Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid : Synthesis , Structural Characterization and Deuterium Isotope Effects

Several phenylenediamine derivatives of dehydroacetic acid, 3-acetyl-4-hydroxy-6-methyl-2Hpyran-2-one have been synthesized and their structure elucidated by using NMR and IR spectroscopies and mass spectrometry. Spectral analyses have pointed toward the localized keto-amine form as the predominant tautomeric form in solution and solid-state which was in agreement with X-ray data. The relatively broad N–H stretching bands observed in IR spectra and significant N–H proton down-field and keto C=O up-field shifts in NMR spectra indicated the presence of intra-molecular hydrogen bonds in all studied compounds. In order to gain further insights into nature of these interactions in solution deuterated isotopomers have been prepared and deuterium isotope effects in C NMR spectra have been determined and analyzed. The magnitude and sign of isotope effects have confirmed the existence of intra-molecular hydrogen bonds. Observed differences in isotope effects reflected different hydrogen bond structure and dynamics in studied compounds. The results presented in this paper might help in better understanding of biological properties of the related compounds.(doi: 10.5562/cca1825)

Generally, enaminones might exist in two tautomeric forms: keto-amine and enol-imine (Figure 2). 12hey have conjugated N-C=C-C=O moiety with two electron-deficient centers at atoms C-1 and C-3, electron rich atom C-2 and an amino/imino group.Hence, they may act as both electrophiles and nucleophiles.A strong intramolecular resonance assisted hydrogen bond (RAHB) was found to be present in both tautomeric forms (Figure 2). 13,14ydrogen bonding is one of the major factors that govern the overall structure and functionality of biologically important molecules. 15−18 Furthermore, X-ray structural analysis proved the existence of ketoamine tautomeric form and intramolecular hydrogen bonds in 1 in solid state. 5The same tautomer was ob- served in the asymmetrical Schiff bases derived from dehydroacetic acid and p-phenylenediamine. 19Since the position of tautomeric equilibria can be crucial for reactivity of these compounds, one of the most important aims of this work was to study solvent influence on tautomeric equilibria and nature of hydrogen bonds in 1-3.
−27 Many isotopes have been investigated, but the most important is deuterium owing to the largest change in mass upon proton/deuterium substitution.Secondary deuterium isotope effects in 13 C NMR spectra (DIE) can be determined as the difference in chemical shifts between carbon substituted with hydrogen δ 13 C(H) and carbon substituted with deuterium δ 13 C(D), where n represents a number of bonds between the investigated nucleus and deuterium: DIE can be intrinsic and equilibrium in origin.They differ in magnitude, sign and temperature dependence.Intrinsic isotope effects can be observed in systems which are not in chemical equilibrium and they are described with a potential energy curve with one minimum.Equilibrium isotope effects, which are described with a two-minimum potential energy curve, exist when isotope exchange alters chemical equilibrium. 13,27The measured deuterium isotope effects can help in elucidating the reactivity of certain functional groups in studied compounds, the nature and strength of possible hydrogen bonds and influence of the solvent.Hence, we prepared deuterated isotopomers 4-6 (Figure 3) and recorded their 13 C NMR spectra.An attention was also paid to conditions of deuteration which might occur at different sites in the molecule yielding various isotopomers.

EXPERIMENTAL Materials
Dehydroacetic acid, ortho-, meta-and para-phenylenediamine, methanol-d 4 , chloroform-d 1 and deuterium oxide were purchased from commercial sources and used without further purification.Chloroform and dichloromethane were dried using P 2 O 5 .Methanol was dried using iodine and magnesium turnings and then distilled.

