Synthesis and spectral properties of fluorescent dyes based on 4-styryl-1,8-naphthalimide

The paper reports on synthesis and spectroscopic study of novel N-butyl-4-styryl-1,8-naphthalimide dyes bearing methoxy (1), dimethoxy (2), and dimethylamino (3) groups in the styryl fragment. It is shown that all synthesized compounds demonstrate positive solvatochromism, high values of Stokes shift in polar solvents, and fluorescence in the long wavelength part of visible range. These facts indicate a potential application of these compounds as fluorescent dyes in the biochemistry. The changes in the dipole moments of the molecules caused by excitation were estimated using Lippert—Mataga equation. The obtained results could be assigned to the formation of the excited states with intramolecular charge transfer. The formation of the twisted states with charge transfer was suggested in the case of compound 3, while the fluorescence quantum yield was significantly reduced in polar protic solvents.

Nowadays, the methods of fluorescence spectroscopy are of great importance for the application in biological and biomedical research. 1 With the appearance of confo cal fluorescence microscopy 2 the organic fluorophores have been used as molecular probes, imaging reagents, and sensors, allowing one to study the structure, dynamics, and function of biological macromolecules. 3- 6 It is known that the emission peaks of the most intensively luminescent organic materials are located in a range of 400-600 nm. This spectral range is characterized by significant absorption of the excitation light by the bio logical sample. In addition, there is relatively strong fluorescent background in this spectral range, which great ly limits the choice of useful photoactive compounds. Cyanine dyes 7-9 are used in many cases for in vivo optical studies, because at relatively small length of conju gated chain (about seven methine groups) their fluores cence maxima may be located near the border of the visible and near infrared (NIR) spectral regions, or even be in the NIR range. The disadvantages of the cyanine dyes include the complexity of their synthesis, relatively low photochemical stability, and low values of the Stokes shift, which usually do not exceed 25 nm. The latter feature of the spectral properties is not desirable when using the confocal fluorescence microscopy, since it leads to a significant reduction in the contrast of the images due to reflection/scattering of the excitation light by the test sample.
The derivatives of imide of naphthalic acid (1,8 naph thalimide) are practically important class of organic fluo rophores, which are used in many areas of science and technology, such as optical brightening, 10 fluorescent crack detection, 11 solar energy conversion, 12 fabrication of the optical memory elements, 13 and electrolumines cent devices. 14 Intensive fluorescence in the visible spec trum, high photostability, and high values of Stokes shift of 1,8 naphthalimides along with the simplicity of syn thetic modifications aimed to molecular structure of this type make these compound attractive for the application as a photoactive component of ratiometric chemosensors suitable for the analysis of ions in living cells 15,16 as well as fluorescent dyes for applications in biology and medi cine. 17, 18 It has been shown in earlier works devoted to the spectroscopic study of 1,8 naphthalimide derivatives that the introduction of polarizing substituents having strong electron donating properties in positions 4 and 5 of the naphthalene nucleus leads to a bathofluoric shift of the emission band. 19,20 In most cases, the 4 substituted derivatives formed at this exhibit blue or yellow green emission (λ max = 450-550 nm). Much rarely the com pounds are found, in which naphthalimide chromophore 1 emits at longer wavelength region of the spectrum (λ max > > 600 nm). 21-23 Exactly such derivatives are of the great est practical interest for the study of biological samples with fluorescent methods.
In this paper, the synthesis and detailed study of spec tral luminescent properties of 1,8 naphthalimides 1-3 (Scheme 1), containing styryl fragment at atom C(4) and differing in the number and nature of the electron donat ing substituent in the phenyl ring, are considered. Such compounds are able to enter cells. They have potential use as in vivo fluorescent imaging reagents. 23 However, their optical properties were investigated in a lesser extent than the properties of 4 amino, 4 (acyl)amino, and 4 alkoxy 1,8 naphthalimides.

