PROCESS FOR THE PREPARATION OF A HYDROGEL, PRO-DRUG CONNECTED TO UMSUPORT AND PHARMACEUTICAL COMPOSI

专利摘要:
"PROMOTIONS IN THE FORM OF HYDROGEL". The present invention relates to a process for the preparation of a hydrogel and to a hydrogel that can be obtained by said process. The present invention further relates to a process for the preparation of a hydrogel-spacer conjugate, to a hydrogel-spacer conjugate obtainable by said process, to a process for the preparation of a prodrug attached to a support and prodrugs attached to a support obtainable by said process, in particular prodrugs attached to a support that provide a controlled or prolonged release of a drug from a support. In addition, the invention relates to the use of hydrogel for the preparation of a prodrug attached to a support.
公开号:BR112015007365B1
申请号:R112015007365-4
申请日:2017-08-22
公开日:2021-02-23
发明作者:Harald Rau；Nicola Bisek；Thomas Knappe；Tobias Voigt；Burkhardt Laufer；Franziska Hahn
申请人:Ascendis Pharma As；
IPC主号:

专利说明:

Field of the Invention
[001] The present invention relates to a process for the preparation of a hydrogel and to a hydrogel that can be obtained by said process. The present invention further relates to a process for the preparation of a hydrogel-spacer conjugate, to a hydrogel-spacer conjugate obtainable by said process, to a process for the preparation of a prodrug attached to a support and prodrugs attached to a support obtainable by said process, in particular prodrugs attached to a support that provide a controlled and / or prolonged release of a drug from a support. In addition, the invention relates to the use of the hydrogel for the preparation of a prodrug attached to a support. Background of the Invention
[002] Conventional hydrogels are three-dimensional polymeric, hydrophilic or amphiphilic networks capable of absorbing large amounts of water. These networks can be composed of several polymers and are insoluble due to the presence of chemical and / or physical covalent cross-links, such as ionic, hydrophobic interactions or entanglements.
[003] Many conventional hydrogels are severely limited in their application. Some hydrogels are used for pharmaceutical applications such as wound closure, tissue engineering or drug delivery. Hydrogels for sealing fabric are disclosed, for example, in International Patent Application Publication No. WO 2008/125655 AI.
[004] In addition, International Patent Application Publication No. WO 99/014259 AI discloses cross-linked PEG hydrogels in which drug molecules are trapped.
[005] The release of drug molecules trapped from similar conventional hydrogels depends on the degradation of the hydrogel and can lead to an explosion release, temporarily causing too high drug levels and also leading to difficulty in predicting drug release. It is desirable to control and / or sustain the release of the drug from a hydrogel. International Patent Application Publication No. WO 06/003014 A2 and No. WO 2011/012715 A1 describe hydrogels as supports in prodrugs attached to a support, in which the biologically active moieties are covalently linked to the hydrogel via reversible prodrugers. Hydrogel-linked prodrugs release the drug with a specific half-life.
[006] However, the hydrogels disclosed in International Patent Application Publication No. WO 2011/012715 A1 are preferably used for the controlled and prolonged release of smaller drug molecules and may not provide sufficient access for larger drug molecules, such as as protein drugs, thereby resulting in a low drug load of similar hydrogels. Detailed Description of the Invention Process for the preparation of a Hydrogel and Hydrogel
[007] Therefore, there is a need for hydrogels that can be used as supports for prodrugs attached to a support, which are suitable for the controlled and prolonged release of larger drug molecules.
[008] Therefore it is an objective of the present invention to overcome at least some of the deficiencies mentioned above and to provide a hydrogel, which can be used as a support for prodrugs attached to a support to which they are suitable for controlled release and / or prolonged use of larger drug molecules.
[009] In one aspect, the present invention relates to a process for the preparation of a hydrogel comprising the steps of: (a) providing a mixture comprising (ai) at least one main chain reagent, wherein at least one reagent the main chain has a molecular weight ranging from 1 to 100 kDa, and comprises at least three amines (-NH2 and / or -NH-); (a-ii) at least one cross-linking reagent, in which at least one cross-linking reagent has a molecular weight ranging from 6 to 40 kDa, at least one cross-linking reagent comprising (i) at least two carbonyloxy groups (- (C = O) -O- or - O- (C = O) -), and additionally (ii) at least two activated final functional groups selected from the group consisting of activated ester groups, activated carbamate groups, activated carbonate groups and activated thiocarbonate groups, and being PEG-based comprising at least 70% PEG; and (a-iii) a first solvent and at least a second solvent, the second solvent of which is immiscible in the first solvent, in a weight ratio of at least one main chain reagent to at least one crosslinking reagent ranging from 1:99 to 99 :1; and (b) polymerize the mixture from step (a) in a suspension polymerization to a hydrogel.
[010] In general, it is known that the design of the crosslinker influences the pore size of a hydrogel, but the expectation was that the longer the crosslinkers were, the more likely they were to form secondary structures which would obstruct access to the internal space of the hydrogel. The aforementioned obstructions would prevent larger protein drugs from penetrating the hydrogel's internal space and the attachment of similar drug molecules would be restricted essentially to the surface and the areas close to the hydrogel's surface. It has now surprisingly been discovered that, despite these expected limitations, large drugs, such as proteins, are able to penetrate the hydrogels of the present invention in amounts that make these hydrogels suitable carriers for prodrugs.
[011] Within the present invention, the terms are used with the following meaning.
[012] As used here, in this patent application, the term "hydrogel" means a hydrophilic or amphiphilic polymeric network composed of homopolymers or copolymers, which is insoluble due to the presence of covalent chemical crosslinks. Physical integrity Hydrogels have a thermodynamic compatibility with water which allows them to swell in an aqueous medium.
[013] As used here, in this patent application, the term "reagent" means a chemical compound which comprises at least one functional group for reaction with the functional group of another reagent or portion.
[014] As used herein, in this patent application, the term "main chain reagent" means a reagent, which is suitable as a starting material for forming hydrogels. As used here, in this patent application, a main chain reagent preferably it does not comprise biodegradable bonds A main chain reagent may comprise a "branching nucleus" which refers to an atom or a portion to which more than one other portion is attached.
[015] As used herein, in this patent application, the term "crosslinking reagent" means a linear or branched reagent, which is suitable as a raw material for crosslinking main chain reagents. Preferably, the crosslinking reagent is a chemical compound A cross-linking reagent comprises at least two biodegradable bonds.
[016] As used here, in this patent application, the term "portion" means a part of a molecule, which lacks one or more atoms compared to the corresponding reagent. If, for example, a reagent of the formula "HXH" reacts with another reagent and becomes part of the reaction product, the corresponding portion of the reaction product has the structure "HX-" or "-X-", while each "-" indicates attachment to another portion. a biologically active portion is released from a prodrug as a drug.
[017] Therefore, the expression "in bound form" is used to refer to the corresponding portion of a reagent, that is, "lysine in bound form" refers to a lysine portion which lacks one or more atoms in the reagent lysine and is part of a molecule.
[018] As used herein, in this patent application, the term "functional group" means a group of atoms which can react with other functional groups. Functional groups include, but are not limited to, the following groups: carboxylic acid (- (C = O) OH), primary or secondary amine (-NH2, -NH-), maleimide, thiol (-SH), sulfonic acid (- (O = S = O) OH), carbonate, carbamate (-O (C = O) N <), hydroxy (-OH), aldehyde (- (C = O) H), ketone (- (C = O) -), hydrazine (> NN <), isocyanate, isothiocyanate, phosphoric acid (-O (P = O) OHOH), phosphonic acid (- O (P = O) OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine.
[019] As used here, in this patent application, the term "activated functional group" means a functional group, which is connected to an activation group, that is, a functional group has been reacted with an activation reagent. Preferred activated groups include, but are not limited to activated ester groups, activated carbamate groups, activated carbonate groups and activated thiocarbonate groups. Preferred activation groups are selected from formulas ((fi) to (f-vi): where

