RU2798085C9 - Prodrug containing a self-cleavable linker - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a prodrug, or a pharmaceutically acceptable salt thereof, containing a drug-linker conjugate D-L, wherein D is a nitrogen-containing polypeptide; L is a biologically inactive linker fragment L¹ represented by the structural formula (I) where the dotted line demonstrates the attachment to the nitrogen of the polypeptide through the formation of an amide bond, where R¹, R^la, R², R^2a, R³, R^3a, X, X¹, X², X³ have the meanings given in the claims, and where L¹ is substituted by 1–4 L²-Z groups, provided that the hydrogen marked with * in formula (I) cannot be substituted by a substituent; where L² represents a single chemical bond or spacer; and Z represents a carrier group such as a linear or branched poly(ethylene glycol) with a molecular weight of 2,000 to 150,000 Da.

EFFECT: obtaining a prodrug containing a self-cleavable linker.

12 cl, 3 dwg, 52 ex

Description

The present invention relates to a prodrug containing a drug-linker conjugate D-L, or a pharmaceutically acceptable salt thereof. The invention also relates to pharmaceutical compositions containing these prodrugs and their use as medicinal products.

To improve the physicochemical or pharmacokinetic properties of a drug in vivo, it can be conjugated to a carrier.

Typically, carriers in drug delivery systems are used either in non-covalent form, with the drug formulated physicochemically as a mixture of carrier and solvent, or by covalently attaching a carrier reagent to one of the functional groups of the drug.

However, the non-covalent method requires encapsulation of the highly potent drug to prevent its uncontrolled, sudden release. When limiting the diffusion of an unbound water-soluble drug molecule, strong van der Waals contacts, often mediated by hydrophobic portions of the molecule, are required. Many conformationally sensitive drugs, such as proteins or peptides, lose their functionality during encapsulation and/or subsequent storage of the encapsulated drug. In addition, such drugs containing amino groups easily enter into side reactions with degradation products of the carrier (see, for example, D.H. Lee et al., J. Contr. Rel, 2003, 92, 291-299). Moreover, the dependence of the mechanism of drug release due to biodegradation may lead to interindividual variability.

Alternatively, the drugs may be conjugated to the carrier via covalent bonds. This method is applicable to various classes of molecules from so-called small molecules, including natural products, up to larger protein molecules. Covalently linked drug-carrier conjugates can be divided into two groups. First, there are conjugates, where a covalent bond between the carrier and the drug typically exists during the drug's duration of action (a "permanent covalent bond"), that is, the drug derivative exhibits its pharmacological effects, which are known for the drug itself. Second, the covalent bond is largely pre-broken, releasing the drug itself, which can exert its known pharmacological effects. In the latter case, the covalent conjugate of the drug with the carrier is called a carrier-linked prodrug or carrier-prodrug.

To ensure cleavage of the covalent bond between the carrier and the drug, easy removal of the bond in vivo is necessary to release the drug (prodrug activation).

Activation of a prodrug may result from enzymatic or non-enzymatic cleavage of the bond between the carrier and the drug molecule, or a sequential combination of both, i.e. through an enzymatic reaction step followed by a non-enzymatic rearrangement.

Enzymatically induced activation of a prodrug is characterized by the fact that in an enzyme-free in vitro environment, such as an aqueous buffer solution, cleavage of, for example, an ester or amide can occur, but the corresponding rate of hydrolysis may be extremely low and therapeutically unsuitable. Esterases or amidases are usually present in the in vivo environment, and these esterases and amidases can lead to significant catalytic acceleration of hydrolysis kinetics, ranging from a twofold increase up to several orders of magnitude. Thus, covalent bond cleavage is controlled primarily by the enzymatic reaction.

The main disadvantage of primarily enzymatic digestion is interindividual variability. Enzyme levels can vary significantly between individuals, resulting in biological variability in prodrug activation by enzymatic hydrolysis. Enzyme levels may also vary depending on the site of administration. For example, subcutaneous injection is known to produce more predictable therapeutic effects in some areas of the body than others. To reduce this unpredictable effect, non-enzymatic degradation or intramolecular catalysis is of particular interest.

Thus, enzyme-independent autocatalytic cleavage of the bond between the support and the biologically active moiety is preferred. In most cases, this is achieved through an appropriately designed linker moiety between the carrier and the biologically active moiety, which is directly attached via a covalent bond to the functional group of the biologically active moiety.

Specific types of linkers are known in the art. Y. Sohma et al. describe in J. Med. Chem. 46 (2003), 4124-4135 ester prodrugs in which the carrier is water soluble and the biologically active moiety is derived from the HIV-1 protease inhibitor KNI-727. The linker moiety used is attached to the biologically active moiety via an ester group. The mechanism of this prodrug system is the activation of the cyclization reaction, resulting in the formation of a cyclic imide to break the ester bonds.

However, it has a disadvantage due to the instability of the ester functional group. Moreover, ester functional groups may provide less chemoselectively accessible groups for conjugating a carrier or linker to a drug.

A.J. Garman et al. (A J. Garman, S. B. Kalindjan, FEBS Lett. 1987, 223 (2), 361-365 1987) use PEG5000-maleic anhydride to reversibly modify the amino groups in tissue-type plasminogen activator and urokinase. Recovery of the functional enzyme from the PEG-uPA conjugate upon incubation in a buffer solution with pH 7.4 through cleavage of the maleamic acid bond proceeds according to first-order reaction kinetics with a half-life of 6.1 hours. The disadvantage of the maleamic acid bond is that the conjugate is not stable at lower pH values. This imposes a limitation on the applicability of the connection of maleamic acid with biologically active substances that are stable at alkaline (high) pH values, since to prevent premature cleavage of the prodrug, purification of the polymer conjugate with the biologically active agent must be carried out under alkaline conditions (high pH values).

WO 2004/108070 describes a prodrug system based on an N,N-bis-(2-hydroxyethyl)glycine (bicine) amide derivative linker. In this system, two PEG carrier molecules are linked to a bicine molecule linked to the amino group of the drug molecule. The first two steps of prodrug activation involve enzymatic cleavage of the first bonds connecting both PEG carrier molecules to the hydroxyl groups of the bicine activating group. This paper describes different linkages between PEG and bicine, resulting in different prodrug activation kinetics. At the second stage of activation of the prodrug, the second bond connecting the bicine-activating group with the amino group of the drug molecule is cleaved. The main disadvantage of this system is the attachment of the polymer to the bicine linker, resulting in a slow rate of hydrolysis of this second bicine amide bond (t _1/2 >3 h in phosphate buffer). As a consequence, the release of the bicine-modified prodrug intermediate may exhibit different pharmacokinetic, immunogenic, toxicological and pharmacodynamic properties compared to the parent native drug molecule.

Another bicine-based system is described in WO 2006/136586.

Thus, there is a need to create alternative carrier-linked prodrugs in which the linker provides autocatalytic cleavage to release the drug in an unmodified form that does not retain linker-derived residues.

It is therefore an object of the present invention to provide such drug-linker conjugates wherein the linker is covalently attached via a cleavable bond to the biologically active moiety (representing the drug once released) and wherein the linker is also covalently attached via a permanent bond directly to the carrier or via a spacer to form a prodrug bound to the carrier.

This problem is achieved by means of a prodrug or a pharmaceutically acceptable salt thereof containing a drug-linker conjugate D-L, where

- D is a nitrogen-containing biologically active fragment; A

- L is a biologically inactive linker fragment of L ¹ represented by formula (I)

where the dotted line shows the attachment of a biologically active fragment to nitrogen through the formation of an amide bond;

X represents C(R ⁴ R ^4a ), N(R ⁴ ), O, C(R ⁴ R ^4a )-C(R ⁵ R ^5a ), C(R ⁵ R ^5a )-C(R ⁴ R ^4a ) , C(R ⁴ R ^4a )-N(R ⁶ ), N(R ⁶ )-C(R ⁴ R ^4a ), C(R ⁴ R ^4a )-O or OC(R ⁴ R ^4a );

X ¹ represents C or S(O);

X ² represents C(R ⁷ , R ^7a ) or C(R ⁷ , R ^7a )-C(R ⁸ , R ^8a );

X ³ represents O, S or N-CN;

R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ⁷ , R ^7a , R ⁸ , R ^8a are independently selected from the group consisting of H and C _1-4 alkyl;

if necessary, one or more pairs of R ^1a /R ^4a , R ^1a /R ^5a , R ^4a /R ^5a , R ^7a /R ^8a form a chemical bond;

if necessary, one or more pairs R ¹ /R ^1a , R ² /R ^2a , R ⁴ /R ^4a , R ⁵ /R ^5a , R ⁷ /R ^7a , R ⁸ /R ^8a together with the atom to which they are attached, combine to form a C _3-7 cycloalkyl or 4-7 membered heterocyclyl;

if necessary, R ⁴ /R ⁶ together with the atoms to which they are attached are combined to form a 4-7-membered heterocyclyl;

if necessary, one or more pairs R ¹ /R ⁴ , R ¹ /R ⁵ , R ¹ /R ⁶ , R ⁴ /R ⁵ , R ⁴ /R ⁶ , R ⁷ /R ⁸ , R ² /R ³ together with atoms , to which they are attached, combine to form cycle A;

if necessary, R ³ /R ^3a , together with the nitrogen atom to which they are attached, are combined to form a 4-7-membered heterocycle;

A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 4-7 membered heterocyclyl and 9-11 membered heterobicyclyl; And

where L ¹ is substituted with 1-4 L ² -Z groups and optionally further substituted, provided that the hydrogen indicated by * in formula (I) is not replaced by a substituent, where

L ² represents a single chemical bond or spacer; And

Z represents a carrier group.

Surprisingly, it has been found that the application of cyclization activation via cyclic imide formation can be extended from ester derivatives to prodrugs linked through an amide bond to a carrier, despite the significantly greater stability of the amide bond under aqueous conditions. It has been noted that N,N'-biscarboxamides linked via one amide bond to a nucleophilic backbone and a drug molecule via a second amide bond exhibit autohydrolysis properties in the range that can be used in prodrug applications. In addition, it has also been discovered that linkers can be designed to include a carrier permanently attached to the N,N'-biscarboxamide motif such that the formation of a cyclic imide can be used as a self-activation basis in the construction of prodrugs linked to the carrier via amide bond.

Examples of such preferred cyclic cleavable bond products are structures with a substituted succinimide or glutarimide ring. A precondition for such activation of the cyclization reaction is the presence of an amine-containing nucleophile in the linker structure and another amide bond, which is not the amide bond in the prodrug, but is an amide bond substituted by a hydrogen atom.

In the event of cleavage of a succinimide- or glutarimide-activated prodrug, the amine-containing nucleophile acts as a neighboring group to enhance the nucleophilicity of the nitrogen contained in the permanent amide bond, which in turn attacks the amide carbonyl group of the prodrug and thus induces intramolecular acylation of the permanent amide bond, leading to to the formation of a cyclic imide ring.

Therefore, preferred linker structures include a permanent bond to a support, an amine-containing nucleophile, and a permanent amide bond with a hydrogen attached to the nitrogen of the amide bond. Prodrugs bound to an appropriate carrier contain a linker containing a bond constant to the carrier, an amine-containing nucleophile and said constant amide bond, and a nitrogen-containing biologically active molecular fragment formed from the drug conjugated to the linker via a cleavable amide bond.

In FIG. Figure 1 shows an example of cleavage leading to the formation of a cyclic imide. The nitrogen of the biologically active moiety is presented as a hydrogen-containing amine, which results in a drug having a primary amine functional group. In this case, also, for example, a secondary amine can be part of the medicinal product. For reasons of simplicity, one to four required substituents of the L ₂ -Z structure, including the support, are not shown.

The preferred properties of the prodrug of the present invention are given by its half-life when hydrolyzed in an aqueous buffer solution at pH 7.4 and 37°C in the range from 1 hour to 3 months; similar rates of hydrolysis under physiological conditions in buffer and blood plasma.

The prodrug of the present invention can exhibit excellent in vivo and in vitro correlation of linker cleavage data, a high degree of enzyme independence, and can be stored at lower pH values (pH dependent cleavage).

The terms used within the scope of the present invention have the following meanings.

“Biologically active moiety D” means the portion of a drug-linker conjugate that, upon cleavage, results in a drug compound D-H of known biological activity.

"Inactive linker" means a linker that does not exhibit the pharmacological effect of a drug derived from a biologically active agent.

"Alkyl" means a straight or branched carbon chain. Each hydrogen carbon atom in an alkyl group can be replaced by a substituent.

"C _1-4 alkyl" means an alkyl chain containing from 1 to 4 carbon atoms, for example, when present at the end of the molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert -butyl, or, for example, -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH(C ₂ H ₅ )-, - C(CH ₃ ) ₂ - if two fragments of the molecule are connected by an alkyl group. Each hydrogen atom at the carbon atom in C ₁ - ₄ alkyl can be replaced by a substituent.

"C _1-6 alkyl" means an alkyl chain containing from 1 to _{6 carbon atoms, for example, if present at the end of the molecule: C 1-4 alkyl} , methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl , sec-butyl, tert-butyl, n-pentyl, n-hexyl or, for example, the group -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH(C ₂ H ₅ )-, -C(CH ₃ ) ₂ -, if two fragments of the molecule are connected by an alkyl group. Each hydrogen atom at the carbon atom in C _1-6 alkyl can be replaced by a substituent.

Accordingly, "C _1-18 alkyl" means an alkyl chain containing from 1 to 18 carbon atoms, and "C _8-18 alkyl" means an alkyl chain containing from 8 to 18 carbon atoms. Accordingly, "C _1-50 alkyl" means an alkyl chain containing from 1 to 50 carbon atoms.

"C _2-50 alkenyl" means a branched or straight alkenyl chain containing from 2 to 50 carbon atoms, for example, when present at the end of the molecule: -CH= _CH2 , -CH=CH- _CH3 , _-CH2 -CH= CH ₂ , -CH=CH-CH ₂ -CH ₃ , -CH=CH-CH=CH ₂ , or, for example, -CH=CH-, if two fragments of the molecule are connected by an alkenyl group. Each hydrogen atom at the carbon atom in C _2-50 alkenyl may be replaced by a substituent, as further specified. Thus, the term "alkenyl" refers to a carbon chain with at least one carbon-carbon double bond. If necessary, one or more triple bonds may occur.

"C _2-50 alkynyl" is a branched or straight alkynyl chain containing from 2 to 50 carbon atoms, for example, if present at the end of the molecule: -C≡CH, -CH ₂ -C≡CH, CH ₂ -CH ₂ - C≡CH, CH ₂ -C≡C-CH ₃ , or, for example, -C≡C-, in the case when two fragments of the molecule are connected by an alkynyl group. Each hydrogen atom at the carbon atom in the C _2-50 alkynyl may be replaced by a substituent, as further specified. Thus, the term "alkynyl" refers to a carbon chain with at least one carbon-carbon triple bond. If necessary, one or more carbon-carbon double bonds may occur.

"C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" means a cyclic alkyl chain containing from 3 to 7 carbon atoms, which may contain at least partially saturated carbon-carbon double bonds, for example, cyclopropyl, cyclobutyl, cyclopentyl , cyclohexyl cyclohexenyl, cycloheptyl. Each hydrogen atom at the cycloalkyl carbon atom can be replaced by a substituent. The term "C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" also includes bridged bicycles similar to norbornane or norbornene. Accordingly, "C _3-5 cycloalkyl" means a cycloalkyl containing from 3 to 5 carbon atoms.

