4-(D-Glucosamino)-7-nitrobenzoxadiazole: synthesis, anomers, spectra, TLC behavior, and applications

A new synthesis of the known fluorescent compound 3 [4-(D-glucosamino)-7-nitro-2,1,3-benzoxadiazol-4-yl] is reported, starting from D -glucosamine and non-fluorescent 4-aryloxy-7-nitrobenzofurazans, 4a-e, 5 . The α and β anomers are easily interconverted but can be separated by TLC (R f β > R f α ). The non-fluorescent new congener 8 {2-[N-(2’,4’,6’-trinitrophenyl)- amino]-2-deoxy- D -glucose} and the related known compound 9 {2-[N-(2’,4’-dinitrophenyl)- amino]-2-deoxy- D -glucose} have anomers that may be seen in NMR spectra, but are too rapidly interconverted for TLC separation. The UV-Vis and fluorescence spectra of 3 depend markedly on solvent polarity. The TLC method allows the analytical determination of glucosamine from pharmaceutical preparations by conversion into 3 and detection by its fluorescence.


Synthesis of compounds 3, 8, and 9
Two synthetic variants, A and B, were adopted for the formation of 3 (Scheme 1).In variant A, the reaction of 1 (as hydrochloride) and 2 takes place in the presence of sodium hydrogen carbonate in methanol at 50ºC, but the difference from the literature 15,16 occurs in the isolation and purification via preparative TLC (yield 40%).In variant B (yield 60%), based upon the previous experience with amino acids, 37 the starting materials were 1 (as hydrochloride) and 4aryloxy-7-nitrobenzofurazans 4a-e or 5, [35][36][37] at 30ºC, also in the presence of NaHCO 3 .In both cases the reactions take place in an S N Ar process (Scheme 1) via a deeper-colored Meisenheimer complex.

NMR Spectra
The 1 H-NMR and 13 C-NMR spectra were recorded in DMSO-d 6 with trifluoroacetic acid (the latter allows a better resolution of hydroxyl protons).In the 1 H-NMR spectra of 3, characteristic peaks for the NBD moiety correspond to aromatic protons H-6' and H-5', and for the glucosamine moiety to protons bonded to carbons 2, 3, 4, 5, and 6.The α anomer is characterized by the H-1 doublet at 5.23 ppm and the β anomer by the H-1 doublet at 4.78 ppm, similarly to the reported values in deuterium oxide (for α, δ = 5.27 ppm, and for β, δ = 4.78 ppm). 15,16The relative amounts at equilibrium are α / β ≈ 1/1, similarly to literature data 15 (per cent ratio α / β = 42/58).In the 13 C-NMR spectra, characteristic peaks for the NBD moiety correspond to aromatic carbons (C-1'a, C-3'a, C-4',5',6',7') and for the glucosamine moiety to all six sp 3 -hybridized carbon atoms. 45n the 1 H-NMR spectra of 8, characteristic peaks for the NBD moiety correspond to aromatic protons H-3' and H-5', and for compound 9 to aromatic protons H-3',5', 6'.In both cases, the H-2,3,4,5,6 peaks are practically identical.For the α anomer of 8, the H-1 doublet appears at 5.13 ppm, and for the β anomer at 4.79 ppm.For the α anomer of 9, the H-1 doublet appears at 5.23 ppm, and for the β anomer at 4.63 ppm.Interestingly, the percent ratio for the two anomers differs markedly: α/β ≈ 95/5 for 8 and α/β ≈ 85/15 for 9.The decreasing α/β anomer ratio 8 > 9 > 3 can be explained by the global electronegativity of the aromatic group which decreases in the same order, and also by the decreasing possibility for an intramolecular hydrogen bond between the axial α-hydroxy group and the nitro-O atom or the heterocyclic N-atom of the aromatic moiety.

