KR20000036015A - Peptide prodrugs containing an alpha-hydroxyacid linker - Google Patents

Abstract

PURPOSE: Provided is a prodrug of pharmaceutically active peptide. A pharmaceutical composition comprising the prodrug and a pharmaceutically acceptable carrier are also provided. The prodrug is used for the preparation of a pharmaceutically composition for use in therapy. CONSTITUTION: Peptide prod rugs 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 carboxyl A, pepsin A, leucine aminopeptidase, alpha -chymotrypsin when masking a pharmaceutically active peptide as a prodrug of the formula (I). The prod rugs of formula (I) are cleaved by the blood plasma enzyme butyryl cholinesterase indicating a readily bio reversibility. It is believed that the stability towards enzymatic cleavage is due to an induced helix-like structure.

Description

Peptide prodrugs containing alpha-hydroxy acid linkers {PEPTIDE PRODRUGS CONTAINING AN ALPHA-HYDROXYACID LINKER}

Numerous pharmaceutically active peptides exist, such as pharmaceutically active peptides naturally present in humans or animals, or synthetic analogs of such peptides. Exemplary examples of such peptides are analgesic active peptide enkephalins that have produced a large number of synthetic analogs. However, the route of peptide administration has been somewhat limited, due strictly to their peptide attributes. In addition, peptides generally have a half-life in the range of several minutes and are rapidly and very effectively degraded by enzymes. Proteases and other proteolytic enzymes are present everywhere, especially in the gastrointestinal tract, and therefore peptides are usually susceptible to degradation in multiple sites upon oral administration and to some extent in the blood, liver, kidney and vasculature. Moreover, a given peptide is usually susceptible to degradation at one or more bonds in the backbone; Each position of hydrolysis is mediated by a constant protease.

Numerous attempts have been made to protect peptides against immature degradation, such as alteration of peptide structure, coadministration of protease inhibitors, or special formulation strategies, but they have only achieved partial success.

The present invention relates to prodrugs of pharmaceutically active peptides having a reduced tendency to hydrolysis.

Summary of the Invention

It has now been found that the pharmaceutically active peptide, at its C-terminus, has the appropriate bioreversible amino acid pre-sequence, making it possible to significantly reduce the peptide's degradation by proteases. Without being bound by any particular model for this effect, the presence of the presequence induces some degree of helical-like composition of the pharmaceutically active peptide, whereby the peptide is directed to the protease as opposed to the random-coiled peptide. It is believed to be less affected. As a result of the construction, the peptide is much more difficult for the protease to degrade. The bioreversible nature of the presequences is obtained by linking the peptides and the presequences by linkers bound to the peptides in a manner different from normal peptide bonds.

Therefore, the present invention

Formula I

X-L-Z

Wherein X is bonded to L in the C-terminal carbonyl function of X;

L is a thermal group containing 3 to 9 skeletal atoms, and the bond between C-terminal carbonyl and L of X is different from C-N amide bond; And

Z is a peptide sequence of 2-20 amino acid units and is bound to L located at the N-terminal nitrogen atom of Z, each amino acid unit being Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys , Arg, His, Met, Orn, and amino acid units of formula II

-NH-C (R ³ ) (R ⁴ ) -C (= O)-

Wherein R ³ and R ⁴ are selected from C _1-6 -alkyl, phenyl, and phenyl-methyl, wherein C _1-6 -alkyl is halogen, hydroxy, amino, cyano, nitro, sulfono, And one to three substituents selected from carboxy, wherein phenyl and phenyl-methyl are C _1-6 -alkyl, C _2-6 -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono And optionally substituted with one to three substituents selected from carboxy, or R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring); To a prodrug or salt thereof of a pharmaceutically active peptide (X-OH), peptide amide (X-NH ₂ ), or peptide ester (X-OR).

The invention also provides for the use of a prodrug of formula (I), for the preparation of a composition for use in the treatment of a prodrug of formula (I), and a pharmaceutical composition comprising the prodrug of formula (I) and a pharmaceutically acceptable carrier It also relates to the composition.

Another aspect of the invention is an immobilized linker-peptide sequence LZ-SSM, where L and Z are as defined above and SSM refers to a solid support, immobilized linker-peptides for the preparation of prodrugs of Formula I A method of making a prodrug of formula (I) consisting of the use of the sequence LZ-SSM and the use of an immobilized linker-peptide sequence LZ-SSM.

Since prodrugs of formula (I) are themselves novel, further aspects of the invention relate to compounds of formula (I).

Detailed description of the invention

Peptides are used in numerous processes, for example, cell-to-cell communication, and some are present in the autonomic and central nervous systems. Some of the central nervous system peptides, and many other peptides, have an important effect on the vasculature and other smooth muscles. These peptides are, for example, vasoconstrictor angiotensin II, vasopressin, endothelin, neuropeptide Y, vasoactive intestinal peptide, substance P, neurotensin, and calcitonin, calcitonin gene-related peptide, and calcitonin gene-related peptide II It includes. Among them, peptides of pharmacy interest include analgesics, antidiabetics, antibiotics, and anesthetic peptides, and thus the peptides may be endorphins, enkephalins, insulin, gramicidine, paracellsine, delta-sleep inducing peptides, ANF , Vasothocin, bradykinin, dinorphine, endothelin, growth hormone release factor, growth hormone release peptide, oxytocin, tachykinin, ACTH, brain natriuretic polypeptide, cholecystokinin, corticotropin release factor, diazepam binding inhibitor Fragment, FMRF-amide, galanine, gastric release polypeptide, gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide-1, glucagon-like peptide-2, LHRH, melanin enrichment hormone, alpha-MSH, morphine regulatory peptide, motiline, Neurokinin, Neuromedin, Neuropeptide K, Neuropeptide Y, PACAP, Pancreatic Polypeptide, Peptide YY, PHM, Secretin, Somatostatin, Substance K, Substance P, TR H, Vascular Bowel Polypeptides, and H.L.Lee, "Peptide and Protein Drug Delivery," Marcel Dekker Inc. 1991, Chapter 9, and references therein, Phoenix Pharmaceuticals, Inc. Biologically active peptides such as those described in "The Peptide Elite", the 1997-1998 catalog, and Bachem, "Feinchemikalien AG", catalog S15-1995.

It should be understood that the aforementioned peptides and also the pharmaceutically active peptide sequences of these peptides may be included in the prodrugs (and compounds) of the present invention.

As used herein, the term "pharmaceutical active peptide sequence" as used in X refers to a naturally occurring or synthetic compound that has two or more amino acid units (preferably three or more amino acid units) and which has a pharmaceutical effect on mammals such as humans. It is intended to mean any peptide or structure containing a peptide. As used herein, the term "amino acid unit" in connection with X is in L-form or corresponding D-form, for example M. Bodanszky and A. Bodanszky, "Performance of Peptide Synthesis", 2.Ed, Springer Aromatic rings, including side chain protected amino acids whose amino acid side chains are protected according to methods known to those skilled in the art of peptide chemistry, as described in Verlag, 1994, and J. Jones, "Chemical Synthesis of Peptides", Clarendon Press, 1991 For example, side chains such as modified tyrosine, which are further substituted with one or more halogens, sulfono groups, nitro groups, and / or phenol groups are converted to ester groups and the like, as well as any naturally occurring or synthetic amino acids. α, β, and γ-amino acids.

The pharmaceutically active peptide sequence X is preferably 2-200 amino acid units, more preferably 2-100 amino acid units (eg 3-100), more preferably 2-50 amino acid units (eg 3 -50 or 4-30), in particular 2-20 amino acid units (e.g. 3-20 or 4-20), especially 2-20 amino acid units (e.g. 3-10 or 4-10), e.g. For example, 2-8 amino acid units (eg 3-8 or 4-8).

In the text, the pharmaceutically active peptide sequence X present as a C-terminal free carboxylic acid such as Leu-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) is represented by X-OH. In a similar manner, a pharmaceutically active peptide sequence X having a C-terminal amide group such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH ₂ ) is represented by X-NH ₂ , And a pharmaceutically active peptide sequence X having a C-terminal ester group is represented by X-OR, where OR is, for example, an alkoxy moiety of an alcohol that forms an ester with the pharmaceutically active peptide sequence X. R refers to C _1-6 -alkyl, aryl such as phenyl, aryl-C _1-6 -alkyl such as benzyl, and the like.

In addition, even if the pharmaceutically active peptide sequence X is linked to the linker via C-terminal carbonyl, the pharmaceutical activity has a C-terminal amide (ie, X-NH ₂ ) or an ester group (ie, X-OR). It is to be understood that any peptide corresponding to a pharmaceutically active peptide having a free C-terminal carboxyl group (ie, X-OH) as well as a peptide corresponding to the peptide is used in the compounds and prodrugs of the present invention.

It is well known that many biologically active peptides also affect their desirable biological effects when present in modified or truncated forms. For example, in the case of insulin, porcine insulin is Ala and the B30 amino acid of human insulin is Thr, and only one amino acid unit is different from human insulin. Despite these differences, pig insulin has been used as an effective diabetes drug for many years. In a similar manner, it was found that the essential features for activity in heptadecanpeptide porcine gastrin I are all contained in the C-terminal tetrapeptide and that essentially all biological effects of neurotensin are related to C-terminal hexapeptide. Furthermore, one or more amide bonds alter, eg, reduced pharmaceutically active peptides often exhibit similar or even enhanced biological activity; For example, the Cys ² ψ [CH ₂ NH] Tyr ³ analog of somatostatin has been found to be a much more potent growth hormone releasing agent than somatostatin itself, and also the transition state analog of angiotensin Leu ¹⁰ ₄ [CH (OH) CH ₂ ] Val ¹¹ was found to show a strong inhibitory effect on aspartamic protease renin. In addition, the term "modified or cleaved analog thereof" refers to one or more carboxamide bonds in reduced form such as, for example, (-CH (OH) -N-), (-CH ₂ -N-) Modifications by altering and / or deleting one or more amino acid units in the sequence of the native peptide, including modifications of side chains, stereochemistry, and backbones of each amino acid unit, such as altering (-C (= O) -N-) Such peptides and (-C (= O) -O), (-C (= O) -CH _2- ), (-CH = CH-), (-PO ₂ -NH-), (SO- It is intended to mean other peptide-binding analogs such as CH _2- ), (SO ₂ -N-), and the like.

As mentioned above, in order to make full use of the present invention the peptide sequence of interest is preferably at least one amide bond (preferably two amide bonds (which is naturally applied to the dipeptides) susceptible to enzymatic degradation. Should be understood as including

The most interesting prospect of the present invention is to find mammals such as humans whose peptides or pharmaceutically active peptide sequences (X-OH) are stable against degradation by proteases and can then be released in an environment where they exhibit pharmacological action or are transported to a desired location. It is possible to produce "peptide prodrugs" for treatment. Although the pharmaceutically active peptide sequence X is preferably released as a free acid (e.g., due to the cleavage of the ester bond between X and L), the free acid has the original pharmaceutically active peptide sequence X as an amide (X-NH). ₂ ) or esters (X-OR) are also believed to have pharmaceutical related effects.

Also in an interesting embodiment, the bond between the C-terminal carbonyl function of X and L may be cleaved by plasma enzymes such as, for example, butyryl cholinesterase, acetylcholinesterase and the like. In particular, the bond between the C-terminal carbonyl function of X and L is a thiol ester bond or an ester bond, preferably an ester bond.

In order to release the pharmaceutically active peptide sequence X-OH, the bond between X and L of the peptide prodrug of the present invention must be cleavable in vivo. It will be understood from the examples provided herein that the bond between X and L (preferably an ester bond) can be cleaved by the enzyme butyryl cholinesterase. Thus, the peptide prodrugs of the invention can be cleaved by, for example, an esterase present in plasma and thereby release the desired pharmaceutically active peptide in the desired place.

Enzyme cleavage rate of the peptide can be adjusted so that the drug consisting of prodrug I has an extended or delayed effect. Control of the cleavage rate can be performed, for example, by increasing or decreasing the bulkyness and / or electron donating effect of the substituents on the L phase.

