BIOCATALYTIC PROCESS FOR THE PREPARATION OF 3-0-ACYL-FLAVONOIDS

Abstract: The present invention provides a process for the preparation of 3 monoesters of flavonoids comprising the following steps: a) chemical esterification of flavonoid with an aliphatic acyl group having from 1 to 18 carbon atoms to give the corresponding peracetylated flavonoid, or, alternatively, partially acylated flavonoid; b) subsequent alcoholysis with an aliphatic alcohol having from 1 to 8 carbon atoms, in the presence of lipase from Mucor miehei in an organic solvent.

Description: BIOCATALYTIC PROCESS FOR THE PREPARATION OF 3-O-ACYL- FLAVONOIDS Flavonoidε are an important class of natural polyphenols present in various plants. Many of them possess important biological activities, such as hepatoprotective , anticholestero- lemic, antineoplastic , antiinflammatory, antiinfluenza, antiulcer, vasoprotective . Some flavonoids exploit inhibiting activity against tyrosine inase and phosphatidyl-inoεitol kinase. Moreover, their antioxidant properties against peroxide radicals are comparable, if not greater, than conventional phenolic antioxidantε . 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 flavonoidε are insoluble in lipophilic media (oils and emulsions) and weakly bioavailable , so that their employment is limited. It is neceεεary to develop a εynthetic process to obtain εelective eεterification 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; Nicoloεi, G . ; Piattelli, M, J. Org . Chem . 1992, 57, 5776-5778) . The proceεs described make use of Pseudomonas cepacia lipase, which catalysed the alcoholyεiε of the acetoxyl group in different poεition, 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 Pεeudomonas cepacia lipase, but the reaction conditions adopted are detrimental to the enzyme and consequently its faεt inactivation iε obεerved. EP 0618203 reports catechins acylated at poεition C-3, prepared by eεterificationε of free catechin catalysed by Streptomyces rachei o Aspergillus niger carboxylesterase . Using this process authors enables for esterε with acetyl, propyl and butyryl groupε . Summary of the Invention It haε now been found that it is possible to obtain 3-monoesterε of flavonoid aε the only reaction product by carrying out the alcoholysis of a peracylated flavonoid in organic solvent in the preεence of Mucor miehei lipaεe . Therefore, it iε an an object of the preεent invention an efficient method of producing 3-monoesters of flavonoidε, comprising: a) chemical eεterification of the flavonoid with an aliphatic acyl group having from 1 to 18 carbon atomε, to the correεponding 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 proceεs of the present invention, the lipase retains its catalytic activity for more process cycles This invention also compriseε new monoeεterε of flavonoidε . Detailed description of the invention The lipaεe from Mucor miehei iε well known since long time, but has never been employed in the eεterification 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 possesεing a hydroxyl function at the C-3 position can be uεed. Exampleε of such flavonoids are: quercetin, galangin, morin, fisetin, kampferolo, kampferide, ( + )-catechin, (- )-catechin, ( + )-epicatechin, ( - )-epicatechin. In a preferred embodiment, the proceεs of the present invention provides the use of the lipase from Mucor miehei adsorbed on εolid support, such aε the commercially available Lipozyme IM from Novo Nordisk or Chirazyme -9 from Boehringer Mannheim. Celite, or other uεual εupportε, can be used too. In a firεt embodiment of the preεent invention, the acylation of flavonoid iε carried out by conventional procedureε. 