Conformational studies for anomers 3α, 3β, 8α, 8β, 9α and 9β
It is well known that the interconversion of glucose anomers (mutarotation) is a general acidbase-catalyzed reaction which requires the simultaneous presence of a base and an acid. 46,47With mineral acids or bases, the protic solvent provides the missing third partner.However, with phenols as acids and pyridines as bases, which do not undergo neutralization, it was proven that when both are present a marked acceleration occurs.An even higher acceleration occurs with 2pyridone derivatives because a bimolecular process replaces the termolecular encounter.
In our case the equilibration of the two anomers is facilitated by the presence of the relatively acidic NH group so that one has to assume that the α/β anomer ratio always corresponds to the equilibrium ratio.
In order to better understand these anomer equilibria, theoretical conformational calculations were undertaken.The molecular geometry was determined by using the Hyperchem program, 48 and the results of the optimization are presented in Fig. 1.The energies computed with programs WinMOPAC 7.21 49 and CODESSA 50 are displayed in Table 1.It can be seen that the two anomers of 3 have higher energy differences (∆E T = E T β -E T α) than the other compounds: ∆E T(3) = -0.98 kcal/mol, ∆E T(8) = -0.17kcal/mol, ∆E T(9) = -0.23 kcal/mol.This fact may provide an explanation for the fact that TLC is able to separate only the anomers of 3. According to the optimized geometry from Figure 1, the α anomer of 3 has an "open" structure favoring stronger interactions with a flat surface, whereas the β anomer has a "closed" structure resembling a sandwich with nearly parallel rings.On considering the possibility of an intramolecular hydrogen bond between the NH group and the glycosidic oxygen for compounds 3, 8, and 9 by means of programs Hyperchem 48 and ArgusLab, 51,52 it was found (Table 2) that the O•••N distance in the 3β anomer is the lowest (closest to 3 Å); 53 α anomers have larger distances.These results may throw light on the differences in retention times due to interactions with Si-OH groups of the silica gel stationary phase.

Thin-layer chromatographic data for anomers of 3
Room-temperature separations by TLC involving silica gel and a mixture of dichloromethane and methanol in three different ratios, presented as (a) -(c) in Table 3, indicated that compound 3 presented two spots (X and Y) irrespective of the synthesis method (A or B), whereas compounds 8 and 9 always gave rise to only one spot, with retention times R f 8 Table 3. TLC behavior (R f ) a,b of compounds 3, c 8, and By using a liquid-liquid partition (silica gel having a covalently-bonded hydrocarbon C 18 chain and aqueous ethanol at three concentrations) it was possible to determine both the TLC ARKAT USA, Inc.
On studying the partition in a solid-liquid system when the solvent is aqueous acetonitrile (17:3 v/v) as mentioned in the literature for 3, 15,16 a single spot was observed.When there is no water in the mobile phase for stationary phase I, and water for stationary phase II, as in Table 5, two spots can be observed.This is in agreement with the idea that the equilibration 3α 3β occurs rapidly in the aqueous mobile phase due to the general acid-base catalysis.
Since reaction rates are also strongly influenced by the temperature, TLC studies in ethyl acetate at various temperatures with two types of stationary phases (I and II) were carried out and the results are displayed in Table 5.][61] e Quantitative (densitometric) TLC analysis (λ max =341nm).
Densitometric TLC determinations show that on summing the chromatographic peak areas, the purity (> 99%) is confirmed.The data presented in Table 5 indicate that for the stationary phase I the spot with higher R f (X) corresponds to the β anomer, which prevails in the equilibrium mixture due to its lower steric hindrance and energy.Recently, literature HPLC data revealed a similar elution time order: t R β > t R α. 16 Our data presented in Table 5 show the influence of lower temperatures regarding the chromatographic resolution for the anomers α and β (the ∆R f value).
For the stationary phase II, due to interactions with the copper complex (known in the case of glucose), [62][63][64] the order of migration of the two anomers is not so clear; we assume that the order in which the two anomers migrate becomes reversed, and that the β/α ratio does not conserve the prevalence of the β anomer in the equilibrium mixture.
The behavior of 3 obtained by synthetic variant B is similar to that via variant A: the purity is again > 99% by densitometric analysis on summing the areas of the two peaks.

