a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,

Aminocoumarin 2 was prepared in high yield via solventless reaction. The structure of the 2 was determined with single crystal X-ray analysis. The detailed NMR and IR spectra were reported for the first time. DFT calculations (B3LYP/6-31+G**) showed good agreement between the theoretical and experimental values for the optimized and X-ray structures, as well as between the vibrational and NMR spectroscopy. The thermodynamic pKBH+ values were calculated using three different methods: Yates and McClelland (with HA acidity functions), Excess Acidity Method and Bunnett and Olsen Method. The experimental and theoretical data presented are consistent with the weak nucleophilic properties of 2.


Introduction
Coumarin and its derivatives from natural products, either semi-synthetic or synthetic, represent one of the most active classes of compounds exhibiting a wide spectrum of biological activity.Additionally, coumarin derivatives are used as additives in food and cosmetic industry.Due to the significance of these compounds, the quest for efficient syntheses of coumarin ring compounds 1 as well as new bioactive derivatives from known coumarins 2 is topic.
From the group of aminocoumarins, 3-aminocoumarin and 7-aminocoumarin derivatives are well studied.The 3-aminocoumarin moiety can be recognized in the molecular structure of several natural antibiotics, such as novobiocin, chlorobiocin, coumermycin, etc.These antibiotics and their derivatives are in the research focus. 3In addition, some simple N-acylderivatives of 3aminocoumarin exhibit an anti-inflammatory activity 4 and antimicrobial activity against the Gram-positive bacteria. 5Most of the publications for 7-aminocoumarin derivatives present interesting photochemical behavior of these compounds 6 and many of them can be used as fluorescent markers. 7Furthermore, some platinum complexes of 7-aminocoumarins have been synthesized and evaluated for their in vitro cytotoxicity against Caco-2T cells. 8Derivatives of 4aminocoumarin 2 were not studied that much, although some of them possess biological activity as well. 9he interesting chemical and physical properties and the pharmacological effects of aminocoumarin derivatives were the main motivation for starting this research.At first, the compound 2 was synthesized as a starting compound for the synthesis of large spectra of new coumarin derivatives.However, during the research we have noticed that the amino group in this compound was very weak nucleophile.In contrast to 3-aminocoumarin, aniline, naphthylamine or similar compounds, which are also weak bases, the amino group of 2 did not react with 3 (Scheme 1; alkyl halides, acyl halides, acetic anhydride, etc) to obtain 4 (N-substituted derivatives).Considering reactions of 2, in few publications only it was presented that instead with nitrogen, electrophiles can form a bond with carbon in position 3 (position C8 in Figure 1). 10To our knowledge, in only three references, reactions of nitrogen in 2 were presented, 11 however, the reported yields of the desired products were very poor.Generally, N-substituted derivatives of 2 have been obtained by the reactions of amino compounds with 4chlorocoumarin 12,10a  Detailed knowledge of the structure and spectral behavior of 2 is a necessary prerequisite for understanding its chemical and biological properties.To our surprise, neither NMR nor IR spectra of the title compound have been studied in detail.The purpose of the present study is to elucidate the structure by single crystal X-ray diffraction, UV, NMR and IR spectroscopy.The experimental data will be accompanied by theoretical prediction at different level of approximation in order to draw out the conclusion on the structure-property relationship.In the molecule of the title compound 2 (Figure 1), the bond lengths and angles are within normal ranges. 14The 4-aminocoumarin is almost a planar molecule (rms of 0.0146 Å for the chromene ring).The deviation of planarity is possibly caused by the intermolecular hydrogen bonding, which builds pseudo-layers parallel to bc plane (Table 2, Figure 2).The pseudo-layers are stacked trough π-π interaction, as evidenced by the distance of ca.3.48 Å between the chromene ring mean planes.
The C7-N1 bond distance (1,335 Å) is just somewhat longer then an average C=N double bond and little bit shorter then C≈N bond in pyridine (1,337 Å), 15 indicating a considerable degree of double bond character.

Infrared spectra
Selected numeric values of measured frequencies together with calculated B3LYP/6-31+G** frequencies and intensities and PED matrix are presented in the Table 3.As one can see, the correlation between experimental and scaled theoretical frequencies is very high.In our study, to determine the types of molecular motion associated with each of the observed experimental bands, the computed vibrational data was used.
The greatest differences between theoretical and experimental IR data correspond to the NH 2 stretching vibrations.The calculations predict that the higher-frequency band (3588 cm -1 ) corresponds to the asymmetric NH 2 mode, whereas the other one (3478 cm -1 ) is assigned to the symmetric one.The measured amino frequencies of 3394 and 3219 cm -1 are significantly lower than theoretically predicted ones, due to the formation of strong hydrogen bonds in solid state.The theory predicted the low intensities of the ν(PhH) bands.In solid state IR spectra it is a problem to distinguish these fundamental bands.
With a qualitative agreement between theory and experiment, the (C=O) band is the strongest one in the spectrum.Experimentally, the very strong band detected at 1634 cm -1 , is considerably lower than the value of 1729 cm -1 , measured for unsubstituted coumarin. 16The lower frequency value is an indication for the occurrence of strong conjugation between the C=O and NH 2 groups.The normal vibration, dominated by ν(C-N) coordinate was predicted near 1400 cm -1 and measured at 1438 cm -1 .For aromatic amines, the frequency of the ν(C-N) band appears at 1360-1250 cm -1 frequency region.The significantly higher frequency in our conjugated molecule is another evidence for the occurrence of charge transfer towards the electron withdrawing CO group.

