WO2006094800A2

WO2006094800A2 - Cascade reaction process

Info

Publication number: WO2006094800A2
Application number: PCT/EP2006/002161
Authority: WO
Inventors: Paula Leandro L. Garcia; Jan C.M. Van Hest; Alan E. Rowan; Joost Nicolaas Hendrik Reek; Aldo Caiazzo
Original assignee: Synthon B.V.
Priority date: 2005-03-04
Filing date: 2006-03-03
Publication date: 2006-09-14
Also published as: WO2006094800A3

Abstract

The invention relates to a process, which comprises converting a chemical substrate into a chemical product by a cascade reaction wherein an enzyme-catalysed chemical transformation reaction and a metal-catalysed chemical transformation reaction are carried out essentially simultaneously.

Description

CASCADE REACTION PROCESS

The present invention deals with a process of converting a chemical substrate into a chemical product by a one-pot combination of an enzyme-catalysed and metal-catalysed chemical transformations.

BACKGROUND OF THE INVENTION The goal of classical organic synthesis is to carry out reactions with the highest possible selectivity and isolated yield, in order to afford new functionalized compounds. Traditionally, multistep processes usually require a sequence of single transformations and the consequent purification of the crude mixtures, which leads to an unavoidable drop in the amount of the recovered product and to remarkably time consuming procedures. On the other side, biologic systems are able to carry out multistep cascade reactions with high efficiency in energy balance and with no interference of the various catalytic sites involved. The combination of the versatility of synthetic organic procedures with the efficiency of biological systems looks appealing to achieve shorter and cheaper routes to functionalized chemicals. But there are problems with such an approach. For example, typically there is a wide difference in conditions required for a chemical transformation (extreme pH, high temperatures, organic solvents) versus a biocatalytic reactions (in most of the cases aqueous solvents, narrow pH and temperature range).

However, the use of enzymes like lipases that can work in apolar organic solvents may overcome these limitations. In particular CAL B, a fraction of the lipase from Candida Antarctica, can catalyze the esterification and amidation reactions in organic solvents and at temperatures higher than 60 °C. These features make CAL B a potential candidate for an employment in a cascade process involving an enzyme-catalyzed chemical transformation reaction, in combination with at least one other chemical reaction.

Several successful examples have been reported where this combination of chemical and biological processes within a cascade reaction has been used. For instance the dynamic kinetic resolutions (DKR) of secondary alcohols or amines lead to high enantiomeric excesses through a combination of metal catalyzed racemization and lipase promoted esterification, wherein the esterification works only with one enantiomer of the racemised substrate. The pioneering work of Reetz (M.T.Reetz, K.Schimossek, Chimia, 1996, 50, 668) in the DKR of 1-phenylethylamine demonstrates the applicability of the lipase-transition metal combination for conversion of amines. Even though it has been mentioned in several reviews (R.Sturmer, Angew .Chem.Int.EngL, 1997, 447; M.T.E1 Gihani, J.M.J. Williams, Curr.Opin.Chem.BioL, 1999, 3, 11), only the dynamic kinetic resolution of an amine has been carried out using systems employing a combination of an enzyme and transition metal catalysts. Thus, these known metal-catalyzed processes within a cascade reaction do not involve a change in chemical structure, i.e. a chemical transformation. A "chemical transformation" as will be used within the present invention is such a change in the structure of the chemical compound, - the substrate-, where at least one covalent bond in the molecule of the substrate is changed to produce a product different in number/arrangement of chemically bound atoms.

SUMMARY OF THE INVENTION

The present invention is based on a finding that it is possible to use metal-catalyzed chemical transformations, such as Pd-catalyzed couplings, in combination with an enzyme- catalyzed chemical transformation, such as amidation, in a one-pot fashion. In this way, two new functionalities may be introduced at different locations on the molecule of the substrate within a single process. This may have a broad application in organic synthesis and may provide for better selectivity, enhanced reaction rate and improved yield in comparison with a traditional sequential arrangement of the both reactions. Accordingly, a first aspect of the present invention relates to a process in which a chemical substrate is converted by a cascade of at least two chemical transformations into a chemical product, wherein the cascade of chemical transformations comprises an essentially simultaneous combination of an enzyme-catalyzed chemical transformation and a metal-catalyzed chemical transformation reactions. The "cascade" represents an arrangement in which the in situ formed product of the first chemical transformation of the substrate is immediately converted by the second, and optionally third, fourth, etc., chemical transformation, into the product of the cascade process. The enzyme-catalyzed reaction is typically an acylation reaction, especially using a lipase as the enzymatic catalyst. In some embodiments the enzyme-catalyzed acylation comprises an amidation of an aminic substrate R-NH2, catalyzed by a lipase, preferably CAL B lipase, wherein R is a carbon-containing organic group having a carbon directly bonded to the nitrogen. The substrate further contains a functional group (i.e. within the R moiety) which is susceptible of undergoing a metal-catalyzed transformation reaction. The metal-catalyzed reaction is typically a substitution or coupling reaction.

