WO2006114096A1

WO2006114096A1 - Pyrrolidinone dipeptide analogs and use thereof as artificial sweeteners

Info

Publication number: WO2006114096A1
Application number: PCT/DK2006/000215
Authority: WO
Inventors: Janne Ejrnæs TØNDER
Original assignee: Danmarks Tekniske Universitet
Priority date: 2005-04-25
Filing date: 2006-04-25
Publication date: 2006-11-02

Abstract

The present invention relates to novel pyrrolidinone derivatives having structural resemblance to certain dipeptide sweeteners such as Aspartame and Alitame. The invention further relates to a method for producing the compounds according to the invention. Further the invention relates to the use of the compounds according to the invention as artificial sweeteners.

Description

Title: Pyrrolidinone dipeptide analogs and use thereof as artificial sweeteners-

Technical Field

The invention provides novel pyrrolidinone derivatives, salts, racemates and stereoisomers thereof, as well as compositions comprising said pyrrolidinone derivatives.

The invention further provides a novel chemical method for the synthesis of the pyrrolidinone derivatives according to the invention.

Further the invention provides the use of the novel pyrrolidinone derivatives as a taste enhancing and/or sweetening additive in compositions for oral consumption.

Background Art

Artificial non-carbohydrate sweeteners are increasingly replacing sugar in consumable products. Non-carbohydrate sweeteners are generally divided into two different structural classes of compounds, the sulfonamides and the dipeptides. Among the sulfonamides are found compounds like saccharin, Acesulfame-K and cyclamate and among the dipeptides are found Alitame and Aspartame. The demand for non-carbohydrate artificial sweeteners have continuously increased in the last decades and are believed to further increase in the future.

Artificial non-carbohydrate sweeteners commonly comprise an anionic group (sulphate in saccharin, Acesulfame-K and cyclamate, carboxylate in Aspartame and Alitame), which is believed to be essential to the taste of the compounds. The dipeptide sweet- eners contain a cationic group and a hydrophobic group (phenyl in Aspartame and tetramethylthietanyl in alitame) as well.

Today the dipeptides such as Aspartame and Alitame are among the most successful artificial sweeteners. Aspartame is already widely used and Alitame has shown promising potential. Previous studies of dipeptide structure-taste relationships have shown that the aspartic acid residue is essential to the taste of the compounds and cannot be replaced by other amino acids. The second peptide residue however may vary significantly and aspartame derivatives containing several different substitutions in the second peptide, e.g. phenylalanine (Aspartame), D-alanyl tetramethylthietanylamide (Alitame), phenylglycine, tyrosine, methionine, glycine methyl ester are known to provide a sweet taste. Different artificial sweeteners have different characteristics and functionalities in relation to for example solution stability and costs of synthesis. Most importantly however the taste profiles of different artificial sweeteners vary significantly. Aspartame has long dominated the commercial market for artificial sweeteners, and consequently the con- sumers have grown accustomed to the taste of Aspartame. However no artificial sweeteners known today exactly match the taste profile of natural sugar and consequently sweeteners are commonly used in mixtures or blends in order to achieve specific taste profiles.

These different taste characteristics of artificial sweeteners highlight the need for the development of new low-calorie compounds useful for altering the taste profile of specific compositions. Especially new artificial sweetening compounds that have structural resemblance to Aspartame would be desirable.

EP 0 483 667 discloses pharmaceutically active compounds among which are compounds comprising an amino moiety coupled via a linking group to a pyrrolidinone ring. These compounds differ structurally from the compounds according to the present invention i.a. in the presence of the linking group.

Disclosure of Invention

It has surprisingly been found that Aspartame analogs comprising a pyrrolidinone ring substituting the aspartic acid residue can be synthesized. Said pyrrolidinone derivates have some structural resemblances to saccharin and in addition is predicted to possess the functional characteristics of the aspartic acid residue of dipeptide sweeteners.

The present invention thus relates to pyrrolidinone derivatives characterized by the general formula (1),

wherein R¹, R², R³, R⁴ and R⁵, which may be identical or different, are selected independently from the group consisting of hydrogen, C₁-C₆ alkyl, OH, NH, SH, 0-(C₁- C₆)-alkyl, N-(CrC₆)-alkyl, S-(C_rC₆)-alkyl, benzyl and aryl, R⁶ is either hydrogen or a protective group, X is selected from the group consisting of residues of amino acids and amino acid derivatives, and wherein said amino group of X is directly coupled to the carbon atom in the 3rd position of the pyrrolidinone ring, as well as salts, racemates and diastereomers thereof.

The term "alkyl" used herein includes C₁-C₆ straight chained, C₁-C₆ branched chain saturated and C₃-C₆ cyclic saturated hydrocarbon groups.

The term "aryl" used herein includes unsubstituted, mono-, di-, or trisubstituted monocyclic, polycyclic, biaryl or heteroaryl aromatic groups covalently attached to any ring position capable of forming a stable covalent bond.

In the present disclosure the nomenclature or naming of the pyrrolidinones and pyrrolidinone derivatives is chosen to closely resembles the peptide nomenclature, using the prefix py before the three letter amino acid code to indicate the pyrrolidinone instead of the amino acid. As is shown below, the naming of protecting groups is identical to the general use in peptides.

Unprotected

H-pyGly-OH H-pyAla-OH H-pyAsp-OH pyGIy pyAla pyAsp

W-Protected

Boc-pyGly-OH Boc-pyAla-OH Boc-pyAsp-OH

Side chain protected

Boc-pyAsp(OBn)-OH When the pyrrolidinones are connected with amino acids in the C-terminus, the nomenclature is as follows: on the enamine form it is simply H-pyAla-Val-OH since the connection is planar. However, on the amine form (after reduction) the stereochemistry of the connection is indicated with cis- or trans-.

Connected with amino acid at the C-terminus

Enamine connection

H-pyAla-Val-OH H-pyAla-Val-OMe

Amine connection

C/s-pyAla-Val-OH Traπs-pyAla-Val-OMe

Best Modes for Carrying out the Invention

In one embodiment according to the invention, the substituent (X), which is directly coupled to the carbon atom in the 3rd position of the pyrrolidinone ring, is selected from the group consisting of residues of amino acids and amino acid derivatives, resulting in a pyrrolidinone dipeptide analog.

Preferred "derivatives" according to the invention are ester and amide derivatives.

Preferably, the substituent (X) is selected from the group consisting of residues of α- amino acids and derivatives thereof as well as D-alanyl tetramethylthietanylamide and derivatives thereof.

Preferably, the substituent (X) is selected from the group consisting of residues of phenylalanine, phenylglycine, tyrosine, methionine and glycine or ester or amide derivatives thereof as well as D-alanyl tetramethylthietanylamide or derivatives thereof.

Even more preferably, said residue of an amino acid or an amino acid derivative, is a residue of phenylalanine or an ester or amide derivative thereof or D-alanyl tetrame- thylthietanylamid or a derivative thereof, resulting in a pyrrolidinone derivative of Aspartame or Alitame, respectively.

