IT202000031025A1 - ENZYMATIC PROCESS FOR THE PREPARATION OF ESTERS OF POORLY WATER-SOLUBLE CARBOXYLIC ACIDS. - Google Patents

Description

Enzymatic process for the preparation of poorly water soluble carboxylic acid esters

The present invention relates to an enzymatic process for the preparation of poorly water soluble carboxylic acid esters. In particular, the present invention relates to an enzymatic process for the preparation of poorly water-soluble carboxylic acid esters with a molecule carrying a hydroxyl group in a monophasic reaction system.

? It is known that the translation of poorly water-soluble drugs into effective medicinal products is an ongoing challenge [1]. Pi? of 40% of the new entities? chemical (NCE) that possess activity? pharmacological, synthesized by superior combinatorial screening programs, are poorly soluble, which ? a major obstacle to formulation development [2][3]. Therefore, drugs with poor solubility in water require new formulation approaches to improve their dissolution rates and bioavailability? oral [4]. Often, the solubility of these drugs? limited in the stomach due to the presence of a carboxylic acid in an aqueous acidic medium [5]. For this reason, one way to increase water solubility is Poorly water-soluble drug forms could be an esterification reaction between its carboxylic acid and an alcohol to yield a drug with enhanced water solubility, designed to have better water availability. oral [6]. This type of modification could lead to the synthesis of a prodrug. A prodrug? a compound that has the ability? to undergo biotransformation before exhibiting its properties? pharmacological [7].

Canonically, the esterification reaction is performed chemically by Fischer esterification [8]. Unlike chemical modifications, biotechnological approaches can represent a ?green? for the synthesis of bioactive derivatives of drugs [9][10]. The ?green? processes they must use environmentally friendly, non-toxic and reusable catalysts to synthesize valuable chemical compounds [11]. For example, ibuprofen ? a poorly water soluble drug. Its esterification with hydrophilic compounds therefore allows to obtain a synthetic molecule enhanced in its water solubility. and consequent bioavailability. This strategy can be developed via a bio-catalyzed esterification reaction. For this purpose, the lipases represent the most biological tool? used for enzymatic esterification. Since the 1990s, lipases (triacylglycerol hydrolase, EC 3.1.1.3) have been used as biocatalysts in industries due to their active activity. in mild reaction conditions, the lack of need? of cofactors, their activity? synthetic in non-aqueous solvents and their wide range of stereospecific substrates [12]. Lipases are widely used as biocatalysts for esterification or transesterification reactions and exist free or in immobilized form [13]. Among the non-immobilized (free) lipases more? commonly used for esterification or transesterification reactions are those of the microorganisms R. miehei [14], C. rugosa [15] and P. cepacian [16]. Immobilized lipases, such as Novozyme 435 (C. antarctica lipase type B, CALB) [17], Lipozyme RM-IM (lipase from R. miehei) and Lipozyme TL-IM (lipase from T. lanuginosus) [18] are also commercially available.

Lipases are hydrophilic molecules with an active site enclosed in a hydrophobic pocket. This hydrophobic pocket ? covered by a polypeptide chain called lid (?lid?), which generally isolates the active site from the reaction medium (in this closed form the lipase is inactive). The lid also has an inner hydrophobic face which interacts with the hydrophobic areas of the active site and a hydrophilic outer face which interacts with the reaction medium. This lid can move forming a large hydrophobic pocket which exposes the active center to the medium, giving rise to the ?open? and active lipase, with the hydrophilic phase of the cap interacting with the protein surface. In a reaction environment, the lipases undergo interfacial activation due to a polarity? opposite between the enzyme (hydrophilic) and the substrates (lipophilic). The reaction takes place at the interface between the aqueous and oily phases, where a conformational change occurs associated with increased activity. enzymatic. The interfaces are therefore key points for the biocatalysis of the lipase and an appropriate site to modulate the activity of the lipase. of lipolysis [19].

The use of solvents in an aqueous-organic biphasic system can thus induce the interfacial activation of lipase. To improve the surface of the interface ? Vigorous mixing of the two phases is recommended, which forms a suspension with a very large interfacial area. Often, however, the biphasic environment can also represent a major disadvantage. The presence of water, in fact, inevitably leads to the hydrolysis reaction of the new esters produced, by virtue of of the stoichiometric equilibrium deducible from the underlying generic esterification reaction between a carboxylic acid and an alcohol, catalysed by lipase:

A specific example of a poorly soluble drug? ibuprofen. Ibuprofen ((R,S)-2-(pisobutylphenyl)-propionic acid) is a traditional non-steroidal anti-inflammatory drug (NSAID) [20] developed in the late 1960s [21] to treat symptoms caused by arthritis, such as swelling, pain and stiffness [22]. Ibuprofen, like ketoprofen, flurbiprofen and other non-steroidal drugs, is widely used for its properties analgesic, anti-inflammatory and antipyretic [23]. ? used in the treatment of pain of mild to moderate intensity, in cases of dysmenorrhea, headache (including migraine), dental pain, post-operative pain, musculoskeletal or joint disorders, including osteoarthritis, rheumatoid arthritis and spondylitis ankylosing [23][24]. The mechanism of action of ibuprofen in different therapeutic contexts? well established. The ibuprofen ? a non-selective reversible inhibitor of cyclooxygenase isoenzymes (COX-1 and COX-2), responsible for the lipase-catalyzed conversion of arachidonic acid to prostaglandins, including thromboxane and prostacyclin [25]. Due to its chiral center, ibuprofen has two enantiomers [26]. However, ? documented that the? activity? therapeutics of ibuprofen? mainly attributed to the enantiomer (S), which ? 160 times more efficacy of the (R)-enantiomer [27]. Considering the high patient compliance, cost effectiveness, sterility constraints? reduced and flexibility, the administration route pi? common for ibuprofen ? oral [28] in the form of tablets, hard capsules or effervescent granules with dosages of 200mg, 400mg, 800mg [29][30]. The dose? of 200-400mg (5-10mg/kg in children), every 4-6 hours up to a maximum of 1.2g per day in adults [31]. Given its solubility? in water of 21mg/L [32]. The ibuprofen ? a drug poorly soluble in water, characterized by bioavailability? oral limited by dissolution [34]. The low dissolution rate of currently available solid dosage forms and the resulting poor bioavailability? of a high oral dose [33] can cause serious undesirable adverse effects. Chronic oral administration of NSAIDs can cause damage to the gastric mucosa, especially gastric ulceration, bleeding and perforation [34]. There are formulations with salt conjugates of ibuprofen (for example arginate, lysinate and sodium) with the aim of increasing the speed? of dissolution in the stomach [35]. The solubility? of ibuprofen ? limited in the stomach, why? it is a carboxylic acid in an aqueous acidic medium [5]. For this reason, a way to increase the solubility? in water of ibuprofen could be an esterification reaction between its carboxylic acid and an alcohol to obtain an enhanced water-soluble prodrug, designed to have better bioavailability? oral [6]. Yeah? In 1980, a toxicological study of several ibuprofen derivatives and formulations showed the ibuprofen ester prodrug guaiacol as a suitable form for this drug, having reduced toxicity. in the rat [36]. The design, synthesis and administration of four prodrugs of ibuprofen and L-ascorbic acid targeting the brain show an increase in ibuprofen levels at the same site [39]. Furthermore, the synthesis and testing of the N,N-disubstituted amino alcohol ester of ibuprofen resulted in a significant reduction of the ulcerogenic effect in the stomach [40].