Results and Discussion
Target 4 styryl substituted derivatives of 1,8 naph thalimide 1-3 were prepared starting from acenaphthene (4) according to Scheme 1. The bromination of 4 to 4 bromo acenaphthene (5) followed by the oxidation led to 4 bromonaphthalic anhydride (6), which further was converted to naphthalimide 7 using n butylamine in etha nol. The introduction of styryl fragment into position 4 of the naphthalimide nucleus at the final step was carried out by the Heck reaction using commercially available styrene derivatives. 24 The structures and compositions of pre pared compounds 1-3 were proved by a combination of physico chemical analytic techniques (see Experimental).
The absorption and fluorescence emission spectra of naphthalimides 1-3 were registered in fifteen different solvents, among them were five protic and ten aprotic solvents. Stokes shifts (Δν), fluorescence quantum yields (ϕ fl ), and excited state lifetimes (τ) were also determined. The data are presented in the Table 1.
The long wavelength band in the absorption spectra of investigated compounds is connected to intramolecular charge transfer (ICT) from electron donating styryl frag ment to carbonyl groups of dicarbonylimide moiety as evidenced by the distribution of electron density in the frontier molecular orbitals (HOМО and LUМО) of 1-3 ( Fig. 1). As examples the absorption and fluorescence spectra of 1-3 in ethyl acetate are presented in the Fig. 2. It is clear from Fig. 2 that an increase of electron donating properties of the substitutent in the position 4 of the naphthalene ring from monomethoxy derivative 1 to dimethoxystyrylnaphthalimide 2 and further to 3 leads to the bathochromic shift, which corresponds to conver gence of energy levels S 0 and S 1 and an increase of the efficiency of the formation of the ICT state.
An increase in the solvent polarity and its ability to form hydrogen bonds also causes displacement of the maxima of absorption bands (λ abs max ) and fluorescence (λ fl max ) to longer wavelength with simultaneous growth of Δν value (see Table 1). Significant solvatochromism and solvatofluorochromism of 4 styrylnaphthalimide deriva tives allowed estimation of the change of dipole moment of these molecules upon the transition into excited state. The Lippert-Mataga equation 25,26 was used for the anal ysis of influence of solvent on the emission spectra of the compounds under investigation: (1) (2) where Δν is the Stokes shift, cm -1 ; Δν abs , Δν fl are the wave numbers corresponding to spectral maxima in ab sorption and fluorescence spectra, respectively, cm -1 ; μ e and μ g are the dipole moments of compound in solu tion in excited and ground states, respectively, D (1 D = = 1•10 -18 cm 5/2 g 1/2 s -1 ); h is the Planck constant (h = 6.626•10 -27 erg s); с is the speed of light in vacuum (с = 2.998•10 10 cm s -1 ); а is the effective radius of Onsager cavity (radius of cavity, where fluorophore is placed), cm; ε and n are the dielectric permittivity and refractive index of solvent, respectively; Δf is the orienta tion polarizability of solvent. Equation (2) is given in CGS system. Notes: ε is the solvent dielectric constant; λ abs max and λ fl max are the maxima in absorption and fluorescence spectra, respectively; ε λ is the extinction coefficient in the point corresponding to λ abs max ; λ ex is the excitation wavelength; Δν is the Stokes shift; ϕ fl is the fluorescence quantum yield; τ is the excited state lifetime.
The values of parameter Δf for used here protic and aprotic solvents and corresponding values of Stokes shifts for compounds 1-3 are represented in the Fig. 3. In considered cases both protic and aprotic solvents demon strate good linear correlation. The changes of dipole mo ments upon the transition into excited state were deter mined using the slope of the lines in the Fig. 3. Calculated values (μ e -μ g ) consisted of 20.78, 21.96, and 22.95 D for 1, 2, and 3, respectively. Half of the distance between N atom or O atom in para position to double bond in styryl fragment and O atom farthest from С(4) substitutent of carbonyl group was taken as the radius of the Onsager cavity. This is similar to approach taken for 4 substituted naphthalimide derivative earlier. The fluorescence quantum yield of compounds 1-3 is changed differently by varying the nature of the solvent. In the case of compounds 1 and 2 the efficiency of radia tive deactivation is approximately the same in both polar and non polar solvents, while for compound 3 the growth of solvent polarity and its proton donating properties causes the fluorescence quenching (see Table 1). A similar de pendence can be traced also for the excited states lifetime values. These observations point to the persistence of radi ative rate constant of studied compounds in solvents of different nature, as well as to suggestion for compound 3 on the existence of relaxation channel, constituting a sig nificant competition to the fluorescence in a polar envi ronment.
