield by column chromatography using
ethyl acetate–petroleum ether (1:20) as an eluent. Melting point:
299–300 ◦C; IR (KBr) vmax: 3432.64 (OH), 2923.45, 1717.98 (OCO),
1632.06 (CO), 1521.64, 1379.94, 1303.88, 1172.45, 1151.44,
1068.49, 882.61, 695.88 cm−1 1H NMR (CDCl3, 400 MHz): ı 2.50
;
4.2.3. Procedure for the synthesis of C-6 and C-8 substituted
methoxycinnamoyl coumarins (6–8)
(s, 3H, C-4 CH3), 3.09 (s, 6H, 2× NCH3), 6.18 (s, 1H, H-3), 6.72
(d, 2H, J = 8.8 Hz, H-3ꢀꢀ and H-5ꢀꢀ), 6.89 (s, 1H, H-8), 7.38 (d, 1H,
J = 14.64 Hz, H-2ꢀ), 7.61 (d, 2H, J = 8.8 Hz, H-2ꢀꢀ and H-6ꢀꢀ), 7.99 (d,
1H, J = 14.68 Hz, H-3ꢀ), 8.12 (s, 1H, H-5), 13.70 (s, 1H, OH); 13C NMR
(CDCl3, 100.5 MHz): ı 18.59, 42.10, 105.56, 111.45, 112.80, 117.58,
118.40, 124.15, 127.70, 129.89, 130.39, 142.50, 151.72, 154.20,
157.63, 159.29, 163.44, 193.33; HRMS: Calculated for C21H19NO4
[M + H]+ 350.1392, found 350.1235.
A mixture of hydroxy cinnamoyl coumarin (0.01 mol), anhy-
drous potassium carbonate (0.025 mol) and methyl iodide
(0.012 mol) in anhydrous dimethyl formamide (25 mL) was stirred
for 14–16 h and the progress of the reaction was monitored on TLC.
The solvent was evaporated under reduced pressure. The resulting
precipitate was washed with water or extracted with ethyl acetate.
4.2.3.1. (E)-6-[3-(4-Dimethylaminophenyl)acryloyl]-7-methoxy-
4-methyl-2H-chromen-2-one (6). The title compound (6) was
obtained from the reaction of (E)-6-[3-(4-dimethylaminophenyl)
4.2.2.2. (E)-6-[3-(4-Dimethylaminophenyl)acryloyl]-3-ethyl-7-
hydroxy-4-methyl-2H-chromen-2-one (3). The title compound
(3) was obtained from the reaction of 6-acetyl-3-
acryloyl]-7-hydroxy-4-methyl-2H-chromen-2-one
(2)
with
methyl iodide as orange-yellow solid in 89% yield. Melting point:
ethyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1b)
with
219–220 ◦C; IR (KBr) vmax: 2923.64, 1725.49 (OCO), 1607.34 (CO),
4-dimethylaminobenzaldehyde as orange-yellow solid in 70%
yield by column chromatography using ethyl acetate–petroleum
ether (1:20) as an eluent. Melting point: 209–210 ◦C; IR (KBr)
1569.11, 1363.70, 1277.58, 1165.45, 1073.93, 987.70, 816.12 cm−1
;
1H NMR (CDCl3, 400 MHz): ı 2.40 (s, 3H, C-4 CH3), 3.03 (s, 6H, 2×
NCH3), 3.93 (s, 3H, OCH3), 6.16 (s, 1H, H-3), 6.78 (d, 2H, J = 8.8 Hz,
H-3ꢀꢀ and H-5ꢀꢀ), 6.86 (s, 1H, H-8), 7.17 (d, 1H, J = 15.4 Hz, H-2ꢀ),
7.49 (d, 2H, J = 8.8 Hz, H-2ꢀꢀ and H-6ꢀꢀ), 7.59 (d, 1H, J = 15.4 Hz,
H-3ꢀ), 7.85 (s, 1H, H-5); 13C NMR (CDCl3, 100.5 MHz): ı 18.73,
40.81, 56.29, 99.69, 112.57, 113.37, 122.12, 126.78, 127.19, 130.45,
144.90, 152.77, 156.74, 160.65, 160.76, 190.72; HRMS: Calculated
for C22H21NO4 [M+Na]+ 386.1368, found 386.3470.