The ibuprofen? been esterified enzymatically with glycerol in solventless-media and ? The kinetic model has been reported [37]. However, this system has several limitations. The presence of water limits the final conversion yield. Glycerol? the polyalcohol pi? short that you can? attack to obtain a prodrug with greater solubility? and ? the only polyalcohol in liquid form. So, this esterification process in solventless media? usable only for this polyalcohol, and ? completely useless for other polyalcohols with a chain pi? long as: erythritol, xylitol and sorbitol. These polyalcohols are in solid form, so they must be solubilized to take part in the enzymatic reaction. However, to produce esters between highly hydrophilic molecules, such as erythritol, xylitol and sorbitol (logP: -1.86 / -3.36) and lipophilic drugs, such as ibuprofen (logP: 3.75), the literature reports biphasic systems solvent/water. In addition to the disadvantages listed above, no n? biphasic esterification systems n? monophasic between the aforementioned polyalcohol and ibuprofen. Indeed, in these conditions, ? difficult an enzymatic reaction between molecules of such polarity? opposite.

In the light of the above, it is therefore evident that the need to provide a process for the enzymatic esterification of poorly water soluble carboxylic acids which overcomes the drawbacks of the known processes.

According to the present invention, a process is provided which overcomes the limitations of current enzymatic esterification strategies and maximizes yields. In particular, the process according to the present invention ? an enzymatic synthesis process catalyzed by lipase in a monophasic system which overcomes the limits of the canonical biphasic system and produces an effective esterification reaction between carboxylic acids which are poorly soluble in water, such as some drugs, and highly hydrophilic molecules, such as solid polyalcohols.

The advantages of the process of the present invention are for example: better solubility? of substrates and of the product, shift of the thermodynamic equilibria (that is, synthesis takes place instead of hydrolysis), possible use of the enzyme directly in an enzymatic phase of a chemical process, and solubilization of hydrophilic and lipophilic compounds in the same organic phase.

The single-phase system according to the present invention ? much more simple in terms of handling the reaction. Therefore, ? far fewer variables need to be considered than the variables in two-phase systems. In the process of the present invention the water is not required, and this shifts the hydrolysis/synthesis balance towards the synthetic behavior of the lipase. The use of a commercially available immobilized enzyme leads to a highly reproducible organic synthesis with high stereo- and regio-specificity. Furthermore, the immobilized enzyme ? stable and perfectly separable from the ground in order to be reused even for more? of 10 cycles.

An example of enzymes suitable for the process of the present invention are lipases, such as CALB. because CALB lacks a lid (?lid?) covering the active site, it doesn't need any interfacial activation, therefore, it can? be used in a one-phase system such as that of the present invention. Other suitable lipases are the lipases from Thermomyces lanuginosa and Rhizomucor miehei, which are considered similar to CALB in terms of their ability to catalytic for different substrates. Furthermore, according to the present invention, preliminary steps for protecting the functional groups are not necessary (while these steps are normally necessary in chemical synthesis, i.e. non-enzymatic synthesis) and no toxic solvents are used. Unlike many enzymatic syntheses reported in the literature, which require reaction times of even 1 week, the process according to the present invention allows a conversion yield ranging from 65 to 100% after only 24 hours. The reaction volumes are small: the esterification can? also be obtained in 2-5 mL reactors or microreactors. This small-scale process can be easily expanded. The process of the present invention advantageously provides a way to solubilize two substrates, i.e. the carboxylic acid and the hydroxyl group-carrying molecule such as a polyalcohol, in a single organic solvent. Therefore, the molecule carrying the hydroxyl group need not be liquid. This system works at low temperatures (40 to 85 ? C), at atmospheric pressure and the reactor is not filled with nitrogen or other gases. Furthermore, the method of the invention shows great flexibility? on different substrates. The specific reaction conditions of the process, in addition to the choice of organic solvent and enzyme, such as preliminary solubilization processes, concentrations, temperatures, stirring, acid to alcohol concentration ratio contribute to the optimization of the process of the present invention in comparison with the state of the art of lipase-catalyzed enzymatic synthesis.

In particular, the process of the invention leads to the enzymatically catalyzed formation of the ester bond between the carboxylic acid of a target molecule and the hydroxyl group of a molecule carrying hydroxyl groups such as an alcohol, even in solid form. According to the present invention, a quantity predetermined amount of organic solvent (e.g. tertiary amyl alcohol: 3 to 5 mL) is placed in a dry reactor. The solvent acts to solubilize both the carboxylic acid (e.g. ibuprofen) and the hydroxyl group-carrying molecule (e.g. a polyalcohol) in a one-phase system. ? is known that the shape of the reactor is important to avoid the mechanical breakage of the catalyst, in particular, the contact between the walls of the reactor and the magnetic stirrer must be almost? null. The magnetic stirrer can be preferably a ?cross? what can? minimize contact with the immobilized catalyst in order to avoid its destruction. To solubilize a highly hydrophilic compound (such as: erythritol, xylitol or sorbitol, but also ascorbic acid) in the solvent (for example t-amyl alcohol) without adding water, ? necessary a preliminary step: a quantity? predetermined amount of polyalcohol (or other types of hydroxyl borne molecules: such as ascorbic acid) is carefully added to the solvent, the reactor is closed to avoid evaporation of the solvent (even if the boiling point of t-amyl alcohol is about 102.4 ?C). The reactor is placed on a magnetic stirrer and the solvent is heated to the desired temperature. Vigorous mixing at high temperatures can aid in the solubilization of the polyalcohol in the solvent such as t-amyl alcohol.