Accordingly, "C _3-10 cycloalkyl" means a cyclic alkyl having from 3 to 10 carbon atoms, for example C _3-7 cycloalkyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. The term "C _3-10 cycloalkyl" also includes at least partially saturated carbomonocycles and bicycles.

"Halogen" means fluorine, chlorine, bromine or iodine. Particularly preferably the halogen is fluorine or chlorine.

"4-7 membered heterocyclyl" or "4-7 membered heterocycle" is a ring containing 4, 5, 6 or 7 ring atoms, which may contain as many double bonds as possible (an aromatic or non-aromatic ring that is fully saturated, partially saturated or unsaturated) in which at least one to 4 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and in which the ring is connected to the rest of the molecule through a carbon or nitrogen atom. Examples of 4-7 membered heterocycles include azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine or homopiperazine.

"9-11 membered heterobicyclyl" or "9-11 membered heterobicycle" is a heterocyclic system consisting of two rings with 9 to 11 ring atoms, in which at least one ring atom is shared by both rings, and which may contain up to the maximum possible number of double bonds (fully saturated, partially saturated or unsaturated aromatic or non-aromatic ring), wherein at least one to 6 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O) ₂ -), oxygen and nitrogen (including =N(O)-), and where said ring is connected to the rest of the molecule through a carbon or nitrogen atom. Examples of a 9-11 membered heterobicycle include indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisooxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline , dihydroisoquinoline, benzazepine, purine or pteridine. The term 9-11 membered heterobicycle also includes two-ring spiro structures, such as 1,4-dioxa-8-azaspiro[4,5]decane, or bridged heterocycles, such as 8-aza-bicyclo[ 3.2.1]octane.

In the case where the compounds of formula (I) contain one or more acidic groups or basic groups, the present invention also contains their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically acceptable salts. Thus, according to the present invention, compounds of formula (I) which contain acidic groups can be used in accordance with the invention, for example, in the form of alkali metal salts, alkaline earth metal salts or ammonium salts. Specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts derived from ammonia or organic amines, such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. Compounds of formula (I) which contain one or more basic groups, i.e. groups which may be protonated may be present and may be used in accordance with the invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid acid, livalinic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids, known to those skilled in the art. If the compounds of formula (I) simultaneously contain acidic and basic groups in the molecule, the present invention also includes, in addition to the above salt forms, internal salts or betaines (zwitterions). The corresponding salts of the compounds according to formula (I) can be prepared by conventional methods known to those skilled in the art, such as, for example, by reacting them with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of formula (I) which, due to their low physiological compatibility, are not directly suitable for use in pharmaceutical preparations, but which can be used, for example, as chemical intermediates or for the preparation of pharmaceutically acceptable salts.

The term "pharmaceutically acceptable" means approved by a regulatory authority such as the European Agency for the Evaluation of Medicinal Products (EMEA) and/or the US Food and Drug Administration (FDA) and/or any other National Regulatory Authority for use in animals. , preferably for humans.

By "pharmaceutical composition" is meant one or more active ingredients in combination with one or more inert ingredients, as well as any product which is formed directly or indirectly as a result of the combination, complexation or aggregation of any two or more of these ingredients, or as a result of the dissociation of one or more more than the specified ingredients, or other types of reactions or interactions of one or more ingredients. Thus, the pharmaceutical compositions of the present invention include any composition obtained by mixing a compound of the present invention with a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier).

The term "excipient" refers to the diluent, adjuvant or carrier through which a therapeutic drug is administered. Examples of such pharmaceutically acceptable excipients include sterile liquid media such as water and oils, including any oil of petroleum, animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil. oil and the like. Water is the preferred excipient when the pharmaceutical composition is administered orally. Saline solutions and aqueous dextrose solutions are preferred excipients when the pharmaceutical composition is intended for intravenous administration. Saline solutions and aqueous solutions of dextrose and glycerol are preferably used as liquid excipients for the preparation of injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol and the like. The composition of the present invention, if desired, may also contain minor amounts of wetting agents or emulsifiers, or buffering agents to stabilize the pH. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition of the present invention can be prepared in the form of a suppository based on traditional binders and fillers such as triglycerides. The oral formulation may include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical excipients are described by E.W. Martin in Remington's Pharmaceutical Sciences. Such compositions typically contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of excipient so as to provide a form for suitable administration to a patient. This composition must satisfy the regimen of its administration.

Preferably X ³ is O.

Preferably X is N( ^R4 ), ^X1 is C, and _X3 is O.

Preferably X ² represents C(R ⁷ R ^7a ).

Preferably L ¹ is selected from the group consisting of

where R represents H or C _1-4 alkyl; Y is NH, O or S; and R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , X, X ¹ , X ² have the above meanings;

Even more preferably, L ¹ is selected from the group consisting of

where R has the above meaning.

At least one (up to four) hydrogen atoms are replaced by an L ² -Z group. In the case where more than one L ² -Z group is present, each L ² and each Z can be selected independently of each other. Preferably, only one L ² -Z group is present, resulting in a structure of the formula DL ¹ -L ² -Z.

Generally, L ² may be connected to L ¹ in any position except the hydrogen substitution position indicated by the symbol * of the structure of formula (I). It is preferable that one to four hydrogen atoms defined directly by the radicals R, R ¹ -R ⁸ , or for example, the hydrogen of C ₁ - ₄ alkyl or additional groups and rings defined by the radicals R and R ¹ -R ⁸ , are replaced by the group L ² -Z.

In addition, L ¹ can be additionally replaced if necessary. Generally, any substituent can be used as long as it does not affect the fundamental principle of bond cleavage.

In a preferred embodiment, one or more additional optional substituents are independently selected from the group consisting of halogen, CN, COOR ⁹ , OR ⁹ , C(O)R ⁹ , C(O)N(R ⁹ R ^9a ), S(O) ₂ N(R ⁹ R ^9a ), S(O)N(R ⁹ R ^9a ), S(O) ₂ R ⁹ , S(O)R ⁹ , N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ), SR ⁹ , N(R ⁹ R ^9a ), NO ₂ , OC(O)R ⁹ , N(R ⁹ )C(O)R ^9a , N(R ⁹ )S(O) ₂ R ^9a , N (R ⁹ )S(O)R ^9a , N(R ⁹ )C(O)OR ^9a , N(R ⁹ )C(O)N(R ^9a R ^9b ), OC(O)N(R ⁹ R ^9a ), T, C _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl, wherein T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more R ¹⁰ , which are the same or different, and where C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-, -O-, -C(O)-, - C(O)N(R ¹¹ )-, -S(O) ₂ N(R ¹¹ )-, -S(O)N(R ¹¹ )-, -S(O) ₂ -, -S(O)-, -N(R ¹¹ S( O) ₂ N(R ^11a )-, -S-, -N(R ¹¹ )-, -OC(O)R ¹¹ , -N(R ¹¹ )C(O), -N(R ¹¹ )S(O ) ₂ -, -N(R ¹¹ )S(O)-, -N(R ¹¹ )C(O)C-, -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O )N(R ¹¹ R ^11a );

R ⁹ , R ^9a , R ^9b are independently selected from the group consisting of H, T, Z and C _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl, where T, C _1-50 alkyl, C _{2 -50} alkenyl and C _2-50 alkynyl are optionally substituted by one or more substituents from R ¹⁰ that are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C(O)O-, -O-, -C(O)-, -C(O)N(R ¹¹ )-, -S(O) ₂ N(R ¹¹ )-, -S(O)N(R ¹¹ )-, -S(O) ₂ -, -S(O)-, -N(R ¹¹ )S(O) ₂ N(R ^11a )-, - S-, -N(R ¹¹ )-, -OC(O)R ¹¹ , -N(R ¹¹ )C(O)-, -N(R ¹¹ )S(O) ₂ -, -N(R ¹¹ ) S(O)-, -N(R ¹¹ )C(O)O-, -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

T is selected from the group consisting of phenyl; naphthyl; indenyl; indanila; tetralinyl; C _3-10 cycloalkyl; 4-7 membered heterocyclyl; or a 9-11 membered heterobicyclyl, wherein T is optionally substituted with one or more substituents from R10, which are the same or different from each other;

R ¹⁰ represents Z, halogen, CN, oxo (=O), COOR ¹² , OR ¹² , C(O)R ¹² , C(O)N(R ¹² R ^12a ), S(O) ₂ N(R ¹² R ^12a ), S(O)N(R ¹² R ^12a ), S(O) ₂ R ¹² , S(O)R ¹² , N(R ¹² )S(O) ₂ N(R ^12a R ^12b ), SR ¹² , N(R ¹² R ^12a ), NO ₂ , OC(O)R ¹² , N(R ¹² )C(O)R ^12a , N(R ¹² )S(O) ₂ R ^12a , N(R ¹² ) S(O)R ^12a , N(R ¹² )C(O)OR ^12a , N(R ¹² )C(O)N(R ^12a R ¹² b), OC(O)N(R ¹² R ^12a ); or C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogen atoms that are the same or different from each other;

R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b are independently selected from the group consisting of H or C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogen atoms, which are the same or different;

The term "interrupted" means that a group is inserted between two carbon atoms or at the end of a carbon chain between a carbon and a hydrogen.

L ² represents a single chemical bond or spacer. In the case where L ² is a spacer, it is preferred that it be one or more of the optional substituents defined above, provided that L ² is replaced by a Z group.

Accordingly, in the case where L ² is not a single chemical bond, L ² -Z represents COOR ⁹ , OR ⁹ , C(O)R ⁹ , C(O)N(R ⁹ R ^9a ), S(O) ₂ N (R ⁹ R ^9a ), S(O)N(R ⁹ R ^9a ), S(O) ₂ R ⁹ , S(O)R ⁹ , N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ), SR ⁹ , N(R ⁹ R ^9a ), OC(O)R ⁹ , N(R ⁹ )C(O)R ^9a , N(R ⁹ )S(O) ₂ R ^9a , N(R ⁹ ) S(O)R ^9a , N(R ⁹ )C(O)OR ^9a , N(R ⁹ )C(O)N(R ^9a R ^9b ), OC(O)N(R ⁹ R ^9a ), T, C _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl, where T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted with one or more R ¹⁰ radicals, which identical or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, - О-, -С(О)-, -C(O)N(R ¹¹ )-, -S(O) ₂ N(R ¹¹ )-, -S(O)N(R ¹¹ )-, -S( O) ₂ -, -S(O)-, -N(R ¹¹ )S(O) ₂ N(R ^11a )-, -S-, -N(R ¹¹ )-, -OC(O)R ¹¹ , -N(R ¹¹ )C(O)-, -N(R ¹¹ )S(O) ₂ -, -N(R ¹¹ )S(O)-, -N(R ¹¹ )C(O)O-, - N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

R ⁹ , R ^9a , R ^9b are independently selected from the group consisting of H, T and C _1-50 alkyl, C _2-50 alkenyl or C _2-50 alkynyl, where T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more R10, which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group , consisting of T, -C(O)O-, -O-, -C(O), -C(O)N(R ¹¹ )-, -S(O) ₂ N(R ¹¹ )-, -S (O)N(R ¹¹ )-, -S(O) ₂ -, -S(O)-, -N(R ¹¹ )S(O) ₂ N(R ^11a )-, -S-, -N( R ¹¹ )-, -OC(O)R ¹¹ , -N(R ¹¹ )C(O)-, -N(R ¹¹ )S(O) ₂ -, -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )- and -OC(O)N(R ¹¹ R ^11a );

T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 4-7 membered heterocyclyl or 9-11 membered heterobicyclyl, where T is optionally substituted with one or more R ¹⁰ which are the same or different;

R ¹⁰ represents Z, halogen, CN, oxo(=O), COOR ¹² ; OR ¹² ; C(O)R ¹² ; C(O)N(R ¹² R ^12a ), S(O) ₂ N(R ¹² R ^12a ), S(O)N(R ¹² R ^12a ), S(O) ₂ R ¹² , S(O)R ¹² , N(R ¹² )S(O) ₂ N(R ^12a R ^12b ), SR ¹² , N(R ¹² R ^12a ), NO ₂ , OC(O)R ¹² , N(R ¹² )C(O) R ^12a , N(R ¹² )S(O) ₂ R ^12a , N(R ¹² )S(O)R ^12a , N(R ¹² )C(O)OR ^12a , N(R ¹² )C(O)N (R ^12a R ^12b ); OC(O)N(R ¹² R ^12a ) or C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogen atoms, which are the same or different;

R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b are independently selected from the group consisting of H, Z or C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogen atoms, which are the same or different ;

provided that one of ^R9 , ^R9a , ^R9b , ^R11 , ^R11a , ^R12 , ^R12a , ^R12b is a Z group.

More preferably, L ² is an alkyl chain containing from 1 to 20 carbon atoms, which is optionally interrupted by one or more groups independently selected from -O- and C(O)N(R ^3aa ); optionally substituted with one or more groups independently selected from OH and C(O)N(R ^3aa R ^3aaa ), and where R ^3aa , R ^3aaa are independently selected from the group consisting of H and C _1-4 alkyl.

Preferably L ² has a molecular weight in the range from 14 g/mol to 750 g/mol.

Preferably L ² is attached to Z via an end group selected from

In case L ² has such a terminal group, it is also preferable that L ² has a molecular weight in the range of 14 g/mol to 500 g/mol, calculated without taking into account such terminal group.

L is preferably represented by formula (Ia)

where R ⁴ , L ² and Z are as defined above, and where R ^3aa , R ^3aaa are independently selected from the group consisting of H and C ₁ - ₄ alkyl? or R ^3aa and R ^3aaa , together with the nitrogen atom to which they are attached, combine to form a 4-7 membered heterocycle. R ⁴ is preferably H or methyl.

L is preferably represented by formula (Ib)

where R ¹ , R ^1a , R ⁴ , L ² and Z are as defined above, and where R ^3aa represents H or C _1-4 alkyl. R ⁴ is preferably H or methyl.

R ¹ in formula (I) is preferably L ² -Z.

R ³ in formula (I) is preferably L ² -Z.

R ³ , R ^3a in formula (I) preferably, together with the nitrogen atom to which they are attached, combine to form a 4-7 membered heterocycle, said heterocycle being substituted with L ² -Z.

D-H is preferably a low molecular weight bioactive agent or biopolymer.

D-H is preferably a biopolymer selected from the group of biopolymers consisting of proteins, polypeptides, oligonucleotides and peptide nucleic acids.

"Oligonucleotides" means DNA, RNA, both single- and double-stranded, siRNA (small interfering RNA), miRNA (microRNA), aptamers, and any chemical modifications thereof, which preferably range from 2 to 1000 nucleotides in length. These modifications include, but are not limited to those that provide other chemical groups that impart additional charge, polarizability, hydrogen bonding, electrostatic interaction and fluxionality to the bases of the ligand nucleic acid or the ligand nucleic acid as a whole. Such modifications include, but are not limited to, modifications at the 2'-position of the ribose sugar, modifications at the 5-position of the pyrimidine, modifications at the 8-position of the purine, modifications to exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodouracil, backbone modifications , methylations, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. Modifications may also include 3' and 5' modifications such as capping and changes in stereochemistry.