Most preferably, said residue of an amino acid or an amino acid derivative, is a residue of phenylalanine or an ester or amide derivative thereof, resulting in a pyrrolidinone de- rivative of Aspartame.

In one embodiment X is a residue of an amino acid derivative carrying an alkyl (AIk)- substitution in the carboxyl group.

In one embodiment X is a residue of an amino acid derivative carrying a methyl (Me)- substitution in the carboxyl group.

In another embodiment X is a residue of an amino acid derivative carrying an ethyl (Et)- substitution in the carboxyl group.

In another embodiment X is a residue of an amino acid derivative carrying a propyl (Pr)-substitution in the carboxyl group.

In a preferred embodiment at least one of R¹, R², R³, R⁴ and R⁵ is hydrogen. Preferably R¹ is hydrogen and preferably one or both of R³ and R⁴ are hydrogen. More preferably R¹, R², R³, R⁴ and R⁵ are all hydrogen.

R⁶ is either hydrogen or a protective group.

In a preferred embodiment according to the invention said protective group is selected from the group consisting of optionally substituted benzyl (Bn), (Ci-C₆)-alkyl, adaman- tyl, tert-butyl (^fBu), cyclohexyl and trialkylsilyl or triarylsilyl.

In a preferred embodiment R⁶ is a group, which results in an anionic group on the pyrrolidinone compounds according to the invention. In a most preferred embodiment R⁶ is hydrogen.

The compounds according to the invention may be either one of the stereoisomers of the pyrrolidinone compound (1) (i.e. the c/s-stereoisomer or the fr-ans-stereoisomer), or it may be a mixture of the stereoisomers.

In one embodiment the compounds according to the invention are the cis- stereoisomers. In one embodiment the compounds according to the invention are the trans- stereoisomers.

In one embodiment the compounds according to the invention may be salts. Preferred salts include alkaline or alkaline earth metal salts of the compounds according to the invention. Preferred salts are the Na, K, Mg and Ca salts of the compounds according to the invention.

R³ and R⁴ may further be absent and replaced by a double bond between the carbon atoms 3 and 4 on the pyrrolidinone ring to which R³ and R⁴ were formerly bound. In this embodiment according to the invention the novel pyrrolidinone derivatives are charac- terized by the general formula (2),

wherein R¹, R², R⁵, R⁶, and X have the meanings as defined above, as well as salts, racemates and diastereomers thereof.

These intermediates are produced according to the invention and are useful in the fur- ther production of the compounds according to the invention.

The invention further provides a method for the production of the pyrrolidinone derivatives of the formula (1 )

wherein R¹, R² , R³, R⁴, R⁵, R⁶ and X has the meaning as defined above, comprising the steps of:

a) Reacting a pyrrolidinone of the general formula (3),

wherein R¹, R², R⁵ and R⁶ has the meaning as defined above, in a condensation reaction, with a compound (H-X) containing a moiety (X) that has any of the meanings defined above, to obtain a product of the formula (2),

b) reducing the product produced in step a), to obtain a product of the formula (1).

c) optionally separating cis- and trans- diastereoisomers by column chromatography,

d) optionally deprotecting the carboxylate group.

Though the above reaction sequence is preferred, the optional reaction step d) may in principle be performed prior to reaction step b). In one embodiment the hydrogen of R³ and/or R⁴ may be substituted with a desired substitution group. This may preferably be done prior to the step a) of the method according to the invention.

The pyrrolidinone (3), which is used as starting material for the production of the com- pounds according to the invention, may be produced following standard literature procedures. The procedure is outlined in the scheme 1 below.

DCC

Scheme 1. Synthesis of py Asp (OBn) from Boc-Asp(OBn)-OH

An example of a preferred starting material for the method according to the invention, an aspartic acid derived pyrrolidinone H-pyAsp(OBn)-OH, (compound of the general formula 3) wherein R¹, R² and R⁵ = H, and R⁶ = Bn) for use according to the invention, may be synthesized by first synthezising Boc-pyAsp(OBn)-OH in quantitative yields from Boc-Asp(OBn)-OH by a standard literature procedure (Jouin, P. et al., J. Chem. Soc, Perkin Trans. 1 , 1987, 1177-1182. Courcambeck, J., et al., J. Chem. Soc, Perkin Trans. 1, 2001, 12, 1421-1430), subsequently deprotecting of the so produced Boc-pyAsp(OBn)-OH with TFA giving the analog of aspartic acid H-pyAsp(OBn)-OH as exemplified in example 1.

The analogs according to the invention may be synthesized from the compounds (3) by a process which, optionally among other steps, involve the above mentioned steps a-d according to the invention.

Such a reaction is exemplified in Scheme 2.

Performing the reaction steps in the sequence a) (shown in scheme 2 as A), b) (shown in scheme 2 as B) and d) (shown in scheme 2 as C) producing the c/s-isomer is preferred as opposed to performing the reaction steps in the sequence a), d) and b), which may result in the trans-isomer.

Scheme 2. Synthesis of dipeptide analogs from H-#yAsp(OBn)-OH (3)

The compound (HX) is preferably a compound selected from the group consisting of amino acids and amino acid derivatives. .More preferably HX is a compound selected from the group consisting of α-amino acids and derivatives thereof as well as D-alanyl tetramethylthietanylamide and derivatives thereof. More preferably HX is a compound selected from the group consisting of phenylalanine, phenylglycine, tyrosine, methionine and glycine or esters or amide derivatives thereof as well as D-alanine tetramethylthietanylamide or derivatives thereof.

Even more preferably HX is phenylalanine or an ester or amide derivative thereof.

HX may be an amino acid derivative carrying a substitution in the carboxyl group.

In one embodiment HX is an amino acid derivative carrying an alkyl (Alk)-substitution in the carboxyl group.

In one embodiment HX is an amino acid derivative carrying a propyl (Pr)-substitution in the carboxyl group.

Preferably HX is an amino acid derivative carrying an ethyl (Et)-substitution in the car- boxyl group.

More preferably HX is an amino acid derivative carrying a methyl (Me)-substitution in the carboxyl group. The compound HX may further be a salt of the above mentioned compounds.

The condensation reaction performed in step a) is preferably performed at a temperature of between 30 - 8O⁰C, more preferably between 40 - 60⁰C, even more preferably about 50⁰C.

In the condensation step (a), there may be a risk of racemisation at the 2-position if the temperature is raised above 50⁰C, but with the reaction conditions used in example 1 below, no racemisation was observed by NMR.

The condensation is followed by a reduction step (b), wherein reduction of the product produced in step a) or possibly a) + c) and/or d) may be achieved using any suitable reduction method. Preferably, said reduction is a Borch reduction performed using sodium cyanoborohydride or the reduction is achieved by hydrogenation over platinum oxide (PtO₂). Borch reduction may be performed according to standard literature procedures (Borch, R. F. et al. J. Am. Chem. Soc. 1971 , 93, 2897-2904).