For example, the temperatures that can be used for the solubilization of polyalcohol erythritol, xylitol, and sorbitol are shown below:

- erythritol: from 55?C to 100?C

- xylitol: from 60?C to 100?C

- sorbitol: from 65?C to 100?C

Once solubilized, the polyalcohol remains in solution even when the temperature of the solvent such as t-amyl alcohol decreases. At this point you can? easily add carboxylic acid such as ibuprofen for its solubilization (100 of 300 mg to 5 mL of t-amyl alcohol). Then, the solvent is allowed to cool to avoid thermal degradation of the enzyme catalyst. When is the temperature of the solvent ? approximately 65 ?C or lower, ? is it possible to load the quantity? predetermined amount of enzyme (range 10 to 55 mg in 5 mL of t-amyl alcohol). Solvent can? be added a certain number of ?molecular sieves? to remove water as a byproduct of bound ester formation (range 50 to 100 mg in 5 mL of t-amyl alcohol). Less water in the system leads to more? easy formation of the bonded ester as shown in the equilibrium reaction above.

The reactor can preferably be placed in a glycerine/silicone oil bath placed on the magnetic stirrer to keep the temperature constant. Some magnetic stirrers have a thermometer which, if placed in the glycerine bath, can auto-set the heating force of the stirrer in order to keep the desired temperature constant over time. The speed? stirring speed should be set from 100 to 200 RMP to avoid mechanical destruction of the catalyst. For example, the acid:alcohol molar ratio can be 5: 1. After 24 hours, or even less, ester is formed which ? purified by the reaction.

? It is important to note that not all organic solvents can lead to this type of esterification process. Indeed, the choice of the right solvent ? basic. Although several solvents have been used in enzymatic synthesis, none are suitable for the process of the present invention. Many apolar organic solvents (up to logP 2) can remove the layer of water which allows for the right enzymatic conformation, causing its denaturation. Many polar solvents cannot solubilize carboxylic acid such as ibuprofen. DMSO deactivates the enzyme. Some organic solvents (such as methanol) are alcohols so? small ones that interact with the active site of the enzyme, inactivating it. Other long-chain alcohols (for example: octanol) can take part in the reaction as a substrate. Furthermore, none of the common organic solvents, which have been tested, can? solubilize both carboxylic acid such as ibuprofen and polyalcohol (log P: 3.75 to -3.36).

According to the present invention, suitable solvents such as t-amyl alcohol have been selected. Also, as previously mentioned, it can a preliminary solubilization step may be required.

Therefore, a specific object of the present invention is a process for the preparation of an ester of a carboxylic acid with a molecule carrying a hydroxyl group, said carboxylic acid being sparingly soluble in water and the molecule carrying a hydroxyl group being a solid compound, in which the process includes or consists of:

a) solubilizing the carboxylic acid and the molecule carrying a hydroxyl group in an organic phase capable of solubilizing both the carboxylic acid and the molecule carrying a hydroxyl group; and b) adding an enzymatic catalyst suitable for catalyzing the esterification, under stirring;

wherein said an organic phase comprises or consists of a tertiary alcohol or a tertiary nitrile having a number of carbon atoms from 4 to 10, preferably from 4 to 8, plus? preferably from 4 to 6, or mixtures thereof, and said enzymatic catalyst ? a serine hydrolase in a catalytically active form.

By "poorly water soluble" is meant a carboxylic acid capable of being solubilized from the organic phase. In particular, a carboxylic acid which is poorly soluble in water ? preferably a carboxylic acid having a solubility? in water that falls within the range of slightly soluble and lower, cio? a solubility? <10 mg/mL (this range also includes water-insoluble carboxylic acids, i.e. carboxylic acids having an aqueous solubility <100 ?g/mL). The carboxylic acid according to the present invention can have a solubility? aqueous from 100 µg/mL to 10 mg/mL.

According to the process of the present invention in step a) the molecule carrying a hydroxyl group can be solubilized in said one organic phase under heating and stirring before adding the carboxylic acid.

The heating temperature depends on the molecule carrying a hydroxyl group. If heating is used, the enzyme catalyst is added when the temperature is lowered to a temperature where the enzyme is released. active. For example, when the enzyme ? CALB, this enzyme? active at or below 80?C. Preferably, CALB can be used at a temperature lower than 70?C, pi? preferably lower than 65°C.

According to the process of the present invention, the ratio of the carboxylic acid to the molecule carrying the hydroxyl group can be vary from 10:1 to 1:10, for example from 5:1 to 1:5.

According to a preferred embodiment, the ratio between the carboxylic acid and the molecule carrying a hydroxyl group can be vary from 10: 1 to 1: 1, for example from 5: 1 to 1: 1, with the exclusion of the ratio 1:1, the cio? you prefer an excess of the carboxylic acid with respect to the quantity? of the molecule carrying a hydroxyl group, for example a ratio of 5:1.

According to the present invention, the carboxylic acid can be selected from the group consisting of ibuprofen, mandelic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, sorbic acid, 3-phenylpropanoic acid, cinnamic acid, 3- (4-hydroxyphenyl) propionic acid, 3,5-dimethoxy-4- hydroxycinnamic, benzoic acid, (2E) -3- (4-hydroxyphenyl) prop-2-enoic acid (p-coumaric acid), 3,4,5-trihydroxybenzoic acid (gallic acid), benzene-1,2-dicarboxylic acid (phthalic acid), 2-hydroxybenzoic acid (salicylic acid), acetylsalicylic acid, succinic acid, stearic acid, tartaric acid, glutamic acid, malonic acid, (2E)-But-2 -endoic acid (fumaric acid), and indomethacin.

According to the present invention, the molecule carrying a hydroxyl group can be a molecule having a LogP value of -4.7 to 0.15, preferably -4.7 to 0, more? preferably from -3.36 to 0, and carrying at least two hydroxyl groups, preferably at least three hydroxyl groups, plus? preferably at least four hydroxyl groups, even more? preferably at least five hydroxyl groups or at least six hydroxyl groups.

In particular, said molecule carrying a hydroxyl group can be selected from the group consisting of a polyalcohol such as sorbitol, xylitol, erythritol, ethylene glycol, ethene-1,1-diol, (2E)-2-butene-1,4-diol, maltitol or isomalt, preferably sorbitol; a sugar such as glucose, fructose, or sucrose; or ascorbic acid.

Table 1 below shows the LogP values of some of the above-mentioned molecules carrying a hydroxyl group.

Table 1

According to the present invention, said tertiary alcohol can be t-amyl alcohol or t-butanol and said tertiary nitrile can? be trimethylacetonitrile.

According to an embodiment of the present invention, said enzymatic catalyst can be an immobilized enzyme catalyst.

According to the process of the present invention, said serine hydrolase can be selected from the group consisting of lipase or serine protease, such as subtilisin or trypsin.