A particular aspect of the present invention relates to a process for making the compound of formula (2)

by the cascade process, which comprises a lipase-catalyzed amidation of substituted benzyl alkyl amines of formula ( 1 )

by an acyl donor, preferably with the compound of formula R₃-COO-R₄, in combination with a transition metal- catalyzed substitution of the group X on the phenyl ring of the compound of formula (1) with the group Y. In this process X represents a halogen, preferably bromine or iodine, R₁ represents a hydrogen or C1-C20 straight, branched or cyclic alkyl group, R₂ represents a hydrogen or C1-C20 straight, branched or cyclic alkyl group, R₃ represents hydrogen, C1-C20 straight, branched or cyclic alkyl, aryl or aralkyl group, R₄ represents hydrogen or Cl -C4 alkyl or C2-C4 alkenyl group, Y represents a group selected from a primary or a secondary amino group, an alkene, particularly styrene group, an alkyne, preferably a phenylalkyne group, or phenyl group, which is optionally substituted by at least one substituent , e.g. with an alkyl group, haloalkyl group, nitro group, alkoxy group etc.. In all embodiments of the invention, the enzyme catalyst is preferably a lipase enzyme, especially the CAL B lipase (Lipase B from Candida Antarctica). Moreover, the CAL B lipase is generally preferably used in an immobilized form. The metal catalyst is generally a transition metal and typically is provided as an organometallic compound. The preferred transition metal is selected from the group consisting of platinum, palladium, rhodium, and nickel, preferably palladium.

DETAILED DESCRIPTION OF THE INVENTION

Within a cascade process, a chemical substrate is transformed into a chemical product by a sequence of (at least) two sequential reactions, one of them immediately following the other. Thus, a substrate is chemically transformed by replacing two leaving atoms or groups Ll, L2 by two new moieties Nl, N2. Schematically, it may be depicted as follows:

Ll-Sub-L2 > Nl-Sub-N2 .

Within the invention, one of the chemical transformations is an enzyme-catalyzed transformation, and another one is a transformation catalyzed by a transition metal catalyst. Both catalysts are present in the reaction medium at the start of the process. Thus, in fact, the above schematically depicted process may comprise, in general, the following sequence (cascade): N₁-SUb — L₂ enzyme metal

The process of the present invention uses a chemical substrate which has at least two types of reactive sites: at least one for the enzyme-catalyzed transformation and at least one for the metal-catalyzed transformation. The enzyme and metal catalysts work at essentially the same reaction conditions, i.e. in the same solvent, under the same environmental pH and at the same temperature range. Surprisingly, it has been discovered that an enzymatic and metallic catalyst can be used concurrently. The reactions proceed essentially simultaneously on the substrate overall, albeit for a given molecule of the substrate, one of the reaction pathways is generally preferred. Nonetheless, the sequential reactions occur at the same time on different sites of the chemical substrate.

The present invention shall be demonstrated on a cascade reaction process, where the enzyme lipase shall be utilized as the catalyst, particularly the CAL B lipase from Candida Antarctica. The use of lipases in organic chemistry is a well-established and successful field of investigation. These enzymes, which can be genetically modified to tune their properties according to the reaction requirements, show a broad range of activity as far as experimental conditions and organic substrates are concerned. In particular there are a large number of reports dealing with the enantioselective esterification of alcohols and amidation of amines in the presence of lipases, which provide relatively inexpensive routes to valuable optically active compounds. The CAL B lipase is known to catalyze reactions in organic solvents, at enhanced temperatures and at pH which is distant from physiological pH.

To further illustrate the invention, which in no way is meant as a limitation, the invention will be discussed with reference to an amine substrate , which further contains at least one leaving group susceptible to undergoing a metal-catalysed chemical substitution reaction. More specifically, a suitable amine is of the general formula (1)

wherein X represents a halogen, preferably bromine or iodine, n is 1 to 3, R₁ represents a hydrogen or C1-C20 straight, branched or cyclic alkyl group, R₂ represents hydrogen or C1-C20 straight, branched or cyclic alkyl group and is preferably hydrogen, or R₁ ad R₂ together form a ring of 3 - 8 carbon atoms. This substrate indeed has two types of reactive centers, one of them being the group(s) X and the second one being the aminic hydrogen. When using the amine of formula (1) as a substrate, the cascade reaction involves a lipase-catalysed amidation of the amine with an acyl donor. The most useful acyl donor is an ester of a suitable acid.