Most preferably the reducing agent used in step (b) to reduce the compounds produced according to step a) or possibly a) + c) and/or d) is sodium cyanoborohydride (NaBH₃CN). If the isomer-selectivity is not satisfying with NaBH₃CN other reduction agents like NaBH₄, NaBH(OAc)₃, (for reference see Abdel-Magid, A. F. et al. J. Org. Chem. 1996, 61, 3849-3862.) Zn(BH₄)₂ (for reference see Ranu, B. C. et al. J. Org. Chem. 1998, 63, 370-373.), or another two step procedure with NaBH₄ in combination with Ti(O¹Pr)₄ (for reference see Neidigh, K. A. et al. J. Chem. Soc, Perkin Trans. 1 , 1998, 2527-2531 ) may be used. In the examples below the Borch reduction is, however, completely diastereoselective (measured by NMR), giving only the cis- diastereomers.

The optional step c) may additionally be performed in order to separate the stereoi- somers of the compounds according to the invention. The step may further be omitted in order to obtain a mixture of diastereoisomers. In principle the step c) may be performed at any point in the procedure such as before or after any of the steps a), b), and d), and is thus not limited to be performed after step b).

Finally, deprotection of protective group R⁶ furnishes the dipeptide analogs according to the invention (1). Deprotection can be performed using standard chemical processes which are well-known to the skilled artesian. For reference see Greene, T. W. and Wuts, P. G. M., "Protective groups in organic synthesis" 3^rd ed. Wiley 1999.

Suitable protective groups used in the inventive method include benzyl (Bn) or substituted benzyl, alkyl, adamantyl, tert-butyl (^fBu), cyclohexyl, trialkylsilyl or triarylsilyl.

In order to obtain the /rans-diastereomers of the compounds according to the invention, the optional step d) may be performed prior to the reduction step b) i.e. the condensed products obtained in step (a) may be deprotected before the Borch reduction, thereby using the carboxylate to direct the reduction.

The optional step d) is preferably performed, thus producing a compound having an anionic carboxylate group conferring a structural resemblance to the dipeptide sweeteners.

The optional steps c) and d) may be performed in any desired sequence and any of the two steps may be omitted if so desired.

The compounds according to the invention possess taste enhancing properties, and may accordingly be used as additives to enhance the taste of suitable compositions. In one embodiment the pyrrolidinone derivatives according to the invention are formulated as a suitable aqueous solution. In another embodiment it is formulated as a solid composition, preferably a powder.

The compounds according to the present invention may be used to enhance the taste of any composition for oral consumption for which enhanced or sweet taste is desired. Such consumable compositions include a variety of compositions such as for example candy, cake, ice cream, youghurt or the like as well as soft drinks and the like.

The compounds according to the invention may be mixed with other compounds according to the invention or with other artificial or conventional sweetening agents or be used in combination with other compounds according to the invention or other artificial or conventional sweetening agents as additives to enhance the taste of suitable compositions.

The compounds according to the invention may be mixed with conventional sweetener, i.e. sweeteners belonging to the group comprising carbohydrates such as sugars and sugar alcohols. Further the compounds according to the invention may be mixed with artificial sweetener, i.e. sweeteners belonging to the group comprising artificial carbohydrate, dipep- tide and sulfonamide sweeteners and derivatives thereof. Preferable additional sweeteners are sweeteners chosen from the group consisting of Aspartame, Alitame, Neo- tame, Saccharine, Acesulfame K and Cyclamate as well as Sucralose or similar carbohydrate sweeteners.

Use of the pyrrolidinone derivatives according to the present invention may provide a reduced calorie content of compositions as compared to compositions sweetened using conventional carbohydrate sweeteners. The inventive compounds, optionally in combination and/or in combination with other conventional or artificial sweeteners, may provide a specific taste profile, i.e. required sweetening and essentially no aftertaste, suitable for specific compositions. Further the compounds according to the invention possess an increased stability, e.g. increased thermal stability in baking and cooking, and may further confer a reduced cost of manufacture.

Examples

Example 1. Preparation of a starting material (3) for the method according to the invention (compound of the general formula 3) wherein R¹, R² and R⁵ = H, R⁶ = Bn).

Preparation of Boc-pyAsp(OBn)-OH (2S)-Λ/-Boc-2-benzyloxycarbonylmethyl-3-hydroxy- 5-oxo-2,5-dihydropyrrole.

A solution of DCC (3.78 g, 18.25 mmol) in CH₂CI₂ (20 ml_) was added drop wise over 20 min to a stirred solution of Boc-Asp(OBn)-OH (5.18 g, 16.0 mmol), Meldrum's acid (2.56 g, 17.8 mmol), DMAP (2.88 g, 23.6 mmol) in CH₂CI₂ (60 ml_) at 0⁰C. A precipitate was formed after 15 min. The mixture was allowed to heat to RT for 4 h, after which it was poured rapidly into EtOAc (cold, 330 mL) and filtered. The filtrate was washed with 5% citric acid (cold, 2 x 210 mL), water (cold, 2 x 210 mL), and brine (cold, 2 * 210 mL), and subsequently dried (Na₂SO₄) and concentrated under reduced pressure to give a yellowish solid (7.18 g). The solid was dissolved in EtOAc (150 mL) heated to 60 °C and subsequently refluxed for 30 min. Evaporation of the solution afforded the de- sired product as a pale yellow semi-crystalline material (6.13 g). The material was of a sufficient purity to carry on to the following step without prior purification.

Preparation of H-pyAsp(OBn)-OH (2S)-2-Benzyloxycarbonylmethyl-3-hydroxy-5-oxo- 2,5-dihydropyrrole.

TFA (4.02 ml, 35.3 mmol) was added by syringe to a solution of the above produced Boc-pyAsp(OBn)-OH (6.06 g, 18.7 mmol) in CH₂CI₂ (45 ml), and stirred for 30 min. The solution was concentrated under reduced pressure followed by co-evaporation with toluene (60 ml). Colmnn chromatography (gradient elution; 33 - 0 % heptane in EtOAc) gave H-pyAsp(OBn)-OH as a yellow oil (2.09 g, 45%).

¹H NMR (300 MHz, CD₃OD): δ 7.32-7.23 (5H, m), 5.13 (1 H, d, J = 9.8 Hz), 5.09 (1 H, d, J = 9.8 Hz), 4.19 (1 H, t, J = 4.4 Hz), 2.96 (1 H, dd, J = 17.6 & 4.6 Hz), 2.83 (1 H, dd, J = 17.6 & 4.2 Hz). ¹³C NMR (75 MHz, CD₃OD): δ 37.0, 56.0, 61.5, 68.0, 129.4, 129.6, 137.0, 171.4, 172.0, 208.7. Mp: 115-117 ⁰C

Example 2. Preparation of novel intermediate compounds of the general formula (2) according to the invention. Intermediate compounds of the general formula (2) according to the invention (compound of the general formula (2) wherein R¹, R² and R⁵ = H, R⁶ = Bn and X = appropriate amino acid or amino acid derivative moity) were prepared by subjecting the above prepared H-pyAsp(OBn)-OH to a condensation reaction with an appropriate amino acid or amino acid derivative.

