Synthesis and analytical characterization of monomeric N-oxidized derivatives of α-tocopheramine

α-Tocopheramine has shown great promises as a stabilizer for synthetic and natural polymers, but is also investigated in various biomedical scenarios. Many studies have been hampered by the fact that the oxidation products of α-tocopheramine have not yet been properly identified and their analytical data are still lacking. In the present study, we synthesized and fully analytically characterized all N-oxidation products that can form upon oxidation of α-tocopheramine in aqueous media, including the hydroxylamine, nitroso, and nitro derivative, in this way providing standards for the identification of the so far elusive byproducts. Synthesis and stability of the derivatives are discussed.


Introduction
As early as 1942, α-tocopheramine (1), the amino analogue of α-tocopherol (2), the main component in vitamin E, has been reported, which differs from its vitamin E parent compound in the presence of an amino function instead of the phenolic OH group [1,2]. Named "tocopheramines" or "aminotocopherols", these aza-derivatives behave similarly to the tocopherols with regard to antioxidant activity, although their general oxidation chemistry can be different. Like tocopherols, tocopheramines are non-toxic and biologically fully compatible [2,3]. They have been studied both with regard to their biological activities, their antioxidant behavior and their general chemistry, but a wide use has been hampered by their limited availability, even if the related activities exceeded those of the corresponding tocopherols in some cases [4,5]. While α-tocopherol is produced on a large industrial scale in well-optimized processes [6], α-tocopheramine still requires comparatively complex approaches which have not yet exceeded the pilot scale [7]. Tocopheramines have been evaluated as food [8] and feed additives [9], surfactants [10], and polymer stabilizers [11]. More recent research has shown amides derived from α-tocopheramine to possess promising anticancer and proapoptotic activities [12,13] which were even superior to the well-studied, structurally related ester α-tocopheryl succinate [14][15][16], and effectiveness in ophthalmological and reproduction studies [17,18]. Also, its activity in stabilizing cellulose solutions in N-methylmorpholine-N-oxide, as used on industrial scale to manufacture cellulosic manmade fibers in the lyocell process, was quite promising [19]. It was inert against the cellulose solvent and effectively counteracted both cellulose degradation and solvent decomposition [20]. It is, therefore, understandable that tocopheramines have become of greater interest in recent years from various points of view.
Oxidation of α-tocopheramine under aqueous conditions or in the presence of water affords α-tocopherylquinone (3) [21,22] as the main product (Scheme 1), but it also generates byproducts, the structure of which is so far not known. Many studies with tocopheramines are hampered by this byproduct formation. Oxidation of α-tocopherol in aqueous medium affords the same main reaction product, compound 3, but the side reactions and byproducts-mainly spiro-dimer 5 [23,24] formed via the transient 5a-ortho-quinone methide intermediate 4 [25,26] are-quite well known as they have been a matter of extensive studies (Scheme 1). By contrast, research on tocopheramines had focused on interaction of tocopheramines with different radicals so far [27], but the minor oxidation products of α-tocopheramine have not yet been identified and characterized; and the corresponding analytical information, in particular NMR and MS data, is nearly completely lacking. Apart from a description of a nitroxide derivative which afforded a well-resolved EPR spectrum [28], and a UV/Vis-active by-product showing red color [29][30][31], none of the byproducts´ structures has been proven and supported by detailed analytical data. However, the identification of the side products and their as standard compounds would be an important prerequisite to usage as polymer stabilizer, and obviously for any biomedical application. It is reasonable to hypothesized that the byproducts formed would be N-oxidized compounds in general, which can be both N-oxygenated, e.g. hydroxylamine, nitroso or nitro derivatives, and N-coupled compounds, e.g. hydrazo, azo or azoxy derivatives, but none of them has ever been directly synthesized or characterized.
In this study, we report the synthesis of all monomeric, i.e. not N-N coupled, N-oxidized derivatives of α-tocopheramine, in an attempt to provide comprehensive analytical data and sufficient amounts in the 100 mg/1 g scale so that they could serve as standards, supporting identification of byproducts in unknown reaction mixtures by compound comparison.