[020] the dashed lines indicate attachment to the rest of the molecule;
[021] b is 1, 2, 3 or 4; and
[022] XH is Cl, Br, I, or F.
[023] Therefore, a preferred activated ester has the formula
[024] - (C = O) -XF, where
[025] XF is selected from the formulas (f-i), (f-ii), (f-iii), (f-iv), (f- v) and (f-vi).
[026] Therefore, a preferred activated carbamate has the formula
[027] -N- (C = O) -XF, where
[028] XF is selected from the formulas (f-i), (f-ii), (f-iii), (f-iv), (f- v) and (f-vi).
[029] Therefore, a preferred activated carbonate has the formula
[030] -O- (C = O) -XF, where
[031] XF is selected from the formulas (f-i), (f-ii), (f-iii), (f-iv), (f- v) and (f-vi).
[032] Therefore, a preferred activated thioester has the formula
[033] -S- (C = O) -XF, where
[034] XF is selected from the formulas (f-i), (f-ii), (f-iii), (f-iv), (f- v) and (f-vi).
[035] Therefore, an "activated end functional group" is an activated functional group which is located at the end of a portion or molecule, that is, it is a terminal activated functional group.
[036] As used here, in this patent application, the term "capping group" means a portion to which it is irreversibly connected, that is, permanently, to a functional group in order to render it unable to react with functional groups of other reagents or portions.
[037] As used here, in this patent application, the term "protection group" means a portion which is reversibly connected to a functional group in order to render it unable to react with, for example, another functional group Suitable alcohol protection groups (-OH) are, for example, acetyl, benzoyl, benzyl, β-methoxyethoxymethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydropyranyl, tetrahydropyranyl. trimethylsilyl, tert-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, tri-isopropylsilyl ether, methyl ether, and ethoxyethyl ether Suitable amine protection groups are, for example, carbobenzyloxy, p-methoxybenzyl carbonyl, tert-butyloxycarbonyl, tert-butyloxycarbonyl, tertiary butyloxycarbonyl acetyl, benzoyl, benzyl, carbamate, 7-methoxybenzyl, 3,4-dimethoxybenzyl, 7-methoxyphenyl, and tosyl Suitable carbonyl protection groups are, for example, acetals and ketals, acylals and dithianes. Suitable carboxylic acid protecting groups are, for example, methyl esters, benzyl esters, tert-butyl esters, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6.-di-tert-butylphenol, silyl esters , orthoesters, and oxazoline. Suitable phosphate protection groups are, for example, 2-cyanoethyl and methyl.
[038] As used here, in this patent application, the terms "elaborate" and "elaboration" refer to the series of manipulations necessary to isolate and purify the one or more products from a chemical reaction, in particular a polymerization.
[039] As used here, in this patent application, the term "polymer" means a molecule comprising structural repeating units, that is, monomers, connected by chemical bonds in a linear, circular, branched, reticulated or dendrimeric way or a combination thereof, which may be of synthetic or biological origin or a combination of both. It is understood that a polymer may also comprise, for example, functional groups or capping portions. Preferably, a polymer has a molecular weight of at least 0.5 kDa, for example, a molecular weight of at least 1 kDa, a molecular weight of at least 2 kDa, a molecular weight of at least 3 kDa or a molecular weight of at least 5 kDa.
[040] As used herein, in this patent application, the term "polymeric" means a reagent or a portion comprising one or more polymers.
[041] A person skilled in the art understands that the polymerization products obtained from a polymerization reaction do not all have the same molecular weight, but instead have a molecular weight distribution. Consequently, the molecular weight ranges, the molecular weights, the monomer number ranges in a polymer and the monomer numbers in a polymer as used here, in this patent application, refer to the average number of molecular weight and the average number of monomers. As used herein, in this patent application, the term "average molecular weight number" means the ordinary arithmetic mean of the molecular weights of the individual polymers.
[042] As used herein, in this patent application, the term "polymerization" or "polymerize" means the process of reacting monomer or macromonomer reagents in a chemical reaction to form polymer chains or networks including, but not limited to, hydrogels .
[043] As used here, in this patent application, the term "macromonomer" means a molecule that was obtained from the polymerization of monomer reagents.
[044] As used here, in this patent application, the term "condensation polymerization" or "condensation reaction" means a chemical reaction, in which the functional groups of two reagents react to form a single molecule, that is, the product of the reaction, and a low molecular weight molecule, for example water, is released.
[045] As used here, in this patent application, the term "suspension polymerization" means a heterogeneous and / or biphasic polymerization reaction, in which the monomer reagents are dissolved in a first solvent, forming the dispersed phase which is emulsified in a second solvent, forming the continuous phase. In the present invention, monomer reagents are at least one main chain reagent and at least one crosslinking reagent. Both the first solvent and the monomer reagents are not soluble in the second solvent. The emulsion referred to is formed by stirring, shaking, exposure to ultrasound or Microsieve ™ emulsification, more preferably by stirring or Microsieve ™ emulsification and more preferably by stirring. This emulsion is stabilized by an appropriate emulsifier. Polymerization is initiated by the addition of a base as an initiator which is soluble in the first solvent. A suitable base commonly known suitable as a primer to be a tertiary base, such as tetramethylethylenediamine (TMEDA).
[046] As used here, in this patent application, the term "immiscible" means the property where two substances capable of combining to form a homogeneous mixture.
[047] As used herein, in this patent application, the term "polyamine" means a reagent or moiety comprising more than one amine (-NH- and / or -NH2), for example, from 2 to 64 amines, the from 4 to 48 amines, from 6 to 32 amines, from 8 to 24 amines, or from 10 to 16 amines Particularly preferred polyamines comprise from 2 to 32 amines.
[048] As used herein, in this patent application, the term "PEG-based comprising at least X% PEG" in relation to a portion or a reagent means that said portion or said reagent comprises at least X% (by weight / weight) of ethylene glycol units (-CH2CH2O-), in which the ethylene glycol units can be arranged en bloc, alternately or can be randomly distributed within the portion or reagent and preferably all ethylene glycol units said portion or said reagent are present in a block; the remaining weight percentage of the PEG-based portion or reagent are other portions specially selected from the following substituents and bonds: • C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 cycloalkyl, 4-heterocyclyl to 7 members, 8 to 11 membered heterobicyclyl, phenyl; naphthyl; indenil; indanyl; and tetralinyl; and • selected connections between the group comprising
where dashed lines indicate attachment to the remainder of the portion or reagent, and R1 and R1a are independently selected from H and C1-6 alkyl.
[049] As used herein, in this patent application, the term "C1-4 alkyl" alone or in combination means a straight or branched alkyl group having 1 to 4 carbon atoms. If present at the end of a molecule, examples of straight and branched C1-4 alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two portions of a molecule are linked by the C1-4 alkyl group, then examples for similar C1-4 alkyl groups are -CH2-, -CH2-CH2-, -CH (CH3) -, -CH2-CH2-CH2-, -CH (C2H5) -, - C (CH3) 2-, -CH2-CH2-CH2-CH2-, and -CH2-CH2-CH2 (CH3) -. Each hydrogen atom in a C1-4 alkyl group can be replaced by a substituent as defined below.
[050] As used herein, in this patent application, the term "C1-6 alkyl" alone or in combination means a straight or branched alkyl group having 1 to 6 carbon atoms. If present at the end of a molecule, examples of straight and branched C1-6 alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2 -methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When two portions of a molecule are linked by the C1-6 alkyl group, then examples for similar C1-6 alkyl groups are -CH2-, -CH2-CH2-, -CH (CH3) -, -CH2-CH2-CH2-, -CH (C2H5) - and -C (CH3) 2-. Each hydrogen atom in a C1-6 alkyl group can be replaced by a substituent as defined below.
[051] Accordingly, as used herein, in this patent application, the term "C1-20 alkyl" alone or in combination means a straight or branched alkyl group having 1 to 20 carbon atoms. The term "C8-18 alkyl" alone or in combination means a straight or branched alkyl group having 8 to 18 carbon atoms. Accordingly, as used herein, in this patent application, the term "C1-50 alkyl" alone or in combination means a straight or branched alkyl group having 1 to 50 carbon atoms. Each hydrogen atom of a C1-20 alkyl group, a C8-18 alkyl group and C1-50 alkyl group can be replaced by a substituent. In each case the alkyl group can be present at the end of a molecule or two portions of a molecule can be connected by the alkyl group.
[052] As used herein, in this patent application, the term "C2-6 alkenyl" alone or in combination means a straight or branched hydrocarbon moiety comprising at least one carbon-carbon double bond having 2 to 6 carbon atoms. If present at the end of a molecule, examples are -CH = CH2, -CH = CH-CH3, -CH2-CH = CH2, -CH = CHCH2-CH3 and - CH = CH-CH = CH2. When two portions of a molecule are linked by the C2-6 alkenyl group, then an example for a similar C2-6 alkenyl is -CH = CH-. Each hydrogen atom in a C2-6 alkenyl group can be replaced by a substituent as defined below. Optionally, one or more triple bonds can occur.
[053] Therefore, as used herein, in this patent application, the term "C2-20 alkenyl" alone or in combination means a straight or branched hydrocarbon residue comprising at least one carbon-carbon double bond having 2 to 20 atoms of carbon. The term "C2-50 alkenyl" alone or in combination means a straight or branched hydrocarbon residue comprising at least one carbon-carbon double bond having 2 to 50 carbon atoms. If present at the end of a molecule, examples are -CH = CH2, -CH = CH-CH3, -CH2-CH = CH2, -CH = CHCH2-CH3 and -CH = CH-CH = CH2. When two portions of a molecule are linked by the alkenyl group, then an example is, for example, -CH = CH-. Each hydrogen atom of a C2-20 alkenyl or C2-50 alkenyl group can be replaced by a substituent as defined below. Optionally, one or more triple bonds can occur.
[054] As used herein, in this patent application, the term "C2-6 alkynyl" alone or in combination means straight or branched hydrocarbon residue comprising at least one carbon-carbon triple bond having 2 to 6 carbon atoms. If present at the end of a molecule, examples are -CHCH, -CH2-C = CH, CH2-CH2-C = CH and CH2-CHC-CH3. When two portions of a molecule are linked by the alkynyl group, then an example is: -C = C-. Each hydrogen atom in a C2-6 alkynyl group can be replaced by a substituent as defined below. Optionally, one or more double bonds can occur.
[055] Therefore, as used herein, in this patent application, the term "C2-20 alkynyl" alone or in combination means a straight or branched hydrocarbon residue comprising at least one carbon-carbon triple bond having 2 to 20 atoms carbon and "C2-50 alkynyl" alone or in combination means a straight or branched hydrocarbon residue comprising at least one carbon-carbon triple bond having 2 to 50 carbon atoms. If present at the end of a molecule, examples are -C ^ CH, -CH2-C = CH, CH2-CH2-CHCH and CH2-C = C-CH3. When two portions of a molecule are linked by the alkynyl group, then an example is -C ^ C-. Each hydrogen atom of a C2-20 alkynyl or C2-50 alkynyl group can be replaced by a substituent as defined below. Optionally, one or more double bonds can occur.
[056] As used herein, in this patent application, the terms "C3-8 cycloalkyl" or "C3-8 cycloalkyl ring" means a cyclic alkyl chain having 3 to 8 carbon atoms, which can be saturated or unsaturated, for example example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl.Each hydrogen atom of a cycloalkyl carbon can be replaced by a substituent as defined below. The term "C3-8 cycloalkyl" or "C3-8 cycloalkyl ring "also includes bicycles forming bridges such as norbonane or norbonene. Therefore," C3-5 cycloalkyl "means a cycloalkyl having 3 to 5 carbon atoms and" C3-10 cycloalkyl "means a cycloalkyl having 3 to 10 carbon atoms.
[057] Therefore, as used here, in this patent application, the term "C3-10 cycloalkyl" means a carbocyclic ring system having 3 to 10 carbon atoms, which can be saturated or unsaturated, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl The term "C3-10 cycloalkyl" also includes carbomono- and - bicycles at least partially saturated.
[058] As used here, in this patent application, the term "halogen" means fluoro, chloro, bromo or iodo. Particularly preferred is fluoro or chloro.
[059] As used herein, in this patent application, the term "4- to 7-membered heterocycly" or "4- to 7-membered heterocycle" means a ring with 4, 5, 6 or 7 ring atoms that can contain up to maximum number of double bonds (aromatic or non-aromatic ring which is totally, partially or unsaturated) in which at least one ring atom up to 4 ring atoms are replaced by a hetero atom selected from the group consisting of sulfur (including - S (O) -, -S (O) 2-), oxygen and nitrogen (including = N (O) -) and where the ring is attached to the rest of the molecule via a carbon or nitrogen atom. Examples for heterocycles 4-7 members include, but are not limited to, azetidine, oxethane, tietan, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, isothiazoline thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazole dine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazol, tetrazoline, tetrazoline, tetrazoline, tetrazoline, tetrazole azepine and homopiperazine. Each hydrogen atom of a 4- to 7-membered heterocyclyl or 4- to 7-membered heterocyclic group can be replaced by a substituent as defined below.
[060] As used herein, in this patent application, the term "8 to 11-membered heterobicyclic" or "8 to 11-membered heterobicycline" means a two-ring heterocyclic system with 8 to 11 ring atoms, where at least one ring atom is shared by both rings and can contain up to the maximum number of double bonds (aromatic or non-aromatic ring which is totally, partially or unsaturated) in which at least one ring atom up to 6 ring atoms be 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 attached to the rest of the molecule through a carbon or nitrogen atom Examples for an 8 to 11 membered heterobicycle are indole, indoline, benzo furan, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, di- hydroquinazoline, quinoline, dihydroquinoline, tetrahydrochloride inoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine and pteridine. The term 8 to 11-membered heterobicycles also includes two-ring spiro structures such as 1,4-dioxa- 8-azaspiro [4.5] decane or linked heterocycles such as 8-aza-bicycle [3.2.1] octane. Each hydrogen atom of an 8 to 11 membered heterobicyclyl group or 8 to 11 membered heterobicyclic group can be replaced by a substituent as defined below.
[061] The term "substituted" means that one or more - H atoms of a molecule or portion are replaced by a different atom or a group of atoms, which are referred to as "substituent". Suitable substituents are selected from the group consisting of halogen; CN; COOR9; OR9; C (O) R9; C (O) N (R9R9a); S (O) 2N 9 9a 9 9a 9 9 9 9a 9b 9 (RR); S (O) N ( RR); S (O) 2R; S (O) R; N (R) S (O) 2N (RR); SR; N (R9R9a); N02; OC (O) R9; N (R9) C (O ) R9a; N (R9) S (O) 2R9a; N (R9) S (O) R9a; N (R9) C (O) OR9a; N (R9) C (O) N (R9aR9b); OC (O) N (R9R9a); T; C1-50 alkyl; C2-50 alkenyl; or C2-50 alkynyl, where T; C1-50 alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally substituted with one or more R10 , which are the same or different and where C1-50 alkyl, C2-50 alkenyl, and C2-50 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 (R11) -; - S (O) 2N (R11) -; -S (O) N (R11) -; -S (O) 2 -; -S (O) -; -N (R11) S (O) 2N (R11a) -; -S-; - N (R11) -; -OC (O) R11; -N (R11) C (O) -; -N (R11) S (O) 2-; -N (R11) S (O) -; - N (R11) C (O) O-; -N (R11) C (O) N (R11a) -; and -OC (O) N (R11R11a); on what
[062] R9, R9a, R9b are independently selected from the group consisting of H; T; and C1-50 alkyl; C2-50 alkenyl; or C2-50 alkynyl, where T; C1-50 alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally substituted with one or more R10, which are the same or different and where C1-50 is alkyl; C2-50 alkenyl; and C2-50 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 (R11) -; -S (O) 2N (R11) -; -S (O) N (R11) -; -S (O) 2-; -ONLY)-; -N (R11) S (O) 2N (R11a) -; -S-; -N (R11) -; -OC (O) R11; -N (R11) C (O) -; - N (R11) S (O) 2-; -N (R11) S (O) -; -N (R11) C (O) O-; -N (R11) C (O) N (R11a) -; and - OC (O) N (R11R11a);
[063] T is selected from the group consisting of phenyl; naphthyl; indenyl; indanila; tetralinyl; C3-10 cycloalkyl; 4- to 7-membered heterocyclyl; or 8 to 11 membered heterobicyclyl, where T is optionally substituted with one or more R10, which are the same or different;
[064] R10 is halogen; CN; oxo (= O); COOR12; OR12; C (O) R12; 12 12a 12 12a 12 12a 12 12 C (O) N (R R); S (O) 2N (R R); S (O) N (R R); S (O) 2R; S (O) R; N (R12) S (O) 2N (R12aR12b); SR12; N (R12R12a); NO2; OC (O) R12; N (R12) C (O) R12a; N (R12) S (O) 2R12a; N (R12) S (O) R12a; N (R12) C (O) OR12a; N (R12) C (O) N (R12aR12b); OC (O) N (R12R12a); or C1-6 alkyl, wherein C1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;
[065] R11, R11a, R12, R12a, R12b are selected independently from the group consisting of H; or C1-6 alkyl, where C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.
[066] In a mode R9, R9a, R9b can be independently of each other H.
[067] In a modality R10 is C1-6 alkyl.
[068] In a T modality it is phenyl.
[069] Preferably, a maximum of 6 -H atoms of a molecule are independently replaced by a substituent, for example, 5 -H atoms are independently replaced by a substituent, 4 -H atoms are independently independently replaced by a substituent, 3 -H atoms are independently replaced by a substituent, 2 -H atoms are independently replaced by a substituent, or 1 -H atom is replaced by a substituent.
[070] As used here, in this patent application, the term "interrupted" means that between two carbon atoms or at the end of a carbon chain between the respective carbon atom and the hydrogen atom one or more atoms are inserted.
[071] As used here, in this patent application, the term "pro-drug" means a compound that undergoes biotransformation before showing its pharmacological effects. Pro-drugs, therefore, can be viewed as biologically active moieties connected to protection groups specialized non-toxic substances used in a transient way to alter or eliminate undesirable properties in the parent molecule, this also includes the reinforcement of desirable properties in the drug and the suppression of undesirable properties.
[072] As used here, in this patent application, the term "prodrug attached to a support" means a prodrug that contains a temporary linkage of a biologically active moiety to a transient support group that produces physiochemical or pharmacokinetic properties improved and can be easily removed in vivo, usually by hydrolytic cleavage.
[073] As used herein, in this patent application, the term "reversible prodruger portion" means a portion which on one end is attached to a biologically active portion D via a reversible connection and on the other end is attached via a permanent bond, which in the present invention is formed by the reaction of an amine functional group of a main chain portion or Ax2 with Ayl, thereby linking the biologically active portion to the hydrogel support in the prodrugs attached to a support of the present invention. A "reversible bond" is a bond that is non-enzymatically hydrolytically degradable, that is, cleavable, under physiological conditions (aqueous buffer at pH 7.4, 37 ° C) with a half-life varying from one hour to twelve months.
[074] In contrast, a "permanent bond" is non-enzymatically hydrolytically degradable under physiological conditions (aqueous buffer at pH 7.4, 37 ° C) with half-lives of more than twelve months.
[075] A "biodegradable bond" is a bond that is enzymatically and / or non-enzymatically hydrolytically degradable, that is, cleavable, under physiological conditions (aqueous buffer at pH 7.4, 37 ° C) with a half-life varying at from one hour to twelve months. Preferably, moreover, a biodegradable bond is non-enzymatically hydrolytically degradable under physiological conditions.
[076] As used here, in this patent application, the term "traceless prodruger" means a reversible prodruger which after cleavage releases the drug in its free form. patent, the term "free form" of a drug means the drug in its pharmacologically active, unmodified form.
[077] As used herein, in this patent application, the term "peptide" means a short polymer of amino acid monomers linked by peptide bonds. The term "polypeptide" means a peptide comprising up to and including 50 amino acid monomers. The term "protein" means a peptide of more than 50 amino acid monomers.
[078] As used here, in this patent application, the term "oligonucleotide" means a short nucleic acid polymer of up to 100 bases.
[079] As used herein, in this patent application, the term "pharmaceutical composition" means one or more active ingredients, and one or more inert ingredients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or dissociation of one or more of the ingredients, or other types of reactions or interactions of one or more of the ingredients. drug attached to a support of the present invention and one or more pharmaceutically acceptable excipients.
[080] As used herein, in this patent application, the term "excipient" refers to a diluent, an adjuvant, or a vehicle with which the drug is administered. The pharmaceutical excipient may be sterile liquids, such as water and oils, including petroleum oils, of animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil and the like . Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol are preferably employed as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol , Propylene, glycol, water, ethanol and the like. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, pH buffering agents, such as, for example, acetate, succinate, tris, carbonate, phosphate, HEPES (4- (2-hydroxyethyl acid) - 1- piperazine ethanesulfonic), MES (2- (N-morpholino) ethanesulfonic acid), or may contain detergents, such as Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids such as, for example, glycine, lysine, or histidine. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, prolonged release formulations and the like. The pharmaceutical composition can be formulated as a suppository, with traditional binders and excipients such as triglycerides. Oral formulation can include routine excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" by EW Martin. The said compositions will contain a therapeutically effective amount of the drug or the biologically active portion, along with a suitable amount of excipient so as to provide the form for proper administration to the patient. The formulation must be suitable for the mode of administration.
[081] In general the term "comprise" or "comprising" also encompasses "consist of" or "consisting of".
[082] Some of the main chain and crosslinker reagents, which can be used as raw material in step (a) of the process are commercially available. In addition, main chain and crosslinker reagents can be prepared according to a method described in the Examples section. A method for the synthesis of a suitable backbone reagent is described in Example 1 of International Patent Application Publication No. WO2011 / 012715A1, which is incorporated by reference here, into this patent application. Example 2 of International Patent Application Publication No. WO2011 / 012715A1 further provides methods for the synthesis of crosslinker reagents of a lower molecular weight whose methods can be changed using routine chemistry knowledge to obtain crosslinker reagents suitable for the present invention. Based on these methods, a person skilled in the art is able to apply routine chemistry knowledge to obtain the main chain and crosslinker reagents used in the present invention.
[083] The mixture from step (a) comprises a first solvent and at least a second solvent. The first solvent referred to is preferably selected from the group comprising dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol and water and mixtures thereof.
[084] At least one main chain reagent and at least one crosslinking reagent are dissolved in the first solvent, that is, the dispersed phase of the suspension polymerization. In one embodiment, the main chain reagent and the crosslinking reagent are dissolved separately, that is, in different containers, using either the same solvent or different solvent and preferably using the same solvent for both reagents. In another embodiment, the main chain reagent and the crosslinking reagent are dissolved together, that is, in the same container and using the same solvent.
[085] A suitable solvent for the main chain reagent is an organic solvent. Preferably, the solvent is selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol and water and mixtures thereof. . Most preferably, the main chain reagent is dissolved in a solvent selected from the group comprising acetonitrile, dimethyl sulfoxide, methanol or mixtures thereof. Most preferably, the main chain reagent is dissolved in dimethylsulfoxide.
[086] In one embodiment the main chain reagent is dissolved in the solvent at a concentration ranging from 1 to 300 mg / ml, more preferably from 5 to 60 mg / ml and most preferably from 10 to 40 mg / ml.
[087] A suitable solvent for the crosslinker reagent is an organic solvent. Preferably, the solvent is selected from the group comprising dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethyl sulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol, water or mixtures thereof. More preferably, the cross-linking reagent is dissolved in a solvent selected from the group comprising dimethylformamide, acetonitrile, dimethyl sulfoxide, methanol or mixtures thereof. Most preferably, the cross-linking reagent is dissolved in dimethylsulfoxide.
[088] In one embodiment the cross-linking reagent is dissolved in the solvent at a concentration ranging from 5 to 500 mg / ml, more preferably from 25 to 300 mg / ml and most preferably from 50 to 200 mg / ml ml.
[089] At least one main chain reagent and at least one crosslinking reagent are mixed in a weight ratio ranging from 1:99 to 99: 1, for example, in a weight ratio ranging from 2:98 to 90:10, in a weight ratio ranging from 3:97 to 88:12, in a weight ratio ranging from 3:96 to 85:15, in a weight ratio ranging from 2: 98 to 90:10 and in a weight ratio ranging from 5:95 to 80:20; particularly preferred in a weight ratio from 5:95 to 80:20, where the first number refers to the main chain reagent and the second number to the crosslinker reagent.
[090] Preferably, the proportions are selected in such a way that the mixture of step (a) comprises a molar excess of amine groups of the main chain reagent compared to the activated final functional groups of the crosslinker reagent. Consequently, the hydrogel resulting from the process of the present invention has free amine groups which can be used to attach other portions to the hydrogel, such as spacers, affinity binders, chelators and / or reversible prodrug chain moieties.
[091] At least one second solvent, that is, the continuous phase of the suspension polymerization, is preferably an organic solvent, more preferably an organic solvent selected from the group comprising C5-30 linear, branched or cyclic alkanes; C5-30 linear, branched or cyclic alkenes; C5-30 linear, branched or cyclic alkynes; linear or cyclic poly (dimethylsiloxanes); C6-20 aromatic hydrocarbons; and mixtures thereof. Even more preferably, at least the second solvent is selected from the group comprising C5-16 linear, branched or cyclic alkanes; toluene; xylene; mesitylene; hexamethyldisiloxane; or mixtures thereof. Most preferably, at least the second solvent selected from the group comprising C7-11 linear alkanes, such as heptane, octane, nonane, decane and undecane.
[092] Preferably, the mixture from step (a) additionally comprises a detergent. Preferred detergents are Cithrol DPHS, Hypermer 70A, Hypermer B246, Hypermer 1599A, Hypermer 2296, or Hypermer 1083. The most preferred is Cithrol DPHS.
[093] Preferably, the detergent has a concentration of 0.1 g to 100 g per 1 L of the total mixture, that is, dispersed phase and continuous phase together. More preferably, the detergent has a concentration of 0.5 g to 10 g per 1 L of the total mixture, and most preferably, the detergent has a concentration of 0.5 g to 5 g per 1 L of the total mixture.
[094] Preferably, the mixture from step (a) is an emulsion.
[095] The polymerization in step (b) is started by adding a base. Preferably, the base is a non-nucleophilic base soluble in alkanes, more preferably the base is selected from N, N, N ', N'-tetramethylethylene diamine (TMEDA), 1,4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1,4-diazabicyclo [2.2.2] octane, 1,1,4,7,10, 10-hexamethyltriethylenetetramine, 1,4,7-trimethyl-1,4,7-triazacyclononane, tris [2- (dimethylamino) ethyl ] amine, triethylamine, diisopropylethylamine (DIPEA), trimethylamine, N, N-dimethylethylamine, N, N, N ', N'-tetramethyl-1,6-hexanediamine, N, N, N', N ", N" -pentamethyldiethylenetriamine, 1,8-diaza-bicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [4.3.0] non-5-eno, and hexamethylenetetramine. Even more preferably, the base is selected from TMEDA, 1,4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1,4-diazabicyclo [2.2.2] octane, 1,1,4,7,10, 10-hexamethyltriethylenetetramine , 1,4,7-trimethyl-1,4,7-triazacyclononane, tris [2- (dimethylamino) ethyl] amine, 1,8-diazabicyclo [5.4.0] undec-7-ene, 1,5-diazabicyclo [ 4.3.0] non-5-ene, and hexamethylenetetramine. Most preferably, the base is TMEDA.
[096] The base is added to the mixture of step (a) in an amount of 1 to 500 equivalents per final functional group activated in the mixture, preferably in an amount of 5 to 50 equivalents, more preferably in an amount of 5 to 25 equivalents and most preferably in an amount of 10 equivalents.
[097] In step (b) of the process, the polymerization of the hydrogel of the present invention is a condensation reaction, which preferably occurs under continuous agitation of the mixture from step (a). Preferably, the tip speed (tip speed = π x rotational speed of the stirrer x diameter of the stirrer) varies from 0.2 to 10 meters per second (m / s), more preferably from 0.5 to 4 m / s and more preferably from 1 to 2 m / s.
[098] In a preferred embodiment of step (b), the polymerization reaction is carried out in a cylindrical vessel equipped with bafles. The ratio of the diameter to the height of the vessel can vary from 4: 1 to 1: 2, more preferably the ratio of the diameter to the height of the vessel varies from 2: 1 to 1: 1.
[099] Preferably, the reaction vessel is equipped with an axial flow stirrer selected from the group comprising pitched blade stirrer, marine type propellant, or Lightnin A-310. Most preferably, the stirrer is a sharp blade stirrer.
[0100] Step (b) can be carried out over a wide temperature range, preferably at a temperature from -10 ° C to 100 ° C, more preferably at a temperature of 0 ° C to 80 ° C, even more preferably at a temperature of 10 ° C to 50 ° C and most preferably at room temperature. "Room temperature" refers to the temperature present in a typical laboratory environment and preferably means a temperature ranging from 17 to 25 ° C.
[0101] Preferably, the hydrogel obtained from the polymerization is a shaped article, such as a coating, mesh, stent, nanoparticle or microparticle. More preferably, the hydrogel is in the form of microparticulate beads having a diameter from 1 to 500 micrometers, more preferably with a diameter from 10 to 300 micrometers, even more preferably with a diameter from 20 and 150 micrometers and most preferably with a diameter from 30 to 130 micrometers. The diameters mentioned above are measured when the hydrogel microparticles are fully hydrated in water.
[0102] In one embodiment, the process for preparing a hydrogel of the present invention further comprises the step of: (c) making the hydrogel.
[0103] Step (c) comprises one or more of the following steps: (c1) removing excess liquid from the polymerization reaction, (c2) washing the hydrogel to remove solvents used during the polymerization, (c3) transferring the hydrogel to inside a buffer solution, (c4) fractionation of size / sieving of the hydrogel, (c5) transfer of the hydrogel into a container, (c6) drying of the hydrogel, (c7) transfer of the hydrogel into a specific solvent suitable for sterilization, and (c8) sterilization of the hydrogel, preferably by gamma radiation.
[0104] Preferably, step (c) comprises all of the following steps: (c1) removing excess liquid from the polymerization reaction, (c2) washing the hydrogel to remove solvents used during the polymerization, (c3) transferring the hydrogel to inside a buffer solution, (c4) size fractionation / sieving of the hydrogel, (c5) transfer of the hydrogel into a container, (c7) transfer of the hydrogel into a specific solvent suitable for sterilization, and (c8) sterilization hydrogel, preferably by gamma radiation. The main chain reagent
[0105] At least one main chain reagent has a molecular weight ranging from 1 to 100 kDa, preferably from 2 to 50 kDa, more preferably from 5 to 30 kDa, even more preferably from 5 to 25 kDa and most preferably from 5 to 15 kDa.
[0106] Preferably, the main chain reagent is based on PEG comprising at least 10% PEG, more preferably comprising at least 20% PEG, even more preferably comprising at least 30% PEG and most preferably comprising at least 40% PEG.
[0107] In one embodiment, the main chain reagent is present in the form of its acidic salt, preferably in the form of an acid addition salt. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, cansilate, citrate, edisylate, esilate, format, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hydrochloride, hydrochloride, hydrochloride, hydrochloride hydrobromide, hydroiodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, saccharate, stearate, succinate, tartarate and tosylate. Particularly preferably, the main chain reagent is present in the form of its hydrochloride salt.
[0108] In one embodiment, at least one main chain reagent is selected from the group consisting of a compound of formula (I)
[0109] B (- (A0) x1 - (SP) x2 - A1- P - A2 - Hyp1) x (I), where
[0110] B is a branching core,
[0111] SP is a spacer portion selected from the group consisting of C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl,
[0112] P is a polymeric PEG-based chain comprising at least 80% PEG, preferably at least 85% PEG, more preferably at least 90% PEG and most preferably at least 95% PEG,
[0113] Hyp1 is a portion comprising an amine (-NH2 and / or -NH-) or a polyamine comprising at least two amines (- NH2 and / or -NH-),
[0114] X is an integer from 3 to 16,
[0115] x1, x2 are independently of each other 0 or 1, with the proviso that x1 is 0, if x2 is 0,
[0116] A0, A1, A2 are independently selected from each other among the group consisting of
wherein R1 and R1a are independently selected from H and C1-6 alkyl; a compound of formula (II)
[0117] Hyp2 - A3 - P - A4 - Hyp3 (II), where
[0118] P is defined as above in the compound of formula (I),
[0119] Hyp2, Hyp3 are independently of each other a polyamine comprising at least two amines (-NH2 and / or -NH), and
[0120] A3 and A4 are selected independently from the group consisting of

wherein R1 and R1a are independently selected from H and C1-6 alkyl; a compound of formula (III)
[0121] P1 - A5 - Hyp4 (III), where
[0122] P1 is a polymeric PEG-based chain comprising at least 80% PEG, preferably at least 85% PEG, more preferably at least 90% PEG and most preferably at least 95% PEG,
[0123] Hyp4 is a polyamine comprising at least three in which R1 and R1a are independently selected from H and C1-6 alkyl; and a compound of formula (IV),
[0124] A5 is selected from the group consisting of
wherein R1 and R1a are independently selected from H and C1-6 alkyl; and a compound of formula (IV),
[0125] T1 - A6 - Hyp5 (IV), where
[0126] Hyp5 is a polyamine comprising at least three amines (-NH2 and / or -NH), and
[0127] A6 is selected from the group consisting of
wherein R1 and R1a are independently selected from H and C1-6 alkyl; and T1 is selected from the group consisting of C1-50 alkyl, C250 alkenyl or C2-50 alkynyl, the fragment of which is optionally interrupted by one or more group (s) selected from -NH-, -N (C1-4 alkyl) -, -O-, -S-, -C (O) -, -C (O) NH-, - C (O) N (C1-4 alkyl) -, -O- C (O) -, -S (O) -, -S (O) 2-, 4- to 7-membered heterocyclyl, phenyl or naphthyl.
[0128] In the sections that follow the term "Hypx" refers to Hyp1, Hyp2, Hyp3, Hyp4 and Hyp5 collectively.
[0129] Preferably, the main chain reagent is a compound of formula (I), (II) or (III), more preferably the main chain reagent is a compound of formula (I) or (III), and the most preferably the main chain reagent is a compound of formula (I).
[0130] In a preferred embodiment, in a compound of formula (I), x is 4, 6 or 8. Preferably, in a compound of formula (I), x is 4 or 8, most preferably, x is 4.
[0131] In a preferred embodiment in the compounds of formula (I) to (IV), A0, A1, A2, A3, A4, A5 and A6 are selected from the group comprising

[0132] Preferably, in a compound of formula (I), A1 is

[0133] Preferably, in a compound of formula (I), A2 is
and A4 is
[0134] Preferably, in a compound of formula (III), A5 is

[0135] Preferably, in a compound of formula (II), A3 is

[0136] Preferably, in a compound of formula (III), A5 is

[0137] Preferably, in a compound of formula (IV), A6 is

[0138] Preferably, in a compound of formula (IV), T1 is selected from H and C1-6 alkyl.
[0139] In one embodiment, in a compound of formula (I), the branching nucleus B is selected from the following structures:


[0140] dashed lines indicate attachment to A0 or, if x1 and x2 are both 0, A1,
[0141] t is 1 or 2; preferably t is 1,
[0142] v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; preferably, v is 2, 3, 4, 5, 6; more preferably, v is 2, 4 or 6; most preferably, v is 2.
[0143] In a preferred embodiment, B has a structure of formula (ai), (a-ii), (a-iii), (a-iv), (av), (a-vi), (a-vii ), (a-viii), (a-ix), (ax), (a-xiv), (a-xv) or (a-xvi). More preferably, B has a structure of formula (a-iii), (a-iv), (av), (a-vi), (a-vii), (a-viii), (a-ix), ( ax) or (a-iv). Most preferably, B has a structure of formula (a-xiv).
[0144] A preferred modality is a combination of B and A0, or, if x1 and x2 are both 0, a preferred combination of B and A1, which is selected from the following structures:
where dashed lines indicate attachment to SP or, if x1 and x2 are both 0, to P.
[0145] More preferably, the combination of B and A0 or, if x1 and x2 are both 0, the combination of B and A1, has a formula structure of formula (bi), (b-iv), (b-vi ) or (b-viii) and most preferably it has a formula structure of formula (bi).
[0146] In an embodiment, x1 and x2 of formula (I) are 0.
[0147] In one embodiment, the polymer chain based on PEG P has a molecular weight from 0.3 kDa to 40 kDa; for example, from 0.4 to 35 kDa, from 0.6 to 38 kDa, from 0.8 to 30 kDa, from 1 to 25 kDa, from 1 to 15 kDa or from from 1 to 10 kDa. Most preferably P has a molecular weight from 1 to 10 kDa.
[0148] In one embodiment, the polymer chain based on PEG P1 has a molecular weight from 0.3 kDa to 40 kDa; for example, from 0.4 to 35 kDa, from 0.6 to 38 kDA, from 0.8 to 30 kDa, from 1 to 25 kDa, from 1 to 15 kDa or at from 1 to 10 kDa. Most preferably P1 has a molecular weight from 1 to 10 kDa.
[0149] In one embodiment, in the compounds of formulas (I) or (II), P has a structure of formula (ci):
wherein it does not vary from 6 to 900, more preferably it does not vary from 20 to 700 and most preferably it does not vary from 20 to 250.
[0150] In one embodiment, in the compounds of formulas (III), P1 has a structure of formula (c-ii):
on what
[0151] does not vary from 6 to 900, more preferably does not vary from 20 to 700 and most preferably does not vary from 20 to 250;
[0152] T0 is selected from the group comprising C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, which is optionally interrupted by one or more group (s) selected from -NH-, - N ( C1-4 alkyl) -, -O-, -S-, -C (O) -, -C (O) NH-, -C (O) N (C1-4 alkyl) -, -OC (O) - , - S (O) - or - S (O) 2-.
[0153] In one embodiment, in the compounds of formulas (I) to (IV), the Hypx portion is a polyamine and preferably comprises in a linked form and, where applicable, in R- and / or S- configuration a portion of the formulas ( di), (d-ii), (d-iii) and / or (d-iv):
on what
[0154] z1, z2, z3, z4, z5, z6 are independently of each other 1, 2, 3, 4, 5, 6, 7 or 8.
[0155] More preferably, Hypx comprises in a linked form and in an R- and / or S-lysine configuration, ornithine, diaminopropionic acid and / or diaminobutyric acid. More preferably, Hypx comprises in linked form and in R- and / or S-lysine configuration.
[0156] Hypx has a molecular weight from 40 kDa to 30 kDa, preferably from 0.3 kDa to 25 kDa, more preferably from 0.5 kDa to 20 kDa, even more preferably from 1 kDa to 20 kDa and the most preferably from 2 kDa to 15 kDa.
[0157] Hypx is preferably selected from the group consisting of a portion of formula (ei)
on what
[0158] p1 is an integer from 1 to 5, preferably p1 is 4, and the dotted line indicates attachment to A2 if the main chain reagent has a structure of formula (I) and A3 or A4 if the main chain reagent has a structure of formula (II);
[0159] a portion of formula (e-ii)
on what
[0160] p2, p3 and p4 are identical or different and each is independently of the others an integer from 1 to 5, preferably p2, p3 and p4 are 4, and the dotted line indicates attachment to A2 if the chain reagent main has a structure of formula (I), A3 or A4 if the main chain reagent has a structure of formula (II), A5 if the main chain reagent has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV);
[0161] a portion of formula (e-iii)
on what
[0162] p5 to p11 are identical or different and each is independently of the other an integer from 1 to 5, preferably p5 to p11 are 4, and
[0163] the dashed line indicates attachment to A2 if the main chain reagent is of formula (I), A3 or A4 if the main chain reagent is of formula (II), A5 if the main chain reagent is of formula (III) and A6 if the main chain reagent is of formula (IV);
[0164] a portion of formula (e-iv)
on what
[0165] p12 to p26 are identical or different and each is independently of the other an integer from 1 to 5, preferably p12 to p26 are 4, and
[0166] the dashed line indicates attachment to A2 if the main chain reagent has a structure of formula (I), A3 or A4 if the main chain reagent has a structure of formula (II), A5 if the reagent from main chain has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV);
[0167] a portion of formula (ev)
on what
[0168] p27 and p28 are identical or different and each is independently of the other an integer from 1 to 5, preferably p27 and p28 are 4,
[0169] q is an integer from 1 to 8, preferably q is 2 or 6 and most preferably q is 6, and
[0170] the dashed line indicates attachment to A2 if the main chain reagent has a structure of formula (I), A3 or A4 if the main chain reagent has a structure of formula (II), A5 if the reagent of the main chain has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV);
[0171] a portion of formula (e-vi)
on what
[0172] p29 and p30 are identical or different and each is independently of the other an integer from 2 to 5, preferably p29 and p30 are 3, and
[0173] the dashed line indicates attachment to A2 if the main chain reagent has a structure of formula (I), A3 or A4 if the main chain reagent has a structure of formula (II), A5 if the reagent of the main chain has a structure of formula (III) and the main chain reagent has a structure of formula (IV);
[0174] where

[0175] independent of the other an integer from 2 to 5, preferably p31 to p36 are 3, and the dotted line indicates attachment to A2 if the main chain reagent has a structure of formula (I), A3 or A4 if the reagent the main chain has a structure of formula (II), A5 if the main chain reagent has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV);
[0176] a portion of formula (e-viii)
on what
[0177] p37 to p50 are identical or different and each is independently of the other an integer from 2 to 5, preferably p37 to p50 are 3, and
[0178] the dashed line indicates attachment to A2 if the main chain reagent has a structure of formula (I), A3 or A4 if the main chain reagent has a structure of formula (II), A5 if the reagent of the main chain has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV); and
[0179] a portion of formula (e-ix):
on what
[0180] p51 to p80 are identical or different and each is independently of the other an integer from 2 to 5, preferably p51 to p80 are 3, and
[0181] the dashed line indicates attachment to A2 if the main chain reagent has a structure of formula (I), to A "or A4 if the main chain reagent has a structure of formula (II), to A5 if the reagent the main chain has a structure of formula (III) and A6 if the main chain reagent has a structure of formula (IV); and
[0182] wherein the portions (e-i) to (e-v) can in each chiral center be in either an R- or S- configuration, preferably all the chiral centers of a portion (e-i) to (e-v) are in the same configuration.
[0183] Preferably, Hypx is has a structure of formulas (ei), (e-ii), (e-iii), (e-iv), (e-vi), (e-vii), (e-viii ) or (e-ix). More preferably, Hypx has a structure of formulas (e-ii), (e-iii), (e-iv), (e-vii), (e-viii) or (e-ix), even more preferably Hypx has a structure of formulas (e-ii), (e-iii), (e-vii) or (e-viii) and most preferably Hypx has a structure of formula (e-iii).
[0184] If Hypx comprises in lysine linked form, it is preferable that it comprises D-lysine, because it has surprisingly been found that hydrogels comprising main chain moieties comprising D-lysine - unlike those comprising L-lysine - are more stable when administered to a patient.
[0185] If the main chain reagent has a structure of formula (I), a preferred portion - A2 - Hyp1 is a portion of the formula
where the dashed line indicates attachment to P; and
[0186] E1 is selected from formulas (e-i) to (e-ix).
[0187] If the main chain reagent has a structure of formula (II) a preferred portion Hyp2 - A3 - is a portion of the formula
on what
[0188] the dashed line indicates attachment to P; and
[0189] E1 is selected from formulas (ei) to (e-ix); and a preferred portion - A4 - Hyp3 is a portion of the formula
on what
[0190] the dashed line indicates attachment to P; and
[0191] E1 is selected from formulas (e-i) to (e-ix).
[0192] If the main chain reagent has a structure of formula (III), a preferred portion - A5 - Hyp4 is a portion of the formula
on what
[0193] the dashed line indicates attachment to P1; and
[0194] E1 is selected from formulas (e-i) to (e-ix).
[0195] More preferably, the main chain reagent has a structure of formula (I) and B has a structure of formula (a-xiv).
[0196] Even more preferably, the main chain reagent has a structure of formula (I), B has a structure of formula (a-xiv), x1 and x2 are 0, and A1 is -O-.
[0197] Even more preferably, the main chain reagent has a structure of formula (I), B has a structure of formula (a-xiv), A1 is -O-, and P has a structure of formula (c-i).
[0198] Even more preferably, the main chain reagent is of formula (I), B is of formula (a-xiv), x1 and x2 are 0, A1 is -O- and P is of formula (c-i).
[0199] Even more preferably, the main chain reagent is of formula (I), B is of formula (a-xiv), x1 and x2 are 0, A1 is -O, P is of formula (ci), A2 is -NH- (C = O) - and Hyp1 is of formula (e-iii).
[0200] Most preferably, the main chain reagent has the following formula:
on what
[0201] does not vary from 10 to 40, preferably from 10 to 30, more preferably from 10 to 20.
[0202] Also preferably, n ranges from 20 to 30 kDa and most preferably n is 28.
[0203] SP of formula (I) is a spacer portion selected from the group comprising C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, preferably SP is -CH2-, -CH2-CH2-, -CH ( CH3) -, -CH2- CH2-CH2-, -CH (C2H5) -, -C (CH3) 2-, -CH = CH- or -CH = CH-, most preferably SP is -CH2-, -CH2 -CH2- or -CH = CH-. The Crosslinking Reagent
[0204] At least one cross-linking reagent comprises at least two carbonyloxy groups (- (C = O) -O- or -O- (C = O) -), which are biodegradable bonds. These biodegradable bonds are necessary to make the hydrogel biodegradable. In addition, at least one cross-linking reagent comprises at least two activated final functional groups which during the polymerization of step (b) react with the amines of at least one main chain reagent.
[0205] The crosslinking reagent has a molecular weight ranging from 6 to 40 kDa, more preferably ranging from 6 to 30 kDa, even more preferably varying from 6 to 20 kDa, even more preferably varying from 6 to 15 kDa and most preferably ranging from 6 to 10 kDa.
[0206] The crosslinking reagent comprises at least two activated final functional groups selected from the group comprising activated ester groups, activated carbamate groups, activated carbonate groups and activated thiocarbonate groups, which during polymerization react with the amine groups of the main chain reagents, forming amide bonds, that is, a main chain portion and a crosslinker portion are preferably connected via an amide bond.
[0207] In a preferred embodiment, the cross-linking reagent is a compound of formula (VI):
wherein each D1, D2 D3 and D4 are identical or different and each is independently selected from the group comprising - O-, -NR5-, -S- and -CR6R6a-;
[0208] each R1, R1a, R2, R2a, R3, R3a, R4, R4a, R6 and R6a are identical or different and each is independently selected from the group comprising -H, -OR7, -NR7R7a, -SR7 and C1-6 alkyl; optionally, each of the R1 / R2, R3 / R4, R1a / R2a, and R3a / R4a pair (s) can independently form a chemical bond and / or each of the R1 / R2 / R2a, R3 pairs / R3a, R4 / R4a, R6 / R6a, R1 / R2, R3 / R4, R1a / R2a, and R3a / R4a are independently linked together with the atom to which they are attached to form a C3-8 cycloalkyl or to form an A ring or are joined together with the atom to which they are attached to form a 4 to 7 membered heterocyclyl or 8 to 11 membered heterocyclyl or adamantyl;
[0209] each R5 is independently selected from -H and C1-6 alkyl; optionally, each of the R1 / R5, R2 / R5, R3 / R5, R4 / R5 and R5 / R6 pair (s) can independently form a chemical bond and / or are joined together with the atom to which they are attached to form a 4- to 7-membered heterocyclyl or 8 to 11-membered heterobicyclyl;
[0210] each R7, R7a is independently selected from H and C1-6 alkyl;
[0211] A is selected from the group consisting of indenyl, indanyl and tetralinyl; P2 is
m ranges from 120 to 920, preferably from 120 to 460 and more preferably from 120 to 230;
[0212] r1, r2, r7, r8 are independently 0 or 1;
[0213] r3, r6 are independently 0, 1, 2, 3, or 4;
[0214] r4, r5 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
[0215] s1, s2 are independently 1, 2, 3, 4, 5 or 6;
[0216] Y1, Y2 are identical or different and each is independently of the other selected from formulas (fi) to (f-vi):
on what
[0217] the dashed lines indicate attachment to the rest of the molecule,
[0218] b is 1, 2, 3 or 4
[0219] XH is Cl, Br, I, or F.
[0220] Preferably, the cross-linking reagent is a compound of formula (V-II):
si L -IS2 (V-II). on what
[0221] D1, D2, D3 and D4 are identical or different and each is independently selected from the group comprising -O-, -NR5-, -S- and -CR5R5a-;
[0222] R1, R1a, R2, R2a, R3, R3a, R4, R4a, R5 and R5a are identical or different and each is independently selected from the group comprising H and C1-6 alkyl; optionally, one or more of the R1 / R1a, R2 / R2a, R3 / R3a, R4 / R4a, R1 / R2, R3 / R4, R1a / R2a, and R3a / R4a pair (s) form a chemical bond or are joined together with the atom to which they are attached to form a C3-8 cycloalkyl or to form an A ring, or are joined together with the atom to which they are attached to form a 4 to 7 membered heterocyclyl or 8 to 11 membered heterocyclyl or adamantyl;
[0223] A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetralinyl;
[0224] P2 is
m ranges from 120 to 920, preferably from 120 to 460 and more preferably from 120 to 230;
[0225] r1, r2, r7, r8 are independently 0 or 1;
[0226] r3, r6 are independently 0, 1, 2, 3, or 4;
[0227] r4, r5 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
[0228] s1, s2 are independently 1, 2, 3, 4, 5 or 6;
[0229] Y1, Y2 are identical or different and each is independently of the other selected from formulas (fi) to (f-vi):
where the dashed lines indicate attachment to the rest of the molecule,
[0230] b is 1, 2, 3 or 4
[0231] XH is Cl, Br, I, or F.
[0232] It is understood that the portions
represent at least two activated final functional groups.
[0233] Preferably, Y1 and Y2 of formula (V-I) and (V-II) have a structure of formula (f-i), (f-ii) or (f-v). More preferably, Y1 and Y2 have a structure of formula (f-i) or (f-ii) and most preferably, Y1 and Y2 have a structure of formula (f-i).
[0234] Preferably, both portions Y1 and Y2 of formula (V-I) and (V-II) have the same structure. More preferably, both portions Y1 and Y2 have the structure of formula (f-i).
[0235] Preferably, r1 of formula (V-I) and (V-II) is 0.
[0236] Preferably, r1 and s1 of formula (V-I) and (V-II) are both 0.
[0237] Preferably, one or more of the pairs R1 / R1a, R2 / R2a, R3 / R3a, R4 / R4a, R1 / R2, R3 / R4, R1a / R2a, and R3a / R4a of formula (VI) and (V -II) form a chemical bond or are joined together with the atom to which they are attached to form a C3-8 cycloalkyl or form an A ring.
[0238] Preferably, one or more of the pairs R1 / R2, R1a / R2a, R3 / R4, R3a / R4a of formula (VI) and (V-II) are joined together with the atom to which they are attached to form a 4- to 7-membered heterocyclyl or 8 to 11-membered heterocyclyl.
[0239] Preferably, the crosslinking reagent of formula (VI) and (V-II) is symmetrical, that is, the portion
has the same structure as the portion

[0240] In a preferred mode s1, s2, r1 and r8 of formula (V-I) and (V-II) are 0.
[0241] In another preferred embodiment s1, s2, r1 and r8 of formula (V-I) and (V-II) are 0 and r4 of formula (V-I) and (V-II) and r5 are 1.
[0242] Preferred cross-linking reagents are of the formula (V-1) to (V-54):






each crosslinking reagent may be in the form of its racemic mixture, where applicable; and
[0243] m, Y1 and Y2 are defined as above.
[0244] Even more preferred cross-linking reagents are of the formula (Va-1) to (Va-54):