Preferably, D-H is a polypeptide selected from the group of polypeptides consisting of adrenocorticotropic hormone ACTH, adenosine deaminase, agalsidase, alpha-1-antitrypsin (AAT), alpha-1-proteinase inhibitor (API), alteplase, amylins (amylin, symlin), anistreplase , ancrodserine protease, antibodies (monoclonal or polyclonal, and fragments or fusion products thereof), antithrombin III, antitrypsins, aprotinin, asparaginase, atosiban, biphalin, bivalirudin, bone morphogenic proteins, bovine pancreatic trypsin inhibitor (BPTI), cadherin fragments, calcitonin (salmon ), collagenase, inhibitor of the complement system with esterase activity C1, conotoxins, fragments of cytokine receptors, DNase, dynorphin A, endorphins, enfuvirtide, enkephalins, erythropoietins, exendins, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, fibrinolysin, fibroblast growth factor (FGF), growth hormone releasing peptide-2 (GHRP2), fusion proteins, follicle stimulating hormone, gramicidin, ghrelin, desacyl ghrelin, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucagon-like peptides, glucocerebrosidase, granulocyte-macrophage colony-stimulating factor (GM-CSF), human heat shock proteins (HSP), phospholipase activating protein (PLAP), human chorionic gonadotropin (hCG), hemoglobins, hepatitis B vaccines, hirudin, human serine protease inhibitor, hyaluronidases, iduronidase, immunoglobulins, influenza vaccines, interleukins (1-alpha, 1-beta, 2, 3, 4, 6, 10, 11, 12, 13, 21), IL-1 receptor antagonist (rhIL-lra), insulins, insulin-like growth factors, insulin-like growth factor binding protein (rhl GFBP), interferons (alpha 2a, alpha 2b, alpha 2c, beta 1a, beta 1b, gamma 1a, gamma 1b), intracellular adhesion molecules, keratinocyte growth factor (KGF) ), glycoprotein P-selectin ligand (PSGL), transforming growth factors, lactase, leptin, leuprolide, levothyroxine, luteinizing hormone, Lyme disease vaccine, natriuretic peptides (ANP, BNP, CNP and their fragments), neuropeptide Y, pancrelipase, pancreatic polypeptide, papain, parathyroid hormone, platelet-derived growth factor (PDGF), pepsin, peptide YY, platelet-activating factor acetyl hydrolase (PAF-AH), prolactin, C protein, thymalfasin, octreotide, secretin, sermorelin, soluble tumor necrosis factor receptor ( TNFR), superoxide dismutase (SOD), somatropins (growth hormones), somatoprim, somatostatin, streptokinase, sucrase, terlipressin, tetanus toxin fragment, thylactase, thrombins, thymosin, thyroid hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor coupled to the Fc region of IgG, tissue plasminogen activator (tPA), thyroid stimulating hormone (TSH), urodilatin, urate oxidase, urokinase, vaccines, vascular endothelial growth factor (VEGF), vasoactive intestinal peptide, vasopressin, ziconotide, lectin and ricin .

Preferably, D-H is a protein produced by recombinant DNA technologies.

Preferably, D-H is a protein selected from the group of proteins consisting of antibody fragments, single chain antigen binding proteins, catalytic antibodies and fusion proteins.

Preferably, D-H is a low molecular weight biologically active agent selected from the group consisting of agents acting on the central nervous system, anti-infective, anti-allergic, immunomodulatory agents, anti-obesity agents, anticoagulants, anti-diabetic agents, anti-tumor, anti-bacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory , steroidal, vasodilator, vasoconstrictor and cardiovascular agents having at least one primary or secondary amino group.

Preferably, D-H is a low molecular weight biologically active agent selected from the group of agents consisting of acarbose, alaproclaate, alendronate, amantadine, amikacin, amineptine, aminoglutethimide, amisulpride, amlodipine, amotosalen, amoxapine, amoxicillin, amphetamine, amphotericin B, ampicillin, ampre navir, amrinona , anileridine, apraclonidine, apramycin, articaine, atenolol, atomoxetine, avizafon, baclofen, benazepril, benserazide, benzocaine, betaxolol, bleomycin, bromfenac, brofaromine, carvedilol, catine, cathinone, carbutamide, cephalexin, clinafloxacin, ciprofloxacin, defer oxamine, delavirdine, desipramine , daunorubicin, dexmethylphenidate, diaphenylsulfone, dizocilpine, dopamine, dobutamine, dorzolamide, doxorubicin, duloxetine, eflornithine, enalapril, adrenaline, epirubicin, ergoline, ertapenem, esmolol, enoxacin, ethambutol, fenfluramine, fenoldopam, fenoterol , fingolimod, flecainide, fluvoxamine, fosamprenavir , frovatriptan, furosemide, fluoxetine, gabapentin, gatifloxacin, gemiflocacin, gentamicin, grepafloxacin, hexylcaine, hydralazine, hydrochlorothiazide, icofungipene, idarubicin, imiquimod, inversin, isoproterenol, isradipine, kanamycin A, ketamine, labet alola, lamivudine, levobunolol, levodopa, levothyroxine, lisinopril, lomefloxacin, loracarbef, maprotiline, mefloquine, melphalan, memantine, meropenem, mesalazine, mescaline, methyldopa, methylenedioxymethamphetamine, metoprolol, milnacipran, mitoxantrone, moxifloxacin, norepinephrine, norfloxacin, nortriptyline, neom icin B, nystatin, oseltamivir, pamidronic acid, paroxetine, pazufloxacin, pemetrexed, perindopril, phenmetrazine, phenelzine, pregabalin, procaine, pseudoephedrine, protriptyline, reboxetine, ritodrine, sabarubicin, salbutamol, serotonin, sertraline, sitagliptin, sotalol, spectinomycin, sulfadiazine, sulfamerazine, sertraline, spr ectinomipine, sulfalene, sulfamethoxazole, tacrine, tamsulosin, terbutaline, timolol, tirofiban, tobramycin, tocainide, tosulfloxacin, trandolapril, tranexamic acid, tranylcypromine, trimerexate, trovafloxacin, valacyclovir, valganciclovir, vancomycin, viomycin, viloxazine and zalygitabine.

Preferably Z is a polymer having a molecular weight of at least 500 Da or a C _8-18 alkyl group.

In a preferred embodiment, Z is selected from the group of optionally cross-linked polymers consisting of poly(propylene glycol), poly(ethylene glycol), dextran, chitosan, hyaluronic acid, alginate, xylan, mannan, carrageenan, agarose, cellulose, starch, hydroxyalkylated starch (HAS) , poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), polyorthoesters, polycarbonates), poly(urethanes), poly(acrylic acids), poly(acrylamides), poly(acrylates), poly(methacrylates), poly (organophosphazenes), polyoxazolines, poly(siloxanes), poly(amides), poly(vinylpyrrolidone), poly(cyanoacrylates), polyesters, poly(iminocarbonates), poly(amino acids), collagen, gelatin, hydrogel or blood plasma protein, and their copolymers.

In a preferred embodiment, Z is a protein.

In a preferred embodiment, Z is a protein selected from the group consisting of albumin, transferrin, immunoglobulin.

In a preferred embodiment, Z is a linear or branched poly(ethylene glycol) with a molecular weight of from 2000 to 150,000 Da.

In an even more preferred embodiment, a prodrug of the present invention is provided wherein DH is a GLP-1 glucagon-like peptide receptor agonist, L is L ¹ represented by formula (I) as above, and Z is a hydrogel. Even more preferably, in formula (I), X is N(R ⁴ ), X ¹ is C, and X ³ is O. In a particularly preferred embodiment, L is represented by formula (Ia) above.

GLP-1 (glucagon-like peptide-1) is one of the intestinal peptide hormones that is released into the circulatory system after eating. It enhances the postprandial release of insulin when carbohydrates (especially carbohydrates) are digested and their blood levels are postprandially elevated. GLP-1 binds to GLP-1 receptor sites located on pancreatic β-cells and increases endogenous cAMP levels in a dose-dependent manner. In isolated rat islets, in the presence of elevated glucose concentrations in the normoglycemic range, GLP-1 stimulates insulin secretion. Previously, the possibility of using GLP-1 in the treatment of patients with type 2 diabetes mellitus was expressed due to the powerful stimulating effect of this insulinotropic peptide for insulin secretion when blood glucose levels increase, and the cessation of its action when returning to a state of normoglycemia. The antidiabetogenic effect of glucagon-like peptide amide-1 (7-36) in normal subjects and patients with diabetes mellitus is described, for example, in the journal N. Engl. J. Med. 326(20): pp. 1316-1322. In vitro and animal studies have shown that GLP-1 improves insulin sensitivity and has an anabolic effect on pancreatic β-cells. In humans, GLP-1 has also been reported to inhibit glucagon secretion, slow gastric emptying, and induce a subjective feeling of fullness, leading to weight loss when taken over several weeks and months.

Exendin-4 is reported to bind to GLP-1 receptors located on pancreatic β-cells and has an affinity 2.5 times higher than GLP-1. In isolated rat islets and β-cells, in the presence of glucose, exendin enhances insulin secretion in a dose-dependent manner. Exendin-4 is a highly active agonist, and the truncated exendin 9-39-amide is an antagonist at the β-cell secreting glucagon-like peptide 1-(7-36) amide receptor (see J. Biol. Chem 268 (26): 19650-19655). In studies in rodents with type 2 diabetes, exendin-4 was shown to be 5,530 times more potent than GLP-1 in lowering blood glucose levels. In addition, the duration of the glucose-lowering effect after a single administration of exendin-4 is significantly longer compared to GLP-1 (see, for example, Diabetes 48(5): 1026-1034). The plasma half-life of exendin-4 in humans was reported to be only 26 minutes. Exendin-4 reduces fasting and postprandial blood glucose levels and reduces energy intake in healthy human volunteers (see, eg, Am. J. Physiol. Endocrinol. Metab. 281(1): E155-61).

The hydrogels used in accordance with this invention are known to those skilled in the art. Suitable hydrogels include those used in WO-A 2006/003014. Accordingly, a hydrogel can be defined as a three-dimensional hydrophilic or amphiphilic polymer network structure capable of absorbing large amounts of water. Such network structures consist of homopolymers or copolymers, and they are insoluble due to the presence of covalent chemical or physical cross-linking units (as a result of ionic, hydrophobic interactions, entanglements). Cross-linking nodes provide network structure and physical integrity. Hydrogels exhibit thermodynamic compatibility with water, which allows them to swell in an aqueous environment. The chains of said network are connected in such a way that there are pores, and that the main fraction of these pores has sizes between 1 nm and 1000 nm.

Another object of the present invention is a pharmaceutical composition containing, in accordance with the present invention, a prodrug or a pharmaceutical salt thereof in combination with a pharmaceutically acceptable excipient.

Another object of the present invention is in accordance with the present invention a prodrug or pharmaceutical composition for use as a drug.

Another aspect of the present invention is a method of treating, regulating, delaying or preventing the development of one or more conditions in a mammalian patient in need of treatment. comprising administering to said patient a therapeutically effective amount of a prodrug of the present invention or a pharmaceutical composition or pharmaceutically acceptable salt thereof.

Still another aspect of the present invention is the chemical precursor prodrug of the formula Act-L, wherein L is as defined above and Act is a leaving group.

Act is preferably a chloride, bromide, fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl, N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl, pentafluorophenoxy, 2-thiooxothiazolidinyl, or N-hydroxysulfosuccinimidyl.

Examples

Materials and methods

Materials: side chain protected exendin-4, (J. Eng et al, J. Biol. Chem. 1992, 267, 11, 7402-7405) on Rink amide resin, side chain protected BNP-32a, (human, in in which Cys 10 and Cys 26 are replaced by Ala) on a chlorotrityl resin, protected in the side chain of BNP-32b (human in which Cys 10 and Cys 26 are replaced by Ala), with a side chain protecting group ivDde on Lys at position 14 on the chlorotrityl resin, and the amide of the side chain-protected fragment of human growth hormone releasing factor 1-29 (GRF(1-29)) on Rink amide resin, which were synthesized using the Fmoc synthesis strategy, were obtained from Peptide Specialty Laboratories GmbH, Heidelberg, Germany. The chains used standard protecting groups, except that for Lys 27 of exendin-4 and Lys 21 of the GRF(1-29) compound, Mmt side chain protecting groups were used.

40 kDa methoxy(polyethylene glycol) maleimide propionamide (a 40 kDa PEG derivative containing a maleimide group) was obtained from Chirotech Technology Ltd, Cambridge, UK.

2-Chlorotrityl chloride resin, Sieber amide resin, and amino acids were obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany. Fmoc-D-homocysteine (Trt)-OH and S-trityl-3-mercaptopropionic acid (TRT-MPA) were obtained from Bachem AG, Bubendorf, Switzerland. O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undeca-ethylene glycol (Fmoc-Pop-OH) was obtained from Polypure AS, Oslo, Norway.

Fmoc-4-(2-aminoethyl)-1-carboxymethylpiperazine (Fmoc-Acp-OH) was purchased from NeoMPC SA, Strasbourg, France. Cis-cyclohexane-1,2-dicarboxylic acid anhydride was obtained from Alfa Aesar GmbH & Co KG, Karlsruhe, Germany.

All other chemicals were obtained from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

Solid-phase synthesis of peptide compounds was carried out on 2-chlorotrityl chloride resin with a load of 1.3 mmol/g resin or Sieber amide resin with a load of 0.55 mmol/g resin. Syringes equipped with polypropylene frits were used as reaction vessels.

Loading of the first amino acid into the resins was carried out in accordance with the manufacturer's instructions.

Removing Fmoc protection:

To remove the Fmoc protecting group, the resin used was mixed with 2/2/96 (v/v/v) piperidine/DBU/DMF (twice for 10 min each) and washed with DMF (ten times).

Removing ivDde protection:

To remove the ivDde protecting group, the resin used was mixed with 98/2 (v/v) DMF/hydrazine hydrate (three times for 10 min each) and washed with DMF (ten times).

Vos protection:

The N-terminus of the peptide was protected with a Boc group by mixing the resin with 30 equiv. (Voc) ₂ O and 60 eq. pyridine in DCM. After 1 hour, the resin is washed with DCM (10 times).

Standard acid binding conditions:

The binding of acids (aliphatic acids, Fmoc-amino acids) with free amino groups on the resin was carried out by mixing the resin with 3 equiv. acid, 3 eq. RuVOR and 6 eq. DIEA for free amino groups on the resin (calculated based on theoretical resin loading) in DMF at room temperature. After 1 hour, the resin is washed with DMF (10 times).

Binding of 3-maleimidopropionic acid:

The coupling of 3-maleimidopropionic acid to free amino groups on the resin was carried out by mixing the resin with 2 eq. acid, 2 eq. OPK and 2 eq. HOBt on free amino groups in DMF at room temperature. After 30 min, the resin is washed with DMF (10 times).

Protocol for standard synthesis of urea derivatives on resin:

The synthesis of urea derivatives on the resin was carried out by mixing the resin with 2.5 eq. bis(pentafluorophenyl)carbonate, 5 eq. diisopropylethylamine (DIEA) and 0.25 eq. dimethylaminopyridine (DMAP) with respect to free amino groups in a 1/1 mixture of dichloromethane and acetonitrile (DCM/ACN) at room temperature. After 15 min, the resin is washed with N,N-dimethylformamide (DMF) (10 times). 5 eq. amine is dissolved in DMF. The mixture was introduced into the resin and stirred for 60 minutes at room temperature. The resin is washed with DMF (10 times).

Cleavage protocol for Sieber amide resin:

Upon completion of the synthesis, the resin was washed with dichloromethane (DCM) (10 times), dried under vacuum and treated several times (five times for 15 minutes) with a mixture of DCM/TES/TFA 97/2/1 (v/v/v). The eluates were combined, volatiles were removed under a stream of nitrogen, and the resulting product was purified by reverse phase HPLC. HPLC fractions containing product were pooled and lyophilized.

