Inhibitors of dipeptidyl peptidase IV

[0001] The present invention relates to a series of novel compounds that are inhibitors of the enzyme dipeptidyl peptidase IV, to pharmaceutical compositions comprising these inhibitors, and the use of such compositions in the treatment of human diseases.

BACKGROUND

[0002] The enzyme dipeptidyl peptidase IV, herein abbreviated DP-IV (and elsewhere as DAP-IV or DPP-IV) and also known by the classification EC.3.4.14.5, is a serine protease that cleaves the N-terminal dipeptide from peptides that begin with the sequence H-Xaa-Pro (where Xaa is any amino acid, although preferably a lipophilic one, and Pro is proline). It will also accept as substrates peptides that begin with the sequence H-Xaa-Ala (where Ala is alanine). DP-IV was first identified as a membrane-bound protein. More recently a soluble form has been identified.

[0003] Initial interest in DP-IV focussed on its role in the activation of T lymphocytes. DP-IV is identical to the T cell protein CD26. It was proposed that inhibitors of DP-IV would be capable of modulating T cell responsiveness, and so could be developed as novel immunomodulators. It was further suggested that CD26 was a necessary co-receptor for HIV, and thus that DP-IV inhibitors could be useful in the treatment of AIDS.

[0004] Attention was given to the role of DP-IV outside the immune system. It was recognised that DP-IV has a key role in the degradation of several peptide hormones, including growth hormone releasing hormone (GHRH) and glucagon-like peptide-1 and -2 (GLP-1 and GLP-2). Since GLP-1 is known to have a potentiating effect on the action of insulin in the control of post-prandial blood glucose levels it is clear that DP-IV inhibitors might also be usefully employed in the treatment of type II diabetes and impaired glucose tolerance. At least two DP-IV inhibitors are currently undergoing clinical trials to explore this possibility.

[0005] Several groups have disclosed inhibitors of DP-IV. While some leads have been found from random screening programs, the majority of the work in this field has been directed towards the investigation of substrate analogs. Inhibitors of DP-IV that are substrate analogs are disclosed in, for example, U.S. Pat. No. 5,462,928, U.S. Pat. No. 5,543,396, WO95/15309 (equivalent to U.S. Pat. No. 5,939,560 and EP 0731789), WO98/119998 (equivalent to U.S. Pat. No. 6,011,155), WO99/46272 and WO99/61431. The most potent inhibitors are aminoacyl pyrrolidine boronic acids, but these are unstable and tend to cydise, while the more stable pyrrolidine and thiazolidine derivatives have a lower affinity for the enzyme and so would require large doses in a clinical situation. Pyrrolidine nitrites appear to offer a good compromise since they have both a high affinity for the enzyme and a reasonably long half-life in solution as the free base. There remains, however, a need for inhibitors of DP-IV with improved properties.

BRIEF DESCRIPTION OF THE INVENTION

[0006] The present invention relates to a series of inhibitors of DP-IV with improved affinity for the enzyme. The compounds can be used for the treatment of a number of human diseases, including impaired glucose tolerance and type II diabetes. Accordingly, the invention further relates to the use of the compounds in the preparation of pharmaceutical compositions, to such compositions per se, and to the use of such compositions in human therapy. The compounds of the invention are described by general formula 1.

[0007] In general formula 1, X1 is selected from —S—, —O—, —SO—, —SO2— and —CH2—; X2 is selected from —O—, —S—, —NH— and —CH2—; X3 is either —NR5— or a >C═O or >C═S group; R1 is either H or —CN; R2 and R3 are independently selected from H and lower alkyl, or together may be —(CH2)p—; R4 is R4A when X3 is —NR5— and R4B when X3 is >C═O or >C═S; R4A is selected from R6R7NC(═O), R6R7NC(═S); R8(CH2)qC(═O), R8(CH2)qC(═S), R8(CH2)qSO2, R8(CH2)qOC(═S) and R8(CH2)qOC(═O); R4B is R6R7N; R5 is H or lower alkyl; R6 and R7 are each independently R8(CH2)q or together they are —(CH2)2—Z—(CH2)2—; R8 is selected from H, alkyl, optionally substituted aryl, optionally substituted aroyl, optionally substituted arylsulphonyl and optionally substituted heteroaryl; Z is selected from a covalent bond, —(CH2)r—, —O—, —SOt— and —N((CH2)qR8)—; n is 0-4; p is 2-5; q is 0-3; r is 1 or 2; and t is 0-2.

DETAILED DESCRIPTION OF THE INVENTION

[0008] In a first aspect, the present invention comprises a series of novel compounds that are inhibitors of the enzyme DP-IV and are useful for the treatment of certain human diseases. The compounds are described by general formula 1.

[0009] In general formula 1, X1 is a divalent group selected from a sulphur atom (—S—), an oxygen atom (—O—), a sulphinyl group (—SO—), a sulphonyl group (—SO2—) and a methylene group (—CH2—). X2 is a divalent group selected from an oxygen atom (—O—), a sulphur atom (—S—) and a methylene group (—CH2—). X3 is either a substituted imino group (—NR5—) or a carbonyl (>C═O) or thiocarbonyl (>C═S) group.

[0010] R1 is either a hydrogen atom (H) or a nitrile group (—CN).

[0011] R2 and R3 may each independently of the other be a hydrogen atom or a lower alkyl group, or together they may be a chain of between two and five methylene units (—(CH2)p— where p is in the range 2-5) so as to form, with the carbon atom to which they are attached, a three, four, five or six-membered ring. The value of m may be 1, 2 or 3. When m is greater than 1 then each CR2R3 unit may be the same or different. For example, when m is 2 then (CR2R3)2 may be CH2CH2, CH2C(Me)2, C(Me)2CH2 and the like.

[0012] The nature of R4 depends on the identity of X3, such that the two groups are linked by an amide (CO—N), thioamide (CS—N) or sulphonamide (SO2—N) bond. So, when X3 is a substituted imino group (—NR5—) then R4 is R4A, where R4A is selected from carbamoyl groups (R6R7NC(═O)), thiocarbamoyl groups (R6R7NC(═S)); optionally modified acyl groups (R8(CH2)qC(═O)), optionally modified thioacyl groups (R8(CH2)qC(═S)), sulphonyl groups (R8(CH2)qSO2), optionally modified (alkyl or aryloxy)carbonyl groups (R8(CH2)qOC(═O)) and optionally modified (alkyl or aryloxy)thiocarbonyl groups(R8(CH2)qOC(═S)). As used herein, the term “optionally modified” is taken to indicate that some embodiments of R8 are beyond the scope of the terms “alkyl”, “acyl” and “aryl”. The scope of the definition of R4A is determined by the scope of the definition of R8. Alternatively, when X3 is a carbonyl (>C═O) or thiocarbonyl (>C═S) group then R4 is R4B, where R4B is a substituted amino group (R6R7N).

[0013] R5 is a hydrogen atom (H) or a lower alkyl group. Preferably, R5 is H.

[0014] R6 and R7 may each independently of the other be R8(CH2)q. Alternatively, they may together be a group —(CH2)2—Z1—(CH2)2— or —CHR9—Z2—CH2—CR10—. Here Z1 is a covalent bond, a methylene or ethylidene group (—(CH2)r— where r is 1 or 2), an oxygen atom (—O—), a sulphur or oxidised sulphur atom (—SOt— where t is zero, 1 or 2) or a substituted imino group (—N((CH2)qR8)—), such that the group NR6R7 is a pyrrolidine, piperidine, perhydroazepine, morpholine, optionally oxidised thiomorpholine or substituted piperazine ring. Z2 is an ortho-phenylene moiety (—C6H4—), such that the group NR6R7 is a tetrahydroisoquinoline.

[0015] R8 is selected from a hydrogen atom (H), a lower alkyl group, a benzo-fused lower cycloalkyl group (such as an indanyl group), an acyl (lower alkyl-CO) group, a di(lower alkyl)amino group, a di(lower alkyl)carbamoyl group, an N-(lower alkyl)piperidinyl group, an optionally substituted α-alkylbenzyl group, an optionally substituted phenyl, naphthyl or heteroaryl group, and an optionally substituted aroyl (aryl-CO) or arylsulphonyl (aryl-SO2) group. In the foregoing, suitable optional substituents are lower alkyl, aryl which may be further substituted with one or more methyl or trifluoromethyl groups, hydroxy, lower alkyloxy, lower alkylsulphonyl, acyl, perfluoroacyl, amino, lower alkylamino, di(lower alkyl)amino, aminoalkylene, fluoro, chloro, bromo, trifluoromethyl, nitro, cyano, carbamoyl, carboxy and lower alkyloxycarbonyl groups. In addition, two adjacent substituents may be linked so as to form a ring fused to the parent aryl or heteroaryl ring.

[0016] R9 and R10 are independently selected from hydrogen, carbamoyl, hydroxymethyl and cyanomethyl groups.

[0017] The integer n is selected from the range zero to 4, and q is selected from the range zero to 3.