Synthesis of Deuterated Isotopomers
Partially deuterated isotopomer of (1) was prepared by dissolving 1 (30 mg) in deuterated methanol (1.0 ml) and by adding a few drops of deuterium oxide.The solution was refluxed for 3 h, evaporated under vacuo to 1/3 of initial volume and the reaction product was rinsed and dried.Yield: 21.5 mg, 71 %.
Partially deuterated isotopomer of (2) was prepared by dissolving 2 (15 mg) in deuterated methanol (1.0 ml) and by adding a few drops of deuterium oxide.Solvent was almost completely evaporated under vacuo and the reaction product at the bottom of the reaction flask was rinsed and dried.Yield: 10.5 mg, 70 %.
Partially deuterated isotopomer of (2) was also prepared by dissolving 2 (15 mg) in the metha-nol/chloroform mixture (v:v = 2:1, 2.0 ml) and by adding a few drops of deuterium oxide.The solution was refluxed for 5 h, evaporated under vacuo to 1/3 of initial volume and the reaction product was rinsed and dried.Yield: 11 mg, 73.3%.
Partially deuterated isotopomer of (3), owing to its low solubility in methanol, was prepared by dissolving 3 (10 mg) in solution of deuterated methanol and chloroform (v:v = 2:1, 2.0 ml) by adding a few drops of deuterium oxide.The solution was refluxed for 5 h and then evaporated under vacuo to 1/3 of the initial volume.Crystals were filtered and dried.Yield: 11 mg, 66.7 %.
To the best of our knowledge, the synthesis of compounds 2- 6 has not yet been reported in the literature.

IR Spectroscopy
IR spectra of compounds 1-3 and deuterated isotopomers 4-6 were recorded on Perkin-Elmer Spectrophotometer 502, with a resolution of 2 cm −1 and 4 scans per spectrum.Samples were prepared by KBr pellets.

Mass spectrometry
Mass spectra were recorded on a Waters Micromass Q-TOF micro instrument equipped with an electrospray ionization (ESI) source and operating in positive ion mode.The instrument parameters were as follows: capillary voltage 3.0 kV, sample cone voltage 18 V, extractor voltage 1 V, source temperature 100 °C, desolvation temperature 150 °C and desolvation gas flow 350 L/h.Samples were dissolved either in a 1:1 (v:v) mixture of acetonitrile and water or in methanol, to a concentration of about 10 µg/mL and introduced into the ESI source from a gastight 250 µL syringe driven by a syringe pump at a flowrate of 10 µL/min.

NMR spectroscopy
One-dimensional ( 1 H and APT) and two-dimensional (COSY, HSQC and HMBC) NMR spectra were recorded on a Bruker Avance 600 spectrometer equipped with an inverse probe and z -gradient accessories with constant magnetic field operating at 14.2 T. Samples were measured at 298 K in 5 mm tubes.CDCl 3 was used as solvent and TMS as internal standard.
Proton spectra with spectral width of 12019.For gHSQC and gHMBC spectra 2048 points in F2 dimension and 512 increments in F1 dimension were used.Spectral width was 12019 Hz in F2 dimension and 36216 Hz in F1 dimension.Relaxation delay was 1 s and number of scans was 128.Digital resolution was 5.87 and 141.48 Hz per point in F2 and F1 dimension, respectively.A delay for long-range coupling was set to 65 ms.