Digestion protocol for 2-chlorotrityl chloride resin:

Upon completion of the synthesis, the resin was washed with DCM, dried under vacuum and treated 2 times for 30 minutes with a mixture of DCM/HFIP 6/4 (v/v). The eluates were combined, volatiles were removed under a stream of nitrogen, and the resulting product was purified by reverse phase HPLC. HPLC fractions containing product were pooled and lyophilized.

Cleavage protocol for Rink amide resin:

Upon completion of the synthesis, the resin was washed with DCM, dried under vacuum and treated with 2 ml of TFA stripping mixture (TFA/TES/H ₂ O/DTT 95/2/2/1) per 100 mg of resin for 60 minutes. Volatile components were removed under a nitrogen stream. Nonpolar side chain products and protecting groups were removed by precipitation of the peptide from diethyl ether. The precipitated product was dried under vacuum and dissolved in a 1/1 mixture of ACN and water, and then purified by reverse phase HPLC.

The amine-containing products obtained as trifluoroacetic acid salts were converted to the corresponding HCl salts using an ion exchange resin (Discovery DSC-SAX, Supelco, USA). This step was carried out when residual TFA was expected to interfere with subsequent coupling reactions, for example.

Purification using reverse phase HPLC:

Reverse-phase HPLC was carried out on a column with C18 silica gel ReproSil-Pur 300 phase ODS-3 5 μm, size 100×20 or 100×40 mm (Dr. Maisch, Ammerbuch, Germany), connected to a Waters 600 HPLC system and a Waters 2487 absorption detector Linear gradients of solution A (0.1% TFA in H ₂ O) and solution B (0.1% TFA in acetonitrile) were used. HPLC fractions containing the product were lyophilized.

Sample analysis: Electrospray ionization mass spectrometry (ESI-MS) was performed on a Waters ZQ 4000 ESI measurement system and, if necessary, mass spectra were interpreted using Waters MaxEnt software.

Size exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with a Superdex200 10/300 column (Amersham Bioscience/GE Healthcare) unless otherwise stated. The mobile phase used was 10 mM sodium phosphate, 140 mM NaCl, pH 7.4, 3 mM EDTA.

Cation exchange chromatography was performed using an Amersham Bioscience AEKTAbasic system equipped with a Source 15S column packed with HR16/10 (Amersham Bioscience/GE Healthcare).

Desalting was performed using an Amersham Bioscience AEKTAbasic system equipped with a HiPrep 26/10 desalting column and 0.1% acetic acid in water as the mobile phase.

In vitro linker hydrolysis and drug release: compounds were dissolved in buffer A (10 mM sodium phosphate, 140 mM NaCl, pH 7.4, 3 mM EDTA) or buffer B (0.1 mM acetate and 3 mM EDTA, pH 4, 0), and then the solution was filtered through a 0.2 μm filter and incubated at 37°C. Time-delayed samples were collected and analyzed by reverse-phase HPLC at 215 nm and ESI-MS. UV signals correlating with the released drug molecule were combined and plotted against incubation time. When prodrug and drug retention times were the same, the mass signal ratio was used to determine the release kinetics.

For hydrogel conjugates, compounds were suspended in buffer A and incubated at 37°C. Analytes were collected after centrifugation of the suspension and analyzed by reverse phase HPLC at 215 nm. UV signals correlating with the released drug molecule were combined and plotted against incubation time.

Curve processing software was used to estimate the appropriate release half-life.

Example 1

Synthesis of a carrier based on fatty acids (1)

1 is synthesized on Sieber amide resin (477 mg, 0.262 mmol), by Fmoc-Lys(ivDde)-OH coupling, Fmoc deprotection, dodecanoic acid coupling, ivDde deprotection, Fmoc-Pop-OH coupling, Fmoc deprotection , coupling of 3-maleimidopropionic acid, cleavage from the resin and subsequent purification as shown above and described in the Materials and Methods section. Yield: 128 mg (0.119 mmol).

MS: m/z 1101.0 = [M+Na] ⁺ (Calculated Mm = 1078.4 g/mol).

Example 2

Synthesis of linker reagent (2)

Linker reagent 2 is synthesized on 3-chlorotrityl chloride resin (300 mg, 0.39 mmol) by loading the resin with Fmoc-Cys (Trt)-OH, deprotecting Fmoc and derivatizing the resin with urea using N,N-dimethylethylenediamine as the amine , cleavage of the product from the resin, as shown above and described in the "Materials and Methods" section. For reverse phase HPLC separation, 0.01% HCl in water was used as solution A and 0.01% HCl in acetonitrile was used as solution B.

Yield: 82 mg of product as HCl salt (0.16 mmol).

MS: m/z 478.2 = [M+H] ⁺ (Calculated Mm = 477.6 g/mol).

Example 3

Synthesis of exendin-4 linked linker intermediate (3)

2 (14 mg, 0.027 mmol), RuBOP (14 mg, 0.027 mmol) and DIEA (17 μl, 0.10 mmol) were dissolved in 0.2 ml anhydrous DMF. The resulting mixture was added to 22 mg of side chain-protected exendin-4 resin (0.1 mmol/g, 2.2 μmol) and stirred vigorously for 30 min at room temperature. The resin is washed with DMF (10 times) and DCM (10 times). 3 is cleaved from the resin and purified by reverse phase HPLC according to the procedure described in the “Materials and Methods” section.

Yield: 1.7 mg 3 as trifluoroacetate salt (0.38 µmol).

MS: m/z 1468.7 = [M+3H] ³ + (Calculated Mm = 4403 g/mol).

Example 4

Synthesis of fatty acid-PEG-linker-exendin-4 conjugate (Compound 4)

3 (1.7 mg, 0.38 mmol) and 1 (0.6 mg, 0.58 mmol) were dissolved in 500 μl of acetonitrile/water 7/3 (v/v). 40 μl of 0.5 M phosphate buffer (pH 7.4) is added and the mixture is incubated at room temperature for 10 min. Conjugate 4 was purified by reverse phase HPLC.

MS: m/z 1828.7 = [M+3H] ³⁺ (Calculated Mm = 5480 g/mol).

Example 5

Synthesis of linker intermediate (5a)

Fmoc-Acp-OH⋅2HCl (100 mg, 0.21 mmol) was suspended in 400 µl DMF/DMSO 1/1 (v/v). S-Tritylcysteamine hydrochloride (75 mg, 0.21 mmol), RuBOP (109 mg, 0.21 mmol) and DIEA (146 μl, 0.86 mmol) were added and the mixture was stirred for 60 min at room temperature. The Fmoc group is removed by adding 75 μl of piperidine and 25 μl of DBU. After 15 minutes, the mixture is hydrolyzed and acidified with acetic acid (AcOH) and the resulting compound is purified by reverse phase HPLC. After lyophilization, 98 mg of product (0.14 mmol, as the double trifluoroacetate salt) is obtained.

MS: m/z 511.6 = [M+Na] ⁺ (Calculated Mm = 488.7 g/mol).

Synthesis of cis-cyclohexane dicarboxylic acid amoxapine monoamide (5b)

Amoxapine (200 mg, 0.64 mmol) and cis-cyclohexane-1,2-dicarboxylic anhydride (108 mg, 0.70 mmol) are dissolved in 700 μl of dry DMF. Pyridine (130 μl, 1.6 mmol) was added and the mixture was stirred for 60 min at room temperature. The mixture was quenched with 2 ml of acetonitrile/acetic acid/water (1/1/1) and purified by reverse phase HPLC. After lyophilization, 344 mg of 5b (0.49 mmol, as trifluoroacetate double salt) is obtained.

MS: m/z 468.5 = [M+H] ⁺ (Calculated Mm = 468.0 g/mol).

Synthesis of linker-amoxapine conjugate (5c)

5b (7 mg, 0.010 mmol) was preactivated by incubation with PyBOP (12.5 mg, 0.024 mmol) and DIEA (5 μL, 0.03 mmol) in 200 μL anhydrous DMF for 45 min at room temperature. 5a (20 mg, 0.028 mmol) and DIEA (15 μl, 0.09 mmol) were added and the mixture incubated for a further 60 min. The mixture was quenched with 0.5 ml of acetonitrile/acetic acid/water (1/1/1) and purified by reverse phase HPLC. After lyophilization, 3 mg 5 s (0.0026 mmol, as the double trifluoroacetate salt) is obtained.

MS: m/z 939.3 = [M+H] ⁺ (Calculated Mm = 938.6 g/mol).

To remove trityl protection, the lyophilisate is incubated in 1 ml HFIP and 3 µl TES for 30 min. The mixture was evaporated and the resulting thiol was purified by reverse phase HPLC. After lyophilization, 2 mg (2.2 µmol, as the double trifluoroacetate salt) of amoxapine-linker conjugate 5c are obtained.

MS: m/z 697.1 = [M+H] ⁺ (= [M+H] ⁺ (Calculated Mw = 696.3 g/mol).

Synthesis of fatty acid-PEG-amoxapine conjugate (5)

Amoxapine-linker conjugate 5c (2 mg, 2.2 µmol) and 1 (3.5 mg, 3.2 µmol) were dissolved in 900 µl of acetonitrile/water 7/3 (v/v). 60 μl of 0.5 M phosphate buffer (pH 7.4) is added and the mixture is incubated at room temperature for 10 min. 5 was purified by reverse phase HPLC. MS:

m/z 1774.9 = [M+H] ⁺ (Calculated Mm = 1774.7 g/mol).

Example 6

Synthesis of linker reagent (Compound 6)

Fmoc-Asp(t-Bu)-OH (411 mg, 1 mmol), HOBt (153 mg, 1 mmol) and DIC (160 μL, 1 mmol) were dissolved in 2 ml DMF and incubated for 10 min at room temperature. Add N,N-dimethylethylenediamine (160 µl, 1.5 mmol) and stir at room temperature for 30 minutes. Acetic acid (300 µl) is added and Fmoc-Asp(t-Bu)-NH-(CH ₂ ) ₂ -N(CH3) ₂ is purified by reverse phase HPLC.

Yield: 220 mg (0.46 mmol)

MS Fmoc-Asp(t-Bu)-NH-(CH ₂ ) ₂ -N(CH3) ₂ : m/z 504.6 = [M+Na] ⁺ (Calculated Mm = 481.6 g/mol).

Fmoc-Asp (t-Bu)-NH-(CH ₂ ) ₂ -N(CH3) ₂ (220 mg, 0.46 mmol) is dissolved in 3 ml of TFA/TES 98/2 (v/v). After 30 min, the solvent was removed under a stream of nitrogen and 6 was purified by reverse phase HPLC using 0.01% HCl in water as solvent A and 0.01% HCl in acetonitrile as solvent B.

Yield: 146 mg (0.32 mmol, as HCl salt).

MS: m/z 426.5 = [M+H] ⁺ (Calculated Mm = 425.5 g/mol).

Example 7

Synthesis of linker reagents 7a and 7b

7a:R=H

7b:CH ₃

Synthesis 7a:

Fmoc-Asp(t-Bu)-OH (300 mg, 0.73 mmol), HOBt (1112 mg, 0.73 mmol) and DIC (117 μl, 0.73 mmol) were dissolved in 2 ml DMF and incubated for 10 min at room temperature. Boc-ethylenediamine (230 mg, 1.44 mmol) was added and the resulting mixture was stirred at room temperature for 30 minutes. Acetic acid (300 µl) is added and Fmoc-Asp(t-Bu)-NH-(CH ₂ ) ₂ -NH-Boc is purified by reverse phase HPLC.

Yield: 205 mg (0.37 mmol)

MS of intermediate: m/z 576.6 = [M+Na] ⁺ (Calculated Mm = 553.7 g/mol).

Fmoc-Asp (t-Bu)-NH-(CH ₂ ) ₂ -NH-Boc (205 mg, 0.37 mmol) was dissolved in 3 ml 98/2 (v/v) TFA/TES. After 30 minutes, the solvent is removed under a stream of nitrogen and the resulting Fmoc-Asp(H)-NH-(CH ₂ ) ₂ -NH ₂ is purified by reverse phase HPLC.

Yield: 140 mg (0.27 mmol, as trifluoroacetate salt)

MS of intermediate: m/z 398.8 = [M+H] ⁺ (Calculated Mm = 397.4 g/mol).

Fmoc-Asp(H)-NH-(CH ₂ ) ₂ -NH ₂ (140 mg, 0.27 mmol, as trifluoroacetate salt) is dissolved in 1 ml DMF and DIEA (140 μl, 0.81 mmol) and boc is added ₂ O (100 mg, 0.46 mmol). The solution is stirred at room temperature for 15 minutes and then acidified with acetic acid (300 μl). 7a was purified by reverse phase HPLC.

Yield 7a: 120 mg (0.24 mmol)

MS 7a: m/z 520.5 = [M+Na] ⁺ (Calculated Mm = 497.6 g/mol).

7b is prepared according to the procedure described above except that in the first stage of the synthesis, H ₂ N-(CH ₂ ) ₂ -N(CH ₃ )-boc is used instead of Boc-ethylenediamine as the amine.

Yield 7b: 115 mg

MS 7b: m/z 534.5 = [M+Na] ⁺ (Calculated Mm = 511.6 g/mol).

Example 8

Synthesis of exendin-linker conjugates (8a, 8b and 8c)

8a: R ¹ = N, R ² = N

8b: R ¹ = H, R ² = CH ₃

8s: R ¹ = CH ₃ , R ² = CH ₃

Synthesis of compound 8a:

7a (30 mg, 60 µmol), HOBt (9 mg, 60 µmol), DIEA (12 µl, 70 µmol) and DIC (10 µl, 63 µmol) are dissolved in 200 µl DMF, and immediately injected into the resin carrying the protected in the side chain of exendin-4, (40 mg, 4 µmol), and incubate for 1 hour at room temperature. The resin is washed ten times with DMF and then incubated for 5 min in 500 μl of 1/1/2 acetic anhydride/pyridine/DMF. The resin is washed 10 times with DMF and the fmoc group is removed. Trt-mercaptopropionic acid is added and 8a is cleaved from the resin and purified by reverse phase HPLC.

Yield: 3.6 mg

MS 8a: m/z 1108.5 = [M+4H] ⁴⁺ , 1477.8 = [M+3H] ³⁺ (Calculated Mm = 4432 g/mol).

8b is obtained according to the procedure for the synthesis of 8a, except that 7b is used instead of 7a.

Yield: 3.5 mg

MS 8b: m/z 1112.5 = [M+4H] ⁴⁺ , 1482.5 = [M+3H] ³⁺ ; (Calculated Mm = 4446 g/mol).

8c is obtained according to the procedure for the synthesis of 8a, except that 6 is used instead of 7a.

Yield: 3.2 mg

MS 8s: m/z 1116.2 = [M+4H] ⁴⁺ , 1487.8 = [M+3H] ³⁺ (Calculated Mm = 4460 g/mol).

Example 9

Synthesis of PEG40kDa-linker-exendin conjugates (9a, 9b and 9c)

9a: R ¹ = H, R ² = H

9b: R ¹ = H, R ² = CH ₃

9s: R ¹ = CH ₃ , R ² = CH ₃

Synthesis 9a:

8a (3.6 mg) was dissolved in 300 μl of a 2/1 water/acetonitrile mixture and 50 mg of PEG40kDa-maleimide was added. Add 100 μl of 0.25 M sodium phosphate buffer, pH 7, and after 5 minutes the solution is acidified with 50 μl of acetic acid.

9a was purified by ion exchange chromatography using 10 mM sodium citrate buffer pH3 as solvent A and 10 mM sodium citrate buffer pH 3 and 1 M NaCl as solvent B and a step gradient (from 0 to 40% solvent B). Fractions containing 9a are salted out and lyophilized.

Yield: 14 mg

9b is synthesized as described above except that 8b is used.