The ester is "split" by the lipase, i.e. the alcoholate moiety of the ester is a leaving group in the amidation reaction. Using esters with good leaving groups, activated esters (for instance vinyl ester), drives the reaction to better yields. We observed that by using methyl esters, better results are obtained than by using ethyl esters. A point of note, however, is that one should be careful and not use too activated esters, since this might lead to problems with the metal catalysis. The acyl-group, which shall be introduced, is not particularly limited within the invention. The lipase may, in essence, amidate the amine substrate by an ester of whatever acid. The ester is generally of formula R₃-COO-R₄, and preferably the R₃ represents a hydrogen, a C1-C20 straight, branched or cyclic alkyl group, an aryl group, or an C7-C10 aralkyl or a C7-C10 alkaryl group; and R₄ preferably represents a C1-C4 alkyl , C7-C10 aralkyl, C2-C4 alkenyl or C2-C4 alkynyl group.

A very suitable acyl donor is, e.g., ethyl acetate , methyl acetate or vinyl acetate , which provides acetamides. As to the amine, the process of the present invention may particularly be used on chiral amines of formula (1) (R₁ in formula (1) is not a hydrogen), since the enantioselectivity properties of CAL B can be utilized. This enzyme shows a preference for the (R)-enantiomer (95% yield). However it is also possible, in case of longer reaction times, to convert the (S)-enantiomer, although in very low rates (14%). It is this enantio- preference that makes this enzyme very attractive for making single enantiomers. Using a racemic 4-bromo-α-methylbenzylamine, the amide obtained by CAL B catalysed amidation has the (R) configuration, and was obtained in 47% yield. Note that, theoretically, the maximum yield that can be obtained using a racemic amine is 50%. In a preferred way, the lipase is used within the cascade reaction in an immobilized form. An example of such form is the commercially available NOVOZYM 435^® (Novozymes A/S), which is the CAL B lipase immobilized on an acrylic resin. Whereas the CAL B itself is thermophilic enough to express its maximum activity around 60 °C, the immobilized version of this enzyme, NOVOZYM 435^®, can be even used up to 100 °C and beyond, thus representing a good choice for the cascade process of the present invention.

A second example is the lipase immobilised within a polymerosome, which is an analogue of a liposome made from artificial polymers. For example the diblock copolymer polystyrene-b-poly (L-isocyanoalanine(2-thiophen-3-yl-ethyl)amide), referred to as PS- PIAT, is able to form very stable and well defined spherical polymerosomes in water and organic solvents. The lipase may be introduced both into the polymerosome membrane and/or into the aqueous pool inside the membrane.

As to the solvent, in principle it is possible to perform the amidation in apolar solvents. The activity of the CAL B enzyme is known to be higher in hydrophobic solvents than in hydrophilic ones. The temperature can effect the enzyme activity. CAL B is very robust (up to 65⁰C it is stable); however, it is still an enzyme, which means that very high temperatures can denaturize the enzyme resulting in a loss of activity, if it is not immobilized.

For the amidation reaction, the enzyme is preferably applied in a water-free and oxygen-free organic solvent (on presence of molecular sieves, under nitrogen or argon atmosphere flow). In order to get good yields (>50%) it is necessary to use an excess of acyl donor (ester) and a certain amount of immobilized enzyme (-100 mg per mmol of amine). As solvents, better yields were obtained when hexane, toluene and ethyl acetate were used. Ethyl acetate gives an extra advantage, since it can be used not only as solvent but also as acyl donor, resulting in very good yields. In this way, the work up of the reaction is also very easy, as the excess of ester may be eliminated by evaporation. The amidation using EtOAc can also be carried out in other solvents, as hexane or toluene, and the amide is also obtained in very good yield.

The second part of the cascade reaction is the metal-catalyzed chemical transformation. Taking the above compound of formula (1) as an example of the substrate for the cascade reaction, the chemical transformation involves a replacement of at least one of the reactive groups X by the moiety Y, by using a corresponding donor of the Y-moiety and a metal catalyst:

cat.

cat.

The donor of the Y-moiety for the metal-catalyzed chemical transformation within the cascade reaction process is not particularly limited within the present invention. The principal requirement however is that the metal-catalyzed chemical transformation is able to proceed at the same conditions as the amidation reaction. Examples of suitable substitution reactions that can be used as the metal-catalyzed chemical transformation include animation, Heck coupling , Suzuki-Miyaura coupling and Sonogashira coupling reactions, but are not limited thereto.

A) An amination with a primary or secondary amine (Buchwald-Hartwig coupling).

Palladium-catalyzed amination of aryl halides has become the most convenient method to prepare aromatic amines. It involves the use of mild conditions and can be a useful tool in the synthesis of pharmaceuticals and special materials. A big effort has been devoted in searching for catalysts which provide high turnover numbers and selectivities.

In general, the source of the Y-moiety which will be introduced into compounds of general formula (1) by the cascade reaction of the present invention is a primary or secondary amine of the formula

wherein A and B are either, independently, hydrogen or (optionally substituted) alkyl/aryl/aralkyl groups of 1-20 carbon atoms, or they together form a ring of 3-20 carbon atoms. A simple example of a useful primary amine is aniline and a simple example of a secondary amine is piperidine.

