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Synthesis and fluorescence properties of

benzoxazole-1,4-dihydropyridine dyads achieved
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Cite this: New J. Chem., 2014,

38, 4607 by a multicomponent reaction†
Ricardo Ferreira Affeldt,a Antônio César de Amorim Borges,a Dennis Russowsky*b
and Fabiano Severo Rodembusch*a

Photoactive 2-(2 0 -hydroxyphenyl)benzoxazole-1,4-dihydropyridine (HBO–DHP) dyads were obtained by a

multicomponent one-pot Hantzsch synthesis using a fluorescent aldehyde, a 1,3-dicarbonylic compound
Received (in Montpellier, France)
and ammonium acetate. The key step in this synthetic methodology was the synthesis of the formyl
13th May 2014, benzoxazole derivative through a Duff-modified functionalization protocol. UV-Vis absorption and
Accepted 2nd July 2014 fluorescence emission spectroscopies were also applied to better understand the photophysics of these
DOI: 10.1039/c4nj00777h compounds. The three novel fluorescent compounds were obtained in moderate yields as stable solids
with absorption in the UV region and emission in the blue-green region. Preliminary results indicate that
www.rsc.org/njc after excitation both HBO and DHP fluorophores behave independently in the HBO–DHP structure.

Introduction
The multicomponent synthesis is an important tool for the
construction of compound libraries with high atom economy,
allowing a large number of derivatives from combinations of
different reagents, also being explored by the combinatorial
chemistry field.1 In contrast to the multistep linear strategy,
these one-pot reactions are versatile and efficient at achieving
small molecules for biological screening and drug design.2
1,4-Dihydropyridines (1,4-DHP) 1 are small molecules that
exhibit pronounced biological activity and are synthesized by the
Hantzsch multicomponent reaction.3 Their activity as calcium
channel modulators has important consequences on the treatment
of heart diseases such as hypertension and angina.4 Other
promising activities include their role as antioxidants, broncho-
dilators, antiartherosclerotic and AD therapeutic agents.5 Scheme 1 Chemical structure of the dihydropyridine core 1, polyhydro-
quinoline 2, polyhydroacridinedione 3 and NADH 4.
The scope of the Hantzsch reaction is not restricted to
1,4-dihydropyridine synthesis only, but polycyclic derivatives of
the aromatic analogues can also be obtained, such as polyhydro- The NADH coenzyme system 4 is structurally related to 1,4-dihydro-
quinolines 2 and polyhydroacridinedione 3 (Scheme 1).6 pyridine compounds found in living cells and is responsible for
electron transport and energy production in metabolic reactions. It
a
possesses two oxidation states – i.e. NAD+ and NADH – that differ in
Grupo de Pesquisa em Fotoquı́mica Orgânica Aplicada, Instituto de Quı́mica/
UFRGS, Av. Bento Gonçalves, 9500. CEP 91501-970, Porto Alegre, RS, Brazil.
one proton and two electrons. NADH is also a powerful natural
E-mail: [email protected] antioxidant. The different photophysical behaviours of the two
b
Laboratório de Sı́ntese Orgânica, Instituto de Quı́mica/UFRGS, Av. Bento species make them useful for protein identification in enzymatic
Gonçalves, 9500. CEP 91501-970, Porto Alegre, RS, Brazil. colorimetric essays.7 Additionally, it can also be applied as a
E-mail: [email protected]
fluorescence sensor for identification of explosives8 and monitoring
† Electronic supplementary information (ESI) available: Spectral and HRMS data
for the new compounds, as well as additional data for fluorophore 9, and details
of biological reactions and microbial fermentations.9
of supplementary photophysical experiments and attempts to achieve the quino- We have recently described a photophysical study of 4-aryl-
line synthesis and salicylic acid formylation. See DOI: 10.1039/c4nj00777h substituted dihydropyridines allied with theoretical calculations,