H and 13 C NMR Spectra
To the best of our knowledge, the full NMR spectra of 2 have not been published yet.Therefore, the experimental and calculated 1 H and 13 C spectra (Table 4) in this paper were included.The spectra were assigned with assistance of 1 and 2D (COSY, HMQC and HMBC) NMR spectra.The calculated spectral data were in agreement with the experimental.Numbering according to Figure 1.

UV spectra
The spectra of 4-aminocoumarin in sulfuric acid solutions, reconstructed in the region from 195 to 400 nm (Figure 3), exhibit four bands resulting from π → π* transitions.With increasing the concentration of the mineral acid, the band with λ max at 209 nm exhibited small hypsochromic and hypochromic effect, i.e. it shifted towards shorter wavelengths and decreased in intensity (Table 5).The absorption band at approximately 228 nm in water appeared only as shoulder, while in sulfuric acid solution it exhibited a hyperchromic effect.The absorption band at 250 nm shifted towards longer wavelengths (257 nm) and increased in intensity.In the same time, bands at longer wavelengths (291 and 304 nm in water) showed both hypsochromic and hyperchromic effects.As a result, the absorption band at longer wavelengths could not be clearly recognized, being submerged (hidden) under the high-intensity band with λ max at 287 nm.

Determination of pK BH +
Acid-base properties of coumarin and its derivatives have been previously studied, but to the best of our knowledge, no protonation data for 2 using UV spectroscopy have been published so far.Structures of protonated forms of coumarin and their hydroxy derivatives have been studied by UV and fluorescence spectroscopy. 19The acid-base properties of 3-formyl-7dialkylaminocoumarins 20 and 3-benzazolylcoumarins 21 have been investigated, too.In order to determine the basicity of 2, the thermodynamic pK BH + values and solvation parameters were calculated using three different methods: Yates and McClelland 22 (with H A acidity functions, where -H A = m*•X + log{c H + }), Excess Acidity Method 23 and Bunnett and Olsen Method. 24onization ratios were calculated from the absorbances of the free and protonated base in 26 different sulphuric acid solutions at several selected wavelengths.Also, they were obtained directly from the coefficients of the first characteristic vector.Characteristic vector analysis was successfully applied to separate the effect of protonation from the medium effect.The first characteristic vector accounts for about 98 % of the variance in the region 195-400 nm.
Protonation parameters (dissociation constant for protonated form of 2 and solvation parameters) calculated from absorbances at reconstructed wavelengths and first characteristic vector are presented in Table 6.
The pK BH + values calculated with all three methods were determined to be between -1.08 and -1.20.Best correlation was obtained using Bunnett and Olsen Method, where average pK BH + is -  The value of the pK BH + of 2 was between values of the pK BH + of benzamide (-1.43 25 ) and acetamide (-0.66 26 ), both with very weak nucleophilic properties of NH 2 group.3-Aminocoumarin (pK a = 1.0 27 ), aniline (pK a = 4.6 28 ), 1-naphthylamine (pK a = 3.9 29 ) and 2naphthylamine (pK a = 4.2 29 ) are weak bases too, but unlike previous compounds they are with positive pK a values, have sufficiently nucleophilic properties to react with alkyl halogenides, carboxylic acid anhydrides, acyl halides, sulfonyl chlorides, etc.

Conclusions
The structure of 2 was determined with single crystal X-ray analysis.The full data from NMR and IR spectra were reported for the first time.The calculated values with quantum chemical methods were in accordance with the experimental.The C7-N1 bond distance indicates a considerable degree of double bond character.The conjugation of NH 2 is additionally confirmed by the infrared spectra.This is most probably the reason for very low nucleophility of nitrogen atom in molecule of 2.Moreover, the values of pK BH + correlate with the week nucleophilic properties of 2.
Considering the above mentioned and our experience in performing reactions with 2, an attempt to synthesize N-substituted 4-aminocoumarins from 2 would be bad strategy.

