MXPA99002149A - Peptide prodrugs containing an alpha-hydroxyacid linker - Google Patents

Abstract

Peptide prodrugs of the general formula (I):X-L-Z, wherein X designates a pharmaceutically active peptide sequence, e.g. Leu-enkephalin;Z designates a peptide pre-sequence of 2 to 20 amino acid units, preferably comprising lysine and glutamic acid;and L is a linking group comprising from 3 to 9 backbone atoms, wherein the bond between the C-terminal carbonyl of X and L is different from a C-N amide bond. Preferably, the bond between X and L is an ester bond. It has been found that it is possible to obtain a remarkable increase in the resistance towards degradation by proteolytic enzymes such as carboxypeptidase A, pepsin A, leucine aminopeptidase,&agr;-chymotrypsin when masking a pharmaceutically active peptide as a prodrug of the formula (I). The prodrugs of formula (I) are cleaved by the blood plasma enzyme butyryl cholinesterase indicating a readily bioreversibility. It is believed that the stability towards enzymatic cleavage is due to an induced helix-like structure.

Description

PEPTIDE DEVICES CONTAINING AN ALPHA-HYDROXYZED LINKER FIELD OF THE INVENTION The present invention relates to prodrugs of pharmaceutically active peptides which have a reduced tendency towards hydrolysis.

BACKGROUND OF THE INVENTION There is a large number of pharmaceutically active peptides, e.g. ex. those that occur naturally in man or in animals, or synthetic analogs of such peptides. An illustrative example of such a peptide is the analgesically active peptide enkephalin which has given rise to a vast number of synthetic analogues. However, precisely because of its peptide nature, the routes of administration thereof have been more or less limited. Thus, the peptides are degraded rapidly and very effectively by enzymes, generally with half-lives in the range of minutes. The proteases and other prololytic enzymes are ubiquitous, particularly in the gastrointestinal tract, therefore the peptides are REF .: 29609 usually susceptible to degradation in multiple sites in oral administration, and to some degree in the blood, liver, kidney, and vascular endothelium. In addition, a given peptide is usually susceptible to degradation in more than one bond in the structure; each hydrolysis locus is mediated by a certain protease.

There have been a number of attempts in the protection of peptides against premature degradation, such as by modification of peptide structure, co-administration of protease inhibitors, or special formulation strategies, but have only been found with limited success.

BRIEF DESCRIPTION OF THE INVENTION It has now surprisingly been found that by equipping a pharmaceutically active peptide, at its C terminus, with a bioreversible amino acid pre-sequence, it is possible to make the peptide significantly less susceptible to degradation by proteases. Without referencing any specific model for this effect, it is believed that the presence of the pre-sequence induces a degree of structuring of a helix-like nature of the pharmaceutically active peptide, whereby the peptide is less susceptible to proteases in contrast to peptides in the random conformation of the winding. As a result of the structuring, the peptide is much more difficult to. Degrade for a protease. The bioreversible property of the pre-sequence is obtained by linking the peptide and the pre-sequence by means of a linking group attached to the peptide in a different manner from a normal peptide bond.

Accordingly, the invention relates to prodrugs of pharmaceutically active peptides (X-OH), peptide amides (X-NH2), or peptide esters (X-OR), wherein the prodrug has the general formula I X - L - Z wherein X binds to L at the C-terminal carbonyl function of X; L is a linking group, comprising -to 9 structural atoms, wherein the bond between the C-terminal carbonyl of X and L is different from a C-N amide bond; Y Z is a peptide sequence of 2-20 amino acid units and binds to L at the N-terminal nitrogen atom of Z, each amino acid unit binds independently of Ala, Leu, Ser, Thr, Tyr, Asn, Gln , Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of formula II -NH-C (R3) (R4) -C (= 0) - II wherein R3 and R4 are independently selected from CX_6 alkyl, -phenyl, and phenyl-methyl, wherein alkyl is optionally substituted with one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfon, and carboxy, and phenyl and phenyl-methyl is optionally substituted with one to three substituents selected from C 1-6 alkyl, C 2- alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or R 3 and R 4 together with the atom of carbon to which a cyclopentyl, cyclohexyl, or cycloheptyl ring is attached; or a salt thereof The present invention also relates to the use of a prodrug of general formula I in therapy, and to the use of a prodrug of general formula I in the preparation of a composition for use in therapy, and a pharmaceutical composition comprising a prodrug of general formula I and a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to an immobilized LZ-SSM linker peptide sequence, wherein L and Z are as defined above, and SSM designates a solid support material, the use of an immobilized linker peptide sequence LZ- SSM for the preparation of a prodrug of general formula I, and methods for the preparation of prodrugs of general formula I comprising the use of an immobilized linker peptide sequence LZ-SSM.

As the prodrugs of general formula I are new per se, a further aspect of the present invention relates to compounds of general formula I.

DETAILED DESCRIPTION OF THE INVENTION The peptides are used in a number of processes, e.g. ex. , cell to cell communication, some that are present in the autonomic and central nervous system. Some of the latter peptides, and a number of other peptides, exert important effects on vascular muscles and other smooth muscles. These peptides include, e.g. ex. , the vasoconstrictors angiotesin II, vasopressin, endothelin, neuropeptide Y, vasoactive intestinal peptide, substance P, neurotensin, and calcitonin, peptide related to the calcitonin gene, and peptide II related to the calcitonin gene. Among other pharmaceutically interesting peptides there may be mentioned the analgesic, antidiabetic, peptides. antibiotics, and anesthetics, etc. and, in this way, the peptide could be or be reminiscent of endorphins, enkephalins, insulin, gramicidin, paracelsin, delta-inducing peptide, ANF, vasotocin, bradykinin, dynorphin, endothelin, growth hormone releasing factor, growth hormone release peptide, oxytocin, tachykinin, ACTH, brain natriuretic polypeptide, cholecystokinin, corticotropin releasing factor, fragment • inhibitor that binds dia-zepam, FMRF-amide -, - galanin, gastric release polypeptide, gastrin, gastrin releasing polypeptide, glucagon, glucagon-like peptide 1, glucagon-like peptide 2, LHRH, melanin-concentrating hormone, alpha-MSH, peptides that modulate morphine, motilin, neurokinins, neuromedins, neuropeptide K, neuropeptide Y, PACAP, pancreatic polypeptide, peptide YY, PHM, secretin, somatostatin, substance K, substance P, THR, vasoactive intestinal polypeptide, and peptides biologically active as described in HL Lee, "Peptide and Protein Drug Delivery", Marcel Dekk'er Inc. 1991, Chapter 9, and references therein, Phoenix Pharmaceuticals, Inc. "The Peptide Elite", 1997-1998 Catalog, and Bachem, "Feinchemikalien AG" , Catalog S15-1995.

It should be understood that the aforementioned peptides as well as the pharmaceutically active peptide sequence of these peptides can be incorporated into the prodrugs (and compounds) of the invention.

In the present context, the term "pharmaceutically active peptide sequence" as applied to X is intended to indicate any peptide or peptide-containing structure, which is presented naturally or synthetically, having two or more units of amino acid (preferably three or more amino acid units) and that exerts a pharmaceutical effect in mammals such as humans. In the present context, the term "amino acid unit" as used in connection with X means any amino acid Oi, β, and? which occurs naturally or synthetically, as well as modified amino acids in the side chain such as modified tyrosines wherein the aromatic ring is further substituted with p. ex. one or more halogens, sulfon groups, nitro groups etc., and / or the phenol group is converted into an ester group, etc., protected amino acids in the side chain, wherein the side chains of the amino acid are protected according to known methods by experts in peptide chemistry, as described in, p. ex. , M. Bodanszky and A. Bodanszky, "The Practice of Peptide Synthesis", 2. Ed, Springer-Verlag, 1994, and J. Jones, "The Chemical Synthesis of Peptides", Clarendon Press, 1991, either in the form L or the corresponding D form.

The X-sequence of pharmaceutically active peptide preferably consists of 2-200 amino acid units, more preferably 2-100 amino acid units (eg 3-100) - even more preferably 2-50 amino acid units (eg 3). -50 or 4-30), in particular 2-20 amino acid units (eg 3-20 or 4-20), especially 2-10 amino acid units (eg, 3-10 or 4-10) ), such as 2-8 amino acid units (eg, 3-8 or 4-8).

In the present context, a pharmaceutically active peptide sequence X which in the native form is presented as the C-terminal free carboxylic acid, such as Leu-encephalin (H-Tyr-Gly-Gly-Phe-Leu-OH), X-OH is denoted. In a similar manner, a pharmaceutically active peptide X sequence with a C-terminal amide group, such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), is denoted as X-NH2 , and a pharmaceutically active peptide X sequence with a C-terminal ester group, is denoted X-OR, where OR, p. ex. is the alkoxy radical of the alcohol which, together with the X sequence of pharmaceutically active peptide, constitutes the ester. R could designate alkyl 1-6, aryl such as phenyl, aryl-Cx_6 alkyl such as benzyl, etc.

Thus, although the pharmaceutically active peptide X sequence is linked to the linker via the carbonyl C-terminus-1, it should be understood that any peptide sequence corresponding to pharmaceutically active peptides having a free C-terminal carboxy group (eg. eg X-OH) as well as peptides corresponding to pharmaceutically active peptides having a C-terminal amide (eg X-NH2) or ester group (eg X-OR) could be used in the compounds and prodrugs of the invention.

It is well known that many biologically active peptides also exert their desired biological effect when presented in a modified or truncated form. In the case of for example insulin, porcine insulin differs from human insulin by only one amino acid unit, amino acid B30 in porcine insulin which is Ala and amino acid B30 in human insulin which is Thr. Despite this difference, porcine insulin has been used as an effective diabetes drug for many years. In a similar manner it has been found that the essential activity characteristics in the porcine gastrin I heptadecapeptide are all contained in the C-terminal tetrapeptide and that essentially all the biological effects of neurotensin are associated with the C-terminal hexapeptide. In addition, pharmaceutically active peptides, wherein one or more amide bonds have been modified, e.g. ex. reduced, they often exhibit a similar or augmented biological activity; for example the analog Cys2 ^ [CH2NH] Tyr3 of somatostat ina was found to be an even more potent growth hormone releasing agent than somatostat ina itself, and also the transition state analog Leu10 ^ [CH (OH) CH2] Val? of angiotensin has been found to show strong inhibitory effect against the Renin protease of aspartic acid. Thus, the term "modified or truncated analogue thereof" is intended to indicate such peptides that are modified by changing and / or deleting one or more amino acid units in the native peptide sequence, which includes modification of the side chain, stereochemistry , and the structure in the individual amino acid units, such as changing one or more carboxamide bonds (-C (= 0) -N-) in p. ex. reduced forms such as (-CH (OH) -N-), (-CH2-N-), and other impeptide peptide bonds such as (-C (= 0) -0), (-C (= 0) - CH2-), (-CH = CH-), (-P02-NH-), (SO-CH, -), (S02-N-), etc.

That is, it should be understood that the peptide sequence in question should preferably comprise at least one amide bond (preferably two amide bonds (this does not apply naturally for a -d-peptide)) susceptible to enzymatic degradation to take full advantage of the present invention. .

The most interesting perspective of the present invention is that it is possible to prepare "peptide prodrugs" for the treatment of mammals, such as humans, which are stabilized towards degradation by proteases and which are subsequently capable of being released in an environment in which the peptide or the pharmaceutically active peptide sequence (X-OH) will exhibit a pharmaceutical action or be transported to the location desired. Although the pharmaceutically active peptide X sequence is preferably released as a free acid (due to cleavage of eg an ester bond between X and L) it is visualized that the free acid could also possess a relevant pharmaceutical effect in cases where the The X sequence of the pharmaceutically active peptide is an amide (X-NH2) or ester (X-OR).

Thus, in an interesting embodiment, the bond between the C-terminal carbonyl function of X and L is capable of being cut by blood plasma enzymes such as p. ex. butyryl cholinesterase, acetyl cholinesterase, etc. In particular, the bond between the C-terminal carbonyl function of X-and L is a -t-iolyester linkage or an ester linkage, preferably an ester linkage.

In order to release the X-OH sequence of pharmaceutically active peptide, the bond between X and L in the peptide prodrugs of the invention must be capable of breaking down in vi. It will be understood from the examples provided herein that the bond between X and L (which is preferably an ester linkage) is capable of being broken down by the butyryl cholinesterase enzyme. Thus, it is visualized that the peptide prodrug of the invention is capable of being broken by p. ex. esterases present in the blood plasma and thereby releasing the desired pharmaceutically active X-OH peptide at a desired location.

The enzymatic cleavage rate of the peptide could be adjusted for a medicament comprising prodrug I to have a prolonged or delayed effect. The adjustment of the break speed could, p. ex. , carried out by increasing or decreasing the volume and / or the electron donation effect of the substituents in L.