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which showed that these systems are close to the well-known paper also deals with the photophysical characterization of
NADH. Their fluorescence emission can be ascribed to a normal the new compounds by means of UV-Visible absorption and
relaxation process or an intramolecular charge transfer in fluorescence emission spectroscopies in solution.
dimethylamino-substituted compounds.10 Albini et al. have also
studied the mechanistic pathways for the excited state of these
dihydropyridines by ultraviolet absorption, fluorescence and Experimental
phosphorescence spectroscopies, leading to different roles of General procedures
substituted 1,4-dihydropyridines.11–14 It is desirable that a
fluorophore shows fluorescence in a well-defined region of All reagents and solvents were purchased from commercial sup-
pliers and used without further purification. Reactions were
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the electromagnetic spectrum and a high value of Stokes’ shift,

i.e. a large separation between emission and absorption maxima monitored using thin-layer chromatography (TLC) carried out on
wavelengths, since the interaction with biological systems can silica gel 60 F254 pre-coated aluminium plates. The visualization
result in a hypsochromic shift of fluorescence favoring self- was achieved under UV light (254 nm) or by staining with I2.
absorption and fluorescence quenching.15 Chromatographic separations were achieved on silica gel columns
Hydroxyphenylbenzazole heterocycles assume an important (70–230 mesh). An ultrasound bath of 40 kHz (50 W) was used for
role as wide-application dyes, hence they show a high Stokes’ mixture homogenization. The 1H and 13C NMR spectra were
shift due to an excited state intramolecular proton transfer recorded in CDCl3 at 300 MHz and 75.4 MHz, respectively. The
(ESIPT) mechanism (Fig. 1), a phototautomerization strongly chemical shifts (d) are reported in parts per million (ppm) relative
influenced by the solvent, in which usually a dual fluorescence to TMS (0.00 ppm) for 1H NMR and to the central line of CDCl3
emission takes place in solution or in the solid state.16 This (77.0 ppm) for 13C NMR. Coupling constants J are reported in hertz
mechanism can be related to a normal species (or enol band) (Hz). The following abbreviations are used for the multiplicities: s,
and a keto tautomer (or ESIPT band). This behavior confers to singlet; d, doublet; dd, double-doublet; dq, double-quartet; t,
these compounds physical and chemical properties that are triplet; q, quartet; m, multiplet; br s, broad signal. FTIR spectra
attractive from both synthetic and technological points of were obtained using KBR pellets with a resolution of 4 cm1
view.17 Fluorescence properties of 1,4-dihydropyridines could be between 400 and 4000 cm1. Fragmentation patterns were
explored for determination and characterization of pharmacological obtained using a quadrupole analyzer equipped with a 70 eV
mechanisms through interactions between molecules and biological electron impact ionization source by direct insertion of samples
systems, such as membrane calcium channels in cardiac cells with diluted in chloroform (1 mL).
fluorimetric essays or other specific targets.18 Spectroscopic grade solvents dichloromethane, acetonitrile,
Considering the high Stokes’ shift originating from 2-(2 0 - 1,4-dioxane and ethanol were used in fluorescence emission
hydroxyphenyl)benzoxazole (HBO), we decided to establish a and UV-Vis absorption spectroscopy measurements. UV-Vis
synthetic methodology for its functionalization to make it a absorption spectra were recorded using a Shimadzu UV-2450
suitable component for one-pot synthesis of new 1,4-dihydro- spectrophotometer, and steady state fluorescence spectra were mea-
pyridine derivatives starting from an ESIPT derivative. This sured using a Shimadzu spectrofluorometer model RF-5301PC.
Spectral correction was performed to enable measurements of true
spectra by eliminating instrumental response such as wavelength
characteristics of the monochromator or the detector using Rhoda-
mine B as an internal standard (quantum counter). The fluorescence
quantum yield (ffl) measurement was made in spectroscopic grade
solvents using the dilute optical methodology. Quinine sulphate
(Riedel) in 1 M H2SO4 (ffl = 0.55) was used as a quantum yield
standard.19 All experiments were performed at room temperature.
High resolution mass spectrometry electrospray ionization (HRMS-
ESI) data, in a positive mode, were collected using a Micromass
Q-Tof instrument. Samples were infused by a 100 mL syringe at a flow
rate 30 mL min1 for all samples. The following typical operating
conditions were used: a capillary voltage of 2980 V, a sample cone
voltage of 30 V, an extraction cone voltage of 3.0 V, and a desolvation
gas temperature of 60 1C. N2 was used as the desolvation gas and
HPLC grade acetonitrile was the solvent used with the samples.