In this case, the metal catalyst is preferably a palladium-containing organometallic complex, e.g. Pd(dba)₂ (dba = dibenzylideneacetone) with a phosphine ligand such as P(t- Bu)₃ or

DPEphos=(bis[2-(diphenylphosphano)phenyl]ether

Xaπtphos=(9,9-dimethyl-4,6-bis[diphenylphosphano]xantene Xantphos and P(tBu)₃ (added as its tetrafluorborate salt) turned out to be the best ligands in terms of activity, although the use OfPtBu₃ led to a slightly lower selectivity than Xantphos, leading to 8% of dehalogenation product.

Anyway, there is one parameter to be taken into account. The amine that is supposed to undergo the metal-catalyzed animation process should be relatively inert to the action of CAL B, at least within the time frame of the cascade reaction. Accordingly, amines that show a high reactivity, e.g. rapid conversion, in the presence of CAL B are generally less suitable for use in the cascade reaction. From this point, aromatic amines such as anilines are good candidates. The results show, e.g., that by the time 4-bromobenzylarnine (Ia) has been completely converted by the CAL B enzyme towards its corresponding amide, the amount of aniline converted is still negligible. This means that aniline can be used in the cascade reaction as the amination reagent on 4-bromobenzylamine (Ia) without the formation of extensive by-products.

On the other hand, experimental results prove that CAL B is a very robust enzyme which can work in the presence of the metal catalyst and that it is a suitable candidate for a one-pot cascade process.

In an example of the amidation/amination cascade reaction, 4-bromobenzylamine of formula (Ia) may be converted by the cascade process using immobilized CAL B lipase and Xanthphos-liganded palladium catalyst Pd(dba)₂ into 4- (phenylamino)benzylacetamide (2a) in toluene and at 100 ⁰C as shown on the following scheme.

100⁰C CO₃

After 24 hrs, the conversion of Ia was 94% while the selectivity of the cascade product 2a was 84%. Using methyl acetate instead of ethyl acetate, the conversion and selectivity were even better. The aniline in the above scheme may be replaced by a substituted aniline. One or more substituents such as an alkyl, alkoxy, nitro or a fluoro group may be used. For instance 2-methyl aniline, 4-methoxy aniline, 3 -nitro aniline and 3- fluoroaniline exhibit similar conversion and selectivity rate as the aniline itself, yielding corresponding compound of formula (2a), where Ph now represents the accordingly substituted phenyl group.

The 4-bromobenzylamine may be replaced by 3-bromobenzylatnine (Ib) in the cascade reaction, yielding 3-(phenylamino)benzylacetamides (2b) in similar degree of conversion and selectivity. These results show that a bio-enzymatic process (CAL B catalyzed amidation) and a palladium catalyzed coupling can occur under the same experimental conditions with no reciprocal inhibition. But the most surprising finding is the apparent improvement of the rate of the animation process, which was pretty poor in the case of a separately investigated animation of p-bromobenzylacetamide in the presence of aniline. Comparative experiments showed that the animation reaction, where NOVOZYM 435^® was added to the mixture, goes faster than the reaction where this additive is absent. This might suggest that a cooperative effect between the enzyme and the palladium catalyst takes place in the reaction mixture. The observed substantive improvement of conversion may be probably caused by the presence of ethanol or methanol, as the side product of the enzyme- catalysed acetylation and of water, as a part of the immobilized lipase . In a model experiment, it was proven that an alcohol and/or water enhances the speed and selectivity of the process . The alcohol probably activates the base ( the cesium carbonate used as a base is apparently partly converted into cesium alkoxide or cesium hydroxide)

Whatever the origin of the cooperative effect might be, it is clear that the presence of NOVOZYM 435^® leads to a stabilization of the active catalyst species, enhancing the rate of the animation process.

B) Heck coupling In this case, the donor of the Y-moiety is an alkene , i.e. a compound of formula

wherein at least one of Zl, Z2, and Z3 groups is an alkyl- or aryl group, whereby the remaining are hydrogens. Typically an alkyl group contains 1 to 20 carbon atoms and an aryl group contains 6 to 20 carbon atoms such as phenyl. Alkyl or aryl groups may be optionally substitured by further groups.

The transition metal-catalysed reaction between the alkylhalogenide and alkene in the presence of a base is known as Heck coupling. In general, electron poor alkenes react more readily and more selectively than electron-rich olefins. Preferably, the alkene compound is a styrene-type compound, which is a donor for a vinylbenzene moiety.

A wide variety of bases can be applied, ranging from inorganic salts to tertiary amines. Triethylamine is a standard base used in the Heck coupling and may be also employed in the cascade reaction process. Usually polar, non-protic solvents give rise to the highest conversions. Concerning the metal catalysts, again the most suitable are palladium catalysts, for instance Pd acetate as Pd source and phosphoramidite derivative or P(o-tol)₃ as ligand.

The reaction is illustrated on the scheme below, using m-iodobenzylamine (Ic) as the starting material, ethyl acetate as the amidation reagent and styrene as the Y-group donor. The product is trans-N-(3-styryl-benzyl)-acetaniide (2c).

