Chemistry and Chemical Technologies/4. Chemical and Pharmaceutical Industry

Dr. Belakhov V.¹, Dr. Botoshansky M.¹, Dr. Goldstein D.², Dr.Sc., Prof. Ionin B.I.³

Towards application of novel stabilized derivatives of vitamin C in cosmetics: synthesis, crystal structure and biological evaluation

of 2-acyl(aryl)-3-phosphoryl derivatives of ascorbic acid

¹Schulich Faculty of Chemistry, Technion – Israel Institute of Technology
Technion City, Haifa 32000, Israel; ²Tagra Biotechnologies Ltd., P.O. Box 8213, 8 Hamlacha Str., Netanya 42293, Israel; ³Department of Organic Chemistry, Saint-Petersburg State Technological Institute 26 Moscowsky Str., Saint-Petersburg, 190013, Russia

Ascorbic acid (AsA, vitamin C) is a vital nutrient for human and has many important functions in its organism. AsA is essential for collagen synthesis and helps maintain the integrity of substances of mesenchymal origin, such as connective tissue, osteoid tissue, and dentin [1, 2]. AsA has been used in recent years as an active ingredient of cosmetics [3–5]. Due to its antioxidant properties, it is considered to confer both antioxidant and photoprotection to skin against free radical attack and UV ray damage [6]. The formulation of pure AsA into a final product, however, presents serious difficulties because it is easily oxidized. In recent years, in order to overcome the problem of the lack of stability of ascorbic acid in its pure form, various semi-synthetic stable derivatives of AsA were prepared by several research groups [7–11].

In this report we presented synthesis and biological evaluation of novel 2-acyl dialkyl(aryl)-3-phosphoryl derivatives 1 of AsA, crystal structure of one of the intermediate compound 2, and biological evaluation of synthesized derivatives 1.

Experimental Chemical Part

¹H NMR spectra were recorded on a Bruker Avance^TM 500 spectrometer, and chemical shifts reported (in ppm) are relative to internal Me₄Si (d =0.0) with CDCl₃ as the solvent, and to HOD (d =4.63) with D₂O as the solvent. ¹³C NMR spectra were recorded on a Bruker Avance^TM 500 spectrometer at 125.8 MHz, and the chemical shifts reported (in ppm) relative to the residual solvent signal for CDCl₃ (d =77.00), or to external sodium 2,2-dimethyl-2-silapentane sulfonate (d =0.0) for D₂O as the solvent. Mass spectra analysis were obtained either on a Bruker Daltonix Apex 3 mass spectrometer under electron spray ionization (ESI), or by a TSQ-70B mass spectrometer (Finnigan Mat). Reactions were monitored by TLC on Silica Gel 60 F₂₅₄ (0.25 mm, Merck), and spots were visualized by charring with a yellow solution containing (NH₄)Mo₇O₂₄^.4H₂O (120 g) and (NH₄)₂Ce(NO₃)₆ (5 g) in 10% H₂SO₄ (800 mL). Flash column chromatography was performed on Silica Gel 60 (70-230 mesh). All reactions were carried out under an argon atmosphere with anhydrous solvents, unless otherwise noted. All chemicals unless otherwise stated, were obtained from commercial sources.

Experimental Biological Part

In order to evaluate the effect of prepared derivatives of AsA 1 on collagen synthesis, cultured human foreskin fibroblasts were placed in 24-well microculture plates in DMEM supplemented with 10% fetal calf serum containing 100 mg/ml b-aminopropionitrile, 10 mCi [2,3-³H]proline, in the presence of either AsA (positive control) or the prepared derivatives of AsA in various concentrations, e.g. from 1mM to 50 mM. The cultures were incubated for 24 hours. The [2,3-³H]proline incorporation into pepsine-resistant salt precipitated extracellular collagen was determined and used as an index of efficiency of the collagen synthesis.

Experimental Crystallographic Part

2-Capryloyl-5,6-O-isopropylidene-L-ascorbic acid (2) was recrystallized from ethyl acetate-hexane (1:1). Intensity data from crystals of compound (2) were collected at 293(2) K on a Nonius KappaCCD diffractometer. Data collection was made by application of the Collect program Nonius-2006 [12]. Data reduction and space group determination were performed using the DENZO HKL-2000 program [13]. The SHELXS-97 program was used for crystal structure solution by application of direct methods [14]. The SHELXL-97 program was used for refinement by full-matrix least squares [15]. The molecular graphics was performed using TEXSAN program (TEXSAN.

Molecular Structure Corporation, 1999).

Results and Discussion

The preparation of title compounds 1 was accomplished in four chemical steps. At the first step, at 0°C 5,6-isopropylidene derivative of AsA was obtained in almost quantitative yield by the treatment with dry acetone excess in the presence of CuSO₄. Regioselective acylation at position 2 of this compound was carried out by using corresponding acyl chlorides in anhydrous pyridine in the presence of catalyst (4-dimethylaminopyridine) at 0°C. Phosphorylation of protected intermediate with dialkyl(aryl)phosphochloridates in anhydrous pyridine at –5°C afforded derivatives of AsA with phosphate group at the position 3. At the last step, deprotection of 5,6-isopropylidene-3-dialkyl(aryl) phosphoryl derivatives of AsA was carried out in mild conditions with dilute HCl at 0°C to furnish the final products 1 with excellent purity and high yields [16].

R = Me, Et, Ph; R¹ = (CH₂)_nMe [n = 6, 14]

Biological tests indicated that studied derivatives of AsA 1 indicated higher level of activity by stimulation of the collagen synthesis in human foreskin fibroblasts in comparison with AsA.

During the last two decades an interest in crystal structures of derivatives of L-ascorbic acid has greatly increased, and interestingly according to Cambridge Structural Database [17] out of 38 known structures more than 60% were established starting 1999. This can be explained by the fact that application of L-ascorbic acid and its derivatives in medicine [1, 2, 6], nutritional industry [18-20] and cosmetics [21-23] has significantly increased. In recent years various research groups have showed that L-ascorbic acid and its derivatives can be considered as potential anticancer [24-27], antitumor [28, 29], antiviral [30-32] and anti-inflammatory agents [33].

Structure of the compound 2, obtained by means of Õ-ray analysis contains two independent very similar molecules in an asymmetric unit of space group P2₁. In one of the molecules capryolyl substituent at the C-2 position is partly disordered. The main difference between the molecules was found in relative conformation of 1,3-dioxalane rings. At the first and the second molecules these rings have inverted boat conformation. As an evidence of this fact, a deviation from the planarity and torsion angles have close absolute values, but with opposite sings. It was found also that dihydrofuranone rings are strictly planar, and in addition a deviation from the mean plane has not increased over 0.0047 Å in both molecules. Relatively strong intermolecular hydrogen bonds interaction includes a hydroxyl group at the position of C(3) and oxygen atoms in the position of C(1) of neighbored furanone rings. Thus, H…O distances equaled 1.835 Å and 1.853 Å for the first molecule and second molecule respectively. The corresponding O^_H…O angles equaled 166.90° and 155.22°, while the O…O distances are 2.640 Å and 2.620 Å for the first molecule and second molecule respectively. The resulting structure may be presented as an infinite chain.

	Crystal structure of compound 2

Conclusions

1. The novel stabilized derivatives of AsA (1) were successfully synthesized with high yields and perfect purity.

2. Biological studies showed that synthesized derivatives of AsA (1) active participated in collagen synthesis of human foreskin fibroblasts.

3. These novel stabilized derivatives of vitamin C can be used for production of various cosmetic products.

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