Syntheses
5-Formylsalicylic acid (6). 22 mL of glacial acetic acid was
Fig. 1 Photophysical pathways for ESIPT-exhibiting dyes: normal (or enol)
added to an equimolar mixture of salicylic acid (5) (14.5 mmol,
emission (green) and ESIPT (or tautomer) emission (red). The asterisk 2.00 g) and hexamethylenetetramine (14.5 mmol, 2.03 g) in a
indicates the excited state. 100 mL round-bottom vessel and the solution was heated to

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reflux for 8 hours, after which 16 mL of hot distilled water and (10 mol%), followed by 2.5 mmol of ethyl acetoacetate (13)
10 mL of concentrated hydrochloric acid were added with (0.317 mL), 1.25 mmol of previously synthesized 9 (0.299 g) and
vigorous stirring. The solution was then cooled to ambient 2.5 mmol of ammonium acetate (12) (0.193 g) were added to a
temperature and the formed precipitate was filtered using a round-bottom vessel. The mixture was dissolved in isopropanol
sintered funnel followed by washings with 50 mL of distilled (10 mL) and refluxed for 3 hours. The solution was cooled and
water (40 1C and ambient temperature). The pale yellow solid the precipitate filtered and washed with cold isopropanol (4 
was dried leading to 0.24 g of 5-formylsalicylic acid in 16% yield 5 mL). The catalyst was separated from the product by redissolution
and its characterization is in agreement with the literature.20 of the solid in dichloromethane and micropore (0.45 mm) filtration.
M.p. 250 1C (decomp.). 1H NMR (DMSO-d6, 300 MHz): d = 7.12 The solution obtained was concentrated and dried leading to 0.26 g
(d, J = 8.5 Hz, 1H); 7.99 (dd, 3J = 8.5 and 4J = 2.0 Hz, 1H); 8.34
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of the pure product (46%), a white solid with blue fluorescence