[0018] Certain compounds are specifically excluded from the scope of the present invention. When X2 is methylene, X3 is NH and R4 is R8(CH2)qO(CO), with q=1, then R8 may not be unsubstituted phenyl or phenyl substituted with a nitro group. It is generally preferred that when X2 is methylene, X3 is NH, R4 is R8(CH2)qO(CO), q is 1 and R8 is a substituted phenyl group then the substituent or substituents should be selected from chloro, methoxy and trifluoromethyl groups.

[0019] In the context of the present disclosure, the term lower alkyl, either by itself or in such combinations as lower alkyloxy, is intended to comprise linear, branched and cyclic saturated hydrocarbon groups of between one and six carbon atoms. Examples of lower alkyl groups include, but are not limited to, methyl, ethyl, isopropyl, tert-butyl, neopentyl, cyclohexyl, cyclopentylmethyl, 2-(cyclopropyl)ethyl, 3,3-dimethylcyclobutyl and bicyclo[3.1.0]hexyl.

[0020] The term heteroaryl includes monocyclic five- and six-membered ring aromatic groups with one or two heteroatoms, which are selected from nitrogen, oxygen and sulphur. Thus, heteroaryl groups include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl and isoxazolyl. Heteroaryl further includes the benzofused derivatives of these rings, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, phthalazinyl, cinnolinyl, indolyl, isoindolyl, benzothiazolyl and the like, and bicyclic groups formed by the fusion of two such monocyclic heteroaromatic groups.

[0021] The term aryl includes phenyl, naphthyl and heteroaryl.

[0022] The compounds of general formula 1 have at least one stereogenic centre and so can exhibit optical isomerism. All such isomers, including enantiomers, diastereomers and epimers are included within the scope of the invention. Furthermore, the invention includes such compounds as single isomers and as mixtures, including racemates. Certain compounds according to general formula 1, including those with a heteroaryl group which carries a hydroxy or amino substituent, can exist as tautomers. These tautomers, either separately or as mixtures, are also considered to be within the scope of the invention.

[0023] The compounds according to general formula 1 have at least one basic functional group. They can therefore form addition salts with acids. Those addition salts that are formed with pharmaceutically acceptable acids are included within the scope of the invention. Examples of suitable acids include acetic acid, trifluoroacetic acid, citric acid, fumaric acid, benzoic acid, pamoic acid, methanesulphonic acid, hydrochloric acid, nitric acid, sulphuric acid, phosphoric acid and the like.

[0024] Certain compounds according to general formula 1 have an acidic group and so are able to form salts with bases. Examples of such salts include the sodium, potassium and calcium salts, which are formed by the reaction of the acid with the corresponding metal hydroxide, oxide, carbonate or bicarbonate. Similarly, tetra-alkyl ammonium salts may be formed by the reaction of the acid with a tetra-alkyl ammonium hydroxide. Primary, secondary and tertiary amines, such as triethylamine, can form addition salts with the acid. A particular case of this would be an internal addition salt formed between an acidic group and the primary amine group of the same molecule, which is also called a zwitterion. Insofar as they are pharmaceutically acceptable, all these salts are included within the scope of the invention.

[0025] In a preferred embodiment of the invention R1 is a nitrile group. Within this embodiment, it is preferred that the stereochemistry of the nitrile group is as shown in general formula 2.

[0026] According to the standard terminology, this is the S configuration when X1 is methylene but the R configuration when X1 is sulphur, oxygen, sulphinyl or sulphonyl.

[0027] In another preferred embodiment, the stereochemistry at the centre adjacent to the primary amine is as shown in general formula 3. This is the S configuration when X2 is an oxygen atom or a methylene or imino group, and the R configuration when X2 is a sulphur atom.

[0028] Within this embodiment, it is more preferred that R1 should be a nitrile group, and more preferred still that it should have the absolute configuration depicted in general formula 4.

[0029] In another preferred embodiment of the invention, m is 1. More preferably m is 1 and R2 and R3 are independently hydrogen atoms or methyl groups. When X2 is a methylene group, it is more preferred that R2 and R3 both be hydrogen. When X2 is an oxygen atom, it is more preferred that one of R2 and R3 be hydrogen and the other a methyl group. When X2 is a sulphur atom, it is more preferred that both R2 and R3 be methyl groups.

[0030] In another preferred embodiment, X1 is either S or methylene. More preferably, X1 is S and R1 is H, or X1 is methylene and R1 is CN.

[0031] In another preferred embodiment, X3 is NH. More preferably, X3 is NH, m is 1, R2 and R3 are both H, X2 is methylene and n is 1 or 2.

[0032] In another preferred embodiment, R4 is R6NHCO or R8CO and R8 is an optionally substituted heteroaryl group. More preferably, R8 is an unsubstituted heteroaryl group, or a heteroaryl group substituted with one or two groups chosen from lower alkyl, lower alkyloxy, fluoro, chloro and trifluoromethyl groups.

[0033] In another preferred embodiment, X3 is CO and R4 is R8NH. More preferably R8 is an optionally substituted heteroaryl group. More preferably still, R8 is an unsubstituted heteroaryl group, or a heteroaryl group substituted with one or two groups chosen from lower alkyl, lower alkyloxy, fluoro, chloro and trifluoromethyl groups.

[0034] In another preferred embodiment, X3 is NH and R4 is selected from R6R7N(CO), R8(CH2)qCO and R8(CH2)qSO2.

[0035] Particularly preferred compounds within the invention include:

[0036] (2S)-1-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine2-carbonitrile,

[0037] (2S)-1-[Nω-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile,

[0038] (2S)-1-[(2′S)-2′-Amino-4′-(pyrazinyl-2″-carbonylamino)butanoyl]pyrrolidine-2-carbonitrile,

[0039] (4R)-3-[Nω-(Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile,

[0040] (2S)-1-[Nω-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile,

[0041] 1-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine,

[0042] (2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,

[0043] 3-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine,

[0044] 1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine, and

[0045] (2S)-1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine2-carbonitrile

[0046] 3-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,

[0047] 3-[Nω-(2-Quinoxaloyl)-L-lysinyl]thiazolidine,

[0048] 3-[Nω-(2-Quinoxaloyl)-L-ornithinyl]thiazolidine,

[0049] (2S)-1-[Nω-(2-Quinoxaloyl)-L-ornithinyl]pyrrolidine-2-carbonitrile,

[0050] 3-[Nω-(6-Methylpyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine,

[0051] 3-[Nω-(Isoquinoline-3-carbonyl)-L-ornithinyl]thiazolidine,

[0052] 3-[Nω-(6-Trifluoromethylnicotinoyl)-L-ornithinyl]thiazolidine,

[0053] (2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile,

[0054] (2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile,

[0055] 3-[Nω-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidine,

[0056] 3-[O-(3-Chlorobenzylcarbamoyl)serinyl]thiazolidine, and

[0057] 3-[(2′S)-2′-Amino-5′-oxo-5′-(1″,2″,3″,4″-tetrahydroisoquinolin-2″-yl)pentanoyl]thiazolidine.

[0058] In a second aspect, the present invention comprises a pharmaceutical composition for human therapeutic use. The composition is characterised in that it has, as an active agent, at least one of the compounds described above. Such a composition is useful in the treatment of human diseases. The composition will generally include one or more additional components selected from pharmaceutically acceptable excipients and pharmaceutically active agents other than those of the present invention.

[0059] The composition may be presented as a solid or liquid formulation, depending on the intended route of administration. Examples of solid formulations include pills, tablets, capsules and powders for oral administration, suppositories for rectal or vaginal administration, powders for nasal or pulmonary administration, and patches for transdermal or transmucosal (such as buccal) administration. Examples of liquid formulations include solutions and suspensions for intravenous, subcutaneous or intramuscular injection and oral, nasal or pulmonary administration. A particularly preferred presentation is a tablet for oral administration. Another preferred presentation, particularly for emergency and critical care, is a sterile solution for intravenous injection.

[0060] The composition comprises at least one compound according to the preceding description. The composition may contain more than one such compound, but in general it is preferred that it should comprise only one. The amount of the compound used in the composition will be such that the total daily dose of the active agent can be administered in one to four convenient dose units. For example, the composition can be a tablet containing an amount of compound equal to the total daily dose necessary, said tablet to be taken once per day. Alternatively, the tablet can contain half (or one third, or one quarter) of the daily dose, to be taken twice (or three or four times) per day. Such a tablet can also be scored to facilitate divided dosing, so that, for example, a tablet comprising a full daily dose can be broken into half and administered in two portions. Preferably, a tablet or other unit dosage form will contain between 0.1 mg and 1 g of active compound. More preferably, it will contain between 1 mg and 250 mg.

[0061] The composition will generally include one or more excipients selected from those that are recognised as being pharmaceutically acceptable. Suitable excipients include, but are not limited to, bulking agents, binding agents, diluents, solvents, preservatives and flavouring agents. Agents that modify the release characteristics of the composition, such as polymers that selectively dissolve in the intestine (“enteric coatings”) are also considered in the context of the present invention, to be suitable excipients.