Results and discussion
Upon oxidation of α-tocopheramine (1) in aqueous medium, formation of quinone 3 is consistently accompanied by byproducts, albeit only in the range of less than 1.5%. This explains on the one hand that these byproducts have so far eluded structure elucidation and, on the other hand, made it clear that this reaction would be inappropriate to provide larger amounts of these target compounds. α-Tocopherol (2) Scheme 1 and α-tocopheramine (1) were considered as suitable starting materials for alternative syntheses, but considering that routes starting from the former would lead via the latter anyway left only the amine 1 as plausible option.
Organic methodology offers a whole wealth of procedures to oxidized aromatic amines to N-oxidized derivatives [32]. In the present case, the choice is more limited due to the labile, oxidant-sensitive nature of the substrate (tocopherol derivative!). Also, all methods which are not selective for one oxidation product, but would yield mixtures (mainly nitroso/nitro), were supposed to be excluded, as well as synthesis protocols operating in aqueous systems, which would yield quinone 3 as the major product. Another constraint was that the synthesis was supposed to follow green chemistry principles, which meant avoiding environmentally malign auxiliaries, solvents [33][34][35] and transition metal catalysts [36,37], if possible. This avoidance was also plausible from the perspective that the substances were to be used in biological systems. These limitations narrowed down the number of promising protocols for the selective conversion of an aromatic amine into either a nitroso-, N-hydroxy-or nitrophenol, from initially more than 40 procedures to rather few based on hydrogen peroxide, peracids and their salts, e.g. peracetic acid, peroxomonosulfate, peroxodisulfate. Organic peroxides, e.g. t-butylhydroperoxide, m-chloroperbenzoic acid, yielded byproducts in the range of 20-30%, which were N-O coupling products with tocopheramine as well as tocopheramine dimers according to MS data, but were not investigated further.
Preliminary screening for the best systems showed the couple of glacial acetic acid and hydrogen peroxide [38] to give promising results. The fact that the 13 C NMR carboxyl resonance in the spectrum of the mixture gave a rather broad signal was an indication that peroxoacetic acid is involved in an equilibrium with acetic acid and may be the active oxidant. This was followed by optimization of the reaction conditions. Apparently, formation of p-tocopherylquinone (3) was hard to suppress and occurred already if traces of water were present, coming, for instance, from the hydrogen peroxide solution used. Hygroscopic additives, such as H 2 SO 4 conc., were no remedy, but just increased the amount of (as yet unidentified) byproducts. In addition, it seemed hard to stop the oxidation at the nitroso stage and avoid "overoxidation" to the nitro compound, although lower temperatures significantly shifted the nitroso/nitro ratio in favor of the former.
The key was to conduct the reaction not with aqueous hydrogen peroxide, but to produce it in situ from suitable salts. Optimum results were provided with sodium percarbonate as H 2 O 2 precursor, which in combination with glacial acetic acid and released from the solid complex by added sulfuric acid reacted quantitatively to a 2:3 molar mixture of 6-desoxy-6-nitroso-α-tocopherol (6) and 6-desoxy-6-nitro-α-tocopherol (7) at room temperature, notably without p-tocopherylquinone (3) being present. Solvents often used in vitamin E chemistry were tested, and both n-hexane and 1,4-dioxane gave similarly good results. However, the latter solvent was considered clearly superior due to the potential neurotoxicity of the former. The amount of H 2 O 2 released can be exactly set by the amount of acid added and can be much better controlled than in the case of aqueous 30% H 2 O 2 . Concentrated H 2 SO 4 in 1,4-dioxane (1 M) gave the best results for that purpose. While sodium percarbonate is completely insoluble and can be added in excess, no free sulfuric acid is present in the system as it reacts immediately with the percarbonate releasing hydrogen peroxide. The presence of glacial acetic acid was imperative to obtain good yields: in its absence roughly 40% of byproducts were obtained, compared to near-quantitative product yields in its presence. This argued once more in favor of peroxoacetic acid as the actual oxidant which brings about the high selectivity, while hydrogen peroxide alone apparently was too harsh an oxidant [39].