In the present context, the term "C ^ g alkyl" used alone or as part of other groups - designates a linear, branched or cyclic saturated hydrocarbon group having from one to six carbon atoms, such as methyl ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl, ter. utilo, n-pentyl, n-hexyl, cyclohexyl, etc. Similarly, the term "C1.5 alkyl" covers a saturated linear, branched or cyclic hydrocarbon group having from one to five carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl, ter. butyl, n-pentyl, isopentyl, cyclopentyl, etc. The term "C1.i alkyl" used alone or as part of other groups designates a straight or branched saturated hydrocarbon group having from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl, ter. butyl, etc., and similarly, the term "C1_3 alkyl" covers a straight or branched saturated hydrocarbon group having one to three carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

In the present context the term "C2_6 alkenyl" designates a hydrocarbon group having from two to six carbon atoms, which could be metal, branched or cyclic and could contain one or more double bonds, such as vinyl, allyl, -butenyl, -2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 4-pentenyl, 3-methyl-1-butenyl, 2-hexenyl, 5-hexenyl, cyclohexenyl, 2,3-dimethyl- 2-butenyl etc. , which could have ci s and / or trans configuration. Similarly, the term "C2_5 alkenyl" designates a hydrocarbon group having from two to five carbon atoms, which could be metal, branched or cyclic and could contain one or more double bonds, such as vinyl, allyl, 1-butenyl, -butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl, 3-methyl-1 -butenyl, cyclopentenyl, etc., which could have cy and / or trans configuration, and the term " C2_4 alkenyl "designates a hydrocarbon group having from two to four carbon atoms, which could be linear or branched and could contain one or more double bonds, such as vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, etc. , which could have ci and / or trans configuration.

The term "alkoxy" means alkyl-oxy.

The term "aryl" is intended to indicate an aromatic, carbocyclic group such as phenyl or naphthyl.

The term "halogen" includes fluorine, chlorine, bromine, and iodine. - --- The term "heteroaryl" includes 5- or 6-membered aromatic onocyclic heterocyclic groups containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur, such as pyrrolyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl. , thiazolyl, triazolyl, pyridyl, and aromatic bicyclic heterocyclic groups containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur, such as quinolinyl.

The peptide sequence Z is the part of compound II responsible for the introduction and / or stabilization of a certain secondary structure in the molecule that will make the compound more stable with respect to degradation by protease, thus, it is believed that Z needs to include at least 2 amino acid units (preferably at least 3 amino acid units) to introduce such a structural element either alone or in combination with the linker L. On the other hand it is also believed that a sequence of more than about 20 amino acid units will not improve more stability. Thus, Z is a peptide sequence of 2-20 amino acid units (preferably 3-20), preferably in the range of 3-15, more preferably 3-9 (such as 4-9), in particular 3-6 amino acid units, such as 4-6 amino acid units.

Each of the amino acid units in the peptide Z sequence are independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acids of formula II as defined here. Preferably, the amino acid units are selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, and Met, more preferably from Glu, Lys, and Met, especially Glu and Lys. The amino acids mentioned above could have D or L configuration, but preferably the aforementioned amino acids have L configuration. Since the X-sequence of pharmaceutically active peptide usually consists of L-amino acids exclusively, it must be expected, in order to preserve the helical structure of the complete prodrug, that a peptide Z sequence consisting only or mainly of L amino acids will be advantageous compared to a peptide Z sequence consisting only or mainly of amino acids D. Furthermore, it is visualized that a peptide Z sequence consisting only or mainly of -amino acids D could exert toxicological effects due to the resistance of D-peptides and D-amino acids with respect to biodegradation.

The amino acid units Z could of course all be different or all identical. However, in interesting embodiments of the present invention, the amino acid units in Z are selected from three different amino acids or from 2 different amino acids, or are identical amino acids, preferably the amino acid units in Z are identical such as (Lys) ) not (Glu) n, where n is an integer in the range of 4 to 6, or a combination of two amino acid units such as (LysGlu) 2, (LysGlu) 3, (GluLys) 2, or (GluLys ) 3, or a combination of three amino acid units, p. ex. Xaa- (Lys) x- (Glu) y, Xaa- (Glu) x- (Lys) y, (Lys) x- (Glu) y-Xaa, (Glu) x- (Lys) y- Xaa, (Lys) ) X-Xaa- (Glu) y, (Glu) y- Xaa- (Lys) y, Xaa-Lys-Glu-Lys, Xaa-Lys-Glu-Lys-Glu, Xaa-Lys-Glu-Lys-Glu- Lys, Xaa-Glu-Lys-Glu, Xaa-Glu-Lys-Glu-Lys, Xaa-Glu-Lys-Glu-Lys-Glu, Lys-Glu-Lys-Xaa, Lys-Glu-Lys-Glu-Xaa, Lys- Glu-Lys-Glu-Lys-Xaa, Glu-Lys-Glu-Xaa, Glu-Lys-Glu-Lys-Xaa, Glu-Lys-Glu-Lys-Glu-Xaa, etc., where x and y are integers in the range of 1 to 4 with the proviso that x + y is at most 5, and Xaa denotes Ala, Leu, -Ser, Thr, Tyr, As-n-Gln, Asp, Arg, His, Met, Orn , and amino acids of formula II as defined herein.

With respect to the peptide sequence Z, it is visualized that the specific amino acid units mentioned as constituents of the peptide sequence Z, p. ex. Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of formula II, are amino acid units that, due to their spherical arrangement around the atom of carbon-, and probably also due to a specific electronic configuration, have certain preferences to participate in, or even stabilize or initiate, helix-like structures. The Chou-Fasman approach (Chou, PY &Fasan, GD Ann .Rev. Bichem 47, 251-276 (1978)) is an attempt to quantify (empirically) the probability of a specific amino acid unit to be involved in a helix structure (expressed as the "Pa conformation parameter"). The Chou and Fasman studies and related studies, however, show that the amino acid units that have a low parameter of Pa, could be found in helices a, but of course not as often as the amino acid units that have a higher Pa. Thus, in the peptide sequence Z, it is considered possible to include a small proportion of amino acid units that are not among the amino acid units selected above as constituents of Z, and still obtain the desired effect of the peptide Z sequence, in the selected amino acid units are believed to compensate for any negative or neutral effect of such an alternative amino acid unit.

Thus, in embodiments that are within the scope of the present invention, it could be realistic to include up to 25% of amino acid units that are not among the preferred amino acids as constituents of Z. (By "25% percent" refers to the number of amino acid units, eg non-alternative amino acid units are allowed in di- and tripeptides, up to an alternative amino acid unit is allowed in tetra-, penta-, hexa-, and hepta-peptides, up to two alternative amino acid units are allowed in octapeptides, etc.) Such alternative amino acid units could be selected from Val, Lie, Pro, Phe, Gly, Trp, as well as N-methyl amino acid units, however, preferably not Pro, Gly and N-methyl amino acid units.

Illustrative examples of the peptide Z sequences are: Lys-Lys-Lys-Lys, Glu-Lys-Lys-Lys, Lys-Glu-Lys-Lys, Lys-Lys-Glu-Lys, Lys-Lys-Lys-Glu, Glu-Glu-Lys-Lys, Glu- Lys-Gly-Lys, Glu-Lys-Lys-Glu, Lys-Glu-Glu-Lys, Lys-Glu-Lys-Glu, Lys-Lys-Glu-GLu, Glu-Glu-Glu-Lys, Glu-Glu- Lys-Glu, Glu-Lys'-Glu-Glu, Lys-Glu-Glu-Glu, Glu-Glu-Glu-Glu, Lys-Lys-Lys-Lys-Lys, Glu-Lys-Lys-Lys-Lys, Lys -Glu-Lys-Lys-Lys, Lys-Lys-Glu-Lys-Lys, Lys-Lys-Lys-Glu-Lys, Lys-Lys-Lys-Lys-Glu, Glu-Glu-Lys-Lys-Lys, Glu -Lys-Glu-Lys-Lys, Glu-Lys-Lys-Glu-Lys, Glu-Lys-Lys-Lys-Glu, Lys-Glu-Glu-Lys-Lys, Lys-Glu-Lys-Glu-Lys, Lys -Glu-Lys-Lys-Glu, Lys-Lys-Glu-Glu-Lys, Lys-Lys-Glu-Lys-Glu, Lys-Lys-Lys-Glu-Glu, Lys-Lys-Glu-Glu-Glu, Lys -Glu-Lys-Glu-Glu, Lys-Glu-Glu-Lys-Glu, Lys-Glu-Glu-Glu-Lys, Glu-Lys-Lys-Glu-Glu, Glu-Lys-Glu-Glu-Lys, Glu -Glu-Lys-Lys-Glu, Glu-Glu-Lys-Glu-Lys, Glu-Glu-Glu-Lys-Lys, Lys-Glu-Glu-Glu-Glu, Glu-Lys-Glu-Glu-Glu, Glu -Glu-Lys-Glu-Glu, Glu-Glu-Glu-Lys-Glu, Glu-Glu-Glu-Glu-Lys, Glu-Glu-Glu-Glu-Glu, Lys-Lys-Lys-Lys-Lys-Lys , Glu-Lys-Lys-Lys-Lys-Lys, Lys-Glu-Lys-Lys-L ys-Lys, Lys-Lys-Glu-Lys-Lys-Lys, Lys-Lys-Lys-Glu-Lys-Lys, Lys-Lys-Lys-Lys-Glu-Lys, Lys-Lys-Lys-Lys-Lys- Glu, Glu-Glu-Lys-Lys-Lys-Lys, Glu-Lys-Glu-Lys-Lys-Lys, Glu-Lys-Lys-Glu-Lys-Lys, Glu-Lys-Lys-Lys-Glu-Lys, Glu-Lys-Lys-Lys-Lys-Glu, Lys- € lu-Glu-Lys-Lys-Lys-, Lys-G? U-Lys-Glu-Lys-Lys, Lys-Glu-Lys-Lys-Glu- Lys, Lys-Glu-Lys-Lys-Lys-Glu, Lys-Lys-Glu-Glu-Lys-Lys, Lys-Lys-Glu-Lys-Glu-Lys, Lys-Lys-Glu-Lys' -Lys-Glu , Lys-Lys-Lys-Glu-Glu-Lye, Lys-Lys-Lys-Glu-Lys' • Glu, Lys-Lys-Lys-Lys-Glu-Glu, Glu-Glu-Glu-Lys-Lys-Lys, Glu-Glu-Lys-Glu-Lys-Lys, Glu-Glu-Lys-Lys-Glu-Lys, Glu-Glu-Lys-Lys-Lys-Glu, Glu-Lys-'Glu-Glu-Lys-Lys, Glu-Lys ^ -Glu-Lys-Glu-Lys, Glu-Lys-Glu-Lys-Lys-Glu, Glu-Lys-Lys- -Glu-Glu-Lys, Glu-Lys-Lys-Glu-Lys-Glu, Glu-Lys-Lys-Lys- -Glu-Glu, Lys-Lys-Lys-Glu-Glu-Glu, Lys-Lys-Glu-Lys-Glu-Glu, Lys-Lys-Glu-Glu-Lys-Glu, Lys-Lys-Glu-Glu-Glu-Lys, Lys-Glu-Lys-Lys-Glu-Glu, Lys-Glu-Lys-Glu-Lys-Glu, Lys-Glu-Lys-Glu-Glu-Lys, Lys -Glu-Glu-Lys-Lys-Glu, Lys-Glu- -Glu-Lys-Glu-Lys, Lys-Glu-Gl u-Glu-Lys-Lys, Lys-Lys-Glu-Glu-Glu-Glu, Lys-Glu-Lys-Glu-Glu-Glu, Lys-Glu-Glu-Lys- • Glu-Glu, Lys-Glu- Glu-Glu-Lys-Glu, Lys-Glu-Glu-Glu-Glu-Lys, Glu-Lys-Lys-Glu-Glu-Glu, Glu-Lys-Glu-Lys-Glu-Glu, Glu-Lys-Glu -Glu-Lys-Glu, Glu-Lys-Glu-Glu-Glu-Lys, Glu-Glu-Lys-Lys-Glu-Glu, Glu-Glu-Lys-Glu-Lys-Glu, Glu-Glu-Lys -Glu-Glu-Lys, Glu-Glu-Glu-Lys-Lys-Glu, Glu-Glu-Glu- • Lys-Glu-Lys, Glu-Glu-Glu-Glu-Lys-Lys, Lys-Glu-Glu- Glu- Glu-Glu, Glu-Lys-Glu-Glu-Glu-Glu, Glu-Glu-Lys-Glu-Glu-Glu, Glu-Glu-Glu-Lys-Glu-Glu, Glu-Glu-Glu- Glu-Lys-Glu, Glu-Glu-Glu-Glu-Glu-Lys, Glu-Glu-Glu-Glu-Glu-Glu.

It should be understood that the C term of Z could be presented in the form of the acid, the amide, or the free ester, e.g. ex. depending on the type of solid support material and cutting conditions used in connection with the synthesis as it will be clear to the art expert.