[0062] The composition may comprise, in addition to the compound of the invention, a second pharmaceutically active agent. For example, the composition may include an anti-diabetic agent, a growth-promoting agent, an anti-inflammatory agent or an antiviral agent. However, it is generally preferred that the composition comprise only one active agent.

[0063] In a third aspect, the invention comprises a use for the compounds and compositions described above for the treatment of human diseases. This aspect can equally be considered to comprise a method of treatment for such diseases. The diseases susceptible to treatment are those wherein an inhibition of DP-IV or CD26 results in a clinical benefit either directly or indirectly. Direct effects include the blockade of T lymphocyte activation. Indirect effects include the potentiation of peptide hormone activity by preventing the degradation of these hormones. Examples of diseases include, but are not limited to, auto-immune and inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis, growth hormone deficiency leading to short stature, polycystic ovary syndrome, impaired glucose tolerance and type 2 diabetes. Particularly preferred is the use of the compounds and compositions for the treatment of impaired glucose tolerance and type 2 diabetes, and equally a method of treatment of these diseases by the administration of an effective amount of a compound or composition as previously described.

[0064] The precise details of the treatment, including the dosing regimen, will be established by the attending physician taking into account the general profile of the patient and the severity of the disease. For diseases such as inflammatory bowel disease that have acute phases of active disease separated by quiescent periods, the physician may select a relatively high dose during the acute phase and a lower maintenance dose for the quiescent period. For chronic diseases such as type 2 diabetes and impaired glucose tolerance, the dosing may need to be maintained at the same level for an extended period. A dosing schedule of one to four tablets per day, each comprising between 0.1 mg and 1 g (and preferably between 1 mg and 250 mg) of active compound might be typical in such a case.

[0065] The compounds according to the invention can be prepared by methods known in the art. The route chosen will depend on the particular nature of the substituents present in the target molecule. In the following general description the synthetic route is outlined for compounds wherein m is 1. Compounds with m=2 or 3 can generally be prepared by analogous routes.

[0066] The starting material will usually be an α,ω-diamino acid derivative 5 or an amino dicarboxylic acid derivative 6.

[0067] PG1 and PG2 are “orthogonal” protecting groups—groups that mask the reactivity of the amine groups and that can each be selectively removed in the presence of the other. Suitable groups are well known in the literature. PG3 and PG4 are protecting groups for carboxylic acids. They are chosen such that they are orthogonal to each other and to the amino protecting groups. Suitable possibilities for PG3 and PG4 are also well known in the literature. Derivatives of diamino acids according to general formula 5 and derivatives of amino dicarboxylic acids according to general formula 6 are either items of commerce, or can be prepared following methods described in the literature. In practice, and depending on the strategy chosen, the starting material will have only two of the three protecting groups present. Either PG3 will be absent to allow the pyrrolidine (or thiazolidine or oxazolidine) residue to be introduced, or PG1 or PG4 will be absent to allow the side chain to be elaborated.

[0068] Scheme A illustrates the introduction of the pyrrolidine (or thiazolidine or oxazolidine) group as the first step in the preparation of the compounds of the invention.

[0069] Compounds 5A and 6A correspond to 5 and 6 with PG3 as a hydrogen atom (i.e. without the protecting group). The free carboxylic acid can be reacted with a pyrrolidine derivative 7 to give the amide 8 or 9. Reaction conditions for achieving this transformation are well known in the literature. Suitable reagents include carbodiimides, phosphorus reagents and alkyl chloroformates, and the reaction is usually catalysed by a tertiary amine such as triethylamine or dimethylaminopyridine.

[0070] The reaction depicted in Scheme A is available for all combinations of R1 and X1. However, for the case where R1 is a nitrile group, or where X1 is a sulphinyl or sulphonyl group, it may be advantageous to modify the strategy as depicted in Schemes B and C.

[0071] In Scheme B, the R1 group is introduced as a primary amide and subsequently transformed into a nitrile by the action of a dehydrating agent such as trifluoroacetic anhydride. In Scheme C, the X1 group is introduced as a thioether and subsequently transformed into a sulphoxide (a=1) or sulphone (a=2) by the action of an oxidant such as sodium periodate. The modification to the strategy afforded by Scheme C is not possible if X2 is a sulphur atom.

[0072] In Scheme D, compound 5D is the diamino acid derivative 5 where the ω-protecting group is a hydrogen atom. The free amine group reacts readily with sulphonyl chlorides, acyl chlorides and thioacyl chlorides, usually in the presence of a tertiary amine, to produce sulphonamides 14, amides 15 and thioamides 16 respectively. The reagents are generally either available per se, or can be prepared from the corresponding acids. The reaction of scheme D is generally applicable to all the variations of the group R8(CH2)q, with the proviso that certain of the substituents contemplated for the phenyl and heteroaryl rings which are options for R8 may require protection. Such substituents and the appropriate protection will generally be obvious to those familiar with the art.

[0073] Reagents: i) COCl2; ii) CSCl2; iii) R8(CH2)qOH; iv) R6R7NH; v) R8(CH2)qOCOCl; vi) R8(CH2)qOCSCl; vii) R6R7NCOCl; viii) R6R7NCSCl.

[0074] Scheme E illustrates the elaboration of 5D to give carbamates and ureas, and their thio analogues. When R5 is other than a hydrogen atom, compound 5D can be converted to the corresponding carbamoyl chloride 17 or thiocarbamoyl chloride 18 by reaction with phosgene or thiophosgene. Other reagents are known in the art to be functionally equivalent to these toxic reagents and they may also be used. When R5 is hydrogen, the intermediate formed is an isocyanate or isothiocyanate, but this behaves functionally as an equivalent of the corresponding chloride. Intermediates 17 and 18 are not normally isolated, but are treated directly with alcohols to give carbamates 19 and thiocarbamates 20. Treatment of these same intermediates with amines leads to the formation of ureas 21 and thioureas 22. Alternatively, 5D may be reacted directly with a chloroformate or chlorothioformate to give the carbamate or thiocarbamate, or, when neither R6 nor R7 is hydrogen, with a chloroformamide or chlorothioformamide to give the urea or thiourea. When R6 or R7 is a hydrogen atom the chloroformamide or chlorothioformamide will tend to be unstable, in which case the isocyanate (eg. R6—NCO) or isothiocyanate (e.g. R6—NCS) is used. As discussed previously for Scheme D, certain substituents within the embodiments of R8 may require appropriate protection.

[0075] Scheme F illustrates the elaboration of the side chain of the amino dicarboxylic acid series. The ω-deprotected acid 6F can be reacted under a variety of conditions with an amine to give amide 23. The condensation may be promoted by a dehydrating reagent such as a carbodiimide or a phosphorus reagent. Alternatively the acid may be converted into a more reactive derivative, such as by treatment with oxalyl chloride or thionyl chloride to give the corresponding acid chloride, which will react directly with an amine. The thioamide 24 may be obtained by treating the amide 23 with Lawesson's reagent.

[0076] When all the groups have been elaborated the final protecting group is removed and the product is isolated and purified using standard techniques.

[0077] These general methods are further illustrated in the following, non limiting examples.

EXAMPLES

[0078] Abbreviations

[0079] The following abbreviations have been used.

1DMFN,N-DimethyformamidehHour(s)hplcHigh pressure liquid chromatographyminMinute(s)pet. etherPetroleum ether fraction boiling at 60-80° C.PyBOP ®(Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphatePyBroP ®Bromotripyrrolidinophosphonium hexafluorophosphateTFATrifluoroacetic acid

Example 1

(2S)-1-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0080]

[0081] A. N-(2-Nitrobenzenesulphenyl)-L-proline

[0082] L-Proline (25 g, 217 mmol) was dissolved in 2M NaOH (110 mL, 220 mmol) and dioxan (120 mL). A solution of 2-nitrobenzenesulphenyl chloride (42 g, 222 mmol) in dioxan (60 mL) was slowly added at the same time as 2M NaOH (110 mL, 220 mmol). After 2 h at room temperature the reaction mixture was poured into water (500 mL) and the solid filtered off. The pH of the filtrate was adjusted to pH3 with 2M HCl and the solution was extracted with ethyl acetate (3×500 mL). The combined organic extracts were washed with water (4×200 mL) and brine (1×200 mL), dried (Na2SO4) and evaporated in vacuo to give an orange solid identified as N-(2-nitrobenzenesulphenyl)-L-proline (58.1 g, 217 mmol, 100%).

[0083] B. N-(2-Nitrobenzenesulphenyl)-L-proline succinimidyl Ester

[0084] N-(2-Nitrobenzenesulphenyl)-L-proline (57.9 g, 216 mmol) was dissolved in CH2Cl2/DMF (9:1, 500 mL). N-Hydroxysuccinimide (37.3 g, 324 mmol) and water-soluble carbodiimide (51.8 g, 260 mmol) were added. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (1000 mL). The solution was washed with water (4×200 mL) and brine (1×200 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow solid identified as N-(2-nitrobenzenesulphenyl)-L-proline succinimidyl ester (78.9 g, 216 mmol, 100%).