Yield: 15 mg

9c is synthesized as described above except that 8c is used.

Yield: 13 mg

Example 10

Synthesis of fatty acid-linker-exendin conjugate (10)

8c (1 mg) is dissolved in 100 μl of a 1/1 acetonitrile/water mixture and add 1 (1 mg) in 100 μl of a 3/1 acetonitrile/water mixture. 100 μl of 0.25 M sodium phosphate buffer was added, the reaction mixture was stirred for 5 min, and the resulting 10 was purified by reverse phase HPLC.

Yield: 1.3 mg

MS 10: m/z 1385.9 = [M+4H] ⁴⁺ , 1846.3 = [M+3H] ³⁺ (Calculated Mm = 5528.3 g/mol).

Example 11

Synthesis of NHS-activated linker reagent (11)

7b (20 mg, 40 µmol), N,N'-dicyclohexylcarbodiimide (10 mg, 48 µmol) and N-hydroxysuccinimide (NHS) (8 mg, 70 µmol) were dissolved in 300 µl anhydrous DCM and stirred at room temperature for 1 hours. The solvent is removed under a stream of nitrogen and 11 is purified by reverse phase HPLC and lyophilized.

Yield: 22 mg (36 µmol)

MS: m/z 631.5 = [M+Na] ⁺ (Calculated Mm = 608.7 g/mol).

Example 12

Synthesis of linker-exendin conjugate (fluorescein) (12a) and linker-GRF conjugate (1-29) (fluorescein) (12b)

12a: R = exendin (εK27-fluorescein)

12b: R=GRF(1-29) (εK21-fluorescein)

6 (60 mg, 130 µmol HCl salt), HOBt (20 mg, 130 µmol), DIEA (40 µl, 230 µmol) and DIC (20 µl, 126 µmol) are dissolved in 700 µl DMF and immediately injected into a protected exendin-4 side chain on the polymer resin (120 mg, 12 μmol), and the resulting reaction mixture was incubated for 1 hour at room temperature. The resin is washed ten times with DMF and then incubated for 5 min in 1 ml of acetic anhydride/pyridine/DMF (1/1/2, v/v/v). The resin is washed ten times with DMF and the Fmoc protecting group is removed. Trt-mercaptopropionic acid is coupled using standard coupling techniques and the resulting resin is washed five times with DMF and then ten times with DCM. The Mmt lysine protecting group at position 27 (Lys 27) was removed by incubating the resin five times in 2 ml of DCM/HFIP 9/1 (v/v) for 5 min. The resin is washed five times with DCM and then ten more times with DMF, after which the resin is injected with 5,6-carboxy-fluorescein-NHS-ester (20 mg, 42 µmol) and DIEA (20 µl, 115 µl) in 300 µl DMF and incubate for 30 min. The resulting 12a was separated from the resin and purified by reverse phase HPLC.

Yield: 12 mg

MS 12a: m/z 1205.9 = [M+4H] ⁴⁺ , 1607.0 = [M+3H] ³⁺ (Calculated Mm = 4818.3 g/mol).

12b was synthesized following the same procedure as 12a except that GRF (1-29) on a polymer resin (120 mg, 12 μmol) was used.

Yield: 11 mg

MS 12b: m/z = 998.6 = [M+4H] ⁴⁺ , 1330.5 = [M+3H] ³⁺ (Calculated Mm = 3989.6 g/mol).

Synthesis of mercaptopropionyl-exendin (fluorescein) (12c) and mercaptopropionyl-GRF (1-29) (fluorescein) (12d)

12c: R = Exendin (εK27-fluorescein)

12d: R=GRF(1-29) (εK27-fluorescein)

Trt-mercaptopropionic acid is coupled using a standard coupling procedure to the side chain-protected moiety of exendin-4 on a polymer resin (120 mg, 12 μmol). Removal of the Mmt-protecting group Lys27 is removed and the 5,6-carboxy-fluorescein-NHS-ester coupling reaction is carried out according to synthesis procedure 12a. 12c is cleaved from the resin and purified by reverse phase HPLC.

Yield: 13 mg

MS 12s: m/z 1545.6 = [M+3H] ³⁺ (Calculated Mm = 4633 g/mol).

12d was prepared following the procedure for the synthesis of 12c, except that GRF (1-29) was used on resin (120 mg, 12 μmol).

Yield: 11 mg

MS 12d: m/z 1269.1 = [M+3H] ³⁺ (Calculated Mm = 3804.3 g/mol).

Example 13

Synthesis of reversible PEG40kDa-linker-exendin (fluorescein) conjugate (13a) and reversible PEG40kDa-linker-GRF (1-29) (fluorescein) conjugate (13b)

13a: R = exendin (ε K27-fluorescein)

13b: R = GRF(1-29) (εK21-fluorescein)

12a (12 mg) was dissolved in 500 μl of 1/1 acetonitrile/water and added 120 mg of PEG40kDa-maleimide in 1 ml of 1/1 acetonitrile/water. Then 300 μl of 0.25 M sodium phosphate buffer pH 7.0 is added and after 10 min the reaction solution is acidified with 300 μl of acetic acid. 13a is purified by cation exchange chromatography, salted out from solution and lyophilized.

Yield: 51 mg

13b is obtained according to the synthesis procedure of 13a, except that 12b is used instead of 12a.

Yield: 46 mg

Synthesis of permanent PEG40kDa-exendin (fluorescein) conjugate (13c) and permanent PEG40kDa-GRF(1-29) (fluorescein) conjugate (13d)

13c: R = exendin (εK27-fluorescein)

13d: R=GRF(1-29) (εK21-fluorescein)

13c is obtained according to the procedure for the synthesis of 13a, except that 12c is used instead of 12a.

Yield: 55 mg

Compound 13d was prepared according to the procedure for the synthesis of 13a, except that 12d was used instead of 12a.

Yield: 45 mg

Example 14

Synthesis of linker-GRF-(1-29) conjugate (14)

14 is prepared following the synthesis procedure of 8c except that side-protected GRF (1-29) is used on the resin.

Yield: 10 mg

MS 14: m/z 908.2 = [M+4H] ⁴⁺ , 1211.2 = [M+3H] ³⁺ (Calculated Mm = 3631.3 g/mol).

Example 15

Synthesis of PEG40kDa-linker-GRF conjugate (1-29) (15)

15 is obtained according to the synthesis procedure 9c except that 14 is used, and to carry out cation exchange chromatography, 10 mM sodium citrate, pH 4, is used as solvent A and 10 mM sodium citrate, pH 4, and 1 M sodium chloride as solvent B.

Yield: 11 mg

Example 16

Synthesis of linker intermediate (16)

N,N-dimethylethylenediamine (198 μl, 1.8 mmol) and NaCNBH ₃ (58 mg, 0.9 mmol) were dissolved in methanol (5 ml) and the solution was adjusted to pH 5.5 by adding acetic acid (250 μl). A suspension of 2,4,6-trimethoxybenzaldehyde (294 mg, 1.5 mmol) in ethanol (5 ml) was added and the reaction mixture was stirred at room temperature for 1 hour. 5N HCl (0.5 ml) was added and the mixture was stirred for a further 12 hours. The solvent is evaporated under reduced pressure, the residue is dissolved in saturated NaHCO ₃ solution and extracted three times with DCM. The combined organic phases are dried over NaSO ₄ and the solvent is evaporated under reduced pressure.

Yield: 303 mg (1.13 mmol)

MS: m/z 269.3 = [M+H] ⁺ (Calculated Mm = 268.4 g/mol)

Example 17

Synthesis of linker 17a and 17b

Synthesis 17a:

Fmoc-Asp(OtBu)-OH (322 mg, 0.78 mmol), Tmob-protected diamine (compound 16) (150 mg, 0.56 mmol), HATU (O-7-azabenzotriazol-1-yl)-N ,N,N',N'-tetramethyluronium hexafluorophosphate) (255 mg, 0.67 mmol) and DIEA (290 µl, 1.68 mmol) were dissolved in DMF (1.5 ml). The mixture was stirred for 30 min, acidified with acetic acid and purified by reverse phase HPLC.

Yield: 463 mg (5.97 mmol, as trifluoroacetate salt, about 90% purity)

MS Fmoc-Asp(OtBu)-N(TMOB)CH ₂ CH ₂ N(CH ₃ ) ₂ : m/z 662.5 = [M+H] ⁺ (Calculated Mm = 661.8 g/mol)

Fmoc-Asp(OtBu)-N(TMOB)CH ₂ CH ₂ N(CH ₃ ) ₂ (225 mg, 0.29 mmol) is dissolved in a solution of piperidine (50 μl) and DBU (15 μl) in DMF (1.5 ml). The reaction mixture is stirred at room temperature for 1.5 hours. AcOH is added and the resulting compound H-Asp(OtBu)-N(TMOB)CH ₂ CH ₂ N(CH ₃ ) ₂ is purified by reverse phase HPLC.

Yield: 114 mg (0.21 mmol, as trifluoroacetate salt)

MS H-Asp(OtBu)-N(TMOB)CH ₂ CH ₂ N(CH ₃ ) ₂ : m/z 462.4 = [M+H] ⁺ (Calculated Mw = 439.6 g/mol)

Trifluoroacetate salt H-Asp(OtBu)-N(Tmob)CH ₂ CH ₂ N(CH ₃ ) ₂ (114 mg, 0.21 mmol) was dissolved in saturated NaHCO ₃ solution (10 ml) and extracted with DCM (3 x 10 ml ). The combined organic layers are dried over NaSO ₄ and the solvent is evaporated under reduced pressure. The residue was dissolved in DMF (1.0 ml), and 6-tritylmethylmercaptohexanoic acid (121 mg, 0.31 mmol), HATU (118 mg, 0.31 mmol) and DIEA (108 μl, 0.62 mmol) were added to the resulting solution ). The mixture is stirred for 30 minutes. AcOH (200 μl) is added and TrtS(CH ₂ ) ₅ CONH-Asp(OtBu)-N(TmOb)CH ₂ CH ₂ N(CH ₃ ) ₂ is purified by reverse phase HPLC.

Yield: 95 mg (0.10 mmol, as trifluoroacetate salt)

MS TrtS(CH ₂ ) ₅ CONH-Asp(OtBu)-N(Tmob)CH ₂ CH ₂ N(CH ₃ ) ₂ : m/z 812.64 = [M+H] ⁺ (Calculated Mm = 812.1 g /mol)

TrtS(CH ₂ ) ₅ CONH-Asp(OtBu)-N(Tmob)CH ₂ CH ₂ N(CH ₃ ) ₂ (95 mg, 0.10 mmol) is dissolved in a mixture of MeOH/H ₂ O 3:1 (1, 0 ml), LiOH (7.4 mg, 0.31 mmol) is added and the reaction mixture is shaken for 5 hours at 60°C. AcOH (100 μl) was added and 17a was purified by reverse phase HPLC.

Yield: 64 mg (0.07 mmol, as trifluoroacetate salt)

MS 17a: m/z 756.5 = [M+H] ⁺ (Calculated Mm = 756.0 g/mol)

17b is synthesized according to the procedure described above, except that in the first stage, Fmoc-NMe-Asp(OtBu)-OH is used instead of Fmoc-Asp(OtBu)-OH.

Yield 17b: 16 mg (18 µmol, as trifluoroacetate salt)

MS 17b: m/z 770.5 = [M+H] ⁺ (Calculated Mm = 770.0 g/mol)

Example 18

Synthesis of linker-BNP conjugates (18a and 18b)

Synthesis 18a:

17a (8.0 mg, 0.01 mmol), RuBOP (5.2 mg, 10 µmol) and DIEA (7 µl, 40 µmol) were dissolved in DMF (400 µl) and the resulting solution was immediately added to the resin bound protected in the side chain of BNP-32a (50 mg, 5 µmol). After incubation for 2 hours at room temperature, the resin is washed ten times with DMF, then another 10 times with DCM and dried under vacuum. The resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 10.6 mg

MS 18a: m/z 930.4 = [M+4H] ⁴⁺ , 1240.1 = [M+3H] ³⁺ (Calculated Mm = 3717.2 g/mol)

18b is synthesized according to the procedure described above except that 17b is used instead of 17a.

Yield: 4.7 mg

MS 18b: m/z 933.9 = [M+4H] ⁴⁺ , 1244.7 = [M+3H] ³⁺ (Calculated Mm = 3731.0 g/mol)

Example 19

Synthesis of PEG40kDa-linker-BNP conjugates (19a and 19b)

18a (5.2 mg) was dissolved in a 1:1 mixture of H ₂ O/acetonitrile containing 0.1% TFA (200 μl). A solution containing PEG40kDa-maleimide (70 mg) in a mixture of 1:1 H ₂ O/acetonitrile (1.5 ml) and phosphate buffer (30 μl, pH 7.4, 0.5 M) was added. The solution is incubated at room temperature, and after 5 min AcOH (30 μl) is added. 19a is purified by cation exchange chromatography, salted out from solution and lyophilized.

Yield: 19.2 mg

19b is obtained according to the procedure for the synthesis of 19a, except that 18b is used instead of 18a.

Example 20

Synthesis of linker 20

Fmoc-Asp(OH)OtBu (100 mg, 0.24 mmol), H ₂ N-(CH ₂ ) ₂ -N(CH ₃ )-boc (36 μl, 0.20 mmol), HATU (92 mg, 0 .24 mmol) and DIEA (105 µl, 0.60 mmol) are dissolved in 1 ml DMF. The mixture was stirred for 1 hour at room temperature, acidified with acetic acid (100 μl) and purified by HPLC.

Yield: 91 mg (0.13 mmol)

MS Fmoc-Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu: 590.3 = [M+Na] ⁺ (Calculated Mm = 567.7 g/mol)

Fmoc-Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu (91 mg, 0.13 mmol) was dissolved in DMF (1.0 ml), piperidine (50 μl) and DBU (15 μl) were added. and the resulting mixture was stirred for 45 minutes at room temperature. Acetic acid (100 μl) was added and the resulting NH ₂ -Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu was purified by reverse phase HPLC.

Yield: 39 mg (0.09 mmol, as trifluoroacetate salt)

MS NH ₂ -Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu: m/z 368.1 = [M+Na] ⁺ (Calculated Mm = 345.4 g/mol)

NH ₂ -Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu (36 mg, 0.09 mmol) was dissolved in DMF (0.5 ml), 6-tritylmethylmercaptohexanoic acid (55 mg) was added to the resulting solution , 0.14 mmol), HATU (53 mg, 0.14 mmol) and DIEA (49 μl, 0.28 mmol). The mixture is stirred for 45 minutes. AcOH (100 μl) is added and TrtS(CH ₂ ) ₅ CONH-Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu is purified by reverse phase HPLC.

Yield: 41 mg (0.06 mmol)

MS TrtS(CH ₂ ) ₅ CONH-Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu: m/z 740.6 = [M+Na] ⁺ (Calculated Mm = 718.0 g/mol )

TrtS(CH ₂ ) ₅ CONH-Asp(NH(CH ₂ ) ₂ N(CH ₃ )-boc)OtBu (41 mg, 0.06 mmol) dissolved in 1:1 dioxane/H ₂ O (1.0 ml ), LiOH (4.1 mg, 0.17 mmol) was added and the mixture was stirred at 60°C for 1 hour. AcOH (50 µl) was added and 20 was purified by reverse phase HPLC.