Structural Characterization
The structures of prepared compounds 1-3 were characterized by IR and NMR spectroscopies and MS spec-trometry.IR spectra revealed characteristic vibrational modes of phenylenediamines and diones.These are displayed in Table 1.
Owing to structural similarity, the IR spectra of 1-3 are very similar in the range 3500-1100 cm −1 .The C-H aromatic stretching vibration in 3 is partially overlapped by a broad N-H stretching vibration band and hence could not be unambiguously assigned in the spectrum.Differences in out of plane bending vibrations are due to different substitution at the benzene ring.The broadening of secondary amine N-H stretching vibration band at ≈ 3450 cm −1 pointed towards the presence of intramolecular hydrogen bonds.This is in agreement with the results obtained by X-ray diffraction analysis of 1. 5 The existence of C=O stretching band of conjugated ketone and lack of O-H stretching mode in 1-3 indicated that the predominant form in solid state is the keto-amine.
The ESI mass spectra of 1-3 showed the existence of precursor ion [M+H] + at m/z = 409 and the expected fragmentation pattern.The precursor ion signal at m/z = 411 in the mass spectra of deuterated compounds 1 (isotopomer 4) and 2 (isotopomer 5a) obtained in methanol indicated the exchange of both N-H groups with deuterium.Surprisingly, precursor ion peaks of deuterated compounds 5b and 6 prepared in a mixture of deuterated methanol and chloroform pointed towards multiple deuteration.This finding will be discussed later on.
The analysis of 1 H and 13 C NMR spectra confirmed the structures of 1-3 in solution and corroborated the presence of the keto-amine form (Table 2).The chemical shifts of C-2(2`) atoms assigned at ≈184.5 ppm corresponded to the keto group of dehydroacetic acid involved in the RAHB.The amino group has been found in proton spectrum at 16.1 ppm.Proton chemical shifts at ≈ 2.2 ppm and ≈ 2.6 ppm were observed for methyl protons H-6(6`) and H-7(7`), respectively.The COSY spectra of 1-3 exhibited correlation peaks corresponding to different substitutions at the benzene ring.Two correlated aromatic proton signals between H-11 (11`) and H-12(12`) with equal integrals revealed ortho-substitution in 1.The H-13(13`) proton singlet at 7.07ppm confirmed the meta-substitution in 2. The absence of correlation peaks between aromatic protons in COSY spectrum of 3 indicated the symmetric parasubstitution at the benzene ring.