It should also be understood that L binds to the N-terminal nitrogen atom of Z, p. ex. the possible types of bond between L and Z are those that involve a nitrogen atom, p. ex. a carboxamide bond (-C (= 0) -N-), a sulfonamide bond (-S0, -N-), or an alkylamine bond (-CN-), a carbamate bond (-0-C (= 0) - N-), a thiocarba bond ato (-SC (= 0) -N-), a urea bond (-NC (= 0) -N-), a thiourea bond ((-NC (, = S) -N-), a thioamide bond (-C (= S) -N-), a .cianomet ilenamino bond (-C (CN) -N-), or an N-methylamide bond (-C (= 0) -N (CH3) -) (In these examples the nitrogen atom (on the right side) arises from Z and the remaining part of the "bond" arises from L.) The preferred bonds are -C (= 0) -N-, -S02-N- , -CN-, -C (= S) -N-, -C (CN) -N-, and -C (= 0) -N (CH3) -, among which -C (= 0) -NH- and -C (= S) -NH- are preferred since they have the geometry of an ordinary peptide bond.

The linker L should preferably be able to participate in a helix type structure initiated or stabilized by Z. Apart from the fact that the link between X and L is not an amide bond, the geometry of L should preferably correspond to the geometry of an amino acid (or two or more amino acids), p. ex. the linker L preferably comprises 3 structural atoms or a multiple one thereof such as 6 or 9 structural atoms. In the present context, the term "structural atoms" when used in-connection with the linker L, therefore refers to the atoms in the linker L that directly link the X-sequence of pharmaceutically active peptide and the pre-sequence Z .

Thus, L is preferably derived from a hydroxy carboxylic acid, in particular an α-hydroxy carboxylic acid. More specifically, L is derived from a hydroxy-carboxylic acid of the general formula HO-CÍR1) (R -) - COOH wherein R1 and R2 are independently selected from H, C2-alkyl, C2_6 alkenyl, aryl, aryl, I rent. Ci_4, heteroaryl, heteroaryl-C1.4 alkyl, or R1 and R2 together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring, where an alkyl or alkenyl group could be -substituted with -u -no to three selected substituents, amino, cyano, halogen, isocyano, isothiocyano, thiocyano, sulfamyl, alkyio Cx_ 4, mono- or di-alkyl C: _.- amino, hydroxy, C-_- alkoxy, aryl, heteroaryl , aryloxy, carboxy, C: 4 alkoxycarbonyl, C, _4 alkylcarbonyloxy, aminocarbonyl, mono- or dialkyl C: _4-aminocarbonyl, mono- or di-alkyl C: _-amino, mono- or di-alkyl C_-amino -alkyl C: _, alkylcarbonylamino C: _4, sulfonyl and sulfino, and wherein an aryl or a heteroaryl group could be substituted with one to three substituents selected from C? _4alkyl, C2_4 alkenyl, nitro, amino, cyano, halogen, isocyanide, isothiocyano, thiocyano, sulfamyl, Ci-4 alkylthio, mono- or di-C 1-4 alkyl-amino, hydroxy, C x 4 alkoxy, aryloxy, carboxy, alkoxycarbonyl C: _4, C4_4alkylcarbonyloxy, aminocarbonyl, mono- or dialkyl4_4-aminocarbonyl, mono- or di_C1-4alkylamino, mono- or di_C1-4alkylamino_C1_alkyl, alkylcarbonylamino Cj_4, sulfone, and sulfino.

From the structural analysis of the pro-drugs of the invention, it is visualized that the additional stabilization of the helical-like structure of the prodrugs that could be achieved when the linker L is derived from a -hydroxy-carboxylic acid that also has a methylene group (-CH2 -) in the-position-oí. Thus, in an interesting embodiment, the linker L is derived from an α-hydroxy carboxylic acid with general formula HO-C (CH 2 -R 5) (R 2) -C00H, wherein R 5 is selected from H, C-alkyl ^, C2_5 alkenyl, aryl, aryl-C3_3alkyl, heteroaryl, neoaryl-C1_3alkyl, where an alkyl or alkenyl group could be substituted with one to three substituents selected from amino, halogen, momo- or di-alkyl C: - α-amino, hydroxy, C 1 -4 alkoxy, aryl, heteroaryl, aryloxy, carboxy, C 1 alkoxycarbonyl, C 1-4 alkylcarbonyloxy, and aminocarbonyl and where an aryl or heteroaryl could be substituted with one to three substituents selected from C: 4 alkyl, C 2-4 alkenyl, nitro, amino, halogen, momo- or di-alkyl examine, hydroxy, C: 4 alkoxy, carboxy, C2_4 alkoxycarbonyl, C4_4 alkoxycarbonyloxy, and aminocarbonyl; and R 2 is as defined above, preferably H, C 6 alkyl, C 6 alkenyl, aryl, aryl C 4 alkyl, heteroaryl, heteroaryl C 1 alkyl, wherein an alkyl or alkenyl group could be substituted with one to three substituents selected from amino, halogen, mono- or di-C 1-4 -alkylamino, hydroxy, Cx_4 alkoxy, aryl, heteroaryl, aryloxy, carboxy, C 1 alkoxycarbonyl, C 1 -C 4 alkylcarbonyloxy, and aminocarbonyl, and where an aryl c heteroaryl could be substituted with one to three substituents selected from alkyl -_.s- C2_4 alkenyl, nitro, amino, halogen, mono- or di-alkyl-test, hydroxy, Cx_4-alkoxy, carboxy, CX_-alkoxycarbonyl, C4_4-alkylcarbonyloxy, and aminocarbonyl.

The aforementioned adjustment of the cutting speed by increasing or decreasing the volume and / or the effect of electron donation of substituents in L could p. ex. carried out by increasing or decreasing the volume and / or the electron donation effect of R1 and / or R2 (or R5).

In particularly interesting embodiments, L is derived from hydroxyacetic acid, (S) - (+) - mandelic acid, L-lactic acid ((S) - (+) - 2-hydroxypropanoic acid), La-hydroxy-butyric acid (acid) (S) -2-hydroxybutanoic), and a-hydroxy-isobutyric acid.

It should be understood that the prodrugs of the invention could also be in the form of a salt thereof. The salts include pharmaceutically acceptable salts, such as acid addition salts and basic salts. Examples of acid addition salts are hydrochloride salts, sodium salts, calcium salts, potassium salts, etc. Examples of basic salts are salts wherein the cation is selected from alkali metals, such as sodium and potassium. , ferrous alkali metals, such as calcium, and ammonium ions + N (R6) 3 (R7), wherein R6 and R7 independently designate optionally substituted alkyl, C2_6 alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are, e.g. ex. these are described in "Remington's Pharmaceutical Sciences" 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, U.S. A., 1985 and more recent editions and in the Encyclopedia of Pharmaceutical Technology.

As mentioned above, the routes of administration of pharmaceutically active peptides have thus been limited more due to the rapid biodegradation by prdteases such as chymotrypsin, trypsin, carboxypept idase A, pepsin, leucine aminopept idase, etc. As will be understood from the examples provided herein, the tendency of prodrugs of general formula I (X-L-Z) for protease catalyzed hydrolysis can be measured directly by means of the in vi tro enzyme test shown in the examples. The tendency of XLZ to resist degradation for example can be expressed as a pseudo-first-order rate constant and / or as the average v-ida of the prodrugs, which could be compared to the corresponding values of X-OH, X- NH2, and / or X-OR. In addition, the ability of the prodrugs of the invention to exert the desired biological effect was tested in various test procedures. in vi tro and i n vi vo. A detailed description of the tests mentioned above is given in the examples.

It has been found that it is possible to obtain a remarkable increase in the half-life (t1 2) of a peptide or pharmaceutically active peptide sequence by protecting the peptide in question as a prodrug according to the invention.

Thus in a preferred embodiment of the invention, the relationship between the half-life of the prodrug in question in the "Hydrolysis test in enzyme solution" as defined herein, and the half-life of the. corresponding peptide (X-OH), in the "Hydrolysis test in enzyme solution", is at least 2, preferably at least 5, and even more preferably at least 10, especially at least 20, when one of the enzymes is used carboxypept idase A and leucine aminopept idase.

The invention also relates to a pharmaceutical composition comprising a prodrug of general formula I as defined above in combination 3C with a pharmaceutically acceptable carrier.

Such compositions could be in a form adapted for oral, parenteral (intravenous, intraperitoneal), rectal, intranasal, dermal, vaginal, buccal, ocular, or pulmonary administration, and such compositions could be prepared in a manner well known to those skilled in the art. , as generally described in "Remington's Pharmaceutical Sciences," 17 Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company,? aston, PA, US A., 1985 and more recent editions and in the monographs of the series "Drugs and the Pharmaceutical Sciences", Marcel Drekker.

The invention also relates to the use of a prodrug of general formula I as defined above or a salt thereof in the preparation of a composition for use in therapy, e.g. ex. in the treatment of conditions in the central nervous system, in vaccine therapy, and in the treatment of HIV, cancer, diabetes, incontinence, hypertension, and as analgesics and contraceptives, - • > and such known indications to be treated by means of therapy comprising the administration of pharmaceutically active peptides The prodrugs of the invention could be prepared by methods known per se in the art. Thus, the peptide X and Z sequences could be prepared by standard peptide preparation techniques such as solution synthesis or solid phase synthesis of the Merrifield type. It is believed that Boc (tertiary butyloxycarbonyl) as well as the Fmoc (9-fluorenylmethyloxycarbonyl) strategies are applicable.

In a possible synthetic strategy, the prodrugs of the invention could be prepared by means of solid phase synthesis by first constructing the peptide Z sequence using the well known standard protection, coupling and deprotection methods, subsequently coupling the linker L group, subsequently coupling sequentially the pharmaceutically active sequence X in the linker L group in a manner similar to the Z construct, and finally cut the total XLZ prodrug of the vehicle.

Another possible strategy is to prepare one or both of the two X and Z sequences separately by solution synthesis, solid phase synthesis, recombinant techniques, or enzymatic synthesis followed by the coupling of the two sequences and the L linker group by methods of condensation of well-known segment, either in solution or using solid-phase techniques or a combination thereof.

In addition, it is envisioned that a combination of the aforementioned strategies could be especially applicable when a modified peptide sequence, e.g. ex. of a biologically active peptide comprising reduced peptide bonds, which is to be coupled to a peptide Z sequence via a linker L. In this case it may be advantageous to first prepare the immobilized LZ fragment by successive coupling of the amino acids (and the linker) and then coupling a complete X sequence of biologically active peptide (prepared in solution or completely or partially using the solid phase techniques) to the LZ fragment.

Thus, the present invention also refers to an immobilized linker-peptide sequence of Prot-LZ-SSM, where L designates a linker of general formula -0-C (R :) (R2) C (= 0), where ' R1 and R2 are as defined above, and Prot designates H or a hydroxy protecting group, wherein the hydroxy protecting group is selected from dimethoxytrityl, monomethoxytrityl, trityl, 9- (9-phenyl) xanthenyl (pixyl), tetrahydropyranol , 'methoxytetrahydro-pyranol, trimethylsilyl, triisopropylsilyl, tert-butyldimethyl-silyl, triti 1 si 1 i 1, phenyl 1 d ime ti 1 si 1 i 1, benzyloxycarbonyl, substituted becyloxycarbonyl ethers, such as 2-bromine benzyloxycarbonyl, tert-butyl ethers, methyl ethers, acetyl, halogen-substituted acetyls, such as chloroacetyl and fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyls, methoxymethyl, benzyl ethers and benzyl ethers, such as 2,6-dichlorobenzyl, etc.; SSM designates a solid support material selected from p. ex. functionalized resins such as polystyrene, polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose, polyethylene, polyethylene glycol inserted in polystyrene, latex, quanta, etc .; and Z is as defined above.

It should be understood that it may be necessary or desirable that the C-terminal amino acid of the Z presequence is bound to the solid support material by means of a common linker such as 2,4-dimethoxy-4'-hydroxy-benzophenone, 4- ( 4-hydroxy-met il-3-methoxyphenoxy) -butyric acid, 4-hydroxy-methyl-ylbenzoic acid, 4-hydroxymethyl-phenoxyacetic acid, 3- (4-hydroxymethylphenoxy) propionic acid and p-i acid (R , S) -a [1- (9H-Fluoren-9-yl) methoxy formamido] -2,4-dimethoxybenzyl] -phenoxy-acetic acid.

Accordingly, the present invention also relates to the use of an immobilized linker-peptide sequence of Prot-LZ-SSM for the preparation of a prodrug according to the invention and to a method for the preparation of a prodrug of a peptide (X-OH ), a peptide amide (X-NH2) or a peptide ester (X-OR) comprising the coupling of the corresponding peptide from an activated C-terminal form (X-Act) to an immobilized linker-peptide sequence HLZ-SSM .