(d, J = 2.0 Hz, 1H); 9.84 (s, 1H). 13C NMR (DMSO-d6, 75.4 MHz): when exposed to UV radiation. M.p. = 233–236 1C. 1H NMR (CDCl3,
d = 114.3; 118.9; 128.9; 134.6; 135.8; 166.4; 171.8; 191.9. 300 MHz): d = 1.27 (t, 6H, J = 7.1 Hz); 2.39 (s, 6H); 4.07–4.22 (m, 4H);
2-(2 0 -Hydroxyphenyl)benzoxazole (8). An equimolar mixture 5.05 (s, 1H); 5.75 (s, 1H); 7.00 (d, J = 8.7 Hz, 1H); 7.38–7.4 (m, 3H);
of salicylic acid (5) (18.3 mmol, 2.53 g) and 2-aminophenol (7) 7.60–7.63 (m, 1H); 7.72–7.73 (m, 1H); 7.83 (d, J = 2.1 Hz, 1H). 13C
(18.3 mmol, 2.00 g) in polyphosphoric acid (10 mL) was stirred NMR (CDCl3, 75.4 MHz): d = 14.5; 19.9; 39.3; 60.0; 104.2; 109.8;
at 180 1C for 5 h. After cooling, the mixture was poured onto ice- 110.8; 117.1; 119.3; 125.1; 125.3; 126.6; 133.9; 139.6; 144.1; 149.3;
cold water and the precipitate was filtered, washed with water 157.4; 163.5; 164.9; 167.8. FTIR (KBr, n = cm1): 3323 (NH), 3096,
and dried in a stove. The solid was purified by column 2980, 1681, 1649, 1548, 1491, 1302, 1212. MS: m/z (%) = 462.2 (10)
chromatography using dichloromethane as an eluant leading [M+], 433.1 (10), 389.3 (19), 252.1 (100), 211.0 (5), 196.0 (25). HRMS
to 2.09 g of the product (54%). M.p. = 125–126 1C. 1H NMR (ESI-qTOF) m/z: [M + H]+ calcd for C26H26N2O6 463.1869; found
(CDCl3, 300 MHz): d = 6.89 (t, J = 8.1 Hz, 1H); 7.01 (dd, 4J = 8.2 463.1865 (Dppm = 0.9).
and 3J = 0.9 Hz, 1H); 7.24–7.35 (m, 3H); 7.46–7.49 (m, 1H); 7.58– Ethyl 4-[2-(20 -hydroxyphenyl)benzoxazolyl]-1,4,5,6,7,8,-hexahydro-
7.62 (m, 1H); 7.90 (dd, 4J = 7.9 and 3J = 1.8 Hz, 1H); 11.37 (br s, 2,7,7-trimethylquinolin-5(1H,4H,6H)-one 3-carboxylate (16). 0.106 g
1H). 13C NMR (CDCl3, 75.4 MHz): d = 110.8; 117.6; 119.4; 119.7; of In/SiO2 (10 mol%), followed by 1.25 mmol of 5,5-dimethyl-
125.1; 125.5; 127.3; 133.7; 140.1; 149.2; 158.9; 163.0; 164.9. cycloexane-1,3-dione (14) (0.175 g), 1.25 mmol of ethyl aceto-
2-(5 0 -Formyl-2 0 -hydroxyphenyl)benzoxazole (9). Method A acetate (13) (0.158 mL), 1.25 mmol of previously synthesized 9
(cyclization): polyphosphoric acid (10 mL) followed by 6.4 mmol of (0.299 g) and 2.5 mmol of ammonium acetate (12) (0.193 g)
5-formylsalicylic acid (6) (1.06 g) and 6.4 mmol of 2-aminophenol (7) were added to a round-bottom vessel. The mixture was dis-
(0.70 g) were added to a round-bottom flask. The mixture was heated solved in isopropanol (10 mL) and refluxed for 3 hours. The
at 170 1C for 5 hours. The crude mixture was poured onto ice-cold solution was cooled and the precipitate filtered and washed
water with vigorous stirring and the precipitate was filtered with cold isopropanol (4  5 mL). The catalyst was separated
yielding a dark brown solid. The solid was extracted by using a from the product by redissolution of the solid in dichloro-
Soxhlet extractor with dichloromethane and then purified by methane and micropore (0.45 mm) filtration. The solution
column chromatography using dichloromethane as the eluent, obtained was concentrated and dried leading to 0.39 g of the
yielding, after solvent evaporation, 0.46 g of a white solid (30%) pure product (66%), a white solid with green fluorescence when
exhibiting intense green fluorescence when exposed to UV exposed to UV radiation. M.p. 4250 1C. 1H NMR (CDCl3,
radiation. Method B (formylation): 10 mL of polyphosphoric 300 MHz): d = 0.96 (s, 3H, CH3); 1.09 (s, 3H, CH3); 1.23 (t, J =
acid (10 mL) followed by 4.7 mmol of previously synthesized 7.1 Hz, 3H, CH3); 2.14–2.40 (m, 4H, 2CH2); 2.4 (s, 3H, CH3); 4.08
HBO (8) (0.99 g) and 5 mmol of hexamethylenetetramine (0.70 g) (dq, J = 1.1 Hz and J = 7.1 Hz, 2H, CH2); 5.08 (s, 1H); 5.97 (s, 1H);
were added to a round-bottom flask. The temperature was raised 6.97 (d, J = 8.7 Hz, 1H); 7.34–7.39 (m, 3H); 7.58–7.61 (m, 1H);
to 100 1C and the mixture stirred for 4 hours. After the heating, 7.69–7.72 (m, 1H); 7.99 (d, J = 2.4 Hz, 1H); 11.34 (br s,1H). 13C
30 mL of ice-cold water was added leading to a white precipitate, NMR (CDCl3, 75.4 MHz): d = 14.2; 19.5; 27.1; 29.4; 32.7; 35.9;
which was then filtered and washed with water before drying. 41.1; 50.6; 59.9; 106.0; 109.6; 110.6; 112.0; 117.0; 119.1; 124.8;
The solid was purified by column chromatography using dichloro- 125.1; 126.5; 133.5; 138.6; 140.1; 143.4; 147.9; 149.1; 157.1;
methane as an eluant leading to 0.38 g of the product (34%). White 163.2; 167.3; 196.0. FTIR (KBr, n = cm1): 3289, 3222, 2963,
needles crystals. M.p. = 165–170 1C. 1H NMR (CDCl3, 300 MHz): d = 1706, 1608, 1487, 1379, 1262. MS: m/z (%) = 472 (39) [M+], 399
7.22 ppm (d, 2H, J = 8.6 Hz); 7.39–7.45 (m, 2H); 7.62–7.66 (m, 1H); (20), 262 (100), 234 (28). HRMS (ESI-qTOF) m/z: [M + H]+ calcd
7.73–7.76 (m, 1H); 7.96 (dd, 3J = 8.6 Hz and 4J = 2.0 Hz); 8.55 (d, J = for C26H28N2O5 473.2076; found 473.2058 (Dppm = 3.8).
2.0 Hz); 9.95 (s, 1H); 12.17 (br s, 1H). 13C NMR (CDCl3, 75.4 MHz): 3,3,6,6-Tetramethyl-9-[2-(20 -hydroxyphenyl) benzoxazolyl]-3,4,6,7-
d = 111.2; 118.6; 119.7; 125.7; 126.3; 129.2; 130.5; 134.3; 139.7; 149.4; tetrahydro acridine-1,8-(2H,5H,9H,10H)-dione (17). 0.106 g of In/
161.9; 163.7; 165.0; 190.2. FTIR (KBr, n = cm1): 3061, 2848, 2735, SiO2 (10 mol%), followed by 2.5 mmol of 5,5-dimethylcyclohexane-
1699, 1630, 1490. MS: m/z (%) = 240.1 (16), 239.1 (100) [M+], 238.1 1,3-dione (14) (0.350 g), 1.25 mmol of previously synthesized 9
(90), 210.2 (10), 182.0 (29), 63.1 (32). HRMS (ESI-qTOF) m/z: [M + H]+ (0.299 g) and 2.5 mmol of ammonium acetate (12) (0.193 g) were
calcd for C14H9NO3 240.0660; found 240.0620 (Dppm = 4.2). added to a round-bottom vessel. The mixture was dissolved in
Diethyl 2,6-dimethyl-4-[2-(2 0 -hydroxyphenyl) benzoxazolyl]- isopropanol (10 mL) and refluxed for 3 hours. The solution was
1,4-dihydropyridine 3,5-dicarboxylate (15). 0.106 g of In/SiO2 cooled and the precipitate filtered and washed with cold isopropanol