As used herein, the term “C _1-6 -alkyl”, used alone or as part of another group, refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, It refers to a straight-chain, branched or cyclic saturated hydrocarbon group having one to six carbon atoms such as n-pentyl, n-hexyl, cyclohexyl and the like. Similarly, the term “C _1-5 -alkyl” refers to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, cyclopentyl, And straight chain, branched or ring saturated hydrocarbon groups having one to five carbon atoms, and the like. The term “C _1-4 -alkyl”, used alone or as part of another group, refers to one such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and the like. Refers to a straight or branched saturated hydrocarbon group having from 4 to 4 carbon atoms, and similarly, the term “C _1-3 -alkyl” has one to three carbon atoms such as methyl, ethyl, n-propyl, and isopropyl It covers a straight or branched saturated hydrocarbon group.

In the text, the term “C _2-6 -alkenyl” refers to vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl having a cis and / or trans structure At least one double bond, such as 2-pentenyl, 4-pentenyl, 3-methyl-1-butenyl, 2-hexenyl, 5-hexenyl, cyclohexenyl, 2,3-dimethyl-2-butenyl, and the like And hydrocarbon groups containing two to five carbon atoms which are straight, branched, or cyclic. Likewise, the term “C _2-5 -alkenyl” refers to vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 4- with cis and / or trans structures Refers to a hydrocarbon group containing two to five carbon atoms that contain one or more double bonds, such as pentenyl, 3-methyl-1-butenyl, cyclopentenyl, and the like, straight chain, branched, or ring, and refers to a "C _2-4 -al Kenyl "is a straight or branched and two to four carbon atom containing one or more double bonds such as vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, etc. having cis and / or trans structures Refer to the group.

The term "alkoxy" means alkyl-oxy.

The term "aryl" is intended to mean aromatic, alicyclic groups such as phenyl or naphthyl.

The term "halogen" includes fluorine, chlorine, bromine, and iodine.

The term "heteroaryl" refers to pyrrolyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazoleyl, thiadiazolyl, triazolyl, nitrogen such as pyridyl And 5- or 6-membered aromatic single ring heterocyclic groups containing 1-4 heteroatoms selected from sulfur and aromatic bicyclic hetero containing 1-6 heteroatoms selected from nitrogen, oxygen and sulfur such as quinolinyl. It includes a cyclic group.

Peptide sequence Z is the portion of compound I responsible for the introduction and / or stabilization of certain secondary structures into molecules that make the compound more stable to degradation by proteases, and Z is also at least two amino acid units (preferably It is contemplated that it is necessary to introduce such structural elements, alone or in combination with linker L, including at least three amino acid units). On the other hand, it is also contemplated that sequences of about 20 amino acid units or more will not further improve stability. Thus, Z is 2-20 amino acid units (preferably 3-20), preferably in the range of 3-15, more preferably 3-9 (eg 4-9), 3-6 amino acid units , 4-6 amino acid units Peptide sequence of 3-6 amino acid units.

Each amino acid unit of peptide sequence Z is 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 herein. . Preferably, the amino acid unit is selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, and Met, more preferably Glu, Lys, and Met, in particular Glu and Lys. The amino acids mentioned above have a D- or L-structure but preferably the above-mentioned amino acids have an L-structure. Since the pharmacologically active peptide sequence X usually consists solely of L-amino acids, in order to preserve the helical structure of the entire prodrug, the peptide sequence Z consisting of only or predominantly L-amino acids consists of only or mainly D-amino acids. It will be advantageous compared to peptide sequence Z. Furthermore, peptide sequence Z, which consists solely or mainly of D-amino acids, affects the toxic effects due to the resistance to D-peptide and D-amino acids to biodegradation.

The amino acid units of Z may, of course, be all different or all the same. However, in an interesting embodiment of the invention, the amino acid unit of Z is selected from three different amino acids or two different amino acids, or is the same amino acid, preferably the amino acid unit of Z is (Lys) n or (Glu) n Where n is an integer ranging from 4 to 6 or a combination of two amino acid units such as (LysGlu) ₂ , (LysGlu) ₃ , (GluLys) ₂ , or (GluLys) ₃ , or 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) x-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, Combination of three amino acid units: 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 from 1 to 4, with x + y being at most 5 and Xaa represents Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Arg, His, Met, Orn, and amino acids of Formula II as defined herein.

With respect to peptide sequence Z, certain amino acid units referred to as components of peptide sequence Z, i.e., 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 have a certain preference for participating in, or stabilizing, or initiating a helix-like structure due to their steric arrangement around the α-carbon atom, and possibly also due to certain electronic structures. The Chou-Fasman approach (Chou, PY & Fasman, GD Ann. Rev. Biochem. 47, 251-276 (1978) describes the potential for special amino acid units to be included in the α-helix structure (denoted as the “structural parameter Pα”). However, Chou and Fasma's research and related studies have found that amino acid units with low parameter Pα are found in the α-helix, but often are not as common as amino acids with higher Pα, of course. Thus, for peptide sequence Z, it would still be desirable to obtain the desired effect from the peptide sequence and still obtain the desired effect from peptide sequence Z, including a small proportion of amino acid units not present in the amino acid units selected above as components of Z. It is thought that it is possible that the selected amino acid unit is produced by compensating for any negative or neutral effects of such alternative amino acid units. Because it is.

Also, in embodiments within the scope of the present invention, it is practical to include up to 25% amino acid units which are not among the preferred amino acids as components of Z ("25%" refers to the number of amino acid units, ie Replacement amino acid units are not allowed in di and tripeptides, up to one replacement amino acid unit is allowed in tetra-, penta-, hexa-, and hepta-peptides, and up to two replacement amino acid units are allowed in octapeptides, etc.) . Such alternative amino acid units are selected from N-methyl amino acid units as well as Val, Ile, Pro, Phe, Gly, Trp, preferably not Pro, Gly and N-methyl amino acid units.

Exemplary examples of peptide sequence Z are as follows:

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, 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-Lys-Lys, Lys-Ly s-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-Glu-Glu-Lys-Lys-Lys, Lys-Glu-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- Lys, 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-Glu-Lys-Lys-Glu, Lys-Glu-Lys-Glu-Glu-Lys, Lys-Glu-Glu-Lys- Glu-Lys, Lys-Glu-Glu-Glu-Lys-Lys, Lys-Lys-Glu-Glu-Glu-Glu, Lys-Gl u-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-terminus of Z may be provided in the form of a free acid, amide, or ester, for example, depending on the type and cleavage conditions of the solid support material used in connection with the synthesis that would be apparent to those skilled in the art.

L is bonded at the N-terminal nitrogen atom of Z, ie a possible bond between L and Z is one containing a nitrogen atom, for example a carboxamide bond (-C (= O) -N- ), Sulfonamide bond (-SO ₂ -N-), or alkylamine bond (-CN-), carbamate bond (-OC (= O) -N-), thiocarbamate bond (-SC (= O) -N-), urea bond (-NC (= O) -N-), thiourea bond (-NC (= S) -N-), thioamide bond (-C (= S) -N-) , Cyanomethyleneamino bond (-C (CN) -N-), or N-methylamide bond (-C (= O) -N- (CH ₃ )-) (in these examples, The nitrogen atom comes from Z and the rest of the "bond" comes from L). Preferred bonds are -C (= 0) -N-, -SO ₂ -N-, -CN-, -C (= S) -N-, -C (CN) -N-, and -C (= 0) -N (CH ₃ )-, of which -C (= O) -NH- and -C (= S) -NH-, are preferred because they have the geometry of ordinary peptide bonds.

The linker L should preferably be able to participate in the helix-like structure initiated or stabilized by Z. In contrast to the fact that the bond 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), in other words, the linker L preferably has three backbones. It consists of atoms or multiples of it like 6 or 9 skeletal atoms. Thus, in the text, when used in connection with a linker L, the term "skeleton atom" refers to an atom in the linker L that directly connects the pharmaceutically active peptide sequence X and the free sequence Z.

In addition, L is preferably derived from hydroxy-carboxylic acids, in particular α-hydroxy-carboxylic acids. More specifically, L is derived from α-hydroxy-carboxylic acid of formula HO-C (R ¹ ) (R ² ) -COOH, wherein R ¹ and R ² are independently H, C _1-6- Alkyl, C _2-6 -alkenyl, aryl, aryl-C _1-4 -alkyl, heteroaryl, heteroaryl-C _1-4 -alkyl or R ¹ and R ² are the carbon atoms to which they are attached Together form a cyclopentyl, cyclohexyl, or cycloheptyl ring, wherein the alkyl or alkenyl group is amino, cyano, halogen, isocyano, isothiocyano, thiocano, sulfamoyl, C _1-4 -alkyl Thio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C _1-4 -alkoxycarbonyl, C _1-4- Alkylcarbonyloxy, aminocarbonyl, mono- or di-C _1-4 -alkyl-aminocarbonyl, mono- or di-C _1-4 -alkyl-amino, mono- or di-C _1-4 -alkyl -Amino-C _1-4 -alkyl, C _1-4- Optionally substituted with one to three substituents selected from alkylcarbonylamino, sulfono, and sulfino, wherein the aryl or heteroaryl group is C _1-4 -alkyl, C _2-4 -alkenyl, nitro, amino, cya Furnace, halogen, isocyano, isothiocyano, thiocyano, sulfamoyl, C _1-4 -alkylthio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4- alkoxy, aryloxy, carboxy, C _1-4 - alkoxycarbonyl, C _1-4 - alkyl carbonyloxy, aminocarbonyl, mono- or di -C _1-4 - alkyl-aminocarbonyl, mono- or di One selected from -C _1-4 -alkyl-amino, mono- or di-C _1-4 -alkyl-amino-C _1-4 -alkyl, C _1-4 -alkylcarbonylamino, sulfono, and sulfino To three substituents.

From the structural analysis of the prodrugs of the present invention, further stabilization of the helix-like structure of the prodrug is achieved when the linker L is derived from an α-hydroxy-carboxylic acid which also has a methylene (-CH _2- ) group at the α-position. I think it is achieved. Thus, in an interesting embodiment, the linker L is derived from α-hydroxy-carboxylic acid having the formula HO-C (CH ₂ -R ⁵ ) (R ² ) -COOH, wherein R ⁵ is H, C _{1- 5} -alkyl, C _2-5 -alkenyl, aryl, aryl-C _1-3 -alkyl, heteroaryl, heteroaryl-C _1-3 -alkyl, wherein the alkyl or alkenyl group is amino, halogen, Mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C _1-4 -alkoxycarbonyl, C _1-4 -alkylcar May be substituted with one to three substituents selected from carbonyloxy, and aminocarbonyl and aryl or heteroaryl is C _1-4 -alkyl, C _2-4 -alkenyl, nitro, amino, halogen, mono- or di- C _1-4 - alkyl-amino, hydroxy, C _1-4 - alkoxy, carboxy, C _1-4 - alkoxycarbonyl, C _1-4 - alkylcarbonyloxy, C _1-4 - alkylcarbonyloxy, And aminocarbonyl; And R ² is as described above, preferably H, C _1-6 -alkyl, C _2-6 -alkenyl, aryl, aryl-C _1-6 -alkyl, heteroaryl, heteroaryl-C _1-4 -Alkyl, wherein the alkyl or alkenyl group is amino, halogen, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C ₁ Or substituted with one to three substituents selected from _-4 -alkoxycarbonyl, C _1-4 -alkylcarbonyloxy, and aminocarbonyl, wherein aryl or heteroaryl is C _1-4 -alkyl, C _{2 -4} -alkenyl, nitro, amino, halogen, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, carboxy, C _1-4 -alkoxycarbonyl, C _1- It may be substituted with one to three substituents selected from ₄ -alkylcarbonyloxy, and aminocarbonyl.

The above-mentioned control of the cleavage rate by increasing or decreasing the bulkyness and / or electron donating effect of substituents on L may be, for example, the bulkyness and / or electron donating of R ¹ and / or R ² (or R ⁵ ). By increasing or decreasing the effect.