S eq , mol. sieves, Novozym 435

Me)₃ only TRANS _(2c)

(1 c) Again, similarly as observed above, the concomitant presence of the enzyme and metal catalysts improves the overall reaction rate in comparison with an arrangement where both reactions would be performed sequentially. In the presence of the zeolite and the enzyme, the Heck reaction was found to proceed within 4 hr in contrast to 5 days without the enzyme and also in 90% yield compared to 30% yield.

In addition, the particular advantage of implementation of the Heck coupling within the cascade reaction process of the present invention is the observation that while an halobenzyl amine provides a cis/trans product, a corresponding amide provides preferably a trans- product.

This advantage is pronounced even further, if a chiral amine of general formula (1) is used as a starting reagent. In this way, the enzyme converts enantioselectively the amine into an amide, activating the Heck reaction which is also stereoselective ending up with a stilbene derivative amide with a chiral center in the R configuration and the double bond in trans configuration.

C) C-C coupling by a Suzuki-Miyaura reaction

The donor of the group Y in the Suzuki-Miyaura coupling reaction is an arylboronic acid, which introduces an aryl, particularly phenyl group as a substituent as the Y group, replacing the halogen, preferably bromine, as the X group.

The cascade reaction process combining CAL B catalyzed amidation and Suzuki- Miyaura coupling on substrates of general formula (1) may proceed under similar experimental conditions as used for the previously described amidation/amination cascade process. The suitable Pd catalyst is, e.g., a combination of the same catalyst precursor as for the above animation reaction while the ligand is preferably a tris-tertbutyl phosphonium tetrafluoroborate salt, a system that releases tris-tertbutyl phosphine directly into the reaction mixture when a base, even as mild as KF, is present .

Using p-bromobenzylamine (Ia) as an example of the substrate of formula (1), the amidation/Suzuki coupling cascade reaction proceeds in toluene as shown on the scheme below.

(Ia) (2d)

The product of the process is p-phenylbenzylacetamide (2d). The conversion is essentially complete within 24 hours. The reaction runs parallelly in both directions

(amidation and coupling vs. coupling and amidation), as intermediates of both particular reactions may be found in the reaction mixture. The relative amount of potassium fluoride in the reaction mixture is important to obtain satisfactory conversion, wherein 2 molar equivalents in respect to the starting (Ib) seem to be optimal. As the source of acyl moiety, ethyl acetate is the compound of the first choice, but methyl acetate and particularly vinyl acetate provide for satisfactory results as well

Accordingly, the phenylboronic acid may be replaced by a substituted phenyl boronic acid. Then the Ph moiety in the compound (2d) shall represent the substituted phenyl group. D) C-C coupling by a Sonogashira reaction

The cascade approach may be used in application of another Pd-catalyzed reaction, the Sonogashira coupling of alkynes with arylbromides.

The application of the approach of the present invention is shown on the following scheme, using 4-bromobenzylamine (Ia) as the substrate and phenylacetylene as the reagent CH₃-COOMe, CAL-B

Pd(dba)₂ _ Ph(o-C₆H₄)P(tBu)₂

(Ia) Cs₂CO₃ toluene, 100⁰C

The process was carried out with 2-(ditertbutylphosphino)biphenyl as ligand and in the absence of any copper precursor, providing the expected final product (2e) in 89 % GC selectivity.

The products of the cascade process of the present invention may be used in various branches of industrial organic chemistry as industrial chemicals. They may be used as such or may be converted into other chemical compounds. For instance, they may serve as intermediates in making agrochemicals, food additives or drugs. In an example, the cascade process of the present invention may be used in the synthesis of repaglinide, a well known antidiabetic. Starting from the amine (Ie), which even may be in a racemic form, repaglinide may be prepared, in the desired conformation, in a single step process employing the cascade reaction comprising a lipase-catalyzed amidation with an ester of corresponding phenylacetic acid and a Pd-catalyzed animation with piperidine:

In another interesting aspect, the resulting amide of general formula (2) can be, if desired, converted to an amine in a separate reaction step, so that the ultimate product of the cascade process and the conversion process is an amine of formula (3).

In comparison with a simple metal-catalyzed replacement of the group X in the compound of formula (1) by a group Y₅ the use of the cascade reaction followed by the amide-amine conversion has several advantages:

The first advantage of the cascade reaction is based on the finding that the lipase- catalyzed amidation sufficiently protects the amino-group in the substrate against the metal-catalyzed autocondensation of the amino-group with the group X, upon formation of side products.

The second advantage is that the amide compound is more reactive for the metal- catalyzed transformation.

The third advantage is that it is possible to prepare enantiomerically enriched products from racemates or racemic mixtures, which is not possible by the simple X-Y transformation on the substrate of formula (1).