Step (a) according to the invention - Condensation

H-pyAsp(OBn)-Phe-OMe ((S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro-1 H- pyrrol-3-ylamino)-3-phenyl-propionic acid methyl ester (2a), an intermediate compound (compound of the general formula (2) wherein R¹, R² and R⁵ = H, R⁶ = Bn and X = a moity consisting of a methyl substituted derivative of Phenylalanine) according to the invention, was produced by stirring a solution of H-pyAsp(OBn)-OH (1.0 equivalent) and the derivative of Phenylalanine H-Phe-OMe-HCI (1.01 equivalents) in MeCN (20 ml/g of H-pyAsp(OBn)-OH) and AcOH (2 ml/g of H-pyAsp(OBn)-OH) with molecular sieves (3A) at 50 ⁰C in a Teflon reactor for 16 hours. The reactor was emptied and the molecular sieves stirred in additional MeOH for 10 minutes. The combined filtrates were co-evaporated twice with toluene (20 ml) affording a brown oil. Purification by column chromatography (EtOAc/MeOH 10:1) gave 0.185 g, (55%) of the desired enamine intermediate of the general formula (2) H-pyAsp(OBn)-Phe-OMe (2a) (NMR data presented below).

¹H NMR (500 MHz, CD₃OD): δ 7.38-7.15 (m, 10H, Ph), 5.17 (d, 1 H, J = 12.3 Hz, PhCH₂), 5.14 (d, 1 H, J = 12.3 Hz, PhCH₂), 4.55 (s, 0.8H, H⁴), 4.37 (dd, 1 H, J = 4.4 & 8.5 Hz, H^α), 4.20 (t, 1 H, J = 7.1 Hz, H²), 3.66 (s, 3H, OMe), 3.18 (dd, 1 H, J = 5.9 & 13.8 Hz, H⁶) 3.03 (dd, 1 H, J = 7.7 & 13.8 Hz, H^6') 2.96 (dd, 1 H, J = 4.4 & 17.0 Hz, H^β), 2.50 (dd, 1 H, J = 8.5 & 17.0 Hz, H^β>). ¹³C NMR (75 MHz, CD₃OD): δ 37.2, 38.9, 52.6, 53.4, 58.5, 67.4, 89.1, 127.5, 128.6, 128.8, 128.89, 128.91 , 129.4, 135.4, 135.7, 164.6, 172.0, 172.1 , 176.1. Mp: 125-128 ⁰C.

The following exemplary compounds (2b— i) were similarly prepared using the indicated appropriate amino acid derivatives following the above procedure:

(2b) H-pyAsp(OBn)-Gly-OMe 2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro-1 H- pyrrol-3-ylamino)-acetic acid methyl ester using the amino acid derivative glycine methyl ester hydrochloride. Yield: 0.539 g, (42%) NMR data presented below. ¹H NMR (300 MHz, CD₃OD): δ 7.38-7.30 (m, 5H, Ph), 5.17 (s, 2H, PhCH₂), 4.55 (s, 0.9H, H⁴), 4.45 (dd, 1 H, J = 4.2 & 8.8 Hz, H²), 3.87 (s, 2H, H^α), 3.72 (s, 3H, OMe), 2.95 (dd, 1 H, J = 4.2 & 16.8 Hz, H⁶), 2.55 (dd, 1 H, J = 8.8 & 16.8 Hz, H^6'). ¹³C NMR (75 MHz, CD₃OD): δ 38.4, 45.4, 51.7, 54.1 , 66.7, 86.8, 128.2, 128.5, 136.1 , 167.3, 170.5, 171.3, 177.7. Mp: 122-125 ⁰C

(2c) H-pyAsp(OBn)-Val-OMe (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro- 1f/-pyrrol-3-ylamino)-3-methyl-butyric acid methyl ester using the amino acid derivative valine methyl ester hydrochloride. Yield: 1.03 g, (72%) NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.40-7.28 (m, 5H, Ph), 5.21 (d, 1 H, J = 12.3 Hz, Ph- CH₂), 5.16 (d, 1H, J = 12.3 Hz, Ph-CH₂), 4.55 (s, 0.9H, H³), 4.45 (dd, 1H, J = 4.4, 8.6 Hz, H⁵), 3.87-3.70 (m, 1 H, H^D), 3.70 (s, 3H, OMe)₁ 3.02 (dd, 1 H, J = 4.4, 17.0 Hz, H⁶), 2.52 (dd, 1H, J = 8.6, 17.0 Hz, H^6'), 2.12 (dd, 1H, J = 6.8, 13.5 Hz, H^β) 1.02-0.90 (m, 6H, Me). ¹³C NMR (75 MHz, CD₃OD): δ 17.6, 18.2, 30.9, 38.6, 51.4, 54.0, 63.5, 66.9, 86.5, 128.2, 128.3, 128.4, 136.1 , 167.2, 171.1 , 172.6, 177.7. Mp: 118-124⁰C

(2d) H-pyAsp(OBn)-Phe-NH₂ (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro- 1H-pyrrol-3-ylamino)-3-phenyl-propionic acid amide using the amino acid derivative phenylalanine amide hydrochloride. Yield: 1.09 g, (70%) NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.39-7.16 (m, 1OH, Ph), 5.15 (s, 2H, PhCH₂), 4.55 (s, 0.9H, H⁴), 4.35 (dd, 1 H, J = 4.2 & 7.8 Hz, H^α), 4.03 (dd, 1 H, J = 5.9 & 8.6 Hz, H²), 3.18 (dd, 1H, J = 5.9 & 13.9 Hz, H⁶), 3.00 (dd, 1 H, J = 4.4 & 16.7 Hz, H^6') 2.96 (dd, 1 H, J = 8.6 & 13.8 Hz, H^β), 2.60 (dd, 1 H, J = 8.0 & 16.7 Hz, H^β). ¹³C NMR (75 MHz, CD₃OD) δ: 37.9, 38.2, 54.1 , 60.0, 66.8, 126.8, 128.1, 128.20, 128.24, 128.38, 128.45, 128.5, 128.6, 128.7, 129.2, 135.9, 137.1 , 171.6. Mp: 115-12O ⁰C.

The compounds 2e-2i below may be produced accordingly

(2e) H-pyAsp(OBn)-Phe-OEt (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro- 1H-pyrrol-3-ylamino)-3-phenyl-propionic acid ethyl ester using the amino acid derivative phenylalanine ethyl ester hydrochloride.

(2f) H-pyAsp(OBn)-Phe-OPr (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro- 1H-pyrrol-3-ylamino)-3-phenyl-propionic acid propyl ester using the amino acid deriva- tive phenylalanine propyl ester hydrochloride. (2g) pyAsp(OBn)-Tyr(Bn)-OMe (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5- dihydro-1H-pyrrol-3-ylamino)-3-(4-benzyloxyphenyl)-propionic acid methyl ester using the amino acid derivative O-benzyl tyrosine methyl ester hydrochloride.