[0085] C. N-(2-Nitrobenzenesulphenyl)-L-prolinamide

[0086] N-(2-Nitrobenzenesulphenyl)-L-proline succinimidyl ester (78.5 g, 215 mmol) was dissolved in dioxan (500 mL). Ammonia (35%, 100 mL) was added, After stirring at room temperature for 2 h the reaction mixture was poured into water (700 mL). The precipitate was filtered off, washed with water (200 mL), dried over P2O5 and recrystallised from ethyl acetate/pet ether to give a yellow solid identified as N-(2-nitrobenzenesulphenyl)-L-prolinamide (49.6 g, 185 mmol, 86%).

[0087] D. (2S)-N-(2-Nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile

[0088] N-(2-Nitrobenzenesulphenyl)-L-prolinamide (49 g, 183 mmol) was dissolved in dry THF(300 mL). The solution was cooled to 0° C., triethylamine (36.7 g, 367 mmol) was added followed by the slow addition of trifluoroacetic anhydride (77 g, 367 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (500 mL), washed with water (1×200 mL) and brine (1×200 mL), dried (Na2SO4) and evaporated in vacuo to give an orange oil which was purified by flash chromatography (eluant: 80% pet ether, 20% ethyl acetate) to give a yellow solid identified as (2S)-N-(2-nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile (38.9 g, 150 mmol, 82%).

[0089] E. (2S)-Pyrrolidine-2-carbonitrile Hydrochloride

[0090] (2S)-N-(2-Nitrobenzenesulphenyl)pyrrolidine-2-carbonitrile (38.5 g, 149 mmol) was dissolved in diethyl ether (200 mL). 4M HCl/Dioxan (150 mL, 600 mmol) was slowly added. After 2 h at room temperature the reaction mixture was poured into diethyl ether (1000 mL). The solid was filtered off, washed with diethyl ether (500 mL) and recrystallised from methanol/diethyl ether to give a white solid identified as (2S)-pyrrolidine-2-carbonitrile hydrochloride (18.9 g, 142.5 mmol, 96%).

[0091] F. (2S)-1-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]-pyrrolidine-2-carbonitrile.

[0092] Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithine (2.5 g, 7.4 mmol) was dissolved in CH2Cl2 (50 mL). This solution was cooled to 0° C., (2S)-pyrrolidine-2-carbonitrile hydrochloride (1.2 g, 9.1 mmol) and PyBOP® (4.3 g, 8.23 mmol) were added, and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 mL). This solution was washed with 0.3M KHSO4 (2×50 mL), sat. NaHCO3 (2×50 mL), water (2×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. This was purified by flash chromatography (eluant: 80% ethyl acetate, 20% pet. ether) to give a colourless oil identified as (2S)-1-[Nα-(tert-butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile (2.98 g, 7.16 mmol, 97%).

[0093] G. (2S)-1-[Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0094] (2S)-1-[Nα-tert-Butyloxycarbonyl-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile (2.8 g, 6.7 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1 h at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a colourless oil identified as (2S)-1-[Nω(-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile trifluoroacetate (1.5 g, 3.48 mmol, 52%).

[0095] [M+H]+=317.3

Example 2

(2S)-1-[Nω-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0096]

[0097] A. (Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide

[0098] Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysine (5 g, 10.7 mmol) was dissolved in CH2Cl2 (100 mL). The solution was cooled to 0° C., L-prolinamide (1.78 g, 11.7 mmol) and PyBOP® (6.7 g, 12.8 mmol) were added, and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 mL). The solution was washed with 0.3M KHSO4 (2×50 mL), sat. NaHCO3 (2×50 mL), water (2×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as (Nα-(tert-butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide (4.05 g, 7.2 mmol, 67%).

[0099] B. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-pyrrolidine-2-carbonitrile

[0100] (Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)-L-prolinamide (3.95 g, 7.02 mmol) was dissolved in dry THF (100 mL). The solution was cooled to 0° C., triethylamine (1.4 g, 14 mmol) was added followed by the slow addition of trifluoroacetic anhydride (2.97 g, 14.1 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (1×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give an orange oil. The residue was purified by flash chromatography (eluant: 60% pet ether, 40% ethyl acetate) to give a colourless oil identified as (2S)-1-(Nα-(tert-butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (3.3 g, 6.11 mmol, 87%).

[0101] C. (2S)-1-(Nα(-tert-Butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile

[0102] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (3.1 g, 5.7 mmol) was dissolved in THF (80 mL). Diethylamine (20 mL) was added. After 2 h at room temperature the solvent was removed in vacuo. The residue was purified by flash chromatography (eluant: 90% chloroform, 7% methanol, 3% triethylamine) to give a colourless oil identified as (2S)-1-(Nα-(tert-butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (1.63 g, 5.03 mmol, 89%).

[0103] D. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile

[0104] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (100 mg, 0.31 mmol) was dissolved in CH2Cl2/DMF (9:1, 20 mL). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (84 mg, 0.62 mmol), water-soluble carbodiimide (76 mg, 0.38 mmol), 2-pyrazinecarboxylic acid (43 mg, 0.35 mmol) and triethylamine (65 mg, 0.65 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). This solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as (2S)-1-(Nα-(tert-butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (124 mg, 0.29 mmol, 93%).

[0105] E. (2S)-1-[Nω-(Pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0106] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl)pyrrolidine-2-carbonitrile (110 mg, 0.26 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1 h at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a colourless oil identified as (2S)-1-[Nω-(pyrazinyl-2-carbonyl)-L-lysinyl]pyrrolidine-2-carbonitrile trifluoroacetate (66 mg).

[0107] [M+H]+=331.1

Example 3

(4R)-3-[Nω-(Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile Trifluoroacetate

[0108]

[0109] A. (4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxamide

[0110] (4R)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxylic acid (12.5 g,54.1 mmol) was dissolved in CH2Cl2/DMF (9:1, 150 mL). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (14.6 g, 108 mmol) and water-soluble carbodiimide (13.0 g, 65 mmol). After 1 h at 0° C. ammonia (35%, 50 mL) was added. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (500 mL). The solution was washed with 0.3M KHSO4 (2×100 mL), sat. NaHCO3 (2×100 mL), water (2×100 mL) and brine (1×100 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as (4R)-3-(tert-butyloxycarbonyl)thiazolidine-4-carboxamide (8.9 g, 38.4 mmol, 71%).

[0111] B. (4R)-Thiazolidine4-carboxamide Hydrochloride

[0112] (4S)-3-(tert-Butyloxycarbonyl)thiazolidine-4-carboxamide (8.6 g, 37.1 mmol) was dissolved in 4M HCl/dioxan (50 mL). After 1 h at room temperature the solvent was evaporated in vacuo to give a white solid identified as (4R)-thiazolidine-4-carboxamide hydrochloride (6.2 g, 36.8 mmol, 99%).

[0113] C. (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]-thiazolidine4-carboxamide

[0114] Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysine (5 g, 10.7 mmol) was dissolved in CH2Cl2 (100 mL). This solution was cooled to 0° C., (4R)-thiazolidine-4-carboxamide hydrochloride (1.78 g, 11.7 mmol) and PyBOP® (6.7 g, 12.8 mmol) were added, and the pH was adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 mL). The solution was washed with 0.3M KHSO4 (2×50 mL), sat. NaHCO3 (2×50 mL), water (2×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as (4R)-3-[Nα-(tert-butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carboxamide (2.81 g, 4.8 mmol, 44%).

[0115] D. (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]-thiazolidine-4-carbonitrile

[0116] (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carboxamide (2.7 g, 4.7 mmol) was dissolved in dry THF (100 mL). The solution was cooled to 0° C., triethylamine (1.0 g, 10 mmol) was added followed by the slow addition of trifluoroacetic anhydride (2.0 g, 9.5 mmol). The pH was adjusted to pH9 with triethylamine. After 30 min the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (1×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 60% pet ether, 40% ethyl acetate) to give a colourless oil identified as (4R)-3-[Nα-(tert-butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (2.14 g, 3.81 mmol, 82%).

[0117] E. (4R)-3-[Nα-(tert-Butyloxycarbonyl)-L-lysinyl]thiazolidine4-carbonitrile

[0118] (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (1.9 g, 3.4 mmol) was dissolved in THF (40 mL). Diethylamine (10 mL) was added. After 2 h at room temperature the solvent was removed in vacuo. The residue was purified by flash chromatography (eluant: 90% chloroform, 7% methanol, 3% triethylamine) to give a colourless oil identified as (4R)-3-[Nα-(tert-butyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (863 mg, 2.5 mmol, 75%).

[0119] F. (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile

[0120] (4R)-3-[Nα-(tert-Butyloxycarbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (100 mg, 0.29 mmol) was dissolved in CH2Cl2 (20 mL). To this solution at 0° C. 2-pyrazinecarboxylic acid (43 mg, 0.35 mmol) and PyBOP® (170 mg, 0.33 mmol) were added and the pH was adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 2% methanol, 98% chloroform) to give a colourless oil identified as (4R)-3-[Nα-(tert-butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (112 mg, 0.25 mmol, 86%).