vmax
:
3432.65 (OH), 2927.55, 1717.96 (OCO), 1629.84 (CO),
1541.87, 1364.38, 1191.06, 1068.96, 816.29 cm−1 1H NMR (CDCl3,
;
400 MHz): ı 1.16 (t, 3H, J = 7.32 Hz, CH3 of ethyl), 2.47 (s, 3H, C-4
CH3), 2.68 (q, 2H, J = 7.32 Hz, CH2 of ethyl), 3.08 (s, 6H, 2× NCH3),
6.71 (d, 2H, J = 8.8 Hz, H-3ꢀꢀ and H-5ꢀꢀ), 6.84 (s, 1H, H-8), 7.38 (d,
1H, J = 14.36 Hz, H-2ꢀ), 7.59 (d, 2H, J = 8.8 Hz, H-2ꢀꢀ and H-6ꢀꢀ), 7.96
(d, 1H, J = 15.4 Hz, H-3ꢀ), 8.10 (s, 1H, H-5), 13.61 (s, 1H, OH); 13C
NMR (CDCl3, 100.5 MHz): ı 13.15, 14.52, 20.94, 40.09, 104.91,
111.76, 113.26, 117.62, 121.94, 125.63, 126.29, 131.09, 145.08,
147.55, 152.64, 156.89, 160.95, 165.43, 192.27; HRMS: Calculated
for C23H23NO4 [M+Na]+ 400.1525, found 400.6265.
4.2.3.2. (E)-6-[3-(4-Dimethylaminophenyl)acryloyl]-
3-ethyl-7-methoxy-4-methyl-2H-chromen-2-one
(7).
The
the
title
reaction
compound
of
(7)
was
obtained
from
(E)-6-[3-(4-dimethylaminophenyl)
(3)
acryloyl]-3-ethyl-7-hydroxy-4-methyl-2H-chromen-2-one
4.2.2.3. (E)-8-[3-(4-Dimethylaminophenyl)acryloyl]-7-hydroxy-
4-methyl-2H-chromen-2-one (4). The title compound (4) was
obtained from the reaction of 8-acetyl-7-hydroxy-4-methyl-
2H-chromen-2-one (1c) with 4-dimethylaminobenzaldehyde
as red solid in 76% yield by crystallization in methylene
with methyl iodide as orange-yellow solid in 82% yield. Melting
point: 206–207 ◦C; IR (KBr) vmax: 2927.37, 1700.98 (OCO), 1603.92
(CO), 1533.33, 1375.51, 1167.59, 1045.62, 809.78 cm−1 1H NMR
;
(CDCl3, 400 MHz): ı 1.15 (t, 3H, J = 7.32 Hz, CH3 of C2H5), 2.41 (s,
3H, C-4 CH3), 2.67 (q, 2H, J = 7.32 Hz, CH2 of C2H5), 3.04 (s, 6H,
2× NCH3), 3.94 (s, 3H, OCH3), 6.68 (d, 2H, J = 8.79 Hz, H-3ꢀꢀ and
H-5ꢀꢀ), 6.86 (s, 1H, H-8), 7.18 (d, 1H, J = 15.38 Hz, H-2ꢀ), 7.49 (d,
2H, J = 8.79 Hz, H-2ꢀꢀ and H-6ꢀꢀ), 7.61 (d, 1H, J = 16.11 Hz, H-3ꢀ), 7.88
(s, 1H, H-5); 13C NMR (CDCl3, 100.5 MHz): ı 13.07, 14.62, 20.86,
40.11, 56.19, 99.36, 111.79, 114.09, 121.60, 122.47, 125.67, 126.77,
127.04, 130.45, 145.30, 146.01, 151.94, 155.08, 159.54, 161.46,
191.28; HRMS: calculated for C24H25NO4 [M+H]+ 392.1862, found
392.4456.