Deuterium Isotope Effects
Deuterium isotope effects have found practical applications as a structural tool for analysis of hydrogen bonds and tautomeric equilibria in solution. 15,20In order to elucidate structure and dynamics of possible hydrogen bonds and influence of the non-polar solvent we prepared deuterated isotopomers of 1-3 in solution of deuterated methanol.According to NMR and MS spectra, deuteration of N-H group was successful for 1 and 2 giving isotopomers 4 and 5a (Figure 3).
An attempt to prepare deuterated compound 3 (isotopomer 6) in deuterated methanol was unsuccessful due to its very low solubility.Hence, isotopomer 6 was prepared in a mixture of deuterated methanol and chloroform (Figure 3).Deuteration of 2 was also performed under these conditions yielding isotopomer 5b (Figure 3). 1 H NMR and mass spectra of isotopomers 4 and 5a revealed that the deuteration occurred at both amino groups.Furthermore, in mass spectra of isotopomers 5b and 6 precursor ion peaks at m/z = 409.2,410.2, 411.1 and 412.2 were observed (Figure 4).These findings pointed towards the formation of multiple deuterated species.The percentage of particular species can be estimated from the peak intensities.Peaks at m/z 411.1 and 412.2 corresponded to three and four deuterium atoms in the molecule which could not be obtained only by deuteration of amino groups.This issue was resolved by a close inspection of 13 C NMR spectra.
Namely, the 13 C NMR spectra of deuterated compounds 5b and 6 prepared in a mixture of deuterated methanol and chloroform exhibited C-D triplets around 19.7 ppm indicating the presence of monodeuterated methyl groups in α-position to amino group (Figure 5).The 13 C NMR spectra recorded with more scans revealed quintets and septets corresponding to two and three deuteriums in the methyl groups, respectively.According to these findings, the following mechanism of deuteration shown in Figure 6 can be proposed.
The mechanism involves hyperconjugation of polar resonant form (c) and the deuteration of methyl groups.The resonant form (c) is stabilized by the partial double bond character of C-N bond, present both in amides and their higher homologues enaminones.The catalytic D + ions might come from the dissociation of CD 3 OD or as a consequence of the decomposition of   − which can be accelerated by heating. 28wing to the higher pK a value of the mixture of deuterated methanol and chloroform in comparison with the pure deuterated methanol, the deuteration of N-H groups is less possible and the main reaction products are 5b and 6.This is in accordance with much smaller deuterium isotope effects measured for these compounds in comparison with 4 and 5a as seen in Table 3.
Hence, the site and selectivity of deuteration depend on solvent properties and reaction conditions.Subsequently, DIEs were determined from mixtures of partially deuterated 4−6 and non-deuterated 1− 3 samples using the equation ( 1).The sign of DIE was determined by quantitative addition of the nondeuterated sample in the NMR tube of the deuterated sample, as demonstrated in Figure 7. DIEs are given in Table 3.
In   high values of DIEs through five bonds observed at C-3(3`) and C-4(4`) in 4 and 5a as well as that at C-2(2`) in 5a are in accordance with this.The values of DIEs at C-10(10`), C-11 (11`) and C-12(12`) are also a consequence of an efficient transfer of isotopic perturbation through conjugated system and hydrogen bonds.The difference in sign and magnitude of DIEs in 4 and 5a could be explained by different structure and dynamics of hydrogen bonds in these molecules.Due to the proximity of keto and amino groups, N-H•••O as well as N-H•••N intra-and inter-molecular hydrogen bonds between two dehydroacetic acid moieties are possible in 4 as also observed in solid state. 5On the other hand, a larger distance between these units in 5a prevents the formation of the corresponding intramolecular hydrogen bonding interactions.
The magnitude of DIEs in 5b and 6 prepared in solution of methanol and chloroform depends on the number of deuterium atoms in the methyl groups.Hence, in 13 C NMR spectra cumulative effects were observed. 22,29The highest value of DIE in both compounds was that observed at the site of deuteration, e.g. at C-7(7`).The triplets arising from a scalar coupling between methyl carbon and directly attached deuterium were observed in 13 C NMR spectra (Figure 5).High values of DIE (22.9 and 24.1 ppb) at atom C-8(8`) in 5b and 6 could be attributed to the proximity of deuterium and are intrinsic in nature.Negative DIEs were observed at carbonyl carbons C-2(2`) and C-5(5`) in 6 whereas no such effects were detected in 5b.It is not quite clear what the reason for this observation is but inter-molecular interactions with other molecules and influence of the solvent which can easily approach these C=O groups cannot be excluded. 30verall values of DIEs in 4-6 are the sum of equilibrium and intrinsic contributions.Generally, equilibrium DIEs have higher magnitude than intrinsic ones. 15,20,27Much larger values of DIEs in isotopomers 4 and 5a with respect to those observed in 5b and 6 can be rationalized by an efficient transmission pathway through intramolecular N−H(D)•••O hydrogen bonds.

Figure 4 .
Figure 4. Precursor ion peaks in the mass spectrum of 6.

Figure 5 .
Figure 5.The methyl region of 13 C NMR APT spectrum of 6.

4
and 5a generally high values of DIE were observed.The high positive values at C-8(8`) in 4 (+320.9ppb) and 5a (+250.0provided further evidence for the localized keto-amine form in solution stabilized by intra-molecular N-H•••O hydrogen bonds.Relatively

Figure 6 .Table 3 .
Figure 6.The mechanism of the methyl group deuteration at position 7.

Figure 7 .
Figure 7. Determination of a deuterium isotope effect at C-2(2`) in 5a.The narrow region APT spectrum of the partially deuterated 5a (a) before and (b) after addition of the nondeuterated isotopomer.
23 Hz, acquisition time of 1.36 s and digital resolution of 0.55 Hz per point were measured with 32 scans.A number of points in time domain was 32 K. APT spectra with spectral width of 35971.22Hz, acquisition time of 0.91 s and digital resolution of 0.55 Hz per point were measured with 51791 scans per spectrum.Number of points in time domain was 64 K. the gCOSY experiment, 2048 points in F2 dimension and 512 increments in F1 dimension were used.Spectra were measured with spectral width of 12019.23 Hz, 4 pulses per increment and relaxation delay of 1 s.Digital resolution was 5.87 Hz per point in F2 and 23.44 Hz per point in F1 dimension.