The versatility of the process of the present invention may be demonstrated on the fact, that the enzyme may catalyse not only the amidation reaction. Any enzymatically convertible reactive substrate may be used in general. For instance, as briefly mentioned above, the lipase within the cascade process may also catalyse a conversion of alcohols into esters by acylation . The acyl donor is an ester of formula R₃-COO-R₄, wherein R₃ represents a hydrgoen, a C1-C20 straight, branched or cyclic alkyl group, an aryl group, or an aralkyl group; and R₄ represents a hydrogen or a C1-C4 alkyl or alkenyl group. The alcohol may typically have from 1 to 20 carbon atoms, which may form an aliphatic or one or more of cyclic chains.

An example of a useful cascade process combines lipase promoted acylation and Cu catalyzed coupling between propargylic alcohols and azides. The propargylic alcohol is generally of the formula

wherein R and/or Rl is hydrogen or an alkyl group.

The two catalyst systems work efficiently in the same pot to provide valuable triazole-containing substrate, with great potential for the production of pharmaceuticals. An example is shown on the following scheme

"Cu(I)", DIPEA

Toluene, AcOR

NOVOZYME 435®, 30 C

The use of ethyl or methyl acetate did not ensure a complete conversion in the acylation steps, which was reached by using vinyl acetate as source of acyl moiety. The regioselectivity in the product 9 was complete. An interesting cooperativity effect may be observed when the reaction is carried out only in the presence of metallic copper shreds and NOVOZYM 435^®. In this case the coupling reaction proceeds along with the acylation step even though there is no diisopropylethylamine (DIPEA) added to the mixture as the base. In this case it is likely that the histidine residues in the enzyme act as the necessary basic sites for forming the intermediate species .

Chiral alkynols ( i.e. having three different substituents in the the position 1 ) may yield the corresponding end product in an enantioselective way. Experimental section a) General considerations

Materials. All chemicals were purchased from commercial sources and used without further purification. NOVOZYM 435^® was purchased from Aldrich, stored at +4 °C and used as received. Solvents were dried prior to their use. Toluene was distilled from sodium. Ethyl acetate was predried on P₂O₅ and then distilled from CaH₂. All experiments were performed using standard Schlenk techniques under an argon atmosphere.

Analytical Techniques. NMR spectra were recorded using CDCl₃ as a standard for ¹H. GC analyses were performed on a Shimadzu GC- 17A instrument, column type DB-I (J & W; 30 m x 0.32 mm). Mass spectra (EI, 70 eV) were recorded on a Apilent Technology 6890/5973 - GC/MS instrument, column type HP - 5 MS (30 m x 0.25 mm). b) Examples Example 1 Amination/amidation cascade reaction

Cesium carbonate (560 mg, 1.72 mmol), Pd(dba)₂ (14 mg, 0.0244 mmol), Xantphos (0.0366 mmol) were inserted into a schlenk under argon atmosphere. The system was then briefly evacuated and backfilled with argon (3 cycles). At this point freshly deoxygenated toluene (6 mL) was inserted into the schlenk under argon and the mixture was briefly stirred (5 seconds). Then brombenzylamine (1) (1.20 mmol), aniline (1.25 mmol), ethyl or methyl acetate (3.60 mmol) and 200 mg NOVOZYME 435^® were inserted into the schlenk under argon atmosphere. The resulted mixture was then heated up to 100 ⁰C and stirred for 20 h, then it was cooled down, filtered and the residue washed with 20 mL of ethyl acetate. The solvents were removed and the the crude oil was eluted on silica (gradient CH₂Cl₂MeOH from 100:0 to 98:2) to provide N-[4-(phenylamino)benzyl]acetamide as a yellow solid (60 % yield). ¹H-NMR (500 MHz, CDCl₃): D 7.26 (dd, , J= 8 Hz, 7.5 Hz, 2H), 7.14 (d, J= 8.5 Hz, 2H), 7.06 (d, J= 8 Hz, 2H), 7.01 (d, J= 7.5 Hz, 2H), 6.93 (t, J= 7.5 Hz, IH), 6.46 (bs, IH), 6.07 (bs, IH), 4.31 (d, J= 5.5 Hz, 2H), 1.97 (s, 3H). ¹³C-NMR (125.7 MHz, CDCl₃): D D 170.5, 143.4, 142.8, 130.7, 129.6, 129.2, 121.1, 118.0, 117.9, 43.527, 23.4. MS (m/z): 240 (100 %), 197 (29 %), 182 (92 %). m.p. 103 ⁰C.

Example Ia

Under essentially the same conditions as in Example 1, various amine nucleophiles were used to replace the aniline in the Example 1. The conversion rates and yield of the corresponding products in the reaction mixture are in the followinmg Table 1 Table 1

Amination/amidation cascade reaction carried out on 4-bromobenzylamine (Ia) in the presence of several amine nucleophiles^a.