The present invention also relates to a further method for the preparation of a prodrug of a peptide (X-OH), a peptide-amide (- X-NH2) or a peptide ester (X-OR) comprising the steps of: a) coupling a protected Na-amino acid in the activated carbonyl form, or a Na dipteido protected the C-terminal activated form for an immobilized linker peptide sequence HLZ-SSM, to thereby form a peptide fragment; Na-protected immobilized, b) removing the N-a protecting group, to thereby form an immobilized peptide fragment having an unprotected N-terminal end, c) coupling an additional N-protected amino acid in the activated carboxyl form, or an additional N-protected a-dipeptide in the C-terminal activated form for the non-protected N-terminal end of the immobilized peptide fragment, and removal / coupling procedure in step b) and c) until the desired sequence of peptide X is obtained, and then d) cutting the prodrug X-L-Z from the solid support material to obtain the prodrug-free in the form of a carboxylic acid, amide or C-terminal ester.

The coupling, removal and cutting step is carried out by methods known to the person skilled in the art in consideration of the protection strategy and the selected solid phase material.

With respect to stabilizing the bonds on the one hand between X and L, and on the other hand between L and Z, of the types indicated above, such bonds could be stabilized by methods known per se for stabilizing thiol ester, ester, carboxamide, sulfonamide, alkylamine, carbamate, thiocarbamate, urea, thiourea, thioa ida, cyanomethyleneamino, or N-methylamide or groupings, see p. ex. J. March, "Advanced Organic Chemistry", 3rd. edition, John Willey & Sons, 1985 as well as references cited therein. So, p. ex. an ester could be formed of an activated derivative (acid halide, acid anhydride, activated ester eg HObt-ester etc.) of the appropriate carboxylic acid by means of the reaction with the relevant hydroxy compound. In addition, a carboxamide could be formed by reacting an activated derivative (acid halide, acid anhydride, activated ester eg HObt-ester etc.) of the appropriate carboxylic acid with the relevant amino compound as known to a person skilled in the art of chemistry. peptides; a sulfonamide could be formed by reacting a sulfonyl chloride with the appropriate amino compound; an alkyl amine bond or grouping could be formed by reacting the appropriate compound bearing a leaving group such as tosyl, halogen, and mesityl on the carbon atom in question with the relevant amino compound in a nucleophilic substitution reaction; a carbamate bond or grouping could be formed by treating the appropriate alcohol with phosgene to provide the corresponding chlorocarbonate which is then reacted with the relevant amino compound; a thiocarbamate bond or grouping could be formed by treating the appropriate alcohol with thiophosgene to provide the corresponding chlorothiocarbonate which is then reacted with the relevant amino compound; a urea bond or grouping could be formed by reacting the appropriate compound carrying an isocyanate group on the carbon atom in question with the relevant amino compound; a thiourea bond or grouping could be formed by reacting the appropriate compound bearing an isothiocyanate group on the carbon atom in question with the relevant amino compound; a t ± -oamide bond or grouping could be formed by reacting the thiono ester of the appropriate carboxylic acid with the relevant amino compound, the thiono ester to be formed p. ex. of the corresponding piperidide.

In addition, it may be necessary or desirable to include side chain protection groups when using amino acid units that carry functional groups that are reactive under the prevailing conditions. The necessary protection scheme will be known to the art expert (see eg M. Bodanszky and A. Bodanszky, "The Practice of Peptide Synthesis" 2. Ed, Springer-Verlag, 1994, and J. Jones, " The Chemical Synthesis of Peptides ", Clarendon Press, 1991).

Thus, the peptide prodrug of the invention could be cut out of the solid support material by means of an acid such as trifluoroacetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogen chloride, hydrogen fluoride, etc. or a base such as ammonia, hydrazine, an alkoxide, such as sodium ethoxide, a hydroxide, such as sodium hydroxide, etc.

As the prodrugs of the invention represent a new class of compounds, a further aspect of the present invention relates to compounds of general formula I X - L - Z wherein X is a peptide sequence that binds to L at the C-terminal carbonyl function of X; L is a linking group, comprising from 3 to 9 structural atoms, wherein the bond between the C-terminal carbonyl of X and L is different from a C-N amide bond; Y Z is a peptide sequence of 2-20 amino acid units and is linked to L at the N-terminal nitrogen atom of Z, each amino acid unit being independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of formula II -NH-C (R3) (R4) -C (= 0) - II wherein R 3 and R 4 are independently selected from C 1, 6 alkyl, phenyl, and phenyl-methyl lov wherein alkyl is optionally substituted with one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfon, and carboxy, and phenyl and phenyl-methyl is optionally substituted with one to three substituents selected from C? _ _alkyl, C C_6 alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfone, and carboxy, or R 3 and R 4 together with the carbon atom at which bind to form a ring of cyclopentyl, cyclohexyl, or cycloheptyl; or a salt of it.

The invention is further illustrated by the following examples.

EXPERIMENTAL Peptide synthesis Procedure in general cough is Abbreviations used: tBu = ter. butyl - - ----- DAMGO = Tyr- (D-Ala) -Gly-? [-C (== 0) -N (CH3) -] Phe-NH-CH2- CH20H DCC = diclohexylcarbodiimide DCM = dichloromethane DIC = diisopropylcarbodiimide DIEA = N, N-diisopropylethylamine DMAP = '4- (N, N-dimethylamino) -pyridine Dhbt-OH = 3, 4 -dih idro- 3-hydroxy-4-oxo- 1, 2, 3-benzotriazine DMF = N, N-dimethylformamide DSIP = Delta Peptide that Induces Sleep, H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH EDT = Ethanedithiol ES-MS = Electrospray Mass Spectrometry Fmoc = 9-fluorenylmethyloxycarbonyl cHex = cyclohexyl HAA = hydroxyacetic acid HMPA = 4-hydroxymethylphenoxyacetic acid Hbot = 1-hydroxybenzotriazole HPLC = high performance liquid chromatography Ma = mandelic acid NHS = N-hydroxy-succinic acid imido ester PEG-PS = polyethylene glycol inserted in polystyrene Pfp = pentafluorophenyl SEM = Standard Error of the Average TFA = trifluoroacetic acid Z = benzyloxycarbonyl Apparatus and synthetic strategy The peptides were batch synthesized in a polyethylene vessel equipped with a polyethylene filter for filtration using 9-fluorenylmethyloxycarbonyl (Fmoc) as the Na-amino protecting group and common protection groups suitable for side chain functionalities (Dryland, A. and Sheppard, RC (1986) J. Chem. Soc., Perkin Trans. 1, 125-137).

Solvents The solvent DMF (N, N-dimethylformamide, Riedel de-Háen, Germany) was purified by passing through a column packed with a strong cation exchange resin (Lewatit S 100 MB / H strong acid, Bayer AG Leverkusen, Germany), and analyzed for free amines before use by addition of 3,4-dihydro-3-hydroxy-4-oxo-l, 2,3-benzotriazine (Dhbt-OH) which gives rise to a yellow color (anion). Dhbt-O-) if free amines are present. The solvent DCM (dichloromethane, analytical grade, Riedel de-Háen, Germany) was used directly without purification.

Amino acids and dipeptides The amino acids protected with Fmoc were purchased in MilliGen (UK) in suitable protected side chain forms. Otherwise the protected amino acids (H-Glu (OtBu) -OtBu; H-Glu (cHex) -OH; Z.-Glu (OtBu) -OH) and the dipeptides Fmoc-Phe-Gly-OH and H-Phe- Leu-OH were purchased in Bachem (Switzerland).

Coupling reagents The diisopropylcarbodiimide coupling reagent (DIC) was purchased from (Riedel de-Haen, Germany) and distilled before use, dicyclohexylcarbodiimide (DCC) was purchased from Merck-Schuchardt, München, Germany, and purified by distillation.

Linkers The linkers are (4-hydroxymethylphenoxy) acetic acid (HMPA), Novabio-Chem, Switzerland, hydroxyacetic acid, (S) - (+) - mandelic acid 99% pure, Aldrich, Germany, and acid (R) - (-) - 98.0% pure mandelic, M &R, England, were coupled to the resin or N terminus of the pre-Z sequence as a 1-hydroxybenzotriazole ester (HObt) developed generated by DIC.

Solid supports The peptides synthesized according to the Fmoc strategy were synthesized in two different types of solid support using 0.05 M or higher concentrations of Fmoc-protected activated amino acid in DMF: 1) PEG-PS (polyethylene glycol inserted in polystyrene; NovaSyn TG resin, 0.29 mmol / g, Novabiochem, Switzerland); 2) NovaSyn K 125 (Kieselguhr supported polydimethacrylamide resin functionalized with sarcosine methyl ester 0.11 mmol / g, Novabiochem, Switzerland).

Catalysts and other reagents Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, and ethylene diamine in Fluka, piperidine and pyridine in Riedel-de Háen, Frankfurt, Germany. 4- (N, -dimethylamino) pyridine (DMAP) was purchased in Fluka, Switzerland and used as a catalyst in coupling reactions involving symmetric anhydrides. Ethanedithiol was purchased at Riedel-de Haen, Frankfurt, Germany. 3, 4-dihydro-3-hydroxy-4-oxo-l, 2,3-benzotriazine (Dhbt-OH) and 1-hydroxybenzotriazole (HObt) were purchased in Fluka, Switzerland. FmocNHS was purchased in Aldrich, Germany.

Enzymes Carboxypept idase A (EC 3.4.17.1) type I of Bovine Pancreas, leucine aminopeptidase (EC 3.4.11.1) type III-CP of Porcine Kidney, butyryl cholinesterase (EC 3.1.1.8) of Horse Serum, a-chymotrypsin ( EC 4.4.21.1) of Bovine Pancreas, and Pepsin A (EC 3.4.23.1) of Porcine Stomach Mucosa Bovine pancreas were obtained from Sigma, UK.

Coupling procedures The first amino acid was coupled as a symmetric anhydride in DMF generated from the appropriate N-a-protected amino acid and BIC or DCC. The following amino acids were coupled as developed HObt esters made of appropriate N-a-protected amino acids and HObt by means of DIC in DMF. The acylations were checked by the ninhydrin test developed at 80 ° C to avoid the deprotection of Fmoc during the test (Larsen, BD and Holm, A., Int. "J. Peptide Protein Res. 43, 1994, 1-9) .

Deprotection of the N-a-amino protective group Deprotection of the Fmoc group was carried out by treatment with 20% piperidine in DMF (1x3 and 1x7 min.), Followed by washing with DMF until no yellow color could be detected (Dhbt-O-) after the addition of Dhbt-OH to the DMF drained.

Separation of the peptide from the resin with acid Peptides were separated from the resins by treatment with 95% trifluoroacetic acid (TFA, Riedel-HSen, Frankfurt, Germany) -water v / v or with 95% TFA and 5% ethanedithiol v / v at t.a. for 2 h. The filtered resins were washed with 95% TFA-water and the filtrates and washes were evaporated under reduced pressure. The residue was washed with ether and dried by freezing with acetic acid-water. The crude product dried by freezing was analyzed by high performance liquid chromatography (HPLC) and identified by electrospray ionization mass spectrometry (ESMS).

ESTER 'of preformed HObt 3 eq. of protected amino acid N-a-amino or hydroxyacetic acid or (S) - (+) - mandelic acid was dissolved in DMF together with 3 eq. of HObt and 3 eq of_ DIC. The solution was left at t.a. for 10 minutes and then added to the resin, which had been washed with a 0.2% solution of Dhbt-OH in DMF before the addition of the preactivated amino acid.

Preformed symmetrical anhydride 6 eq. of protected amino acid 'N-a-amino was dissolved in DCM and cooled to 0 ° C. DCC (3 eq.) Was added and the reaction continued for 10 min. The solvent was removed in vacuo and the remainder was dissolved in DMF. The solution was filtered and immediately added to the resin followed by 0.1 eq. of DMAP.

Estimation of coupling performance of the first protected amino acid N-a-amino 3-5 mg of resin-peptide protected with dry Fmoc were treated with 5 ml of 20% piperidine in DMF for 10 min at t.a. and the UV absorption and for the dibenzofulven-piperidine adduct was estimated at 301 nm. The yield was determined using a calculated coefficient of extension e30i based on a standard Fmoc-Ala-OH.

Peptide synthesis in PepSvn K resin Dry PepSyn K (ca 500 mg) was covered with ethylenediamine and left at t.a. All night long. The resin was drained and washed with DMF 10 x 15 ml, 5 min each. After draining, the resin was washed with 10% DTEA in DMF v / v (2 x 15"mi, 5 min each) and finally washed with DMF - until no yellow color could be detected by addition of Dhbt- OH to drained DMF 3 eq of HMPA 3 eq of HObt and 3 eq of DIC were dissolved in 10 ml of DMF and left for activation for 10 mi-n, after which the mixture was added to the resin and the coupling continued for 24 h.The resin was drained and washed with DMF (10 x 15 ml 5 min each), and the acylation was verified by the ninhydrin test.The first amino acid was coupled as the preformed symmetric anhydride (see above), and the coupling yields were estimated as described above.In all cases it was greater than 70% .The synthesis was then continued as "batch".

