Synthesis and fluorescent properties of new derivatives of 4-amino-7-nitrobenzofurazan

The following new compounds were obtained by reacting 4-chloro-7-nitrobenzofurazan (NBD-Cl, 1 ) with five primary amines: 3b with a benzo-crown ether 18C6; 3c with an N-( α -naphthyl)- ethylenediamine group; 3d , with a 2,2,6,6-tetramethylpiperidin-N-oxyl group; 3e , with an α - picolyl group; and 3f , derived from tris(hydroxymethyl)aminomethanol. Also, from the reaction of 1 with N-methylhydroxylamine an N-hydroxy-N-methyl-NBD derivative ( 3g ) was prepared. All these six new NBD derivatives 3b-g were studied (in comparison with the known compound 3a prepared from 1 and aniline) for their physical and chemical properties, with special emphasis on hydrophobicity, UV-Vis, fluorescence, using also structural studies trough QSPR.


NMR Spectra of compounds 3a-g
The NMR data of compounds 3a-c, 3e-g (Table 2) confirm the proposed structure.Assignments in Table 2 are using the atom numbering indicated in Table 1.No NMR data are reported for the paramagnetic compound 3d.Compound 3c, with two amino groups, is converted into an ammonium salt by protonation of the naphthylamino group (the strongly electronwithdrawing NBD group cancels the basicity of the adjacent amino group).The changes in the NMR spectra are evident -there are significant differences for C-9 and protons H-8 and H-9 in the ethylene group, small changes for H-11 in the naphthyl group, and no changes in the NBD moiety.

Hydrophobic/hydrophilic balance of compounds 3a-g
All biological uses of chemical compounds depend on how they interact with biomembranes, and such interactions are governed by the hydrophobic/hydrophilic balance, so that we had to include such effects in the present study.Following previous reports, 25,26 the hydrophobic/hydrophilic balance of compounds 3a-g was studied experimentally by reverse phase TLC (RP-TLC), a simple, efficient, and precise method.The molecular hydrophobicity R M0 was determined by means of equations ( 1) and ( 2), using the data presented in Table 3.

Electronic absorption spectra and fluorescence of compounds 3a-g UV-Vis spectra
Compounds 3a-g are reddish or brown in crystalline state, and their solutions in organic solvents are yellow, orange, or red.All are soluble in absolute ethanol, so that one can make comparisons between their electronic absorption bands.As seen from Table 4, all compounds present a strong band in the visible region (λ max = 457 -483 nm) due to the NBD chromophore. 3,25The differences are due to extended conjugation with the acceptor NBD group 10,24,36 for aromatic susbtituents at the amino group (3a, 3b, which absorb at higher wavelengths), whereas the remaining compounds having alkyl, hydroxy, or aralkyl groups absorb at lower wavelengths.Calculated Mulliken net atomic charges on the amino nitrogen (NAC N ) using the AM1 algorithm for molecular geometries, 37 and the CODESSA program 38 are presented in Table 5 together with the values found by a simple linear correlation, eq. ( 3), where NAC N is the net atomic charge for the nitrogen atom, and SD is the standard deviation (calculated and experimental values had two decimals).

General characteristics for the fluorescence of compounds 3a-g
,12,15,16 Among compounds 3a-g, only compounds 3e-g are strongly fluorescent in solid state and in most solvents.Compounds 3a and 3d are weakly fluorescent in solid state and in most solvents.Compound 3b is not fluorescent either pure or as complex with KClO 4 .Compound 3c is not fluorescent in solid state, but is weakly fluorescent in some solvents (e. g. dichloromethane, benzene, and toluene); a more detailed account will be seen below.
where: τ 0 is the lifetime, ν is the wavenumber of the maximum of the absorption band, n is the refractive index of the solvent (1.3595 for ethanol), I F is the fluorescence intensity, ε is the molar absorption coefficient, and τ = τ 0 • Ф.
[46]  As discussed above, the electronic absorption spectra, for the three compounds 3d,3e,3g that have a significant fluorescence, it was possible to correlate the fluorescence lifetime τ (which involves also the quantum yield) with the calculated net atomic charge for the amino nitrogen atom (NAC N ) by the equation ( 5), as seen in Table 7.

Fluorescence of compound 3d
The paramagnetic compound 3d is weakly fluorescent due to intermolecular quenching.The EPR spectrum has three lines (Figure 2) due to a hyperfine coupling with a N = 14.79 Gauss (in methylene chloride) in agreement with that of 4-amino-TEMPO. 47With an excess of ascorbic acid in absolute ethanol, the solution becomes strongly fluorescent in a few minutes (the intensity of the fluorescence increases about six times, as seen in Figure 3), due to the formation of hydroxylamine 4 (Scheme 2).Compound 4 was detected by TLC (R f 3d = 0.907, R f 4 =0.372, on silica gel with methylene chloride:methanol 9.5:0.5 v/v).The process described in Scheme 2 is reversible, because oxidation of 4 (with PbO 2 , Ag 2 O, KMnO 4 , even with air) produces 3d.These fluorescence and paramagnetic properties of compound 3d may lead to applications as a molecular probe for biological redox processes.