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(4  5 mL). The catalyst was separated from the product by

redissolution of the solid in dichloromethane and micropore
(0.45 mm) filtration. The solution obtained was concentrated and
dried leading to 0.30 g of the pure product (50%), a white solid with
blue fluorescence when UV exposed. M.p. = 212–215 1C. 1H NMR
(CDCl3, 300 MHz): d = 1.15 (s, 6H, 2CH3); 1.35 (s, 6H, 2CH3); 2.33–
2.55 (m, 8H, 4CH2); 5.53 (s, 1H); 7, 03 (d, J = 8.7 Hz, 1H); 7. 16 (d, J =
8.7 Hz); 7.36–7.38 (m, 2H); 7.48–7.51 (m, 1H); 7.71–7.74 (m, 1H);
7.78–7.81 (m, 1H); 11.23 (br s, 1H, NH); 12.01 (s, 1H, OH). 13C NMR
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(CDCl3, 75.4 MHz): d = 27.4; 30.2; 31.6; 32.3; 48.7; 47.3; 110.6; 115.7;
117.5; 119.5; 125.2; 125.5; 126.8; 129.2; 132.4; 140.4; 149.2; 157.0; Fig. 2 Proposed HBO–DHP structure.
163.1; 165.0; 169.7; 180.9. FTIR (KBr, n = cm1): 2958, 2925, 1593,
1372. MS: m/z (%) = 361.1 (100) [M+], 318.0 (8), 277.0 (16), 263.0 (9),
249.0 (30), 140.0 (13), 83.0 (49). HRMS (ESI-qTOF) m/z: [M + H]+ calcd
for C30H30N2O4 483.2284; found 483.2293 (Dppm = 1.9).