Synthesis of the batch peptide continued in PepSvn K The resin (ca. 500 mg) with the first bound amino acid was placed in a polyethylene container equipped with a polypropylene filter for filtration, and the Fmoc group deprotected as described above. The remaining amino acids according to the sequence were coupled as Fmoc-protected preformed, if a protected side chain is necessary, HObt esters (3 eq.) In DMF (5 ml) were prepared as described above. The couplings were continued for 2 h unless otherwise specified. The excess reagent was then removed by washing with DMF (12 min, flow rate 1 ml / min) All acylations were verified by the ninhydrin-a test developed at 8-0 ° C. After completing the synthesis the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (5 × 5 ml, 1 min each) and finally diethyl ether (5 × 5 ml, 1 min each) and It dried in va cuo.

Synthesis of the batch peptide in PEG-PS The NovaSyn TG resin (250 mg, 0.27-0.29 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration. The resin was distended in DMF (5 ml), and treated with 20% piperidine in DMF to ensure the presence of non-protonated amino groups in the resin. The resin was drained and washed with DMF until a yellow color could not be detected after the addition of Dhbt-OH to the drained DMF. HMPA (3 eq.) Was coupled as a preformed HObt ester as described above and the coupling was continued for 24 h. The resin was drained and washed with DMF (5 x 5 ml, 5 min each) and the acylation was verified by the ninhydrin test. The first amino acid was coupled as a preformed symmetric anhydride as described above. The coupling yields of the first amino acids protected with Fmoc were estimated as described above. In all cases it was greater than 60%. The following amino acids according to the sequence were coupled as preforms protected with Fmoc, if necessary a protected side chain, esters HObt - (3 eq.) As described above. The links were continued for 2 h unless otherwise specified. The resin was drained and washed with DMF (5 x 5 ml, 5 min each) to remove excess reagent. All acylations were verified by the ninhydrin test developed at 80 ° C. After completion of the synthesis the peptide-resin was washed with DMF (3x5 ml, 5 min each), DCM (3x5 ml, 1 min each) and finally diethyl ether (3x5 ml, 1 min each) and dried in go cuo.

HPLC conditions The isocratic HPLC analysis was preformed in a Shimadzu system consisting of an LC-6A pump, a MERCK HITACHI L-400 UV sensor operated at 215 nm and a Rheodyne 7125 injection valve with a cycle of 2, 20, or 100 μl . The column used for isocratic analysis was a Spherisorb ODS-2 (100 x 3 mm, 5-μ particles). HPLC analysis using gradients was developed on a MERCK HITACHI L-6200 Intelligent pump, an M &C RCK HITACHI L-400-UV sensor operated at 215 nm and a Rheodyne 7125 injection valve with a 20 μl cycle. In the column used was a Rescorce? RPC of 1 mi.

Buffer A was 0.1% vol of TFA in water and buffer 90% vol of acetonitrile, 9.9% vol of water and 0.1% vol of TFA. The buffers were pumped into the column at a flow rate of 1.3-1.5 ml / min using the following gradient for peptide analysis 1. Linear gradient of 0% - 100% B (30 min), for enzymatic studies 2. Gradient linear of 40 - 100% of B (15 min), 3. Linear gradient of 10 - 40% of B (15 min), or 4. Linear gradient of 0-50% of B (15 min). The mobile phase used for isocratic analysis could be mentioned under the description of the individual experiments.

Mass spectroscopy The mass spectrum was obtained on a Finnigan Mat LCQ instrument equipped with an electro-spread probe (ES-MS) (ESI).

Synthesis of individual peptide peptides is 1. Peptide synthesis of H-Tyr-Glv-Glv-Phe-Leu-Gluc-OH in NovaSvn TentaGel Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described according to "PEG-PS batch peptide synthesis" until the end of the Glu6 pre-sequence. The following amino acids forming the Leu-enkephalin sequence were coupled preformed with Fmoc, if necessary a protected side chain, esters HObt (3 eq.) In DMF (5 ml) generated by means of DIC. Before each of the last five couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to follow the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The acylations were then verified by the ninhydrin test developed at 80 ° C as described above. After the synthesis was complete, the peptide-resin was washed with DMF (3 × 5 ml, 1 min each), DCM (3 × 5 ml, 1 min each), diethyl ether (3 × 5 ml, 1 min each) and dried in va cuo The peptide was separated from the resin as described above and freeze-dried with acetic acid. The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and sequences protected with Fmoc. The purity was found to be greater than 90% and the identity of the peptide was confirmed by ES-MS. Performance of 76%. 2. Synthesis of H-Tyr-Glv-Glv-Phe-Leu-HAA-Glu ^ -OH in a PepSvn K resin Dry PepSyn K (ca 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated with ethylenediamine as described above. The first glutamic acid units forming the pre-sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.). The acylations were verified by the ninhydrin test developed at 80 ° C as described above. The Fmoc group was deprotected as previously described. After finishing the pre-sequence the deprotected peptide-resin was reacted with 6 eq. hydroxyacetic acid as a pre-activated HObt ester as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min). The acylation was verified by the ninhydrin test. The next amino acid according to the sequence (leucine) was coupled as preformed symmetric anhydride as described above and the reaction was continued for 2 h. The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min). A small resin sample was removed to verify coupling performance, which was estimated as described above, and found to be 90%. The synthesis was then continued by splitting the Fmoc group as described above.

The next coupling according to the sequence was to avoid the formation of preformed diketopiperazine as a dipeptide coupling. Thus F oc-Gly-Phe-OH was coupled as a preformed HObt ester (3 eq.) In DMF "(5 ml) prepared as described above for 2 h.The excess reagent was removed after washing c-on DMF ( 12 min flow rate 1 ml / min) and the acylation was verified by the ninhydrin test developed at 80 ° C as described above The Fmoc group was then removed by treatment with 20% piperidine in DMF as described The remaining amino acids according to the sequence were coupled as preformed with Fmoc, if necessary a protected side chain, HObt esters (3 eq.) with the addition of 1 eq of Dhbt-OH in DMF (2 ml) for 2 hours. The acylation was then verified by the ninhydrin test developed as described above The remaining amino acids according to the sequence were coupled as Pfp esters protected with Fmoc (3 eq.) with the addition of Dhbt-OH (1 eq. ) in DMF (2 ml) The excess reagent was removed by washing with DMF (1 ml). 2 min flow rate 1 ml / min) and the acylations were verified by the ninhydrin test developed at 80 ° C as described above. The Fmoc group was deprotected as described above. After completion of the synthesis the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as described above and was freeze-dried with ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous, the purity was found to be greater than 80%, the identity of the peptide was confirmed by ESMS, and the yield 58%. 3. Synthesis of H-Tyr-Glv-Glv-Phe-Leu- ((S) - (+) - Ma) -Glu, - OH in a PepSvn K resin.

Dry PepSyn K (ca 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated with ethylenediamine as described above. The first 6 glutamic acids that form the pre-sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.). The acylations were verified by the ninhydrin test developed at 80 ° C as described above. The Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resin was reacted with 6 eq. of (S) - (+) - mandelic acid as a pre-activated HObt ester as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min). The a-e-ylation was verified by the ninhydrin test. The next amino acid according to the sequence (leucine) was coupled as preformed symmetric anhydride as described above and the reaction was continued for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min). A small resin sample was then removed to verify coupling performance, which was estimated as described above, and found to be 85%. The synthesis was then continued by cutting the Fmoc group as described above.

The next coupling according to the sequence was to avoid the formation of diketopiperazine developed as a dipeptide coupling. Thus Fmoc-Gly-Phe-OH was coupled as a preformed HObt ester (3 eq.) In DMF (5 ml) prepared as described above for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min) and the acylation was verified by the ninhydrin test developed at 80 ° C as described above. The Fmoc group was then removed with 20% piperidine in DMF as described above. The remaining amino acids according to to the sequence s-e coupled with preformed HObt e-s-teres protected with Fmoc (3 eq.) with the addition of -1 eq of Dhbt-OH in DMF (2 ml) for 2 h. The acylation was verified by the ninhydrin test developed as described above. The remaining amino acids according to the sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.) In DMF (2 ml). The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min) and the acylations were checked by the ninhydrin test developed at 80 ° C as described above. The Fmoc group was deprotected as described above. After the synthesis was completed the peptide-resin was washed with DMF (10 min., flow rate 1 ml / min), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried in va cuo.

The peptide was separated from the resin as described above and dried by freezing with ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous, the purity was found to be greater than 80%, the identity of the peptide was confirmed by ESMS, and the yield 57%.

The prodrug H-Tyr-Gly-Gly-Phe-Leu- ((R) - (-) - Ma) -Glu6-OH was prepared as described above for 34. Synthesis of H-Tyr-Glv-Glv-Phe-Leu (S) - (+) - Ma) -Lvs, -OH in a PeoSvn K resin.

Dry PepSyn K (ca 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated with ethylenediamine as described above. The first 6 Usinas forming the pre-sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.). The acylations were verified by the ninhydrin test performed at 80 ° C as described above. The Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resin was reacted with 6 eq. (S) - (+) - mandelic acid as a HObt ester preactivated as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min). The acylation was verified by the ninhydrin test. The next amino acid according to the sequence - (leucine) was coupled as preformed symmetric anhydride as described above and the reaction was continued for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min). A small sample of resin was removed to verify coupling performance which was estimated as described above, and found to be -85%. The synthesis was then continued by removal of the Fmoc group as described above.

The next coupling according to the sequence was to avoid the formation of diketopiperazine developed as a dipeptide coupling. In this way Fmoc-Gly-Phe-OH was coupled as a preformed HObt ester (3 eq.) In DMF (5 ml) prepared as described above for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min) and the acylation was verified by the ninhydrin test performed at 80 ° C as described above. The Fmoc group was then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to the sequence were coupled with preformed HObt esters protected with Fmoc (3 eq.) With the addition of 1 eq of-Dhbt-OH in DMF (2-m-1) for 2 h. Acylation was verified by the ninhydrin test performed as described above.

The remaining amino acids according to the sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.) In DMF (2 ml). The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min) and the acylations were checked by the ninhydrin test performed at 180 ° C as described above. The Fmoc group was deprotected as described above. After completion of the peptide-resin synthesis was washed with DMF (10 min, flow rate 1 ml / min), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as described above and dried by freezing with ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous, the purity was found to be greater than 80%, and the identity of the peptide was confirmed by ESMS, and the yield of 57%.

The prodrug H-Tyr-Gly-Gly-Phe-Leu- ((R) - (-) - Ma) -Lys6-0H was prepared as described above for 4.

. Synthesis of H-Tyr-Glv-Glv-Phe-Leu- ((S) - (+) - Ma) - (LysGlu) -.- OH in a PepSvn K resin.

Dry PepSyn K (ca 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated with ethylenediamine as described above. The first 6 amino acids forming the pre-sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.). The acylations were verified by the ninhydrin test performed at 80 ° C as described above. The Fmoc group was deprotected as described above. After finishing the pre-sequence the deprotected peptide-resin was reacted with 6 eq. of acid (s) - (+) - mandelic ester as a HObt ester preactivated as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min). The acylation was verified by the ninhydrin test. The next amino acid according to the sequence (leucine) was coupled as preformed symmetric anhydride as described above and the reaction was continued for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min). A small sample of resin was removed to verify coupling performance, which was estimated as described above, and found to be 85%. The synthesis was then continued by removal of the Fmoc group as described above.

The next coupling according to the sequence was to avoid the diketopiperazine formation performed as a dipeptide coupling. Thus F oc-Gly-Phe-OH was coupled as a preformed HObt ester (3 eq.) In DMF (5 ml) prepared as described above for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate 1 ml / min) and the acylation was verified by the ninhydrin test performed at 80 ° C as described above. The Fmoc group was then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to the sequence were coupled with preformed HObt esters protected with Fmoc (3 eq.) With the addition of 1 eq of Dhbt-OH in DMF (2 ml) for 2 h. The acylation was verified by the ninhydrin test performed as described above. The remaining amino acids according to the sequence were coupled as Pfp esters protected with Fmoc (3 eq.) With the addition of Dhbt-OH (1 eq.) In DMF (2 ml). The excess reagent was removed by washing with DMF (12 min flow rate 1 ml / min) and the acylations were checked by the ninhydrin test performed at 80 ° C as described above. The Fmoc group was deprotected as described above. After the synthesis was complete the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried in va cuo.

The peptide was separated from the resin as described above and dried by freezing with ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous, the purity was found to be greater than 80%, the identity of the peptide was confirmed by ESMS, and the yield 63%.

The prodrug H-Tyr-Gly-Gly-Phe-Leu- ((R) - (-) - Ma) - (LysGlu) 3-0H was prepared as described above for 5. 6. Synthesis of Fmoc-Phe-Leu-HAA-Glu ^ -OH peptide in Novasvn Tentasel.

Resin Novasyn TG dry (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described under the "PEG-PS batch peptide synthesis" until Glu6 pre-sequence was completed. The peptide-resin was then reacted with 6 eq. of hydroxyacetic acid as a HObt ester preactivated as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate of 1 ml / min). The acylation was verified by the ninhydrin test. The next amino acid according to the sequence (leucine) was coupled with preformed symmetrical anhydride as described above and the reaction was continued for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate of 1 ml / min). A small sample of resin was removed to verify the coupling performance, which was estimated as described above, and - s-e found to be -100%. The next amino acid according to the sequence was coupled as the preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before the last two couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The acylations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as previously described and dried by freezing from ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the Fmoc-protected sequences. The purity was found to be greater than 90% and the identity of the peptide was confirmed by ES-MS. Performance 83%. - ------ 7. Peptide Synthesis of Fmoc-Phe-Leu- ((S) - (+) - Ma) -Glug-OH in NovaSvn TentaGel.