UV-Vis and fluorescence spectra of compound 3 (α + β)
Compound 3 in crystalline state and in solution is yellow-orange and strongly fluorescent.The longest-wavelength absorption band presents in various solvents a positive solvatochromy: λ max increases with increasing values of Reichardt's empirical solvent polariy parameter, 65 as one can observe from Table 6.
The fluorescence characteristics, namely λ ex , λ em , the quantum yield Ф, natural lifetime τ 0 and calculated lifetime τ, also depend on the solvent polarity: with increasing E T (30), Ф and τ (which includes parameter Ф) decrease.From Figure 2, one can see that in aqueous ethanol the fluorescence intensity decreases with decreasing ethanol concentration.Also, with decreasing ethanol concentration, one observes a decrease of the fluorescence parameters Ф, τ 0 and τ, in agreement with qualitative literature data about the fluorescence of 3 (α + β) in water. 16hese findings are useful for applications of 3 as a fluorescent probe for assaying glucosamine, because the adequate choice of solvent is critical for obtaining a satisfactory intensity of fluorescence.Table 6.UV-Vis spectral data and the fluorescence characteristics: λ ex , λ em , quantum yield (Ф), natural lifetime (τ 0 ) and calculated lifetime (τ) in various solvents for compound 3 Solvent (E T (30)) 65 λ max (nm) ε × 10 where: τ 0 is the lifetime, ν is the wavenumber of the maximum absorption band, n is the refractive index of the solvent (1.3595 for ethanol), I F is the fluorescence intensity and ε, the molar absorption coefficient.g τ = τ 0 × Ф.

Analysis of glucosamine in pharmaceutical preparations
As mentioned earlier, salts of D-glucosamine with hydrochloric or sulfuric acid are used therapeutically for treatment and functional maintenance of cartilage in joints, and under the form of nutritional supplements. 10,67Often they are associated with chondroitin sulfate. 67,68or analytical and bioanalytical qualitative or quantitative determinations of 3 (α + β) 8,15-33 one may use TLC as a simpler alternative to electrophoresis 34 or HPLC. 16The procedure involves reacting 5 to 10 mg of powdered pharmaceutical preparation either with 1 or with 4a, in conditions described in the Experimental Part, followed by TLC analysis for qualitative analysis (one or two spots depending on the adsorbant and solvent) or by quantitative densitometry when the detection involves UV-Vis or fluorescence spectroscopy.

Conclusions
NMR spectra of the fluorescent compound 3 (synthesized by a novel method using glucosamine 1 and 7-aryloxy-4-nitrobenzofurazans 4 or 5) provided evidence for the two α and β anomers.They could be visualized by TLC under special conditions of solid phase and solvent mixture.The less sterically hindered β anomer is present in slighly higher concentration in the equilibrium mixture.By contrast, the α and β anomers of analogous polynitrophenyl-substituted compounds 8 and 9 cannot be separated by TLC although their presence is proved by NMR spectra; in this case, probably owing to the higher electronegativity and better hydrogen-bond acceptor properties of ortho-situated nitro groups, the α anomer appears to predominate in the equilibrium mixture.The solvent polarity influences markedly the UV-Vis and fluorescence spectra of 3 (α + β).The TLC method may be applied for determining the presence of glucosamine in pharmaceutical preparations.

Experimental Section
General Procedures.Commercial products were employed: D-glucosamine hydrochloride 1 (Acros Organics), 2 (Aldrich), 4a-e and 5, [35][36][37] 6 (Merck), 7, 69 TLC analytical silica gel plates GF 254 , TLC preparative silica gel plates PLC-F 254 , TLC analytical silica gel plates RP-18F 254 and HPTLC analytical plates CHIR (Merck). 1 H-NMR and 13 C-NMR spectra were recorded with a Varian Gemini 300BB spectrometer (300MHz for 1 H and 75MHz for 13 C).We used Camag Software 1992 scanner II -Switzerland for densitometric TLC analysis.Temperatures were recorded with a digital termometer Diplex (-40ºC to 200ºC).The absorption spectra were recorded with Perkin Elmer Lambda 35 UV-vis spectrometer; conditions are specified in Table 6.For fluorescence spectra, a Perkin-Elmer 204 spectrofluorimeter was used; conditions are specified in Table 6.For fluorescence, an excitation lamp (Xe, 150 W) interfaced with the computer was used, allowing a pre-established data reading time of 0.5 s; IR spectra were recorded with FTIR spectrophotometer Bruker-Model Vertex 70, using ATR techniques.Melting points have been recorded in open capillary with Electrothermal IA 9000 Series of digital melting point instruments.

