Many of them possess important biological activities, such as hepatoprotective, anticholestero- lemic, antineoplastic, antiinflammatory, antiinfluenza, antiulcer, vasoprotective. Some flavonoids exploit inhibiting activity against tyrosine kinase and phosphatidyl-inositol kinase. Moreover, their antioxidant properties against peroxide radicals are comparable, if not greater, than conventional phenolic antioxidants.

Many of these chemical and biological properties are strictly correlated to the position of hydroxyl groups on the flavane skeleton.

Flavane Due to their polyhydroxylated nature flavonoids are insoluble in lipophilic media (oils and emulsions) and weakly bioavailable, so that their employment is limited.

It is necessary to develop a synthetic process to obtain selective esterification of hydroxyl group at the C-3 position, without involving the other phenolic groups in the molecule.

Regioeselective alcoholysis of peracetylated flavonoids have been reported by Natoli et al. (Natoli, M; Nicolosi, G.; Piattelli, M, J. Org. Chem. 1992,57, 5776-5778). The process described make use of Pseudomonas

cepacia lipase, which catalysed the alcoholysis of the acetoxyl group in different position, but not at C-3; nevertheless the 3-acetoxyl derivative is recovered in very low yield.

Lambusta e al., Synthesis 1993,1155-1158 reported the preparation of (+)-3-0-acetylcatechin by alcoholysis of peracetylated (+)-catechin in the presence of Pseudomonas cepacia lipase, but the reaction conditions adopted are detrimental to the enzyme and consequently its fast inactivation is observed.

EP 0618203 reports catechins acylated at position C-3, prepared by esterifications of free catechin catalysed by Streptomyces rachei o Aspergillus niger carboxylesterase. Using this process authors enables for esters with acetyl, propyl and butyryl groups.

Summary of the Invention It has now been found that it is possible to obtain 3-monoesters of flavonoid as the only reaction product by carrying out the alcoholysis of a peracylated flavonoid in organic solvent in the presence of Mucor miehei lipase.

Therefore, it is an an object of the present invention an efficient method of producing 3-monoesters of flavonoids, comprising: a) chemical esterification of the flavonoid with an aliphatic acyl group having from 1 to 18 carbon atoms, to the corresponding peracylated flavonoid, or alternatively partially acylated flavonoid; b) alcoholysis of the above ester with an aliphatic alcohol having from 1 to 8 carbon atoms or with a polyol in the presence of Mucor miehei lipase in an

organic solvent.

Advantageously, according to the process of the present invention, the lipase retains its catalytic activity for more process cycles This invention also comprises new monoesters of flavonoids.

Detailed description of the invention The lipase from Mucor miehei is well known since long time, but has never been employed in the esterification of phenols or hydrolysis of peracylated phenols.

According to the present invention, the alcoholysis of flavonoids, partially or exhaustively acylated, is carried out in an organic solvent.

Preferred examples of solvent can be aliphatic and aromatic hydrocarbons, halogenated hydrocarbons, ethers, more preferably terbutyl methyl ether.

All the flavonoids possessing a hydroxyl function at the C-3 position can be used. Examples of such flavonoids are: quercetin, galangin, morin, fisetin, kampferolo, kampferide, (+)-catechin, (-)-catechin, (+)-epicatechin, (-)-epicatechin.

In a preferred embodiment, the process of the present invention provides the use of the lipase from Mucor miehei adsorbed on solid support, such as the commercially available Lipozyme IM from Novo Nordisk or Chirazyme L-9 from Boehringer Mannheim. Celite, or other usual supports, can be used too.

In a first embodiment of the present invention, the acylation of flavonoid is carried out by conventional procedures. For example, the acylating agent can be an

acyl halide, preferably acyl chloride. Examples of C2-C18 aliphatic acyl group can be, in the context of this invention, acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, heptadecanoyl, oleoyl, stearoyl.

Palmitoyl is preferred.

The next step is an alcoholysis carried out in organic solvent using an aliphatic alcohol possessing from 1 to 8 carbon atoms, as above disclosed.

Examples of this aliphatic alcohol are methanol, ethanol, propanol, isopropanol, n-butanol, t-butanol, amyl alcohol, n-hexanol, n-octanol. n-Butanol is preferred. Examples of polyols are glycerine and glycols.

The product of interest, namely the flavonoid 3- monoester, is recovered from the reaction mixture by conventional procedures, well known to the person skilled in the art. By suitably selecting the acyl group and the aliphatic alcohol, the desired product can be recovered by cooling the reaction mixture.

The process according to the present invention has produced new 3-monoesters of quercetin, as shown in formula 1: 1 in which R is an acyl group of 3-18 carbon atoms.

Of galangin, as shown in formula 2:

in which R is an acyl group of 3-18 carbon atoms.

Of morin, as shown in formula 3: 3 in which R is an acyl group of 2-18 carbon atoms.

Of fisetin, as shown in formula 4: 4 in which R is an acyl group of 2-18 carbon atoms.