[0121] G. (4R)-3-[Nω-Pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile Trifluoroacetate

[0122] (4R)-3-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile (110 mg, 0.26 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1 h at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a colourless oil identified as (4R)-3-[Nω-(pyrazinyl-2-carbonyl)-L-lysinyl]thiazolidine-4-carbonitrile trifluoroacetate (57 mg).

[0123] [M+H]+=349.1

Example 4

(2S)-1-[Nω-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile Dihydrochloride

[0124]

[0125] A. (2S)-1-[N-(tert-Butyloxycarbonyl)-Oω-methyl-L-glutamyl]pyrrolidine-2-carbonitrile

[0126] N-(tert-Butyloxycarbonyl)-Oω-methyl-L-glutamic acid (1.0 g, 3.83 mmol) was dissolved in CH2Cl2/DMF (9:1, 20 mL). To this solution at 0° C. were added 1-hydroxybenzotriazole hydrate (788 mg, 5.84 mmol), water-soluble carbodiimide (877 mg, 4.38 mmol), (2S)-pyrrolidine-2-carbonitrile hydrochloride (609 mg, 4.6 mmol) and triethylamine (65 mg, 0.65 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL), this solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 50% ethyl acetate, 50% pet. ether) to give a brown oil identified as (2S)-1-[N-(tert-butyloxycarbonyl)-Oω-methyl-L-glutamyl]pyrrolidine-2-carbonitrile (290 mg, 0.86 mmol, 22%).

[0127] B. (2S)-1-[N-(tert-Butyloxycarbonyl)-L-glutamyl]pyrrolidine-2-carbonitrile

[0128] (2S)-1-[N-(tert-Butyloxycarbonyl)-Oω-methyl-L-glutamyl]pyrrolidine-2-carbonitrile (250 mg, 0.74 mmol) was dissolved in dioxan (5 mL). 1M Lithium hydroxide (1.1 mL, 1.1 mmol) was added. After 1 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). This solution was washed with 1M KHSO4 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo to give a colourless oil identified as (2S)-1-[N-(tert-butyloxycarbonyl)-L-glutamy]pyrrolidine-2-carbonitrile (200 mg, 0.61 mmol, 83%).

[0129] C. (2S)-1-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile

[0130] (2S)-1-[N-(tert-Butyloxycarbonyl)-L-glutamyl]pyrrolidine-2-carbonitrile (30 mg, 0.093 mmol) was dissolved in CH2Cl2/DMF (9:1, 10 mL). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (21 mg, 0.16 mmol), water-soluble carbodiimide (21 mg, 0.105 mmol), 3-(aminomethyl)pyridine (11 mg, 0.1 mmol) and triethylamine(20 mg, 0.2 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. The residue was purified by flash chromatography (eluant: 5% methano, 97% chloroform) to give a colourless oil identified as (2S)-1-[Nα-(tert-butyloxycarbonyl)-Nω-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile (16.6 mg, 0.04 mmol, 44%).

[0131] D. (2S)-1-[Nω-(Pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile Dihydrochloride

[0132] (2S)-1-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidin-2-carbonitrile (17 mg, 0.04 mmol) was dissolved in 4M HCl/dioxan (5 mL). After 1 h at room temperature the solvent was removed in vacuo to give a white solid identified as (2S)-1-[Nω-(pyridyl-3-methyl)-L-glutaminyl]pyrrolidine-2-carbonitrile dihydrochloride (17 mg, 0.04 mmol, 100%).

[0133] [M+H]+=316.2

Example 5

1-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine Trifluoroacetate

[0134]

[0135] A. 1-[Nω-(Benzyloxycarbonyl)-Nα-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine

[0136] Nω-(Benzyloxycarbonyl)-Nα-(tert-butyloxycarbonyl)-L-ornithine (5.49 g, 15 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 mL). To this solution at 0° C. was added 1-hydroxybenzotriazole hydrate (3.37 g, 22 mmol), water-soluble carbodiimide (3.46 g, 18 mmol), pyrrolidine (1.28 g, 18 mmol) and triethylamine (200 mg, 20 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (200 mL). The solution was washed with 0.3M KHSO4 (2×50 mL), sat. NaHCO3 (2×50 mL), water (2×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 90% ethyl acetate, 10% pet. ether) to give a colourless oil identified as 1-[Nω-(benzyloxycarbonyl)-Nα-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine (5.15 g, 12.3 mmol, 82%).

[0137] B. 1-[Nα-(tert-Butyloxycarbonyl)-L-ornithinyl]pyrrolidine

[0138] 1-[Nω-(1-(Benzyloxycarbonyl)-Nα-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine (2.15 g, 5.13 mmol) was dissolved in methanol (80 mL). This solution was hydrogenated over 10% Pd/C (400 mg). After 2 h the catalyst was filtered off and washed with methanol (50 mL). The combined filtrates were evaporated in vacuo to give an off white solid identified as 1-[Nα-(tert-butyloxycarbonyl)-L-ornithinyl]pyrrolidine (1.35 g, 4.74 mmol, 94%).

[0139] C. 1-[Nα-tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine

[0140] 1-[Nα-(tert-Butyloxycarbonyl)-L-ornithinyl]pyrrolidine (100 mg, 0.35 mmol) was dissolved in CH2Cl2 (20 mL). To this solution at 0° C. were added PyBroP® (195 mg, 0.4 mmol), 2-pyrazinecarboxylic acid (50 mg, 0.4 mmol) and triethylamine (100 mg, 1.0 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give a sticky white solid identified as 1-[Nα-(tert-butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine (90 mg, 0.25 mmol, 66%).

[0141] D. 1-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine Trifluoroacetate

[0142] 1-[Nα-(ert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine (90 mg, 0.23 mmol) was dissolved in 4M HCl/dioxan (15 mL). After 45 min at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a colourless oil identified as 1-[Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]pyrrolidine trifluoroacetate (51 mg).

[0143] [M+H]+=292.1

Example 6

3-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine trifluoroacetate

[0144]

[0145] A. 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine

[0146] Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-ornithine (2.73 g, 6 mmol) was dissolved in CH2Cl2/DMF (9:1, 100 mL). To this solution at 0° C. were added 1-hydroxybenzotriazole hydrate (1.53 g, 10 mmol), water-soluble carbodiimide (1.34 g, 7 mmol), thiazolidine (1.28 g, 18 mmol) and triethylamine (80 mg, 8 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (100 mL). The solution was washed with 0.3M KHSO4 (2×25 mL), sat. NaHCO3 (2×25 mL), water (2×25 mL) and brine (1×25 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 75% ethyl acetate, 25% pet. ether) to give a white solid identified as 3-[Nα-(tert-butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine (2.55 g, 4.85 mmol, 81%).

[0147] B. 3-[Nα-(tert-Butyloxycarbonyl)-L-ornithinyl]thiazolidine

[0148] 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(9-fluorenylmethyloxycarbonyl)-L-ornithinyl]thiazolidine (1.15 g, 2.13 mmol) was dissolved in acetonitrile (20 mL). Diethylamine (5 mL) was added. After 90 min at room temperature the solvent was removed in vacuo and the residue was purified by flash chromatography (eluant: 90% chloroform, 7% methanol, 3% triethylamine) to give a pale yellow oil identified as 3-[Nα-(tert-butyloxycarbonyl)-L-ornithinyl]thiazolidine (530 mg, 1.67 mmol, 78%).

[0149] C. 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine

[0150] 3-[Nα-(tert-Butyloxycarbonyl)-L-ornithinyl]thiazolidine (80 mg, 0.27 mmol) was dissolved in CH2Cl2 (20 mL). To this solution at 0° C. were added PyBroP® (146 mg, 0.3 mmol), 2-pyrazinecarboxylic acid (37 mg, 0.3 mmol) and triethylamine (90 mg, 0.9 mmol). After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 3% methanol, 97% chloroform) to give a sticky white solid identified as 3-[Nα-(tert-butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine (45 mg, 0.11 mmol, 41%).

[0151] D. 3-[Nω-(Pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine Trifluoroacetate

[0152] 3-[Nα-(-(tert-Butyloxycarbonyl)-Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine (45 mg, 0.11 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 min at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a colourless oil identified as 3-[Nω-(pyrazinyl-2-carbonyl)-L-ornithinyl]thiazolidine trifluoroacetate (14 mg).

[0153] [M+H]+=310.0

Example 7

(2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0154]

[0155] A. (2S)-1-[S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0156] S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteine (660 mg, 2.26 mmol) was dissolved in CH2Cl2 (30 mL). To this solution at 0° C. were added (2S)-pyrrolidine-2-carbonitrile hydrochloride (250 mg, 1.89 mmol) and PyBOP® (1.3 g, 2.49 mmol), and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (150 mL). The solution was washed with 0.3M KHSO4 (2×30 mL), sat. NaHCO3 (2×30 mL), water (2×30 mL) and brine (1×30 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 75% ethyl acetate, 25% pet. ether) to give a colourless oil identified as (2S)-1-[S-(acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]-pyrrolidine-2-carbonitrile (650 mg, 1.76 mmol, 78%).