In a particularly interesting embodiment, L is hydroxyacetic acid, (S)-(+)-mandelic acid, L-lactic acid ((S)-(+)-2-hydroxypropanoic acid), L-α-hydroxy- Butyric acid ((S) -2-hydroxybutane nonacid), and α-hydroxy-isobutyric acid.

It is to be understood that the prodrugs of the present invention also exist in their salt form. Salts include pharmaceutically acceptable salts such as acid addition salts and basic salts. Examples of acid addition salts are hydrochlorides, sodium salts, calcium salts, and the like. Examples of basic salts are salts in which the cation is selected from alkali metals such as sodium and potassium, alkaline earth metals such as calcium, and ammonium ions ⁺ N (R ⁶ ) ₃ (R ⁷ ), where R ⁶ and R ⁷ are independently Refers to C _1-6 -alkyl, C _2-6 -alkenyl, optionally substituted aryl, optionally substituted heteroaryl. Other examples of pharmaceutically acceptable salts are described, for example, in "Remington's Pharmaceutical Sciences" 17. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and the more recent editions and Encyclopedia of Pharmaceutical Technology.

As mentioned above, the route of administration of pharmaceutically active peptides has been rather limited due to rapid biodegradation by proteases such as chymotrypsin, trypsin, carboxypeptidase A, pepsin, leucine aminopeptidase, and the like. As understood from the examples provided herein, the tendency of prodrugs of Formula I (XLZ) to withstand protease-catalyzed hydrolysis can be measured directly by the in vitro enzyme assay shown in the Examples. The propensity of XLZ to withstand degradation can be expressed, for example, as a pseudo-first-order rate constant and / or as a half-life of the prodrug, compared to the corresponding values of X-OH, X-NH ₂ , and / or X-OR do. Furthermore, the ability of the prodrugs of the present invention to affect the desired biological effect has been tested with various in vitro and in vivo assay procedures. Detailed description of the above-mentioned test is given in the Examples.

It has been found that by protecting the peptide of interest as a prodrug according to the invention it is possible to obtain a significant increase in the half-life (T _3/2 ) of the peptide or pharmaceutically active peptide sequence.

Thus, in a preferred embodiment of the present invention, as defined herein, between the half-life of the prodrug of interest in "hydrolysis in enzyme solution test" and the half-life of the peptide (X-OH) in "hydrolysis in enzyme solution test". The ratio of is at least 2, preferably at least 5, and even more preferably at least 10, especially at least 20 when using one of the enzymes carboxypeptidase A and leucine aminopeptidase.

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

Such compositions may be in a form adapted for oral, parenteral (intravenous, intraperitoneal), rectal, intranasal, skin, vaginal, mouth, ocular, or pulmonary administration, and such compositions include, for example, " Remington's Pharmaceutical Sciences ", 17. Ed. In a manner well known to those skilled in the art, as generally described in Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 and later editions and in the papers of the "Drugs and the Pharmaceutical Sciences" series, Marcel Dekker. Can be prepared.

The present invention provides treatments, including, for example, treatment of diseases of the central nervous system, vaccine therapy and the treatment of HIV, cancer, diabetes, incontinence, hypertension, and administration of analgesics and contraceptives, and pharmaceutically active peptides. The use of salts of prodrugs or prodrugs of formula (I) or salts thereof as defined above in the manufacture of a composition for use in the treatment of such indications known to be treated by.

Prodrugs of the present invention are prepared by methods known in the art. Peptide sequences X and Z are also prepared by standard peptide-making techniques such as solution synthesis or Merrifield-type solid phase synthesis. It is believed that the Boc (tert-butyloxycarbonyl) strategy as well as Fmoc (9-fluorenylmethyloxycarbonyl) are applicable.

In one possible synthesis strategy, the prodrugs of the present invention first construct peptide sequence Z using well-known standard protection, and then undergo a coupling and deprotection process, followed by coupling of the linker L, followed by the configuration of Z in succession. In a similar manner it is prepared by solid phase synthesis by coupling the pharmaceutically active sequence X on the linking group L and finally cleaving the entire prodrug XLZ from the carrier.

Another possible strategy is to prepare one or both of the two sequences X and Z separately by solution synthesis, solid phase synthesis, recombination techniques, or enzymatic synthesis, either in solution or using solid phase techniques, or a combination thereof, followed by well known fragments. Condensation binds the two sequences and the linking group L.

Furthermore, for example, a combination of the above mentioned strategies can be considered particularly applicable when a peptide sequence modified from a biologically active peptide comprising reduced peptide bonds is linked to peptide sequence Z via a linker L. In this case, first, a fragment LZ immobilized by continuous coupling of amino acids (and linkers) is prepared, and then a complete biologically active peptide sequence X (prepared in solution or sufficiently or partially using solid phase technology) is added to the fragment LZ. Coupling may be advantageous.

Accordingly, the present invention relates to an immobilized linker-peptide sequence Prot-LZ-SSM, where L refers to a linker of formula -OC (R ¹ ) (R ² ) C (= 0), wherein R ¹ and R ² is as defined above and Prot refers to H or a hydroxy protecting group wherein the hydroxy protecting group is dimethoxytrityl, monomethoxytrityl, trityl, 9- (9-phenyl) xanthenyl (picyl ), Tetrahydropyranol, methoxytetrahydropyranol, trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triethylsilyl, phenyldimethylsilyl, benzyloxycarbonyl, 2-bromo benzyloxycarbonyl, tert-butylether, methyl ether, substituted benzyloxycarbonylether such as acetyl, halogen substituted acetyl, isobutyryl, pivaloyl, benzoyl and substituted benzoyl, methoxymethyl, benzyl ether such as chloroacetyl and fluoroacetyl And halogen values such as 2,6-dichlorobenzyl Benzyl ether; SSM refers to a solid support material selected from functionalized resins such as, for example, polystyrene, polyacrylamide, polydimethylacrylamide, polyethylene glycol, latex, dinavid, and the like; And Z is as defined above.

The C-terminal amino acid of presequence Z is 2,4-dimethoxy-4'-hydroxy-benzophenone, 4- (4-hydroxy-methyl-3-methoxyphenoxy) -butyric acid, 4-hydroxy- Methylbenzoic acid, 4-hydroxy methyl-phenoxyacetic acid, 3- (4-hydroxymethylphenoxy) propionic acid, and p-[(R, S) -a [1- (9H-fluoren-9-yl) It is attached to the solid support by a conventional linker such as methoxyformamido] -2,4-dimethoxybenzyl] -phenoxy-acetic acid.

As a result, the present invention relates to the use of the immobilized linker-peptide sequence Port-LZ-SSM for the preparation of prodrugs according to the invention, wherein the C-terminal activation form (X) is immobilized to the immobilized linker-peptide sequence HLZ-SSM. A method for preparing a prodrug of a peptide (X-OH), a peptide amide (X-NH2), or a peptide ester (X-OR) comprising coupling a corresponding peptide of -Act).

The present invention also provides a) coupling an immobilized linker peptide sequence HLZ-SSM with an N-α-protected amino acid in carboxy active form or an N-α-protected dipeptide in C-terminal activated form, thereby immobilizing Forming an N-α-protected peptide fragment,

b) removing the N-α-protecting group, thereby forming an immobilized peptide fragment having an unprotected N-terminal end,

c) coupling an additional N-α-protecting amino acid of the carboxyl-activated form or an additional N-α-protecting dipeptide of the C-terminal active form to the unprotected N-terminal end of the immobilized peptide fragment, and Repeat the removal / coupling process in steps b) and c) until peptide sequence X is obtained,

d) a peptide comprising the steps of cleaving the prodrug XLZ from the solid support material to cleave the prodrug XLZ from the solid support material to obtain free prodrug in the form of a C-terminal carboxylic acid, amide, or ester (X- OH), peptide amide (X-NH ₂ ), or other methods of making prodrugs of peptide esters (X-OR).

The binding, removal and cleavage steps are performed by methods known to those skilled in the art, taking into account the protection strategy and the selected solid phase material.

With regard to establishing a bond between X and L on the one hand and L and Z on the other hand of the type indicated above, such a bond may be a thiol ester, ester, carboxamide, sulfonamide, alkylamine, carbamate , Thiocarbamate, urea, thiourea, thioamide, cyanomethyleneamino, or N-methylamide bonds or by known methods for establishing groups (eg, J. March, "Advanced Organic Chemistry" , 3rd edition, John Wiley & Sons, 1985 and references cited therein). Thus, for example, esters can be formed from the appropriate carboxylic acid activation derivatives (acid halides, acid anhydrides, activated esters such as HObt-esters, etc.) by reaction with a suitable hydroxy compound.

Likewise, carboxamides can be formed by reacting appropriate amino compounds known to those skilled in the art in peptide chemistry with the appropriate carboxylic acid activation derivatives (acid halides, acid anhydrides, activated esters, e.g. HObt-esters, etc.). have; Sulfonamides are formed by reacting a suitable amino compound with sulfonyl chloride; Alkylamine bonds or groups are formed by reacting a suitable amino compound in a nucleophilic substitution with a suitable compound having leaving groups such as tosyl, halogen, and mesityl on the carbon atom of interest; Carbamate bonds or groups are formed by treating a suitable alcohol with phosgene to give the corresponding chlorocarbonate and then reacting with a suitable amino compound; Thiocarbamate bonds or groups are formed by treating a suitable alcohol with thiophosphene to give the corresponding chlorothiocarboxylic acid and then reacting with a suitable amino compound. Urea bonds or groups can be formed by reacting a suitable compound having an isocyanate group on the carbon atom of interest with a suitable amino compound; Thiourea bonds or groups are formed by reacting a suitable amino compound with a suitable compound having an isothiocyanate group on the carbon atom of interest; Thioamide bonds or groups are formed by reacting a thionoester of a suitable carboxylic acid with a suitable amino compound, which is formed from the corresponding piperidine.

Furthermore, it is necessary or desirable to include side chain protecting groups when using amino acid units having functional groups reactive under dominant conditions. The necessary degree of protection will be known to those skilled in the art (eg, M. Bodanszky and A. Bodanszky, "Performance of Peptide Synthesis", 2. Ed, Springer-Verlag, 1994, and J. Jones, "Chemical Synthesis of Peptides", See Clarendon Press, 1991).

Thus, the peptide prodrugs of the present invention are acids such as trifluoroacetic acid, trifluoromethanesulfonic acid, hydrogen bromide, hydrogen chloride, hydrogen fluoride, and the like, or bases such as ammonia, alkoxides such as hydrazine, sodium ethoxylate, sodium hydroxide and It is cleaved from the solid support material by the same hydroxide or the like.

Since the prodrugs of the present invention represent a new class of compounds, yet another aspect of the present invention relates to compounds of formula (I) or salts thereof.

Formula I

X-L-Z

Wherein X is a peptide sequence that binds to L in the C-terminal carbonyl function of X;

L is a thermal group containing 3 to 9 skeletal atoms, and the bond between C-terminal carbonyl and L of X is different from C-N amide bond; And

Z is a peptide sequence of 2-20 amino acid units and is bound to L located at the N-terminal nitrogen atom of Z, each amino acid unit being Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys , Arg, His, Met, Orn, and amino acid units of formula II

Formula II

-NH-C (R ³ ) (R ⁴ ) -C (= O)-

Wherein R ³ and R ⁴ are selected from C _1-6 -alkyl, phenyl, and phenyl-methyl, wherein C _1-6 -alkyl is halogen, hydroxy, amino, cyano, nitro, sulfono, And one to three substituents selected from carboxy, wherein phenyl and phenyl-methyl are C _1-6 -alkyl, C _2-6 -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono And optionally substituted with one to three substituents selected from carboxy, or R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring) .

The invention is further described by the following examples.