Yield: 31 mg (0.05 mmol)

MS 20: m/z 684.5 = [M+Na] ⁺ (Calculated Mm = 661.9 g/mol)

Example 21

Synthesis of linker-exendin conjugate (21)

Resin-bound, side chain-protected exendin (50 mg, 5 µmol) with an Mmt-protecting group at Lys27 is first protected at the N-terminus with a Boc group (see Materials and Methods) and then incubated five times (5 min each). 2 ml of mixture DCM/HFIP 9/1 (v/v) to remove the Mmt protecting group from Lys27. Compound 20 (6.6 mg, 10 µmol), RuBOP (5.2 mg, 10 µmol) and DIEA (7 µl, 40 µmol) were dissolved in DMF (400 µl) and immediately added to the resin. After incubation for 3 hours at room temperature, the resin is washed ten times with DMF and ten times with DCM and dried under vacuum. The resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 2.4 mg

MS 21: m/z 1497.2 = [M+3H] ³⁺ (Calculated Mm = 4488.0 g/mol)

Example 22

Synthesis of fatty acid-linker-exendin conjugate (22)

21 (2.6 mg) is dissolved in 200 μl of acetonitrile/water mixture, 1/1, and compound 1 (0.8 mg) is added in 400 μl of acetonitrile/water mixture, 7/3. 100 μl of 0.25 M sodium phosphate buffer was added, the reaction mixture was stirred for 5 min, after which 22 was purified by reverse phase HPLC.

Yield: 2.4 mg

MS 22: m/z 1388.3 = [M+4H] ⁴⁺ ; 1857.1 = [M+3H] ³⁺ (Calculated Mm = 5566.4 g/mol).

Example 23

Synthesis of precursor 23

6-Tritylmercaptohexanoic acid (200 mg, 0.51 mmol), (PfpO) _2CO (202 mg, 0.51 mmol) and collidine (340 μl, 2.65 mmol) are dissolved in DMSO (1 ml) and stirred in for 30 minutes at room temperature. The mixture was added to a solution of Fmoc-Lys-OH (170 mg, 0.46 mmol) in H ₂ O/pyridine/tBuOH (3:03:01, 6 ml). The reaction mixture is then heated at 60°C for 2 hours, then diluted with EtOAc, extracted twice with 0.1 M H ₂ SO ₄ , treated twice with brine and dried over Na ₂ SO ₄ . The solvent was distilled off under reduced pressure and the residue was purified by reverse phase HPLC.

Yield: 109 mg

MS 23: m/g 741.3 = [M+H] ⁺ (Calculated Mm = 741.0 g/mol)

Example 24

Linker synthesis (24a -24c)

24a: R1 = H, R2 = boc

24b: R1 = Me, R2 = boc

24c: R1 = R2 = Me

23 (186 mg, 0.25 mmol) and DIEA (160 μl, 0.92 mmol) were dissolved in DCM (2 ml), added to 2-chlorotrityl chloride resin (312 mg, 1.3 mmol/g) and stirred for 45 min at room temperature. Methanol (0.6 ml) is added and the resin is incubated for a further 15 minutes. The resin is washed with DCM (10 times) and DMF (10 times). Removal of Fmoc protection and formation of urea is carried out in accordance with general methods of synthesis (see Materials and Methods) by reacting with N-boc-ethylenediamine (57 μl, 0.34 mmol). The resulting product is cleaved from the resin and purified by the reverse method. phase HPLC.

Yield: 14 mg

MS 24a: m/z 705.4 = [M+H] ⁺ , 727.3 = [M+Na] ⁺ (Calculated Mm = 704.9 g/mol)

Compound 24b was prepared following the procedure for synthesis 24a except that N-boc-N-methylethylenediamine was used instead of N-boc-ethylenediamine.

Yield: 21 mg

MS 24b: m/z 719.3 = [M+H] ⁺ , 741.4 = [M+Na] ⁺ (Calculated Mm = 719.0 g/mol)

24c is prepared according to the procedure for synthesis 24a except that N,N-dimethylethylenediamine is used instead of N-boc-ethylenediamine.

Yield: 10 mg

MS 24s: m/z 633.2 = [M+H] ⁺ (Calculated Mm = 632.9 g/mol)

Example 25

Synthesis of exendin-linker conjugates (25a-25c)

24a (7.0 mg, 0.01 mmol), RuBOP (5.2 mg, 10 µmol) and DIEA (7 µl, 40 µmol) were dissolved in DMF (250 µl), the resulting solution was immediately added to the resin bound side chain protected exendin (50 mg, 5 µmol), and incubated for 2 hours at room temperature. The resin is washed with DMF (10 times), DCM (10 times) and dried under vacuum. The resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 1.6 mg

MS 25a: m/z 1511.8 = [M+3H] ³⁺ (Calculated Mm = 4530.8 g/mol)

25b is prepared according to the synthesis procedure of 25a, except that 24b is used instead of 24a.

Yield: 4.3 mg

MS 25b: m/z 1516.3 = [M+3H] ³⁺ (Calculated Mm = 4544.8 g/mol)

25c is prepared according to the procedure for the synthesis of 25a, except that 24c is used instead of 24a.

Yield: 1.3 mg

MS 25s: m/z 1520.4 = [M+3H] ³⁺ (Calculated Mm = 4558.8 g/mol)

Example 26

Synthesis of fatty acid-linker conjugates (26a-26c)

25a (1.6 mg) is dissolved in 200 μl of acetonitrile/water, 1/1, and compound 1 (0.11 mg) in 200 μl of acetonitrile/water, 7/3 is added. 30 μl of 0.25 M sodium phosphate buffer was added to the reaction mixture, the reaction mixture was stirred for 5 min, after which 26a was purified by reverse-phase HPLC.

MS of compound 26a: m/z 1870.0 = [M+3H] ³⁺ (Calculated Mm = 5609.2 g/mol).

26b is prepared according to the synthesis procedure of 26a, except that 25b is used instead of 25a.

MS 26b: m/z 1875.9 = [M+3H] ³⁺ , 1406.7 = [M+4H] ⁴⁺ , (Calculated Mm = 5623.2 g/mol)

26 s is obtained according to the procedure for the synthesis of 26a, except that 25c is used instead of 25a.

MS 26s: m/z 1879.4 = [M+3H] ³⁺ , 1410.5 = [M+4H] ⁴⁺ , (Calculated Mm = 5637.2 g/mol)

Example 27

Synthesis of PEG20kDa-linker-exendin conjugates (Compound 27a-27c)

25a (2.0 mg) is dissolved in a mixture of H ₂ O /MeCN, 1:1, containing 0.1% TFA (200 μl). A solution of PEG40kDa-maleimide (18 mg) in 1:1 H ₂ O/MeCN (1 ml) and phosphate buffer (15 μl, pH 7.4, 0.5 M) was added. The solution is incubated at room temperature, after 5 min AcOH (20 μl) is added and 27a is purified by cation exchange chromatography, salted out and lyophilized.

27b is prepared according to the procedure for the synthesis of 27a, except that 25b is used instead of 25a.

27c is prepared according to the procedure for the synthesis of 27a, except that 25c is used instead of 25a.

Example 28

Synthesis of linker 28

6-Bromohexanoyl chloride (46 µl, 0.31 mmol) is dissolved in 0.2 ml CH ₂ Cl ₂ and added to a solution containing H ₂ N-CH ₂ -CH ₂ -STrt (100 mg, 0.28 mmol), DIEA (97 µl, 0.56 mmol) in CH ₂ Cl ₂ (0.8 ml). The mixture is stirred for 2 hours at room temperature. The reaction mixture was acidified with AcOH (50 μl) and the solvent was distilled off under vacuum. After purification of the residue on silica gel (heptane/EtOAc = 1:1), the compound Br-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt is obtained.

Yield: 137 mg (0.276 mmol, 98%)

MS Br-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt: 518.9 = [M+Na] ⁺ , (Calculated Mm = 496.5 g/mol)

N-boc-ethylenediamine (81 μl, 0.51 mmol) is added to a solution containing Br-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt (230 mg, 0.46 mmol) and Na ₂ CO ₃ (196 mg, 1.85 mmol) in DMF (0.8 ml). The reaction mixture is stirred for 10 hours at 70°C. After cooling to room temperature, the mixture was diluted with 4 ml of MeCN/H ₂ O, 25:75 in the presence of 1% TFA and, after purification by reverse phase HPLC, Boc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₅ was obtained. -CONH-(CH ₂ ) ₂ -STrt.

Yield: 189 mg (0.27 mmol, 59%, as trifluoroacetate salt)

MS compound Boc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₅ CONH-(CH ₂ ) ₂ -STrt: 576.5 = [M+H] ⁺ , (Calculated Mm = 575.5 g/mol )

Boc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt (189 mg, 0.27 mmol) and HCHO (35% aqueous solution, 113 μl) are dissolved in MeCN (1.5 ml) and CNBH ₃ (34 mg, 0.54 mmol) was added to the resulting mixture. The reaction mixture is stirred for 5 hours at room temperature. After completion of the reaction (monitored by mass spectrometry), the solution is diluted with H ₂ O (5 ml) and extracted with CH ₂ Cl ₂ (three times 5 ml). The combined organic layers are dried over MgSO ₄ , filtered and the solvent is distilled off under vacuum. After purification of the residue by reverse phase HPLC, Boc-NH-(CH ₂ ) ₂ -N(CH ₃ )-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt is obtained.

Yield: 62.8 mg (0.11 mmol, 39%)

MS Boc-NH-(CH ₂ ) ₂ -N(CH ₃ )-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt: 590.6 = [M+H] ⁺ , (Calculated Mm = 589, 0 g/mol)

Boc-NH-(CH ₂ ) ₂ -N(CH ₃ )-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt (62.8 mg, 0.11 mmol) was dissolved in THF (6 ml) and add a solution of HCl in dioxane (130 μl, 4 M solution). The reaction mixture is stirred for 12 hours at room temperature. 200 μl of HCl in dioxane are added and the solvent is distilled off under vacuum. After purification of the residue by reverse phase HPLC, a mixture of H ₂ N-(CH2) ₂ -N(CH ₃ -(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt and the unreacted starting compound Boc-NH-(CH ₂ ) ₂ -N(CH ₃ )-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt

Yield: 32.8 mg (0.062 mmol, 44%, as hydrochloride salt) of compound 28 and 14.7 mg (0.025 mmol, 23%, as trifluoroacetate salt) of starting compound

MS 28: 490.5 = [M+H] ⁺ , (Calculated Mm = 489.0 g/mol)

Example 29

General procedure for the synthesis of carbolic acid-substituted BNP precursors (29a and 29b)

Cis-cyclohexane-1,2-dicarboxylic acid anhydride (231 mg, 1.5 mmol) and pyridine (271 µl, 2 mmol) were dissolved in DCM (2 ml) and the resulting solution was immediately added to the resin bound to the side-protected BNP-32a chains (300 mg). After incubation for 1 hour at room temperature, the resulting product is washed 10 times with DCM and dried under vacuum.

The side chain-protected resin-bound fragment of BNP-32 bearing an ivDde protecting group at Lys 14 is first protected with a Boc group at the N-terminus, deprotected at Lys14 (see Materials and Methods), and then reacted with cis-cyclohexane-1,2-dicarboxylic acid anhydride as described above for 29a.

Example 30

Synthesis of BNP linker thiols (30a and 30b).

Compound 28 H ₂ N-(CH ₂ ) ₂ -N(CH ₃ )-(CH ₂ ) ₅ -CONH-(CH ₂ ) ₂ -STrt (5.2 mg, 0.01 mmol), RuBOR (5.2 mg, 0.01 mmol) and DIEA (7.0 µl, 0.04 mmol) were dissolved in DMF (300 µl) and the resulting solution was added to the side chain-protected resin-bound BNP 29a (50 mg, 0.005 mmol) . After incubation for 2 hours at room temperature, the resin is washed with DMF (10 times), DCM (10 times) and dried under vacuum. The resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 9.8 mg

MS 30a: m/z 947.6 = [M+4H] ⁴⁺ , 1263.1 = [M+3H] ³⁺ (Calculated Mm = 3786.3 g/mol)

30b is prepared as described above except that the resin-bound BNP derivative 29b is used instead of 29a.

Yield: 7.4 mg

MS of compound 30b: m/z 947.5 = [M+4H] ⁴⁺ , 1263.0 = [M+3H] ³⁺ (Calculated Mm = 3786.3 g/mol)

Example 31

Synthesis of PEG40kDa-linker-BNP conjugates (31a and 31b)

30a (4 mg) is dissolved in a 1:1 mixture of H ₂ O/MeCN containing 0.1% TFA (200 μl). Add a solution of PEG40kDa-maleimide (42.2 mg) in a mixture of H ₂ O/MeCN, 1:1 (1 ml) and phosphate buffer (15 μl, pH 7.4, 0.5 M). The resulting solution is incubated at room temperature, after 5 min AcOH (20 μl) is added and 31a is purified by cation exchange chromatography, salted out and lyophilized.

Yield: 2.0 mg

31b is obtained according to the synthesis procedure of 31a, except that 30b is used instead of 30a.

Yield: 16.8 mg

Example 32

Synthesis of linker 32

3-Bromopropionyl chloride (62.5 µl, 0.62 mmol) is dissolved in 0.5 ml CH ₂ Cl ₂ and added to a solution of H ₂ N-CH ₂ -CH ₂ -STrt (200 mg, 0.56 mmol), DIEA (196 µl, 1.1 mmol) in CH ₂ Cl ₂ (1 ml). The mixture is stirred for 1 hour at room temperature. The reaction mixture was acidified with AcOH (100 μl) and the solvent was removed in vacuo. After purification of the residue on silica gel (heptane/EtOAc = 1:1), Br-(CH ₂ ) ₂ CONH-(CH ₂ ) ₂ -STrt is obtained.

Yield: 223 mg (0.49 mmol, 87%)

MS Br-(CH ₂ ) ₂ CONH-(CH ₂ ) ₂ -STrt: 478.7 = [M+Na] ⁺ , (Calculated Mm = 454.7 g/mol)

N-Alloc-ethylenediamine hydrochloride salt (43.5 mg, 0.24 mmol) and DIEA (38 μl, 0.22 mmol) are added to the solution containing Br-(CH ₂ ) ₂ CONH-(CH ₂ ) ₂ -STrt (100 mg, 0.22 mmol) and Na ₂ CO ₃ (93 mg, 0.87 mmol) in DMF (1 ml). The reaction mixture is stirred for 10 hours at a temperature of 70°C. After cooling to room temperature, the reaction mixture was diluted with 4 ml (MeCNZH ₂ O = 25:75 in the presence of 0.1% TFA) and purified by HPLC to give Alloc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₂ -CONH -(CH ₂ ) ₂ -STrt.

Yield: 61 mg (0.096 mmol, 44%, as trifluoroacetate salt)

MS Alloc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₂ -CONH-(CH ₂ ) ₂ -STrt: 540.8 = [M+Na] ⁺ , (Calculated Mm = 517.8 g/mol )

Alloc-NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₂ -CONH-(CH ₂ ) ₂ -STrt (60.9 mg, 0.096 mmol) was dissolved in CH ₂ Cl ₂ and Boc ₂ O (42 mg, 0.19 mmol). The solution is stirred for 20 hours at room temperature. Upon completion of the reaction, the reaction mixture was quenched by adding 70 μl of ASON and the solvent was evaporated under vacuum. The residue was diluted with 4 ml MeCNZH ₂ O (25:75, in the presence of 0.1% TFA) and after purification by reverse phase HPLC, Alloc-NH-(CH ₂ ) ₂ -N(Boc)-(CH ₂ ) ₂ - was obtained. CONH-(CH ₂ ) ₂ -STrt.