Of kaempferol, as shown in formula 5: in which R is an acyl group of 2-18 carbon atoms.

Of kaempferide, as shown in formula 6:

in which R is an acyl group of 2-18 carbon atoms.

Of (+)-catechin, as shown in formula 7: in which R is an acyl group of 2-18 carbon atoms.

Of (-)-catechin, as shown in formula 8: in which R is an acyl group of 2-18 carbon atoms.

Of (-)-epicatechin, as shown in formula 9: in which R is an acyl group of 2-18 carbon atoms.

Of (+)-epicatechin, as shown in formula 10: in which R is an acyl group of 2-18 carbon atoms.

These compounds are useful as antioxidants.

To provide an example of the experimental process, the specific case of quercetin is reported here.

Quercetin (1) is esterified by a conventional chemical process using an aliphatic acyl group having 1- 18 carbon atoms, to give the corresponding pentacyl derivative (step A). These esters are subjected to alcoholysis with an aliphatic alcohol, having from 1 to 8 <BR> <BR> <BR> carbon atoms, in the presence of Mucor miehei lipases (Lipozyme IM, Chirazyme L-9, or adsorbed on inert support) using tert-butyl methyl ether as the solvent (step B).

Alcoholysis leads to monoesters, in which all the phenolic hydroxyl functions are free, whereas the alcoholic group, at position C-3, remains esterified. It should be noted that the ester undergoing alcoholysis may have the hydroxyl groups on the A or B ring partially esterified. This is achieved by esterification with insufficient amounts of acylating agent or by a conventional protection process. Moreover, it is possible to use mixed esters in which hydroxyls in different positions are esterified by different acyls. In this way a number of advantages are realised. For example, alcoholysis of partial esters occurs faster. In the case of the mixed esters, if the desired 3-monoester possesses a long chain acyl group, it is advantageous to have all the other hydroxyls esterified with short chain acid groups, thus facilitating the conditions of the final alcoholysis.

The Mucor miehei lipase employed in immobilised form such as Lipozyme IM retains its activity throughout at least 10 cycles of use.

The use of lipases from porcine pancreas, Pseudomonas cepacia, Chromobacterium viscosum, Candida cylindracea, Candida antarctica, Aspergillus niger, Rhizopus niveus, Rhizopus javanicus, Rhizopus delemar, Candida lipolitica, Penicillium roqueforti, Penicillium cyclopium and esterase from porcine liver give scarce conversion or do not catalyse the alcoholysis at all.

The following example further illustrates the invention.

Example: Quercetin (1 g, 3.3 mmol) was dissolved in t-butyl

methyl ether (50 mL) containing triethylamine (5 ml) and palmitoyl chloride (5.2 ml 7.2 mmol) was added at room temperature. After 12 hours the solution was acidifie with dil. H2SO4 and extracted with CH2C12 three times.

The obtained organic phase was taken to dryness to furnish 4.75 grams of pentapalmitoyl quercetin (96% yield).

Lipozyme IM (2.5 grams) was added to a solution of tert-butyl methyl ether (250 ml) containing pentapalmitoyl quercetine (3 g, 2.9 mmol) and n-butanol (1 1,10.9 mmol) and the suspension was shaken (300 rpm) at 45°C. After 5 days the reaction was stopped and the enzyme filtered off. The solvent was removed under vacuum and the residue added to hexane (200ml). On cooling the solution, 1 g of 3-0-palmitoylquercetin (92% yield) was obtained as a low-melting solid.

1H-nmr: 1HNMR (CD3Cl3): 8 0.89 (bt,-CH3), 1.30 (bs,- (CH2) 13-), 1. 73 (bt,-CH2CO-), 6.31 (d, J= 2.2 Hz, H6), 6.55 (d, J= 2.2 Hz, H8), 7.02 (d, J= 8.2 Hz, H5'), 7.40 (dd, J= 2.2,8.2 Hz, H6'), 7.47 (d, J=2.2 Hz, H2').

In the same way the following quercetin 3-monoesters have been prepared.

Acyl group R Substrate Product Quercetin Quercetin Pentacylated 3-0-acylated Acetyl C2H30 C25H20O12 C17H12O8 Propionyl C3H50 C30H30O12 C18H14O8 Butyryl C4H70 C35H40012 C1gHl608 Valeryl C5H9O C40H50O12 C20H18O8 Hexanoyl C6H11O C45H60012C21H2008 Heptanoyl C7Hi30 C5oH70012 C22H2208 Octanoyl C8H15O C55H80012 C23H2408 Nonanoyl C9H17° C60H90012 C24H2608 Decanoyl CloHgO C65H100O12 C25H28O8 Lauroyl c12H230 C75H120012 C27H3208 Miristoyl c14H270 C85Hl400l2C29H3608 Heptadeca-C17H330 C1ooH170012 C32H4208 noyl Oleoyl C18H330 C105H170O12C33H42O8 Stearoyl C18H35O C105H180O12 C33H44O8