In particular, said lipase pu? be selected from the group consisting of C. antarctica lipase B, lipase from R. miehei, lipase from T. lanuginosus or C. antarctica lipase A, preferably C. antarctica lipase B, plus? preferably an immobilized C. antarctica lipase B.

CALB shows a Ser-His-Asp catalytic triad in its active site, with a very small lid (?lid?) that is not ? capable of completely isolating the active site.

Examples of commercially available lipases which can be used according to the present invention are Lipozyme? TL 100L (from Thermomyces lanuginosa), Lipozyme? CALB L (not immobilized, ie free, CALB), Lipozyme? CALA L and Novocor? AD L (not immobilized, ie free, lipase A from Candida antarctica), Lipozyme? 435 (a recombinant lipase from Candida antarctica, expressed in Aspergillus niger and immobilized on Lewatit VP OC 1600).)

According to an embodiment of the present invention, step a) and/or step b) can comprise the addition of one or more? molecular sieves to remove water from the reaction medium. For example, the following molecular sieves can be used: molecular sieves, 4?, spheres, 4-8 mesh; molecular sieves, 3?, spheres, 4-8 mesh; molecular sieves, 3 ?, spheres, 8-12 mesh.

Furthermore, the present invention relates to an ester of a poorly water soluble carboxylic acid with a solid hydroxyl group carrier molecule, wherein said ester is? chosen from the group consisting of:

ibuprofen erythritol ester, such as 2,3,4-trihydroxybutyl 2- (4-isobutylphenyl) propanoate, for example (2R, 3S) -2,3,4-trihydroxybutyl 2- (4-isobutylphenyl) propanoate;

ibuprofen xylitol ester, such as 2,3,4,5-tetrahydroxypentyl 2- (4-isobutylphenyl) propanoate, such as (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl 2- (4-isobutylphenyl) propanoate ;

ibuprofen sorbitol ester, such as 2,3,4,5,6-pentahydroxyl 2- (4-isobutylphenyl)propanoate, for example (2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyl 2 - (4-isobutylphenyl)propanoate;

ester of mandelic acid with ascorbic acid, such as 2- (3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl) -2-hydroxyethyl 2-hydroxy-2-phenylacetate, e.g. (S) -2 - ((R) -3,4-dihydroxy-5-oxo-2,5-dihydrofuran -2-yl) -2-hydroxyethyl 2-hydroxy-2-phenylacetate;

ursodeoxycholic acid erythritol ester, such as 2,3,4-trihydroxybutyl 4- (3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate, e.g. (2S , 3R) -2,3,4-trihydroxybutyl (R) -4 - ((3R, 5S, 7S, 8R, 9S, 10S, 13R, 14S, 17R) -3,7-dihydroxy-10,13-dimethylhexadecahydro- 1H-cyclopenta [a]phenanthrene-17-yl) pentanoate;

ursodeoxycholic acid xylitol ester, such as 2,3,4,5-tetrahydroxypentyl 4-(3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-17-yl)pentanoate, e.g. ( 2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (R) -4 - ((3R, 5S, 7S, 8R, 9S, 10S, 13R, 14S, 17R) -3,7-dihydroxy-10 ,13 -dimethylhexadecahydro-1H-cyclopenta [a]phenanthrene-17-yl) pentanoate;

ursodeoxycholic acid sorbitol ester, such as 2,3,4,5,6-pentahydroxyhexyl 4-(3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate, for example ( 2R,3R,4R,5S)-2,3,4,5,6-pentahydroxyhexyl (R)-4-((3R,5S,7S,8R,9S,10S, 13R,14S,17R)-3,7 -dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate;

deoxycholic acid erythritol ester, such as 2,3,4-trihydroxybutyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate, e.g. (2S , 3R) -2,3,4-trihydroxybutyl (R) -4 - ((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,12-dihydroxy-10,13-dimethylhexadecahydro- 1H-cyclopenta [a]phenanthrene-17-yl) pentanoate;

deoxycholic acid xylitol ester, such as 2,3,4,5-tetrahydroxypentyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate, e.g. ( 2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (R) -4 - ((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,12-dihydroxy-10 ,13 -dimethylhexadecahydro -1H-cyclopenta [a]phenanthrene-17-yl) pentanoate;

deoxycholic acid sorbitol ester, such as 2,3,4,5,6-pentahydroxyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate, such as example ( 2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl (R) -4 - ((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3 ,12 -dihydroxy-10,13-dimethylhexadecahydro -1H-cyclopenta [a]phenan-17-yl) pentanoate;

sorbic acid erythritol ester, such as 2,3,4-trihydroxybutyl (2E, 4E) -hexa-2,4-dienoate, e.g. (2S, 3R) -2,3,4-trihydroxybutyl (2E, 4E ) -hex -2,4-dienoate;

xylitol ester of sorbic acid, such as 2,3,4,5-tetrahydroxypentyl (2E, 4E) -hexa-2,4-dienoate, such as (2S, 3S, 4R) -2,3,4,5- tetrahydroxypentyl (2E,4E)-hexa-2,4-dienoate;

sorbitol ester of sorbic acid, such as 2,3,4,5,6-pentahydroxyl (2E, 4E) -hexa-2,4-dienoate, such as (2R, 3R, 4R, 5S) -2,3, 4, 5,6-pentahydroxyl (2E, 4E) -hexa-2,4-dienoate;

3-phenylpropanoic acid erythritol ester, such as 2,3,4-trihydroxybutyl 3-phenylpropanoate, e.g. (2S,3R)-2,3,4-trihydroxybutyl 3-phenylpropanoate;

3-phenylpropanoic acid ester with xylitol, such as 2,3,4,5-tetrahydroxypentyl 3-phenylpropanoate, e.g. (2S,3S,4R)-2,3,4,5-tetrahydroxypentyl 3-phenylpropanoate;

Sorbitol ester of 3-phenylpropanoic acid, such as 2,3,4,5,6-pentahydroxyl 3-phenylpropanoate, e.g. (2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl 3 -phenylpropanoate;

erythritol ester of 3- (4-hydroxyphenyl) propionic acid, such as 2,3,4-trihydroxybutyl 3- (4-hydroxyphenyl) propanoate, e.g. (2S, 3R) -2,3,4-trihydroxybutyl 3- ( 4-hydroxyphenyl)propanoate;