(2h) pyAsp(OBn)-Met-OMe (S)-2-((S)-2-Benzyloxycarbonylmethyl-5-oxo-2,5-dihydro- 1H-pyrrol-3-ylamino)-4-methylthio-butyric acid methyl ester using the amino acid derivative methionine methyl ester hydrochloride.

(2i) pyAsp(OBn)-D-Ala-NH(CH(CMe₂)₂S) {(S)-5-Oxo-3-[(R)-1 -(2,2 ,4,4-tetramethyl- thietan-3-ylcarbamoyl)-ethylamino] 2,5-dihydro-1/7-pyrrol-2-yl}-acetic acid benzyl ester using the amino acid derivative D-AIa-NH(CH(CMe₂^S), which was synthesized follow- ing a published procedure (Brennan, T. M. et al. US4411925, 1980).

Example 3. Preparation of the novel compounds of the general formula (1 ) according to the invention (compounds of the general formula (1) wherein R¹- R⁵ = H, R⁶ = Bn and X = appropriate amino acid or amino acid derivative moiety) from the intermediate compound (2a-i) according to the invention produced as described in example 2.

Step b) according to the invention - reduction

The below mentioned compounds (c/s-1a - i) were produced by adding NaBH₃CN (6.2 equivalents) to solutions of each of the above mentioned compounds (2a-i) (1.0 equivalent) in MeOH (200 ml/g of 2a-i) and AcOH (20 ml/g of 2a-i) and the reaction mixture was left to stir for 24 hours. Water (10 ml) and CH₂CI₂ (10 ml) was added, the layers separated, and the aqueous phase was extracted with CH₂CI₂ (3 * 20 ml).

The combined organic phases were dried (Na₂SO₄) and evaporated under reduced pressure. Toluene (20 ml) was added and the solution was evaporated to give the crude product containing the compounds according to the invention, primarily in the form of the c/s-stereoisomer.

Step c) - column chromatography

Purification by column chromatography (using varying concentrations of MeOH in EtOAc), afforded the desired purified c/s-isomers; (c/s-1 a) c/s-pyAsp(OBn)-Phe-OMe (S)-2-((2S,3S>2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-3-phenyl-propionic acid methyl ester. Yield: 0.167 g, (50%) using 7 - 15 % MeOH in EtOAc. NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.38-7.15 (m, 1OH, Ph), 5.16 (d, 1 H₁ J = 12.3 Hz, PhCH₂), 5.11 (d, 1H, J = 12.3 Hz, PhCH₂) 3.74-3.67 (m, 1H, H³), 3.58 (s, 3H, OMe),

3.58 (dd, 1 H, J = 6.2 & 13.1 Hz, H^α), 3.18-3.12 (rri, 1 H, H²), 2.90 (d, 2H, J = 7.1 Hz,

H^β), 2.70 (dd, 1 H, J = 6.1 & 16.7, H⁶), 2.57 (dd, 1H, J = 7.0 & 17.0 Hz, H⁴), 2.52 (dd,

1 H, J = 7.6 & 17.0 Hz, H⁴), 2.02 (dd, 1 H, J = 5.4 & 17.1 Hz, H^6'). ¹³C NMR (75 MHz,

CD₃OD) δ: 37.0, 38.6, 39.3, 51.1 , 57.5, 60.9, 66.5, 126.6, 128.2, 128.3, 128.4, 129.2, 136.2, 137.4, 171.2, 174.7, 177.1. HRMS (El⁺): m/z calculated for C₂₃H₂₇N₂O₅ (MH⁺)

411.1922, found 411.1920. Sample was dissolved in formic acid/MeOH.

(c/s-1 b) c/s-pyAsp(OBn)-Gly-OMe 2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-acetic acid methyl ester. Colorless powder, Yield: 90 mg, (26%) using EtOAc/MeOH 15:1 as eluent. NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.38-7.30 (m, 5H, Ph), 5.16 (d, 1H, J = 12.5 Hz, PhCH₂), 5.12 (d, 1 H, J = 12.5 Hz, PhCH₂) 3.80 (dt, 1 H, J = 3.4 & 6.7 Hz, H³), 3.70 (s, 3H, OMe), 3.46 (d, 1 H, J = 17.5 Hz, H^α), 3.38 (d, 1 H, J = 17.5 Hz, H^α), 3.32-3.23 (m, 1 H, H²), 2.77 (dd, 1 H, J = 6.4 & 16.7 Hz, H⁴) 2.68 (dd, 1 H, v/ = 7.7 & 17.4 Hz, H⁶), 2.60 (dd, 1 H, J = 6.9 & 16.8 Hz, H^4'), 2.15 (dd, 1 H, J = 4.3 & 17.3 Hz, H^6'). ¹³C NMR (75 MHz, CD₃OD): δ 36.7, 38.6, 51.2, 57.1 , 58.2, 66.5, 128.2, 128.3, 128.5, 136.1 , 171.2, 172.6, 177.2. HRMS (El⁺): m/z calculated for C₁₆H₂₀N₂O₅ 320.1372, found 320.1372

(c/s-1 c) C/s-pyAsp(OBn)-Val-OMe (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-3-methyl-butyric acid methyl ester. Pale yellow oil, yield: 0.163 g, (52%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD) δ: 7.40-7.30 (m, 5H, Ph), 5.18 (d, 1 H, J = 12.3 Hz, Ph- CH₂), 5.13 (d, 1 H, J = 12.3 Hz, Ph-CH₂), 3.76 (ddd, 1 H, J = 4.3, 6.7, 10.7 Hz, H⁵), 3.70 (s, 3H, OMe), 3.22-3.04 (m, 1 H, H⁴), 3.03 (d, 1 H, J = 5.9 Hz, H^σ), 2.72 (dd, 1 H, J = 6.2, 16.7 Hz, H⁶), 2.60 (dd, 1H, J = 6.9, 16.7 Hz, H^6'), 2.55 (dd, 1 H, J = 7.6, 17.0 Hz, H³), 2.10 (dd, 1 H, J = 5.4, 17.0 Hz, H^3'), 1.89-1.73 (m, 1 H, H^β), 0.94-0.86 (m, 6H, Me). ¹³C NMR (75 MHz, CD₃OD) δ: 17.7, 18.4, 31.5, 37.2, 38.8, 57.7, 58.0, 65.0, 66.5, 128.1, 128.2, 128.4, 136.2, 171.2, 175.4, 177.2. HRMS (El⁺): m/z calculated for C₁₉H₂₆N₂O₅ 362.1842, found 362.1843. (c/s-1 d) C/s-pyAsp(OBn)-Phe-NH₂ (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-3-phenyl-propionic acid amide. Colorless oil, yield: 0.027 g, (34%). NMR data presented below.