Yield: 53.3 mg (0.086 mmol, 89%)

MS Alloc-NH-(CH ₂ ) ₂ -N(Boc)-(CH ₂ ) ₂ -CONH-(CH ₂ ) ₂ -STrt: 640.6 = [M+Na] ⁺ , (Calculated Mm = 617.9 , g/mol)

Alloc-NH-(CH ₂ ) ₂ -N(Boc)-(CH ₂ ) ₂ -CONH-(CH ₂ ) ₂ -STrt (48.3 mg, 0.078 mmol) was dissolved in THF, triethylammonium formate (62 μl) was added. and Pd( _PPh3 ) ₄ (16 mg). The solution is stirred for 12 hours at room temperature with mass spectrometric monitoring of the reaction progress. Upon completion of the reaction, the solvent is evaporated under vacuum. The residue was dissolved in MeCN/H ₂ O (50:50, with 0.1% TFA) and purified by reverse phase HPLC to give H ₂ N-(CH ₂ ) ₂ -N(Boc)-(CH ₂ ) ₂ - CONH-(CH ₂ ) ₂ -STrt (31).

Yield: 20.1 mg (0.031 mmol, 40%, as trifluoroacetate salt)

MS 31: 534.6 = [M+H] ⁺ , 556.6 = [M+Na] ⁺ , (Calculated Mm = 533.5 g/mol)

Example 33

Synthesis of BNP linker thiols (Compound 33a and 33b)

Compound 33a is prepared according to the procedure for the synthesis of compound 30a, except that compound 32 is used instead of compound 28.

Yield: 8.0 mg

MS 33a: m/z 933.5 = [M+4H] ⁴⁺ , 1244.3 = [M+3H] ³⁺ (Calculated Mm - 3729.9 g/mol)

33b is obtained according to the synthesis procedure of 30b, except that 32 is used instead of 28.

Yield: 5.0 mg

MS 33b: m/z 933.5 = [M+4H] ⁴⁺ , 1244.3 = [M+3H] ³⁺ (Calculated Mm = 3715.9 g/mol)

Example 34

Synthesis of PEG40kDa-linker-BNP conjugates (34a n 34b)

33a (4.3 mg) was dissolved in a 1:1 mixture of H ₂ O/MeCN containing 0.1% TFA (200 μl). Add a solution of PEG40kDa-maleimide (42.2 mg) in a mixture of H ₂ O/MeCN, 1:1 (1 ml) and phosphate buffer (20 μl, pH 7.4, 0.5 M). The resulting solution is incubated at room temperature, after 5 min AcOH (20 μl) is added and 34a is purified by cation exchange chromatography, salted out and lyophilized.

Yield: 9.7 mg

34b is prepared according to the synthesis procedure of 34a, except that 33b is used instead of 33a.

Yield: 11.5 mg

Example 35

Synthesis of linker 35

Bromoacetyl bromide (54 µl, 0.62 mmol) is dissolved in 0.5 ml CH ₂ Cl ₂ and added to a solution containing H ₂ N-CH ₂ -CH ₂ -STrt (200 mg, 0.56 mmol) and DIEA (196 µl, 1.1 mmol) in CH ₂ Cl ₂ (1 ml). The mixture is stirred for 1 hour at room temperature. The reaction mixture is acidified with acetic acid (100 μl) and the solvent is evaporated under vacuum. After purification, the residue is purified on silica gel (heptane/EtOAc = 1:), obtaining Br-CH ₂ -CONH-(CH ₂ ) ₂ -STrt.

Yield: 245 mg (0.55 mmol, 99%)

MS Br-CH ₂ -CONH-(CH ₂ ) ₂ -STrt: 462.4 = [M+Na] ⁺ , (Calculated Mm = 440.4 g/mol)

N-Alloc-ethylenediamine hydrochloride salt (45 mg, 0.25 mmol) and DIEA (79 μl, 0.45 mmol) are added to a solution containing Br-CH ₂ -CONH-(CH ₂ ) ₂ -STrt (100 mg, 0.23 mmol) in DMF (1 ml). The reaction mixture is stirred for 10 hours at a temperature of 70°C. After cooling to room temperature, the reaction mixture is diluted with H ₂ O/Et ₂ O (1:1, 40 ml) and the resulting layers are separated. The aqueous layer is extracted several times with Et ₂ O. The combined organic layers are dried over MgSO ₄ , filtered and the solvent is evaporated in vacuo. The residue is purified on silica gel (DCM/MeOH = 95:5) to give Alloc-NH-(CH ₂ ) ₂ -NH-CH ₂ -CONH-(CH ₂ ) ₂ -STrt.

Yield: 94 mg (0.186 mmol, 82%, contains residual DMF)

MS Alloc-NH-(CH ₂ ) ₂ -NH-CH ₂ -CONH-(CH ₂ ) ₂ -STrt: 526.8 = [M+Na] ⁺ , (Calculated Mm = 503.8 g/mol)

Alloc-NH-(CH ₂ ) ₂ -NH-CH ₂ -CONH-(CH ₂ ) ₂ -STrt (94 mg, 0.186 mmol, with DMF remaining) was dissolved in CH ₂ CI ₂ and Boc ₂ O ( 81 mg, 0.37 mmol). The solution is stirred for 20 hours at room temperature. Upon completion of the reaction, the reaction mixture was quenched by adding 100 μl of AcOH and the solvent was evaporated under vacuum. The residue is diluted with 4 ml MeCN/H ₂ O (25:75, in the presence of 0.1% TFA) and after purification by reverse phase HPLC, Alloc-NH-(CH ₂ ) ₂ -N(Boc)-CH ₂ -CONH is obtained -(CH ₂ ) ₂ -STrt.

Yield: 34.7 mg (0.057 mmol, 26%)

MS Alloc-NH-(CH ₂ ) ₂ -N(Boc)-CH ₂ -CONH-(CH ₂ ) ₂ -STrt: 603.9 = [M+Na] ⁺ (Calculated Mm = 603.9 g/mol)

Alloc-NH-(CH ₂ ) ₂ -N(Boc)-CH ₂ -CONH-(CH ₂ ) ₂ -STrt (34.7 mg, 0.048 mmol) was dissolved in THF, triethylammonium formate (38 μl) and Pd ( PPh ₃ ) ₄ (5 mg). The solution is stirred for 12 hours at room temperature with mass spectrometric monitoring of the reaction progress. Upon completion of the reaction, the solvent is evaporated under vacuum. The residue was dissolved in MeCN/H ₂ O (50:50, with 0.1% TFA) and purified by reverse phase HPLC to give H ₂ N-(CH ₂ ) ₂ -N(Boc)-CH ₂ -CONH-( CH ₂ ) ₂ -STrt connections 35.

Yield: 12.6 mg (0.019 mmol, 42%, as trifluoroacetate salt)

MS 35: 520.1 = [M+H] ⁺ , 542.2 = [M+Na] ⁺ , (Calculated Mm = 519.2 g/mol)

Example 36

Synthesis of BNP linker thiols (36a and 36b)

36a is obtained according to the method of synthesis of 30a, except that instead of 28, 35 is used.

Yield: 9.1 mg

MS 36a: m/z 930.0 = [M+4H] ⁴⁺ , 1239.6 = [M+3H] ³⁺ (Calculated Mm = 3715.9 g/mol)

36b is prepared according to the procedure for the synthesis of 30b, except that compound 35 is used instead of compound 28.

Yield: 8.0 mg

MS 36b: m/z 929.5 = [M+4H] ⁴⁺ , 1239.5 = [M+3H] ³⁺ (Calculated Mm = 3715.9 g/mol)

Example 37

Synthesis of PEG40kDa-linker-BNP conjugates (31a and 31b)

Compound 36a (4.2 mg) was dissolved in a 1:1 H ₂ O/MeCN mixture containing 0.1% TFA (200 μl). Add a solution of PEG40kDa-maleimide (68 mg) in a mixture of H ₂ O/MeCN, 1:1 (1 ml) and phosphate buffer (20 μl, pH 7.4, 0.5 M). The resulting solution is incubated at room temperature, after 5 min AcOH (20 μl) is added and 37a is purified by ion exchange chromatography, salted out and lyophilized.

Yield: 16 mg

37b is synthesized in the same manner as 37a, except that 36b is used instead of 36a.

Yield: 18.5 mg

Example 38

Synthesis of linker-exendin conjugates

Linker-exendin conjugates are prepared according to general synthetic methods C, D, E or F.

Method A

Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2 mmol) are dissolved in 0.3 ml of dry DMF. The resulting mixture was added to the side chain protected exendin-4 resin (2 μmol) and stirred for 30 min at room temperature. The resin is washed with DMF (10 times). RuBOP (0.1 mmol) and diamine (0.1 mmol) are dissolved in 0.3 ml of dry DMF. The resulting mixture is introduced into the resin and stirred for 30 minutes at room temperature. The resin is washed with DMF (10 times). The resulting exendin-linker conjugates are cleaved from the resin and purified by reverse phase chromatography according to the procedure described in the “Materials and Methods” section.

Method B

The synthesis was carried out according to Method A except that diacid (0.2 mmol), HOBt (0.2 mmol), DIC (0.2 mmol), and collidine (0.4 mmol) were used instead of diacid anhydride and pyridine.

Method C

Synthesis: diamine (0.6 mmol) is dissolved in 1 ml of dry DCM and diacid anhydride (0.4 mmol) is added to the resulting solution. The mixture is stirred for 60 minutes at room temperature. The DCM is evaporated, the residue is dissolved in a mixture of ACN/water/AcOH, the resulting amino acid is purified by reverse phase chromatography and lyophilized.

The resulting amino acid (0.1 mmol), HOBt (0.1 mmol), DIC (0.1 mmol) and collidine (0.2 mmol) were dissolved in 0.3 ml of dry DMF. The resulting mixture was added to exendin-4 resin (2 μmol) and stirred for 30 min at room temperature. The resin is washed with DMF (10 times). The resulting exendin-linker conjugates are cleaved from the resin and purified by reverse phase chromatography according to the procedure described in the “Materials and Methods” section.

Method D

Synthesis: Diacid anhydride (0.2 mmol) and pyridine (0.2 mmol) are dissolved in 0.3 ml of dry DMF. The resulting mixture was added to exendin-4 resin (2 μmol) and stirred for 30 min at room temperature. The resin is washed with DMF (10 times). RuBOR (0.1 mmol), HOBt (0.1 mmol) and collidine (0.4 mmol) are dissolved in 0.3 ml of dry DMF. The resulting mixture is introduced into the resin and stirred vigorously for 30 minutes at room temperature. The resin is washed with DMF (10 times). Diamine (0.1 mmol) and DIEA (0.3 mmol) are dissolved in a mixture of 0.4 ml DMF and 0.4 ml ethanol. The resulting mixture is added to the resin and stirred for 30 minutes at room temperature. The resin is washed with DMF (10 times). The resulting exendin-linker conjugates are cleaved from the resin and purified by reverse phase chromatography according to the procedure described in the “Materials and Methods” section.

Method E

Synthesis: Fmoc-amino acid (0.1 mmol), RuBOP (0.1 mmol) and DIEA (0.2 mmol) are dissolved in 0.3 ml of dry DMF. The resulting mixture was added to exendin-4 resin (2 μmol) and stirred for 30 min at room temperature. The Fmoc protecting group is removed by incubating the resin in a mixture of DMF/piperidine, 4/1 (v/v) 2 times for 10 minutes. The resin is washed with DMF (10 times) and DCM (10 times). Paranitrophenyl chloroformate (0.1 mmol) is dissolved in 0.3 ml of dry THF and DIEA (0.2 mmol). The resulting mixture is added to the resin and stirred for 30 minutes at room temperature. The resin is washed with DCM (10 times). Diamine (0.1 mmol) is dissolved in 0.3 ml DMF. The mixture is added to the resin and stirred for 30 minutes at room temperature. The resin is washed with DMF (10 times). The resulting exendin-linker conjugates are cleaved from the resin and purified by reverse phase chromatography according to the procedure described in the “Materials and Methods” section.

Method F

The synthesis is carried out according to Method A, followed by Fmoc deprotection and acetylation: The Fmoc protecting group is removed by incubating the resin in a mixture of DMF/piperidine, 4/1 (v/v) 2 times for 10 min. The resin is washed with DMF (10 times). Acetylation is carried out by incubating the resin in a mixture of acetic anhydride/pyridine/DMF (1/1/2, (v/v/v) for 30 minutes. The resulting resin is washed with DMF (10 times).

The resulting exendin-linker conjugates are cleaved from the resin and purified by reverse phase chromatography according to the procedure described in the “Materials and Methods” section.

More detailed information regarding the synthesized compounds, starting materials, synthesis method, molecular weight (Mm) and mass spectrometry data are given in Fig. 2.

Example 39

Synthesis of hydrogel-linker-exendin conjugates (39)

Maleimide-functionalized hydrogel microparticles are synthesized by the method described in EP 1625856 A1.

30 mg of maleimide-modified hydrogel microparticles (loading 40 μmol/g, 1.2 μmol) are reacted with 6 mg of compound 25a (1.32 μmol, 1.1 equiv) in 600 μl of acetonitrile/50 M phosphate buffer (pH 7 ,4), 20/80 (v/v) for 10 minutes to form hydrogel microparticles 39 loaded with exendin-linker conjugate. The loaded hydrogel 39 is washed 5 times with a 50/50 (v/v) acetonitrile/water mixture and three times with water.

Example 40

Synthesis of linker 40

Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 µl, 1.0 mmol) were dissolved in DCM (2 ml), the resulting mixture was added to 2-chlorotrityl chloride resin (312 mg, 1.3 mmol/g ) and stir for 45 minutes at room temperature. Methanol (0.6 ml) is added and the resin is incubated for another 15 minutes. The resin is washed with DCM (10 times) and DMF (10 times). Fmoc deprotection and urea formation are achieved in accordance with the general synthesis procedure (see section "Materials and Methods") by reacting with linker intermediate 5a, the resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 53 mg

MS of compound 40: m/z 604.4 [M+H] ⁺ (Calculated Mm = 603.8 g/mol)

Example 41

Synthesis of exendin-linker conjugate (41)

Compound 40 (as hydrochloride salt, 14.0 mg, 0.02 mmol), RuBOP (10.2 mg, 0.02 mmol) and DIEA (17 µl, 0.1 mmol) were dissolved in DMF (300 µl), the resulting solution was immediately added to the side-chain protected resin-bound exendin-4 (100 mg, 10 μmol) and incubated for 4 hours at room temperature. The resin is washed with DMF (10x), DCM (10x) and dried under vacuum. The resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 5.4 mg

MS 40: m/z 1510.9 = [M+3H] ³⁺ (Calculated Mm = 4530.1 g/mol)

Example 42

Synthesis of fatty acid-linker conjugate (42)

41 (1.6 mg) is dissolved in 200 μl of acetonitrile/water mixture, 3/1, and compound 1 (0.11 mg) in 200 μl of acetonitrile/water mixture, 3/1 is added to the resulting solution. Then 30 μl of 0.25 M sodium phosphate buffer was added, the reaction mixture was stirred for 5 minutes, after which 42 was purified by reverse phase HPLC.

MS 42: m/z 1870.2 = [M+3H] ³⁺ (Calculated Mm = 5608.4 g/mol).

Example 43

Synthesis of hydrogel-linker-exendin conjugate (43)

43 is obtained according to the synthesis method of 39, except that instead of 25, 41 is used.

Example 44

Linker synthesis (44)

44 is obtained according to the method of synthesis of 40, except that 28 is used instead of 5a.

Yield: 74 mg

MS 44: m/z 605.4 = [M+3H] ³⁺ (Calculated Mm = 604.8 g/mol)

Example 45

Synthesis of exendin-linker conjugate (45)

45 is obtained according to the synthesis method of 41, except that 44 is used instead of 40.