Peptide synthesis

General procedure

Abbreviations used:

tBu = tert-butyl

DAMGO = Tyr- (D-Ala) -Gly-ψ [-C (= O) -N (CH ₃ )-[Phe-NH-CH ₂ -CH ₂ OH

DCC = dicyclohexylcarbodiimide

DCM = dichloromethane

DIC = diisopropylcarbodiimide

DIEA = N, N-diisopropylethylamine

DMAP = 4- (N, N-dimethylamino) -pyridine

Dhbt-OH = 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

DMF = N, N-dimethylformamide

DSIP = delta-sleep inducing peptide,

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH

EDT = ethanedithiol

ES-MS = Electrospray Mass Spectroscopy

Fmoc = 9-fluorenylmethyloxycarbonyl

cHex = cyclohexyl

HAA = hydroxyacetic acid

HMPA = 4-hydroxymethylphenoxyacetic acid

HObt = 1-hydroxybenzotriazole

HPLC = high performance liquid chromatography

Ma = mandelic acid

NHS = N-hydroxy-succinic acid imido ester

PEG-PS = Graft Polyethyleneglycol in Polystyrene

Pfp = pentafluorophenyl

SEM = standard deviation of the mean

TFA = trifluoroacetic acid

Z = benzyloxycarbonyl

Device and Synthesis Strategy

Peptides were synthesized batchwise in polyethylene containers equipped with polypropylene filter paper for filtration using 9-fluorenylmethyloxycarbonyl (Fmoc) as the N-α-amino protecting group and suitable conventional protecting groups for side chain functionality ( Dryland, A. and Sheppard, RC (1986) J. Chem. Soc., Perkin Trans. 1, 125-137).

menstruum

Solvent DMF (N, N-dimethylformamide, Riedel de-Haen, 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). 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) with yellow (Dhbt-O-anion) when free amine is present Free amines were analyzed before. Solvent DCM (dichloromethane, analytical grade, Riedel de-Haen, Germany) was used directly without purification.

Amino acids and dipeptides

Fmoc-protected amino acids in the appropriate side chain protection form were purchased from MilliGen (UK). Otherwise protected amino acids (H-Glu (OtBu) -OtBu; H-Glu (cHex) -OH; Z-Glu (OtBu) -OH) and dipeptides Fmoc-Phe-Gly-OH and H-Phe-Leu-OH Was purchased from Bachem (Switzerland).

Binder

Binder diisopropylcarbodiimide (DIC) was purchased from Riedel de-Haen, Germany and distilled before use, dicyclohexylcarbodiimide (DCC) was purchased from Merck Schuchardt, Munich, Germany and purified by distillation.

Linker

Linker (4-hydroxymethylphenoxy) acetic acid (HMPA), Novabiochem, Switzerland; Hydroxyacetic acid, (S)-(+)-mandelic acid purity 99%, Aldrich, Germany, and (R)-(-)-mandelic acid purity 98.5%, M & R, UK Preformed 1- produced by DIC Hydroxybenzotriazole (HObt) was bound to the N-terminus of presequence Z or to the resin.

Solid support

Peptides synthesized according to the Fmoc-strategy were synthesized on two different types of supports using concentrations of Fmoc-protecting activated amino acids in concentrations greater than 0.05 M in DMF: 1) PEG-PS (polyethylene glycol grafted onto polystyrene; NovaSyn TG Resin, 0.29 mmol / g, Novabiochem, Switzerland); 2) NovaSyn K 125 (Kieselguhr supported polydimethylacrylamide resin functionalized with sarcosine methyl ester 0.11 mmol / g; Novabiochem, Switzerland)

Catalyst and other reagents

Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany, ethylenediamine was purchased from Fluka, piperidine and pyridine from Riedel-de Haen, Frankfurt, Germany. 4- (N, N-dimethylamino) pyridine (DMAP) was purchased from Fluka, Switzerland and used as catalyst for coupling reactions involving symmetric anhydrides. Ethandithiol was purchased from Riedel de-Haen, Frankfurt, Germany. 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) and 1-hydroxybenzotriazole (HObt) were obtained from Fluka, Switzerland. FmocNHS was purchased from Aldrich, Germany.

enzyme

Carboxypeptidase A (EC 3.4.17.1) type I from bovine pancreas, Leucine aminopeptidase (EC 3.4.11.1) from porcine kidney Type III-CP, butyryl cholinesterase from horse serum (EC 3.1.1.8) ), 5-chymotrypsin (EC 4.4.21.1) from bovine pancreas, and pepsin A (EC 3.4.23.1) from porcine gastric mucosa, bovine pancreas were obtained from Sigma, UK.

Joining Procedure

The first amino acid was coupled as a symmetric anhydride in the appropriate N-α-protected amino acid and DMF generated from DIC or DCC. The following amino acids were coupled by DIC in DMF as appropriate preformed HObt esters made from HObt with the appropriate N-α-protected amino acids. Acylation was checked by a ninhydrin test performed at 80 ° C. to prevent Fmoc deprotection during the test (Larsen, BD and Holm, A., Int. J. Peptide Protein Res. 43, 1994, 1-9). .

Deprotection of N-α-amino protecting groups

Deprotection of the Fmoc group was carried out by treatment with 20% piperidine (1 × 3 and 1 × 7 min) in DMF, and DMFt-OH was added to the discharged DMF until no yellow (Dhbt-O-) was detected. Washed with.

Cleavage of Peptides from Resin with Acid

Peptide from resin by treatment with 95% trifluoroacetic acid (TFA, Riedel-de Haen, Frankfurt, Germany) -water v / v or 95% TFA and 5% ethanedithiol v / v at room temperature for 2 hours. Was cut. The filtered resin was washed with 95% TFA-water and the filtrate and washes were evaporated under reduced pressure. The residue was washed with ether and lyophilized from acetic acid-water. Crude dry products were analyzed by high performance liquid chromatography (HPLC) and confirmed by electrospray ionization mass spectroscopy (ESMS).

Preformed HObt-ester

Three equivalents of N-a-amino protected amino acid or hydroxyacetic acid or (S)-(+)-mandelic acid were dissolved in DMF with three equivalents of HObt and three equivalents of DIC. The solution was left at room temperature for 10 minutes and then added to the resin washed with a 0.2% Dhbt-OH solution in DMF before adding the preactivated amino acid.

Preformed Symmetric Anhydride

Six equivalents of N-a-amino protected amino acid were dissolved in DCM and cooled to 0 ° C. DCC (3 equiv) was added and the reaction continued for 10 minutes. The solvent was removed in vacuo and the residue was dissolved in DMF. The solution was filtered and immediately added to the resin followed by 0.1 equivalent of DMAP.

Measurement of Coupling Yield of the First N-α-Amino Protected Amino Acids

3-5 mg of dry Fmoc protected peptide-resin was treated with 5 ml of 20% piperidine in DMF for 10 minutes at room temperature and the UV absorbance for the dibenzopulbene-piperidine adduct was measured at 301 nm. The yield was determined using the calculated expansion coefficient ε ₃₀₁ based on the Fmoc-Ala-OH standard.

Peptide Synthesis on PepSyn K Dendritic

Dry PepSyn K (about 500 mg) was covered with ethylenediamine and left overnight at room temperature. The resin was drained and washed with 10 × 15 ml DMF (5 min each). After draining, Dhbt-OH was added to the drained DMF and the resin was washed with 10% DIEA (2 × 15 ml, 5 min each) in DMF v / v until finally yellow was not detected and finally with DMF. Three equivalents of HMPA, three equivalents of HObt and three equivalents of DIC were dissolved in 10 ml of DMF and left for 10 minutes for activation, then the mixture was added to the resin and the coupling continued for 24 hours. The resin was drained and washed with DMF (10 × 15 ml, 5 minutes each) and acylation was checked by ninhydrin test. The first amino acid was coupled as preformed symmetric anhydride (see above) and the coupling yield was measured as described above. This yield was better than 70% in all cases. The synthesis was then continued in "batch".

Continuous Batch Peptide Synthesis on PepSyn K

The resin with the first amino acid (about 500 mg) was placed in a polyethylene vessel equipped with a filtration polypropylene filter to deprotect the Fmoc group as described above. The remaining amino acids according to sequence were coupled as preformed and Fmoc protected, if desired side chain protected HObt ester (3 equiv) in DMF (5 ml) prepared as described above. Coupling continued for 2 hours unless otherwise specified. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). All acylation was checked by the ninhydrin test run at 80 ° C. After completion of the synthesis, the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (5 x 5 ml, 1 min each) and finally diethyl ether (5 x 5 ml, 1 min each) and vacuum dried.

Batch Peptide Synthesis on PEG-PS

NovaSyn TG resin (250 mg, 0.27-0.29 mmol / g) was placed in a polyethylene container equipped with a filtration polypropylene filter. The resin was swollen in DMF (5 ml) and treated with 20% piperidine in DMF to confirm the presence of aprotic amino groups on the resinous phase. After adding Dhbt-OH to the drained DMF, the resin was drained and washed with DMF until yellow could not be detected. HMPA (3 equiv) was coupled as a preformed HObt-ester as described above and the coupling was continued for 24 hours. The resin was drained and washed with DMF (5 × 5 ml, 5 minutes each) and acylation was checked by ninhydrin test. The first amino acid was coupled as symmetric anhydride preformed as described above. Coupling yield of the first Fmoc protected amino acid was evaluated as described above. This yield was better than 60% in all cases. The next amino acid according to sequence was coupled as preformed and Fmoc protected, if necessary side chain protected HObt ester (3 equiv) as described above. Coupling was continued for 2 hours unless otherwise specified. The resin was drained and washed with DMF (5 × 5 ml, 5 minutes each) to remove excess reagent. All acylation was checked by the ninhydrin test run at 80 ° C. After completion of the synthesis, the peptide-resin was washed with DMF (3 × 5 ml, 5 min each), DCM (3 × 5 ml, 1 min each) and finally with diethyl ether (3 × 5 ml, 1 min each) and vacuum dried.

HPLC conditions

Isocratic HPLC analysis was performed on a Shimadzu system consisting of an LC-6A pump, a MERCK HITACHI L-4000 UV detector operating at 215 nm, and a Rheodyne 7125 injection valve with 2, 20 or 100 μl loops. The column used for isocratic analysis was Spherisorb ODS-2 (100 × 3 mm; 5 μm particles). HPLC analysis using gradients was performed with a MERCK-HITACHI L-6200 intelligent pump, a MERCK-HITACHI L-4000 UV detector operating at 215 nm and a Rheodyne 7125 injection valve with 20 μl loop. The column used was 1 ml of Rescorce ^™ RPC.

Buffer A was 0.1 volume% TFA in water and Buffer B was 90 volume% acetonitrile, 9.9 volume% water and 0.1 volume% TFA. The buffer was pumped through the column at a flow rate of 1.3-1.5 ml / min using the following gradient for peptide analysis. Linear gradient 0% -100% B (30 min) for enzyme studies, 2. Linear gradient 40-100% B (15 min), 3.Linear gradient 10-40% B (15 min), or 4. Linear gradient 0-50% B (15 minutes). The mobile phase used for isocratic analysis will be mentioned in the description of each experiment.

Mass spectroscopy

Mass spectra were obtained on a Finnigan Mat LCQ instrument equipped with an electrospray (ESI) probe (ES-MS).

Peptide Synthesis of Each Peptide

1. Peptide synthesis of H-Tyr-Gly-Gly-Phe-Leu-Glu ₆ -OH on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis for PEG-PS" until the presequence Glu ₆ was terminated. It was. The next amino acid forming the Leu-enkephalin sequence was coupled as a preformed, Fmoc protected, if desired side chain protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before each final five couplings to cause the yellow color to disappear as the coupling reaction proceeded. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. Acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin and lyophilized from acetic acid as described above. The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity was found to be better than 90% and peptide identification was confirmed by ES-MS. Yield 76%.

2. Synthesis of H-Tyr-Gly-Gly-Phe-Leu-HAA-Glu ₆ -OH on PepSyn K resin.

Dry PepSyn K (about 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated with ethylenediamine as described above. The first six glutamic acid units forming the presequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv). Acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After termination of the presequence, the deprotected peptide-resin was reacted with 6 equivalents of hydroxyacetic acid as a HObt-ester preactivated as described above and the coupling was continued for 24 hours. Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min). Acylation was checked by ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 2 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed to check the coupling yield and evaluated as described above and found to be 90%. Synthesis was then continued by cleavage of the Fmoc group as described above.