¹H NMR (300 MHz₁ CD₃OD): δ 7.15-7.40 (m, 1OH, Ph), 5.15 (d, 1 H, J = 12.4 Hz, Ph- CH₂), 5.11 (d, 1H, J = 12.4 Hz, Ph-CH₂), 3.68 (ddd, 1 H, J = 4.6, 5.7 Hz, 10.3, H⁵), 3.38

(dd, 1 H, J = 6.2, 7.8 Hz, H^α), 3.09-3.12 (m, 1 H, H⁴), 2.98 (dd, 1 H, J = 6.2, 13.5 Hz, H^β),

2.76 (dd, 1 H, J = 7.8, 13.5 Hz, H^β), 2.70 (dd, 1 H, J = 5.7, 16.6 Hz, H⁶), 2.53 (dd, 1 H, J

= 7.2, 16.7 Hz, H^6'), 2.48 (dd, 1 H, J = 7.6, 17.1 Hz, H³), 1.88 (dd, 1 H, J = 5.6, 17.1 Hz,

H^3'). ¹³C NMR (75 MHz, CD₃OD): δ 36.8, 38.4, 39.4, 50.7, 57.4, 61.7, 66.5, 67.4, 126.6, 128.11 , 128.14, 128.2, 128.3, 128.38, 128.39, 128.42, 128.49, 129.19, 129.21 , 136.2,

137.7, 171.2, 176.5, 177.0. HRMS (El+): m/z calculated for C₂₂H₂₅N₃O₄ 395.1845, found 395.1846

The compounds c/s-1 e - c/s-1 i below may be produced accordingly

(c/s-1 e) C/s-pyAsp(OBn)-Phe-OEt (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-3-phenyl-propionic acid ethyl ester.

(c/s-1 f) C/s-pyAsp(OBn)-Phe-OPr (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolidin-3-ylamino)-3-phenyl-propionic acid propyl ester.

(c/s-1 g) C/s-pyAsp(OBn)-Tyr(Bn)-OMe (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5- oxo-pyrrolidin-3-ylamino)-3-(4-benzyloxyphenyl)-propionic acid methyl ester.

(c/s-1 h) C/s-pyAsp(OBn)-Met-OMe (S)-2-((2S,3S)-2-Benzyloxycarbonylmethyl-5-oxo- pyrrolin-3-ylamino)-4-methylthio-butyric acid methyl ester.

(c/s-1 i) C/s-pyAsp(OBn)-D-Ala-NH(CH(CMe₂)₂S) {(2S,3S)-5-Oxo-3-[(K)-1 -(2,2,4,4- tetramethyl-thietan-S-ylcarbamoyO-ethylaminoJ-pyrrolidin^-ylJ-acetic acid benzyl ester.

Example 4. Preparation of c/s-isomers of the preferred compounds of the general for- mula (1 ) according to the invention, wherein R⁶ is hydrogen (compounds of the general formula 1 ) wherein R¹- R⁶ = H and X = appropriate amino acid or amino acid derivative moiety).

step d) - Hydrogenolysis Hydrogen (1 atm.) was applied to a solution of each of the compounds (c/s-1a - i) in methanol containing Pd/C (5%, 30 mg/100 mg c/s-1a - i), and the reaction was stirred until TLC (EtOAc) showed, that it had run to completion. Filtration through celite and concentration in vacuo provided the desired product (cis-1j - r):

(c/s-1j) c/s-pyAsp-Phe-OMe (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid methyl ester. Pale yellow oil, yield: 0.037 g, (54%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.30 - 7.10 (m, 5H, Ph), 3.73-3.65 (m, 1 H, H⁵), 3.63-3.57 (m, 1 H, H^σ), 3.61 (s, 3H, OMe), 3.21-3.13 (m, 1 H, H⁴), 2.95 (dd, 2H, J = 2.5, 6.6 Hz, H^β), 2.62 (dd, 1H₁ J = 6.2, 16.9 Hz, H⁶), 2.56 (dd, 1H, J = 8.0, 17.2 Hz, H³), 2.47 (dd, 1H, J = 7.1 , 16.9 Hz, H^6'), 2.05 (dd, 1 H, J = 5.4, 17.1 Hz, H^3'). ¹³C NMR (75 MHz, CD₃OD): δ 29.5, 36.8, 38.8, 39.2, 51.1 , 57.5, 61.0, 126.6, 128.0, 128.3, 128.8, 129.1, 137.3, 173.0, 174.5, 177.0.

(c/s-1 k) c/s-pyAsp-Gly-OMe 2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3-ylamino)- acetic acid methyl ester. Pale yellow oil, yield: 0.016 g, (26%). NMR data presented below. The spectra are very complicated due to the presence of rotamers.

¹H NMR (300 MHz, CD₃OD): δ 8.22 (s, 0.7H, NH), 8.08 (s, 0.3H, NH), 4.48-4.40 (m, 1 H, H⁵), 4.28 (s, 0.8H), 4.17 (d, 1 H, J = 17.4 Hz, H^α), 4.10 (d, 1 H, J = 17.4 Hz, H^α'), 4.01-3.95 (m, 1H, H⁴), 3.76 (s, 0.8H, OMe), 3.73 (s, 3H₁ OMe), 2.80-2.52 (m, 4H). ¹³C NMR (75 MHz, CD₃OD): δ 31.9, 34.2, 36.8, 40.8, 42.3, 51.1, 51.7, 54.2, 55.0, 58.9, 63.9, 164.4, 169.7, 171.8, 177.3. MS (El⁺, direct inlet): 44, 57, 69, 95, 112, 127, 149, 155, 172, 230.

(c/s-11) c/s-pyAsp-Val-OMe (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-methyl-butyric acid methyl ester. Colorless oil, yield: 0.023 g, (79%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 3.74-3.68 (m, 1H, H⁵), 3.72 (s, 3H, OMe), 3.13-3.00 (m, 1 H, H⁴), 3.03 (d, 1 H, J = 6.0, H^α), 2.55 (dd, 1H, J = 7.6, 17.0 Hz, H³), 2.50 (dd, 1 H, J = 6.4, 15.9 Hz, H⁶), 2.40 (dd, 1H, J = 7.3, 15.9 Hz, H^6'), 2.10 (dd, 1H, J = 5.6, 16.9 Hz, H³), 2.00-1.86 (m, 1H, H^β), 1.00-0.90 (m, 6H, Me). ¹³C NMR (75 MHz, CD₃OD): δ 17.9, 18.3, 31.5, 37.2, 41.7, 51.0, 58.4, 58.6, 65.1 , 175.3, 176.5, 177.0. (c/s-1 m) c/s-pyAsp-Phe-NH₂ (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid amide. Colorless oil, yield 0.010 g, (59%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.32-7.15 (m, 5H, Ph), 3.68-3.63 (m, 1 H, H⁵), 3.36 (dd, 1 H, J = 6.3, 7.7 Hz, H^α), 3.10-3.04 (m, 1 H, H⁴), 3.00 (dd, 1 H, J = 6.2, 13.5 Hz, H^β), 2.78 (dd, 1 H, J = 7.8, 13.4 Hz, H^β'), 2.50 (dd, 1 H, J = 7.7, 17.0 Hz, H³), 2.42 (dd, 1 H, J = 6.7, 15.3 Hz, H⁶), 2.32 (dd, 1 H₁ J = 7.4, 15.3 Hz, H⁶ ), 1.82 (dd, 1 H, J = 5.6, 17.0 Hz, H³). ¹³C NMR (75 MHz, CD₃OD): δ 22.6, 29.6, 37.0, 39.4, 58.1 , 59.0, 61.9, 126.6, 128.4, 129.2, 137.7, 176.3, 176.8, 178.6.