[0157] B. (2S)-1-[S-(Acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0158] (2S)-1-[S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (610 mg, 1.65 mmol) was dissolved in trifluoroacetic acid (30 mL). After 1 h at room temperature the solvent was removed in vacuo to give a colourless oil identified as (2S)-1-[S-(acetylaminomethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile trifluoroacetate (620 mg, 1.61 mmol, 98%).

[0159] [M+H]+=271.0

Example 8

(2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0160]

[0161] A. (2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile

[0162] S-(Acetylaminomethyl)-N-(tert-butyloxycarbonyl)penicillamine (720 mg, 2.25 mmol) was dissolved in CH2Cl2 (30 mL). To this solution at 0° C. were added (2S)-pyrrolidine-2-carbonitrile hydrochloride (270 mg, 2.04 mmol) and PyBOP® (1.3 g, 2.49 mmol), and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (150 mL). The solution was washed with 0.3M KHSO4 (2×30 mL), sat. NaHCO3 (2×30 mL), water (2×30 mL) and brine (1×30 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 75% ethyl acetate, 25% pet. ether) to give a colourless oil identified as (2S)-1-[(2′R)-3′-(acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile (742 mg, 1.86 mmol, 83%).

[0163] B. (2S)-1-[(2′-R)-3′-(Acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0164] (2S)-1-[(2′R)-3′-(Acetylaminomethylthio)-2′-(tert-butyloxycarbonylamino)-3′-methylbutanoyl]pyrrolidine-2-carbonitrile (710 mg, 1.78 mmol) was dissolved in trifluoroacetic acid (30 mL). After 1 h at room temperature the solvent was removed in vacuo to give a colourless oil identified as (2S)-1-[(2′R)-3′-(acetylaminomethylthio)-2′-amino-3′-methylbutanoyl]pyrrolidine-2-carbonitrile trifluoroacetate (560 mg, 1.36 mmol, 76%).

[0165] [M+H]+=299.1

Example 9

(2S)-1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0166]

[0167] A. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile

[0168] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile was prepared by the method described for the lysine derivative in Example 2.

[0169] B. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)-pyrrolidine-2-carbonitrile

[0170] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile (80 mg, 0.26 mmol) was dissolved in CH2Cl2 (20 mL). To this solution was added 2-chloropyridine-3-carbonyl chloride (55 mg, 0.32 mmol) and the pH adjusted to pH9 with triethylamine. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 95% ethyl acetate, 5% pet. ether) to give a colourless oil identified as (2S)-1-(Nα-(tert-butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile (60 mg, 0.14 mmol, 53%).

[0171] C. (2S)-1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0172] (2S)-1-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile (60 mg, 0.14 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1 h at room temperature the solvent was removed in vacuo. The residue was purified by

[0173] [M+H]+=299.1

Example 9

(2S)-1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0174]

[0175] A. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile

[0176] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile was prepared by the method described for the lysine derivative in Example 2.

[0177] B. (2S)-1-(Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)-pyrrolidine-2-carbonitrile

[0178] (2S)-1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile (80 mg, 0.26 mmol) was dissolved in CH2Cl2 (20 mL). To this solution was added 2-chloropyridine-3-carbonyl chloride (55 mg, 0.32 mmol) and the pH adjusted to pH9 with triethylamine. After 18 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 95% ethyl acetate, 5% pet. ether) to give a colourless oil identified as (2S)-1-(Nα-(tert-butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine-2-carbonitrile (60 mg, 0.14 mmol, 53%).

[0179] C. (2S)-1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0180] (2S)-1-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile (60 mg, 0.14 mmol) was dissolved in trifluoroacetic acid (5 mL). After 1 h at room temperature the solvent was removed in vacuo. The residue was purified by preparative hplc (Vydac C18, 5 to 50% 0.1% TFA/acetonitrile into 0.1% TFA/water over 40 min at 3 mL/min). Fractions containing the product were lyophilised to give a white solid identified as (2S)-1-[Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine-2-carbonitrile trifluoroacetate (52 mg).

[0181] [M+H]+=350.1

Example 10 3

1-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine Hydrochloride

[0182]

[0183] A. 1-(Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine

[0184] 1-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)pyrrolidine (20 mg, 0.069 mmol) was dissolved in CH2Cl2 (5 mL). To this solution was added 2-chloropyridine-3-carbonyl chloride (14 mg, 0.076 mmol) and the pH adjusted to pH9 with triethylamine. After 1 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 10% methanol, 90% dichloromethane) to give a colourless oil identified as 1-(Nα-(tert-butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)pyrrolidine (19 mg, 0.045 mmol, 63%).

[0185] B. 1-[Nω-(2Chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine Hydrochloride

[0186] 1-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine (19 mg, 0.045 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 min at room temperature the solvent was removed in vacuo to give a white solid identified as 1-[Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]pyrrolidine hydrochloride (15 mg).

[0187] [M+H]+=325.1

Example 11

3-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine Hydrochloride

[0188]

[0189] A. 3-(Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)thiazolidine

[0190] 3-(Nα-(tert-Butyloxycarbonyl)-L-ornithyl)thiazolidine (136 mg, 0.45 mmol) was dissolved in CH2Cl2 (10 mL). To this solution was added 2-chloropyridine3-carbonyl chloride (88 mg, 0.5 mmol) and the pH adjusted to pH9 with triethylamine. After 1 h at room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). The solution was washed with 0.3M KHSO4 (2×20 mL), sat. NaHCO3 (2×20 mL), water (2×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo. The residue was purified by flash chromatography (eluant: 1.5% methanol, 98.5% dichloromethane) to give a colourless oil identified as 3-(Nα-(tert-butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithyl)thiazolidine (30 mg, 0.068 mmol, 15%).

[0191] B. 3-[Nω-(2-Chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine Hydrochloride

[0192] 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine (30 mg, 0.068 mmol) was dissolved in 4M HCl/dioxan (10 mL). After 45 min at room temperature the solvent was removed in vacuo to give a white solid identified as 1-[Nω-(2-chloropyridyl-3-carbonyl)-L-ornithinyl]thiazolidine hydrochloride (25 mg).

[0193] [M+H]+=342.1

Example 12

(2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2carbonitrile Trifluoroacetate

[0194]

[0195] A. S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine

[0196] N-(tert-Butyloxycarbonyl)-L-cysteine (3.5 g, 15.8 mmol), benzyl 2-bromoacetate (3.7 g, 16.1 mmol) and triethylamine (1.8 g, 18.0 mmol) were dissolved in THF (100 mL). After 18 hours at room temperature the reaction mixture was diluted with ethyl acetate (100 mL), washed with 0.3M KHSO4, sat NaHCO3, water and brine, dried (Na2SO4) and evaporated. The residue was purified by flash chromatography (eluant 95% chloroform, 4% methanol, 1% acetic acid) yielding a colourless oil identified as S-(benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine (5.2 g, 14.1 mmol, 89%).

[0197] B. (2S)-1-[S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0198] S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteine (5.10 g, 13.8 mmol) was dissolved in CH2Cl2 (200 mL). This solution was cooled to 0° C., (2S)-pyrrolidine-2-carbonitrile hydrochloride (2.1 g, 15.8 mmol) and PyBOP (8.0 g, 15.3 mmol) were added, and the pH was adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (150 mL). This solution was washed with 0.3M KHSO4 (1×50 mL), sat. NaHCO3 (1×50 mL), water (1×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. This was purified by flash chromatography (eluant: 40% ethyl acetate, 60% pet. ether) to give a colourless oil identified as (2S)-1-[S-(benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (5.82 g, 13.0 mmol, 94%).

[0199] C. (2S)-1-[N-(tert-Butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0200] (2S)-1-[S-(Benzyloxycarbonylmethyl)-N-(tert-butyloxycarbonyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (1.31 g, 2.9 mmol) was dissolved in THF(100 mL). Aqueous lithium hydroxide (1M, 3.5 mL, 3.5 mmol) was added. After 3 hours at room temperature the reaction mixture was diluted with ethyl acetate (100 mL), washed with 1M citric acid, water and brine, dried (Na2SO4) and evaporated in vacuo to give a colourless oil. This was purified by flash chromatography (eluant: 97% chloroform, 2% methanol, 1% acetic acid) to give a colourless oil identified as (2S)-1-[N-(tert-butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (860 mg, 2.4 mmol, 82%).

