WO2008136037A2

WO2008136037A2 - Chemical-catalytic method for the peracylation of oleuropein and its products of hydrolysis

Info

Publication number: WO2008136037A2
Application number: PCT/IT2008/000303
Authority: WO
Inventors: Antonio Procopio; Giovanni Sindona; Nicola Costa; Marco Gaspari; Monica Nardi
Original assignee: Università Degli Studi Magna Graecia Di Catanzaro; Universita' Della Calabria
Priority date: 2007-05-04
Filing date: 2008-05-05
Publication date: 2008-11-13
Also published as: EP2235032A2; WO2008136037A3

Abstract

The method, object of the present invention, concerns the peracylation of oleuropein and its products of hydrolysis: The method makes use of the excellent properties as Lewis acid catalysts of halides and tryphilates of lanthanides (III). The component is placed to react, in the presence of catalytic quantities of Lewis acid, directly with an acylating agent containing at least one acylic group R, where R is H, an alkylic radical of 1-31 atoms of linear or branched carbon, an alkenylic radical containing up to 31 atoms of carbon or an arylic group. The procedures for the extraction and the successive hydrolysis of the oleuropein for the synthesis of its aglycon and the hydroxytyrosol, resolve the problems tied to the quantitative yield of the products and to the use of highly-toxic and expensive catalysts. Furthermore, the innovative and inventive contribution is given by the peracylation of the oleuropein and its products of synthesis, aglycon and hydroxytyrosol, that supply a new class of molecule, biologically active as anti-oxidants and anti-inflammatory ones. The proven anti-oxidant activity of oleuropein and its derivates leads to the hypothesis that they could also act as protectors against oxidative stress at the level of the central nervous system, one of the causal factors of Parkinson's disease. The molecules examined are all good protectors against oxidative stress and the greater efficiency of the peracylated derivatives is presumably due to their greater lipophilicity and the possibility of penetrating the cellular membrane.

Description

Chemical-catalytic method for the peracylation of oleuropein and its

products of hydrolysis Technical field of the invention

The method, object of the present invention, regards a method for the chemical manipulation of oleuropein and its synthetic products using Lewis acidic catalysts with a low environmental impact, in order to obtain a new class of molecules that are biologically active, such as anti-oxidants and anti-inflammatory ones. State of the art

An ever increasing interest for phenolic compounds is due to their anti-oxidant properties and their many beneficial effects on human health. The phenolic compounds of the olive are distributed in all the parts of the plant, but their nature and concentration vary greatly among the various tissues.

In Olea Enropaea we find hydroxytyrosol and oleuropein (Fig. 1) that represent the predominant phenolic compound and which can reach concentrations of 140 mg/g in dried green olives and 60-90 mg/g in the dried leaves; studies in vitro have demonstrated that oleuropein and hydroxytyrosol carry out an anti-tumour activity.

Notwithstanding this, it is rare to find oleuropein in extra-virgin olive oil and many authors have suggested that this is due to the various decays to which the same oleuropein is subject during the working of the olives, when, an endogenous β- glucosidase selectively hydrolyses the glycosidic bond of the oleuropein transforming it into its aglycon (Fig.2), a complex mixture of tautomers with multiple biological activities.

These compounds protect against the formation of artereo-sclerotic lesions; furthermore in vitro studies have demonstrated that these derivatives carry out various biological activities (anti-microbe, in anti-platelet drugs, with vasodilators, with hypertension, with hyperglycaemia, etc.) that very often are connected to their activities as scavengers of free radicals.

To form its aglycon the selective hydrolysis of the glycosidic bond of oleuropein is a process that occurs naturally in the olive drupes, caused by the endogenous β- glucosidase.

It is difficult, however, to reproduce said selective hydrolysis synthetically, due to the presence, on the molecule of oleuropein, of multiple acidic functions or faint traces of such, other than the glycoside bond. On the other hand, methods of controlled chemical manipulation capable of supplying its derivatives do not exist.

In this sector diverse techniques of hydrolysis are well-known, such as:

• Acid hydrolysis: The oleuropein (0.5g) is dissolved in 100 ml Of H₂SO₄ IN and heated to 100°C for 1 hour. The mixture of reaction is therefore cooled, brought to pH 2.0 and extracted with ethyl acetate.^" The basic product thus obtained, purified by chromatography gave hydroxytyrosol (65 mg), elenoic acid (15 mg) and the aglycon of the oleuropein (14 mg) in very low yields.