According to current concepts, to explain the low ϕ fl values for fluorophores of ICT type in polar protic sol vents the model that postulates the formation of a twisted state of the charge transfer (twisted intramolecular charge transfer -TICT) 32 can be involved. This model assumes the existence of two energy minima on the potential ener gy surface of the excited state: the first one corresponds to locally excited state with quasi planar arrangement of π system and the donor group (Me 2 NC 6 H 4 in the case of naphthalimide 3); the second one corresponds to a state with a charge transfer from a donor to an acceptor group with quasi perpendicular location of π system and the donor group. In the frameworks of TICT model the for mation of twisted state with charge transfer includes two steps: the charge transfer and following rotation of donor in respect to the rest of the molecule. TICT model has been suggested for the description of two band fluores cence of 4 N,N dimethylaminobenzonitrile. 33,34 How  ever, in most cases the TICT states relax nonradiative and their formation decreases the quantum yield and fluores cence decay time. 32 Apparently, the transition to TICT state is more probable for compound 3 than for com pounds 1 and 2 because 4 (dimethylamino)styryl group, having the highest electron donating effect, contributes significantly to the charge separation during photoexcita tion. It should be stressed out again that another factor (in addition to the electronic effects of substituents in the chromophore), which promote the formation of the twist ed state is the solvation. In a strongly polar solvent the formation of non fluorescent TICT form may be the dom inant process. In particular, this results in a significant decrease in fluorescence quantum yield and lifetime of the excited state for compound 2 in methanol (see Table 1). Thus, in the course of this work the naphthalimides 1-3, differing in the structure of the styryl moiety, were synthesized. The analysis of spectral luminescent proper ties showed that all three compounds are ICT fluoro phores and exhibit significant solvatochromism. The ab sorption maxima (400-420 nm) and the fluorescence maxima (500-570 nm) of monometoxy derivative 1 are located at approximately the same wavelengths as in the case of 4 aminonaphthalimide. 19 As it is noted above, 4 aminonaphthalimide has substantial limitations for us ing as a fluorescent component of molecular devices in biochemical research. 4 (N,N Dimethylamino)styryl substituted compound 3 is the most long wavelength flu orophore. However, the fluorescence quantum yield of this compound is low in proton polar environment due to the high probability of formation of twisted state with charge transfer. At the same time, it was shown that the presence of two electron donating methoxy groups in the phenyl nucleus in compound 2 leads to bathofluoric shift of the maximum to a value of 550-630 nm while main taining high fluorescent signal intensity in polar solvents. Experimental 1 Н NMR spectra were registered using Avance 400 and Avance 600 spectrometers (Bruker). The solutions were prepared in deuter ated benzene, dimethylsulfoxide, and chloroform. The chemical shift of nuclei 1 H and 13 С were determined with deviation of 0.01 ppm in respect to the residue signals of solvents and recalculat ed to internal standard (ТМS). The coupling constants were mea sured with a precision of 0.1 Hz. Numeration of carbon atoms in naphthalimide nucleus, styryl fragment, and butyl group, used for the description of 1 Н and 13 С NMR spectra of compounds 1-5 are represented in Scheme 1. The assignment of signals was carried out using two dimensional methods HSQC, HMBС, and 1 Н COSY with pulse field gradients.