The dried NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batch peptide synthesis in PEG-PS" until the Glu6 pre-sequence was finished. The peptide-resin was then reacted with (S) - (+) - mandelic acid 6 eq. As a pre-activated HObt ester as described above and the coupling was continued for 24 h. The excess reagent was removed by washing with DMF (12 min flow rate of 1 ml / min). The acylation was verified by the ninhydrin test. The next amino acid according to the sequence (leucine) was coupled as the symmetric anhydride was preformed as described above and the reaction was continued for 2 h. The excess reagent was then removed by washing with DMF (12 min flow rate of 1 ml / min). A small sample of resin was removed to verify coupling performance, which was estimated as described above, and it was found that it was 90%. The next amino acid according to the sequence was coupled as the preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before the last two couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 ml, 5 min each). The acylations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as previously described and dried by freezing from ammonium bicarbonate (0.1 M). The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the Fmoc-protected sequences. The purity was found to be greater than 90% and the identity of the peptide was confirmed by ES-MS. Performance 71%. 8. Synthesis of H-Tyr-Glv-Glv-Phe-Leu-Lvs.-OH in NovaSvn TentaGel.

The dried NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batch peptide synthesis in PEG-PS" until the Lys6 pre-sequence was finished. The following amino acids that form the Leu-encephalin sequence were coupled as preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before each of the last five couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The acylations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5-ml, 1 min each-tmo), diethyl ether (3x5 ml, 1 min each) and dried in va cuo The peptide was separated from the resin as previously described and dried by freezing from acetic acid. The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the Fmoc-protected sequences. The purity was found to be greater than 98% and the identity of the peptide was confirmed by ES-MS. Performance 84%. 9. Synthesis of the peptide H-Trp-Ala-Glv-Glv-Asp-Ala-Ser-Glv-Glu-Gluc-OH in NovaSvn TentaGel.

The dried NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batch peptide synthesis in PEG-PS" until the Glu6 pre-sequence was finished. The following amino acids forming the DSIP sequence were coupled as the preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before each of the last nine couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color -as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The acylations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as previously described and dried by freezing from acetic acid. The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the Fmoc-protected sequences. The purity was found to be greater than 98% and the identity of the peptide was confirmed by ES-MS. Performance 80%.

. Synthesis of the peptide H-Trp-Ala-Gly-Glv-Asp-Ala-Ser-Glv-Glu- (LvsGlu), -OH in NovaSvn TentaGel.

The dried NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a polypropylene filter for filtration and treated as described b-garlic for "PEG-peptide synthesis in batch. -PS "until the pre-sequence (LysGlu) 3 is finished. The following amino acids forming the DSIP sequence were coupled as the preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before each of the last nine couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The acylations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as previously described and dried by freezing from acetic acid. The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the Fmoc-protected sequences. The purity was found to be greater than 98% and the identity of the peptide was confirmed by ES-MS. Rendimie-nto 91%. 11. Synthesis of the peptide H-Tro-Ala-Glv-Glv-Asp-Ala-Ser-Gly-Glu-OH (DSIP) in NovaSyn TentaGel.

The dried NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a polypropylene filter for filtration and treated as described under "batch peptide synthesis in PEG-PS" . The first amino acid was coupled as the preformed symmetric anhydride as previously described. The coupling yields of the first amino acids protected with Fmoc were estimated as previously described. It was in all cases better than 60%. The following amino acids forming the DSIP sequence were coupled as the preformed HObt esters protected with Fmoc (3 eq.) In DMF (5 ml) generated by means of DIC. Before each of the last eight couplings the resin was washed with a solution of Dhbt-OH (80 mg in 25 ml), to continue with the disappearance of the yellow color as the coupling reaction proceeded. When the yellow color was no longer visible, the couplings were interrupted by washing the resin with DMF (5 x 5 ml, 5 min each). The -acilations were then verified by the ninhydrin test performed at 80 ° C as previously described. After the synthesis was complete, the peptide-resin was washed with DMF (3x5 ml, 1 min each), DCM (3x5 ml, 1 min each), diethyl ether (3x5 ml, 1 min each) and dried up in va cuo The peptide was separated from the resin as previously described and dried by freezing from acetic acid. The crude product dried by freezing was analyzed by HPLC and found to be homogeneous without elimination and the o-protected F sequences. The purity was found to be greater than 98% and the identity of the peptide was confirmed by ES-MS. Performance 78%.

General Synthesis 12. Synthesis of Fmoc-Phe-Leu-OH 1. 0 g (3.6 mmol) of H-Phe-Leu-OH was dissolved in 25 ml of 10% w / w sodium carbonate and 25 ml of dioxane was added. To this mixture was added dropwise 1.24 g (3.68 mmol) FmocNHS dissolved in 10 ml of dioxane. The resulting mixture was stirred overnight at room temperature. The dioxane was removed by evaporation and the resulting aqueous-basic solution was extracted 3 times with ether (5 ml each). The pH was adjusted to 2 by adding HCl (1 M) and the aqueous phase was extracted 3 times with ethyl acetate (20 ml each). The combined ethyl acetate phase was washed 3 times with water (10 ml each), and evaporated to dryness. The rest was crystallized by adding petroleum ether. Yield 1.45 g (86%). Pf. 153-158; MS calculated 500.59, found MH + 501.3. 13. Synthesis of Z-Glu (OtBu) -Glu (OtBu) -OtBu 1. 14 g (3.38 mmol) of Z.-Glu (OtBu) -OH, 0.457 g of HObt (3.38 mmol) and 520 μl of DIC (3.38 mmol) was dissolved in 10 ml of THF and pre-activated for 10 min. added to a solution of 1.0 g (3.38 mmol) of H-Glu (OtBu) -OtBu, HCl in 10 ml of THF. The mixture was stirred overnight at room temperature. The solvent was removed by evaporation, and the residue was dissolved in DCM (10 ml) and extracted 3 times with 10% v / v aqueous acetic acid (10 ml each) and 3 times with sodium bicarbonate. water at 10% w / v (10 ml each) and finally 2 times with water (10 ml each). The organic phase was dried with sodium sulfate, filtered and evaporated. The residue was used directly as a colorless solid without further purification. '"14. Synthesis of Z-Glu-Glu-OH The oil of 13 was dissolved in 20 ml of 50% v / v TFA-DCM and stirred for 2 h at room temperature. The solution was evaporated to dryness and the rest was crystallized from petroleum ether. Yield 1.2 g (86.5%). Mp. 174-176 ° C; MS: Calculated 410.4, found MH + 411.0.

Hydrolysis in the enzyme solution test The decomposition of the peptide prodrug (XLZ) and the corresponding peptide (X-OH) is studied at 37 ° C in a buffer solution with 0.05 M phosphate. The buffer solutions contain leucine aminopeptidase (25 u / ml) at pH 7.4, or carboxypeptidase A (25 u / ml) at pH 7.4. Decomposition is initiated by the addition of an aliquot (~ 10 ~ 7-10-8 mol) of a stock solution or peptide prodrug, respectively, to the test solution giving a total volume of ~ 5 ml of the mixture of reaction which is maintained in a bath with water at 37 ° C. At appropriate time intervals in 50 μl s "e *" samples, they are removed and analyzed by reverse phase HPLC as described above without prior protein precipitation. The pseudo first order rate constants for the degradation of the peptide prodrug (XLZ) and the corresponding peptide (X-OH) are determined from the slopes (eg kobs) of the linear plots of the logarithm of the derivative concentration residual (upper HPLC peaks) against time using the formula t1 / 2 = (ln2) / (kobs). The relationship between the half-life of the prodrug and the corresponding peptide is calculated according to the formula: ratio = (t1 / 2 (prodrug)) / (t1 / 2 (X-0H)).

Kinetic Measurements Hi dróli si s in the sun cushion The decomposition of some of the peptide prodrugs was studied in buffer solutions of phosphate buffer or aqueous carbonate with a total buffer concentration of 0.1 - 0.05 M. To maintain a constant ionic strength (μ) of 0.5 a quantity of chloride was added of potassium wings buffer solutions "unless otherwise stated." The temperature was maintained at 37 ° C during the degradation studies and the pH was adjusted by adding hydrochloric acid (4M) or sodium hydroxide (2M). were carried out at pH 2, 7.4 and 11.

Decomposition rates were determined using the reverse phase of HPLC. The mobile phase systems used for the isocratic separation were 20% acetonitrile, 79.9% water 0.1% trifluoroacetic acid or 10% acetonitrile, 89.9% water 0.1% trifluoroacetic acid. When using a linear gradient (40-100% B in 30 min) of buffer A was 0.1% TFA in water v / v and buffer B was 90% acetonitrile, 9.9% water, 0.1% TFA v / v .

Hi dróli si s in sol uci on of the enzyme Decomposition of the peptides and peptide prodrugs was studied at 37 ° C in a 0.05 M phosphate buffer solution containing leucine aminopeptidase (25 u / ml) at pH 7.4, carboxypeptidase A (25 u / ml) at pH 7.4 , aq-uimiotripsina (25 - u / ml) at pH 7.4, pepsin A (25 u / ml) at pH 2.0, or butyryl cholinesterase (at two concentrations: 25 and 50 u / ml) at pH 7.4. Decomposition was initiated by adding an aliquot i ~ 10"7-10" 8 mol) of a stock solution of the peptide or peptide prodrug to the test solution which gives a total volume of reaction mixture of ~ 5 ml which is kept in a water bath at 37 ° C and at appropriate intervals the ~ 50 μl samples were removed and analyzed by reverse phase HPLC as described above without previous protein precipitation. The pseudo first-order velocity constants for the impairments were determined from the slopes (eg kobs) of the linear plots of the logarithm of the concentration of the residual derivative (upper HPLC peaks) against time using the formula t1 2 = (ln2) / (kobs). The individual test conditions are given in the following examples.

Hydrolysis in plasma sol ution Decomposition of the peptides and peptide prodrugs was studied at 37 ° C in 80% human plasma. Decomposition was initiated by adding an aliquot (~ 10 ~ 7-10"8 mol) of a stock solution of the peptide to the test solution which gives a total volume of reaction mixture of -50 ml which was maintained in a bath with water at 37 ° C and at appropriate intervals the 50 μl samples were removed and the samples were treated with 50 μl of 2% (w / v) solution of zinc sulfate in methanol water (1: 1 v / v) to deprotect the samples and stop the reactions. After immediate centrifugation for 30 min. at 13,000 rpm., 20 μl of the clear supernatant was analyzed by reverse phase HPLC as described above. The pseudo-first order rate constants for the impairments were determined from the slopes (eg kobs) of the linear graphs of the logarithm of the concentration of the residual derivative (upper HPLC peaks) versus time using the formula t1 / 2 = (ln2) / (kobs). The individual test conditions are given in the following examples.

H-Tyr-Gly-Gly-Phe-Leu- (Glu) 6-OH: Hydrolysis in buffer solution The degradation of H-Tyr-Gly-Gly-Phe-Leu (Glu) s-OH (~ 5 x 10"6 M) was studied in different aqueous buffers (0.1 M) as described above. pH = 2, pH = 7.4 and pH = 11. The peptide was found stable at the pH values mentioned above, thus only ~ 5% of the peptide was degraded during a period of 24 h.

Hi dróli si s in l eucina aminopepti dasa The degradation of H-Tyr-Gly-Gly-Phe-Leu (Glu) 6-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing leucine aminopeptidase (25 u / ml) it was studied as described above. The pseudo first order rate constant was determined as described above and found to be 5.2 x 10"3 min-1. The half-life was calculated at 133 min.

Hydrolyses in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu (Glu) 6-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing carboxypeptidase A (25 u / ml) it was studied as described above. The peptide was characterized as stable. Approximately 15% of the peptide was degraded over a period of 24 h.

H-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH Hidróli si s in l eucina aminopepti dasa The degradation of H-Tyr-Gly-Gly-Phe-Leu (Lys) 6-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing leucine aminopeptidase (25 u / ml) it was studied as described above. The pseudo first order rate constant was determined as described above and was found to be 3.6 x 10 ~ 3 min. "1 The half-life was calculated at 191 min.

Hi dróli si s in pepsin A The degradation of H-Tyr-Gly-Gly-Phe-Leu- (Lys) 6-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 2.0) containing pepsin A (25 u / ml) it was studied as described above. The pseudo first order rate constant was determined as described above and found to be 1.2 x '10"3 min" 1. The half-life was calculated at 580 min.