3- (4-Hydroxyphenyl)propionic acid xylitol ester, such as 2,3,4,5-Tetrahydroxypentyl 3- (4-Hydroxyphenyl)propanoate, e.g. (2S, 3S, 4R)-2,3,4,5 - Tetrahydroxypentyl 3- (4-hydroxyphenyl) propanoate;

sorbitol ester of 3- (4-hydroxyphenyl) propionic acid, such as (2,3,4,5,6-pentahydroxyphenyl 3- (4-hydroxyphenyl) propanoate, e.g. (2R, 3R, 4R, 5S) -2 ,3,4,5,6-pentahydroxyl 3- (4-hydroxyphenyl) propanoate;

erythritol ester of 3,5-dimethoxy-4-hydroxycinnamic acid, such as 2,3,4-trihydroxybutyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate, e.g. (2S, 3R) -2,3,4-trihydroxybutyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate;

3,5-dimethoxy-4-hydroxycinnamic acid xylitol ester, such as 2,3,4,5-tetrahydroxypentyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate, e.g. (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate;

sorbitol ester of 3,5-dimethoxy-4-hydroxycinnamic acid, such as 2,3,4,5,6-pentahydroxyl (E) -3(4-hydroxy-3,5-dimethoxyphenyl) acrylate, e.g. (2R , 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyphenyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate.

The present invention also relates to a pharmaceutical composition comprising or consisting of an ester as defined above in combination with one or more? excipients and/or adjuvants.

A further object of the present invention ? an ester as defined above or a pharmaceutical composition as defined above for use in anti-inflammatory therapy when the carboxylic acid is ibuprofen.

A further object of the present invention ? an ester as defined above or a pharmaceutical composition as defined above for use in the treatment of gallstones when the carboxylic acid is ursodeoxycholic acid or deoxycholic acid.

Among the esters of the present invention:

- sorbic acid esters can be used as antimicrobial agents, as food preservatives;

- mandelic acid esters can be used in cosmetics, for example they can be used as anti-aging agents;

- esters of aromatic acids such as 3-phenylpropanoic acid or 3- (4-hydroxyphenyl) propionic acid can be used in cosmetics, as food additives or in pharmaceuticals;

- 3,5-dimethoxy-4-hydroxycinnamic acid esters can be used in matrix for spectrophotometric techniques, such as MALDI mass spectrometry;

- indomethacin esters can be used as non-steroidal anti-inflammatory drugs;

- fumaric acid esters can be used in the pharmaceutical and/or food industry;

- malonic acid esters can be used for the preparation of flavors and fragrances;

- glutamic acid esters can be used as food additives;

- tartaric acid esters can be used as antioxidant agents;

- stearic acid esters can be used as surfactants;

- can succinic acid esters be used in the food industry, for example as acidity regulators? and flavoring agents;

- esters of salicylic or acetylsalicylic acids can be used as anti-inflammatory agents;

- phthalic acid esters can be used as plasticizers;

- esters of gallic acid or pcumaric acid can be used as antioxidant agents.

Based on the above, the process of the present invention can be used both for the production of known molecules and for the synthesis of new derivatives, such as the three ibuprofen esters shown in Figure 1. These three molecules, subject to patent, are potential candidates for in vivo and in vitro experimentation as new anti-inflammatory agents with better bioavailability? instead of ibuprofen. As mentioned above, sorbitol is a polyalcohol that can? be used as a hydrophilicity enhancer. Sorbitol ((2R, 3R, 4R, 5S) -hexane-1,2,3,4,5,6-hexol), less commonly known as glucitol.

The developed synthesis protocol can be used for low-cost, simple-scale "green" syntheses for the production of highly pure and selective pharmaceutical esters. The three chemical substances, synthesized with the protocol in question, can find application as new non-steroidal anti-inflammatory drugs for the treatment of pain (headache, dysmenorrhea, dental pain) up to musculoskeletal disorders such as osteoarthritis and rheumatoid arthritis. This technique can be exploited by various sectors, from the pharmaceutical to the food sector. This method can easily obtain prodrugs, preservatives or aromatic esters increased in solubility.

This invention will come now described in an illustrative, but non-limiting manner, according to preferred embodiments thereof, with particular reference to the examples and accompanying drawings, in which:

- figure 1 shows a scheme of formation of the ester catalysed by the lipase linked between ibuprofen and 3 different solid polyalcohols: a) ibuprofen erythritol ester; b) ibuprofen xylitol ester; c) ibuprofen sorbitol ester;

- figure 2 shows an esterification reaction scheme between ibuprofen and ascorbic acid;

- figure 3 shows an esterification scheme of mandelic acid with ascorbic acid;

- Figure 4 shows a scheme of formation of the ester catalyzed by the lipase linked between ursodeoxycholic acid (UDCA) and 3 different solid polyalcohols: a) UDCA erythritol ester; b) UDCA xylitol ester; c) UDCA sorbitol ester;

- Figure 5 shows a scheme of formation of the ester catalyzed by the lipase linked between deoxycholic acid (DCA) and 3 different solid polyalcohols: a) DCA erythritol ester; b) DCA xylitol ester; c) DCA sorbitol ester;

- figure 6 shows a scheme of formation of the ester catalyzed by the lipase bound between sorbic acid and 3 different solid polyalcohols: a) erythritol ester of sorbic acid; b) xylitol ester of sorbic acid; c) sorbitol ester of sorbic acid;

- figure 7 shows a scheme of formation of the ester catalyzed by lipase linked between 3-phenylpropanoic acid and 3 different solid polyalcohols: a) erythritol ester of 3-phenylpropanoic acid; b) xylitol ester of 3-phenylpropanoic acid; c) sorbitol ester of 3-phenylpropanoic acid;

- figure 8 shows the reaction between 3-(4-hydroxyphenyl) propionic acid with 3 polyalcohols (erythritol, xylitol and sorbitol);

- figure 9 shows the reaction between 3,5-dimethoxy-4-hydroxycinnamic acid with 3 polyalcohols (erythritol, xylitol and sorbitol);

- Figure 10 shows the reaction between benzoic acid with 3 polyalcohols (erythritol, xylitol and sorbitol).

EXAMPLE 1: Process according to the present invention for the preparation of ibuprofen esters.

A pattern of lipase catalyzed ester formation bonded between ibuprofen and 3 different solid sugar alcohols? shown in figure 1: a) ibuprofen ester with erythritol b) ibuprofen ester with xylitol c) ibuprofen ester with sorbitol.

The following ranges were tested:

- reaction volumes: 3 to 10 mL of t-amyl alcohol - enzymatic concentration: 5 to 18 gL-1

- concentration of molecular sieves: from 10 to 32 gL-1 - speed? stirring speed: from 150 to 220 RPM

a) Ibuprofen erythritol ester: (2R, 3S) -2,3,4-trihydroxybutyl 2- (4-isobutylphenyl) propanoate

b) ibuprofen xylitol ester: (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl 2- (4-isobutylphenyl) propanoate

c) Ibuprofen sorbitol ester: (2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyl 2- (4-isobutylphenyl) propanoate

An esterification reaction scheme between ibuprofen and ascorbic acid? shown in figure 2.