Results and discussion

Synthesis
We first attempted to apply aminobenzazoles on benzoxazolyl-
quinoline synthesis without success using multicomponent
and even classical cyclization protocols involving anilines, such
as Skaup glycerol acidic reactions and Combes, Doebner-Miller
and Conrad-Limpach approaches (see ESI†).
Therefore it was decided to bypass this drawback by preparing a
formyl derivative which has, to the best of our knowledge, not been
described in the literature, although 3,5-formyl-hydroxyphenyl-
benzoxazole has been recently described as an intermediate for a
fluorescent pyrophosphate sensor.21 In Hantzsch multicomponent
condensation giving 1,4-dihydropyridines, it has been observed that
aromatic and heteroaromatic aldehydes bearing both electron- Scheme 2 Reaction pathways for obtaining formyl functionalized HBO
(9). Conditions: (i) Hexamethylenetetramine (HMTA), acetic acid (AcOH),
withdrawing as well as electron-releasing substituted groups do
reflux, 8 h; (ii) and (iii) 7, polyphosphoric acid (PPA), 170 1C, 5 h, (iv) HMTA,
not significantly affect the reaction yields.22 On the other hand, PPA, 100 1C, 4 h.
the synthesis of 2-(2 0 -hydroxyphenyl)benzoxazole (HBO) is well
established in the literature.23 In this way, a synthetic method
was proposed to afford functionalization of HBO by introducing mixture of the 3-formyl and 5-formyl derivatives (11 and 6, respec-
a formyl group, which was predicted to react with two equiva- tively) could be obtained, whose separation was quite difficult.
lents of a 1,3-dicarbonyl compound and ammonia, aiming for Therefore, a simple procedure was chosen: filtering the solid pre-
the synthesis of fluorescent dyad HBO–DHP, the structure of cipitate after hydrolysis and washing with hot water portions. This
which is depicted in Fig. 2. method has proved to be useful since 5-formylsalicylic acid (6) is less
In this way, as depicted in Scheme 2, two different methodologies soluble in water than 3-formylsalicylic acid (11); however, product 6
(routes A and B) were used to synthesize the formyl functionalized was obtained in low yield (16%).
1
fluorophore 9. Duff’s formylation for salicylic acid (5) using hexam- H-NMR analysis of the pale yellow precipitate produced a
ethylenetetramine (HMTA) involves stable and low-cost reagents, spectrum in agreement with that of pure 5-formylsalicylic acid
although low yields are usually obtained.20 The procedure was (6). Pure 3-formylsalicylic acid (11) could also be obtained by
employed for salicylic acid (5) and later for condensation of recrystallization (m.p. = 178–179 1C). It is worth noting that no
5-formylsalicylic acid (6) with 2-aminophenol (7). At the same formylated product was observed when the reaction was carried
time, formylation of HBO 8 obtained by condensation of in pure water up to 16 hours.
salicylic acid (5) and 2-aminophenol (7) was tested. The condensation procedure to yield the novel fluorescent
It was expected that the rate of Duff’s formylation reaction formylated compound 9 was adapted from the literature,23 i.e.
was highly dependent on the successful hydrolysis of iminium reacting the previously synthesized 5-formylsalicylic acid (6)
cation 10 formed by protonated HMTA and the activated and 2-aminophenol (7) in polyphosphoric acid (PPA) at 170 1C
phenol derivative in acidic media (Scheme 3). We performed for 5 hours. The product was purified by column chromatography
this reaction by refluxing salicylic acid (5) and an excess of yielding (9) (yield 30%), a white solid with green fluorescence, quite
HMTA in acetic acid for 8 hours. After several attempts, a similar to the unfunctionalized HBO fluorophore 8. It was expected

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Scheme 3 Iminium hydrolysis in Duff’s formylation of salicylic acid (5).