Experimental Section
General.Ammonium acetate and 4-hydroxycoumarin were purchased commercially and used without further purification.Melting points were determined on a Reichert hot-stage apparatus.The crystallographic analysis was carried out on an Enraf-Nonius CAD4 diffractometer, using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) at room temperature.An orange irregular block was cut off from a large crystal and selected for measurement of the intensities.Unit cell parameters were determined from centering 22 reflections in the θ range (17.94-19.58)ºand refined by the least-squares method.Maximum 2θ was 27.98º and scan mode: ω/2θ.Three standard reflections were monitored every 500 reflections during data collection and no significant intensity decay was observed.All diffracted intensities were corrected for Lorentz and polarization effects. 30No absorption correction was employed.After merging of equivalent reflections, 1836 independent reflections were obtained (R int = 0.0896), which were used for the solution and refinement of the structure.The structure was solved by direct methods and was refined by the full-matrix least-squares method using SHELXS97 and SHELXL97 computer programs, 31 respectively, in the space group C 2/c (no.15).All non-hydrogen atoms were refined with anisotropic displacement parameters.H atoms were placed at idealized positions using standard geometric criteria.The final refinement of the structure converged to the final indices R1 = 0.0578 and wR2 = 0.1538 for 1836 reflections with [I > 2σ(I)].The ORTEP program 32 was used to generate the ellipsoid plot and the figures involving H-bonds and packing were drawn using Mercury. 33Further relevant crystallographic data are summarized in Table 7.The FTIR spectra (4000-400 cm -1 ) were recorded at ambient temperature as KBr pellets on Perkin-Elmer System 2000.The NMR spectra were run on a Bruker DRX 250 spectrometer using standard Bruker software in solvent DMSO-d 6 .The residual solvent signal was used as an internal standard for the 1 H (δ = 2.5 ppm) and 13 C (δ = 39.5 ppm) NMR spectra.
The UV spectra of the 2 (altogether 26 samples) and appropriate blanks were recorded in a range of sulfuric acid solutions from 0.0 to 15.0 mol dm −3 .The UV spectra were recorded, immediately after preparing the solutions, on a Varian Cary 50 Spectrophotometer in 1 cm quartz cell, with resolution of 1 nm, in the range from 190 to 400 nm, at room temperature.Since the absorption spectra are affected by medium effects (well-defined isosbestic points have not been observed), corrections were made by means of the characteristic vector analysis using the developed procedure in MathCad environment, on preprocessed experimental data (all normalized to unit area under the curves).The ab initio restricted Hartree-Fock (RHF) method and DFT are used to obtain equilibrium geometry of 4-aminocoumarin molecule.All calculations have been performed with the standard GAUSSIAN software (AIX, version 1998). 34DFT method, employed in the present study is B3LYP-Becke's three parameter hybrid method 35 using the correlation functional of Lee, Yang and Parr. 36The standard 6-31+G** basis set was applied in all calculations.The local minimum was verified by establishing that the matrix of the energy second derivatives (Hessian) has no negative eigenvalues.The theoretical vibrational spectra were interpreted by means of potential energy distributions (PEDs) using VEDA 4 program. 18For a better correspondence between experimental and calculated values, we modified the results using the empirical scaling factors. 171H and 13C NMR chemical shifts were calculated by using the GIAO method 37 at the HF/6-311+G** level of theory (reference compound TMS was calculated at the same level); a solvent was not considered.

4-Aminocoumarin (2).
A mixture of well powdered 4-hydroxycoumarin (1,07 g, 0,0066 mol) and ammonium acetate (7,87 g, 0,1 mol) was melted in an oil bath (max.130 ºС).Liquid mixture was stirred 3 hours and was left to cool to ambient temperature.At the cooled mixture, water was added.Crude product (max.74 %) was isolated as yellow crystals by simple filtration.First purification was made by dissolving the crystals in ethanol and precipitation with water.

Supplementary material
CCDC 754032 contains the supplementary crystallographic data for this paper.These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033).

Figure 1 .
Figure 1.The molecular structure of the title molecule with the atom-numbering scheme; displacement ellipsoids are drawn at the 50 % probability level.

Figure 2 .
Figure 2. Crystal packing along the c axis showing the propagation of the pseudo-layers propagating parallel to bc plane.

Figure 3 .
Figure 3. Changes in the reconstructed UV spectra of 4-aminocoumarin as a function of sulfuric acid concentration from 0.0 to 15.0 mol dm −3

Table 1 .
Selected bond lengths and angles from crystallographic data and comparison with the theoretical data a

Table 3 .
Selected theoretical and experimental vibrational frequencies (ν in cm -1 ) and IR integrated intensities (A in km.mol -1 ) of 4-aminocoumarin 18ibrational modes: ν, stretching; δ, in plane and out of plane deformations; τ, torsion; The numbers before the mode symbols indicate % contribution (10 or more) of a given mode to the corresponding normal vibration, according to the potential energy distribution matrix.18

Table 5 .
Experimental transitions in the UV spectra of unprotonated and protonated form of 4aminocoumarin (c = 4.93•10 -5 mol dm -3 ) 1911.The slope of the log I vs. H A (calculated using the m* = 0.3) was close to 1.The results indicate that the acidity function H A satisfactorily describes the protonation equilibria for the entire acidity range.The obtained φ(1-m*) values presented in Table6were positive, as expected for oxygen Brønsted bases.The site of protonation is probably the carbonyl oxygen, according to the previous theoretical studies of coumarin and derivatives.19

Table 6 .
Protonation parameters for 2 calculated from reconstructed wavelengths and first characteristic vector