The compounds c/s-1 n - c/s-1 r below may be produced accordingly

(c/s-1 n) c/s-pyAsp-Phe-OEt (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid ethyl ester.

(c/s-1 o) c/s-pyAsp-Phe-OPr (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid propyl ester.

(c/s-1 p) c/s-pyAsp-Tyr-OMe (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-(4-hydroxyphenyl)-propionic acid methyl ester.

(c/s-1 q) c/s-pyAsp-Met-OMe (S)-2-((2S,3S)-2-Carboxymethyl-5-oxo-pyrrolin-3- ylamino)-4-methylthio-butyric acid methyl ester.

(c/s-1 r) c/s-pyAsp-D-Ala-NH(CH(CMe₂)₂S) {(2S,3S)-5-Oxo-3-[(ft)-1 -(2,2,4,4- tetramethyl-thietan-3-ylcarbamoyl)-ethylamino]-pyrrolidin-2-yl}-acetic acid.

Example 5. Preparation of intermediate compounds of the general formula (2) according to the invention, wherein R⁶ is hydrogen (compounds of the general formula 2) wherein R¹, R², R⁵ and R⁶= H and X = appropriate amino acid or amino acid derivative moity).

The below mentioned intermediate compounds (2j— r of the general formula 2) were produced by subjecting the above mentioned intermediate compounds (2a-i) to a hy- drogenolysis as described above (step d)): (2j) pyAsp-Phe-OMe (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 f/-pyrrol-3- ylamino)-3-phenyl-propionic acid methyl ester. Colorless oil, yield: 0.090 g, (75%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 7.32-7.15 (m, 5H₁ -Ph)₁ 4.55 (s, 0.2H, H³), 4.40-4.26 (m, 1 H, H⁵), 4.22-4.12 (m, 1 H, H^α), 3.68 (s, 3H, OMe), 3.18 (dd, 1 H, J = 5.5, 13.7 Hz, H^β), 3.03 (dd, 1 H, J = 7.7, 13.3 Hz, H^β'), 2.90-2.72 (m, 1 H, H⁶), 2.51-2.33 (m, 1 H, H⁶). ¹³C NMR (75 MHz, CD₃OD): δ 37.1 , 51.6, 59.0, 126.9, 128.4, 128.5, 129.1 , 136.6, 167.1 , 172.1 , 177.7.

(2k) pyAsp-Gly-OMe 2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 H-pyrrol-3-ylamino)- acetic acid methyl ester. Colorless oil, yield: 0.015 g, (28%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 4.50 (s, 0.2H, H³), 4.43 (t, 1 H, J = 6.9 Hz, H⁵), 3.90 (s, 2H, H^α), 3.72 (s, 3H, OMe), 2.60 (dd, 1 H, J = 6.1 , 16.4 Hz, H⁶), 2.48 (dd, 1 H, J = 5.4, 16.2 Hz, H^6').

(2I) pyAsp-Val-OMe (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1H-pyrrol-3- ylamino)-3-methyl-butyric acid methyl ester. Colorless oil, yield: 0.015 g, (12%). NMR data presented below.

¹H NMR (300 MHz, CD₃OD): δ 4.45 (s, 0.8H, H³), 4.50-4.34 (m, 1 H, H^α), 3.70 (s, 3H, OMe), 2.95-2.76 (m, 1 H, H⁶), 2.59-2.48 (m, 1 H, H⁶ ), 2.23-2.18 (m, 1 H, H^β), 1.10-0.93 (m, 6H, Me). ¹³C NMR (75 MHz, CD₃OD): δ 17.6, 18.3, 30.9, 51.4, 54.3, 63.5, 168.0, 172.5, 172.7.

The compounds 2m - 2r below may be produced accordingly

(2m) pyAsp-Phe-NH₂ (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 W-pyrrol-3- ylamino)-3-phenyl-propionic acid amide.

(2n) pyAsp-Phe-OEt (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1H-pyrrol-3- ylamino)-3-phenyl-propionic acid ethyl ester.

(2o) pyAsp-Phe-OPr (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 H-pyrrol-3- ylamino)-3-phenyl-propionic acid propyl ester. (2p) pyAsp-Tyr-OMe (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 H-pyrrol-3- ylamino)-3-(4-hydroxyphenyl)-propionic acid methyl ester.

(2q) pyAsp-Met-OMe (S)-2-((S)-2-Carboxymethyl-5-oxo-2,5-dihydro-1 H-pyrrol-3- ylamino)-4-methylthio-butyric acid methyl ester.

(2r) pyAsp-D-Ala-NH(CH(CMe₂)₂S) {(S)-5-Oxo-3-[(R)-1-(2,2,4,4-tetramethyl-thietan-3- ylcarbamoyl)-ethylamino] 2,5-dihydro-1 H-pyrrol-2-yl}-acetic acid.

Example 6. Preparation of frans-isomers of the preferred compounds of the general formula (1 ) according to the invention, wherein R⁶ is hydrogen (compounds of the general formula 1 ) wherein R¹- R⁶ = H and X = appropriate amino acid or amino acid derivative moiety).

The below mentioned compounds (trans-ij-r of the general formula 1) may be produced by subjecting the above mentioned intermediate compounds (2j-r) to a reduction as described above (step b):

(trans-V]) frans-pyAsp-Phe-OMe (S)-2-((2S,3R)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid methyl ester.

(frans-1 k) frans-pyAsp-Gly-OMe 2-((2S,3R)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-acetic acid methyl ester.

(/rans-11) frans-pyAsp-Val-OMe (S)-2-((2S,3ft)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-methyl-butyric acid methyl ester.

(trans-1m) frans-pyAsp-Phe-NH₂ (S)-2-((2S,3R)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid amide.

(trans-ϊ n) frans-pyAsp-Phe-OEt (S)-2-((2S,3R)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid ethyl ester.

(frans-1 o) fr-ans-pyAsp-Phe-OPr (S)-2-((2S,3f?)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-phenyl-propionic acid propyl ester.

(trans-ϊ p) fr-ans-pyAsp-Tyr-OMe (S)-2-((2S,3/^:?)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-3-(4-hydroxyphenyl)-propionic acid methyl ester. (trans-iq) trans-pyAsp-Met-OMe (S)-2-((2S,3R)-2-Carboxymethyl-5-oxo-pyrrolidin-3- ylamino)-4-methylthio-butyric acid methyl ester.

