Substituent and Solvent Effects on the Spectral Properties of 3-Substituted Derivatives of 4-Hydroxycoumarin

Solvent and substitution effects on the UV/Vis spectroscopic and fluorescence behaviour of seven synthesized 3-substituted 4-hydroxycoumarin derivatives were tested. The tested compounds were dissolved in ethyl acetate, acetonitrile, and dimethyl sulfoxide. Absorption and emission spectra were recorded in the range of 200–800 nm. All tested 4-hydroxycoumarin derivatives showed good absorption in a wide range of 200–550 nm, depending on the properties of the substituents on the benzene ring of the cinnamoyl moiety and the type of solvent. In comparison to the unsubstituted analogue, compounds with an electron-donating group exhibited bathochromically shifted UV/Vis absorption and emission spectra. The highest fluorescence quantum yield was observed for compounds with dimethylamino and acetamido groups as substituents at the benzene ring. Considering that both substitution and solvent affect the absorption and emission spectra of the tested compounds, it can be concluded that judiciously selecting these parameters can improve their absorption and fluorescence properties, making them suitable for various analytical uses.


Introduction
2][13][14][15][16] Coumarin derivatives fluorescence is relatively photostable, and their excitation and emission maxima are suitable to use for measurements in cellular components, tissues, and biological fluids. 17The importance of coumarin compounds as fluorescent agents is derived from their stability and relatively easy synthesis. 18ny coumarin derivatives show a characteristic fluorescence in basic and neutral solutions and in concentrated sulphuric acid, especially in the UV region.This property is particularly pronounced in 7-hydroxycoumarin derivatives, and is associated with a change in its polarisation in alkaline solution and in sulphuric acid. 19The fluorescence intensity is significantly influenced by the position of substitution as well as the properties of the substituent itself.The double bond on the pyrone ring absorbs light with a wavelength greater than 300 nm, so it can be concluded that complete aromatisation and delocalisation of the double bond at the 3,4-position of the lactone ring do not occur. 20Three major tautomeric forms of 3-substituted-4-hydroxy coumarin can be formed. 21The occurrence of tautomerism for 4-, 5-, and 7-hydroxycoumarins was also observed. 22,23The introduction of electron acceptor groups in position 3 on the coumarin ring (carbonyl, cyano group) leads to the transfer of electrons in the pyrone ring, which causes fluorescence even in neutral solutions.If these groups are in position 4, no fluorescence occurs. 24The oxygen atom at the 3-carbonyl group acts as a hydrogen bond acceptor and donating group and at the 7-position enhances the fluorescence emission. 25,26Murata et al., examined coumarin derivatives with both donating groups at the 6-and 7-positions and an electron-withdrawing group at the 3-position, which develop intense fluorescence. 27Previous investigations of the fluorescent properties of coumarin derivatives showed that the position of the absorption and emission spectra depend on the position and type of substituents introduced, and the intensity of the emission depends on the pH. 28The introduction of donating groups at the 6-and 7-positions along with a withdrawing group at the 3-position, leads to the development of intense fluorescence. 27 this study, 4-hydroxycoumarin derivatives, (1-7), containing a chalcone moiety with electron-withdrawing or electron-donating substituents were spectroscopically investigated.

Material and methods
The compounds investigated in this study were synthesized according to the procedure described by Zavrsnik et al. 29 using chemicals of analytical grade.Briefly, the first step was the acylation of 4-hydroxycoumarin with acetic acid and phosphorous oxychloride.By reaction with 3-acetyl-4-hydroxycoumarin, and with appropriate aromatic aldehydes with pyridine and piperidine as catalysts, seven 4-hydroxycoumarin derivatives were prepared with a phenyl-prop-2-enoyl moiety at position 3.

Substituent and Solvent Effects on the Spectral Properties of 3-Substituted Derivatives of 4-Hydroxycoumarin
The synthesis procedure is shown in Scheme 1.All the tested compounds, except compound 5, were obtained as yellow crystals.Compound 5 was obtained as a red crystal.Analytical data of synthesized compounds (IR and NMR) were presented in our previous paper. 30emical names of synthesized compounds are presented in Table 1.
After optimisation of measurement methods, the tested compounds were dissolved in ethyl acetate (ETAC), acetonitrile (ACN), and dimethyl sulfoxide (DMSO), with their concentrations in the 10 −6 M range for UV/Vis and 10 −7 M for fluorescence measurements.The UV/Vis absorption spectra were recorded on a Shimadzu 1601 UV/Vis spectrophotometer in the range of 200-800 nm, using a 1 cm quartz cuvette.Fluorescence was measured on a Shimadzu RF-5301PC spectrofluorophotometer with Panorama fluorescence 1.1 software in a 1 cm cuvette in the right-angle arrangement.Excitation and emission slits were both 5 nm.Fluorescence spectra were taken at excitation into the maximum of the longest wavelength absorption band of each tested compound.The temperature of the samples was controlled using a circulating water bath during the absorption and fluorescence spectra measurements; therefore, the ambient temperature was used.
Molar extinction coefficients (ε) were determined by linear least squares fitting of Beer's Law plots of absorbance ver-sus concentration.Fluorescence quantum yields (Ф) were determined using the Eq.(1).
where A is the absorbance at the excitation wavelength, F is the area under the fluorescence curve, and n is the refraction index.Subscripts s and x refer to the standard and sample of unknown quantum yield, respectively. 31e relative fluorescence quantum yield was determined using quinine sulphate in 0.1 M (Ф = 0.58) as a standard for compounds 1, 2, 3, 4, 6, and 7.The standard for compound 5 was Rhodamine B in water (Ф = 31). 31Results and discussion