Yield: 6.0 mg

MS 45: m/z 1511.3 = [M+3H] ³⁺ (Calculated Mm = 4531.1 g/mol)

Example 46

Synthesis of fatty acid-linker conjugate (46)

46 is obtained according to the synthesis method of 42, except that 45 is used instead of 41.

MS of compound 46: m/z 1870.5 3 = (Calculated Mm = 5609.5 g/mol).

Example 47

Synthesis of linker intermediate 47

Trityl sulfide (247 mg, 0.89 mmol) was suspended in 1 ml DMSO. DBU (152 μl, 1.02 mmol) and 6-bromohexan-1-ol (173 mg, 0.96) were added and the resulting mixture was stirred for 5 min at room temperature. The reaction mixture is dissolved in 20 ml of ethyl acetate and washed with 1 N H ₂ SO ₄ (2x) and brine (3x). The organic layer is dried (Na ₂ SO ₄ ) and the volatile components are evaporated in vacuo. The resulting product is purified by flash chromatography on silica gel (heptane/AcOEt, 1/1).

Yield: 283 mg (S-trityl)-6-mercaptohexan-1-ol.

(S-Trityl)-6-mercaptohexan-1-ol (466 mg, 1.24 mmol) was dissolved in a solvent mixture containing 3.5 ml DCM, 0.5 ml DMSO and 0.6 ml NEt ₃ and then reacted the mixture is cooled in an ice bath. SO ₃ -pyridine (408 mg, 2.57 mmol) was suspended in 0.5 ml DMSO and added to the reaction mixture. The ice bath was removed and the reaction mixture was stirred for 60 minutes at room temperature. The reaction mixture is then dissolved in 20 ml Et ₂ O and extracted using 1 N H ₂ SO ₄ (2x) and brine (3x). The organic layer is dried (Na ₂ SO ₄ ) and the volatile components are evaporated in vacuo. The resulting product is purified by flash chromatography on silica gel (heptane/AcOEt 1/1).

Yield: 390 mg of compound 47 in the form of (S-trityl)-6-mercaptohexane-1-ala.

MS 47: m/z 243.1 = [Trt] ⁺ 413.1 = [M+K] ⁺ (Calculated Mm = 374.4 g/mol)

Example 48

Linker synthesis (48)

Fmoc-Ala-OH (250 mg, 0.8 mmol) and DIEA (170 µl, 1.0 mmol) were dissolved in DCM (2 ml), the resulting mixture was added to 2-chlorotrityl chloride resin (312 mg, 1.3 mmol/ d) and stir for 45 minutes at room temperature. Methanol (0.6 ml) is added and the resin is incubated for another 15 minutes. The resin is washed with DCM (10x) and DMF (10x). Removal of Fmoc protection and formation of urea is ensured in accordance with the general synthesis procedure (see section “Materials and Methods”) by reacting the resin with ethylenediamine. For reductive alkylation, compound 47 (299 mg, 0.8 mmol) and Na(OAC) ₃ BH (340 mg, 1.6 mmol) were dissolved in 0.5 ml DMF, 0.5 ml methanol and 10 μl AcOH, added to resin and stir for 2 hours at room temperature. The resin is washed with DMF (10x) and DCM (10x). Boc anhydride protection is accomplished by vigorously stirring the resin in a solution containing Boc anhydride (218 mg, 1.0 mmol) and DIEA (170 μl, 1.0 mmol) in DCM. The resin is washed with DCM (10x) and the resulting product is cleaved from the resin and purified by reverse phase HPLC.

Yield: 34 mg

MS 48: m/z 634.2 [M+H] ⁺ (Calculated Mm = 633.9 g/mol)

Example 49

Synthesis of exendin-linker conjugate (49)

49 is obtained according to the synthesis method of 41, except that 48 is used instead of 40.

Yield: 4.8 mg

MS 49: m/z 1487.3 = [M+3H] ³⁺ (Calculated Mm = 4460.0 g/mol)

Example 50

Synthesis of hydrogel-linker-exendin conjugate (50)

50 is obtained according to the method of synthesis of 39, except that instead of 25a, 49 is used.

Example 51

Release kinetics in vitro

Release of drug molecule from 38a-38z, 38aa-38ab, 4, 5, 9a, 9b, 9c, 10, 13a, 15, 19a, 19b, 22, 26a-26c, 31a, 31b, 34a, 34b, 37a, 37b , 42, 43, 46 and 50 are carried out by hydrolysis in a buffer solution at pH 7.4 and 37°C or pH 4 and 37°C, as described in the Materials and Methods section.

Example 52

Correlation analysis of in vivo - in vitro/in vivo release kinetics

In vivo release kinetics were determined by comparing the pharmacokinetic parameters of compound 13a with those of compound 13c and compound 13b with compound 13d, respectively, after intravenous administration in the rat. Animal studies were performed at Heidelberg Pharma AG, Heidelberg, Germany.

Compound 13a (27 mg) was dissolved in 3.5 ml of PBS and 500 μl of the resulting solution was administered intravenously to each of the six test rats. Male SD rats weighing approximately 270 g were used. Blood samples were collected at T = 0.2 hours, 24 hours, 32 hours, 48 hours, 72 hours, 96 hours, 120 hours and 168 hours, after which plasma samples were obtained blood, which were analyzed by fluorescein fluorescence on a Perkin-Elmer LS 50B spectrometer.

The pharmacokinetics of compound 13c was determined according to the procedure described for compound 13a. The pharmacokinetics of compounds 13b and 13d were determined as described for compound 13a, except that 20 mg of compounds 13b and 13d, each in 2.5 ml PBS, and four rats were used.

The hydrolysis half-life of the linker was calculated based on the ratio of fluorescence of compound 13a versus fluorescence of 13c and 13b versus 13d, respectively, at the corresponding time points.

The in vivo hydrolysis half-life of the linker was determined to be 115 and 160 hours for compounds 13a and 13b, respectively, which is an excellent correlation with the results obtained for the in vitro hydrolysis linker half-life of 120 and 160 hours for compounds 13a and 13b, respectively.

Fig. 3 presents data for in vivo and in vitro cleavage of the linker bond with compound 13b, where in vivo (triangles) and in vitro (diamonds) cleavage kinetics are shown in semi-logarithmic form.

Abbreviations:

Acp - 4-(2-aminoethyl) - 1-carboxymethyl-piperazine;

AcOH - acetic acid;

Boc - tert-butoxycarbonyl;

Dab - 2,4-diaminobutyric acid;

DBU - 1,3-diazabicyclo[5.4.0]undecene;

DCM - dichloromethane;

Dda - dodecanoic acid;

DIC - diisopropylcarbodiimide;

DIEA - diisopropylethylamine;

DMAP - dimethylaminopyridine;

DMF - N,N-dimethylformamide;

DMSO - dimethyl sulfoxide;

EDTA - ethylenediaminetetraacetic acid;

Eq - stoichiometric equivalent;

Fmoc - 9-fluorenylmethoxycarbonyl;

HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

HFIP - hexafluoroisopropanol;

HEPES - N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid;

HOBt - N-hydroxybenzotriazole;

ivDde - 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl;

LCMS - liquid chromatography combined with mass spectrometry;

Mal 3-maleimidopropionyl;

Mmt - 4-methoxytrityl;

MS - mass spectrum;

MW - molecular weight;

n.d. - not installed;

PfpOH - pentafluorophenol;

RuBOR-benzotriazol-1-yl-oxy-Tris-pyrrolidinophosphonium hexafluorophosphate;

RP-HPLC - reverse-phase high-performance liquid chromatography (RP-HPLC);

RT - room temperature;

SEC - size exclusion chromatography of molecules;

Suc - succinimidopropionyl;

TCP - 2-chlorotityl chloride resin;

TES - triethylsilane;

TMOV - 2,4,6-trimethoxybenzyl;

TFA - trifluoroacetic acid;

THF - tetrahydrofuran;

UV - ultraviolet;

VIS - visual.

Claims (51)

1. A prodrug or a pharmaceutically acceptable salt thereof, consisting of a drug-linker conjugate D-L, where -D is a nitrogen-containing polypeptide; A -L represents -L ¹ represented by formula (I) where the dotted line shows the attachment to the nitrogen of the polypeptide through the formation of an amide bond; X represents C(R ⁴ R ^4a ), N(R ⁴ ), O, C(R ⁴ R ^4a )-C(R ⁵ R ^5a ), C(R ⁵ R ^5a )-C(R ⁴ R ^4a ) , C(R ⁴ R ^4a )-N(R ⁶ ), N(R ⁶ )-C(R ⁴ R ^4a ), C(R ⁴ R ^4a )-O or OC(R ⁴ R ^4a ); X ¹ represents C; X ² represents C(R ⁷ R ^7a ) or C(R ⁷ R ^7a )-C(R ⁸ R ^8a ); X ³ represents O; R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , R ^4a , R ⁵ , R ^5a , R ⁶ , R ⁷ , R ^7a , R ⁸ , R ^8a are independently selected from the group consisting from H and C _1-4 alkyl; alternatively, one or more pairs of R ^1a /R ^4a , R ^1a /R ^5a , R ^4a /R ^5a , R ^7a /R ^8a form a chemical bond; alternatively, one or more pairs of R ¹ /R ^1a , R ² /R ^2a , R ⁴ /R ^4a , R ⁵ /R ^5a , R ⁷ /R ^7a , R ⁸ /R ^8a , together with the atom to which they are attached, combine with the formation of C _3-7 cycloalkyl or 4-7 membered heterocyclyl; alternatively one or more pairs of R ¹ /R ⁴ , R ¹ /R ⁵ , R ¹ /R ⁶ , R ⁴ /R ⁵ , R ⁴ /R ⁶ , R ⁷ /R ⁸ , R ² /R ³ together with atoms, to which they are attached, combine to form ring A; alternatively, R ³ /R ^3a , together with the nitrogen atom to which they are attached, combine to form a 4-7 membered heterocycle; A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 4-7 membered heterocyclyl and 9-11 membered heterobicyclyl; And where L ¹ is replaced by 1-4 L ² -Z groups, provided that the hydrogen indicated by * in formula (I) cannot be replaced by a substituent, where L ² represents a single chemical bond or alkyl chain having from 1 to 20 carbon atoms, which is optionally interrupted by one or more groups independently selected from -O- and C(O)N(R ^3aa ); optionally substituted with one or more groups independently selected from OH and C(O)N(R ^3aa R ^3aaa ), and wherein R ^3aa , R ^3aaa are independently selected from the group consisting of H and C _1-4 alkyl; And L ² is attached to Z through the end group, selected from And Z is a linear or branched poly(ethylene glycol) with a molecular weight ranging from 2000 to 150,000 Da. 2. The prodrug according to claim 1, wherein X is N(R ⁴ ). 3. The prodrug according to claim 1, where X ² represents C(R ⁷ , R ^7a ). 4. The prodrug according to claim 1, where L ¹ is selected from the group consisting of: where R represents H or C _1-4 alkyl; Y is NH, O or S; a R ¹ , R ^1a , R ² , R ^2a , R ³ , R ^3a , R ⁴ , X, X ¹ , X ² have the meaning specified in paragraph 1. 5. The prodrug according to claim 1, where L ¹ is selected from the group consisting of: where R has the meaning specified in paragraph 4. 6. Prodrug according to claim 1, where L ² has a molecular weight in the range from 14 to 750 g/mol. 7. Prodrug according to claim 1, where L is represented by formula (Ia) where R ⁴ , L ² and Z have the meaning specified in paragraph 1, and where R ^3aa , R ^3aaa are independently selected from the group consisting of H and C _1-4 alkyl, or R ^3aa and R ^3aaa together with a nitrogen atom, to which they are attached, combine to form a 4-7-membered heterocycle. 8. Prodrug according to claim 1, where L is represented by formula (Ib) where R ¹ , R ^1a , R ⁴ , L ² and Z have the meaning specified in paragraph 1, and where R ^3aa represents H or C _1-4 alkyl. 9. A prodrug according to claim 1, wherein R ¹ in formula (I) is L ² -Z. 10. The prodrug according to claim 1, wherein R ³ in formula (I) is L ² -Z. 11. A prodrug according to claim 1, wherein R ³ , R ^3a in formula (I) together with the nitrogen atom to which they are attached combine to form a 4-7 membered heterocycle and where said heterocycle is substituted with L ² -Z. 12. The prodrug according to claim 1, where D-H is a polypeptide selected from the group consisting of adrenocorticotropic hormone ACTH, adenosine deaminase, agalsidase, alpha-1-antitrypsin (AAT), alpha-1-proteinase inhibitor (API), alteplase, amylins , anistreplase, ancrod-serine protease, antibodies, antithrombin III, antitrypsins, aprotinin, asparaginase, atosiban, bifalin, bivalirudin, bone morphogenic proteins, bovine pancreatic trypsin inhibitor (BPTI), cadherin fragments, calcitonin, collagenase, complement inhibitor with esterase activity C1 , conotoxins, cytokine receptor fragments, DNase, dynorphin A, endorphins, enfuvirtide, enkephalins, erythropoietins, exendins, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, fibrinolysin, fibroblast growth factor (FGF), growth hormone-releasing peptide -2 (GHRP2), fusion proteins, follicle-stimulating hormones, gramicidin, ghrelin, desacyl ghrelin, granulocyte colony-stimulating factor (G-CSF), galactosidase, glucagon, glucagon-like peptides, glucocerebrosidase, granulocyte-macrophage colony-stimulating factor (GM-CSF), proteins human heat shock protein (HSP), phospholipase activating protein (PLAP), human chorionic gonadotropin (hCG), hemoglobins, hepatitis B vaccines, hirudin, human serine protease inhibitor, hyaluronidases, iduronidase, immunoglobulins, influenza vaccines, interleukins, receptor antagonist IL-1 (rhIL-Ira), insulins, insulin-like growth factors, insulin-like growth factor binding protein (rhIGFBP), interferons, intracellular adhesion molecule, keratinocyte growth factor (KGF), P-selectin glycoprotein ligand (PSGL), transforming growth factors , lactase, leptin, leuprolide, levothyroxine, luteinizing hormone, Lyme disease vaccine, natriuretic peptides, neuropeptide Y, pancrelipase, pancreatic polypeptide, papain, parathyroid hormone, platelet-derived growth factor (PDGF), pepsin, peptide YY, acetylhydrolase platelet-activating factor (PAF-AH), prolactin, C-protein, thymalfasin, octreotide, secretin, sermorelin, soluble tumor necrosis factor receptor (TNFR), superoxide dismutase (SOD), somatropin, somatoprim, somatostatin, streptokinase, sucrase, terlipressin, tetanus toxin fragment, thylactase, thrombins, thymosin, thyroid hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor coupled to the Fc region of IgG, tissue plasminogen activator (tPA), thyroid stimulating hormone (TSH), urodilatin, urate oxidase, urokinase , vaccines, vascular endothelial growth factor (VEGF), vasoactive intestinal peptide, vasopressin, ziconotide, lectin and ricin. 13. Prodrug according to claim 12, where amylins are selected from amylin and symlin; and/or the antibodies are selected from monoclonal or polyclonal antibodies and fragments or fusion products thereof; and/or calcitonin is salmon calcitonin; and/or interleukins selected from interleukin 1-alpha, 1-beta, 2, 3, 4, 6, 10, 11, 12, 13 and 21; and/or interferons selected from interferon alpha 2a, alpha 2b, alpha 2c, beta 1a, beta 1b, gamma 1a and gamma 1b); and/or natriuretic peptides are selected from ANP, BNP, CNP and fragments thereof.

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