Mass spectra with electrospray ionization at the atmosphere pressure (electrospray ionization method, ESI) were registered in the mode of total scanning of positive ions masses with tandem dynamic mass spectrometer Finnigan LCQ Advantage. Melting point were determined in glass capillary using Mel Temp device  1, 2, and 3 The absorption spectra were measured using two channel spec trometer Varian Cary 5G. The fluorescence spectra were measured using spectrofluorimeter FluoroMax 3 at temperature 20±1 °С. Observed fluorescence was registered perpendicular to exciting beam. The fluorescence spectra were corrected in respect to sensi tivity of the photomultiplier. The solvents of spectroscopic grade (Sigma Aldrich, purity not less than 99.5%) were used for the ab sorption and fluorescence spectra. The spectra were registered in solvents with a concentration of ∼10 -6 mol L -1 .
The fluorescence quantum yields were determined in air satu rated solutions at temperature 20±1 °С in respect to coumarin 481 standard in acetonitrile (ϕ fl = 0.08). 35 To calculate quantum yield following equation was used 36 (3) where ϕ fl i and ϕ fl 0 are the quantum yields, D i and D 0 are the absorptions, S i and S 0 are the integrals of fluorescence spectra of analyzed compound and standard, respectively; n i and n 0 are the refractive indices of solvents for studied compound and standard, respectively.
Time dependent fluorescence spectra were registered using a system consisting of spectrograph Chromex 250, connected to Hamamatsu 5680 streak camera with the module of fast scanning M5676 with a time resolution of 2 ps. Excitation pulses were gener ated using light optical parametric oscillator pumped by the funda mental emission of femtosecond laser system Ti-sapphire Femto power Compact Pro. All values of lifetime of excited states were obtained using depolarized excitation light. Maximum pulse energy of the fluorescence excitation was not higher than 100 nJ, average power was 0.1 mW, pulse repetition rate was 1 kHz. The excitation beam of 0.1 mm diameter was focused onto quartz cuvette (l = 10 mm). The analysis of florescence kinetics was carried out by curve fitting using method of Levenberg-Marquardt least squares applying the solutions of differential equations describing the be havior of one of the excited state in time without taking into account changes in the population of the ground state according to the equation (4) where I(t) is the intensity of fluorescence, τ is the lifetime of the excited state, Gauss is the Gauss profile of exciting pulse, t 0 is the time position of maximum, Δt is the pulse width, A is the pulse amplitude. Initial conditions were I(-∞) = 0. By selecting the solu tion the values of χ 2 (Pearson criterion) was always smaller than 1•10 -4 , and correlation coefficient R > 0.999. Error of calculation of the lifetime did not exceed 1%. Fluorescence accumulation time in the experiments did not exceed 90 s. The registration of the fluorescence spectra with time resolution has been made on the equipment of the Aquitaine Waves and Matter Laboratory, Univer sity of Bordeaux/CNRS (France).
Quantum chemical calculations were carried out with half empirical method РМ6 37 using the software package МОРАС 2012. Iterative procedure was continued as long as the molecular energy difference of two successive iterations did not exceed 0.01 kcal mol -1 . Influence of solvent nature was taken into account in accordance with the model COSMO (Сonductor like Screening Model), included in МОРАС 2012. It was assumed in the calcula tions that the solvent has a dielectric constant ε = 20 and refractive index n such that n 2 = 2. 6 Bromobenzo[d,e]isochromene 1,3 dione (6). The mixture of 12.5 g (0.053 mol) 5 brormoacenaphtene (5) and 125 mL of acetic acid was heated to 90 °С. To this stirred solution 78.0 g (0.262 mol) Na 2 Cr 2 O 7 •2H 2 O was added by small portions in order to maintain temperature not higher than 90 °С. Then reaction mass was heated to reflux and was kept at reflux with condenser for 5.5 h. Afterwards, the reaction mass was cooled to room temperature and poured into 750 mL of water. Formed precipitate was separated by the filtration and washed on the filter by diluted hydrochloric acid and then by water. This precipitate was suspended in 500 mL of 10% aqueous solution of Na 2 CO 3 and boiled for 1 h. Obtained solution was cooled down to room temperature and insoluble precipitate was separated by filtration. The filtrate was acidified by concentrat ed hydrochloric acid, formed precipitate of 4 bromonaphtalic acid was separated by filtration, washed with water and dried. In order to obtain anhydride the product was recrystallized from acetic acid. The yield was 6.