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that an inverse procedure starting with the HBO synthesis and its
further formylation could improve the yields in both reaction steps
(Scheme 2, route B).
Precursor 8 was prepared as reported in the literature24 (yield
57%) and the formylation methodology was investigated in three
acidic solvents. The results are summarized in Table 1. PPA was
found to be suitable for both steps of cyclization and formylation.
The structure of 9 was confirmed by 1H and 13C NMR, FTIR and MS
analyses (see ESI†). The signal pattern in the region between 7 and Scheme 4 Multicomponent synthesis of HBO–DHPs 15–17.
9 ppm in the 1H NMR spectrum (Fig. 3) was crucial for the
identification of the formyl group in the 50 position of the phenolic
ring. The novel fluorophore precursor shows good thermal stability moiety,25 for further investigations, two N-heterocycles, 16
and no decomposition was observed up to 250 1C. and 17, bearing the HBO moiety and the DHP core were
The Hantzsch three-component synthesis of 4-(2-(20 -hydroxy- synthesized using the same methodology. The compounds were
phenyl)benzoxazolyl)-1,4-dihydropyridine 15 was performed by obtained with significant yields (66% and 50% for 16 and 17,
mixing one equivalent of 9 with ammonium acetate (12) and two respectively) without any additional chromatographic purification.
equivalents of ethyl acetoacetate (13), employing a In/SiO2 All synthesized novel heterocycles were white solids and fluorescent
heterogeneous Lewis-acid catalyst in refluxing isopropanol in the blue-green region when exposed to UV 365 nm radiation
(Scheme 4).22 The product was isolated after 3 hours as a blue (solid or in solution).
fluorescent solid in moderate yield (46%) by simply filtering the
heterogeneous isopropanol mixture. It is worth mentioning that Photophysics
when acetylacetone was used, a complex mixture was obtained. The photophysical study of the synthesized compounds was
Considering the antimicrobial activity of decahydroacridine- performed in solution (105 M) using four different organic
diones and hexahydroquinolines with a fused dimedone solvents with a wide range of dielectric constants. This investigation
was focused on the influence of the structural changes, as well as the
Table 1 Experimental conditions for the HBO’s formylation oxidation state of the dihydropyridine core in the ESIPT emission.
Fig. 4 presents the UV-Vis absorption and fluorescence emission
Entry Solvent Time (h) Temp. (1C) Yield (%)
spectra of 9. The fluorescence emission spectra were obtained using
1 Acetic acid (AcOH) 6 Reflux 8 the absorption maxima as the excitation wavelengths. The relevant
2 Trifluoroacetic acid (TFAA) 6 Reflux 15
3 Polyphosphoric acid (PPA) 4 100 34 photophysical data are summarized in Table 2.
The UV-Vis absorption spectra of 9 in all solvents exhibit an
intense band located at 266 nm and a red-shifted one at 320–335 nm
with molar absorptivity coefficient, e, values in agreement with p–p*
transitions. No significant solvatochromism in the ground state was
observed for this dye.
The fluorescence emission spectra show a major emission
band located at 474–483 nm. As already presented in the
literature, the measured Stokes’ shift (B9150 cm1) can be
ascribed to the ESIPT mechanism.17 Due to this dye the ground-
state enol-cis conformer (N) absorbs UV radiation leading to the
singlet excited state enol-cis (N*), which tautomerizes to the
excited-state keto (T*). This conversion is responsible for energy
loss with a large Stokes’ shift, and the excited keto decays to the
ground state (T) emiting fluorescence. The single red-shifted
emission band ascribed to (T)S0 ’ (T*)S1 and consequently the
absence of a blue-shifted band from the locally excited enol
Fig. 3 1
H NMR spectrum of 9. species (N)S0 ’ (N*)S1 is quite similar to that of the HBO

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Fig. 4 Normalized UV-Vis absorption and fluorescence emission spectra

of 9 in different solvents.

Table 2 Relevant UV-Vis absorption and fluorescence emission data of 9,

where labs and lem are the absorption and emission maxima, respectively
in nanometers, and e is the molar extinction coefficient in 103 L mol1 cm1

Solvent labs (e) lem DlST a (cm1)

CHCl3 334(9.68), 272(19.1) 474 8843
MeCN 330(13.3), 266(28.9) 478 9382
EtOH 332(10.9), 269(18.9) 475 9068
1,4-Dioxane 333(13.6), 265(28.5) 483 9326
a
Stokes’ shift was obtained from the difference of the emission and
absorption maxima (DlST = lem  labs).