Influence of the substituent and solvent properties on UV/Vis absorption
Numerous studies of the spectroscopic properties of a large number of substituted coumarin derivatives have shown two absorption maxima.One from the coumarin ring around 250-280 nm, and other at a longer wavelength originating from the substituent. 32Considering the type of substituents on cinnamoyl moiety, the tested compounds were divided into two groups: compounds with withdrawing substituents, such as chlorine and nitro group at the benzene ring, and compounds with electron-donating substituents acetamido, metoxy, dimethylamino, and hidroxy.In order to measure the effects of substituent and solvent, changes in the absorption properties of these compounds were observed in comparison to compound 1, the benzene ring of which was not substituted.The absorption spectra of compound 1 in ACN and DMSO, showed two bands with maxima between 260 and 280 nm and 350 and 370 nm, while in ETAC, compound 1 had only one maximum at about 370 nm (Fig. 1).It was expected that the maximum at longer wavelength depended on the substitution on the benzene ring of the cinnamoyl moiety; therefore, the focus was on the changes in the position of the maximum at longer wavelength.
Comparing the absorption maxima of compound 1 and compound 2 with electron-withdrawing substituent chlorine, the difference in absorption maxima in all solvents was ± 3 nm.Substitution with nitro group caused a hypsochromic shift of the absorption maximum in ETAC (Δλ = 9 nm), while in DMSO, the absorption maximum was shifted bathochromically (Δλ = 8 nm).In ACN there were no changes in the position of the absorption maxima.Due to the presence of electron-donating substituents, longer absorptions were shifted bathochromically in all solvents in comparison with compound 1.This shift was largest for compound 5, with Δλ = 123 nm in ETAC, Δλ = 144 nm in ACN, and Δλ = 167 nm in DMSO (Fig. 2).

Fluorescence properties
Studies on the fluorescence properties of synthesized derivatives showed changes in fluorescence properties. 34,35t is also known that the substitution of a coumarin moiety with a benzene ring in the 3-position enhances the emission yield. 32For fluorescence spectroscopy of tested compounds, the excitation spectra were similar to the ab-sorption spectra, and emission occurred at the maximum absorption.Our results showed that the fluorescence of the tested compounds depended on the solvent properties and the substituents on the benzene ring.Table 2 summarizes the spectral data based on absorption and emission for the parent compound 1 and all the tested compounds.
It can be seen that compound 1 exhibited zero fluorescence in all solvents.
Compounds with chlorine and nitro groups had no fluorescence in ETAC and DMSO, as could be expected, since they are known as fluorescence quenchers. 36In the presence of nitro group, the absence of fluorescence can be explained by the S 1 → S 0 internal conversion as a result of the strong influence of nitro group as the electron-withdrawing group. 31wever, in ACN, the compound with the nitro group showed fluorescence, which can be explained by the influence of solvents.
In polar solvents, the n → π* transition shifts towards higher energies and π → π* towards lower energies, which can result in the appearance of or increase in the intensity of emission. 31The appearance of fluorescence could be caused by donor-acceptor crossings between the compound and the triple bond in ACN.
Electron-donating substituents, such as acetamido (compound 3), methoxy (compound 4), dimethylamino (compound 5), and hydroxy (compound 6) lead to the appearance of fluorescence in all solvents.The presence of free electrons on the electron-donor substituents should not affect the π → π* transition, which is responsible for the emissions of the tested compounds.However, based on the results, it is possible to conclude that the emission was caused by interaction with aromatic ring π-electrons.This intramolecular charge transfer requires that the substituent be planar in relation to the aromatic ring.If they are not planar, the number of conjugations is reduced, which causes a reduction in emission intensity. 35,36Departure from coplanarity with the aromatic ring is also pronounced with alkoxy groups.Alkoxy groups exhibit significant deviation from planarity with the aromatic ring, which was the case with compound 4 with a methoxy group. 31The results obtained for compound 4, which had a methoxy group as a substituent, showed that compound 4 had a lower fluorescence quantum yield than compounds 5 and 6, which were substituted with dimethylamino and hydroxy groups, respectively (Table 2).
In ACN, the fluorescence of all tested compounds was observed, which could be explained by donor-acceptor transitions between compounds and the triple bond in ACN (Fig. 3).
As seen from Fig. 4, compound 5 with a dimethylamino group in ETAC and compound 3 with an acetamido group in ACN had the highest fluorescence quantum yield.The fluorescence quantum yield of compound 5 was three times higher in ETAC than in ACN (Table 2).The present study indicates that the UV/Vis and fluorescence properties of synthesized derivatives (1-7) are dependent on the properties of the substituents attached to the cinnamoyl and solvent properties.
As shown in the present paper, by judiciously choosing the substituent on the phenyl ring of cinnamoyl moiety of 4-hydroxycoumarin derivatives, it is possible to obtain a sufficient bathochromic shift of the UV/Vis band and fluorescence transition to render these compounds useful for analytical applications.

Table 2 -
Summary of the UV/Vis and fluorescence spectral data