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 glycolε. The product of intereεt, namely the flavonoid 3- monoeεter, iε recovered from the reaction mixture by conventional procedureε, well known to the perεon skilled in the art. By suitably selecting the acyl group and the aliphatic alcohol, the deεired product can be recovered by cooling the reaction mixture. The proceεε according to the preεent invention haε produced new 3-monoeεters 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 iε an acyl group of 3-18 carbon atoms Of morin, as εhown in formula 3: in which R iε an acyl group of 2-18 carbon ato ε Of fiεetin, aε εhown in formula 4: in which R is an acyl group of 2-18 carbon atoms Of kae pferol, aε εhown in formula 5: in which R iε an acyl group of 2-18 carbon atomε Of kaempferide, aε shown in formula 6: in which R iε an acyl group of 2-18 carbon atomε, Of ( + )-catechin, aε εhown in formula 7: in which R iε an acyl group of 2-18 carbon atomε Of (- )-catechin, as εhown in formula 8: in which R is an acyl group of 2-18 carbon atomε Of ( - )-epicatechin, aε εhown in formula 9: in which R iε an acyl group of 2-18 carbon atoms Of ( + )-epicatechin, as shown in formula 10 10 in which R iε an acyl group of 2-18 carbon atomε. Theεe compoundε are uεeful as antioxidantε . To provide an example of the experimental proceεε, the εpecific caεe of quercetin iε reported here. Quercetin (1) iε eεterified by a conventional chemical proceεs using an aliphatic acyl group having 1- 18 carbon atomε, to give the corresponding pentacyl derivative (step A). These esters are subjected to alcoholyεis with an aliphatic alcohol, having from 1 to 8 carbon atomε, in the presence of Mucor mi ehei lipaseε ( ipozyme IM, Chirazyme L-9, or adsorbed on inert support) using tert-butyl methyl ether as the solvent Alcoholysis leads to monoesters, in which all the phenolic hydroxyl functions are free, whereas the alcoholic group, at position C-3, re ainε esterified. It should be noted that the ester undergoing alcoholyεiε may have the hydroxyl groupε on the A or B ring partially esterified. This is achieved by eεterification with inεufficient amounts of acylating agent or by a conventional protection procesε. Moreover, it iε poεεible to uεe mixed eεterε in which hydroxylε in different poεitionε are esterified by different acyls. In thiε way a number of advantageε are realiεed. For example, alcoholyεiε of partial esters occurs faεter. In the case of the mixed esterε, if the deεired 3-monoeεter posεeεεeε a long chain acyl group, it iε advantageous to have all the other hydroxyls esterified with short chain acid groups, thuε facilitating the conditionε of the final alcoholysiε . The Mucor miehei lipase employed in immobilised form εuch aε Lipozyme IM retains its activity throughout at least 10 cycles of use. The uεe of lipaεeε from porcine pancreaε, Pεeudo onas cepacia, Chromobacteriu viscoεum, Candida cylindracea, Candida antarctica, Aεpergilluε niger, Rhizopuε niveuε, Rhizopuε javanicuε, Rhizopuε delemar, Candida lipolitica, Penicillium roqueforti, Penicillium cyclopium and eεteraεe 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 m ) containing triethylamine (5 ml) and palmitoyl chloride (5.2 ml 7.2 mmol) waε added at room temperature. After 12 hours the solution was acidified with dil. H2SO4 and extracted with CH2CI2 three times. The obtained organic phase was taken to drynesε 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 suspenεion waε shaken (300 rpm) at 45 β C. After 5 days the reaction waε εtopped and the enzyme filtered off. The εolvent was removed under vacuum and the reεidue added to hexane (200ml). On cooling the εolution, 1 g of 3-O-palmitoylquercetin (92% yield) waε obtained as a low-melting εolid. 1 H-nmr: ^NMR (CD3CI3): δ 0.89 (bt, -CH3), 1.30 (bε, - (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-monoesterε have been prepared. Acyl group R Subεtrate Product Quercetin Quercetin Pentacylated 3-O-acylated Acetyl C H 3 0 C25H20O12 C17H12O8 Propionyl C3H5O C30H30O12 C18H14O8 Butyryl C4H7O C35H40O12 C19H16O8 Valeryl C5H9O C40H50O12 C20HI8O8 Hexanoyl C6H11O C45H60O12 C21H20O8 Heptanoyl C7H13O C50H70O12 C22H2208 Octanoyl C8H15O C55H8OO12 C23H24O8 Nonanoyl C9H17O C60H90O12 C2 H26O8 Decanoyl C10H9O C65H100O12 C25H2808 Lauroyl C12H23O C75H120O12 C27H3208 Miriεtoyl C14H270 C85H140O12 C29H3608 Heptadeca- C17H33O C100H170O12 C32H42O8 noyl Oleoyl C18H330 C105H170O12 C33H42O8 Stearoyl C18H350 C105H180O12 C33H44O8