[0201] D. (2S)-1[N-(Tert-Butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile

[0202] (2S)-1-[N-(tert-Butyloxycarbonyl)-S-(carboxymethyl)-L-cysteinyl]pyrrolidine-2-carbonitrite (150 mg, 0.42 mmol) was dissolved in CH2Cl2 (20 mL). This solution was cooled to 0° C., 3-(aminomethyl)pyridine (53 mg, 0.5 mmol) and PyBOP (270 mg, 0.52 mmol) were added, and the pH adjusted to pH9 with triethylamine. After 18 h at 0° C. to room temperature the solvent was removed in vacuo and the residue was taken up in ethyl acetate (70 mL). This solution was washed with 0.3M KHSO4 (1×20 mL), sat. NaHCO3 (1×20 mL), water (1×20 mL) and brine (1×20 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. This was purified by flash chromatography (eluant: 96% chloroform, 4% methanol) to give a colourless oil identified as (2S)-1-[N-(tert-butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (170 mg, 0.38 mmol, 91%).

[0203] E. (2S)-1-[S-(3-Picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile Trifluoroacetate

[0204] (2S)-1-[N-(tert-Butyloxycarbonyl)-S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile (130 mg, 0.29 mmol) was dissolved in trifluoroacetic acid (10 mL). After 1 hour at room temperature the solvent was removed in vacuo to give a white solid identified as (2S)-1-[S-(3-picolylcarbamoylmethyl)-L-cysteinyl]pyrrolidine-2-carbonitrile trifluoroacetate (116 mg,0.25 mmol, 86%).

[0205] [M+H]+=348.2

Example 13

3-[Nω-(2-Quinoxaloyl)-L-lysinyl]thiazolidine Hydrochloride

[0206]

[0207] A. 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-quinoxaloyl)-L-lysinyl]thiazolidine

[0208] 3-[Nα-(tert-Butyloxycarbonyl)lysinyl]thiazolidine (128 mg, 0.4 mmol) was dissolved in CH2Cl2 (10 mL). 2-Quinoxaloyl chloride (85 mg, 0.44 mmol) and potassium carbonate (45.8 mg, 0.3 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and the solvent removed in vacuo. The residue was purified by flash chromatography (eluant: 99.5% chloroform, 0.5% methanol) to give a colourless oil identified as 3-[Nα-(tert-butyloxycarbonyl)-Nω-(2-quinoxaloyl)-L-lysinyl]thiazolidine (140 mg, 0.296 mmol, 74%).

[0209] B. 3-[Nω-(2-Quinoxaloyl)-L-lysinyl]thiazolidine Hydrochloride

[0210] 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(2-quinoxaloyl)-L-lysinyl]thiazolidine (140 mg, 0.296 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1 hour at room temperature the solvent was removed in vacuo to give a white solid identified as 3-[Nω-(2-quinoxaloyl)-L-lysinyl]thiazolidine hydrochloride (128 mg, 0.296 mmol, 100%).

[0211] [M+H]+=374.2

Example 14

3-[Nω-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidine Hydrochloride

[0212]

[0213] A. 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine

[0214] 3-Hydroxypyridine (14.9 mg, 0.138 mmol) was dissolved in CH2Cl2 (20 mL). Phosgene (20% solution in toluene, 0.335 mL, 0.685 mmol) and pyridine (14 mg, 0.182 mmol) were added at 0° C. After 90 mins the solvent was removed in vacuo and the residue dissolved in CH2Cl2 (20 mL). 3-[Nα-(tert-Butyloxycarbonyl)ornithinyl]thiazolidine (42 mg, 0.138 mmol) and triethylamine (28 mg, 0.28 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours and the solvent removed in vacuo. The residue was purified by flash chromatography (eluant: 97% chloroform, 3% methanol) to give a colourless oil identified as 3-[Nα-(tert-butyloxycarbonyl)-Nω-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine (16 mg. 0.038 mmol, 27%).

[0215] B. 3-[Nω-(3-Pyridyloxycarbonyl)-L-ornithinyl]thiazolidine Hydrochloride

[0216] 3-[Nα-(tert-Butyloxycarbonyl)-Nω-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine (16 mg, 0.038 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1 hour at room temperature the solvent was removed in vacuo to give a white solid identified as 3-[Nω-(3-pyridyloxycarbonyl)-L-ornithinyl]thiazolidine hydrochloride (14 mg, 0.038 mmol, 100%).

[0217] [M+H]+=325.1

Example 15

3-[O-(3-Chlorobenzylcarbamoyl)serinyl]thiazolidine Hydrochloride

[0218]

[0219] A. 3-[N-(tert-Butyloxycarbonyl)-L-serinyl]thiazolidine

[0220] N-(tert-Butyloxycarbonyl)-L-serine (2.1 g, 10.2 mmol) was dissolved in CH2Cl2/DMF (9:1,50 mL). Thiazolidine (650 mg, 11.2 mmol), hydroxybenzotriazole(2.8 g, 20.7 mmol) and water soluble carbodiimide (3.9 g, 19.5 mmol) were added at 0° C. The pH was adjusted to pH8 with N-methylmorpholine. The reaction mixture was stirred at room temperature for 18 hours, the solvent removed in vacuo and the residue was taken up in ethyl acetate (150 mL). This solution was washed with 0.3M KHSO4 (1×50 mL), sat. NaHCO3 (1×50 mL), water (1×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give a white solid identified as 3-[N-(tert-butyloxycarbonyl)-L-serinyl]thiazolidine (2.15 g, 7.78 mmol, 76%).

[0221] B. 3-[N-(tert-Butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine

[0222] 3-[N-(tert-Butyloxycarbonyl)-L-serinyl]thiazolidine (110 mg, 0.48 mmol) was dissolved in DMF (10 mL) and 1,1′-carbonyl-diimidazole (71 mg, 0.43 mmol) was added. After 2 hours at room temperature 3-chlorobenzylamine (62 mg, 0.4 mmol) was added. After a further 18 hours EtOAc (200 mL) was added. This solution was washed with 0.3M KHSO4 (1×50 mL), sat. NaHCO3 (1×50 mL), water (4×50 mL) and brine (1×50 mL), dried (Na2SO4) and evaporated in vacuo to give a yellow oil. This was purified by flash chromatography (eluant: 40% ethyl acetate, 60% pet. ether) to give a colourless oil identified as 3-[N-(tert-butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine (158 mg, 0.36 mmol, 90%).

[0223] C. 3-[O-(3-Chlorobenzylcarbamoyl)-L-serinyl]thiazolidine Hydrochloride

[0224] 3-[N-(tert-Butyloxycarbonyl)-O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine (140 mg, 0.32 mmol) was dissolved in 4M HCl/dioxan (20 mL). After 1 hour at room temperature the solvent was removed in vacuo to give a white solid identified as 3-[O-(3-chlorobenzylcarbamoyl)-L-serinyl]thiazolidine hydrochloride (115 mg, 0.3 mmol, 94%).

[0225] [M+H]+=344.1

[0226] The Examples set out in the following Tables were prepared by analogous methods to the above.

2TABLE 1Examples 16-162

ExamplenR8(CH2)qR1X116 17 18 191 2 1 2

CN CN H HCH2CH2S S20 21 22 231 2 1 2

CN CN H HS S CH2CH224 25 26 271 2 1 2

CN CN H HCH2CH2S S28 29 30 311 2 1 2

CN CN H HCH2CH2S S32 33 34 351 2 1 2

CN CN H HCH2CH2S S36 37 381 2 1

CN CN HCH2CH2S39 40 41 421 2 1 2

CN CN H HCH2CH2S S43 44 45 461 2 1 2

CN CN H HCH2CH2S S47 48 49 501 2 1 2

CN CN H HCH2CH2S S51 52 53 541 2 1 2

CN CN H HCH2CH2S S55 56 57 581 2 1 2

CN CN H HCH2CH2S S59 60 61 621 2 1 2

CN CN H HCH2CH2S S63 64 65 661 2 1 2

CN CN H HCH2CH2S S67 68 69 701 2 1 2

CN CN H HCH2CH2S S71 72 73 741 2 1 2

CN CN H HCH2CH2S S75 76 77 781 2 1 2

CN CN H HCH2CH2S S79 80 81 821 2 1 2

CN CN H HCH2CH2S S83 84 85 861 2 1 2

CN CN H HCH2CH2S S87 88 89 901 2 1 2

CN CN H HCH2CH2S S91 92 93 941 2 1 2

CN CN H HCH2CH2S S95 96 97 981 2 1 2

CN CN H HCH2CH2S S99 100 101 1021 2 1 2

CN CN H HCH2CH2S S103 104 105 1061 2 1 2

CN CN H HCH2CH2S S107 108 109 1101 2 1 2

CN CN H HCH2CH2S S111 112 113 1141 2 1 2

CN CN H HCH2CH2S S115 116 117 1181 2 1 2

CN CN H HCH2CH2S S119 120 121 1221 2 1 2

CN CN H HCH2CH2S S123 124 125 1261 2 1 2

CN CN H HCH2CH2S S1271

HS128 1291 2

H HS S130 1311 2

H HS S1321

HS1331

HS1341

HS1351

HS1361

HS1371

HS1381

HS1391

HS1401

HS1411

HS1421

HS1431

HS1441

HCH21451

HCH21461

HCH21471

HCH21481

HS149 1501 2

H HS S151 1511 2

H HS S1531

HS1541

HS1551

HS1561

HS1571

HS1581

HS1591

HS1601

HS1611

HS1621

HCH2

[0227]