For example, (S) -2 - ((R) -3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl) -2-hydroxyethyl 2- (4-isobutylphenyl) propanoate ? obtained under the following conditions:

EXAMPLE 2: Process according to the present invention for the preparation of the mandelic acid ester.

The esterification of mandelic acid with ascorbic acid? shown in Fig. 3. In particular, (S) -2 - ((R) -3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl) -2-hydroxyethyl Il 2-hydroxy-2- Phenylacetate is obtained under the following conditions:

EXAMPLE 3: Process according to the present invention for the preparation of UDCA esters

A pattern of lipase catalyzed ester formation bonded between ursodeoxycholic acid (UDCA) and 3 different solid polyalcohols? shown in figure 5 : a) erythritol ester UDCA; b) UDCA xylitol ester; c) UDCA sorbitol ester.

a) UDCA ester with erythritol: (2S, 3R) -2,3,4-trihydroxybutyl (R) -4 - ((3R, 5S, 7S, 8R, 9S, 10S, 13R, 14S, 17R) -3, 7-Dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-17-yl)pentanoate.

The concentration of erythritol ? limited to 20 mg to bring the solubilization of a quantity? 5 mol greater of UDCA in t-amyl alcohol.

The same esterification pu? be obtained with other polyalcohols according to the modalities? described above in order to obtain:

b) UDCA xylitol ester: (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (R) -4 - ((3R, 5S, 7S, 8R, 9S, 10S, 13R, 14S, 17R) - 3,7-dihydroxy-10,13-dimethylhexadecahydro-1H cyclopenta [a]phenanthrene-17-yl) pentanoate and

c) UDCA sorbitol ester: (2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl (R) -4 - ((3R, 5S, 7S, 8R, 9S, 10S, 13R, 14S , 17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H cyclopenta[a]phenanthrene-17-yl)pentanoate.

EXAMPLE 4: Process according to the present invention for the preparation of DCA esters

Figure 5 shows a pattern of lipase catalyzed ester formation bonded between deoxycholic acid (DCA) and 3 different solid polyalcohols:

a) erythritol ester DCA:

(2S, 3R) -2,3,4-trihydroxybutyl (R) -4 - ((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,12-dihydroxy-10,13- dimethylhexadecahydro-1Hcyclopenta[a]phenanthrene-17-yl) pentanoate;

b) DCA xylitol ester:

(2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (R) -4 - ((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3,12-dihydroxy- 10,13-dimethylhexadecahydro-1Hcyclopenta [a]phenanthrene-17-yl) pentanoate;

c) DCA sorbitol ester:

(2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl (R) -4 -((3R, 5R, 8R, 9S, 10S, 12S, 13R, 14S, 17R) -3, 12-Dihydroxy-10,13-dimethylhexadecahydro-1Hcyclopenta [a]phenanthrene-17-yl) pentanoate.

DCA esters can be synthesized analogously to the described method of the present invention considering its molecular weight of 392.6 g/mol.

EXAMPLE 5: Process according to the present invention for the preparation of sorbic acid esters

A pattern of lipase catalyzed ester formation bonded between sorbic acid and 3 different solid polyalcohols? shown in figure 6: a) ester of sorbic acid with erythritol; b) xylitol ester of sorbic acid; c) sorbitol ester of sorbic acid.

a) sorbic acid erythritol ester:

(2S,3R)-2,3,4-trihydroxybutyl (2E,4E)-hexa-2,4-dienoate

b) ester of sorbic acid with xylitol:

(2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (2E, 4E) -hexa-2,4-dienoate

c) sorbitol ester of sorbic acid:

(2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl (2E, 4E) -hexa-2,4-dienoate

EXAMPLE 6: Process according to the present invention for the preparation of cinnamic acid esters

Figure 7 shows a pattern of bonded lipase catalyzed ester formation between 3-phenylpropanoic acid and 3 different solid polyalcohols:

a) 3-phenylpropanoic acid ester with erythritol: (2S, 3R) -2,3,4-trihydroxybutyl 3-phenylpropanoate; b) 3-phenylpropanoic acid ester with xylitol: (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl 3-phenylpropanoate;

c) 3-phenylpropanoic acid ester with sorbitol: (2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl 3-phenylpropanoate.

The following forms of cinnamic acid are also esterifiable with the method of the invention.

Figure 8 shows a pattern of lipase catalyzed ester formation bonded between 3(4-hydroxyphenyl) propionic acid - (floretic acid) and 3 different solid polyalcohols:

a) 3- (4-hydroxyphenyl) propionic acid erythritol ester: (2S, 3R) -2,3,4-trihydroxybutyl 3- (4-hydroxyphenyl) propanoate;

b) 3-(4-Hydroxyphenyl)propionic acid xylitol ester: (2S,3S,4R)-2,3,4,5-tetrahydroxypentyl 3-(4-phydroxyphenyl)propanoate;

c) 3-(4-Hydroxyphenyl)propionic acid sorbitol ester: (2R,3R,4R,5S)-2,3,4,5,6-pentahydroxyhexyl 3-(4-hydroxyphenyl)propanoate;

and a lipase catalyzed ester formation pattern linked between the reaction of 3,5-dimethoxy-4-hydroxycinnamic acid - synaptic acid and 3 different solid polyalcohols ? shown in figure 9:

a) 3,5-dimethoxy-4-hydroxycinnamic acid erythritol ester: (2S, 3R) -2,3,4-trihydroxybutyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate ;

b) 3,5-dimethoxy-4-hydroxycinnamic acid xylitol ester: (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl (E) -3- (4-hydroxy-3,5- dimethoxyphenyl) acrylate;

c) 3,5-dimethoxy-4-hydroxycinnamic acid sorbitol ester: (2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl (E) -3- (4-hydroxy- 3,5 -dimethoxyphenyl) acrylate

can? be synthesized in a similar way to the described method considering its molecular weight of 166.17 g/mol and 224.21 g/mol respectively.

EXAMPLE 7: Process according to the present invention for the preparation of esters of benzoic acids.