Fmoc-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) 6-OH: Hydrolysis in butyryl or cholinesterase The hydrolysis of the ester bond generated via the mandelic acid in the peptide prodrug Fmoc-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) 6-0H (~ 10-5 M) it was studied in 0.05 M phosphate buffers (pH = 7.4) containing butyryl cholinesterase (50 u / ml) as described above. The half-life was estimated at t1 / 2 = 212 min as described by the general procedures. Based on the recovery of Fmoc-Phe-Leu-OH the half-life was estimated at t1 / 2 = 164 min. The difference in the values is probably due to the further degradation of Fmoc-Phe-Leu-OH at the urethane linkage in the Fmoc protecting group.

Fmoc-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-OH Hydrolysis in butyryl or cholinesterase Hydrolysis of the ester-bond generated by hydroxyacetic acid in the peptide prodrug Fmoc-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-OH (~ 10 ~ 5 M) was studied in phosphate buffer solutions 0.05 M (pH = 7.4) containing butyryl cholinesterase (50 u / ml) as described above. Based on the recovery of Fmoc-Phe-Leu-OH the half-life was estimated at t1 / 2 = 144 min.

Fmoc-Phe-Leu-OH: Hi dróli si s in bu tiril or chinesterase The degradation of Fmoc-Phe-Leu-OH (~ 5 x 10 -5 M) was studied in 0.05 M phosphate buffer solutions (pH = 7.4) containing butyryl cholinesterase (50 u / ml) as described above. The half-life was estimated at t1 2 = 25 h.

H-Tyr-Gly-Gly-Phe-Leu-. { acid (S) - (+) - mandelic) - (Glu) 6-OH: Hi dróli si s in sol uci ón amorti guadora The degradation of H-Tyr-Gly-Gly-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) ^ OH (1 x 10-5 M) was studied in different aqueous buffers (0.1 M) as described above. Decomposition was continued at pH = 2, pH = 7.4 and pH = 11. The peptide was characterized as stable at the pH values mentioned above, thus less than 5% of the peptide was degraded over a period of 24 h.

Hydrolyses in lucine aminopeptidase The degradation of H-Tyr-Gly-Gly-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) g-OH (1 x 10"5 M) was studied in 0.05 phosphate buffer solutions M (pH 7.4) containing leucine aminopeptidase (25 u / ml) as described above The pseudo first order rate constant was determined as previously described and the half-life was calculated at 93 min.

Hi dróli si s in carboxypepti da sa A The degradation of H-Tyr-Gly-Gly-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) g-OH (1 x 10"5 M) was studied in 0.05 phosphate buffer solutions M (pH 7.4) containing leucine carboxypeptidase (25 u / ml) as described above The pseudo first order rate constant was determined as previously described and the half-life was calculated at 10.3 min.

Hi dróli si s in bu tiril or colines terasa The hydrolysis of the ester bond generated via the mandelic acid in the peptide prodrug H-Tyr-Gly-Gly-Phe-Leu- ((S) - (+) - mandelic acid) - (Glu) 6-0H ( 1.75 x 10"5 M) was studied in 0.05 M phosphate buffered solutions (pH 7.4) containing butyryl cholinesterase (50 u / ml) as described above.The half-life was estimated at t 1/2 50.2 min.

H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-OH: Hi dróli si s in sol uci ón amorti guadora The decomposition of H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid - (Glu) 6-OH (1 x 10"5 M) was studied in different aqueous buffers (0.1 M) as described above. Decomposition was continued at pH = 2, pH = 7.4 and pH = 11. The peptide was characterized as stable at the pH values mentioned above, thus less than 5% of the peptide was degraded over a period of 24 h.

Hydrolyses in leucine aminopeptidase The degradation of H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid - (Glu) 6-0H (1 x 10"5 M) was studied in 0.05 M phosphate buffers (pH 7.4) containing leucine aminopeptidase (25 u / ml) as described above The pseudo first order rate constant was determined as previously described and the half-life was calculated at 16.1 min.

Hydrolysis in carboxypeptidase A The degradation of H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-OH (1 x 10"5 M) was studied in 0.05 M phosphate buffer solutions (pH 7.4) containing carboxypeptidase A (25 u / ml) as described above The rate constant was characterized as stable, thus less than 13% of the peptide was degraded over a period of 24 h.

Hydrolyses in butyryl or cholinesterase The hydrolysis of the ester-linked union via the hydroxyacetic acid in the peptide prodrug H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-OH (1.75 x 10"5 M) was studied in 0.05 M phosphate buffered solutions (pH 7.4) containing butyryl cholinesterase (25 u / ml or 50 u / ml) as described above.The half-life was estimated using 25 u of butyryl cholinesterase / ml at t1 / 2 119.3 min and ti / 2 86.6 min when 50 u / ml was used.

Hi dróli si s in human plasma The decomposition of the peptide prodrug H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid) - (Glu) 6-0H was studied by adding 1 ml of 0.05 M phosphate buffer solutions (pH = 7.4) of the peptide (7). x 10"5 M) at 37 ° C for 4 ml of human plasma and treated as described above The pseudo first order rate constant was determined as previously described and the half-life was calculated at t1 / 2 = 19.2 min Z-Glu-Glu-OH Hidróli sis en carboxipepti dasa A The degradation of Zr-Glu-Glu-OH (1 x - 10"5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing carboxypeptidase A (25 u / ml) was studied as described above. Peptide was characterized as stable for a period of 24 h.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH Hi dróli si s in carboxypepti dasa A The degradation of H-Trp-AIA-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH (1 x 10-5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) ) containing carboxypeptidase A (25 u / ml) was studied as described above. The pseudo first order rate constant was determined as previously described and the half-life was calculated at 396 min.

Hidróli si s in l eucina aminopepti da sa The degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH (~ 10-5 .M) in 0.05 M phosphate buffer solutions (pH = 7.4) ) containing leucine aminopeptidase (25 u / ml) was studied as described above. The pseudo first order rate constant was determined as previously described and the half-life was calculated at 145 min.

Hidróli si s en a-quimi otripsina The degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-0H (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing a-chymotrypsin (25 u / ml) was studied as described above. The pseudo first order rate constant was determined as previously described and the half-life was calculated at 613 min.

Hydrole si s in pepsin A The degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) 3-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 2.0) containing pepsin A (25 u / ml) was studied as described above. The peptide was characterized as stable.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) 6-OH Hydrolysis in a-quimi otripsina The degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) 6-0H (~ 10"5M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing a Chymotrypsin (25 u / ml) was studied as described above The pseudo first order rate constant was determined as previously described and the half-life was calculated at 523 min.

H-Trp-Ala-Gly-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH Hi dróli si s in l eucina aminopepti da sa The degradation of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH (~ 10 ~ 5 M) in 0.05 M phosphate buffer solutions (pH = 7.4) containing α-leucine aminopeptidase ( 25 u / ml) was studied as described above. The half-life was calculated to be less than 20 min.

In V ± txo Studies Acti viid 1 of the receiver opi oi of μ The affinity of the prodrugs of the invention for the μ-opioid receptor in the calf brain was determined as described by Kristensen et al. . (1994) [K. Kristensen, C.B. Cristensen, L.L. Cristrup, and L.C. Nielsen (1994). The mul mu2, delta, kappa opioid receptor binds the stereoisomer profiles of methadone and morphine. Li faith Sci. 56, PL45-PL50.]. The activity of the prodrugs was determined in fresh solutions and solutions stored for 20 h at room temperature.

The experimental results are summarized in Table 1.

Table 1. Inhibition of 3H-DAMGO (2 nM) Acti vi dad 2 of the receiver opi oi of u: The affinity of the prodrugs of the invention was determined as μ-opioid receptor agonists using the in vi tro model of the mouse vas deferens described by Kramer et al. (1997) [T.H. Kramer, H. Bartosz-Becho ski, P. Davis, V.J. Hruby, and F. Porreca (1997). The extraordinary potency of a new delta opioid receptor agonist is due in part to increased efficiency. Life Sci. 61: 2, 129-135]. The activity of the prodrugs was determined in fresh solutions and solutions stored for 48 h at room temperature.

The experimental results are summarized in Table 2 and 3.

Table 2. Activity of Defensive Conduits e u - e n c e .na 'S) - (-r) -Ma) - aa (LysGlu) s-OH Leu-enkephalin-OH? aa aaa reduction to 100 nM: < 25%; aa: < fifty%; aaa: < 75% Table 3. Activity of the Deferent Conduits Peptide / prodrug IC..0 ± SEM (nM) Leu-enkephalin- OH 41 + 13 In Vivo Studies Analgesic activity Attempts to determine in vivo activity in the mouse were carried out using the mesh shock model described by Swedberg (1994) [M.D. Swedberg (1994). The analgesic test of mesh shock in mouse: pharmacological characterization of latency at the threshold of vocalization as an index of antinociception. J. Pharma col. Exp. Ther. 269: 3, 1021-1028.]. The experimental results are summarized in Table 4.

Table 4. Analgesic Activity in the Mouse NT: Not tested. NA: Not active. WA: weakly active at 20 mg / kg. conclusion Because native enkephalin degrades with a half-life of 6.0 minutes in 80% human plasma, with a half-life of 10.0 minutes in aminopeptidase (20 u / ml), and with a half-life of 2.0 minutes in carboxypeptidase ( 1 u / ml) (see GJ Rasmussen and H. Bundgaard, In t. J. Phar. , 79, pp 113-122 (1991)), it is concluded that the invention provides significant protection of a peptide sequence compared to the native peptide sequence. This is also corroborated by the results obtained by DSIP; Native DSIP degrades with a half-life of less than 20 minutes in the leucine aminopeptidase (25 u / ml), while DSIP- (LysGlu) 3-OH degrades with a half-life of 145 minutes under identical conditions. In general, the half-lives of the pre-sequence containing the DSIP molecules in solutions containing a-chymotrypsin or carboxypeptidase A (25 u / ml) were in the order of several hours. Although the native DSIP has not been tested under these conditions, it should be expected that the corresponding half-lives are significantly lower than the values obtained from the presequence containing the DSIP molecules as is properly established that the native DSIP degrades rapidly in the extracts of plasma and tissue (see HL Lee, "Peptide and Protein Drug Delivery", Marcel Dekker Inc. 1991, Chapter 9).

In addition, the prodrugs of peptides tested were all cut by butyryl cholinesterase indicating adequate bioreversibility.

From the in vi tro tests carried out, it can be concluded that the pre-sequence significantly influences the biological activity. The results presented in Tables 1, 2 and 3 clearly indicate that the prodrugs of the invention have a reduced affinity with respect to the μ-opioid receptor compared to native Leu-enkephalin. In this way, in order to exert the desired activity, in this case the opioid receptor μ is bound, the prodrug must be hydrolyzed eg. by enzymes from blood plasma such as butyryl cholinesterase to release the pharmaceutically active native peptide.

The difference between the results obtained from the freshly prepared solutions and the solutions maintained at room temperature for 20 or 48 h, could be due to two different factors: The solutions containing the prodrugs of the invention could be hydrolyzed to some degree when stored during 20 or 48 h whereby the native Leu-enkephalin is released. However, as shown by the kinetic measurements, the prodrugs of the invention are stable over the entire pH range. The most pronounced effect observed is when the pre-sequence (Glu) 6 is applied. Thus, a more plausible explanation is the low solubility in water of the prodrugs that contain (Glu) 6: It is very likely that due to the low kinetic solutions, only a fraction of the prodrug dissolves in the solutions prepared freshly. However, when left in solution for 20 or 48 hours, the compounds will dissolve slowly and therefore increase the available amount of the active substance in the tests.

From the studies of analgesia activity in vi it can be concluded that the pre-sequence in addition to the linker is of importance. Apparently, the presequence of (Lys) 6 positively charged in combination with the (S) enantiomer of mandelic acid exhibited a desired effect while the enkephalin containing the presequence (Lys) 6 in combination with the (R) enantiomer of mandelic acid did not showed no activity In addition, the enkephalin prodrugs with the electroneutral pre-sequence (LysGlu) 3 either in combination with (R) or (S) mandelic acid did not show the desired effect. In conclusion, the combination of the linker and the pre-sequencing is of importance in p. ex. the ability of the prodrugs of the invention to cross biological barriers such as the blood-brain barrier, and the present invention opens the package insert for the transport of prodrugs to the desired region by selecting an appropriate combination of the linker and the pre-sequence.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Having described the invention as above, the content of the following is claimed as property.

Claims (30)

1. A prodrug of a pharmaceutically active peptide (X-OH), peptide amide (X-NH2), or peptide ester (X-OR), wherein the prodrug has the general formula I X-L-Z I characterized in that X binds to L at the C-terminal carbonyl function of X; L is a linking group, comprising from 3 to 9 structural atoms, wherein the bond between the C-terminal carbonyl of X and L is different from a C-N amide bond; Y Z is a peptide sequence of 2-20 amino acid units and is linked to L at the N-terminal nitrogen atom of Z, each amino acid unit being independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of formula II -NH-C (R3) (R4) -C (= 0) - II wherein R3 and R4 are independently selected from phenyl alkyl, and phenyl-methyl, wherein C-L6 alkyl is optionally substituted with one to three selected substituents of halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is optionally substituted with one to three substituents selected from C2_6 alkenyl alkyl, halogen, hydroxy, amino, cyano, nitro, sulfon, and carboxy , or R3 and R4 together with the carbon atom to which they are linked to form a cyclopentyl, cyclohexyl, or cycloheptyl ring; or a salt of it

2. A prodrug as claimed in claim 1, characterized in that the amino acid units in Z are selected from three or two different amino acids, or are identical amino acids.