Peroxomonosulfate and peroxodisulfate were too strong oxidants as well and gave only the nitro derivative in moderate yields, perborates gave yields below 45%, and the hydrogen peroxide urea complex caused extensive formation of unknown byproducts and chromophores. Another advantage of the percarbonate / glacial acetic acid / H 2 SO 4 / dioxane system was that simply by adjusting the temperature the nitroso / nitro selectivity could be completely tuned (Scheme 2). At −5 °C, only the nitroso derivative 6 was formed, even if the oxidant was present in moderate (threefold) molar excess. At 50 °C, only the nitro-compound 7 was obtained. Hence, no different setup for obtaining the nitroderivative other than simply working at higher temperature than for the nitroso compound was required [40]. Both reactions provided virtually quantitative yields so that subsequent chromatographic separation was unnecessary. Also the work-up is strikingly easy: it is just done by removal of solids by filtration and solvent evaporation, which is a Scheme 2 distinct plus if the standard compounds are to be synthesized in labs not fully equipped for synthetic organic chemistry operations.
The best access to the N-hydroxy-α-tocopheramine (8) compound seemed to be the reduction of either nitro or nitroso derivative, as oxidative reactions starting from the amine would always be threatened by overoxidation to the nitroso or nitro compounds. The reduction of nitro-derivative 7 by SmI 2 gave an excellent 88% yield with 12% of amine 1 as the side product. However, the use of the toxic transition metal catalyst did not seem optimal to us. The alternative, the conventional reduction method with Zn/aq. NH 4 Cl, provided initially fair yields of about 60% which were further improved to 74% of 8 by working in glacial acetic acid, although still being accompanied by significant amine formation (26% of 1). Exchanging Zn for Mg as the reductant, adding a drop of gaseous HCl in MeOH before the metal addition and working at -20 °C throughout, was the key in avoiding overreduction to the amine and getting quantitative yields of hydroxylamine. The practical advantage of the approach is that it can be conducted as one-pot procedure starting from amine 1 via nitro derivative 7 (Scheme 2). Table 1 provides the NMR data of the N-oxidized compounds 6-8 in comparison to the starting amine 1. In 1 H NMR the compounds can be distinguished by the effect of the 5-substituents of the protons of the aromatic methyl groups, which-by analogy to tocopherols-is most pronounced for C-5a, followed by C-7a and C-8b. In 13 C NMR, the nitroso derivative 6 is distinguished by a large C-6 downfield shift of more than 30 ppm, compared to 15 ppm for the nitro-derivative 7 and 11 ppm for the hydroxylamine 8. For the nitroso and nitro derivatives, the two orthopositions (C-5 and C-7) as well as the para-position (C-8a) are affected by downfield shifts, which are not seen for the hydroxylamino derivative, of which the shifts were closer to the starting amine 1.
All three derivatives were completely stable when stored under an inert gas in the dark; no chemical changes were detectable after storage over more than 8 months under these conditions. The same is true for degassed (oxygenfree) solutions in n-hexane or 1,4-dioxane. All three derivatives 6-8 are tolerant towards acids (1 M H 2 SO 4 , glacial acetic acid, phosphate buffer pH 1 in aqueous methanol), and the nitro-derivative 7 and hydroxylamine derivative 8 also towards bases and alkali (triethylamine, 1 M NaOH). The nitroso-derivative 6, when treated with alkali (1 M NaOH in aqueous methanol), provided a single compound which was identified as the spiro-dimer of α-tocopherol (5), which is usually formed by dimerization of the ortho-quinone methide 4 derived from α-tocopherol, a very common intermediate in vitamin E chemistry, see Scheme 1. It was logical to assume that this ortho-quinone methide intermediate would also be involved in the present case, albeit coming from a different precursor. When repeating the reaction with the 15 N-labeled model compound 6-( 15 N-amino)-2,2,5,7,8pentamethylchroman (1a) at r.t. in the NMR tube, an intermediate at − 42.6 ppm was observed, which changed into a stable shift at − 276.2 ppm. While the first shift is in the typical range of oximes, the second value originates from free hydroxylamine, which was unambiguously proven by spiking with the authentic compound. The nitroso-compound 6, upon alkali treatment, would thus undergo a tautomeric [1,5]-sigmatropic proton shift involving the C-5a methyl group, leading to a transient ortho-quinone methide oxime (9), in fact the oxime of ortho-quinone methide 4, see Scheme 3. The special activation of the C-5a methyl group and its preference over the alternative ortho-methyl group at C-7a is well known in vitamin E chemistry and has been accounted for by the theory of strain-induced bond localization (SIBL) [26,41]. The ortho-quinone methide involving C-5a experiences less strain than the alternative C-7a-orthoquinone methide and is just energetically favored. The same would be true also for their oximes; note that a distinction between E-or Z-configuration of the oxime intermediate cannot be made based on the present data. In the presence of water, the oxime releases hydroxylamine to form 4 which immediately dimerizes to the spiro-dimer 5 (see above). In α-tocopherol chemistry, this dimerization is an astonishingly clean process which gives the neat spirodimer in quantitative yield, and the same course was observed in the present case, making alkali treatment of the nitroso-compound 6 an elegant route to spiro-dimer 5 starting from tocopheramine material.