Synthesis of compounds 3 (α + β), 8(α + β) and 9(α + β). General procedure
Variant A was applied for obtaining compounds 3, 8, and 9. Starting from D-gluocosamine 1 (as hydrochloride) and halogen derivatives 2, 6, 7 (molar ratio 1:1) in methanol (5 mL for one gram of reactant mixture) and sodium hydrogen carbonate (2.5 moles for each mole of reactant 1) the reaction mixture was stirred at 50ºC (for 3 during 1 h, for 8 during 24 h, and for 9 during 7 days).The reaction mixture was filtered and the filtrate was evaporated under vacuum.The residue was purified by preparative TLC using preparative silica gel TLC plates PLC-F 254 (Merck): for 3, the mobile phase was dichloromethane: methanol 8.5:1.5, v/v; for 8 and 9, dichloromethane: methanol 9:1, v/v.Fluorescence detection was used at 360 nm for 3, and at 254 nm for 8 and 9.The area of maximum concentration was retained and extracted in a Soxhlet with dichloromethane:methanol (9:1, v/v), followed by evaporation under vacuum.With TLC using silica gel GF 254 (Merck) analytical plates, methylene chloride:methanol 8.5:1.5 v/v, two spots were detected by densitometric analysis.Variant B was used only for 3 (α + β) starting from D-glucosamine 1 (as hydrochloride) and 4a (molar ratio 1:1.2) in the presence of NaHCO 3 in methanol (5 g for one gram of reactant mixture) at 30˚C for 24 h.The reaction mixture was then worked up as indicated above, and the product 3 was indistinguishable from that obtained by variant A. With compounds 4b -4e or 5, similar results were obtained.

Determination of D-glucosamine (1) from pharmaceutical preparations
Finely ground (5 to 10 mg) preparation was suspended in 2 mL of methanol and stirred at 50˚C for 10 min.The filtered solution was treated with a slight excess of NaHCO 3 for neutralizing the acids accompanying the D-glucosamine and/or chondroitin, stirred for 15 min.then treated with two molar equivalents of 2 or 4a and stirred at 50˚C for another 10 min.(when using 2) or 30 min.(when using 4a).One more mL of methanol was added to the reaction mixture.Then 5 µL of this solution was subjected to TLC in one of the following alternatives: (i) analytical silica gel plates RP-18F 254 (Merck), mobile phase ethanol:water (6:3, v/v), producing one spot; (ii) HPTLC analytical plates CHIR (Merck), mobile phase H 2 O:EtOH (9:1, v/v), producing two spots; (iii) analytical silica gel plates GF 254 (Merck), mobile phase dichloromethane:methanol (8.5:1.5,v/v),producing two spots.The detection was achieved either by UV-Vis at 254 nm, or by fluorescence at 360 nm.

Figure 1 .
Figure 1.Optimized structures (with the MM+ force field from the Hyperchem 48 program) for α and β anomers corresponding to compounds 3, 8 and 9.
= molecular hydrophobicity, where R M0 is the R M value of the organic component extrapolated to zero concentration in the organic:water mixture; b = the change in the R M value caused by increasing the concentration (K) of the organic component in the mobile phase; R = the correlation coefficient for parameters R M0 and b described by ecuation R M = R M0 + bK [where R M = log(1/(R f -1)]

Table 2 .
Distance between the nitrogen atom and the glycosidic oxygen computed by means of the Hyperchem program