The next coupling along the sequence is to prevent diketopiperazine formation, which is performed as a dipeptide coupling. Thus Fmoc-Gly-Phe-OH was coupled for 2 hours as preformed HObt ester (3 equiv) in DMF (5 ml) prepared as described above. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to sequence were coupled as preformed, Fmoc protected, if necessary side chain protected HObt esters (3 equivalents) with addition of 1 equivalent Dhbt-OH in DMF (2 ml) for 2 hours. Acylation was checked by the ninhydrin test performed as described above. The remaining amino acids along the sequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv) in DMF (2 mL). Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After completion of the synthesis, the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3 x 5 ml, 1 min each), diethyl ether (3 x 5 ml, 1 min each) and vacuum dried.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). The crude lyophilized product was analyzed by HPLC and found to be homologous, purity was found to be better than 80%, peptide identification was confirmed by ESMS and yield was 58%.

3. Synthesis of H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-Ma) -Glu ₆ -OH on PepSyn K resin.

Dry PepSyn K (about 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated with ethylenediamine as described above. The first six glutamic acids forming the presequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv). Acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After termination of the presequence, the deprotected peptide-resin was reacted with 6 equivalents (S)-(+)-mandelic acid as a preactivated HObt-ester as described above and the coupling continued for 24 hours. Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min). Acylation was checked by ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 2 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed to check coupling yield and evaluated as described above to find 85%. Synthesis was then continued by cleavage of the Fmoc group as described above.

The next coupling along the sequence is to prevent diketopiperazine formation, which is performed as a dipeptide coupling. Thus Fmoc-Gly-Phe-OH was coupled for 2 hours as preformed HObt ester (3 equiv) in DMF (5 ml) prepared as described above. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to sequence were coupled for 2 hours as preformed and Fmoc protected HObt ester (3 equiv) with the addition of 1 equiv Dhbt-OH in DMF (2 mL). Acylation was checked by the ninhydrin test performed as described above. The remaining amino acids along the sequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv) in DMF (2 mL). Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After completion of the synthesis, the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3 x 5 ml, 1 min each), diethyl ether (3 x 5 ml, 1 min each) and vacuum dried.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). The crude lyophilized product was analyzed by HPLC and found to be homologous. The purity was higher than 80%. The peptide was identified by ESMS and the yield was 57%.

Prodrug H-Tyr-Gly-Gly-Phe-Leu-((R)-(-)-Ma) -Glu ₆ -OH was prepared as described above for 3.

4. Synthesis of H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-Ma) -Lys ₆ -OH on PepSyn K resin.

Dry PepSyn K (about 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated with ethylenediamine as described above. The first six lysines forming the presequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv). Acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After termination of the presequence, the deprotected peptide-resin was reacted with 6 equivalents of (S)-(+)-mandelic acid as the preactivated HObt-ester as described above and the coupling was continued for 24 hours. Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min). Acylation was checked by ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 24 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed to check the coupling yield and evaluated as described above and found to be 85%. Synthesis was then continued by cleavage of the Fmoc group as described above.

The next coupling along the sequence is to prevent diketopiperazine formation, which is performed as a dipeptide coupling. Thus Fmoc-Gly-Phe-OH was coupled for 2 hours as preformed HObt ester (3 equiv) in DMF (5 ml) prepared as described above. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to sequence were coupled for 2 hours as preformed and Fmoc protected HObt ester (3 equivalents) with the addition of 1 equivalent of Dhbt-OH in DMF (2 ml). Acylation was checked by the ninhydrin test performed as described above. The remaining amino acids along the sequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv) in DMF (2 mL). Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After completion of the synthesis, the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3 x 5 ml, 1 min each), diethyl ether (3 x 5 ml, 1 min each) and vacuum dried.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). HPLC analysis of the crude lyophilized product showed homology and purity was better than 80%. Identification of the peptide was confirmed by ESMS and the yield was 57%.

Prodrug H-Tyr-Gly-Gly-Phe-Leu-((R)-(-)-Ma) -Lys ₆ -OH was prepared as described for 4 above.

5. Synthesis of H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-Ma)-(LysGlu) ₃ -OH on PepSyn K resin.

Dry PepSyn K (about 500 mg, 0.1 mmol / g) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated with ethylenediamine as described above. The first six amino acids forming the presequence were coupled as Fmoc protected Pfp ester (3 equiv) with the addition of Dhbt-OH (1 equiv). Acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After termination of the presequence, the deprotected peptide-resin was reacted with 6 equivalents of (S)-(+)-mandelic acid as the preactivated HObt-ester as described above and the coupling was continued for 24 hours. Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min). Acylation was confirmed by the ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 2 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed to check the coupling yield and evaluated as described above and found to be 85%. Synthesis was then continued by cleavage of the Fmoc group as described above.

The next coupling along the sequence is to prevent diketopiperazine formation, which is performed as a dipeptide coupling. Thus Fmoc-Gly-Phe-OH was coupled for 2 hours as preformed HObt ester (3 equiv) in DMF (5 ml) prepared as described above. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were then removed by treatment with 20% piperidine in DMF as described above. The remaining amino acids according to sequence were coupled for 2 hours as preformed Fmoc protected HObt ester (3 equivalents) with the addition of 1 equivalent of Dhbt-OH in DMF (2 ml). Acylation was checked by the ninhydrin test performed as described above. The remaining amino acids according to sequence were coupled as Fmoc protected Pfp Estre (3 equiv) with the addition of Dhbt-OH (1 equiv) in DMF (2 mL). Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min) and acylation was checked by the ninhydrin test run at 80 ° C. as described above. Fmoc groups were deprotected as described above. After completion of the synthesis, the peptide-resin was washed with DMF (10 min, flow rate 1 ml / min), DCM (3 x 5 ml, 1 min each), diethyl ether (3 x 5 ml, 1 min each) and vacuum dried.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). The crude lyophilized product was analyzed by HPLC and found to be homologous. The purity was better than 80%, the identification of peptide was confirmed by ESMS, and the yield was 63%.

Prodrug H-Tyr-Gly-Gly-Phy-Leu-((R)-(-)-Ma)-(LysGlu) ₃ -OH was prepared as described above for 5.

6. Synthesis of Fmoc-Phe-Leu-HAA-Glu ₆ -OH on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis on PEG-PS" until the termination of presequence Glu ₆ . . The peptide-resin was then reacted with 6 equivalents of hydroxyacetic acid as pre-activated HObt-ester as described above and the coupling continued for 24 hours. Excess reagent was removed by DMF washing (12 min, flow rate 1 ml / min). Acylation was checked by ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 2 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed and the coupling yield was checked and evaluated as described above and found to be -100%. The next amino acid according to sequence was coupled as preformed and Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH (80 mg / 25 ml) solution before the last two couplings to allow the yellow color to disappear as the coupling reaction proceeds. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity was better than 90% and peptide identification was confirmed by ES-MS. Yield 83%.

7. Peptide synthesis of Fmoc-Phe-Leu-((S)-(+)-Ma) -Glu ₆ -OH on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis on PEG-PS" until the termination of presequence Glu ₆ . . The peptide-resin was then reacted with 6 equivalents of (S)-(+)-mandelic acid as pre-activated HObt-ester as described above and the coupling continued for 24 hours. Excess reagent was removed by DMF wash (12 min, flow rate 1 ml / min). Acylation was checked by ninhydrin test. The next amino acid (leucine) according to the sequence was coupled as a preformed symmetric anhydride as described above and the reaction continued for 2 hours. Excess reagent was then removed by DMF wash (12 min, flow rate 1 ml / min). A small amount of resin sample was removed to check the coupling yield and evaluated as described above and found to be 90%. The next amino acid according to sequence was coupled as preformed and Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before the last two couplings to allow the yellow color to disappear as the coupling reaction proceeds. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin as described above and lyophilized from ammonium bicarbonate (0.1M). The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity appeared to be better than 90% and the identification of peptides was confirmed by ES-MS. Yield 71%.

8. Peptide synthesis of H-Tyr-Gly-Gly-Phe-Leu-Lys _6- OH on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis on PEG-PS" until the termination of Presequence Lys ₆ . The next amino acid forming the Leu-enkephalin sequence was coupled as preformed, Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before each last five couplings so that the yellow color disappeared as the coupling reaction proceeded. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin and lyophilized from acetic acid as described above. The crude lyophilized product was analyzed by HPLC and found homologous without deletion and Fmoc protected sequences. Purity appeared to be better than 98% and the identification of peptides was confirmed by ES-MS. Yield 84%.

9. Peptide synthesis of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-Glu ₆ -OH on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene vessel equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis on PEG-PS" until the termination of presequence Glu ₆ . . The next amino acid forming the DSIP sequence was coupled as a preformed, Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before each last 9 couplings so that the yellow color disappeared as the coupling reaction proceeded. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin and lyophilized from acetic acid as described above. The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity was better than 98% and the identification of peptides was confirmed by ES-MS. Yield 80%.

10. Peptide Synthesis of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (LysGlu) ₃ -OH on NovaSyn TentaGel

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a filtration polypropylene filter and described in "Batch Peptide Synthesis on PEG-PS" until the termination of Presequence (LysGlu) ₃ . Treated. The next amino acid sequence forming the DSIP surplus was coupled as a preformed and Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before each last 9 couplings so that the yellow color disappeared as the coupling reaction proceeded. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin and lyophilized from acetic acid as described above. The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity was better than 98% and peptide identification was confirmed by ES-MS. Yield 91%.

11. Peptide synthesis of H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH (DSIP) on NovaSyn TentaGel.

Dry NovaSyn TG resin (0.29 mmol / g, 250 mg) was placed in a polyethylene container equipped with a filtration polypropylene filter and treated as described in "Batch Peptide Synthesis on PEG-PS". The first amino acid was coupled as symmetric anhydride preformed as described above. Coupling yield of the first Fmoc protected amino acid was evaluated as described above. This yield was better than 60% in all cases. The next amino acid forming the DSIP sequence was coupled as a preformed, Fmoc protected HObt ester (3 equiv) in DMF (5 ml) produced by DIC. The resin was washed with Dhbt-OH solution (80 mg / 25 ml) before each last eight couplings to allow the yellow color to disappear as the coupling reaction proceeded. The coupling was stopped by washing the resin with DMF (5 × 5 ml, 5 minutes each) when the yellow was no longer visible. The acylation was then checked by the ninhydrin test run at 80 ° C. as described above. After completion of the synthesis, 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 vacuo.

Peptides were cleaved from the resin and lyophilized from acetic acid as described above. The crude lyophilized product was analyzed by HPLC and found homologous without deletions and Fmoc protected sequences. Purity was better than 98% and the identification of peptides was confirmed by ES-MS. Yield 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% sodium carbonate w / v and 25 ml of dioxane was added. To this mixture was added dropwise 1.24 g (3.68 mmol) of FmocNHS dissolved in 10 ml of dioxane. The resulting mixture was stirred at rt overnight. Dioxane was evaporated off in vacuo and the resulting aqueous basic solution was extracted three times with ether (5 ml each). HCl (1M) was added to adjust the pH to 2 and the aqueous phase was extracted three times (20 ml each) with ethyl acetate. The combined ethyl acetate phases were washed three times with water (10 ml each) and evaporated to dryness. Petroleum ether was added to crystallize the residue. Yield 1.45 g (86%). Mp. 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 HObt (3.38 mmol) and 520 μl of DIC (3.38 mmol) were dissolved in 10 ml of THF and preactivated for 10 minutes and in 10 ml of THF. 1.0 g (3.38 mmol) of H-Glu (OtBu) -OtBu, HCl solution was added. The mixture was stirred overnight at room temperature. The solvent was evaporated in vacuo and the residue was dissolved in DCM (10 ml), 3 times with 10% acetic acid v / v (10 ml each), 3 times with 10% sodium bicarbonate water w / v (10 ml each) and finally Extracted twice with water (10 ml each). The organic phase was dried over sodium sulphate, filtered and evaporated. The remaining colorless oil was used directly without further purification.