(trans-1 r) frans-pyAsp-D-Ala-NH(CH(CMe₂)₂S) {(2S,3/?)-5-Oxo-3-[(R)-1 -(2,2,4,4- tetramethyl-thietan-3-ylcarbamoyl)-ethylamino]-pyrrolidin-2-yl}-acetic acid.

Claims

1 . Pyrrolidinone derivatives characterized by the general formula (1),

wherein R¹, R², R³, R⁴ and R⁵, which may be identical or different, are selected independently from the group consisting of hydrogen, linear, branched or, if applicable, cyclic C₁-C₆ alky], OH, NH, SH, O-(C_rC₆)-alkyl, N-(C_rC₆)-alkyl, S-(C_rC₆)-alkyl, benzyl and aryl, R⁶ is either hydrogen or a protective group, X is selected from the group con- sisting of residues of amino acids and amino acid derivatives, and wherein said amino group of X is directly coupled to the carbon atom in the 3rd position of the pyrrolidinone ring, as well as salts, racemates and diastereomers thereof.

2. Pyrrolidinone derivatives according to claim 1 characterized in that X is selected from the group consisting of residues of α-amino acids and derivatives of α- amino acids as well as D-alanyl tetramethylthietanylamide or derivatives thereof.

3. Pyrrolidinone derivatives according to claims 1 or 2, characterized in that X is selected from the group consisting of residues of phenylalanine, phenylglycine, tyrosine, methionine and glycine or esters or amide derivatives thereof as well as D-alanyl tetramethylthietanylamide or derivatives thereof.

4. Pyrrolidinone derivatives according to claim 3 characterized in that X is a residue of phenylalanine or an ester or amide derivative thereof.

5. Pyrrolidinone derivatives according to any of the above claims 1 - 4, characterized in that X is a residue of an amino acid or an ester or amide derivative thereof carrying an alkyl (Alk)-substitution in the carboxyl group.

6. Pyrrolidinone derivatives according to claim 5, characterized in that X is a resi- due of an amino acid or an ester or amide derivative thereof carrying a propyl (Pr)- substitution in the carboxyl group.

7. Pyrrolidinone derivatives according to claim 5, characterized in that X is a residue of an amino acid or an ester or amide derivative thereof carrying an ethyl (Et)- substitution in the carboxyl group.

8. Pyrrolidinone derivatives according to claim 5, characterized in that X is a residue of an amino acid or an ester or amide derivative thereof carrying a methyl (Me)- substitution in the carboxyl group.

9. Pyrrolidinone derivatives according to any of the above claims characterized in that R⁶ is either hydrogen or a protective group selected from the group consisting of optionally substituted benzyl (Bn)₁ (C_rC₆)-all<yl, adamantyl, tert-butyl (^fBu), cyclohexyl, trialkylsilyl and triarylsilyl.

10. Pyrrolidinone derivatives according to any of the above claims characterized in that at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is hydrogen.

11. Pyrrolidinone derivatives according to any of the above claims, characterized in that R⁶ is hydrogen.

12. Pyrrolidinone derivatives according to any of the above claims, characterized in that R¹ is hydrogen.

13. Pyrrolidinone derivatives according to any of the above claims, characterized in that R³ and/or R⁴ is/are hydrogen.

14. Pyrrolidinone derivatives according to any of the above claims, characterized in that R² and/or R⁵ is/are hydrogen.

15. Pyrrolidinone derivatives characterized by the general formula (2),

wherein R¹, R², R⁵, R⁶, and X have the meanings as defined in any of claims 1-14, and wherein the amino group of X is directly coupled to the carbon atom in the 3rd position of the pyrrolidinone ring, as well as salts, racemates and diastereomers thereof.

16. A composition comprising one or more pyrrolidinone derivatives according to any of claims 1 - 14.

17. The composition according to claim 16 further comprising at least one conven- tional sweetening agent.

18. The composition according to claim 17 wherein said conventional sweetener is an artificial sweetening agent chosen among the group comprising Aspartame, Alitame, Neotame, Saccharine, Acesulfame K, Cyclamate and Sucralose.

19. A method for producing a pyrrolidinone derivative of the formula (1),

wherein R¹, R², R³, R⁴, R⁵, R⁶ and X has the meaning as defined in claims 1-14, and wherein the amino group of X is directly coupled to the carbon atom in the 3rd position of the pyrrolidinone ring, comprising the steps of:

a) Reacting a pyrrolidinone of the general formula (3),

wherein R¹, R², R⁵ and R⁶ has the meaning as defined in claims 1-14, in a condensation reaction, with a compound (HX) containing a moiety (X) that has the meaning as defined in any of the claims 1 -14, to obtain a product of the formula (2),

b) reducing the product of the formula (2) produced in step a) to obtain a product of the formula (1) wherein R³ and R⁴ are hydrogen and, in either order,

c) optionally separating cis- and trans- diastereoisomers,

d) optionally deprotecting the carboxylate group.

20. The method according to claim 19 wherein HX is a compound selected from the group consisting of α-amino acids and derivatives of α-amino acids as well as D- alanyl tetramethylthietanylamide or derivatives thereof.

21. The method according to claim 19 or 20, wherein HX is a compound selected from the group consisting of phenylalanine, phenylglycine, tyrosine, methionine and glycine or esters or amide derivatives thereof as well as D-alanyl tetramethylthietanyl- amide or derivatives thereof.

22. The method according to any of claims 19 - 21 , wherein HX is phenylalanine or an ester or amide derivative thereof.

23. The method according to any of claims 19 - 22 wherein HX is an amino acid or an ester or amide derivative thereof carrying an alkyl (Alk)-substitution in the car- boxyl group.

24. The method according to claim 23, wherein HX is an amino acid or an ester or amide derivative thereof carrying a propyl (Pr)-substitution in the carboxyl group.

25. The method according to claim 23, wherein HX is an amino acid or an ester or amide derivative thereof carrying a methyl (Me)-substitution in the carboxyl group.

26. The method according to claim 23, wherein HX is an amino acid or an ester or amide derivative thereof carrying an ethyl (Et)-substitution in the carboxyl group.

27. The method according to any of claims 19 - 26 wherein the reduction method in step b) is a reduction using sodium cyanoborohydride.

28. The method according to any of claims 19 - 26 wherein the reduction method in step b) is a hydrogenation over platinum oxide (PtO₂).

29. The method according to any of claims 19-28 wherein the step c) is performed by column chromatography.

30. The method according to claim 19 - 29, wherein the temperature in step a) is between 30 - 80⁰C, preferably between 40 - 60°C, even more preferably about 5O⁰C.

31. A use of a pyrrolidinone derivative according to any of claims 1 - 14 as a taste enhancer in compositions for oral consumption.

32. The use of a pyrrolidinone derivative according to claim 1 - 14 as a sweetening agent in compositions for oral consumption.