chloride–methanol. Melting point: 226–228 ◦C; IR (KBr) vmax
3417.24 (OH), 2912.68, 1721.91 (OCO), 1609.85 (CO), 1574.87,
1488.84, 1366.18, 1167.45, 1031.73, 823.38, 699.07 cm−1 1H NMR
:
;
(CDCl3, 400 MHz): ı 2.40 (d, 3H, J = 0.3 Hz, C-4 CH3), 3.04 (s, 6H, 2×
NCH3), 6.14 (d, 1H, J = 0.3 Hz, H-3), 6.68 (d, 1H, J = 8.8 Hz, H-3ꢀꢀ and
H-5ꢀꢀ), 6.90 (d, 1H, J = 9.5 Hz, H-6), 7.60–7.63 (m, 3H, H-2ꢀꢀ, H-6ꢀꢀ and
H-5), 7.99 (d, 1H, J = 14.6 Hz, H-2ꢀ), 8.09 (d, 1H, J = 15.4 Hz, H-3ꢀ),
14.40 (s, 1H, OH); 13C NMR (CDCl3, 100.5 MHz): ı 19.32, 40.07,
110.01, 110.81, 111.87, 115.21, 120.31, 122.68, 130.21, 131.47,
147.81, 152.51, 153.19, 154.72, 159.71, 167.50, 192.45; HRMS:
Calculated for C21H19NO4 [M−H]+ 348.1314, found 348.2266.
4.2.3.3. (E)-8-[3-(4-Dimethylaminophenyl)acryloyl]-7-
methoxy-4-methyl-2H-chromen-2-one
(8). The
title
compound (8) was obtained from the reaction of
(E)-8-[3-(4-dimethylaminophenyl)acryloyl]-7-hydroxy-4-
4.2.2.4. (E)-8-[3-(4-Dimethylaminophenyl)acryloyl]-3-ethyl-7-
hydroxy-4-methyl-2H-chromen-2-one (5). The title compound
(5) was obtained from the reaction of 8-acetyl-3-
methyl-2H-chromen-2-one (4) with methyl iodide as
orange-yellow solid in 80% yield. Melting point: 199–200 ◦C;
IR (KBr) vmax: 2914.32, 1722.62 (OCO), 1599.47 (CO), 1564.33,
ethyl-7-hydroxy-4-methyl-2H-chromen-2-one
(1d)
with
4-dimethylaminobenzaldehyde as red solid in 76% yield
1383.12, 1286.05, 1179.71, 1098.30, 987.00, 816.04 cm−1 1H NMR
;

P. Yadav et al. / Journal of Photochemistry and Photobiology A: Chemistry 280 (2014) 39–45
45
(CDCl3, 400 MHz): ı 2.42 (s, 3H, C-4 CH3), 3.02 (s, 6H, 2× NCH3),
3.88 (s, 3H, OCH3), 6.13 (s, 1H, H-3), 6.62 (d, 2H, J = 8.76 Hz, H-3ꢀꢀ
and H-5ꢀꢀ), 6.83 (d, 1H, J = 16.12 Hz, H-2ꢀ), 6.93 (d, 1H, J = 8.8 Hz,
H-6), 7.19 (d, 1H, J = 15.36 Hz, H-3ꢀ), 7.38 (d, 2H, J = 8.8 Hz, H-2ꢀꢀ and
H-6ꢀꢀ), 7.61 (d, 1H, J = 8.76 Hz, H-5); 13C NMR (CDCl3, 100.5 MHz):
ı 18.73, 40.05, 56.31, 107.41, 111.64, 112.69, 113.97, 118.17,
121.98, 123.30, 125.93, 130.57, 147.86, 151.21, 151.86, 152.12,
159.17, 160.29, 191.81; HRMS: calculated for C22H21NO4 [M+H]+
364.1549, found 364.4527.
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Acknowledgments
Financial support from the University of Delhi, Council of Scien-
tific and Industrial Research (CSIR, New Delhi) CSIR Award No. -For
PY-09/045(1095)/2011-EMR-I For MK-09/045(1060)/2011-EMR-I,
and the Indo-German Science and Technology Center (IGSTC) is
gratefully acknowledged. This research was also supported in part
by an appointment to the Faculty Research Participation Program at
the US Army Natick Soldier Research, Development and Engineer-
ing Center (NSRDEC) administered by the Oak Ridge Institute for
Science and Education through an interagency agreement between
the US Department of Energy and NSRDEC. The authors are thankful
to the Department of Biotechnology, India for the CREST Award to
SKS, and CSIR for the award of Junior/Senior Research Fellowships
to PY and MK. JK and SS would like to thank Center for Advanced
Materials, and Sukant Tripathy Summer Fellowship.
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