MeO- 82^C

95

-NH,

98°

O₂N > 99

^aReactions carried out at 100 ⁰C in toluene; concentration of Ia: 0.21 rnM; amine nucleophile: 1.05 eq; catalyst precursor: Pd(dba)₂ (2%); XANTPHOS (3%); Cs₂CO₃ as base (1.4 eq); NOVOZYME 435^® (100 mg); AcOMe (3 eq). ^bDetermined via GC analysis of the crude reaction mixtures after 24 h of stirring. ^cDetermined via ¹H-NMR analysis of the crude reaction mixtures after 24 h of stirring.. Example 2

Suzuki coupling/amidation cascade reaction

Phenyl boronic acid (148 mg, 1.21 mmol), potassium fluoride (188 mg, 3.23 mmol), Pd(dba)₂ (12.5 mg, 0.0217 mmol) and P⁴Bu₃H⁺ BF₄ ^" (8 mg, 0.0270 mmol) were inserted into a schlenk under argon atmosphere. The system was then briefly evacuated and backfilled with argon (3 cycles). At this point freshly deoxygenated toluene (6 niL) was inserted into the schlenk under argon and the mixture was briefly stirred (5 seconds). Then 4-bromobenzylamine (Ia) (1.08 mmol), ethyl acetate (3.60 mmol) and 200 mg NOVOZYME 435^® were inserted into the schlenk under argon atmosphere. The resulted mixture was then heated up to 100 ⁰C and stirred for 20 h, then it was cooled down, filtered and the residue washed with 20 mL of ethyl acetate. The solvents were removed and the the crude oil was eluted on silica (gradient CH₂Cl₂/Me0H from 100:0 to 98:2) to provide N-[4-(phenylamino)benzyl]acetamide as a white solid (60% yield). ¹H-NMR (500 MHz, CDCl₃): D 7.57 (m, 4H), 7.45 (t, J= 7.5 Hz, 2H), 7.36 (m, 3H), 5.73 (bs, IH), 4.49 (d, J= 5.5 Hz, 2H), 2.06 (s, 3H). ¹³C-NMR (125.7 MHz, CDCl₃): D 170.3, 140.9, 140.8, 137.4, 129.0, 128.6, 127.7, 127.6, 127.3, 43.7, 23.5. MS (m/z): 225 (100 %), 182 (66 %). m.p. 182 °C (lit. 180-182 ⁰C).

Example 3

Sonogashira coupling/amidation cascade reaction

Cesium carbonate (560 mg, 1.72 mmol), 2-(ditertbutylphosphino)biphenyl (22.5 mg, 0.0756 mmol), Pd(dba)₂ (15.5 mg, 0.0270 mmol) were inserted into a schlenk under argon atmosphere. The system was then briefly evacuated and backfilled with argon (3 cycles). At this point freshly deoxygenated toluene (6 mL) was inserted into the schlenk under argon and the mixture was briefly stirred (5 seconds). Then 4-bromobenzylamine (Ia) (1.08 mmol), phenylacetylene (1.51 mmol), 0.30 mL methyl acetate (3.60 mmol) and 200 mg NOVOZYM 435^® were inserted into the schlenk under argon atmosphere. The resulted mixture was then heated up to 100 ⁰C and stirred for 20 h, then it was cooled down, filtered and the residue washed with 20 mL of ethyl acetate. The solvents were removed and the the crude oil was eluted on silica (gradient CH₂Cl₂/Me0H from 100:0 to 98:2) to provide N-[4-(2-phenylethynyl)benzyl]acetamide as a white solid

¹H-NMR (500 MHz, CDCl₃): D 7.53 (m, 2H), 7.50 (d, J= 8.0 Hz, 2H), 7.35 (m, 3H), 7.26 (d, J= 8.0 Hz, 2H), 5.81 (bs, IH), 4.45 (d, J= 6.0 Hz, 2H), 2.04 (s). ¹³C-NMR (125.7 MHz, CDCl₃): D 170.1, 138.7, 132.1, 131.8, 128.6, 128.5, 128.0, 123.4, 122.8, 89.8, 89.2, 43.7, 23.5. MS (mlz): 249 (100 %), 206 (66 %), 191 (30 %), 178 (31 %). m.p. 156 ⁰C.

The invention having been thus described, it will be obvious to the worker skilled in the art that the same may be varied in many ways without departing from the spirit of the invention and all such modifications are included within the scope of the present invention as set forth in the following claims.

Claims

L A process, which comprises converting a chemical substrate into a chemical product by a cascade reaction wherein an enzyme-catalysed chemical transformation reaction and a metal-catalysed chemical transformation reaction are carried out essentially simultaneously.

2. The process according to claim 1 in which the enzyme used as a catalyst is a lipase.

3. The process according to claim 1 or 2 wherein the chemical substrate is an amine, which further contains at least one leaving group susceptible to undergoing a metal- catalysed chemical substitution reaction.