Figure 10 shows a pattern of lipase catalyzed ester formation bonded between benzoic acid and 3 different solid polyalcohols (erythritol, xylitol and sorbitol):

a) benzoic acid ester with erythritol: (2S, 3R) -2,3,4-trihydroxybutyl benzoate

b) benzoic acid ester with xylitol: (2S, 3S, 4R) -2,3,4,5-tetrahydroxypentyl benzoate

c) benzoic acid ester with sorbitol: (2R, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyl benzoate

In connection with the previous examples 1-7, the evidence for effective lipase-catalyzed esterification ? was obtained by chromatographic assays (TLC, column chromatography, HPLC, uHPLC), <1>H- and <13>C-NMR and MS analysis. Spectroscopic analyzes demonstrate ester bond formation for enzymatically synthesized molecules. Esters have shown a greater affinity? for the stationary phase compared to the mobile phases of the chromatographies used in the analyses. This ? the proof of how the greater polarity? of the newly formed esters lower the logP compared to the starting acid forms, increasing the solubility? in compound water.

EXAMPLE 8: In vitro analysis to test the activity antimicrobial of the sorbic acid esters obtained by the process of the present invention.

The in vitro analysis? been performed to test the activity? antimicrobial (MIC, Disk Diffusion) of food preservative esters enzymatically esterified with the method according to the present invention. These esters can be advantageously used to enhance the activity? antimicrobial of poorly water soluble food and beverage preservatives.

Materials and methods

? been used the technique Disk Diffusion (DD), which allows the identification of the sensitivity? of various microorganisms against pharmaceutical compounds, and ? adequate when the resistance mechanism occurs due to the destruction of the antimicrobial agent by the microorganism. The bacterial species was Streptomyces griseus while the choice of the fungal species? fallout on Saccharomyces cerevisiae. The cultures were maintained in the laboratory as agar slants on a suitable culture medium: i.e. GYM (glucose-yeast-malt extract) for Streptomyces griseus and YMB (mannitol yeast broth) for yeast. To evaluate the activity Biologics, mother cultures of each organism were prepared in Petri dishes and grown to confluence.

So, the following compounds have been tested for their activity? antimicrobial: sorbic acid ester with erythritol, sorbic acid ester with xylitol and sorbic acid ester with sorbitol obtained according to the process of the present invention (see Example 5). In particular, appropriate agar medium (GYM or YMB) and different amounts were added to other Petri dishes. of the tested compounds. The final concentrations achieved are shown in Table 2.

Three replicates were used for each concentration. 100 ?l of the suspension containing 10<6 >unit? colony forming (CFU)/mL of microbial cells were spread on Petri dishes with appropriate agar-based growth medium (GYM or YMB). The compounds were solubilized using a 50% ethanol solution. ? been prepared a negative control by adding quantity? equal to this solution. The surface of the agar? been first inoculated with the specific test organism. Then, 5 mm diameter filter paper discs were lightly impregnated using 10 µL of drug derivative solution and placed on the agar surface. Clotrimazole [1 mg/mL] and chloramphenicol [2 mg/mL] were added to the positive controls, while solvent discs were made as negative controls. Were the cultured plates held upside down during incubation at 28? C for 6 days. The areas where the microorganism growth ? been blocked by the antibacterial agents are called zones of inhibition. To determine the activity antibacterial, zones of inhibition diameters for the test organism were measured in millimeters (mm), including a disc diameter of 5 mm, and compared to negative controls. To measure the diameter of the zone of inhibition, ImageJ ? been used as an image analyzer. Digital images of Petri dishes were analyzed after software calibration. The calibration step correlates the pixels of a reference in the image with the real-world distance measurement (mm). Have zones of inhibition been selected and the diameter of the selection ? been displayed in the "results" window of the software.

In addition, minimum inhibitory concentration (MIC) assessments were performed. The MIC? commonly defined as the concentration pi? of compound that completely inhibited (MIC100) or reduced to 50% (MIC50) clearly visible microbial growth after the entire incubation period, which was 6 days. These studies on newly formed sorbic acid esters will be enhanced with solubility tests. in water and stability? at different pH values.

Results

The results obtained showed higher inhibition zones (for the disk diffusion test) for the sorbic acid esters obtained according to the process of the present invention, with respect to the zones obtained for the non-esterified sorbic acid. Therefore, these results show that the esters according to the present invention have a greater activity? antimicrobial than the active ingredient of non-esterified sorbic acid.

Furthermore, also the minimum inhibitory concentrations (MIC) obtained show that lower concentrations of sorbic acid esters according to the present invention are required to inhibit the growth of the microorganism with respect to the concentrations required by the active ingredient in acid form.

EXAMPLE 9: In vitro analysis to test the activity anti-inflammatory properties of the ibuprofen esters obtained by the process of the present invention.

? an in vitro analysis was performed to test the activity? anti-inflammatory properties of the ibuprofen esters enzymatically esterified with the method according to the present invention.

In particular, the following esters obtained according to the present invention were tested:

Ibuprofen Erythritol Ester

Ibuprofen Xylitol Ester

Ibuprofen sorbitol ester

Ibuprofen ascorbic acid ester

Moreover, ? Ibuprofen glycerol ester was also tested.

The activity? anti-inflammatory of the aforementioned compounds? been tested against the canonical NSAIDs (ibuprofen).

Materials and methods

The cell line (ATCC? CRL2777?) selected? was cultured in 12-well plates. The next day the cells were treated with NSAIDs and with an equimolar concentration of the esters. TNFalpha, ? been used as an inflammatory stimulant. Total RNA, extracted using the TRIzol reagent, is been reverse transcribed. The cDNA? was the template for real-time PCR quantification. The quantity? relative of each mRNA studied? was normalized to GAPDH as a housekeeping gene, and data were analyzed according to the 2-??CT method.

Results

Preliminary data on the in vitro test of the NSAID derivatives (ibuprofen esters) synthesized with the method of the present invention demonstrate the same anti-inflammatory effects of the acid forms but with better solubility? in water. For esters with greater solubility? the anti-inflammatory effect is even better than the usual NSAIDs.