• Alkaline hydrolysis: The oleuropein (Ig) is dissolved in 50 ml of methanol. The stirred solution is cooled to 10°C and treated with approximately 2g of potassium hydroxide in tablets. The mixture of reaction is allowed to warm to room temperature and after about 6 hours, the same mixture is diluted with approximately 60 ml of HCl 6N (pH of about 7.5) and then dried by evaporation. The chromatographic purification of the mixture of products thus obtained gave the oleoside as main product.

These techniques present, however, some inconveniences or problems linked to the scarce quantitative yields of aglycon of the oleuropein, and to the used catalyst that result toxic and non-recyclable.

Some solutions have been proposed to partially avoid the above-mentioned drawbacks, the object of the Patent request US6117844 having as its basic element the following technique:

• Enzymatic hydrolysis with glycosidase: the oleuropein (5g) is dissolved in 500 ml^bf buffer water at pH 5.0 and treated with glycosidase until the mixture of reaction monitored by t.l.c. indicates the complete disappearance of the oleuropein. After the classic treatment of the mixture of reaction, the chromatographic purification gives the various products of the hydrolysis of the oleuropein.

Neither the state of the technique in use at present nor the existing patented solutions overcome the above-cited critical aspects, however. Presentation of the invention

The present invention proposes to overcome the difficulties and the disadvantages present in the solutions at use at present.

In particular, the discovery object of the present application, can be considered a technique that overcomes and resolves the problems tied to the quantitative yield of the products and to the use of non-recyclable catalysts, that are highly toxic and expensive.

The principal aim of the invention is to create a chemical-catalytic method for the peracylation of oleuropein and of its synthetic products, characterised by the fact that it comprises the following phases:

• oleuropein or one of its products from hydrolisis is placed to react in the presence of Lewis acid catalysts directly with acylating agents that contain at least one acylic group R, where R is H, an alkenilic radical of 1-31 linear or branched atoms of carbon, an alkenilic radical containing up to 31 atoms of carbon or an aryl group;

• the reaction is treated by adding a volume of hydrolysis agents of the acylating type (alcohol, water, etc.) that is agitated, at the end of which the solvent is dried under reduced pressure, leaving a residue.

The residue is revived in an organic solvent and extracted at least twice with water; the organic phases collected are dried on anhydrous Na2SO4 and evaporated; the basic product obtained is purified by flash chromatography on a column of silica gel.

Another characteristic is given by the fact that the Lewis acid catalysts are halides and triflates of lanthanides (III).

Another characteristic is given by the fact that the residue is revived in an organic solvent and is extracted three times with water.

Another characteristic is given by the fact that the oleuropein is manipulated to synthesize its aglycon and the hydroxy-tirosol.

Another characteristic is given by the fact that the manipulation of the oleuropein comprises the following phases:

• hi order to chemically degrade it in a controlled manner, the oleuropein is made to react in a reflux aqueous organic solvent in the presence of triflates or halides of lanthanides (III) as Lewis acid catalysts.

• the reaction is terminated by adding a few ml of H2O and the product is extracted three times in CH2CI2;

• The united organic phases are dried on anhydrous Na2SO4 and evaporated; Another characteristic is given by the fact that the Lewis acid catalysts are halides and triflates (trifluoromethanesulfonates) of lanthanides (III).

Above all, the innovative and inventive contribution is given by the peracylation of the oleuropein and its synthetic products, the aglycon and the hydroxytyrosol, that provide a new class of molecule that are biologically active as anti-oxidants and antiinflammatory.

After having extracted the oleuropein from the leaves of the olive the procedure, object of patent application, consists of a first phase characterised by the hydrolysis of the oleuropein finalised by the synthesis of its aglycon and/or the hydroxytyrosol.

The characterisation of the aglycon demonstrates that what is chromatographically separable as a single fraction is, actually, a mixture of at least three tautomeric forms (compounds 4-6, Figure 3), plus a hydrated form (compound 8, Figure 3) and a methanolated form (compound 7, Figure 3), as has been ascertained by NMR structural analysis and by mass spectrometry.