Fluorescence of compound 3c
In absolute ethanol, compound 3c is not fluorescent, but in less polar solvents (benzene, toluene) a weak fluorescence (Table 8) due to the NBD group was detected (λ ex =450 nm, λ em =505 -512 nm).
In acetic acid which has the same polarity as absolute ethanol, a weak fluorescence has also been observed.However, the fluorescence increases significantly in the presence of strong acids such as trifluoroacetic acid and 4-toluenesulfonic acid (Table 9), when the α-naphthylamino group becomes protonated affording cation 5 (Scheme 3).Trifluoroacetic acid introduces a significant hypsochromic shift (16 nm) in the visible spectrum, and the protonated compound 5 has the highest value for Ф (Table 9).In compound 3c there is an electron-acceptor NBD group (A) and a π-electron-donor moiety (D) represented by the α-naphthylamino group, linked together by a flexible ethylenediamino chain.An intramolecular D-A interaction will quench the fluorescence, but the protonation cancels the donor effect of the donor group.
By simulating the molecular geometry using the Hyperchem force field MM+, 48 it was possible to simulate the closed-sandwich geometry of 3c as a consequence of the intramolecular D-A interaction.As seen in an earlier Section, NMR data (Table 2) confirm the structure of the salt 5 , and its geometry appears as an open structure without such an intramolecular D-A interaction (Scheme 3 and Fig. 4).Qualitative experiments with compound 3c evidenced the fluorescence-enhancing effect of inorganic acids (e.g.HCl, H 2 SO 4 , H 3 PO 4 , HPO 3 , H 4 [Si(W 3 O 10 ) 4 ]) or organic acids (e.g.bile acids, nicotinic acid, sulfanilic acid, salicylic acid, tannic acid).Compound 3c does not become fluorescent in the presence of benzoic, ascorbic, or caprilic acids, as well as α-amino acids (i.e.leucine, alanine, phenylalanine, glycine, thyrosine, glutamic acid, arginine, ornitine).

Fluorescence of compound 3f
It was shown earlier that compounds 3e, 3f, and 3g have the highest fluorescence in the series examined in this report.The hydrophobicity of these compounds decreases in the order 3e > 3g > 3f.The last compound is actually amphiphilic due to the presence of the hydrophobic NBD moiety, and the hydrophilic tris(hydroxymethyl) group.We examined the behavior of the fluorescence of 3f in aqueous ethanol as a function of the ethanol concentration.As shown in Fig. 5, the fluorescence intensity raises markedly with an increasingly higher ethanol content (about 20 times from 20% to 96% ethanol).5][56][57][58] Thus, compound 3f may be useful as a fluorescent probe for exploring how the stronger non-covalent interactions (hydrogen bonds, hydrophobic interactions, donor-acceptor or charge transfer interactions) behave for biomolecules such as glycoproteins, glycolipids, lectins.More generally, all strongly fluorescent compounds 3e, 3f, 3g may be useful as molecular fluorescent probes for antibody-antigen biochemical species that manifest affinity for 2,4-dinitrophenyl groups, which are similar to the NBD moiety. 41,59

Conclusions
The present study was undertaken in order to obtain new 4-amino-7-nitro-NBD derivatives 3b-3g by reacting NBD-Cl with corresponding amines.The known 4-anilino derivative 3a, which is weakly fluorescent, was the reference compound.With a benzo-crown structure, 3b has ionophoric character.The weakly fluorescent N-α-naphthyl-N'-NBD-ethylenediamino derivative 3c becomes intensely fluorescent on treatment with strong acids, as the result of a change in geometry that cancels the intramolecular fluorescence quenching.The weakly fluorescent paramagnetic derivative 3d with an amino-TEMPO nitroxide group becomes intensely fluorescent on reduction with ascorbic acid yielding the corresponding hydroxylamine derivative.Compounds 3e with an α-picolyl group and 3g with a hydroxylamino group are strongly fluorescent.Derivative 3f with a tris(hydroxymethyl) group has an amphiphilic character and may be useful as a molecular probe for studying emulsions and micelles.Other derivatives (3b-3d) may be useful as biochemical fluorescent probes.

Experimental Section
General Procedures.Chemicals (amines 2a-2g) and NBD-Cl (1) were Aldrich commercial products.The 1 H-NMR and 13 C-NMR spectra were recorded with a Varian Gemini 300BB spectrometer at 300 MHz for protons and 75 MHz for 13 C. Electronic absorption spectra were recorded with a Perkin-Elmer Lambda UV-Vis spectrophotometer, and fluorescence with a Perkin-Elmer 204 spectrofluorimeter using an excitation lamp (Xe, 150 W) interfaced with the computer, allowing a pre-established data reading time of 0.5 s.EPR spectra were recorded using a Jeol JES FA100 spectrometer.IR spectra were recorded with a Bruker FTIR spectrophotometer Model Vertex 70, using ATR technique.Melting points have been recorded in open capillary with Electrothermal's IA 9000 Series of digital melting point instruments.

Reduction of compound 3d to 4 (Scheme 2)
A six-fold molar excess of ascorbic acid was added to the solution of 3d in absolute ethanol under stirring at room temperature till TLC shows the disappearance of 3d and the complete

Figure 3 .
Figure 3. Variation of the fluorescence intensity (I F ) during the reduction of 3d (in absolute ethanol) with an excess of ascorbic acid.

Table 1 .
Structures of the NBD derivatives 3a -g

Table 4 .
UV-Vis spectral data of compounds 3a-g in absolute ethanol

Table 5 .
Net atomic charges on the amino nitrogen (NAC N ), and λ max (exp. in Table4and calc.with eq. 3, in nm) for compounds 3a-g in absolute ethanol a see Table4

Table 7 .
Calculated values of net atomic charge on the amino nitrogen (NAC N ) by CODESSA program and τ (exp. in Table6and calc.witheq. 5) for compounds 3e-g a) See Table6

Table 8 .
The effect of solvent polarity on the absorption and fluorescence spectra of compound 3c (using λ ex = 450 nm)