Claims (16)

1) A process for the preparation of an ester of a carboxylic acid with a molecule carrying a hydroxyl group, said carboxylic acid being sparingly soluble in water and the molecule carrying a hydroxyl group being a solid compound, wherein the process comprises or consists of: a) solubilizing the carboxylic acid and the molecule carrying a hydroxyl group in an organic phase capable of solubilizing both the carboxylic acid and the molecule carrying a hydroxyl group; and b) addition of an enzymatic catalyst suitable for catalysing the esterification, under stirring; wherein said an organic phase comprises or consists of a tertiary alcohol or a tertiary nitrile having a number of carbon atoms from 4 to 10, preferably from 4 to 8, plus? preferably from 4 to 6, or mixtures thereof, and said enzymatic catalyst ? a serine hydrolase in a catalytically active form. 2) Process according to claim 1, wherein in step a) the molecule carrying a hydroxyl group ? solubilized in said one organic phase under heating and stirring before adding the carboxylic acid. 3) Process according to any one of claims 1-2, wherein the ratio between the carboxylic acid and the molecule carrying a hydroxyl group varies from 10:1 to 1:10, such as for example from 5:1 to 1:5 . 4. Process according to claim 3, wherein the ratio between the carboxylic acid and the molecule carrying a hydroxyl group varies from 10:1 to 1:1, such as for example from 5:1 to 1:1, with the exclusion of 1:1 ratio. 5) Process according to any one of claims 1-4, wherein the carboxylic acid is selected from the group consisting of ibuprofen, mandelic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, sorbic acid, 3-phenylpropanoic acid, cinnamic acid, 3 - (4-hydroxyphenyl)propionic acid, 3,5-dimethoxy-4-hydroxycinnamic acid , benzoic acid, (2E) -3- (4-hydroxyphenyl) prop-2-enoic acid, 3,4,5-trihydroxybenzoic acid, benzene-1,2-dicarboxylic acid, 2-hydroxybenzoic acid, acetylsalicylic acid, succinic acid , stearic acid, tartaric acid, glutamic acid, malonic acid, (2E)-But-2-enedioic acid and indomethacin. 6) Process according to any one of claims 1-5, wherein the molecule carrying a hydroxyl group ? a molecule having a LogP value of -4.7 to 0.15, preferably -4.7 to 0, plus? preferably from -3.36 to 0, and carrying at least two hydroxyl groups, preferably at least three hydroxyl groups, plus? preferably at least four hydroxyl groups, even more? preferably at least five hydroxyl groups or at least six hydroxyl groups. 7) Process according to claim 6, wherein said molecule carrying a hydroxyl group ? selected from the group consisting of a polyalcohol such as sorbitol, xylitol, erythritol, ethylene glycol, ethene-1,1-diol, (2E)-2-butene-1,4-diol, maltitol or isomalt, preferably sorbitol; a sugar such as glucose, fructose, or sucrose; or ascorbic acid. 8) Process according to any one of claims 1-7, wherein said tertiary alcohol is t-amyl alcohol or t-butanol and called tertiary nitrile ? trimethylacetonitrile. 9) Process according to any one of claims 1-8, wherein said enzymatic catalyst ? an immobilized enzyme catalyst. 10) Process according to any one of claims 1-9, wherein said serine hydrolase ? selected from the group consisting of lipase or serine protease, such as subtilisin or trypsin. 11) Process according to claim 10, wherein said lipase ? selected from the group consisting of C. antarctica lipase B, lipase from R. miehei, lipase from T. lanuginosus or C. antarctica lipase A, preferably C. antarctica lipase B, plus? preferably immobilized C. antarctica lipase B. 12) Process according to any one of claims 1-11, wherein step a) and/or step b) comprise adding one or more? molecular sieves to remove water from the reaction medium. 13) Ester of a carboxylic acid poorly soluble in water with a molecule carrying a solid hydroxyl group, in which said ester ? chosen from the group consisting of: ester of ibuprofen with erythritol, such as 2,3,4-trihydroxybutyl 2-(4-isobutylphenyl)propanoate; ester of ibuprofen with xylitol, such as 2,3,4,5-tetrahydroxypentyl 2-(4-isobutylphenyl)propanoate; ester of ibuprofen with sorbitol, such as 2,3,4,5,6-pentahydroxyl 2- (4-isobutylphenyl) propanoate; ester of mandelic acid with ascorbic acid, such as 2-(3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl 2-hydroxy-2-phenylacetate; ester of ursodeoxycholic acid with erythritol, such as 2,3,4-trihydroxybutyl 4-(3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate; ester of ursodeoxycholic acid with xylitol, such as 2,3,4,5-tetrahydroxypentyl 4-(3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate; ester of ursodeoxycholic acid with sorbitol, such as 2,3,4,5,6-pentahydroxyl 4-(3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate; ester of deoxycholic acid with erythritol, such as 2,3,4-trihydroxybutyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate; ester of deoxycholic acid with xylitol, such as 2,3,4,5-tetrahydroxypentyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenan-17-yl)pentanoate; ester of deoxycholic acid with sorbitol, such as 2,3,4,5,6-pentahydroxyl 4-(3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoate; ester of sorbic acid with erythritol, such as 2,3,4-trihydroxybutyl (2E, 4E) -hexa-2,4-dienoate; ester of sorbic acid with xylitol, such as 2,3,4,5-tetrahydroxypentyl (2E, 4E) -hexa-2,4-dienoate; ester of sorbic acid with sorbitol, such as 2,3,4,5,6-pentahydroxyl (2E, 4E) -hexa-2,4-dienoate; ester of 3-phenylpropanoic acid with erythritol, such as 2,3,4-trihydroxybutyl 3-phenylpropanoate; 3-phenylpropanoic acid ester with xylitol, such as 2,3,4,5-tetrahydroxypentyl 3-phenylpropanoate; 3-phenylpropanoic acid ester with sorbitol, such as 2,3,4,5,6-pentahydroxyl 3-phenylpropanoate; 3-(4-hydroxyphenyl)propionic acid ester with erythritol, such as 2,3,4-trihydroxybutyl 3-(4-hydroxyphenyl)propanoate; 3-(4-hydroxyphenyl)propionic acid ester with xylitol, such as 2,3,4,5-tetrahydroxypentyl 3-(4-hydroxyphenyl)propanoate; 3-(4-hydroxyphenyl)propionic acid ester with sorbitol, such as (2,3,4,5,6-pentahydroxyphenyl 3-(4-hydroxyphenyl)propanoate; 3,5-dimethoxy-4-hydroxycinnamic acid ester with erythritol, such as 2,3,4-trihydroxybutyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate; 3,5-dimethoxy-4-hydroxycinnamic acid ester with xylitol, such as 2,3,4,5-tetrahydroxypentyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate; 3,5-dimethoxy-4-hydroxycinnamic acid ester with sorbitol, as 2,3,4,5,6-pentahydroxyl (E) -3- (4-hydroxy-3,5-dimethoxyphenyl) acrylate. 14) Pharmaceutical composition comprising or consisting of an ester as defined in claim 13 in combination with one or more? excipients and/or adjuvants. 15) Ester as defined in claim 13 or pharmaceutical composition as defined in claim 14 for use in anti-inflammatory therapy when the carboxylic acid is? ibuprofen. 16. Ester as defined in claim 13 or pharmaceutical composition as defined in claim 14 for use in the treatment of gallstones when the carboxylic acid is? ursodeoxycholic acid or deoxycholic acid.