The components of the mixture, visible by HPLC analysis as five distinct peaks are due to, on the one hand, the tautomeric equilibrium of a dialdehydic form (compound 5, Figure 3), capable of rapidly inter-converting itself into its enolic form (compound 4, Figure 3) and into the closed form (compound 6, Figure 3), and on the other hand from the nucleophilic attack from the water (present in the environment of the reaction) and from methanol (present in the eluent phase) on the aldehyde carbon, to give the hydrated form (compound 7, Figure 3) and the methanolated form (compound 8, Figure

3).

The ¹H-NMR analysis of the mixture demonstrates that the tautomeric form present in greatest quantity at equilibrium is the dialdehydic form, as seen from the doublet at 9.56 and 9.53 ppm, characteristic of the two aldehydic protons, while the peaks which are characteristic of the two remaining tautomers can also be seen in lesser quantities.

From the LC/MS-ESI analysis can be noted, finally, the presence of three significant peaks of three different m/z values, that are attributable to the proton forms of the tautomers (all the tautomers have the same mass and therefore contribute to the same peak), of the hydrated molecule and the methanolated molecule.

The hydroxytyrosol, however, is obtained by known techniques. It will later be used as the crude base (with a yield of 90%) for the acetylation, while only a small part is cromato graphically purified (eluent mixture CH₂Cl₂/Me0H 9.5/0.5 v/v).

After obtaining the oleuropein, its aglycon and the hydroxytyrosol, the procedure consists of a second phase, characterised by the peracylation of the above-mentioned components to obtain, respectively, the oleuropein acylate (Figure 6 shows as an example, the acetylisation) and a new class of molecules that are biologically active as anti-oxidants or anti-inflammatory ones (Figure 5).

In particular, for the peracylation of the above-mentioned components, the synthetic strategy takes profit from the excellent properties of the halides and triflates of lanthanides (IΙI)cas Lewis acid catalysts. The component is placed to react, in the presence of catalytic quantities of Lewis acid, directly with an acylating agent containing at least one acylic group R, where R is H, an alkylic radical of 1-31 atoms of linear or branched carbon, an alkenylic radical containing up to 31 atoms of carbon or an arylic group.

In the mixture obtained from the peracylation of the aglycon at least 5 compounds that are diversely acylated are present (componds 6 a-e, Figure 5); the peracetylated compounds 6d and 6e with the highest molecular weight deriving, one from a methanolated form and the other from a hydrated form of the initial aglycon, are the primary products of the reaction; the diacetylated compound 6a is present only in small quantities in the mixture; the acetylisation effectively freezes the composition of equilibrium of the aglycon at the beginning of the reaction and the products of the acetylisation do not interconvert with each other during the course of the reaction itself. The proven anti-oxidant activity of oleuropein and its derivatives has led to the hypothesis that they could also act as a protection against oxidative stress at the level of the central nervous system, one of the causal factors of Parkinson's disease. The acetylated derivatives of the oleuropein, the aglycon and the hydroxytyrosol are more efficient with respect to the non-acetylated molecules, with a particular relevancy for the acetylated aglycon, which has a protection factor that is higher than 80% at the concentration of only lμg/μL; at the concentration of 10 μg/μL all the acetylated molecules have a total protection factor while the aglycon and hydroxytyrosol demonstrate a survival percentage greater than, or equal to, 80%. It can be deduced that the molecules examined are all good protectors against oxidative stress and the greater efficiency of the peracetylated derivatives is presumably due to their greater lipophilicity and the possibility to penetrate the cellular membrane. The use of Lewis acid catalysis in the hydrolysis of the oleuropein for the synthesis of its aglycon and of the hydroxytyrosol presents numerous advantages as given here following:

• Synthetic: the derivation of the oleuropein makes use of innovative and renewable methods such as "green" processes, to save or eliminate organic solvents at the stage of synthesis and in the recovery of the catalysts, which are also of low-toxicity, at the treatment stage. The catalysts used are recyclable, of low-toxicity, economic, and are used precisely in catalytic quantities, presenting, therefore, an unquestionable advantage with regard to sulphuric acid or alkaline hydroxides used in high concentrations to obtain the aglyconic product in very low yield.

• quantitative: the Lewis acid catalysts proposed cause the selective hydrolysis of the glycosidic bond present in the molecule of the oleuropein thus permitting almost quantitative yields of the product;

• Environmental: the olive leaves, which until now represented waste in the olive- culture process of extraction of extra-virgin olive oil, become a source of primary material.