14. Synthesis of Z-Glu-Glu-OH:

The oil from 13 was dissolved in 20 ml of 50% TFA-DCM v / v and stirred at room temperature for 2 hours. The solution was evaporated to dryness and the residue crystallized from petroleum ether. Yield 1.2 g (86.5%). Mp. 174-176 ° C .; MS: calcd 410.4, found MH + 411.0.

Hydrolysis in Enzyme Solution Test

The degradation of the peptide prodrug (XLZ) and the corresponding peptide (X-OH) was studied in 0.05M phosphate buffer at 37 ° C. The buffer solution contains leucine aminopeptidase (25 u / ml) at pH 7.4, or Carboxypeptidase A (25 u / ml) at pH 7.4. Decomposition is initiated by adding the aliquots (~ 10 ^-7 -10 ^-8 mol) from the peptide or peptide prodrug stock solutions to the test solution so that the total volume of the reaction mixture is -5 ml, respectively, and maintained in a 37 ° C water bath. 50 μl of the sample is collected at appropriate time intervals and analyzed by reverse phase HPLC as described above without pre-precipitation of the protein. Pseudo-first-order rate constants for degradation of peptide prodrugs (XLZ) and corresponding peptides (X-OH) are given by the formula t _1/2 = (ln2) / (k _obs ) concentration of residual derivatives over time (HPLC). Determine from the slope of the logarithmic straight line (ie k _obs ) with respect to the peak height). The ratio of the half-life of the prodrug and the corresponding peptide is calculated according to the formula: ratio = (t _1/2 (prodrug)) / (t _1/2 (X-OH)).

Kinetic measurement

Hydrolysis in Buffer

The degradation of a portion of the peptide prodrug was studied with an aqueous solution of phosphoric acid or carbonate buffer with a total buffer concentration of 0.1-0.05M. To maintain a constant ionic strength of 0.5, a calculated amount of potassium chloride was added to the buffer unless otherwise stated. During the decomposition study, the temperature was maintained at 37 ° C. and pH was adjusted by addition of hydrochloric acid (4M) or sodium hydroxide (2M). Hydrolysis experiments were performed at pH 2, 7.4 and 11.

The rate of degradation was determined using reverse phase HPLC. The mobile phase system used for isocratic separation was 20% acetonitrile, 79.9% water, 0.1% trifluoroacetic acid or 10% acetonitrile, 89.9% water, 0.1% trifluoroacetic acid. Buffer A was 0.1% TFA v / v in water and buffer B was 90% acetonitrile, 9.9% water, 0.1% TFA v / v when using a linear gradient (40-100% B, 30 minutes).

Hydrolysis in Enzyme Solution

Degradation of peptides and peptide prodrugs was performed at 37 ° C. with 0.05M phosphate buffer solution containing leucine aminopeptidase (25 u / ml), pH 7.4, Carboxypeptidase A (25 u / ml), pH 7.4. Study in α-chymotrypsin (25 u / ml), pepsin A at pH 2.0 (25 u / ml), or butyryl cholinesterase at pH 7.4 (two concentrations: 25 and 50 u / ml). Decomposition was initiated by adding an aliquot (˜10 ⁻⁷ −10 ⁻⁸ mol) from the stock solution of the peptide or peptide prodrug to the test solution so that the total volume of the reaction mixture was ˜50 ml and maintained in a 37 ° C. water bath with appropriate intervals. Samples of ˜50 μl were collected and analyzed by reverse phase HPLC as described above without pre-precipitation of protein. The pseudo-first-order rate constant for decomposition is the slope of the logarithmic straight line (ie k _obs ) with respect to the concentration of residual derivative (HPLC peak height) over time using the equation t _1/2 = (ln2) / (k _obs ). Determined from Each analysis condition is shown in the following Examples.

Hydrolysis in Plasma Solution

Degradation of peptides and peptide prodrugs was studied in 80% human plasma at 37 ° C. Aliquots (~ 10 ^-7 -10 ^-8 mol) from the stock solutions of the peptide were added to the test solution so that the total volume of the reaction mixture was -5 ml, initiation of decomposition and maintained in a 37 ° C. water bath, with appropriate intervals of -50 μl. Samples were recovered and the samples were treated with 50 μl of a 2% (w / v) zinc sulfate solution in methanol-water (1: 1 v / v) to deproteinize and stop the reaction. Immediately centrifuged at 13000 rpm for 3 minutes, 20 μl of clear supernatant was analyzed by reversed phase HPLC as described above. The pseudo-first-order rate constant for decomposition is the slope of the logarithmic straight line (ie k _obs ) with respect to the concentration of residual derivative (HPLC peak height) over time using the equation t _1/2 = (ln2) / (k _obs ). Determined from Each analysis condition is shown in the following examples.

H-Tyr-Gly-Gly-Phe-Leu- (Glu) ₆ -OH:

Hydrolysis in Buffer

The degradation of H-Tyr-Gly-Gly-Phe-Leu- (Glu) ₆ -OH (˜5 × 10 ⁻⁶ M) was studied in different aqueous buffers (0.1M) as described above. Degradation occurred at pH = 2, pH = 7.4 and pH = 11. The peptide appeared to be stable at this pH value, so only ˜5% of the peptide was degraded over 24 hours.

Hydrolysis in Leucine Aminopeptidase

Of H-Tyr-Gly-Gly-Phe-Leu- (Glu) ₆ -OH (˜10 ^-5 M) in 0.05 M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) The degradation was studied as described above. The pseudo first order rate constant was determined as described above and found to be 5.2 × 10 ⁻³ min ⁻¹ . The half life was calculated to be 133 minutes.

Hydrolysis in Carboxypeptidase A

H-Tyr-Gly-Gly-Phe-Leu- (Glu) ₆ -OH (˜10 ^-5 M) in 0.05M phosphate buffer solution (pH = 7.4) containing Carboxypeptidase A (25 u / ml) The degradation was studied as described above. The peptide appeared to be stable. Approximately 15% of peptides degraded over 24 hours.

H-Tyr-Gly-Gly-Phe-Leu- (Lys) ₆ -OH

Hydrolysis in Leucine Aminopeptidase

H-Tyr-Gly-Gly-Phe-Leu- (Lys) ₆ -OH (˜10 ^-5 M) in 0.05 M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) The degradation was studied as described above. The pseudo-first-order rate constant was determined as described above and found 3.6 × 10 ⁻³ min ⁻¹ . The half life was calculated to be 191 minutes.

Hydrolysis in Pepsin A

Decomposition of H-Tyr-Gly-Gly-Phe-Leu- (Lys) _6- OH (˜10 ^-5 M) in 0.05M phosphate buffer solution (pH = 2.0) containing pepsin A (25 u / ml) The study was as follows. The pseudo-first order rate constant was determined as described above and found 1.2 × 10 ⁻³ min ⁻¹ . The half life was calculated to be 580 minutes.

Fmoc-Phe-Leu-((S)-(+)-mandelic acid)-(Glu) ₆ -OH:

Hydrolysis in Butyryl Cholinesterase

Hydrolysis of the Ester Bonds Produced by Mandelic Acid at Peptide Prodrug Fmoc-Phe-Leu-((S)-(+)-Mandelic Acid)-(Glu) ₆ -OH (˜10 ^-5 M) was described above. The study was carried out in 0.05M phosphate buffer solution (pH = 7.4) containing butyryl cholinesterase (50 u / ml). Half-life was evaluated as t _1/2 = 212 minutes as described in the general method. Based on the recovery of Fmoc-Phe-Leu-OH, the half-life was estimated at t _1/2 = 164 minutes. The difference in the values is probably due to the further degradation of Fmoc-Phe-Leu-OH in the urethane which is a bond in the Fmoc protecting group.

Fmoc-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH:

Hydrolysis in Butyryl Cholinesterase

Hydrolysis of the ester bonds produced by hydroxyacetic acid in the peptide prodrug Fmoc-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH (˜10 ^-5 M) was as described above with butyryl cholines. Studies were performed in 0.05 M phosphate buffer solution (pH = 7.4) containing terase (50 u / ml). Based on the recovery of Fmoc-Phe-Leu-OH, the half-life was estimated to t _1/2 = 144 minutes.

Fmoc-Phe-Leu-OH:

Partial in butyryl cholinesterase.

The degradation of Fmoc-Phe-Leu-OH (˜5 × 10 ⁻⁵ M) in 0.05 M phosphate buffer solution (pH = 7.4) containing butyryl cholinesterase (50 u / ml) was studied as described above. . The half life was estimated at t _1/2 = 25 hours.

H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-mandelic acid)-(Glu) ₆ -OH:

Hydrolysis in Buffer

Decomposition of H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-mandelic acid)-(Glu) ₆ -OH (1 × 10 ^-5 M) was carried out using a different aqueous buffer ( 0.1M). Degradation occurred at pH = 2, pH = 7.4 and pH = 11. The peptide appeared to be stable at this pH value, so less than 5% of the peptide was degraded over 24 hours.

Hydrolysis in Leucine Aminopeptidase

H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-mandelic acid)-() in 0.05M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) Glu) ₆ -OH (1 x 10 ^-5 M) was studied as described above. The pseudo first order rate constant was determined as described above and the half life was calculated to be 93 minutes.

Hydrolysis in Carboxypeptidase A

H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-mandelic acid)-() in 0.05M phosphate buffer solution (pH = 7.4) containing carboxypeptidase A (25 u / ml) Glu) ₆ -OH (1 x 10 ^-5 M) was studied as described above. The pseudo-first order rate constant was determined as described above and the half life was calculated to be 10.3 minutes.

Hydrolysis in Butyryl Cholinesterase

Ester produced by mandelic acid at the peptide prodrug H-Tyr-Gly-Gly-Phe-Leu-((S)-(+)-mandelic acid)-(Glu) ₆ -OH (1.75 × 10 ^-5 M) The hydrolysis of the bonds was studied in 0.05M phosphate buffer solution (pH = 7.4) containing butyryl cholinesterase (50 u / ml) as described above. The half life was t _1/2 = 50.2 minutes.

H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH:

Hydrolysis in Buffer

The degradation of H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH (1 × 10 ^-5 M) was studied in different aqueous buffers (0.1 M) as described above. Degradation occurred at pH = 2, pH = 7.4 and pH = 11. The peptide was found to be stable at the pH values described above, so less than 5% of the peptide was degraded over 24 hours.

Hydrolysis in Leucine Aminopeptidase

H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH (1) in 0.05 M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) Decomposition of ^x10-5 M) was studied as described above. The pseudo first order rate constant was determined as described above and the half life was calculated to be 16.1 minutes.

Hydrolysis in Carboxypeptidase A

H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH (1) in 0.05M phosphate buffer solution (pH = 7.4) containing carboxypeptidase A (25 u / ml) Decomposition of ^x10-5 M) was studied as described above. The peptide appeared to be stable, so less than 13% of the peptide degraded over 24 hours.

Hydrolysis in Butyryl Cholinesterase

The hydrolysis of the ester bonds produced by hydroxyacetic acid in the peptide prodrug H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) ₆ -OH (1.75 × 10 ^-5 M) is described in detail. The study was conducted in 0.05M phosphate buffer solution (pH = 7.4) containing butyryl cholinesterase (25u / ml or 50u / ml) as described. The half-life was calculated to be t _1/2 = 119.3 minutes using butyryl cholinesterase 25u / ml and t _1/2 = 86.6 minutes using 50u / ml.

Hydrolysis in human plasma

Degradation of peptide prodrug H-Tyr-Gly-Gly-Phe-Leu- (hydroxyacetic acid)-(Glu) _6- OH was carried out in 4 ml of human plasma with 1 ml of peptide (7 × 10 ⁻⁵ M) at 37 ° C. Of 0.05M phosphate buffer solution (pH = 7.4) was added and studied as described above. The pseudo-first order rate constant was determined as described above and the half-life was calculated as t _1/2 = 19.2 minutes.