3

TABLE 2

Examples 163-250

Example
n
R6R7N
R1
X1

163 164 165 166
1 2 1 2

CN CN H H
CH2CH2S S

167 168 169 170
1 2 1 2

CN CN H H
S S CH2CH2

171 172 173 174
1 2 1 2

CN CN H H
CH2CH2S S

175 176 177 178
1 2 1 2

CN CN H H
CH2CH2S S

179 180 181 182
1 2 1 2

CN CN H H
CH2CH2S S

183 184 185 186
1 2 1 2

CN CN H H
CH2CH2S S

187 188 189 190
1 2 1 2

CN CN H H
CH2CH2S S

191
1
NH2
CN
CH2

192
2

CN
CH2

193
1

H
S

194
2

H
S

195 196 197 198
1 2 1 2

CN CN H H
CH2CH2S S

199 200 201 202
1 2 1 2

CN CN H H
CH2CH2S S

203 204 205 206
1 2 1 2

100

CN CN H H
CH2CH2S S

207 208 209 210
1 2 1 2

101

CN CN H H
CH2CH2S S

211 212 213 214
1 2 1 2

102

CN CN H H
CH2CH2S S

215 216 217 218
1 2 1 2

103

CN CN H H
CH2CH2S S

219 220 221 222
1 2 1 2

104

CN CN H H
CH2CH2S S

223 224 225 226
1 2 1 2

105

CN CN H H
CH2CH2S S

227 228 229 230
1 2 1 2

106

CN CN H H
CH2CH2S S

231 232 233 234
1 2 1 2

107

CN CN H H
CH2CH2S S

235 236 237 238
1 2 1 2

108

CN CN H H
CH2CH2S S

239 240 241 242
1 2 1 2

109

CN CN H H
CH2CH2S S

243 244 245 246
1 2 1 2

110

CN CN H H
CH2CH2S S

247 248 249 250
1 2 1 2

111

CN CN H H
CH2CH2S S

[0228]

4

TABLE 3

Examples 251-266

112

Example
n
R6R7N
R1
X1

251 252 253 254
1 2 1 2

113

CN CN H H
CH2CH2S S

255 256 257 258
1 2 1 2

114

CN CN H H
CH2CH2S S

259 260 261 262
1 2 1 2

115

CN CN H H
CH2CH2S S

263 264 265 266
1 2 1 2

116

CN CN H H
S S CH2CH2

[0229]

5

TABLE 4

Examples 267-318

117

Example
n
R8(CH2)q
R1
X1

267 268
1 2

118

CN CN
CH2CH2

269 270
1 2

119

CN CN
S S

271 272
1 2

120

CN CN
CH2CH2

273 274
1 2

121

CN CN
CH2CH2

275 276
1 2

122

CN CN
CH2CH2

277 278
1 2

123

CN CN
CH2CH2

279 280
1 2

124

CN CN
CH2CH2

281 282
1 2

125

H H
CH2CH2

283 284
1 2

126

H H
CH2CH2

285 286
1 2

127

H H
CH2CH2

287 288
1 2

128

H H
S S

289 290
1 2

129

H H
S S

291 292
1 2

130

H H
S S

293 294
1 2

131

CN CN
CH2CH2

295 296
1 2

132

CN CN
CH2CH2

297 298
1 2

133

CN CN
CH2CH2

299 300
1 2

134

CN CN
CH2CH2

301 302
1 2

135

CN CN
CH2CH2

303 304
1 2

136

CN CN
CH2CH2

305 306
1 2

137

CN CN
CH2CH2

307 308
1 2

138

H H
S S

309 310
1 2

139

H H
S S

311 312
1 2

140

H H
S S

313 314
1 2

141

H H
S S

315 316
1 2

142

H H
S S

317 318
1 2

143

H H
S S

[0230]

6

TABLE 5

Examples 319-378

144

Example
R4B
R1
X1

319
NH2
CN
CH2

320

145

CN
CH2

321

146

CN
CH2

322

147

CN
CH2

323

148

CN
CH2

324

149

CN
CH2

325

150

CN
CH2

326

151

CN
CH2

327

152

H
CH2

328

153

H
CH2

329

154

H
S

330

155

H
S

331

156

H
S

332

157

H
S

333

158

CN
CH2

334

159

CN
CH2

335

160

CN
S

336

161

CN
S

337

162

CN
CH2

338

163

CN
CH2

339

164

CN
CH2

340

165

CN
CH2

341

166

H
S

342

167

H
S

343

168

H
S

344

169

H
S

345

170

H
S

346

171

H
S

347

172

H
S

348

173

H
S

349

174

H
S

350

175

H
S

351

176

H
S

352

177

H
S

353

178

H
S

354

179

H
S

355

180

H
S

356

181

H
S

357

182

H
S

358

183

H
S

359

184

H
S

360

185

H
S

361

186

H
CH2

362

187

H
CH2

363

188

H
S

364

189

H
S

365

190

H
S

366

191

H
S

367

192

H
S

368

193

H
S

369

194

H
S

370

195

H
S

371

196

H
S

372

197

H
S

373

198

H
S

374

199

H
S

375

200

H
S

376

201

H
CH2

377

202

H
CH2

378

203

H
CH2

[0231]

7

TABLE 6

Examples 379-418

204

Example
n
R8(CH2)q
R1
X1

379 380
1 2

205

CN CN
CH2CH2

381 382
1 2

206

CN CN
CH2CH2

383 384
1 2

207

CN CN
CH2CH2

385 386
1 2

208

CN CN
CH2CH2

387 388
1 2

209

CN CN
CH2CH2

389 390
1 2

210

CN CN
CH2CH2

391 392
1 2

211

CN CN
CH2CH2

393 394
1 2

212

CN CN
CH2CH2

395 396
1 2

213

H H
CH2CH2

397 398
1 2

214

H H
S S

399 400
1 2

215

CN CN
S S

401 402
1 2

216

CN CN
CH2CH2

403 404
1 2

217

CN CN
CH2CH2

405 406
1 2

218

CN CN
CH2CH2

407 408
1 2

219

CN CN
CH2CH2

409 410
1 2

220

CN CN
CH2CH2

411 412
1 2

221

CN CN
CH2CH2

413 414
1 2

222

H H
S S

415 416
1 2

223

H H
S S

417 418
1 2

224

H H
S S

[0232]

8

TABLE 7

Examples 419-438

225

Example
m
R6R7N

419
1

226

420
1

227

421
1

228

422
1

229

423
1

230

424 425 426
1 2 3

231

427 428 429
1 2 3

232

430 431 432
1 2 3

233

433 434
2 3

234

435 436
2 3

235

437 438
2 3

236

[0233]

9

TABLE 8

Examples 439-450

237

Example
n
R8(CH2)q
R1
X1

439 440
1 2

238

CN CN
CH2CH2

441 442
1 2

239

CN CN
CH2CH2

443 444
1 2

240

H H
S S

445 446
1 2

241

CN CN
CH2CH2

447 448
1 2

242

CN CN
CH2CH2

449 450
1 2

243

CN CN
CH2CH2

Example 451

Determination of Activity

[0234] Compounds were assayed as inhibitors of DP-IV according to the methods described in WO95/15309. All the compounds described in the foregoing Examples were competitive inhibitors of DP-IV with Ki values less than 300 nM.

Example 452

Determination of Activity in Vivo

[0235] The anti-diabetic action of selected compounds was demonstrated in Zucker obese rats using a standard oral glucose tolerance test. Control rats were given a solution of glucose by oral gavage, and plasma glucose levels were determined. These rats demonstrated a significant hyperglycaemia. Compounds according to the present invention were dissolved in glucose solution at various concentrations, such that the rats could be given varying doses of the compound simultaneously with the glucose challenge. The hyperglycaemic excursion was reduced in a dose-dependent manner in animals receiving between 0.1 and 100 mg/kg of DP-IV inhibitor.

Example 453

Pharmaceutical Formulation

[0236] Tablets containing 100 mg of the compound of Example 1 as the active agent are prepared from the following:

10Compound of Example 1200.0gCorn starch71.0gHydroxypropylcellulose18.0gCarboxymethylcellulose calcium13.0gMagnesium stearate3.0gLactose195.0gTotal500.0g

[0237] The materials are blended and then pressed to give 2000 tablets of 250 mg, each containing 100 mg of the compound of Example 1.

[0238] The above demonstrates that the compounds according to the present invention are inhibitors of DP-IV and would accordingly be expected to be useful as therapeutic agents for the treatment of impaired glucose tolerance, type II diabetes, and other diseases where inhibition of this enzyme leads to an improvement in the underlying pathology or the symptoms.

[0239] The present invention is further defined in the following Claims.

Inhibitors of dipeptidyl peptidase IV

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Date Filed

Date Published

Inventors

CPC

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Abstract

Description

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Priority Claims (1)

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