To provide additional structural information about the N-oxidized derivatives, the above syntheses were repeated with the model compound 1a carrying a methyl group instead of the isoprenoid side chain and an 15 N-isotopic label, which was available from previous work [42]. 1a is the amino analogue of the well-known truncated α-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol [43]. The N-oxidized derivatives (nitroso: 6a, nitro: 7a, hydroxylamino: 8a) showed characteristic 15 N NMR shifts, all in the expectable ranges, and thus confirmed the 1 H and 13 C NMR data, as shown in Scheme 4. Note the characteristic, large positive 15 N shift of the nitroso derivative. To rationalize reactivity and NMR shift effects of the nitrogen substituents it is helpful to think of nitroso, nitro, and hydroxylamino groups as hetero-analogues of carbonyl (C=O), carboxyl (COOH), and hydroxymethyl (CH 2 OH) moieties, respectively.

Conclusion
The optimized synthetic procedures provided access to the monomeric N-oxidized derivatives 6-8 of α-tocopheramine (1) in quantitative yields, without the need for chromatographic purification and avoiding solvents and auxiliaries that would contradict green chemistry principles. Compounds 6-8 can alternatively be perceived as the 6-desoxy-6-nitroso, 6-desoxy-6-nitro, and 6-desoxy-6-hydroxylamino derivatives, respectively, of α-tocopherol (2). With these compounds, standards are accessible that will allow easy and fast identification of the byproducts in oxidation reactions of α-tocopheramine, even if they are formed only in trace amounts. While the nitroso, nitro, and hydroxylamine derivatives are the dominant byproducts of oxidations carried out in aqueous media, such as under physiological conditions, some dimers of α-tocopheramine, probably of the hydrazo, azo or azoxy type, dominate under non-aqueous conditions, e.g. when tocopheramine is applied as a polymer (melt) stabilizer. The structural identification and synthesis of these dimeric N-oxidation products will be the topic of an upcoming account.

Experimental
All chemicals were commercial products, of the highest purity available and used without further purification. HPLC-grade solvents were used for all extractions and workup procedures. Bidistilled water was used for all aqueous extractions and for all aqueous solutions. 1,4-Dioxane, n-heptane, ethyl acetate, and toluene used in chromatography were distilled before use. α-Tocopheramine (1) was of the [R,R,R]-type, maintenance of stereochemical integrity over the reactions performed was not further checked, however.
All reactions involving non-aqueous conditions were conducted in oven-dried (140 °C, overnight) glassware under an argon atmosphere. TLC was performed using Merck silica get 60 F 254 pre-coated plates, and flash chromatography on Baker silica gel (40 µm particle size). All products were purified to homogeneity by TLC / GC analysis; yields refer to isolated, pure products with satisfying elemental analysis data (± 0.2). Elemental analyses were performed at the Microanalytical Laboratory of the University of Vienna. Melting points, determined on a Kofler-type micro hot stage with Reichert-Biovar microscope, are corrected. 1 H NMR spectra were recorded at 300.13 MHz for 1 H and at 75.47 MHz for 13 C NMR in CDCl 3 if not otherwise stated. Chemical shifts, relative to TMS as internal standard, are given in δ values, coupling constants in Hz. 13 C peaks were assigned by means of APT, HMQC, and HMBC spectra, "d.i." denoting peaks with double intensity.