Z-Glu-Glu-OH:

Hydrolysis in Carboxypeptidase A

Degradation of Z-Glu-Glu-OH (1 × 10 ⁻⁵ M) in 0.05M phosphate buffer solution (pH = 7.4) containing Carboxypeptidase A (25 u / ml) was studied as described above. The peptide appeared to be stable over 24 hours.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) ₃ -OH

Hydrolysis in Carboxypeptidase A

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) _{3 in} 0.05M phosphate buffer solution (pH = 7.4) containing Carboxypeptidase A (25 u / ml) Decomposition of —OH (˜10 ⁻⁵ M) was studied as described above. The pseudo first order rate constant was determined as described above and the half life was calculated to be 396 minutes.

Hydrolysis in Leucine Aminopeptidase

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) _{3 in} 0.05M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) Decomposition of —OH (˜10 ⁻⁵ M) was studied as described above. The pseudo first order rate constant was determined as described above and the half life was calculated to be 145 minutes.

Hydrolysis in α-chymotrypsin

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) ₃ -in 0.05M phosphate buffer solution (pH = 7.4) containing α-chymotrypsin (25u / ml) Decomposition of OH (˜10 ⁻⁵ M) was studied as described above. The pseudo-first order rate constant was determined as described above and the half life was calculated to be 613 minutes.

Hydrolysis in Pepsin A

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Lys-Glu) ₃ -OH in 0.05M phosphate buffer solution (pH = 7.4) containing pepsin A (25 u / ml) The degradation of ˜10 ⁻⁵ M) was studied as described above. The peptide appeared to be stable.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) ₆ -OH

Hydrolysis in α-chymotrypsin

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu- (Glu) ₆ -OH in 0.05M phosphate buffer solution (pH = 7.4) containing α-chymotrypsin (25u / ml) The degradation of ˜10 ⁻⁵ M) was studied as described above. The pseudo first order rate constant was determined as described above and the half life was calculated to be 523 minutes.

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH

Hydrolysis in Leucine Aminopeptidase

H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-OH (˜10 ^-5 ) in 0.05M phosphate buffer solution (pH = 7.4) containing leucine aminopeptidase (25 u / ml) The degradation of M) was studied as described above. The half life was calculated to be less than 20 minutes.

In vitro studies

μ-opioid receptor activity-1

The affinity of the prodrugs of the invention for c-opioid receptors in calf brain is described by Kristensen et al. (1994) [K. Kristensen, CB Christensen, LL Christrup, and LC Nielsen (1994). The mu ₁ , mu ₂ , delta, kappa opioid receptor binding profiles of methadone stereoisomers and morphine. Life Sci. 56, PL45-PL50.]. The activity of prodrugs was determined in freshly prepared solutions and in solutions stored at room temperature for 20 hours. Experimental data is shown in Table 1.

Inhibition of 3H-DAMGO (2nM) compound IC ₅₀ ± SEM (nM) 2 hours 20 hours Leu-enkephalin-((R)-(-)-Ma)-(Lys) ₆ -OH 100 ± 11 200 ± 61 Leu-enkephalin-((S)-(+)-Ma)-(Lys) ₆ -OH 150 ± 73 110 ± 9 Leu-enkephalin-((R)-(-)-Ma)-(Glu) ₆ -OH 3500 ± 3200 220 ± 42 Leu-enkephalin-((S)-(+)-Ma)-(Glu) ₆ -OH 1200 ± 910 190 ± 55 Leu-enkephalin- (R (-)-Ma)-(LysGlu) ₃ -OH 1200 ± 920 170 ± 63 Leu-enkephalin- (S (+)-Ma)-(LysGlu) ₃ -OH 450 ± 210 230 ± 73 Leu-enkephalin-OH 97 ± 9 56 ± 14 Met-enkephalin-OH 30 30 Naloxone 9 ± 1 4 ± 2 DAMGO 7 ± 1 8 ± 3

μ-opioid receptor activity-2

The affinity of the prodrugs of the invention as μ-opioid receptor agonists is described by Kramer et al. (1997) [T.H. Kramer, H. Bartosz-Bechowski, P. Davis, V.J. Hruby, and F. Porreca (1997). Extraordinary potency of a novel delta opioid receptor agonist is due in part to increased efficacy. Life Sci. 61: 2, 129-135.] Was used using the isolated mouse in vitro model in vitro. The activity of prodrugs was determined in freshly prepared solutions and in solutions stored for 48 hours at room temperature. Experimental data are shown in Tables 2 and 3.

Fertility tube Peptides / Prodrugs Newly created solution 48 hour storage solution Leu-enkephalin-((R)-(-)-Ma)-(Lys) ₆ -OH a aa Leu-enkephalin-((S)-(+)-Ma)-(Lys) ₆ -OH aa aa Leu-enkephalin-((R)-(-)-Ma)-(Glu) ₆ -OH aaa aaa Leu-enkephalin-((S)-(+)-Ma)-(Glu) ₆ -OH aaa aaa Leu-enkephalin-((R)-(-)-Ma)-(LysGlu) ₃ -OH aa aa Leu-enkephalin-((S)-(+)-Ma)-(LysGlu) ₃ -OH aa aa Leu-enkephalin-OH aaa aaa % Reduction (100 nM): a: <25%; aa: <50%; aaa: <75%

Fertility tube Peptides / Prodrugs IC ₅₀ ± SEM (nM) Leu-enkephalin-((R)-(-)-Ma)-(Lys) ₆ -OH 590 ± 200 Leu-enkephalin-((S)-(+)-Ma)-(Lys) ₆ -OH 240 ± 130 Leu-enkephalin-((R)-(-)-Ma)-(Glu) ₆ -OH 110 ± 23 Leu-enkephalin-((S)-(+)-Ma)-(Glu) ₆ -OH 58 ± 11 Leu-enkephalin-((R)-(-)-Ma)-(LysGlu) ₃ -OH 140 ± 11 Leu-enkephalin-((S)-(+)-Ma)-(LysGlu) ₃ -OH 200 ± 26 Leu-enkephalin-OH 41 ± 13

In vivo research

Painless active

Attempts to determine the in vivo activity of mice are described in Swedberg (1994) [M.D. Swedberg (1994). The mouse grid-shock analgesia test: pharmacological characterization of latency to vocalization threshold as an index of antinociception, J. Pharmacol. Exp. Ther. 269: 3, 1021-1028.] Using the grid-shock model described. The experimental results are shown in Table 4.

Mouse painless active Prodrug Intraperitoneal Intravenous Leu-enkephalin-((R)-(-)-Ma)-(Lys) ₆ -OH NA NT Leu-enkephalin-((S)-(+)-Ma)-(Lys) ₆ -OH WA WA Leu-enkephalin-((R)-(-)-Ma)-(Glu) ₆ -OH NT NT Leu-enkephalin-((S)-(+)-Ma)-(Glu) ₆ -OH NT NT Leu-enkephalin-((R)-(-)-Ma)-(LysGlu) ₃ -OH NA NT Leu-enkephalin-((S)-(+)-Ma)-(LysGlu) ₃ -OH NA NT NT: Not tested. NA: Inactive. WA: weakly active (at 20 mg / kg).

conclusion

Natural enkephalins degrade into half-life of 6.0 minutes in 80% human plasma, half-life of 10.0 minutes in aminopeptidase (20 u / ml), and half-life of 2.0 minutes in carboxypeptidase (1 u / ml) (GJ Rasmussen and H. Bundgaard, Int. J. Pharm., 79, pp 113-122 (1991)), and concluded that the present invention provides significant protection of peptide sequences compared to native peptide sequences. This is further confirmed by the results obtained for DSIP, that is, natural DSIP degrades with less than 20 minutes half-life in leucine aminopeptidase (25 u / ml), while DISP- (LysGlu) ₃ -OH is 145 minutes under the same conditions. Decompose into half-life of. In general, the half-life of the presequence containing DISP molecules in a solution containing α-chymotrypsin or carboxypeptidase A (25 u / ml) was about several hours. Although natural DSIP has not been tested under these conditions, the corresponding half-life is expected to be significantly lower than that obtained for presequence-containing DSIP molecules, as it has been confirmed that natural DSIP degrades rapidly in both plasma and tissue extracts (HL Lee, "Peptide and Protein Drug Delivery", Marcel Dekker Inc. 1991, Chapter 9). Moreover, all tested peptide prodrugs were immediately cleaved by butyryl cholinesterase, indicating bioreversibility.

From the in vitro tests performed, it can be concluded that the presequence significantly affects bioactivity. The results shown in Tables 1, 2 and 3 clearly show that the prodrugs of the invention have a reduced affinity for the μ-opoid receptor as compared to native Leu-enkephalin. Thus, when bound to the μ-opoid receptor to exert the desired activity, the prodrug will be hydrolyzed by a plasma enzyme such as, for example, butyryl cholinesterase to release the natural pharmaceutically active peptide. This must be this.

The difference between the results obtained from the freshly prepared solution and the solution stored at room temperature for 20 or 48 hours may be due to two different factors. That is, the solution containing the prodrug of the present invention can be somewhat hydrolyzed by releasing natural Leu-enkephalin when stored for 20 or 48 hours. However, as shown by the kinetic measurements, the prodrugs of the present invention are stable over the entire pH range. Most sustained significant effects are observed with the application of preliminary sequence (Glu) ₆ . Thus, a more plausible explanation is the rather low water solubility of (Glu) ₆ -containing prodrugs. Ie due to the slow solution kinetics it is believed that only fractions of the prodrug are dissolved in the freshly prepared solution. However, when left in solution for 20 or 48 hours, the compound will dissolve slowly, thereby increasing the effective amount of active material in the analyte.

From in vivo analgesia studies, it can be concluded that linkers as well as pre-sequences are important. Clearly, positively charged presequences (Lys) ₆ in combination with (S) enantiomers of maleic acid have the desired effect, while enkephalins containing presequences (Lys) ₆ in combination with (R) enantiomers of maleic acid No activity was shown. Moreover, enkephalin prodrugs with the electric neutral presequence (LysGlu) ₃ in combination with (R) or (S) mandelic acid did not show the desired effect. In conclusion, the combination of linker and presequences is important in terms of the ability of the prodrugs of the invention to cross biological barriers, such as, for example, blood-brain-barriers, and the present invention can be achieved by selecting the appropriate combination of linkers and presequences. It includes the possibility of transferring the prodrug to the desired area.

