HK1208694B - Hydrogel prodrugs - Google Patents

Description

Hydrogel prodrugs

Technical Field

The present invention relates to a process for preparing hydrogels and to hydrogels obtainable by said process. The 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 carrier-linked prodrug and to a carrier-linked prodrug obtainable by said process, in particular to a carrier-linked prodrug providing a controlled and/or sustained release of a drug from a carrier. In addition, the invention relates to the use of a hydrogel for the preparation of a prodrug linked to a carrier.

Background

Conventional hydrogels are three-dimensional hydrophilic or amphiphilic polymer networks capable of absorbing large amounts of water. These networks may comprise various polymers and are insoluble due to covalent chemical and/or physical cross-linking, e.g. the presence of ionic, hydrophobic interactions or entanglements.

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 tissue sealing are for example disclosed in WO 2008/125655 a 1.

In addition, WO 99/014259A 1 discloses cross-linked PEG hydrogels in which drug molecules are entrapped.

The release of entrapped drug molecules from such conventional hydrogels depends on the degradation of the hydrogel and can lead to sudden release, temporarily resulting in too high a drug level and difficulty in predicting drug release. It is desirable to control and/or maintain the release of the drug from the hydrogel. WO06/003014 a2 and WO2011/012715a1 describe hydrogels as carriers in prodrugs for linking carriers, wherein a biologically active moiety is covalently linked to the hydrogel by a reversible prodrug linker. Such prodrugs attached to hydrogels release the drug controllably and with a specific half-life.

However, the hydrogels disclosed in WO2011/012715a1 are preferred for controlled and sustained release of smaller drug molecules and do not provide sufficient access to larger drug molecules, such as protein drugs, thus resulting in a low drug loading of such hydrogels.

Detailed Description

Method for producing hydrogel and hydrogel

Thus, there is a need for hydrogels that can be used as carriers for carrier-linked prodrugs suitable for controlled and sustained release of larger drug molecules.

It is therefore an object of the present invention to overcome at least some of the above disadvantages and to provide a carrier for a prodrug that can be used as a linking carrier for the controlled and/or sustained release of larger drug molecules.

In one aspect, the present invention relates to a method of making a hydrogel, the method comprising the steps of:

(a) providing a mixture comprising:

(a-i) at least one framing agent, wherein the at least one framing agent has a molecular weight of 1-100kDa and comprises at least 3 amines (-NH)₂and/or-NH-);

(a-ii) at least one crosslinker agent, wherein the at least one crosslinker agent has a molecular weight of 6-40kDa, wherein the at least one crosslinker agent comprises:

(i) at least 2 carbonyloxy (- (C ═ O) -O-or-O- (C ═ O) -), and additionally

(ii) At least 2 reactive functional end groups selected from: an active ester group, an active urethane group, an active carbonate group and an active thiocarbonate group,

and is PEG-based comprising at least 70% PEG; and

(a-iii) a first solvent and at least a second solvent, said second solvent being immiscible in the first solvent,

wherein the weight ratio of the at least one framing agent to the at least one cross-linking agent is 1:99 to 99: 1; and

(b) polymerizing the mixture of step (a) to form a hydrogel in a suspension polymerization.

In general, the design of the crosslinking agents is known to affect the pore size of the hydrogel, but it is expected that the longer the crosslinking agents, the more likely they will form secondary structures that will block access to the interior space of the hydrogel. This obstruction will prevent the larger protein drug from entering the interior space of the hydrogel and the attachment of the drug molecules will be limited primarily to the surface and the area near the hydrogel surface. It has now surprisingly been found that despite these anticipated limitations, large drugs, such as proteins, can be incorporated into the hydrogels of the present invention in amounts that make these hydrogels suitable as prodrug carriers.

Within the present invention, the terms are used in the following meanings.

As used herein, the term "hydrogel" means a hydrophilic or amphiphilic polymer network composed of homopolymers or copolymers that are insoluble due to the presence of covalent chemical crosslinks. Crosslinking provides network structure and physical integrity. Hydrogels exhibit thermodynamic compatibility with water, which allows them to swell in aqueous media.

The term "reagent" as used herein means a compound comprising at least one functional group that reacts with a functional group of another reagent or moiety.

The term "backbone reagent" as used herein means a reagent suitable as a starting material for forming a hydrogel. As used herein, the backbone reagent preferably does not comprise a biodegradable linkage. The backbone reagent may comprise a "branched core," which refers to an atom or moiety to which more than one other moiety is attached.

The term "crosslinker agent" as used herein means a linear or branched agent suitable as a starting material for crosslinking a backbone agent. Preferably, the crosslinker agent is a linear compound. The crosslinker agent comprises at least 2 biodegradable linkages.

The term "moiety" as used herein means a portion of a molecule that lacks one or more atoms as compared to a corresponding agent. If, for example, a reagent of the formula "H-X-H" reacts with another reagent and becomes part of a reaction product, the corresponding moiety of the reaction product has the structure "H-X-" or "-X-", with each "-" being represented on another moiety. Thus, the biologically active moiety is released from the prodrug as a drug.