Claims: Claims 1. A proceεε for the preparation of 3 monoeεters of flavonoids comprising the following stepε: a) chemical eεterification of flavonoid with an aliphatic acyl group having from 1 to 18 carbon atomε to give the correεponding peracetylated flavonoid, or, alternatively, partially acylated flavonoid; b) subsequent alcoholysis with an aliphatic alcohol having from 1 to 8 carbon atoms, in the presence of lipase from Mucor miehei in an organic solvent. 2. A process according to claim 1, in which the flavonoid is selected from the group comprising quercetin, galangin, morin, fisetin, kampferolo, kampferide, ( + )-catechin, (- )-catechin, ( + )-epicatechin, (-)-epicatechin. 3. A proceεs according to claims 1 or 2 , in which the aliphatic acyl is εelected from the group compriεing acetyl, propionyl, butyryl, valeryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl , lauroyl, miriεtoyl, palmitoyl, heptadecanoyl , oleoyl , stearoyl. 4. A proceεε according to anyone of claims 1-3, in which the lipase from Mucor mi ehei iε εupported . 5. A proceεε according to claim 4, in which the lipaεe is supported on celite. 6. A proceεs according to anyone of claims 1-5, in which the solvent iε a halogenated aliphatic hydrocarbon, an aromatic hydrocarbon, an ether. 7. A proceεε according to claim 6, in which the εolvent iε tert-butyl methyl ether. 8. A process according to anyone of claims 1-7, in which, in step a) the esterification iε partial and is performed with insufficient acylating agent. 9. A proceεs according to anyone of claims 1-7, in which, in step a) the esterification is partial and is performed by previously protecting the hydroxyl groups and subsequentdeprotection. 10. A proceεs according to anyone of claims 1-7, in which, in εtep a) the eεterification is partial and is carried out in succesεive εtepε, with different acylating agentε, to give mixed eεterε. 11. Compoundε of formula 1 1 in which R iε an acyl group of 3-18 carbon atomε. 12. Compound according to claim 11, in which R iε palmitoyl . 13. Compounds of formula 2; 2 in which R is an acyl group of 3-18 carbon atoms 14. Compounds of formula 3; in which R is an acyl group of 2-18 carbon atoms . 15. Compounds of formula 4; in which R iε an acyl group of 2-18 carbon atoms . 16. Compoundε of formula 5; in which R is an acyl group of 2-18 carbon atoms . 17. Compounds of formula 6; in which R iε an acyl group of 2-18 carbon atomε 18. Compoundε of formula 7; in which R iε an acyl group of 2-18 carbon atomε . 19. Compoundε of formula 8; 8 in which R iε an acyl group of 2-18 carbon atoms. 20. Compounds of formula 9; in which R is an acyl group of 2-18 carbon atoms . 21. Compounds of formula 10; 10 in which R iε an acyl group of 2-18 carbon atomε. 22. Uεe of the compoundε of claimε 11-21 aε antioxidants

Inventors: LAMBUSTA DANIELA, NICOLOSI GIOVANNI, PATTI ANGELA, PIATTELI MARIO, LAMBUSTA, DANIELA, NICOLOSI, GIOVANNI, PATTI, ANGELA, PIATTELI, MARIO
Applicants: CONSIGLIO NAZIONALE RICERCHE, LAMBUSTA DANIELA, NICOLOSI GIOVANNI, PATTI ANGELA, PIATTELI MARIO, RAO ERBE DI RAO FELICE, CONSIGLIO NAZIONALE DELLE RICERCHE, LAMBUSTA, DANIELA, NICOLOSI, GIOVANNI, PATTI, ANGELA, PIATTELI, MARIO, RAO-ERBE DI RAO FELICE
Application year: 1998
Main classification: C12P17
IPC codes: C07D311, C09K15, C12P7, C12P17