where each crosslinking reagent may be in the form of its racemic mixture, where applicable; and
[0245] m, Y1 and Y2 are defined as above.
[0246] It has been surprisingly discovered that the use of crosslinking reagents with branches, that is, residues other than H, in the alpha carbon of the carbonyloxy group leads to the formation of hydrogels which are more resistant against enzymatic degradation, such as degradation through esterases.
[0247] Similarly, it was surprisingly found that the fewer atoms between the (C = O) of a carbonyloxy group and the (C = O) of the adjacent activated ester, activated carbamate, activated carbonate or activated thiocarbamate, more resistant to degradation are the resulting hydrogels, as well as more resistant to degradation through esterases.
[0248] Therefore, cross-linking reagents V-11 to V-54, V-1, V-2, Va-11 to Va-54, Va-1 and Va-2 are preferred cross-linking reagents. The cross-linking reagents Va-11 to Va-54, Va-1 and Va-2 are the most preferred cross-linking reagents. The most preferred is the crosslinker reagent Va-14.
[0249] In another embodiment, the crosslinking reagents V-1, V-2, V-5, V-6, V-7, V-8, V-9, V-10, V-11l, V-12, V-13, V-14, V-15, V-16, V17, V-18, V-19, V-20, V-21, V-22, V-23, V-24, V-25, V-26, V-27, V-28, V-29, V-30, V-31, V-32, V-33, V-34, V-35, V-36, V-37, V- 38, V-39, V40, V-41, V-42, V-43, V-44, V-45, V-46, V-47, V-48, V-49, V-50, V- 51, V-52, V-53 to V-54 are preferred cross-linking reagents. More preferably, at least one cross-linking reagent is of formula V-5, V-6, V-7, V-8, V-9, V-10, V-14, V-22, V-23, V-43 , V-44, V-45 or V46, and most preferably, at least one crosslinking reagent is of formula V-5, V-6, V-9 or V-14.
[0250] In another modality, the crosslinking reagents Va-1, Va-2, Va-5, Va-6, Va-7, Va-8, Va-9, Va-10, Va-11, Va-12, Va-13, Va-14, Va-15, Va-16, Va-17, Va-18, Va-19, Va-20, Va-21, Va-22, Va-23, Va- 24, Va- 25, Va-26, Va-27, Va-28, Va-29, Va-30, Va-31, Va-32, Va-33, Va-34, Va-35, Va-36, Va-37, Va-38, Va-39, Va-40, Va-41, Va-42, Va-43, Va-44, Va-45, Va-46, Va-47, Va-48, Va-49, Va- 50, Va-51, Va-52, Va-53 to Va-54 are even more preferred cross-linking reagents. More preferably, at least one crosslinking reagent is of the formula Va-5, Va-6, Va-7, Va-8, Va-9, Va-10, Va-14, Va-22, Va-23, Va- 43 , Va-44, Va-45 or Va-46, and most preferably, at least one crosslinking reagent is of the formula Va-5, Va-6, Va-9 or Va-14.
[0251] The preferred embodiments of the compound of formula (VI) and (V-II) as mentioned above apply, therefore, to the preferred compounds of formulas (V-1) to (V-54) and the most preferred compounds of formulas (Va-1) to (Va-54).
[0252] In another aspect, the present invention relates to a hydrogel that can be obtained by a process of the present invention as defined above.
[0253] The hydrogel contains from 0.01 to 1 mmol / g of primary amine groups (-NH2), more preferably, from 0.02 to 0.5 mmol / g of primary amine groups and the more preferably from 0.05 to 0.3 mmol / g of primary amine groups. The term "X mmol / g of primary amine groups" means that 1 g of dry hydrogel comprises X mmol of primary amine groups. Measurement of the amine content of the hydrogel is performed according to Gude et al. (Letters in Peptide Science , 2002, 9 (4): 203-206, which is incorporated by reference in its entirety) and is also described in detail in the Examples section.
[0254] Preferably, the term "dry" as used herein, in this patent application, means having a residual water content of a maximum of 10%, preferably less than 5% and more preferably less than 2% (determined according to Karl Fischer). The preferred method of drying is lyophilization. Process for the preparation of a hydrogel-spacer conjugate
[0255] In another aspect, the present invention relates to a process for the preparation of a hydrogel-spacer conjugate comprising the step of: (d) reacting the hydrogel of step (b) or (c) with a spacer reagent of formula (VI)
[0256] Ax1-S0-Ax2 (VI), where
[0257] S0 is selected from the group comprising C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl, the fragment of which is optionally interrupted by one or more group (s) selected from -NH-, -N ( C1-4 alkyl) -, -O-, -S, -C (O) -, -C (O) NH, -C (O) N (C1-4 alkyl) -, -OC (O) -, - S (O) -, -S (O) 2-, 4- to 7-membered heterocyclyl, phenyl and naphthyl;
[0258] Ax1 is a functional group for reaction with an amine group of the hydrogel; and
[0259] Ax2 is a functional group;
[0260] in the presence of a solvent to obtain a hydrogel-spacer conjugate.
[0261] Preferably, Ax1 is selected from the group comprising activated carboxylic acid; Cl- (C = O) -; NHS- (C = O) -, where NHS is N-hydroxysuccinimide; ClSO2-; R1 (C = O) -; I-; Br-; Cl-; SCN-; and CN-, where
[0262] R1 is selected from the group comprising H, C1-6 alkyl, alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 7-membered heterocyclyl, 8 to 11-membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl , and tetralinyl.
[0263] Most preferably, Ax1 is an activated carboxylic acid.
[0264] Suitable activation reagents to obtain the activated carboxylic acid are for example N, N'-dicyclohexyl-carbodiimide (DCC), 1-ethyl-3-carbodiimide (EDC), benzotriazole-1-yl-oxitripyrrolidinophosphonium hexafluorophosphate (PyBOP ), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), 1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbene (COMU), 1-hydroxybenzotriazol (HOBT), 1-hydroxy-azat-7-hydroxy O- (6-chlorobenzotriazol-1-yl) -N, N, N ', N'- tetramethyluronium (HCTU), 1-H-benzotriazolium (HBTU), (O- (7-azabenzotriazol-1-) hexafluorophosphate il) -N, N, N ', N'-tetramethyluronium (HATU), and O- (benzotriazol-1-yl) -N, N, N', N'-tetramethyluronium (TBTU) tetrafluoroborate These reagents are available commercially and are generally known to the specialist.
[0265] Preferably, Ax2 is selected from the group comprising -maleimide, -SH, -NH2, -SeH, -N3, -C ^ CH, - CR1 = CR1aR1b, -OH, - (CH = X0) -R1, - (C = O) -S-R1, - (C = O) -H, -NH-NH2, - O-NH2, -Ar-X0, -Ar-Sn (R1) (R1a) (R1b), -Ar -B (OH) (OH),
and with optional protection groups; on what
[0266] X0 is -OH, -NR1R1a, -SH, and -SeH,
[0267] Air is selected from phenyl, naphthyl, indenyl, indanyl, and tetralinyl, and
[0268] R1, R1a, R1b are independently selected from the group comprising H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 7-membered heterocyclyl, heterobicyclic 8 to 11 members, phenyl, naphthyl, indenyl, indanyl, and tetralinyl.
[0269] More preferably, Ax2 is selected from -NH2, maleimide and thiol and most preferably Ax2 is maleimide. Equally preferred is thiol (-SH).
[0270] Suitable reaction conditions are described in the Examples sections and are known to a person skilled in the art.
[0271] Step (d) of the process can be performed in the presence of a base. Suitable bases include customary inorganic or organic bases. These preferably include hydrides, hydroxides, amides, alkoxides, acetates, carbonates or bicarbonates of alkali metals or alkaline earth metals such as, for example, sodium hydride, sodium amide, sodium methoxide, sodium ethoxide, tert-butoxide potassium, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium acetate, potassium acetate, calcium acetate, ammonium acetate, sodium carbonate, potassium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium carbonate , and tertiary amines such as trimethylamine, triethylamine, tributylamine, N, N-dimethylaniline, N, N-dimethylbenzylamine, pyridine, N-methylpiperidine, N-methylmorpholine, N, N-dimethylaminopyridine, diazabicyclooctane (DABCO), diazab N, N-diisopropylethylamine (DIPEA), diazabicycloundecene (DBU) or collidine.
[0272] Process step (d) can be carried out in the presence of a solvent. Suitable solvents for carrying out step (d) of the process of the invention include organic solvents. These preferably include water and aliphatic, alicyclic or aromatic hydrocarbons such as, for example, petroleum ether, hexane, heptane, cyclohexane, methylcyclohexane, benzene, toluene, xylene or decalin; halogenated hydrocarbons such as, for example, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or trichloroethane; alcohols such as methanol, ethanol, n- or i-propanol, n-, i-, sec- or tert-butanol, ethanediol, propane-1,2-diol, ethoxyethanol, methoxy ethanol, diethylene glycol mono methyl ether, ether dimethyl, diethylene glycol; acetonitrile, N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N, N-dimethylacetamide, nitromethane, nitrobenzene, hexamethylphosphoramide (HMPT), 1,3-dimethyl-2-imidazolidinone (DMI ), α, 3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone (DMPU), ethyl acetate, acetone, butanone; ethers such as diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl t-amyl ether, dioxane, tetrahydrofuran, 1,2-dimethoxyethane, 1,2-diethoxyethane or anisol; or mixtures thereof. Preferably, the solvent is selected from water, acetonitrile or N-methyl-2-pyrrolidone.
[0273] In another aspect, the present invention relates to a hydrogel-spacer conjugate that can be obtained through the process described above. Process for the preparation of a prodrug attached to a support
[0274] In another aspect, the present invention relates to a process for the preparation of a linked prodrug comprising the step of: (e) reacting the hydrogel of step (b) or (c) or the hydrogel conjugate spacer from step (d) with a prodrug chaining reagent and biologically active portion of formula (VII)
[0275] Ay1-L-D (VII), where
[0276] Ay1 is a functional group for reaction with an amine of the hydrogel of step (b) or (c) or for reaction with the functional group Ax2 of the hydrogel-spacer conjugate of step (d),
[0277] L is a prodrugger;
[0278] D is a biologically active portion; in the presence of a solvent, to obtain a prodrug attached to a support.
[0279] The step (e) of the process can be carried out in the presence of a solvent. Suitable solvents for carrying out step (e) of the process of the invention include organic solvents. These preferably include water and aliphatic, alicyclic or aromatic hydrocarbons such as, for example, petroleum ether, hexane, heptane, cyclohexane, methylcyclohexane, benzene, toluene, xylene or decalin; halogenated hydrocarbons such as, for example, chlorobenzene, dichlorobenzene, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or trichloroethane; alcohols such as methanol, ethanol, n- or i-propanol, n-, i-, sec- or tert-butanol, ethanediol, propane-1,2-diol, ethoxyethanol, methoxy ethanol, diethylene glycol mono methyl ether, ether dimethyl, diethylene glycol; acetonitrile, N-methyl-2-pyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N, N-dimethylacetamide, nitromethane, nitrobenzene, hexamethylphosphoramide (HMPT), 1,3-dimethyl-2-imidazolidinone (DMI ), 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone (DMPU), ethyl acetate, acetone, butanone; ethers such as diethyl ether, diisopropyl ether, methyl t-butyl ether, methyl t-amyl ether, dioxane, tetrahydrofuran, 1,2-dimethoxyethane, 1,2-diethoxyethane or anisol; or mixtures thereof. Preferably, the solvent is selected from water, acetonitrile or N-methyl-2-pyrrolidone.
[0280] If the prodrug chaining reagent and biologically active portion of formula (VII) reacts with an hydrogel amine from step (b) or (c), then Ay1 is maleimide or OH-, preferably maleimide.
[0281] If the prodrug chaining reagent and biologically active portion of formula (VII) reacts with Ax2 of the hydrogel-spacer conjugate from step (d), the structure of Ay1 depends on the structure of Ax2 with which Ay1 reacts . Ax2 / Ay1 preferred pairs are selected from the following:


on what
[0282] X0 is -OH, -NR1R1a, -SH, and -SeH;
[0283] R1, R1a, R1b are independently selected from the group comprising H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 7-membered heterocyclyl, heterobicyclic 8 to 11 members, phenyl, naphthyl, indenyl, indanyl, and tetralinyl; and
[0284] Air is selected from phenyl, naphthyl, indenyl, indanyl, and tetralinyl.
[0285] Most preferably, Ay1 is selected from -SH or - maleimide and most preferably Ay1 is -SH and, therefore, a preferred Ax2 is -maleimide or -SH and a more preferred Ax2 is - maleimide.
[0286] In a particularly preferred embodiment Ax2 is a thiol and Ay1 of formula (VII) is of formula (VIIa)
[0287] T-PG0 - S- (VIIa), where
[0288] T is H or a portion of a label;
[0289] PG0 is a sulfur-activating portion; and
[0290] S is sulfur.
[0291] In a PG0 modality of formula (VIIa) it is selected from the group consisting of
wherein the dashed lines marked with an asterisk indicate attachment to T of formula (VIIa) and unmarked lines indicate attachment to the sulfur of formula (VIIa);
[0292] Air is an aromatic portion which is optionally additionally substituted;
[0293] R01, R03, R04 are independently a chemical bond or are C1-50 alkyl; C2-50 alkenyl; or C2-50 alkynyl, where C1-50 is alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally substituted with one or more R3, which are the same or different and where C1-50 is alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, -C (O) O-; -O-; -C (O) -; -C (O) N (R4) -; -S (O) 2N (R4) -; -S (O) N (R4) -; -S (O) 2-; -ONLY)-; - N (R4) S (O) 2N (R4a) -; -S-; -N (R4) -; -OC (O) R4; -N (R4) C (O) -; -N (R4) S (O) 2-; - N (R4) S (O) -; -N (R4) C (O) O-; -N (R4) C (O) N (R4a) -; and -OC (O) N (R4R4a);
[0294] R02 is -H; C1-50 alkyl; C2-50 alkenyl; or C2-50 alkynyl, where C1-50 is alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally substituted with one or more R3, which are the same or different and where C1-50 is alkyl; C2-50 alkenyl; and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -Q-, -C (O) O-; -O-; -C (O) -; -C (O) N (R4) -; -S (O) 2N (R4) -; -S (O) N (R4) -; -S (O) 2-; -ONLY)-; -N (R4) S (O) 2N (R4a) -; -S-; -N (R4) -; -OC (O) R4; -N (R4) C (O) -; -N (R4) S (O) 2-; -N (R4) S (O) -; -N (R4) C (O) O-; -N (R4) C (O) N (R4a) -; and -OC (O) N (R4R4a);
[0295] Q is selected from the group consisting of phenyl; naphthyl; indenyl; indanila; tetralinyl; C3-10 cycloalkyl; 4- to 7-membered heterocyclyl; and 8 to 11 membered heterobicyclyl, where T is optionally substituted with one or more R3, which are the same or different;
[0296] R3 is halogen; -CN; oxo (= O); -COOR5; -OR5; -C (O) R5; - C (O) N (R5R5a); -S (O) 2N (R5R5a); -S (O) N (R5R5a); -S (O) 2R5; -S (O) R5; - N (R5) S (O) 2N (R5aR5b); -SR5; -N (R5R5a); -NO2; -OC (O) R5; -N (R5) C (O) R5a; -N (R5) S (O) 2R5a; -N (R5) S (O) R5a; -N (R5) C (O) OR5a; -N (R5) C (O) N (R5aR5b); -OC (O) N (R5R5a); or C1-6 alkyl, where C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and
[0297] R4, R4a, R5, R5a, R5b are independently selected from the group consisting of -H; or C1-6 alkyl, where C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.
[0298] Preferably, R01 is C1-6 alkyl. Even more preferably, R01 is selected from -CH2-, -CH2-CH2-and -CH2- CH2-CH2-.
[0299] Preferably, R02 is selected from H and C1-6 alkyl.
[0300] Preferably, R03 is C1-6 alkyl.
[0301] Preferably, R04 is C1-6 alkyl.
[0302] Preferably, Ar is selected from the group
and in which dashed lines marked with an asterisk indicate attachment to T of formula (VIIa) and unmarked dashed lines indicate attachment to the rest of PG0 of formula (VIIa);
[0303] W is independently of each other O, S, or N;
[0304] W 'is N; and where Ar is optionally substituted with one or more substituents independently selected from the group consisting of NO2, CI and F.
[0305] Most preferably, PG0 selected from the group consisting of
wherein the dashed lines marked with an asterisk indicate attachment to T of formula (VIIa) and unmarked lines indicate attachment to the sulfur of formula (VIIa); and Ar, R01, R02, R03 and R04 are used as above.
[0306] Most preferably, PG0 of formula (VIIa) is
where the dashed line marked with an asterisk indicates attachment to T of formula (VIIa) and the unmarked dashed line indicates attachment to the sulfur of formula (VIIa).
[0307] Most preferably, PG0 of formula (VIIa) is where the dashed line marked with an asterisk indicates attachment to T of formula (VIIa) and the unmarked dashed line indicates attachment to the sulfur of formula (VIIa).

[0308] In a preferred embodiment T of formula (VIIa) is H.
[0309] In another embodiment, T of formula (VIIa) comprises a polymeric portion. Preferably, T of formula (VIIa) comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl choline, poly (acrylic acids), poly (acrylates), poly (acrylamides), poly (alkyloxy) polymers, poly (amides ), poly (amidoamines), poly (amino acids), poly (anhydrides), poly (aspartamides), poly (butyric acids), poly (glycolic acids), polybutylene terephthalates, poly (caprolactones), poly (carbonates), poly ( cyanoacrylates), poly (dimethylacrylamides), poly (esters), poly (ethylenes), poly (alkylene glycols), poly (ethylene oxides), poly (ethyl phosphates), poly (ethyloxazolines), poly (glycolic acids), poly (hydroxyethyl) acrylates), poly (hydroxyethyl-oxazolines), poly (hydroxymethacrylates), poly (hydroxypropylmethacrylamides), poly (hydroxypropyl methacrylates), poly (hydroxypropyloxazolines), poly (imino carbonates), poly (lactic acids), poly (lactic-co-glycolic) acids), poly (methacrylamides), poly (methacrylates), poly (methyloxazolines), poly (organophosphasenes), poly (ortho esters), poly (oxazolines), poly (propylene glycols), poly (siloxanes), poly (urethanes), poly (vinyl alcohols), poly (vinyl amines), poly (vinyl methyl ethers), polyvinylpyrrolidones), silicones, celluloses, carbomethyls celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other polymers based on carbohydrates and carbohydrate-based polymers and carbohydrate-based polymers and carbohydrate-based polymers and carbohydrate-based polymers and carbohydrate-based polymers, and carbohydrate-based polymers and carbohydrate-based polymers. themselves.
[0310] If T of formula (VIIa) is a polymeric portion, it is preferred that T has a molecular weight of at least 1 kDa, preferably of at least 3 kDa and most preferably of at least 5 kDa. If T of formula (VIIa) is a polymeric portion, it is preferable that it has a molecular weight of a maximum of 1000 kDa, for example, a maximum of 800 kDa, a maximum of 500 kDa, a maximum of 250 kDa, a maximum of 200 kDa, or at most maximum 100 kDa.
[0311] In another embodiment, T of formula (VIIa) comprises an affinity binder. Preferably, T of formula (VIIa) comprises, more preferably T is an affinity binding moiety selected from the group consisting of 4-aminobenzamidine, 3- (2'-aminobenzhydryloxy) tropane, ε-aminocaproyl-p-chlorobenzylamide, 1- amino-4- [3- (4,6-dichlorotriazin-2-ylamino) -4-sulfophenylamino] anthraquinone-2-sulfonic, 2- (2'-amino-4'-methylphenylthio) -N, N -dimethylbenzylamine, angiopoietin-1, aptamers, arotinoid acid, avidin, biotin, calmodulin, cocaethylene, cytosporone B, N, N-dihexyl-2- (4-fluorophenyl) indole-3-acetamide, N, N-dipropyl-2- (4-chlorophenyl) -6,8-dichloro-imidazo [1,2-a] pyridine-3-acetamide, 5-fluoro-2'-deoxyuridine 5 '- (p-aminophenyl) monophosphate, S-hexyl-L- glutathione, (S, S) -4-phenyl-α- (4-phenyloxazolidin-2-ylidene) -2-oxazoline-2-acetonitrile, Pro-Leu-Gli hydroxamate, 2- (4- (2- (trifluoromethyl) ) phenyl) piperidine-1-carboxamido) benzoic, trimethyl (m-aminophenyl) ammonium chloride, urocortin III, cofactors such as adenosine triphosphate, s-adenosyl methionine, ascorbic acid, cobalamin, coenzyme A, coenzyme B, coenzyme M, coenzyme Q, coenzyme F420, cytidine triphosphate, flavin mononucleotide, flavin adenine dinucleotide, glutathione, heme, lipoamide, menaquinone, methanofuran, methanofuran, methanofuran, , nucleotide sugars, 3'-phosphoadenosine-5'-phosphosulfate, pyridoxal phosphate, polyhistidines, pyrroloquinoline quinone, riboflavin, streptavidin, tetrahydrobiopterine, tetrahydromethanopterin, tetrahydro-folic acid, BCC vehicle protein ), chitin-binding protein, FK506-binding protein, FLAG tag, green fluorescent protein, glutathione-S-transferase, hemagglutinin, maltose-binding protein, myc tag, NusA, protein C epitope, S-tag tag, tag strep-tag, thioredoxins, triazines and antibody fragments.
[0312] If T of formula (VIIa) comprises an affinity ligand, it is preferred that the affinity ligand is a polyhistidine.
[0313] In another embodiment, T of formula (VIIa) comprises a charged portion. Preferably, T of formula (VIIa) comprises at least one positive and / or negative charge. It is understood that the number of positive and negative charges for T is unequal to ensure that T is a charged molecule.
[0314] Preferably, T of formula (VIIa) comprises at least one positive or negative charge, for example, at least two positive or negative charges, at least three positive or negative charges, at least four positive or negative charges, at least five positive or negative charges, at least six positive or negative charges, at least seven positive or negative charges, at least eight positive or negative charges, at least nine positive or negative charges, at least eleven positive charges or negative, at least twelve positive or negative charges, at least thirteen positive or negative charges, at least fourteen positive or negative charges or at least fifteen positive or negative charges.
[0315] More preferably, T of formula (VIIa) comprises at least one positive charge, such as a positive charge, two positive charges, three positive charges, four positive charges, five positive charges, six positive charges, eight positive charges, eight positive charges, nine positive charges, ten positive charges, eleven positive charges, twelve positive charges, thirteen positive charges, fourteen positive charges or fifteen positive charges. More preferably, T of formula (VIIa) comprises a positive charge, two positive charges, three positive charges, four positive charges, five positive charges, six positive charges, seven positive charges or eight positive charges. More preferably, T of formula (VIIa) comprises two positive charges, three positive charges, four positive charges, five positive charges or six positive charges.
[0316] Preferably, at least one positive charge of T of formula (VIIa) is provided by an ammonium or phosphonium.
[0317] Preferably, T of formula (VIIa) comprises a polyamide containing at least one quaternary ammonium residue and / or at least one preferably comprising amine functional groups. Preferably, T of formula (VIIa) comprises at least one quaternary ammonium residue and / or at least one protonated ammonium residue. Even more preferably, T of formula (VIIa) comprises four quaternary ammonium residues and / or four protonated ammonium residues.
[0318] Preferably, T of formula (VIIa) comprises in a linked form a polyamine. More preferably, T of formula (VIIa) comprises in a bound form a polyamine selected from ethylene diamine, 1,3-diaminopropane, cadaverine, putrescine, spermine, T of formula (VIIa)
[0319] Even more preferably, T of formula (VIIa) comprises a portion of formula (a):
where the dashed line indicates attachment to PG0;
[0320] R1, R1a, R1b, R2, R2a, R2b, R3, R3a, R3b, R4, R4a, R4b are independently of each other H or methyl;
[0321] each m is independently of each other 1, 2, 3, 4, 5, 6, 7, or 8;
[0322] each n is independently of one another 1, 2, 3, 4, 5, 6, 7, or 8;
[0323] each x is independently of one another 1, 2, 3, 4, 5, 6, 7 or 8:
[0324] each y is independently of the other 0, 1, 2, 3, 4, 5, 6, 7 or 8; and
[0325] SP is a portion of spacer.
[0326] Preferably, the portion of formula (a) is symmetric, that is, the portion
is the same as the portion