• Pharmaceutical: oleuropein and its derivatives, already preliminarily tested in vivo in laboratories as protectors against oxidative stress induced by Paraquat, will be subjected to biological tests in vitro and in vivo to demonstrate their activity as non-selective inhibitors of cyclo-oxygenase enzymes COX-I e COX- 2 and as the antiviral and antitumour entities.

In Figure 1 the Olea Europaea molecule is shown, and in which we find the oleuropein that represents the predominant phenolic compound and the hydroxytyrosol.

In Figure 2 the oleuropein is shown after having undergone, when an endogenous β- glucosidase that selectively hydrolyses the glicosidic bond of the oleuropein itself, the transformation of its aglycon, a complex mixture of tautomers with multiple biological activities.

In Figure 3 the procedure of synthesis of the oleuropein to obtain its aglycon as a mixture of three tautomeric forms, a hydrated form and a methanolated form is shown. In Figure 4 the hydroxytyrosol is shown, and which represents the hydroxy-phenolic part of the oleuropein.

In Figure 5 the procedure of synthesis of the oleuropein to obtain the hydroxytyrosbl and the successive acetylisation is shown.

In Figure 6 the acetylisation of the aglycon and the obtaining of diverse contents is shown.

The discovery, it must be noted, is not limited to the description given, but may be perfected and modified by those skilled in the art, without, however, departing from the limits of patent.

The present invention permits numerous advantages and to overcome difficulties that cannot be overcome with the systems on sale at present.

METHOD OF USE OF THE DISCOVERY

The discovery is illustrated in the following example that in no way limits the aim of the present invention.

EXAMPLE l

Acylation of oleuropein

500 mg of oleuropein (28.73 mmol) are placed in a 100 ml round bottomed flask with 10.0 ml of acetic anhydride and 3.0 mol % of catalyst. Then, it is magnetically stirred at room temperature.

After 2 hours 20.0 millilitres of methanol are added, and the solution is left to cool. All the solvent evaporates, and the residue, revived with dichloromethane is dried on sodium sulphate. The crude product, obtained after filtering and the evaporation of the solvent, is purified on a chromatographic column (eluent mixture dichloromethane/methanol = 9.50/0.50), obtaining a yield of 67%.

Claims

1. Chemical-catalytic method for the peracylation of oleuropein and its products of hydrolysis, characterised by the fact that it comprises the following phases:

• oleuropein or one of its hydrolysis products is placed to react in the presence of

Lewis acid catalyst directly with acylating agents containing at least one acylic group R, where R is H, an alkylic radical of 1-31 atoms of linear or branched carbon, an alkenylic radical containing up to 31 atoms of carbon or an arylic group;

• the reaction is treated with the addition of a volume of hydrolysing agents of the acylant type (alcohol, water, etc.) and is stirred, at the end of which the solvent is dried under reduced pressure, leaving a residue;

• The residue is revived in an organic solvent and extracted with water at least twice; the organic phases gathered are dried on anhydrous Na2SO4 and evaporated; the raw product obtained is purified by flash chromatography on a column of silica gel.

2. Chemical-catalytic method for the peracylation of oleuropein and of its hydrolysis products according to claim 1, characterised by the fact that the Lewis acid catalysts are halides and triflates of lanthanides (III).

3. Chemical-catalytic method for the peracylation of oleuropein and of its products of synthesis, according to claims 1 or 2, characterised by the fact that the residue is revived in an organic solvent and extracted three times with water.

4. Chemical-catalytic method for the peracylation of oleuropein and of its products of synthesis, according to any of the preceding claims, characterised by the fact that the oleuropein is manipulated to syήthesise its aglycoh arid the hydroxy-tirosol.

5. Chemical-catalytic method for the peracylation of oleuropein and of its products of synthesis, according to the preceding claim, characterised by the fact that the manipulation of the oleuropein comprises the following phases:

• in order to chemically degrade in a controlled manner, the oleuropein is made to react in a reflux aqueous organic solvent, in the presence of Lewis acid catalysts of triflates or halides of lanthanides (III).

• the reaction is terminated by adding several ml of H2O and the product is extracted three times in CH2CI2 ;

• the united organic phases are dried on anhydrous Na2SO4 and evaporated;

6. Chemical-catalytic method for the peracylation of oleuropein and of its products of synthesis, according to the preceding claim, characterised by the fact that the Lewis acid catalysts are halides and triflates of lanthanides (III).