3. A prodrug as claimed in claim 1 or 2, characterized in that the amino acid units in Z are independently selected from Glu, Met and Lys, in particular from Glu and Lys.

4. A prodrug as claimed in any of the claims, characterized in that the pharmaceutically active peptide (X-OH), the peptide amide (X-NH2), or the peptide ester (X-R) consists of 2-200 amino acid units.

5. A prodrug as claimed in any of the preceding claims, characterized in that Z consists of 3-15 amino acid units.

6. A prodrug as claimed in any of the preceding claims, characterized in that the bond between the C-terminal carbonyl function of X and L is capable of being cut by enzymes from the blood plasma.

7. A prodrug as claimed in any of the preceding claims, characterized in that the bond between the C-terminal carbonyl function of X and L is a thiol ester bond or an ester bond.

8. A prodrug as claimed in any of the preceding claims, characterized in that the bond between L and the N-terminal nitrogen atom in Z is a carboxamide bond (-C (= 0) -N-), a sulfonamide bond (-S02-N-), an alkylamine bond (-CN-), a carbamate bond (-0-C (= 0) -N-), a thiocarbamate bond (-SC (= 0) -N- ), a urea linkage (-NC- (= 0) -N-), a thioamide linkage (-C (= S) -N-), a cyanomethyleneamino bond (-C (CN) -N), or a linkage of N-methylamide (-C (= 0) -N (CH 3) -).

9. A prodrug as claimed in any of the preceding claims, characterized in that L is derived from a hydroxy carboxylic acid.

10. A prodrug as claimed in any of the preceding claims, characterized in that L is derived from an α-hydroxy carboxylic acid.

11. A prodrug as claimed in claim 10, characterized in that L is derived from an α-hydroxy carboxylic acid of the general formula HO-C (R 1) (R 2) -COOH wherein R 1 and R 2 are independently selected from H, alkenyl alkyl C2_6, aryl, aryl-C1_4alkyl, heteroaryl, heteroaryl-Cx_4alkyl, or R1 and R2 together with the carbon atom to which they are attached form a ring of cyclopentyl, cyclohexyl, or cycloheptyl, where an alkyl or alkenyl group could be substituted with one to three substituents selected from amino, cyano, halogen, isocyano, isothiocyano, thiocyano, sulfamyl, Cx_4alkaryl, mono- or di-C1-4alkylamino, hydroxy, C4alkoxy, aryl, heteroaryl, aryloxy, carboxy, C4_alkoxycarbonyl, C1_4alkylcarbonyloxy , aminocarbonyl, mono- or dialkyl C1-4-aminocarbonyl, mono- or di-alkyl Cx_4-amino, mono- or di-alkyl C ^ -amino-C1-4alkyl, alkylcarbonylaminoC4-4, sulphono and sulfino, and where an aryl or heteroaryl group could be substituted with one to three substituents selected from C, _4 alkyl, C2_4 alkenyl, nitro, amino, cyano, halogen, isocyano, isothiocyano, thiocyano, sulfamyl, Cn-4 alkylthio, mono- or di- -C 1-4 alkyl-apino, hydroxy, C 4 -alkoxy, aryloxy, carboxy, C 1-4 alkoxycarbonyl, C 1 -C 4 alkylcarbonyloxy, aminocarbonyl, mono- or dialkyl-1,4-aminocarbonyl, mono- or di-C 1-4 -alkylamino, mono- or di-C1-4alkylaminoC1-4alkyl, C5alkylaminocarbonylamino, sulphono, and sulfino.

12. A prodrug as claimed in any of the preceding claims, characterized in that L is derived from hydroxyacetic acid, (S) - (+) - mandelic acid, L-lactic acid (acid - * (S.). - (+) - 2-hydroxypropanoic acid), La-hydroxy-butyric acid (acid (S) -2-hydroxybutanoic), and a-hydroxy-isobutyric acid

13. A prodrug as claimed in claim 11, characterized in that L is derived from an α-hydroxy carboxylic acid of general formula H0-C (CH2-R5) (R2) -COOH, wherein R5 is selected from H, C-alkyl ^, C2_5 alkenyl, aryl, arylC1_3alkyl, heteroaryl, heteroarylC1_alkyl, where an alkyl or alkenyl group could be substituted with one to three substituents selected from amino, halogen, mono- or di-C1-4 alkyl -amino, hydroxy, C ^ alkoxy, aryl, heteroaryl, aryloxy, carboxy, C ^ alkoxycarbonyl, C ^ alkylcarbonyloxy, and aminocarbonyl, and where an aryl or heteroaryl could be substituted with one to three substituents selected from C ^ alkyl, C2_4 alkenyl, nitro, amino, halogen, mono- or di-alkyl, hydroxy, alkoxy, Ca 4, carboxy, C 4 alkoxycarbonyl, C 4 alkoxycarbonyloxy, and aminocarbonyl; and R2 is as defined in claim 11.

14. A prodrug as claimed in any of the preceding claims, characterized in that X is the peptide sequence of an -enkephalin, angiotesin II, vasopressin, endothelin, neuropeptide Y, vasoactive intestinal peptide, substance P, neurotensin, endorphins, insulin, gramicidin, paracelsin, delta-inducing peptide, ANF, vasotocin, bradykinin, dynorphin, endothelin, growth hormone releasing factor, growth hormone release peptide, oxytocin, calcitonin, peptide related to the calcitonin gene, peptide II related to the calcitonin gene, growth hormone release peptide, tachykinin, ACTH, brain natriuretic polypeptide, cholecystokinin, corticotropin releasing factor, diazepam-binding inhibitor fragment, FMRF-amide, galanin, gastric-release polypeptide, gastrin, polypeptide that releases gastrin, glucagon, glucagon-like peptide 1, p 2-glucagon-like peptide, LHRH, melanin-concentrating hormone, alpha-MSH, peptides that modulate morphine, motilin, neurocínin, neuromedin, neuropeptide K, neuropeptide Y, PACAP, pancreatic polypeptide, peptide YY, PHM, secretin, somatostatin, substance K , substance P, THR, vasoactive intestinal polypeptide, or any modified or truncated analogue thereof.

15. A prodrug according to any of the preceding claims, characterized in that the ratio between the half-life of the prodrug in question in the "hydrolysis test in enzyme solution", as defined herein, and the corresponding half-life of the peptide ( X-OH), in the "hydrolysis test in enzyme solution", is at least 2, when the enzyme carboxypeptidase A is used.

16. A prodrug according to claim 15, characterized in that the ratio is at least 5.

17. A prodrug according to claim 16, characterized in that the ratio is at least 10.

18. A prodrug according to any of the preceding claims, characterized in that the ratio between the half-life of the prodrug in question in the "hydrolysis test in enzyme solution", as defined herein, and the corresponding half-life of the peptide (X -OH), in the "hydrolysis test in enzyme solution", is at least 2, when the enzyme aminopeptidase is used.

19. A prodrug according to the re-v-indication 18, characterized in that the ratio is at least 5.

20. A prodrug according to claim 19, characterized in that the ratio is at least 10.

21. A pharmaceutical composition, characterized in that it comprises a prodrug as defined in any of claims 1-20, and a pharmaceutically acceptable carrier.

22. A prodrug as defined in any of claims 1-20, characterized in that it is used in therapy.

23. The use of a prodrug as defined in any of claims 1-20, characterized in that it is used for the preparation of a pharmaceutical composition for use in therapy.

24. An immobilized linker-peptide sequence of Prot-LZ-SSM, characterized in that L denotes a linker of the general formula HO-C (R1) (R2) -COOH wherein R1 and R2 are independently selected from H, C6 alkyl, C2_6 alkenyl , aryl, aryl-Cx_4 alkyl, heteroaryl, heteroaryl-Cx_4 alkyl, or R1- and R2 together with the carbon atom to which they bind form a cyclopentyl, cyclohexyl, or cycloheptyl ring, where an alkyl or alkenyl group could to be substituted with one to three substituents selected from amino, cyano, halogen, isocyano, isothiocyano, thiocyano, sulfamyl, Cx_4_alkto, mono- or di-Cx_4-amino alkyl, hydroxy, Cx_4 alkoxy, aryl, heteroaryl, aryloxy, carboxy, alkoxycarbonyl Cx_4, C 4_4 alkylcarbonyloxy, aminocarbonyl, mono- or dialkyl Cx_4-aminocarbonyl, mono- or di-Cx_4-amino alkyl, mono- or di-alkyl Cx_4-amino-alkyl Cx_4, alkylcarbonylamino Cx_4, sulfono and sulfino, and where an aryl group or a heteroaryl could be substituted with one to three substituents selected from Cx_4 alkyl, C2_4 alkenyl, nitro, amino, cyano, halogen, isocyano, isothiocyan, thiocyano, sulfamyl, CX_4 alkylthio, mono- or di-Cx_4-amino alkyl, hydroxy, Cx_4 alkoxy, aryloxy, carboxy, alkoxycarbonyl Cx_4, C4_4alkylcarbonyloxy, aminocarbonyl, mono- or dialkyl_4-aminocarbonyl, mono- or di_C1_4_alkylamino, mono- or di_alkylCx_4-amino-Cx_4alkyl, alkylcarbonylamino Cx_4, sulfono, and sulfino, Z designate a sequence of peptide comprising 2-20 amino acid units, each amino acid unit being independently selected from Ala, Leu, -Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn , and amino acid units of formula II • NH-C (R3) (R4) -C (= 0) - II wherein R3 and R4 are independently selected from CX_6 alkyl, phenyl, and phenyl-methyl, wherein Cx_6 alkyl is optionally substituted with one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfon, and carboxy, and phenyl and phenylmethyl is optionally substituted with one to three substituents selected from CX_6 alkyl, C2_6 alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfone, and carboxy, or R3 and R4 together with the carbon atom to which they are linked to forming a cyclopentyl, cyclohexyl, or cycloheptyl ring; SSM designates a solid support material; and Prot designates H or a hydroxy protecting group.

25. An immobilized linker sequence-Prot-LZ-SSM peptide according to claim 24, characterized in that the solid support material (SSM) is selected from polystyrene, polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose, polyethylene, polyethylene glycol inserted in polystyrene, latex, and accounts. - -

26. The use of an immobilized linker sequence-Prot-L-Z-SSM peptide for the preparation of a prodrug of a peptide, a peptide amide, or a peptide ester.

27. A method for the preparation of a prodrug of a peptide (X-OH), a peptide amide (X-NH2), or a peptide ester (X-OR), characterized in that it comprises coupling the corresponding peptide in an activated form C -terminal (X-Act) to an immobilized peptide linker sequence HLZ-SSM.

28. A method for the preparation of a prodrug of a peptide (X-OH), a peptide amide (X-NH2), or a peptide ester (X-OR), characterized in that it comprises the steps of: a) coupling a protected Na-amino acid in the activated carbonyl form, or a protected Na dipétide in the C-terminal activated form for an immobilized HLZ-SSM linker peptide sequence, to thereby form an immobilized Na-protected peptide fragment , b) removing the N-a protecting group, to thereby form an immobilized peptide fragment having an unprotected N-terminal end, c) coupling an additional N-protected amino acid in the activated carboxyl form, or an additional N-protected a-dipeptide in the C-terminal activated form for the non-protected N-terminal end of the immobilized peptide fragment, and removal / coupling procedure in step b) and c) until the desired sequence of peptide X is obtained, and then d) separating the prodrug X-L-Z from the solid support material to obtain the free prodrug in the form of a carboxylic acid, amide or C-terminal ester.

29. A method according to claim 28, characterized in that the N-a-protective group is selected from t er. but i loxycarbonyl and 9-fluorenylmethyloxycarbonyl.

30. A compound of general formula I X-L-Z characterized in that X is a peptide sequence that binds to L at the C-terminal carbonyl function of X; L is a linking group, comprising from 3 to 9 structural atoms, wherein the bond between the C-terminal carbonyl of X and L is different from a C-N amide bond; Y Z is a peptide sequence of 2-20 amino acid units and is linked to L at the N-terminal nitrogen atom of Z, each amino acid unit being independently selected from Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of formula II -NH-C (R3) (R4) -C (= 0) II wherein R3 and R4 are independently selected from CX_6 alkyl, phenyl, and phenyl-methyl, wherein Cx_6 alkyl is optionally substituted with one to three substituents selected from halogen, hydroxy, amino, cyano, nitro, sulfon, and carboxy, and phenyl and phenylmethyl is optionally substituted with one to three substituents selected from CX_6 alkyl, C2_6 alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and carboxy, or, R3 and R4 together with the carbon atom to which they are linked to form a ring of cyclopentyl, cyclohexyl, or cycloheptyl; or a salt of it