Claims (30)

Formula I Formula I X-L-Z Wherein X is bonded to L in the C-terminal carbonyl function of X; L is a linking group containing 3 to 9 backbone atoms, and the bond between C-terminal carbonyl and L of X is different from a C-N amide bond; And Z is a peptide sequence of 2-20 amino acid units and is bound to L located at the N-terminal nitrogen atom of Z, each amino acid unit being Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys , Arg, His, Met, Orn, and amino acid units of formula II Formula II -NH-C (R ³ ) (R ⁴ ) -C (= O)- Wherein R ³ and R ⁴ are selected from C _1-6 -alkyl, phenyl, and phenyl-methyl, wherein C _1-6 -alkyl is halogen, hydroxy, amino, cyano, nitro, sulfono, And one to three substituents selected from carboxy, wherein phenyl and phenyl-methyl are C _1-6 -alkyl, C _2-6 -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono And optionally substituted with one to three substituents selected from carboxy, or R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring); A prodrug or salt thereof of a pharmaceutically active peptide (X-OH), peptide amide (X-NH ₂ ), or peptide ester (X-OR). The prodrug of claim 1, wherein the amino acid unit of Z is selected from three or two different amino acids or is the same amino acid. Prodrug according to claim 1 or 2, characterized in that the amino acid units of Z are independently selected from Glu, Met, and Lys, in particular Glu and Lys. The method according to any one of claims 1 to 3, wherein the pharmaceutically active peptide (X-OH), peptide amide (X-NH ₂ ), or peptide ester (XR) consists of 2 to 200 amino acid units. Pro-drugs. The prodrug according to any one of claims 1 to 4, wherein Z is composed of 3 to 15 amino acid units. The prodrug according to any one of claims 1 to 5, wherein the bond between the C-terminal carbonyl function of X and L can be cleaved by plasma enzyme. The prodrug according to any one of claims 1 to 6, wherein the bond between the C-terminal carbonyl function of X and L is a thiol ester bond or an ester bond. 8. The bond of claim 1 wherein the bond between the N-terminal nitrogen atoms at L and Z is a carboxyimide bond (—C (═O) —N—), a sulfonamide bond (—SO ₂ −). N-), alkylamine bond (-CN-), carbamate bond (-OC (= O) -N-), thiocarbamate bond (-SC (= O) -N-), urea bond (- NC (= O) -N-), thioamide bond (-C (= S) -N-), cyanomethyleneamino bond (-C (CN) -N-), or N-methylamide bond (-C And (= O) -N (CH ₃ )-). The prodrug according to any one of claims 1 to 8, wherein L is derived from hydroxy-carboxylic acid. 10. The prodrug according to any one of claims 1 to 9, wherein L is derived from α-hydroxy carboxylic acid. The compound of claim 10, wherein 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 H, C _1-6 -Alkyl, C _2-6 -alkenyl, aryl, aryl-C _1-4 -alkyl, heteroaryl or heteroaryl-C _1-4 -alkyl, or R ¹ and R ² together with the carbon atom to which they are attached Forming a cyclopentyl, cyclohexyl, or cycloheptyl ring, wherein the alkyl or alkenyl group is amino, cyano, halogen, isocyano, isothiocyano, thiocyano, sulfamoyl, C _1-4 -alkyl Thio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C _1-4 -alkoxycarbonyl, C _1-4- Alkylcarbonyloxy, aminocarbonyl, mono- or di-C _1-4 -alkyl-aminocarbonyl, mono- or di-C _1-4 -alkyl-amino, mono- or di-C _1-4 -alkyl -amino -C _1-4 - alkyl, C _1-4 - alkyl car Amino, phono alcohol, and which is substituted by one to three substituents selected from alcohol Pino, and here, an aryl or heteroaryl group is C _1-4 - alkyl, C _2-4 - alkenyl, nitro, amino, cyano, , Halogen, isocyano, isothiocyano, thiocyano, sulfamoyl, C _1-4 -alkylthio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy , aryloxy, carboxy, C _1-4 - alkoxycarbonyl, C _1-4 - alkyl carbonyloxy, aminocarbonyl, mono- or di -C _1-4 - alkyl-amino-carbonyl, mono- or di One selected from -C _1-4 -alkylamino, mono- or di-C _1-4 -alkyl-amino-C _1-4 -alkyl, C _1-4 -alkylcarbonylamino, sulfono, and sulfino A prodrug characterized by being substituted with three substituents. The compound according to any one of claims 1 to 11, wherein L is hydroxyacetic acid, (S)-(+)-mandelic acid, L-lactic acid ((S)-(+)-2-hydroxypropanoic acid ), L-α-hydroxy-butyric acid ((S) -2-hydroxybutanoic acid), and a prodrug derived from α-hydroxy-isobutyric acid. 12. The compound of claim 11, wherein L is derived from α-hydroxy carboxylic acid of the general formula HO-C (CH ₂ -R ⁵ ) (R ² ) -COOH, wherein R ⁵ is H, C _1-5 -alkyl , C _2-5 -alkenyl, aryl, aryl-C _1-3 -alkyl, heteroaryl, heteroaryl-C _1-3 -alkyl, wherein the alkyl or alkenyl group is amino, halogen, mono- or di -C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C _1-4 -alkoxycarbonyl, C _1-4 -alkylcarbonyloxy, and Substituted with one to three substituents selected from aminocarbonyl, wherein aryl or heteroaryl is C _1-4 -alkyl, C _2-4 -alkenyl, nitro, amino, halogen, mono- or di-C _1- Substituted with one to three substituents selected from ₄ -alkyl-amino, hydroxy, C _1-4 -alkoxy, carboxy, C _1-4 -alkylcarbonyl, C _1-4 -alkylcarbonyloxy, and aminocarbonyl Become; And R ² is as defined in claim 11. The method of claim 1, wherein X is enkephalin, angiotensin II, vasopressin, endothelin, neuropeptide Y, angiogenic intestinal peptide, substance P, neurotensin, endorphin, insulin, gramicidine, para Celsine, Delta-Sleep Inducing Peptide, ANF, Vasothocin, Bradykinin, Dinorphine, Growth Hormone Release Factor, Growth Hormone Release Peptide, Oxytocin, Calcitonin, Calcitonin Gene Related Peptide, Calcitonin Gene Related Peptide II, Growth Hormone Release Peptide, Takkinin, ACTH, Cerebral Natriuretic Polypeptide, Cholecystokinin, Corticotropin Release Factor, Diazepam Binding Fragment, FMRF-amide, Galinine, Gastric Release Peptide, Gastrin, Gastrin Release Peptide, Glucagon, Glucagon-Like Peptide- 1, Glucagon-Like Peptide-2, LHRH, Melanin-Enriched Hormone, Alpha-MSH, Morphine Regulatory Peptides, Motilin, Neurokinin, Neuromedin, Neuropeptide A prodrug characterized in that it is a peptide sequence of de K, neuropeptide Y, PACAP, pancreatic polypeptide, peptide YY, PHM, secretin, somatostatin, substance P, TRH, angiogenic intestinal polypeptide, or any modified or truncated analog thereof. . 15. The method according to any one of claims 1 to 14, wherein when using enzyme carboxypeptidase A, as defined herein, the half-life of the prodrug in the "hydrolysis in the enzyme solution test" and the hydrolysis in the "enzyme solution test" And a ratio between the half-lives of the peptide (X-OH) in the degradation. 16. The prodrug of claim 15, wherein the ratio is at least 5. 17. The prodrug of claim 16, wherein the ratio is at least 10. 18. The method according to any one of claims 1 to 17, when using the enzyme leucine aminopeptidase, as defined herein, the half-life of the prodrug in the "hydrolysis in the enzyme solution test" and the hydrolysis in the "enzyme solution test". Prodrug, wherein the ratio between the half-lives of the peptide of interest is at least two. 19. The prodrug of claim 18, wherein the ratio is at least 5. 20. The prodrug of claim 19, wherein the ratio is at least 10. A pharmaceutical composition comprising the prodrug as defined in any one of claims 1 to 20 and a pharmaceutically acceptable carrier. A prodrug as defined in any one of claims 1 to 20 for use in therapy. Use of a prodrug as defined in any one of claims 1 to 20 for the preparation of a pharmaceutical composition for use in therapy. Immobilized Linker-Peptide Sequence Prot-L-Z-SSM. Where L refers to a linker of the formula -OC (R ¹ ) (R ² ) -C (= 0)- Wherein R ¹ and R ² are independently from H, C _1-6 -alkyl, C _2-6 -alkenyl, aryl, aryl-C _1-4 -alkyl, heteroaryl, heteroaryl-C _1-4 -alkyl Or selected from R ¹ and R ² together with the carbon atoms to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring, wherein the alkyl or alkenyl group is amino, cyano, halogen, isocyano, isothio Cyano, thiocyano, sulfamyl, C _1-4 -alkylthio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryl, heteroaryl, aryloxy, carboxy, C _1-4 - alkoxycarbonyl, C _1-4 - alkyl carbonyloxy, aminocarbonyl, mono- or di -C _1-4 - alkyl-aminocarbonyl, mono- or di -C _1-4 From one to three substituents selected from -alkyl-amino, mono- or di-C _1-4 -alkyl-amino-C _1-4 -alkyl, C _1-4 -alkylcarbonylamino, sulfono, and sulfino substitution Wherein the aryl or heteroaryl group is C _1-4 -alkyl, C _2-4 -alkenyl, nitro, amino, cyano, halogen, isocyano, isothiocyano, thiocano, sulfamoyl, C _1-4 -alkylthio, mono- or di-C _1-4 -alkyl-amino, hydroxy, C _1-4 -alkoxy, aryloxy, carboxy, C _1-4 -alkoxycarbonyl, C _1-4 -Alkylcarbonyloxy, aminocarbonyl, mono- or di-C _1-4 -alkyl-aminocarbonyl, mono- or di-C _1-4 -alkyl-amino, mono- or di-C _1-4- May be substituted with one to three substituents selected from alkyl-amino-C _1-4 -alkyl, C _1-4 -alkylcarbonylamino, sulfono, and sulfino; Z refers to a peptide sequence consisting of 2 to 20 amino acid units, wherein each amino acid unit is Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and II, amino acid unit Formula II -NH-C (R ³ ) (R ⁴ ) -C (= O)- Wherein R ₃ and R ₄ are selected from C _1-6 -alkyl, phenyl, and phenyl-methyl, wherein C _1-6 -alkyl is halogen, hydroxy, amino, cyano, nitro, sulfono, And one to three substituents selected from carboxy, wherein phenyl and phenyl-methyl are C _1-6 -alkyl, C _2-6 -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono And optionally substituted with one to three substituents selected from carboxy, or R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring); ; SSM refers to a solid support material; Prot refers to H or hydroxy protecting group. 25. The solid support material (SSM) of claim 24, wherein the solid support material (SSM) is selected from polystyrene, polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose, polyethylene, polyethylene glycol grafted to polystyrene, latex, and dinavid. Immobilized linker-peptide sequence Prot-LZ-SSM. Use of the immobilized linker-peptide sequence Prot-L-Z-SSM for the preparation of prodrugs of peptides, peptide amides, or peptide esters. Peptides (X-OH), peptide amides (X-NH ₂ ), or peptide esters (X) comprising binding the corresponding peptides of the C-terminal activated form (X-Act) to the immobilized linker-peptide sequence HLZ-SSM -OR) method for producing a prodrug. a) coupling an immobilized linker peptide sequence HLZ-SSM with an N-a-protected amino acid in carboxy active form or an N-a-protected dipeptide in C-terminal activated form, thereby immobilizing an immobilized N-α- Forming a protected peptide fragment, b) removing the N-α-protecting group, thereby forming an immobilized peptide fragment having an unprotected N-terminal end, c) coupling an additional N-α-protecting amino acid of the carboxyl-activated form or an additional N-α-protecting dipeptide of the C-terminal active form to the unprotected N-terminal end of the immobilized peptide fragment, and Repeat the removal / coupling process in steps b) and c) until peptide sequence X is obtained, d) a peptide comprising the steps of cleaving the prodrug XLZ from the solid support material to cleave the prodrug XLZ from the solid support material to obtain free prodrug in the form of a C-terminal carboxylic acid, amide, or ester (X- OH), peptide amide (X-NH ₂ ), or peptide ester (X-OR). The method of claim 28, wherein the N-α-protecting group is selected from tert-butyloxycarbonyl and 9-fluorenylmethyloxycarbonyl. Compound of Formula (I) or salt thereof Formula I X-L-Z Wherein X is a peptide sequence that binds to L in the C-terminal carbonyl function of X; L is a thermal group containing 3 to 9 skeletal atoms, and the bond between C-terminal carbonyl and L of X is different from C-N amide bond; And Z is a peptide sequence of 2-20 amino acid units and is bound to L located at the N-terminal nitrogen atom of Z, each amino acid unit being Ala, Leu, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys , Arg, His, Met, Orn, and amino acid units of formula II Formula II -NH-C (R ³ ) (R ⁴ ) -C (= O)- Wherein R ³ and R ⁴ are selected from C _1-6 -alkyl, phenyl, and phenyl-methyl, wherein C _1-6 -alkyl is halogen, hydroxy, amino, cyano, nitro, sulfono, And one to three substituents selected from carboxy, wherein phenyl and phenyl-methyl are C _1-6 -alkyl, C _2-6 -alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono And optionally substituted with one to three substituents selected from carboxy, or R ³ and R ⁴ together with the carbon atom to which they are attached form a cyclopentyl, cyclohexyl, or cycloheptyl ring) .