spectra.24 These results strongly suggest that the formyl group

in the 5 0 -position of the phenolic ring does not favour an
equilibrium between the conformers in solution in the ground
state, as observed for amino derivatives in the same position.16
Excitation spectra show similar profiles to the absorption
curves (not shown, see ESI†), where the structured bands
ascribed to the HBO (322 and 334 nm) and benzoxazolyl Fig. 5 Normalized UV-Vis absorption and fluorescence emission spectra
moieties (B270 nm) can be observed. of 15–17 in different solvents.
Fig. 5 presents the absorption and emission spectra of HBO–
DHPs 15–17 in solution. The relevant data from the photophysical
study of these compounds are summarized in Table 3. the so-called short wavelength (SW) and long wavelength (LW),
The compounds exhibit absorption maxima in the UV region respectively. In these compounds, the fluorescence emission
(334–340 nm) with electronic transitions ascribed to p–p*. can be influenced by internal electron transfer and/or the
Changes in the solvent polarity, where only a slight solvato- energy transfer character of the corresponding DHP core
chromism was observed, as well as modifications in the DHP (blue-shifted band)11–14 or by deactivation of the keto tautomer,
core, seem not to affect the photophysics of these compounds which arises from the ESIPT mechanism (red-shifted band). It
in the ground state. The fluorescence emission was also is worth mentioning that the normal emission from enol
obtained using the absorption maxima as the excitation wave- conformers was discarded since the measured Stokes’ shifts
lengths. The compounds in the solid state exhibit emission in was much higher (B5200–7000 cm1) than those attributed to
the blue-green region (Fig. 6). this emission in similar compounds (B3500 cm1).16,17 An
Although ESIPT emission could be observed in the studied exception can be observed in the case of compound 17 in
HBO–DHPs, as already observed for compound 9, the fluores- acetonitrile, where the blue-shifted band could be related to
cence spectra of compounds 15–17 seem to be more complex the normal emission. The nature of the emission spectra of
being dependent on both the solvent polarity and the DHP these compounds indicates that both HBO and DHP behave,
moiety. Generally, an emission spectrum presents a dual after excitation, as independent fluorophores in the HBO–DHP
fluorescence emission located at around 425 nm and 488 nm, structure. Concerning the intricate photophysics of the DHPs,11–14

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NJC Paper

Table 3 Relevant UV-Vis absorption and fluorescence emission data of the acetate. The first attempt at using different ESIPT fluorescent amino
HBO–DHPs 15–17, where the concentration is B106 mol L1, labs and lem derivatives as nitrogen sources did not provide the desired
are the absorption and emission maxima, respectively in nanometers, DlST is
cyclization compounds. A new photoactive aldehyde derivative
the Stokes’ shift, e is the molar extinction coefficient in 103 L mol1 cm1 and
ffl is the fluorescence quantum yield of 2-(20 -hydroxyphenyl)benzoxazole was successfully obtained
through a Duff-modified functionalization protocol.
lem DlST (cm1) The new HBO–DHPs were obtained in moderate yields as
Solvent labs (e) SW LW SW LW ffl thermal and photostable solids. The photophysical study in
15 solution shows an absorption maximum in the UV region and
CHCl3 339(24.1) 424 483 5914 8795 0.19 fluorescence emission in the blue-green region depending on
MeCN 336(10.2) 433 — — 6667 0.07
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the compound. Preliminary results indicate that after excitation

EtOH 336(15.5) 441 — — 7086 0.11
1,4-Dioxane 339(24.5) 426 485 6024 8880 0.11 both HBO and DHP behave as independent fluorophores in the
HBO–DHP structure. Since a solid synthetic methodology to
16 incorporate ESIPT compounds into the DHP structures could
CHCl3 339(24.1) 424 483 5914 8795 0.31
MeCN 336(10.2) 433 — — 6667 0.08 be established, additional experiments are in progress to better
EtOH 336(15.5) 441 — — 7086 0.08 understand the photophysics of these dyes.
1,4-Dioxane 339(24.5) 426 485 6024 8880 0.11

17
CHCl3 340(8.17) — 499 — 7140 0.35 Acknowledgements
MeCN 337(13.7) 382 492 3456 9348 0.23
EtOH 334(21.3) — 484 — 9279 0.33 The authors thank the Conselho Nacional de Desenvolvimento
1,4-Dioxane 339(23.9) — 506 — 9736 0.17 Cientı́fico e Tecnológico (CNPq) and the Instituto Nacional de
Inovação em Diagnósticos para Saúde Pública (INDI-Saúde) for
financial support.

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