4. The process according to claim 3, wherein the amine chemical substrate has the general formula ( 1 )

wherein R₁ represents a hydrogen or a C1-C20 straight, branched or cyclic alkyl group; R₂ represents hydrogen or a C1-C20 straight, branched or cyclic alkyl group or R₁ ad R₂ together form a ring of 3 - 8 carbon atoms ; and X represents a halogen, preferably bromine or iodine and n is 1 to 3 .

5. The process according to claims 1-4 wherein the enzyme-catalysed chemical transformation is an amidation with an acyl donor.

6. The process according to claim 5 wherein the acyl donor is an ester of formula R₃- COO-R₄, wherein R₃ represents a hydrogen, a C1-C20 straight, branched or cyclic alkyl group, an aryl group, an C7-C10aralkyl or a C7-C10 alkaryl group; and R₄ represents a Cl-C4 alkyl, C7-C10 aralkyl, C2-C4 alkenyl or C2-C4 alkynyl group .

7. The process according to claims 2-6 wherein the lipase is a CAL B lipase.

8. The process according to claim 7 wherein the CAL B lipase is in an immobilized form.

9. The process according to claims 4-8 wherein the metal-catalyzed chemical transformation is a substitution of the group X by a group Y selected from a primary or a secondary amino group, an alkene, particularly styrene group, an alkyne, preferably a phenylalkyne group and a phenyl group which is optionally substituted by at least one substituent , e.g. with an C1-C20 straight, branched or cyclic alkyl group, halo C1-C20 straight, branched or cyclic alkylgroup, nitro group, C1-C20 straight, branched or cyclic alkoxy group.

10. The process according to claim 9 wherein the metal catalyst is an organometallic compound containing a metal selected from the group consisting of platinum, palladium, rhodium, and nickel, and preferably palladium.

11. A process for making the compound of formula (2)

by a cascade process, which comprises a lipase-catalyzed amidation of substituted benzyl alkyl amines of formula (1)

by an acyl donor, preferably a compound of formula R₃-COO-R₄, in combination with a transition metal-catalyzed substitution of the group X with the group Y; wherein X represents a halogen, preferably bromine or iodine, R₁ represents a hydrogen or C1-C20 straight, branched or cyclic alkyl group, R₂ represents a hydrogen or C1-C20 straight, branched or cyclic alkyl group, R₃ represents hydrogen, C1-C20 straight, branched or cyclic alkyl or phenyl group, R₄ represents hydrogen or C1-C4 alkyl or C2- C4 alkenyl group, Y represents a group selected from a primary or a secondary amino group, an alkene, particularly a styrene group, alkyne, particularly a phenylalkyne group and phenyl group, which may be optionally substituted.

12. The process according to claim 11, wherein the lipase enzyme is a CAL B lipase (Lipase B from Candida Antarctica).

13. The process according to claim 12, wherein the CAL B lipase is in an immobilized form.

14. The process according to claims 11-13, wherein the transition metal catalyst is an organometallic compound, wherein the metal is selected from a group comprising platinum, palladium, rhodium, or nickel, preferably palladium.

15. A process which comprises subjecting a chemical substrate to an enzyme- catalyzed acylation reaction and to a metal-catalyzed substitution reaction essentially simultaneously to produce an acylated and substituted product.

16. The process according to claim 15, wherein said enzyme-catalyzed acylation reaction is carried out using a Lipase enzyme and an ester reagent of the formula R₃-COO- R₄ wherein R₃ represents hydrogen, C1-C20 straight, branched or cyclic alkyl or phenyl group, and R₄ represents hydrogen or C1-C4 alkyl or C2-C4 alkenyl group.

17. The process according to claim 15 or 16, wherein said metal-catalyzed substitution reaction is selected from the group consisting of an amination reaction with a primary or secondary amine, a Heck coupling with an alkene, Sonogashira coupling with an alkyne and a Suzuki-Miyaura coupling reaction with an arylboronic acid.

18. The process according to claim 1, 2 or 15 wherein the chemical substrate is an C1-C20 straight, branched or cyclic alcohol, which further contains a leaving group susceptible to undergoing a metal-catalysed chemical substitution reaction.

19. The process according to claim 18, wherein the enzyme-catalysed chemical transformation is an acylation with an acyl donor.

20. The process according to claim 19 wherein the acyl donor is an ester of formula R₃-COO-R₄, wherein R₃ represents a hydrgoen, a C1-C20 straight, branched or cyclic alkyl group, an aryl group, or an aralkyl group; and R₄ represents a hydrogen or a C1-C4 alkyl or alkenyl group.

21. The process according to claims 18-20 wherein the lipase is a CAL B lipase.

22. The process according to claims 18-21 wherein the CAL B lipase is in an immobilized form.

23. The process according to claim 18-22 wherein the alcohol is of the formula

wherein R and/or Rl is hydrogen or an alkyl group

24. The process according to claim 23 wherein the metal-catalysed chemical transformation is copper-catalysed coupling of an azide on a triple bond under formation of a 1,2,3 -triazole nucleus.