[0327] In an embodiment R1, R1a, R1b of formula (a) are all methyl.
[0328] In another embodiment R1 of formula (a) is H and R1a and R1b of formula (a) are both methyl.
[0329] In a modality R2, R2a, R2b of formula (a) are all methyl.
[0330] In another embodiment R2 of formula (a) is H and R2a and R2b of formula (a) are both methyl.
[0331] In an embodiment R3, R3a, R3b of formula (a) are all methyl.
[0332] In another embodiment R3 of formula (a) is H and R3a and R3b of formula (a) are both methyl.
[0333] In an embodiment R4, R4a, R4b of formula (a) are all methyl.
[0334] In another embodiment R4 of formula (a) is H and R4a and R4b of formula (a) are both methyl.
[0335] Preferably, m of formula (a) is 1, 2, 3, 4, 5 or 6. More preferably, m of formula (a) is 2, 3, 4, or 5, even more preferably, m of formula (a) is 3, 4 or 5 and most preferably, m of formula (a) is 4.
[0336] Preferably n of formula (a) is 1, 2, 3, 4, 5 or 6. More preferably, n of formula (a) is 2, 3, 4, or 5, even more preferably, n of formula (a) is 2, 3 or 4 and most preferably, n of formula (a) is 3.
[0337] Preferably, x of formula (a) is 1, 2, 3, 4, 5 or 6. More preferably, n of formula (a) is 1, 2, 3, or 4, even more preferably, x of formula (a) is 1, 2, or 3 and most preferably, x of formula (a) is 1.
[0338] Preferably, y of formula (a) is 1, 2, 3, 4, 5 or 6. More preferably, y of formula (a) is 1, 2, 3, or 4, even more preferably, y of formula (a) is 1, 2, or 3 and most preferably, y of formula (a) is 1.
[0339] In a preferred embodiment, R1, R1a, R1b, R2, R2a, R2b, R3, R3a, R3b, R4, R4a, R4b are methyl; m is 4; n is 3; y is 1 and x is 1.
[0340] In another preferred embodiment, R1, R2, R3, R4 are H; R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b are methyl, m is 4; n is 3; y is 1 and x is 1.
[0341] Preferred counter ions for T of formula (I) are Cl-, TFA- and S04-.
[0342] If Ay1 is of formula (VIIa) and T is a tag portion, then T can be used to purify biologically active prodrug-linking reagents of formula (VII) which have a defined number of Ay1 portions -L per D portion of a mixture of prodrug-linking reagents and biologically active portion of formula (VII) in which each D is connected to x Ay1-L portions and where x is a positive integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. T can therefore be used to isolate monoconjugates from the prodrug-binding reagent and biologically active portion of formula (VII).
[0343] The method of isolating some monoconjugates from the biologically active prodrug-linker depends on the T-tag portion of formula (VIIa).
[0344] If T of formula (VIIa) is a polymeric portion with a molecular weight of at least 10% (by weight / weight) of D of formula (VII), the isolation step is preferably chromatography by size exclusion.
[0345] If T of formula (VIIa) comprises an affinity binder, the isolation step is preferably affinity chromatography.
[0346] If T of formula (VIIa) comprises a charged portion, the isolation step is preferably ion exchange chromatography.
[0347] In a preferred embodiment T of formula (VIIa) is a charged portion and the isolation step is preferably ion exchange chromatography.
[0348] Preferably, step (e) of the process comprises the step of reacting a hydrogel-spacer conjugate of step (d) with a pro-drug reagent-biologically active portion of formula (VII).
[0349] Suitable reaction conditions are described in the Examples sections and are known to a person skilled in the art.
[0350] Consequently, the process for preparing a prodrug attached to a support preferably comprises the steps of: (d) reacting the hydrogel of step (b) or (c) with a spacer reagent of formula (VI)
[0351] Ax1-S0-Ax2 (VI), where
[0352] S0 is selected from the group comprising C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl, the fragment of which is optionally interrupted by one or more group (s) selected from -NH-, -N ( C1-4 alkyl) -, -O-, -S, -C (O) -, -C (O) NH, - C (O) N (C1-4 alkyl) -, -OC (O) -, - S (O) -, -S (O) 2-, 4- to 7-membered heterocyclyl, phenyl and naphthyl;
[0353] Ax1 is a functional group for reaction with an amine group of the hydrogel; and
[0354] Ax2 is a functional group;
[0355] in the presence of a solvent to obtain a hydrogel-spacer conjugate; and (e) reacting the hydrogel-spacer conjugate of step (d) with a biologically active prodrug-linking reagent of formula (VII)
[0356] Ay1-L-D (VII), where
[0357] Ay1 is a functional group for reaction with the functional group Ax2 of the hydrogel-spacer conjugate of step (d),
[0358] L is a prodrugger;
[0359] D is a biologically active portion; in the presence of a solvent, to obtain a prodrug attached to a support.
[0360] Preferred combinations of Ax2 and Ay1 are as revealed above.
[0361] The pro-drug chaining reagent or the pro-drug chaining portion as part of the pro-drug chaining reagent and biologically active portion may have the structure of any pro-drug chaining portion known in the art.
[0362] Preferably, the prodrug chaining reagent or the prodrug chaining portion as part of the prodrug chaining reagent and biologically active portion is a traceless reagent or chaining prodrug portion.
[0363] A preferred prodruger is disclosed and can be obtained as described in International Patent Application Publication No. WO 2005/099768 A2. Therefore, a pro-drug chaining reagent and preferred biologically active moiety Ay1-LD has a structure of formula hi) or (h-ii):
on what
[0364] Ay1 is a functional group as defined in step (e),
[0365] D is a biologically active moiety which is connected to L via an amine group of the corresponding drug;
[0366] X is a spacer portion such as R5-Y6,
[0367] Y1, Y2 are independently O, S or NR6,
[0368] Y3, Y5 are independently O or S,
[0369] Y4 is O, NR6 or C (R7) (R8) -,
[0370] Y6 is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon bonds or any heteroatom containing a free electron pair or is absent,
[0371] R2, R3 are independently selected from hydrogel, linear, branched or cyclic substituted or unsubstituted alkyl or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryl, cyan, nitro, halogen, carboxy, carboxylalkyl , alkylcarbonyl or carboxamidoalkyl;
[0372] R4 is selected from hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted linear, branched or cyclic linear, branched or cyclic heteroalkyloxy, substituted or cyclic heteroalkyloxy or unsubstituted, aryloxy or heteroaryloxy, cyan, halogen,
[0373] R5 is selected from linear, branched or cyclic substituted or unsubstituted alkyl or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls,
[0374] R6 is selected from hydrogel, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryls, substituted or unsubstituted heteroaryls,
[0375] R7, R8 are independently selected from hydrogen, linear, branched or cyclic substituted or unsubstituted or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls, carboxyalkyl, alkylcarbonyl, carboxamidoalkyl, cyan or halogen,
[0376] W is selected from substituted or unsubstituted linear, branched or cyclic alkyl, substituted or unsubstituted aryls, linear, branched or cyclic heteroalkyl, substituted or unsubstituted heteroaryls,
[0377] Nu is the nucleophile,
[0378] n is zero or a positive integer, and
[0379] Ar is a multi-substituted aromatic hydrocarbon or a multi-substituted aromatic heterocycle.
[0380] It is understood that in formulas (h-i) and (h-ii) L corresponds to the portion linking Ay1 and D.
[0381] Preferably, R2, R3, R4, R5, R6, R7 and R8 of formula (hi) and (h-ii) are independently selected from H, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl.
[0382] Preferably, Y6 of formula (h-i) and (h-ii) is C1-20 alkyl, C2-20 alkenyl or C2-20 alkynyl.
[0383] Preferably, Nu of formula (h-i) and (h-ii) is selected from the group of nucleophiles consisting of primary, secondary and tertiary amino groups, thiol, carboxylic acid, hydroxylamine, hydrazine and nitrogen containing heteroaryl.
[0384] Preferably, W of formula (hi) and (h-ii) is - (CR9R10) b-, where R9 and R10 are independently selected from H, C1-6 alkyl, C2-6 alkenyl and C2- 6 alkynyl and where b is 1, 2, 3, 4 or 5.
[0385] Preferably, n of formula (h-i) and (h-ii) is 0, 1 or 2, more preferably, n is 0 or 1 and most preferably n is 0.
[0386] Preferably, Ar of formula (hi) and (h-ii) is selected from among

[0387] Other preferred prodrug chains are disclosed and can be obtained as described in International Patent Application Publication No. WO 2006/136586 A2. Accordingly, a pro-drug chaining reagent and preferred biologically active portion Ay1-LD has a structure of formula (h-iii), (h-iv) or (hv):
on what
[0388] Ay1 is a functional group as defined in step (e);
[0389] D is a biologically active moiety which is connected to L via an amine group of the corresponding drug forming an amide bond;
[0390] X is a spacer portion such as R13-Y1;
[0391] Y1 is O, S, NR6, succinimide, maleimide, unsaturated carbon-carbon bonds or any hetero-atom containing a free electron pair or is absent;
[0392] R13 is selected from linear, branched or cyclic substituted or unsubstituted alkyl or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls;
[0393] R2 and R3 are independently selected from hydrogen, acyl groups, or protection groups for hydroxyl groups;
[0394] R4 to R12 are independently selected from hydrogen, linear, branched or cyclic substituted or unsubstituted or heteroalkyl, aryls, substituted aryls, substituted or unsubstituted heteroaryls, cyan, nitro, halogen, carboxy, carboxamide.
[0395] It is understood that in formulas (h-iii), (h-iv) and (h-v) L corresponds to the portion linking Ay1 and D.
[0396] Preferably, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 of formula (h-iii), (h-iv) and (hv) are independently selected from H , C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl.
[0397] Preferably, in formulas (h-iii), (h-iv) and (h-v) Y1 is C1 20 alkyl, C2-20 alkenyl or C2-20 alkynyl.
[0398] Another preferred prodruger is disclosed and can be obtained as described in International Patent Application Publication No. WO 2009/095479 A2. Therefore, a pro-drug chaining reagent and preferred biologically active moiety Ay1-LD has a structure of formula (h-vi):
on what
[0399] Ay1 is a functional group as defined in step (e);
[0400] D is a biologically active moiety which is connected to the rest of the molecule via an aromatic amine of the corresponding drug forming an amide bond;
[0401] C (R4R4A); C (R4R4A);
[0402] X1 is C; the use);
[0403] X2 is C (R7, R7A); or C (R7, R7a) -C (R8, R8A);
[0404] R1, R1a, R2, R2A, R3, R3A, R4, R4A, R5, R5A, R6, R7, R7A, R8, R8A are independently selected from the group consisting of H; and C1-4 alkyl;
[0405] optionally, one or more of the pairs R1a / R4A, R1a / R5A, R4A / R5A, R4A / R5A, R7A / R8A form a chemical bond;
[0406] optionally, one or more of the pairs R1 / R1a R2 / R2A, R4 / R4A, R5 / R5A, R7 / R7A, R8 / R8A are joined together with the atom to which they are attached to form a C3-8 cycloalkyl ; or 4- to 7-membered heterocyclyl;
[0407] optionally, one or more of the pairs R1 / R4, R1 / R5, R1 / R6, R4 / R5, R7 / R8, R2 / R3 are joined together with the atoms to which they are attached to form an A ring; optionally, R3 / R3A are joined together with the nitrogen atom to which they are attached to form a 4- to 7-membered heterocycle;
[0408] A is selected from the group consisting of phenyl; naphthyl; indenyl; indanila; tetralinyl; C3-10 cycloalkyl; 4- to 7-membered heterocyclyl; and 8 to 11 membered heterobicyclyl;
[0409] as long as a hydrogen of R1, R1a, R2, R2A, R3, R3A, R4, R4A, R5, R5A, R6, R7, R7A, R8 or R8A is replaced by Ay1.
[0410] It is understood that in the formula (h-vi) L corresponds to the portion linking Ay1 and D.
[0411] Another preferred prodruger is revealed and can be obtained as described in International Patent Application Publication Nos. WO 2011/012721 A1 and WO 2011/012722 A1. As a result, a preferred biologically active prodrug-binding reagent reagent Ay1-LD has a formula structure (h-vii):

[0412] where
[0413] D is a biologically active moiety which is connected to the rest of the molecule via an aromatic amine of the corresponding drug forming an amide bond;
[0414] Ay1 is a functional group as defined in step (e);
[0415] X1 is C (R1Rla) or a cyclic fragment selected from C3 8 cycloalkyl, 4- to 7-membered heterocyclyl, phenyl, naphthyl, indenyl, indanyl, tetralinyl, or 8 to 11-membered heterobicyclyl, where
[0416] in the case that X1 is a cyclic fragment, the said cyclic fragment is incorporated into L1 through two atoms of the adjacent ring and the ring atom of X1, which is adjacent to the carbon atom of the amide bond, is also a carbon atom;
[0417] X2 is a chemical bond or is selected from C (R3R3a), N (R3), O, C (R3R3a) -C (R4R4a), C (R3R3a) -N (R4), N (R3) -C (R4R4a), C (R3R3a) -O, or OC (R3R3a), where
[0418] if X1 is a cyclic fragment, X2 is a chemical bond, C (R3R3a), N (R3) or O;
[0419] optionally, if X1 is a cyclic fragment and X2 is C (R3R3a), the order of fragment X1 and fragment X2 within L1 can be changed and the cyclic fragment is incorporated into L1 through two ring atoms adjacent.
[0420] R1, R3 and R4 are independently selected from the group consisting of H, C1-4 alkyl and -N (R5R5a);
[0421] R1a, R2, R3a, R4a and R5a are independently selected from the group consisting of H, and C1-4 alkyl;
[0422] R5 is C (O) R6;
[0423] R6 is C1-4 alkyl;
[0424] optionally, one of the pairs R1a / R4a, R3a / R4a or R1a / R3a forms a chemical bond;
[0425] as long as a hydrogen of R1, R1a, R2, R2a, R3, R3a, R4, R4a, R5, R5a or R6 is replaced by Ay1.
[0426] It is understood that in the formula (h-vii) L corresponds to the portion linking Ay1 and D.
[0427] Another preferred prodruger is disclosed and can be obtained as described in International Patent Application Publication No. WO 2011/089214 A1. Therefore, a pro-drug chaining reagent and preferred biologically active portion Ay1-LD has a structure of formula (h-viii):

[0428] Ay1 is a functional group as defined in step (e);
[0429] D is a biologically active portion which is connected to the rest of the molecule via an aromatic hydroxyl (-OH) of the corresponding drug forming a carbamate bond;
[0430] R1 is selected from the group consisting of C1-4 alkyl; heteroalkyl; C3-8 cycloalkyl; and

[0431] R2, R2a, R3 and R3a are independently selected from hydrogen, C1-4 substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl;
[0432] each d is independently 2, 3 or 4;
[0433] as long as a hydrogen of R1, R2, R2a, R3, or R3a is replaced by Ay1.
[0434] It is understood that in the formula (h-viii) L corresponds to the portion linking Ay1 and D.
[0435] Another preferred prodruger is disclosed and can be obtained as described in International Patent Application Publication No. WO 2011/089216 A1. Accordingly, a pro-drug chaining reagent and preferred biologically active portion Ay1-LD has a formula structure (h-ix):
on what
[0436] Ay1 is a functional group as defined in step (e);
[0437] D is a biologically active moiety which is connected to the rest of the molecule via an aliphatic amine of the corresponding drug forming an amide bond;
[0438] X1 is selected from O, S or CH-R1a;
[0439] R1 and R1a are selected independently from H, OH, CH3;
[0440] R2, R2a, R4 and R4a are independently selected from H and C1-4 alkyl,
[0441] R3 and R3a are independently selected from H, C1-4 alkyl, and R5;
[0442] R5 is selected from among
where dashed lines indicate attachment to the rest of the portion, provided that a hydrogen of R1, R1a, R2, R2a, R3, R3a, R4, R4a and R5 is replaced by Ay1.
[0443] It is understood that in the formula (h-ix) L corresponds to the portion linking Ay1 and D.
[0444] Preferably, R3 of formula (h-ix) is H and R3a of formula (h-ix) is R5.
[0445] Preferably, one of R4 / R4a of formula (h-ix) is H.
[0446] Optionally, one or more of the pairs R3 / R3a, R4 / R4a, R3 / R4 of formula (h-ix) can independently form one or more cyclic fragments selected from C3-8 cycloalkyl, heterocyclyl from 4 to 7 limbs, or 8 to 11-membered heterobicyclyl.
[0447] Optionally, R3, R3a, R4 and R4a of formula (h-ix) are additionally substituted with C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, phenyl, 4- to 7-membered heterocycle or halogen.
[0448] Another preferred prodruger is disclosed and can be obtained as described in International Patent Application Publication No. WO 2011/089215 A1. Therefore, a pro-drug chaining reagent and preferred biologically active portion Ay1-LD has a structure of formula (hx):
on what
[0449] Ay1 is a functional group as defined in step (e);
[0450] D is a biologically active moiety which is connected to the rest of the molecule via an aromatic amine of the corresponding drug forming an amide bond;
[0451] R1, R1a, R2, R3, R3a, R4 and R4a are independently selected from H and C1-4 alkyl;
[0452] optionally, any two of R1, R1a, R2, R3, R3a, R4 and R4a can independently form one or more cyclic fragments selected from C3-8 cycloalkyl, 4- to 7-membered heterocyclyl, phenyl, naphthyl, indenyl , indanyl, tetralinyl, or 8 to 11 membered heterobicyclyl;
[0453] optionally, R1, R1a, R2, R3, R3a, R4 and R4a are additionally substituted with a substituent selected from the group comprising C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, heterocyclyl 4 to 7 members, phenyl, naphthyl, indenyl, indanyl, tetralinyl, or 8 to 11 membered heterobicyclyl;
[0454] as long as a hydrogen of R1, R1a, R2, R3, R3a, R4 and R4a is replaced by Ay1.
[0455] It is understood that in the formula (h-x) L corresponds to the portion linking Ay1 and D.
[0456] Another preferred prodruger is disclosed and can be obtained as described in Patent Publication No. PCT / EP2012 / 065748. Therefore, a preferred biologically active pro-drug-reagent reagent Ay1-LD has a formula structure (h-xi):
on what
[0457] Ay1 is a functional group as defined in step (e);
[0458] D is a biologically active portion which is connected to the rest of the molecule via a carboxylic acid (- (C = O) -OH) group of the corresponding drug forming a carboxylic ester bond;
[0459] R1 is selected from the group of unsubstituted alkyl; substituted alkyl; unsubstituted phenyl; substituted phenyl; unsubstituted naphthyl; substituted naphthyl; unsubstituted indenyl; substituted indenyl; unsubstituted indanila; substituted indanila; unsubstituted tetralinyl; substituted tetralinyl; C3-10 unsubstituted cycloalkyl; Substituted cycloalkyl C310; 4- to 7-membered unsubstituted heterocyclyl; 4- to 7-membered heterocyclyl substituted; 8 to 11-membered unsubstituted heterobicyclyl; and 8 to 11-membered substituted heterobicyclyl;
[0460] R2 is selected from H, unsubstituted alkyl, and substituted alkyl;
[0461] R3 and R4 are independently selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl;
[0462] and is 0 or 1;
[0463] optionally, R1 and R3 are joined together with the atoms to which they are attached to form an A ring;
[0464] A is selected from the group consisting of C3-10 cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; and 8 to 11 membered aliphatic heterobicyclyl, where A is unsubstituted or substituted;
[0465] Q is selected from the group comprising C1-50 alkyl, C2-50 alkenyl or C2-50 alkynyl, the fragment of which is optionally interrupted by one or more group (s) selected from -NH-, -N ( C1-4 alkyl) -, -O-, -S-, -C (O) -, -C (O) NH-, - C (O) N (C1-4 alkyl) -, -OC (O) - , -S (O) -, -S (O) 2-, 4- to 7-membered heterocyclyl, phenyl or naphthyl.
[0466] It is understood that in the formula (h-xi) L corresponds to the portion linking Ay1 and D.
[0467] Another preferred prodruger is revealed and can be obtained as described in European Patent Publication No. EP12165516. Accordingly, a pro-drug chaining reagent and preferred biologically active portion Ay1-LD has the structure of formula (h-xii):
on what
[0468] Ay1 is a functional group as defined in step (e);
[0469] D is a biologically active portion which is connected to the rest of the molecule via a hydroxyl group of the corresponding drug forming an ester or carbamate bond
[0470] Y is -C (R1) (R1a) -; or -N (R1) -;
[0471] X is -C (R4) (R4a) -; -N (R4) -; -O-; -C (R4) (R4a) -C (R5) (R5a) -; - C (R4) (R4a) -N (R6) -; -N (R6) -C (R4) (R4a) -; -C (R4) (R4a) -O-; -O-C (R4) (R4a) -; - C (O) -N (R6) -; or -N (R6) -C (O) -;
[0472] X1 is either -
[0473] X2 is -C (R7) (R7A) -; or -C (R7) (R7a) -C (R8) (R8A) -;
[0474] X3 is = O; = S; or = N-CN;
[0475] R1 R1a R2 R2A R3 R3A R4 R4A R5 R5A R6 R7 R7A R8 R8A are independently selected from the group consisting of H; C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-20 heteroalkyl and Y1- T; and independently none, one or more of the pairs R1a / R4A, R1a / R5A, R4A / R5A, R7A / R8A are absent and the corresponding carbon atoms to which they are attached form a cis double bond;
[0476] Y1 is a chemical bond or C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl;
[0477] T is selected from the group consisting of phenyl; naphthyl; indenyl; indanila; tetralinyl; C3-10 cycloalkyl; 4- to 7-membered heterocyclyl; or 8 to 11 membered heterobicyclyl, where T is optionally substituted with one or more R9, which are the same or different;
[0478] R9 is halogen; -CN; oxo (= O); -C (O) OH; -OH; -S (O) 2NH2; - S (O) NH2; -S (O) 2OH; -S (O) OH; -SH; -NH2; -N02; C1-6 alkyl, or C1-10 heteroalkyl;
[0479] optionally, one or more of the pairs R1 / R1a, R1 / R4, R1 / R6, R1 / R5, R2 / R2A, R2 / R3, R4 / R4A, R4 / R5, R5 / R5A, R7 / R7A, R7 / R8, R8 / R8A are joined together with the atom to which they are attached to form a T ring;
[0480] optionally, R3 / R3A are joined together with the nitrogen atom to which they are attached to form a 4- to 7-membered heterocycle;
[0481] as long as a hydrogen of R1, R1a, R2, R2A, R3, R3A, R4, R4A, R5, R5A, R6, R7, R7A, R8 or R8A is replaced by Ay1.
[0482] It is understood that in the formula (h-xii) L corresponds to a portion linking Ay1 and D.
[0483] In a preferred embodiment, the biologically active prodrug-binding reagent Ay1-L-D is of formula (h-i) or (h-ii).
[0484] In another preferred embodiment, the biologically active prodrug-linking reagent Ay1-L-D is of formula (h-vi).
[0485] Any drug that comprises at least one functional group can be combined with a reagent prodruger to obtain a biologically active prodrug reagent-reagent Ay1-L-D. The drug referred to is selected from the group comprising polypeptides, proteins, and oligonucleotides. Preferably, the drug is a protein.
[0486] Preferably, D has a molecular weight ranging between 2 and 500 kDa, more preferably between 5 and 250 kDa, more preferably between 5 and 100 kDa and most preferably between 10 and 60 kDa.
[0487] In one embodiment the drug is a protein drug. Preferably, the drug is a protein drug that modulates the activity of one or more of the selected biological targets among basic fibroblast growth factors (bFGF), acidic fibroblast growth factors (aFGF), transforming alpha growth factors (TGFa) , transforming growth factors beta (TGFβ), platelet-derived growth factor (PDGF), angiogenin, platelet-derived endothelial cell growth factor (PD-ECGF), interleukin-1 (IL-1) interleukin-8 (IL -8), interleukin- 12, vascular endothelial growth factor (VEGF), angiopoietin-I, Del-I, follistatin, granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), leptin, midquine, placental growth factor, pleiotrophin (PTN), progranulin, proliferin, tumor necrosis factor-alpha (TNF-alpha), angioarrestin, angio statin (plasminogen fragment), antiangiogenic anti-thrombin III, cartilage-derived inhibitor gem (CDI), fragment of complement CDS9, endostatin (fragment of collagen XVIII), fragment of fibronectin, gro-beta, heparinases, fragment of heparin hexassaccharide, human chorionic gonadotropin (hCG), alpha / beta / gamma interferon, inducible protein interferon (IP-IO), kringle S (plasminogen fragment), metalloproteinase inhibitors (TIMPs), 2-methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, platelet factor 4 (PF4), 16 kD fragment of prolactin, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S, thrombospondin-I (TSP-I), vasculine statin, and vasostatin (fragment of calreticulin), prostaglandin, growth hormone, growth factor similar to insulin -I (IGF-I), sphingosine-1-phosphate, factor D, TP801, complement inhibitors, including CI, C3 and C5, α2 adrenergic agonist, mTOR, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor ( BDNF), neurotrophic factor I am derived from glial cells (GDNF), growth factor derived from lens epithelium (LEDGF), stem stem viability factor (RdCVF), factor derived from pigmentary epithelium (PEDF).
[0488] If the drug is a protein, it is preferably selected from the group consisting of ACTH, adenosine deaminase, agalsidase, albumin, alpha-1 antitrypsin (AAT), alpha-1 proteinase inhibitor (API), alglucosidase, alteplase, anistreplase , ancrod serine protease, antibodies (monoclonal or polyclonal and fragments or fusions), antithrombin III, antitrypsins, aprotinin, asparaginases, biphaline, bone morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, encephalins, erythalins, erythalins, factor , factor VIII, factor VIlla, factor IX, fibrinolysin, fusion proteins, follicle stimulating hormones, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucagon-like peptides such as GLP-1, glucocerebrosidase, stimulating factor of colonies of granulocyte macrophages (GM-CSF), chorionic gonadotropin (hCG), hemoglobins, hepatitis B vaccines, hirudin, hyaluronidases, idunonidase, immunoglobulins, influenza, interleukins (1 alpha, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-1 receptor antagonist (rhlL-1ra), insulins, interferons (alpha 2a, alpha 2b, alpha 2c, beta 1a, beta 1b, gamma 1a, gamma 1b), keratinocyte growth factor (KGF), lactase, leuprolide, levy thyroxine, luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF, pepsin, phospholipase activating protein (PLAP), platelet activating factor alkethylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD), somatropins (growth hormone), somatostatin, streptokinase, sucrase, fragment of tetanus toxin, tilactase, thrombin, thymosin, thyroid stimulating hormone, thyrotropin, transforming growth factors, tumor necrosis factor (TNF), TNF-IgG Fc receptor, tissue plasminogen activator (tPA), transferrin, TSH, urate oxidase and urokinase.
[0489] If the drug is an antibody, it can be a monoclonal or polyclonal antibody or a fragment or fusion thereof. Preferred antibody fragments are selected from the group comprising Fab (fragment, antigenic binding), F (ab) 2, Fc (fragment, crystallizable) fragments, pFc 'fragment, Fv (fragment, variable), scFv (single chain variable fragment) ), di scFv / diabodies, bi-specific T cell engager, CDRs (complementarity determining regions), single domain antibodies (sdABs / Nanobodies), heavy chains (α, δ, ε, Y, μ) or fragments of heavy chain, light chains (À, K) or light chain fragments, VH fragments (variable region of the heavy chain), VL fragments (variable region of the light chain), VHH fragments and VNAR fragments.
[0490] If the drug is an affinity scaffold protein, it is preferably selected from the group comprising shark-derived affinity scaffold proteins, Kunitz domain-derived affinity scaffold proteins, centirine-derived affinity scaffold proteins, proteins ubiquitin-derived affinity scaffold proteins, lipocalin-derived affinity scaffold proteins, ankyrin-derived affinity scaffold proteins, disulfide-rich affinity scaffold proteins, fibronectin-derived affinity scaffold proteins, scaffold proteins of affinity and antibody fragments derived from cameloids, affinity scaffold proteins and antibody fragments derived from llama, transferrin-derived affinity scaffold proteins, and pumpkin-type protease inhibitors with affinity scaffold-derived proteins cysteine knot.
[0491] In another aspect, the present invention relates to a prodrug attached to a support which can be obtained by a process for the preparation of a prodrug attached to a support of the present invention.
[0492] Said prodrug attached to a support releases drug molecules with a half-life ranging from 1 hour to twelve months, for example, from 6 hours to twelve months, from twelve hours to eleven months, from one day to ten months, from three days to nine months, from six days to nine months, from one week to nine months, from two weeks to seven months, from three weeks to eight months, from four weeks to eight months, from six weeks to seven months, from eight weeks to seven months, from ten weeks to six months, from twelve weeks to six months or from sixteen weeks to five months.
[0493] Another aspect of the present invention is a pharmaceutical composition comprising the prodrugs attached to a support of the present invention or a pharmaceutical salt thereof together with a pharmaceutically acceptable excipient.
[0494] Yet another aspect of the present invention is a prodrug attached to a support of the present invention or a pharmaceutical composition comprising the prodrug attached to a support of the present invention for use as a medicament.
[0495] Yet another aspect of the present invention is a method of treating, controlling, delaying or preventing in a mammalian patient, preferably a human being, who needs treatment for one or more conditions comprising administering to said patient a therapeutically effective amount of prodrug attached to a support of the present invention or pharmaceutical composition comprising the prodrug attached to a support of the present invention or a pharmaceutically acceptable salt thereof. Material Examples and Material Methods:
[0496] Amino 4-arm PEG5000 was obtained from JenKem Technology, Beijing, People's Republic of China. Cithrol ™ DPHS was obtained from Croda International Pic, Cowick Hall, United Kingdom. Cis-1,4-cyclohexanedicaboxylic acid was obtained from TCI EUROPE N.V., Boerenveldseweg 6 - Haven 1063, 2070 Zwijndrecht, Belgium.
[0497] Isopropylmalonic acid was obtained from ABCR GmbH & Co. KG, 76187 Karlsruhe, Germany.
[0498] Glutaric acid monobenzyl ester was obtained from IRIS Biotec GmbH, 95615 Marktredwitz, Germany.
[0499] N- (3-maleimidopropyl) -21-amino-4,7, 10,13,16,19-hexaoxa-heneicosanoic acid pentafluorophenyl ester (maleimide-NH-PEG6-PFE) and N- acid pentafluorophenyl ester (3-maleimidopropyl) - 39-amino-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa- nonatriacontanoic (maleimide-NH-PEG12-PFE) was obtained from Biomatrik Inc., Jiaxing, People's Republic of China.
[0500] Oxyma pure and Fmoc-Z-Asp (OtBu) -OH were purchased from Merck Biosciences GmbH, Schwalbach / Ts, Germany.
[0501] (5-methyl-2-oxo-1,3-dioxol-4-yl) -methyl-4-nitrophenyl carbonate was purchased from Chemzon Scientific Inc., Lachine, QC, Canada.
[0502] All other chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany. Methods:
[0503] RP-HPLC was performed on a 100x20 mm or 100x40 mm C18 ReproSil-Pur 300 ODS-3 5μ column (Dr. Maisch, Ammerbuch, Germany) connected to a Waters 600 or 2535 HPLC System and Waters 2487 or 2489 detector Absorbance, respectively. Linear gradients of solution A (0.1% TFA in H2O) and solution B (0.1% TFA in acetonitrile) were used. HPLC fractions containing product were combined and lyophilized.
[0504] Scintillation chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane, ethyl acetate, and methanol as eluents. The products were detected at 254 nm. For products with no absorbance above 240 nm, the fractions were screened by LC / MS.
[0505] LC analytical ultra-performance (UPLC) was performed in a Waters Acquity system equipped with a Waters BEH300 C18 column (2.1 x 50 mm, 1.7 μm particle size) coupled to an LTQ mass spectrometer Orbitrap Discovery by Thermo Scientific.
[0506] HPLC- Electrospray ionization -Mass spectrometry (HPLC-ESI-MS) was performed on a Waters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQ Orbitrap Discovery high resolution / high precision mass spectrometer equipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.1 x 50 mm, 300 A, 1.7 μm, flow: 0.25 mL / min; solvent A: UP-H2O + 0.04% TFA, solvent B: UP -Acetonitrile + 0.05% TFA.
[0507] The MS spectra of PEG products showed a series of portions (CH2CH2O) n due to the polydispersity of the PEG raw materials. For easier interpretation, only a single representative m / z signal is provided in the examples. Example 1 Synthesis of the main chain reagent 1a, 1g, and 1h:


[0508] Main chain reagent 1a has been synthesized as described in example 1 of International Patent Application Publication No. WO 2011/012715 Al with the exception of the use of Boc-DLys (Boc) -OH instead of Boc-LLys (Boc) -OH. MS: m / z 888.50 = [M + 10H +] 10+ (calculated = 888.54)


[0509] The main chain reagent 1g was synthesized from amino 4-arm PEG5000 1b according to the following scheme:

[0510] For the synthesis of compound 1b, amino 4-arm PEG5000 (molecular weight of about 5350 g / mol, 10.7 g, 2.00 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (4.73 g, 12.0 mmol) were dissolved in 43 mL of DCM (anhydrous) and DIPEA (3.10 g, 24.0 mmol, 4.18 mL) was added at room temperature. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (5.30 g, 16.0 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triazanonane (0.33 g, 1.0 mmol) was added. After complete dissolution, the reaction mixture was filtered and the solvent was evaporated at room temperature.
[0511] The residue was dissolved in 40 ml of iPrOH and diluted with 320 ml of MTBE. The product was precipitated overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 200 ml of cooled MTBE (0 ° C). The product was vacuum dried overnight. Yield of 11.1 g (83%) of white solid 1b. MS: m / z 1112.86 = [M + 6H] 6+ (calculated = 11 113.04).
[0512] For the synthesis of compound 1c, the boc-protected compound 1b (11.1 g, 1.66 mmol) was dissolved in 40 ml of 3 M HCl in MeOH and stirred for 20 min at 45 ° C, and then for 10 min at 55 ° C. For precipitation, 10 mL of MeOH and 200 mL of MTBE were added and the mixture was stored for 16 h at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3 and washed with 200 ml of cooled MTBE (0 ° C). The product was vacuum dried overnight. Yield 9.14 g (89%) of white powder 1c (HCl salt). MS: m / z 979.45 = [M + 6H] 6+ (calculated = 979.55).
[0513] For the synthesis of compound 1d, compound 1c (9.06 g, 1.47 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (6.95 g, 17.6 mmol) were dissolved in 50 mL of DCM (anhydrous) and DIPEA (4.56 g, 35.3 mmol, 6.15 mL) was added at room temperature. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (7.80 g, 23.5 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triazanonane (0.49 g, 1.5 mmol) was added. After complete dissolution, the solvent was evaporated at room temperature.
[0514] The residue was dissolved in 35 ml of iPrOH at 40 ° C and diluted with 200 ml of MTBE. The product was precipitated overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 200 ml of cooled MTBE (0 ° C). The product was vacuum dried overnight to provide 1d as a white solid. Yield of 11.6 g (90%) of white solid 1d. MS: m / z 1248.08 = [M + 7H] 7+ (calculated = 1248.27).
[0515] For the synthesis of compound 1e, the boc-protected compound 1d (11.4 g, 1.31 mmol) was dissolved in 40 ml of 3 M HCl in MeOH and stirred for 20 min at 45 ° C, and then for 10 min at 55 ° C. For precipitation, 10 mL of MeOH and 200 mL of MTBE were added and the mixture was stored for 16 h at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3 and washed with 200 ml of cooled MTBE (0 ° C). The product was vacuum dried overnight to provide white powder 1e. Yield 7.60 g (75%) of white powder 1e (HCl salt). MS: m / z 891.96 = [M + 8H] 8+ (calculated = 892.13).
[0516] For the synthesis of compound 1f, compound 1e (7.56 g, 0.980 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (9.27 g, 23.0 mmol) were dissolved in 250 mL of DCM (anhydrous) and DIPEA (6.08 g, 47.0 mmol, 8.19 mL) was added at 35 ° C. After 10 min, 1,9-bis-boc-1,5,9-triazanonane (5.30 g, 16.0 mmol) was added and the mixture was stirred for 15 min. Then additional 1,9-bis-boc-1,5,9-triaza-nonane (0.33 g, 1.0 mmol) was added. After complete dissolution, the solvent was evaporated at room temperature.
[0517] The residue was dissolved in 250 ml of iPrOH at 60 ° C and diluted with 1350 ml of MTBE. The product was precipitated overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 400 ml of cooled MTBE (0 ° C). The product was vacuum dried overnight to provide 1f as a glassy solid. Yield of 11.1 g (83%) of glassy solid 1f. MS: m / z 1312.01 = [M + 10H] 10+ (calculated = 1312.21).
[0518] For the synthesis of the main chain reagent 1g, the boc-protected compound 1f (7.84 g, 0.610 mmol) was dissolved in 16 ml of MeOH at 37 ° C and 55 ml of a pre-cooled solution of 4 M HCl (4 ° C) in dioxane was added at room temperature. The mixture was stirred without cooling for 20 min. After 20 min, 110 mL of 3 M HCl in MeOH was added. The solution was partitioned into 24 Falcon tubes (50 ml) and precipitated with the addition of 40 ml of cold MTBE (-20 ° C) to each Falcon tube. After centrifugation at 3214 rcf for 1 min, the supernatant was decanted and the glassy solid was dissolved in 5 ml of MeOH per Falcon tube and precipitated by adding 40 ml of cold MTBE (-20 ° C) to each Falcon tube again. The supernatant was discarded and the remaining solid was vacuum dried overnight. Yield of 5.74 g (87%) of white glassy solid 1g (HCl salt). MS: m / z 965.46 = [M + 10H] 10+ (calculated = 965.45).

[0519] The 1h main chain reagent was synthesized as described for compound 1e in example 1 of International Patent Application Publication No. WO 2011/012715 A1 with the exception of using Boc-DLys (Boc) -OH instead of Boc-LLys (Boc) -OH. MS: m / z 848.52 = [M + 8H +] 8+ (calculated = 848.57) Example 2 Synthesis of crosslinker reagents 2d, 2g, rac-2k, rac-2o, 2s, 2v, rac-2y, 2ac, 2ag, and 2ak
[0520] Crosslinker reagent 2e was prepared from monobenzyl ester of azelaic acid and PEG 10000 according to the following scheme:

[0521] For the synthesis of monobenzyl ester of azelaic acid 2a, a mixture of azelaic acid (37.6 g, 200 mmol), benzyl alcohol (21.6 g, 200 mmol), p-toluenesulfonic acid (0.80 g , 4.2 mmol), and 240 mL of toluene was refluxed for 7 h in a Dean-Stark equipment. After cooling, the solvent was evaporated and 300 ml of sat. NaHCO3 were added. This mixture was extracted with 3 x 200 ml of MTBE. The combined organic phases were dried over Na2SO4 and the solvent was evaporated. The product was purified on 2 x 340 g of silica using ethyl acetate / heptane (10:90 ^ 25:75) as the eluant. The eluent was evaporated and the residue was vacuum dried overnight. Yield of 25.8 g (46%) of colorless oil 2a. MS: m / z 279.16 = [M + H] + (calculated = 279.16).
[0522] For the synthesis of compound 2b, monobenzyl ester of azelaic acid 2a (3.90 g, 14.0 mmol) and PEG 10,000 (40.0 g, 4.00 mmol) were dissolved in 64 mL of dichloromethane and cooled with an ice bath. A solution of DCC (2.89 g, 14.0 mmol) and DMAP (0.024 g, 0.020 mmol) in 32 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0523] The residue was dissolved in 65 ml of dichloromethane and diluted with 308 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 250 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 40.8 g (97%) of white powder 2b. MS: m / z 835.50 = [M + 14H] 14+ (calculated = 835.56).
[0524] For the synthesis of compound 2c, compound 2b (40.6 g, 3.86 mmol) was dissolved in methyl acetate (250 mL) and 203 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield of 37.2 g (93%) of glassy solid 2c. MS: m / z 882.53 = [M + 13H] 13+ (calculated = 882.51).
[0525] For the synthesis of compound 2d, compound 2c (32.0 g, 3.10 mmol) and TSTU (3.73 g, 12.4 mmol) were dissolved in 150 ml of dichloromethane at room temperature. Then DIPEA (1.60 g, 12.4 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered and the filtrate was diluted with 170 ml of dichloromethane, washed with 140 ml of a solution of 750 g of water / 197 g of NaCl / 3 g of NaOH. The organic phase was dried over MgS04 and the solvent was evaporated in vacuo.
[0526] The residue was dissolved in 200 ml of toluene, diluted with 180 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 100 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 28.8 g (88%) of white powder 2d. MS: m / z 795.47 = [M + 15H] 15+ (calculated = 795.54).
[0527] The crosslinker reagent 2g was prepared from monobenzyl ester of azelaic acid and PEG6000 according to the following scheme:

[0528] For the synthesis of compound 2e, monobenzyl ester of azelaic acid 2a (6.50 g, 23.3 mmol) and PEG 6000 (40.0 g, 6.67 mmol) were dissolved in 140 ml of dichloromethane and cooled with an ice bath. A solution of DCC (4.81 g, 23.3 mmol) and DMAP (0.040 g, 0.33 mmol) in 40 ml of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo. The residue was dissolved in 70 ml of dichloromethane and diluted with 300 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 500 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 41.2 g (95%) of white powder 2e. MS: m / z 833.75 = [M + 8H] 8+ (calculated = 833.75).
[0529] For the synthesis of compound 2f, compound 2e (41.2 g, 6.32 mmol) was dissolved in methyl acetate (238 ml) and ethanol (40 ml), then 400 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield 38.4 g (96%) of vitreous solid 2f. MS: m / z 750.46 = [M + 9H] 9+ (calculated = 750.56).
[0530] For the synthesis of compound 2g, compound 2f (38.2 g, 6.02 mmol) and TSTU (7.25 g, mmol) were dissolved in 130 ml of dichloromethane at room temperature. Then DIPEA (3.11 g, 24.1 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered, the filtrate was diluted with 100 ml of dichloromethane and washed with 200 ml of a solution of 750 g of water / 197 g of NaCl / 3 g of NaOH. The organic phase was dried over MgSO4 and the solvent was evaporated in vacuo.
[0531] The residue was dissolved in 210 ml of toluene, diluted with 430 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 450 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 35.8 g (91%) of white powder 2g. MS: m / z 857.51 = [M + 8H] 8+ (calculated = 857.51).
[0532] The crosslinker reagent 2k was prepared from isopropylmalonic acid monobenzyl ester and PEG 10000 according to the following scheme:

[0533] For the synthesis of monobenzyl ester of rac-2h isopropylmalonic acid, isopropylmalonic acid (35.0 g, 239 mmol), benzyl alcohol (23.3 g, 216 mmol) and DMAP (1.46 g, 12.0 mmol) were dissolved in 100 ml of acetonitrile. The mixture was cooled to 0 ° C with an ice bath. A solution of DCC (49.4 g, 239 mmol) in 150 ml of acetonitrile was added within 15 min at 0 ° C. The ice bath was removed and the reaction mixture was stirred overnight at room temperature, then the solid was filtered. The filtrate was evaporated at 40 ° C in vacuo and the residue was dissolved in 300 ml of MTBE. This solution was extracted with 2 x 300 ml of aqueous sat. of NaHCO3, then the combined aqueous phases were acidified to pH = 1 to 3 using 6 N hydrochloric acid. The resulting emulsion was extracted with 2 x 300 ml of MTBE and the solvent was evaporated. The combined organic phases were washed with 200 ml of aqueous sat. NaCl and dried over MgSO4. The product was purified on 340 g of silica using ethyl acetate / heptane (10:90 ^ 20:80) as the eluent. The eluent was evaporated and the residue was vacuum dried overnight. Yield of 9.62 g (17%) of colorless rac-2h oil. MS: m / z 237.11 = [M + H] + (calculated = 237.11).
[0534] For the synthesis of the rac-2i compound, rac-2h isopropylmalonic acid monobenzyl ester (945 mg, 4.00 mmol) and PEG 10000 (10.0 g, 4.00 mmol) were dissolved in 20 mL of dichloromethane and cooled with an ice bath. A solution of DCC (825 mg, 4.00 mmol) and DMAP (6 mg, 0.05 mmol) in 10 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0535] The residue was dissolved in 20 ml of dichloromethane and diluted with 150 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 500 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 9.63 g (92%) of rac-2i white powder. MS: m / z 742.50 = [M + 16H] 16+ (calculated = 742.51).
[0536] For the synthesis of the rac-2j compound, the rac-2i compound (3.38 g, 0.323 mmol) was dissolved in methyl acetate (100 mL) and 105 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield 3.25 g (98%) of rac-2j glassy solid. MS: m z 731.25 = [M + 16H] 16+ (calculated = 731.25).
[0537] For the synthesis of the rac-2k compound, the rac-2j compound (3.10 g, 0.302 mmol) and TSTU (0.364 g, 1.21 mmol) were dissolved in 15 mL of dichloromethane at room temperature. Then DIPEA (0.156 g, 1.21 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered and the filtrate was washed with 2 x 10 ml of a 0.5 M phosphate buffer, pH = 6.5. The organic phase was dried over MgSO4 and the solvent was evaporated in vacuo. The residue was dissolved in 20 ml of toluene, diluted with 10 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 250 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 2.66 g (84%) of rac-2k white powder. MS: m / z 743.37 = [M + 16H] 16+ (calculated = 743.38).
[0538] The rac-2o crosslinker reagent was prepared from cis-1,4-cyclohexanedicarboxylic acid and PEG 10000 according to the following scheme:


[0539] For the synthesis of rac-2l cis-1,4-cyclohexanedicarboxylic acid monobenzyl ester, cis-1,4-cyclohexanedicarboxylic acid (20.0 g, 116 mmol), benzyl alcohol (11.3 g, 105 mmol ) and DMAP (710 mg, 5.81 mmol) were dissolved in 200 ml of THF. The mixture was cooled to 0 ° C with an ice bath. A solution of DCC (49.4 g, 239 mmol) in 100 mL of THF was added within 15 min at 0 ° C. The ice bath was removed and the reaction mixture was stirred overnight at room temperature, then the solid was filtered. The filtrate was evaporated at 40 ° C and the residue was dissolved in 300 ml of MTBE. This solution was extracted with 2 x 300 ml of aqueous sat. of NaHCO3, then the combined aqueous phases were acidified to pH = 1 to 3 using 6 N hydrochloric acid. The resulting emulsion was extracted with 2 x 300 ml of MTBE and the solvent was evaporated. The combined organic phases were washed with 200 ml of aqueous sat. NaCl and dried over MgS04. The product was purified on 340 g of silica using ethyl acetate / heptane (10:90 ^ 20:80) as the eluent. The eluent was evaporated and the colorless oily residue crystallized during vacuum drying overnight. Yield of 4.82 g (16%) of colorless rac-2l crystals. MS: m / z 263.13 = [M + H] + (calculated = 263.13).
[0540] For the synthesis of the rac-2m compound, cis-1,4-cyclohexanedicarboxylic acid monobenzyl ester rac-21 (2.10 g, 8.00 mmol) and PEG 10,000 (20.0 g, 10.0 mmol ) were dissolved in 50 ml of dichloromethane and cooled with an ice bath. A solution of DCC (1.65 g, 8.00 mmol) and DMAP (0.012 g, 0.10 mmol) in 25 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0541] The residue was dissolved in 55 ml of dichloromethane and diluted with 300 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 250 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 18.2 g (87%) of white powder rac-2m. MS: m / z 745.76 = [M + 16H] 16+ (calculated = 745.77).
[0542] For the synthesis of the rac-2n compound, the rac-2m compound (9.00 g, 0.857 mmol) was dissolved in methyl acetate (100 mL) and 157 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield of 8.83g (100%) of rac-2n vitreous solid. MS: m z 734.50 = [M + 16H] 16+ (calculated = 734.50).
[0543] For the synthesis of the rac-2o compound, the rac-2n compound (8.92 g, 0.864 mmol) and TSTU (1.04 g, 3.64 mmol) were dissolved in 35 ml of dichloromethane at room temperature. Then DIPEA (0.447 g, 3.46 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered and the filtrate was washed with 2 x 10 ml of a 0.5 M phosphate buffer, pH = 6.5. The organic phase was dried over MgSO4 and the solvent was evaporated in vacuo.
[0544] The residue was dissolved in 50 ml of toluene, diluted with 25 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 400 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 7.62 g (84%) of rac-2o white powder. MS: m / z 702.60 = [M + 16H] 16+ (calculated = 702.59).
[0545] The crosslinker reagent 2s was prepared from monobenzyl ester of submeric acid and PEG 10000 according to the following scheme:


[0546] 2p Submeric acid monobenzyl ester was synthesized from submeric acid and benzyl alcohol, therefore to azelaic acid monobenzyl ester 2a.
[0547] Compound 2q was therefore synthesized for the PEG 2b derivative from 2p submeric acid monobenzyl ester and PEG 10000.
[0548] Compound 2r was therefore synthesized for the PEG 2c derivative from compound 2q.
[0549] For the synthesis of compound 2s, compound 2r (18.0 g, 1.74 mmol) and p-nitrophenyl carbonate (2.12 g, 6.76 mmol) were dissolved in 70 ml of acetonitrile at room temperature . Then DIPEA (0.90 g, 6.76 mmol) in 1.0 ml of dichloromethane was added and the mixture was stirred for 17 h. The mixture was diluted with 270 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 200 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 17.6 g (96%) of light yellow powder 2s. MS: m / z 822.20 = [M + 14H] 14+ (calculated = 822.25).
[0550] The crosslinker reagent 2v was prepared from 2p submeric acid monobenzyl ester and PEG6000 according to the following scheme:

[0551] Compound 2t was therefore synthesized for the PEG 2b derivative from 2p submeric acid monobenzyl ester and PEG6000.
[0552] Compound 2u was therefore synthesized for the PEG 2c derivative from compound 2t.
[0553] To 0.38 mmol) and then dissolved DIPEA (0.20 g, 1.52 mmol) in 0.14 mL of dichloromethane was added and the mixture was stirred for 1 h. The mixture was diluted with 26 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 100 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 2.37 g (95%) of light yellow powder 2v. MS: m / z 774.34 = [M + 9H] 9+ (calculated = 77)
[0554] The crosslinker reagent 2y was prepared from isopropylmalonic acid monobenzyl ester and PEG8000 according to the following scheme:

[0555] For the synthesis of the rac-2w compound, rac-2h isopropylmalonic acid monobenzyl ester (2.25 g, 9.50 mmol) and PEG 8000 (19.0 g, 2.38 mmol) were dissolved in 100 mL dichloromethane and cooled with an ice bath. A solution of DCC (1.96 g, 9.50 mmol) and DMAP (14 mg, 0.12 mmol) in 10 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0556] The residue was dissolved in 40 ml of dichloromethane and diluted with 270 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 500 ml of cooled MTBE (-20 ° C).
[0557] The product was vacuum dried overnight. Yield of 18.5 g (92%) of white powder rac-2w. MS: m / z 737.43 = [M + 13H] 13+ (calculated = 737.42).
[0558] For the synthesis of the rac-2x compound, the rac-2w compound (18.4 g, 2.18 mmol) was dissolved in methyl acetate (160 mL) and 254 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield 17.7 g (98%) of rac-2x glassy solid. MS: m / z 723.51 = [M + 13H] 13+ (calculated = 723.55).
[0559] For the synthesis of the rac-2y compound, the rac-2x compound (13.6 g, 1.65 mmol) and TSTU (1.96 g, 6.60 mmol) were dissolved in 60 ml of dichloromethane at temperature environment. Then DIPEA (852 mg, 6.60 mmol) was added and the mixture was stirred for 45 min. The resulting suspension was filtered, the filtrate was diluted with 70 ml of ethyl acetate and washed with 70 ml of a 0.5 M phosphate buffer, pH = 6.5. The organic phase was dried over MgS04 and the solvent was evaporated in vacuo. The residue was dissolved in 80 ml of toluene, the remaining solid was filtered and washed with 20 ml of toluene. The combined toluene fractions were diluted with 35 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 600 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 12.1 g (87%) of rac-2y white powder. MS: m z 738.51 = [M + 13H] 13+ (calculated = 738.49).
[0560] Crosslinker reagent 2ac was prepared from sebacic acid monobenzyl ester and PEG 10000 according to the following scheme:

[0561] For the synthesis of sebacic acid monobenzyl ester 2z, a mixture of sebacic acid (20.2 g, 100 mmol), benzyl alcohol (10.8 g, 100 mmol), p-toluenesulfonic acid (0.40 g , 2.1 mmol), and 120 mL of toluene was refluxed for 19 h in a Dean-Stark equipment. After cooling, the solvent was evaporated and 150 ml of sat. NaHCO3 were added. This mixture was extracted with 3 x 100 ml of MTBE. The combined organic phases were dried over Na2SO4 and the solvent was evaporated. The product was purified on 340 g of silica using ethyl acetate / heptane (10:90 ^ 25:75) as the eluent. The eluent was evaporated and the residue was vacuum dried overnight. Yield of 13.4 g (46%) of colorless oil 2z. MS: m / z 293.16 = [M + H] + (calculated = 293.16).
[0562] For the synthesis of compound 2aa, sebacic acid monobenzyl ester 2z (1.02 g, 3.50 mmol) and PEG 10,000 (10.0 g, 1.00 mmol) were dissolved in 40 ml of dichloromethane and cooled with an ice bath. A solution of DCC (722 mg, 3.5 mmol) and DMAP (6.1 mg, 0.05 mmol) in 12 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0563] The residue was dissolved in 12 ml of dichloromethane and diluted with 45 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 250 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 9.92 g (94%) of white powder 2aa. MS: m / z 793.48 = [M + 15H] 15+ (calculated = 793.56).
[0564] For the synthesis of compound 2ab, compound 2aa (9.83 g, 0.93 mmol) was dissolved in methyl acetate (100 mL) and 123 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield 9.14 g (95%) of glassy solid 2ab. MS: m / z 840.51 = [M + 14H] 14+ (calculated = 840.44).
[0565] For the synthesis of compound 2ac, compound 2ab (9.00 g, 0.87 mmol) and TSTU (1.05 g, 3.47 mmol) were dissolved in 30 ml of dichloromethane at room temperature. Then DIPEA (449 mg, 3.47 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered and the filtrate was diluted with 30 ml of dichloromethane, washed with 60 ml of a solution of 750 g of water / 197 g of NaCl / 3 g of NaOH. The organic phase was dried over MgSO4 and the solvent was evaporated in vacuo.
[0566] The residue was dissolved in 45 ml of toluene, diluted with 35 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 350 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 8.29 g (90%) of white powder 2ac. MS: m z 794.47 = [M + 15H] 15+ (calculated = 794.48).
[0567] The 2ag crosslinker reagent was prepared from undecanedioic acid monobenzyl ester and PEG 10000 according to the following scheme:

[0568] For the synthesis of monobenzyl ester of undecanedioic acid 2ad, a mixture of undecanedioic acid (21.6 g, 100 mmol), benzyl alcohol (10.8 g, 100 mmol), p-toluenesulfonic acid (0.40 g , 2.1 mmol), and 120 mL of toluene was refluxed for 19 h in a Dean-Stark equipment. After cooling, the solvent was evaporated and 150 ml of sat. NaHCO3 were added. This mixture was extracted with 3 x 100 ml of MTBE. The combined organic phases were dried over Na2SO4 and the solvent was evaporated. The product was purified on 340 g of silica using ethyl acetate / heptane (10:90 ^ 25: 75) as the eluent. The eluent was evaporated and the residue was vacuum dried overnight. Yield of 14.8 g (48%) of colorless oil 2ad. MS: m / z 307.19 = [M + H] + (calculated = 307.19).
[0569] For the synthesis of compound 2ae, monobenzyl ester of undecanedioic acid 2ad (1.07 g, 3.50 mmol) and PEG 10,000 (10.0 g, 1.00 mmol) were dissolved in 40 ml of dichloromethane and cooled with an ice bath. A solution of DCC (722 mg, 3.5 mmol) and DMAP (6.1 mg, 0.05 mmol) in 12 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0570] The residue was dissolved in 13 ml of dichloromethane and diluted with 43 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 250 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 10.0 g (95%) of white powder 2ae. MS: m / z 792.48 = [M + 15H] 15+ (calculated = 792.49).
[0571] For the synthesis of compound 2af, compound 2ae (9.83 g, 0.93 mmol) was dissolved in methyl acetate (100 mL) and 103 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield of 8.90 g (92%) of vitreous solid 2af. MS: m / z 780.47 = [M + 15H] 15+ (calculated = 780.47).
[0572] For the synthesis of compound 2ag, compound 2af (8.75 g, 0.84 mmol) and TSTU (1.01 g, 3.37 mmol) were dissolved in 30 ml of dichloromethane at room temperature. Then DIPEA (435 mg, 3.37 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered and the filtrate was diluted with 30 ml of dichloromethane, washed with 60 ml of a solution of 750 g of water / 197 g of NaCl / 3 g of NaOH. The organic phase was dried over MgS04 and the solvent was evaporated in vacuo.
[0573] The residue was dissolved in 38 ml of toluene, diluted with 30 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 350 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield 7.51 g (84%) of white powder 2 g. MS: m z 793.48 = [M + 14H] 14+ (calculated = 793.38).
[0574] The crosslinker reagent was prepared from glutaric acid monobenzyl ester and PEG 10000 according to the following scheme:

[0575] For the synthesis of compound 2ai, monobenzyl ester of glutaric acid 2ah (1.95 g, 8.78 mmol) and PEG 10000 (25.1 g, 2.51 mmol) were dissolved in 70 ml of dichloromethane and cooled with an ice bath. A solution of DCC (1.81 g, 8.78 mmol) and DMAP (15.3 mg, 0.13 mmol) in 18 mL of dichloromethane was added. The ice bath was removed and the mixture was stirred at room temperature overnight. The resulting suspension was cooled to 0 ° C and the solid was filtered. The solvent was evaporated in vacuo.
[0576] The residue was dissolved in 70 ml of dichloromethane and diluted with 360 ml of MTBE at room temperature. The mixture was stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 500 ml of cooled MTBE (-20 ° C).
[0577] The product was vacuum dried overnight.
[0578] Yield of 24.9 g (95%) of white powder 2ai. MS: m / z 740.76 = [M + 16H] 16+ (calculated = 740.76).
[0579] For the synthesis of compound 2aj, compound 2ai (24.9 g, 2.39 mmol) was dissolved in methyl acetate (220 mL) and 302 mg of palladium on charcoal was added. Under an atmosphere of hydrogen at room pressure, the mixture was stirred overnight at room temperature. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated and dried in vacuo overnight. Yield of 24.3 g (99%) of 2aj glassy solid. MS: m / z 729.50 = [M + 16H] 16+ (calculated = 729.49).
[0580] For the synthesis of compound 2ak, compound 2aj (23.9 g, 2.33 mmol) and TSTU (2.81 g, 9.33 mmol) were dissolved in 100 mL of dichloromethane at room temperature. Then DIPEA (1.21 g, 9.33 mmol) was added and the mixture was stirred for 1 h. The resulting suspension was filtered and the filtrate was diluted with 20 ml of dichloromethane, washed with 120 ml of a solution of 750 g of water / 197 g of NaCl / 3 g of NaOH. The organic phase was dried over MgSC4 and the solvent was evaporated in vacuo.
[0581] The residue was dissolved in 125 ml of toluene, diluted with 100 ml of MTBE at room temperature and stored overnight at -20 ° C. The precipitate was collected by filtration through a Por glass filter. 3, and washed with 650 ml of cooled MTBE (-20 ° C). The product was vacuum dried overnight. Yield of 22.5 g (92%) of white powder 2ak. MS: m / z 741.63 = [M + 16H] 16+ (calculated = 741.63). Example 3 Preparation of hydrogel beads 3a, 3b, 3c, and 3d containing free amino groups.
[0582] In a 250 mL cylindrical reactor with a 60 mm diameter bottom outlet, equipped with breathers, a 218 mg emulsion of Cithrol ™ DPHS in 100 mL undecane was stirred with an isojet stirrer, diameter 50 mm at 580 rpm, at room temperature. A solution of 250 mg of 1a and 2205 mg of 2d in 22.1 g of DMSO was added and stirred for 10 min at room temperature to form a suspension. 1.1 mL of TMEDA was added to perform polymerization. The mixture was stirred for 16 h. 1.7 ml of acetic acid were added and then after 10 min, 100 ml of a 15 wt% solution of sodium chloride in water was added. After 10 min, the stirrer was stopped and the phases were left to separate. After 2 h, the aqueous phase containing the hydrogel was drained.
[0583] For fractionation of the size of the beads, the water-hydrogel suspension was diluted with 40 mL of ethanol and sieved wet over steel sieves of 125, 100, 75, 63, 50, 40, and 32 μm using a machine control screen Retsch AS200 for 15 min. The sieving amplitude was 1.5 mm, the water flow was 300 mL / min. The bead fractions that were retained on the 63 and 75 μm sieves were pooled and washed 3 times with 0.1% AcOH, 10 times with ethanol and dried for 16 h at 0.1 mbar to provide 670 mg of 3a as a White powder.
[0584] The content of amino groups of the hydrogel was determined to be 0.145 mmol / g by conjugating a fmoc-amino acid to the free amino groups on the hydrogel and subsequent fmoc determination.
[0585] 3b was prepared as described for 3a with the exception of applying a stirrer speed of 560 rpm, using 350 mg of 1a, 2548 mg of 2g, 26.1 g of DMSO, 257 mg of Cithrol ™ DPHS , 1.5 mL of TMEDA, and 2.4 mL of acetic acid, producing 550 mg of 3b as a white powder, free amino groups at 0.120 mmol / g.
[0586] 3c was prepared as described for 3a with the exception of applying a stirrer speed of 560 rpm, using 250 mg of 1a, 3019 mg of rac-2k, 32.7 g of DMSO, 290 mg of Cithrol ™ DPHS, 1.1 mL of TMEDA, and 1.7 mL of acetic acid, yielding 770 mg of 3c as a white powder, free amino groups at 0.126 mmol / g.
[0587] 3d was prepared as described for 3a with the exception of using 250 mg of 1a, 2258 mg of rac-2o, 22.6 g of DMSO, 222 mg of Cithrol ™ DPHS, 1.1 ml of TMEDA, and 1.7 mL of acetic acid, producing 186 mg of 3d as a white powder, free amino groups at 0.153 mmol / g.
[0588] 3e was prepared as described for 3a with the exception of using 250 mg of 1a, 2168 mg of rac-2y, 21.8 g of DMSO, 215 mg of Cithrol ™ DPHS, 1.1 ml of TMEDA, 1 , 7 mL of acetic acid, producing 710 mg of 3e as a white powder, free amino groups at 0.154 mmol / g.
[0589] 3f was prepared as described for 3a with the exception of applying a stirrer speed of 400 rpm, using 290 mg of the main chain reagent that was described in example 1 of International Patent Application Publication No. WO 2011/012715 A1 as 1g, 2281 mg of 2v, 15.8 g of DMSO, 290 mg of Cithrol ™ DPHS, 2.2 ml of TMEDA, 3.3 ml of acetic acid, sifting over 250, 180 steel sieves , 125, 90, and 63 μm, the product was collected on a 180 μm sieve. The elaboration was producing 820 mg of 3f as a light yellow powder, free amino groups at 0.108 mmol / g.
[0590] 3g was prepared as described for 3a with the exception of applying a stirrer speed of 500 rpm, using 300 mg of the main chain reagent that was described in example 1 of International Patent Application Publication No. WO 2011/012715 A1 as 1g, 2520 mg of 2s, 32.4 g of DMSO, 300 mg of Cithrol ™ DPHS, 2.2 ml of TMEDA, 3.4 ml of acetic acid, yielding 770 mg of 3g as a yellow powder of course, free amino groups at 0.175 mmol / g.
[0591] 3h was prepared as described for 3a with the exception of using 200 mg of 1a, 1995 mg of 2ac, 19.8 g of DMSO, 195 mg of Cithrol ™ DPHS, 0.9 ml of TMEDA, 1.4 mL of acetic acid, producing 650 mg of 3h as a white powder, free amino groups at 0.131 mmol / g.
[0592] 3i was prepared as described for 3a with the exception of using 150 mg of 1a, 1591 mg of 2ag, 15.7 g of DMSO, 154 mg of Cithrol ™ DPHS, 0.7 ml of TMEDA, 1.0 mL of acetic acid, producing 300 mg of 3i as a white powder, free amino groups at 0.123 mmol / g.
[0593] 3j was prepared as described for 3a with the exception of applying a stirrer speed of 570 rpm, using 360 mg of 1h, 2567 mg of 2ak, 26.3 g of DMSO, 257 mg of Cithrol ™ DPHS , 2.2 mL of TMEDA, 3.4 mL of acetic acid, yielding 744 mg of 3j as a white powder, free amino groups at 0.097 mmol / g. Example 4 Synthesis of chaining reagent 4c
[0594] The chaining reagent 4c was synthesized according to the following scheme:
Synthesis of 4th:
[0595] Fmoc-L-Asp (OtBu) -OH (1.00 g, 2.43 mmol) was dissolved with DCC (0.70 g, 3.33 mmol) in DCM (25 mL). Pure oxyma (0.51 g, 3.58 mmol) and collidine (0.50 mL, 3.58 mmol) were added in one portion and a solution of N-Boc-ethylenediamine (0.41 g, 2.56 mmol) ) in DCM (15 mL) was added slowly. After stirring the mixture for 90 min at room temperature, the precipitate formed was filtered and the filtrate was washed with aqueous HCl (0.1 M, 50 ml). The aqueous layer was extracted with DCM (2 x 20 ml) and the combined organic fractions were washed with sat. aqueous (3 x 25 ml) and brine (1 x 50 ml), dried over Na2SO4, filtered and concentrated in vacuo. The crude solid was purified by scintillation chromatography. The intermediate N-boc-N '- (N-fmoc-4-terc.-butyl-L-aspartoyl) -ethylenediamine was obtained with white solid (0.98 g, 1.77 mmol, 73%). MS: m / z 554.29 = [M + H] +, (calculated = 554.29). N-boc-N '- (N-fmoc-4-terc.-butyl-L-aspartoyl) -ethylenediamine (0.98 g, 1.77 mmol) was dissolved in THF (15 mL), DBU (0.31 mL) was added and the solution was stirred for 12 min at room temperature. The reaction was quenched with AcOH (0.5 ml), concentrated in vacuo and the residue was purified by scintillation chromatography to provide 4a (0.61 g, 1.77 mmol, 73% over 2 steps) as a white solid. MS: m / z 332.38 = [M + H] +, (calculated = 332.22). Synthesis of 4b:
[0596] 6-Acetylthiohexanoic acid (0.37 g, 1.95 mmol) was dissolved in DCM (19.5 mL) and pure Oxyma (0.35 g, 2.48 mmol) and DCC (0.40 g, 1.95 mmol) added in one portion. The solution was stirred for 30 min at room temperature, filtered, and the filtrate was added to a solution of 4a (0.61 g, 1.77 mmol) in DCM (10.5 ml). DIPEA (0.46 mL, 2.66 mmol) was added to the solution and the reaction was stirred for 2 h at room temperature. The solution was washed with aqueous H2SO4 (0.1 M, 2 x 30 ml), sat. NaHCO3. aqueous (2 x 20 ml) and brine (1 x 20 ml). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The crude material was purified by scintillation chromatography to provide N-boc-N '- (N-6-acetylthiohexyl-4-terc.-butyl-L-aspartoyl) -ethylenediamine (0.65 g, 1.30 mmol, 73 % over 2 steps) as a white solid. MS: m z 504.27 = [M + H] +, (calculated = 504.28). N-boc-N '- (N-6-Acetylthiohexyl-4-tert.-butyl-L-aspartoyl) -ethylenediamine (0.60 g, 1.18 mmol) was dissolved in TFA (5 mL) and TES (0 , 13 mL) and water (0.13 mL) were added. The mixture was stirred for 30 min at room temperature. TFA was removed in a stream of N2, and crude 4b dissolved in H2O / ACN 1: 1 and purified by RP-HPLC. Yield: 0.39 g, 0.85 mmol (TFA salt), 72%. MS: m / z 348.25 = [M + H] +, (calculated = 348.16). Synthesis of 4c:
[0597] 4b (TFA salt, 0.38 g, 0.80 mmol) was dissolved in DMF (5 mL) and (5-methyl-2-oxo-1,3-dioxol-4- (4-nitrophenyl carbonate) il) -methyl (0.26 g, 0.88 mmol) and DIPEA (0.28 ml, 1.6 mmol) were added. The resulting suspension was diluted with DCM (5 ml) and stirred for 3 h at room temperature. More DIPEA (0.28 mL, 1.6 mmol) was added and stirring continued for 2 h. DCM was concentrated in vacuo, the residue was diluted with 3: 1 H2O / ACN and purified by RP-HPLC to provide N- (5-methyl-2-oxo-1,3-dioxol-4-yl) -methyl- oxocarbonyl-N '- (N-6-acetylthiohexyl-L-aspartyl) -ethylenediamine (0.31 g, 0.62 mmol, 77%) as a colorless oil. MS: m / z 504.16 = [M + H] +, (calculated = 504.17).
[0598] N- (5-methyl-2-oxo-1,3-dioxol-4-yl) -methyl oxocarbonyl-N '- (N-6-acetylthiohexyl-L-aspartyl) -ethylene-diamine (150 mg, 0.30 mmol) was dissolved in DCM (17.5 mL) and NHS (41 mg, 0.36 mmol), DCC (74 mg, 0.36 mmol) and DMAP (4 mg, 0.03 mmol) were added in one serving. The reaction was stirred for 1 h at room temperature and the resulting suspension was filtered. The precipitate was washed with a small amount of DCM and the combined filtrates were concentrated in vacuo. 4c was purified by RP-HPLC to provide a colorless oil (144 mg, 0.24 mmol, 80%). MS: m / z 601.18 = [M + H] +, (calculated = 601.18). Example 5 Preparation of hydrogel beads functionalized with maleimide 5a
[0599] 259.3 mg of dry hydrogel beads 3a was incubated for 15 min in 10 ml of 1% n-propylamine in NMP and then washed twice with 1% n-propylamine in NMP and twice with 2% DIPEA in PWN. 171 mg of maleimide-NH-PEG12-PFE was dissolved in 1 ml of NMP and added to the washed hydrogel beads 3a. The hydrogel suspension was incubated for 2 h at room temperature. The resulting maleimide-functionalized hydrogel beads 5a were washed five times each with NMP, 20 mM succinate, 1 mM Na2EDTA, 0.01% Tween20, pH 3.0, water, and 0.1% acetic acid , 0.01% Tween20. Preparation of hydrogel beads functionalized with maleimide 5b
[0600] 117.7 mg of dry hydrogel beads 3a were dilated in 5 ml of NMP and washed five times with NMP and five times with 2% DIPEA in NMP. 5 eq (56 mg) of maleimide-NH-PEG6-PFE (based on the amine content of the hydrogel beads) were dissolved in 0.5 ml of NMP and added to the washed hydrogel beads 5b. The hydrogel suspension was incubated for 2.5 h at room temperature. The resulting maleimide-functionalized hydrogel beads were washed five times each with NMP and thereafter with 0.1% acetic acid, 0.01% Tween20. Example 6 Transient synthesis of Lucentis-chained-hydrogel 6c transient
[0601] 4.6 mg of Lucentis (represented in the scheme below as Lucentis-NH2) (460 μL of 10 mg / mL of Lucentis in 10 mM of histidine, 10% by weight of α, α-trehalose, 0.01% Tween20, pH 5.5) was exchanged with buffer to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, pH 7.4 and the Lucentis concentration was adjusted to 16, 4 mg / ml. 6 mg of Linker 4c reagent was dissolved in 100 μL of DMSO to produce a concentration of 100 mM. 1 molar equivalent of chaining reagent 4c relative to the amount of Lucentis was added to the Lucentis solution. The reaction mixture was mixed carefully and incubated for 5 min at room temperature. Then, 2 additional molar equivalents of chaining reagent 4c were added to the Lucentis solution in 1 molar equivalent steps and after adding each equivalent the reaction mixture was incubated for 5 min at room temperature producing an unmodified Lucentis mixture and the Lucentis-chained 6a protected monoconjugate.
[0602] The pH of the reaction mixture was adjusted to pH 6.5 by adding 1 M sodium citrate, pH 5.0 and Na2EDTA was added to a final concentration of 5 mM. In order to remove the protective groups of 6a, 0.5 M NH2OH (dissolved in 10 mM sodium citrate, 140 mM sodium chloride, 5 mM Na2EDTA, pH 6.5) was added to a final concentration 45 mM and the deprotection reaction was incubated at room temperature for 4 h, producing the Lucentis-chained 6b monoconjugate. The Lucentis and Lucentis monoconjugate-6b mixture was buffered to 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na2EDTA, 0.01% Tween 20, pH 6.5 and the total concentration of the two Lucentis species was adjusted to 11.8 mg / mL. The mono-conjugated content of Lucentis-chandler 6b in the mixture was 20% as determined by ESI-MS.
[0603] 4 mg of the Lucentis / Lucentis monoconjugate-6b mixture in 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 5 mM Na2EDTA, 0.01% Tween 20, pH 6.5 was added to 1 mg of hydrogel beads functionalized with maleimide 5a and incubated overnight at room temperature producing the transient Lucentis-chain-hydrogel 6c prodrug.
Example 7 In vitro release kinetics - determination of in vitro half-life
[0604] Produce 6c Lucentis-chained-hydrogel (containing approximately 1 mg of Lucentis) was washed five times with 60 mM sodium phosphate, 3 mM Na2EDTA, 0.01% Tween20, pH 7.4 and finally suspended in 1 mL of the aforementioned buffer. The suspension was incubated at 37 ° C. The suspension buffer was changed after different time intervals and analyzed by HPLC-SEC at 220 nm. The peaks corresponding to released Lucentis were integrated and the total released Lucentis was plotted against the total incubation time. Curve adjustment software was applied to determine the first order cleavage rates. Example 8 Preparation of hydrogel beads 8a and 8b containing free amino groups
[0605] 8a was prepared as described for 3a with the exception of using 400 mg of [PEG1250-LLys-LLys2-LLys4 (NH2) 8] 4, 5082 mg of 2s, 33.5 g of DMSO, 400 mg of Cithrol ™ DPHS, 2.3 mL of TMEDA, and 4.8 mL of acetic acid, yielding 1460 mg of 8a as a white powder, free amino groups at 0.057 mmol / g.
[0606] 8b was prepared as described for 3a with the exception of using 290 mg of [PEG1250-LLys-LLys2-LLys4 (NH2) 8] 4, 2281 mg of 2v, 26.1 g of DMSO, 257 mg of Cithrol ™ DPHS, 1.7 mL of TMEDA, and 3.5 mL of acetic acid, producing 820 mg of 8b as a white powder, free amino groups at 0.108 mmol / g.
[0607] 8c was prepared as described for 3a with the exception of using 250 mg of [PEG1250-LLys-LLys2-LLys4 (NH2) 8] 4, 2168 mg of rac-2y, 21.8 g of DMSO, 215 mg of Cithrol ™ DPHS, 1.1 ml of TMEDA, and 1.7 ml of acetic acid, producing 810 mg of 8c as a white powder, free amino groups at 0.154 mmol / g. Example 9 Preparation of hydrogel conjugated to biotin 9a and 9b
[0608] 11 mg of 8a were transferred to a syringe equipped with a filter frit. Hydrogel beads 8a were washed three times each with NMP and H2O / ACN / TFA (1/1 / 0.002; in vol / vol / vol), respectively. 1.14 mg of biotinamidohexanoic acid N-hydroxysuccinimide ester (2.5 μmol) was dissolved in 250 μl of H2O / ACN / TFA (1/1 / 0.002; in v / v / v), added to the washed hydrogel beads 8a and incubated for 5 min at room temperature with shaking (15 rpm). 50 μl of 0.5 M sodium phosphate, pH 7.4 was added and the reaction mixture was incubated for 60 min at room temperature with shaking (15 rpm). The resulting biotin-conjugated hydrogel 9a was washed five times each with H2O / ACN / TFA (1/1 / 0.002; in v / v / v) and with 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 10% ACN, pH 7.4, respectively.
[0609] 9b was prepared according to 9a with the exception that 11.3 mg of hydrogel beads 8b and 2.2 mg of N-hydroxysuccinimide ester of biotinamidohexanoic acid (4.8 μmol) were used. Example 10 Preparation of streptavidin-biotin hydrogel 10a and 10b
[0610] 6 mg of streptavidin was dissolved in 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 10% ACN, pH 7.4, added to biotin conjugated hydrogel 9a (prepared from 11 mg of 8a) and incubated for 60 min at room temperature with shaking (15 rpm) producing streptavidin-biotin hydrogel 10a. 10a was washed twice with 850 μl of 10 mM sodium phosphate, 2.7 mM potassium chloride, 140 mM sodium chloride, 10% ACN, pH 7.4 and the wash fractions were combined. The amount of streptavidin bound to the biotin-conjugated hydrogel was indirectly quantified by photometric determination at 280 nm of the streptavidin concentration in the combined wash fractions and was considered to be 2.6 mg.
[0611] 10b was prepared according to 10a with the exception that hydrogel conjugated to biotin 9b (prepared from 11.3 mg of 8b) and 8.1 mg of streptavidin were used. The amount of streptavidin bound to the biotin 9b-conjugated hydrogel was considered to be 0.5 mg. Example 11 Blocked hydrogel beads 11
[0612] Hydrogel beads were synthesized according to the procedure described in example 3c and functionalized with maleimide groups according to the procedure described in example 5b. Thereafter, 4 ml of the 10 mg / ml hydrogel suspension was transferred into a 20 ml syringe equipped with a frit. The solvent was expelled and the hydrogel was washed 10 times with 5 ml of 10 mM histidine / 10 wt% α, α-trehalose / 0.01% Tween 20 / pH 5.5. The solvent was expelled and 5 ml of 1 mM β-mercaptoethanol in 10 mM histidine / 10 wt% α, α-trehalose / 0.01% Tween 20 / pH 5.5 were extracted into the syringe. The resulting suspension was left to incubate at room temperature under gentle agitation for 5 min. The solvent was discarded and the hydrogel was treated 9 additional times with 5 ml of 1 mM β-mercaptoethanol in 10 mM histidine / 10 wt% α, α-trehalose / 0.01% Tween 20 / pH 5, 5. The solvent was discarded each time. The hydrogel beads were then washed 10 times with each time 5 ml of 10 mM histidine / 10% by weight of α, α-trehalose / 0.01% Tween 20 / pH 5.5, the solvent was discarded each time. The hydrogel beads were then washed ten times with each time 5 ml of PBS-T / pH 7.4, the solvent was discarded each time. Finally, fresh PBS-T / pH 7.4 was extracted into the syringe and the suspension was transferred into a Falcon tube to provide 11.

权利要求:
Claims (34)
[0001]
1. Process for the preparation of a hydrogel, characterized by the fact that it comprises the steps of: (a) providing a mixture comprising (ai) at least one reagent in the main chain, in which at least one reagent in the main chain has a weight molecular ranging from 1 to 100 kDa, and comprises at least three amines (-NH2 and / or -NH-); (a-ii) at least one cross-linking reagent, in which at least one cross-linking reagent has a molecular weight ranging from 6 to 40 kDa, at least one cross-linking reagent comprising (i) at least two carbonyloxy groups (- (C = O) -O- or -O- (C = O) -), and additionally (ii) at least two activated final functional groups selected from the group consisting of activated ester groups, activated carbamate groups, activated carbonate groups and activated thiocarbonate groups, and being PEG-based comprising at least 70% PEG; and (a-iii) a first solvent and at least a second solvent, the second solvent of which is immiscible in the first solvent, in a weight ratio of at least one main chain reagent to at least one crosslinking reagent ranging from 1:99 to 99: 1; and (b) polymerizing the mixture from step (a) in a suspension polymerization to a hydrogel; wherein at least one main chain reagent is selected from the group consisting of a compound of formula (I): B (- (A0) x1 - (SP) x2 - A1- P - A2 - Hyp1) x (I), where B is a branching core, SP is a spacer portion selected from the group consisting of C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl, P is a polymeric PEG-based chain comprising at least 80% of PEG, preferably at least 85% PEG, more preferably at least 90% PEG and more preferably at least 95% PEG, Hyp1 is a moiety comprising an amine (-NH2 and / or -NH-) or a polyamine comprising at least two amines (- NH2 and / or -NH-), x is an integer from 3 to 16, x1, x2 are independently of each other 0 or 1, with the proviso that x1 is 0, if x2 is 0, A0, A1, A2 are independently selected from the group consisting of:
[0002]
Process according to claim 1, characterized in that the mixture of step (a) still comprises a detergent.
[0003]
Process according to claim 1 or 2, characterized by the fact that the polymerization in step (b) is initiated by adding a base.
[0004]
Process according to claim 3, characterized in that the base is selected from N, N, N ', N'-tetramethylethylene diamine, 1,4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1,4 - diazabicyclo [2.2.2] octane, 1,1,4,7,10,10-hexamethyltriethylenetetramine, 1,4,7-trimethyl-1,4,7-triazacyclononane, tris [2- (dimethylamino) ethyl] amine, triethylamine, diisopropylethylamine, trimethylamine, N, N-dimethylethylamine, N, N, N ', N'-tetramethyl-1,6-hexanediamine, N, N, N', N ", N" - pentamethyldiethylenetriamine, 1,8 -diazabicycles [5.4.0] undec-7-ene, 1,5-diazabicycles [4.3.0] non-5-ene, and hexamethylenetetramine.
[0005]
Process according to any one of claims 1 to 4, characterized in that the mixture of step (a) is an emulsion.
[0006]
Process according to any one of claims 1 to 5, characterized by the fact that it also comprises the step of: (c) preparation of the hydrogel.
[0007]
Process according to any one of claims 1 to 6, characterized by the fact that Hyp1, Hyp2, Hyp3 and Hyp4 are selected from the group consisting of a portion of formula (ei) NH2
[0008]
Process according to any one of claims 1 to 7, characterized in that the main chain reagent is a compound of formula (I).
[0009]
Process according to any one of claims 1 to 8, characterized in that the branching core B is selected from the following structures:
[0010]
10. Process according to claim 9, characterized by the fact that B is of formula (a-xiv).
[0011]
11. Process according to any one of claims 1 to 10, characterized by the fact that A0 is
[0012]
Process according to any one of claims 1 to 11, characterized in that x1 and x2 are 0.
[0013]
Process according to any one of claims 1 to 12, characterized in that P has a structure of formula (ci) in which n varies from 6 to 900, more preferably n varies from 20 to 700 and most preferably n ranges from 20 to 250.
[0014]
Process according to any one of claims 1 to 13, characterized in that the portion - A2 - Hyp1 is a portion of the formula
[0015]
Process according to any one of claims 1 to 14, characterized in that the main chain reagent has the following formula:
[0016]
16. Process according to claim 15, characterized by the fact that n ranges from 20 to 30 and most preferably n is 28.
[0017]
Process according to any one of claims 1 to 16, characterized in that the main chain reagent is present in the form of its acidic salt.
[0018]
18. Process according to any one of claims 1 to 17, characterized in that the cross-linking reagent is a compound of formula (V-II):
[0019]
19. Process according to any one of claims 1 to 18, characterized in that the cross-linking reagent is of formula (Vl) to (V-54):
[0020]
20. Process according to any one of claims 1 to 19, characterized in that the hydrogel obtained from the polymerization is a molded article.
[0021]
21. Process according to claim 20, characterized in that the hydrogel is in the form of microparticular beads having a diameter of 1 to 500 micrometers.
[0022]
22. Hydrogel, characterized by the fact that it can be obtained by a process as defined in any one of claims 1 to 21.
[0023]
23. Process for the preparation of a hydrogel-spacer conjugate, characterized by the fact that it comprises the step of: (d) reacting the hydrogel as defined in claim 22 with a spacer reagent of the formula (VI) Ax1-S0-Ax2 ( VI), where S0 is selected from the group comprising C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl, the fragment of which is optionally interrupted by one or more group (s) selected from -NH-, - N (C1-4 alkyl) -, -O-, -S, -C (O) -, -C (O) NH-, - C (O) N (C1-4 alkyl) -, -OC (O) -, -S (O) -, -S (O) 2-, 4- to 7-membered heterocyclyl, phenyl and naphthyl; Ax1 is a functional group for reaction with an amine group of the hydrogel; and Ax2 is a functional group; in the presence of a solvent to obtain a hydrogel-spacer conjugate.
[0024]
24. Process according to claim 23, characterized by the fact that Ax1 is selected from the group comprising activated carboxylic acid; Cl- (C = O) -; NHS- (C = O) -, where NHS is N-hydroxysuccinimide; CISO2-; R1 (C = O) -; I-; Br-; Cl-; SCN-; and CN-, where R1 is selected from the group comprising H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, 4- to 7-membered heterocyclyl, 8 to 11-membered heterocyclyl, phenyl , naphthyl, indenyl, indanyl, and tetralinyl.
[0025]
25. Process according to claim 23 or 24, characterized in that Ax2 is selected from the group comprising -maleimide, -SH, -NH2, -SeH, -N3, -C ^ CH, - CR1 = CR1aR1b, - OH, - (C = O) -S-R1, - (C = O) -H, -NH-NH2, -O-NH2, -Ar-X0, - Ar-Sn (R1) (R1a) (R1b) , -Ar- B (OH) (OH),
[0026]
26. Hydrogel-spacer conjugate, characterized by the fact that it can be obtained by the process as defined in claim 24 or 25.
[0027]
27. Process for the preparation of a prodrug attached to a support, characterized in that it comprises the step of: (e) reacting a hydrogel as defined in claim 22 or a hydrogel-spacer conjugate as defined in claim 27 with a prodruger reagent and biologically active portion of formula (VII) Ay1-LD (VII), where Ay1 is a functional group for reaction with an amine of the hydrogel of step (b) or (c) or for reaction with the functional group Ax2 of the hydrogel-spacer conjugate of step (d), L is a prodruger; D is a biologically active portion; in the presence of a solvent, to obtain a prodrug attached to a support.
[0028]
28. Process according to claim 27, characterized by the fact that Ax2 / Ay1 are selected from the following:
[0029]
29. Process according to claim 27 or 28, characterized in that Ax2 is a thiol and Ay1 is of formula (VIIa) T-PG0 - S- (VIIa), where T is H or a label portion; PG0 is a sulfur-activating portion; and S is sulfur.
[0030]
Process according to any one of claims 27 to 29, characterized in that D has a molecular weight ranging between 2 and 500 kDa, more preferably between 5 and 250 kDa, more preferably between 5 and 100 kDa and most preferably between 10 and 60 kDa.
[0031]
31. Process according to any of claims 27 to 30, characterized in that D is a monoclonal or polyclonal antibody or a fragment or fusion thereof.
[0032]
32. Support-linked prodrug, characterized by the fact that it can be obtained by a process for the preparation of a support-linked prodrug as defined in any of claims 28 to 31.
[0033]
33. Pharmaceutical composition, characterized in that it comprises the prodrug attached to a support as defined in claim 32, or a pharmaceutical salt thereof together with a pharmaceutically acceptable excipient.
[0034]
34. A prodrug attached to a support according to claim 32 or a pharmaceutical composition according to claim 33, characterized in that it is for use as a medicine.

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WO2022043493A1|2020-08-28|2022-03-03|Ascendis Pharma Oncology Division A/S|Glycosylated il-2 proteins and uses thereof|

法律状态:
2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C08J 3/24 (2006.01), C08G 83/00 (2006.01), A61K 47 |

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2019-07-02| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-03-24| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-09-01| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-12-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-23| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 08/10/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP12188228.6|2012-10-11|

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EP12188228|2012-10-11|

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PCT/EP2013/070962|WO2014056926A1|2012-10-11|2013-10-08|Hydrogel prodrugs|

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