Thus, the phrase "in bound form" is used to refer to the corresponding moiety of an agent, i.e., "lysine in bound form" refers to a lysine moiety that lacks one or more atoms of a lysine agent and is part of a molecule.

The term "functional group" as used herein means an atomic group that can react with other functional groups. Functional groups include, but are not limited to, the following: carboxylic acid (- (C ═ O) OH), primary or secondary amine (-NH)₂-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 (C ═ O) -, and (d) hydroxy (- (OH) H)>N-N<) Isocyanates, isothiocyanates, phosphoric acid (-O (P ═ O) OHOH), phosphonic acid (-O (P ═ O) OHH), haloacetyl, alkyl halides, acryloyl, aryl fluoride, hydroxylamine, disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, oxetanes and aziridines.

The term "reactive functional group" as used herein means a functional group that is attached to an activating group, i.e., the functional group is reacted with an activating reagent. Preferred reactive functional groups include, but are not limited to, reactive ester groups, reactive urethane groups, reactive carbonate groups, and reactive thiocarbonate groups. Preferred activating groups are selected from the formulae ((f-i) to (f-vi):

wherein:

the dotted line indicates attachment to the rest of the molecule;

b is 1,2, 3 or 4; and is

X^HIs Cl, Br, I or F.

Thus, preferred active esters have the formula:

-(C＝O)-X^F

wherein:

X^Fselected from the group consisting of formulas (f-i), (f-ii), (f-iii), (f-iv), (f-v), and (f-vi).

Thus, preferred reactive carbamates have the formula:

-N-(C＝O)-X^F

wherein:

X^Fselected from the group consisting of formulas (f-i), (f-ii), (f-iii), (f-iv), (f-v), and (f-vi).

Thus, preferred activated carbonates have the formula:

-O-(C＝O)-X^F

wherein:

X^Fselected from the group consisting of formulas (f-i), (f-ii), (f-iii), (f-iv), (f-v), and (f-vi).

Thus, preferred activated thioesters have the formula:

-S-(C＝O)-X^F，

wherein:

X^Fselected from the group consisting of formulas (f-i), (f-ii), (f-iii), (f-iv), (f-v), and (f-vi).

Thus, a "reactive terminal functional group" is a reactive functional group located at the end of a structural moiety or molecule, i.e., a terminal reactive functional group.

The term "end-capping group" as used herein means a moiety that is irreversibly, i.e., permanently, attached to a functional group such that it cannot react with the functional group of another reagent or moiety.

The term "protecting group" as used herein means a moiety which reversibly attaches to a functional group such that it cannot react with, for example, another functional group suitable alcohol (-OH) protecting groups are, for example, acetyl, benzoyl, benzyl, β -methoxyethoxymethyl, dimethoxytrityl, methoxymethyl, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, trityl, trimethylsilyl, t-butyldimethylsilyl, tri-isopropylsiloxymethyl, triisopropylsilyl ether, methyl ether and ethoxyethyl ether suitable amine protecting groups are, for example, benzyloxyformyl, p-methoxybenzylcarbonyl, t-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3, 4-dimethoxybenzyl, p-methoxyphenyl and tosyl suitable carbonyl protecting groups are, for example, acetal and ketal, acylal and dithiane suitable carboxylic acid protecting groups are, for example, benzyl, t-butyl, 2, 6-dimethylphenol, 2, 6-di-tert-butylphenol, di-butyl-phenol and di-tert-butylphenolAn oxazoline. Suitable phosphate protecting groups are for example 2-cyanoethyl and methyl.

The term "work-up" as used herein refers to the series of operations required to isolate and purify the product of a chemical reaction, particularly a polymerization.

The term "polymer" as used herein means a molecule comprising repeating structural units (i.e., monomers) linked by chemical bonds in a linear, cyclic, branched, crosslinked, or dendritic fashion, or a combination thereof, which may be of synthetic or biological origin, or a combination of both. It is to be understood that the polymer may, for example, further comprise functional groups or end-capping moieties. Preferably, the polymer has a molecular weight of at least 0.5kDa, such as a molecular weight of at least 1kDa, a molecular weight of at least 2kDa, a molecular weight of at least 3kDa or a molecular weight of at least 5 kDa.

The term "polymeric" as used herein means an agent or moiety comprising one or more polymers.

It will be understood by those skilled in the art that the polymerization products obtained from the polymerization reaction do not all have the same molecular weight but exhibit a molecular weight distribution. Thus, as used herein, the molecular weight ranges, molecular weights and monomer number ranges in the polymer and the monomer number index average molecular weight and number average of monomers in the polymer. The term "number average molecular weight" as used herein means the ordinary arithmetic mean of the molecular weights of the individual polymers.

The term "polymerization" as used herein means the process of reacting a monomer or macromer reagent in a chemical reaction to form a polymer chain or network, including but not limited to a hydrogel.

The term "macromonomer" as used herein means a molecule resulting from the polymerization of a monomeric reagent.

The term "polycondensation" or "condensation reaction" as used herein means a chemical reaction in which the functional groups of two reagents react to form one single molecule, i.e., a reaction product, and release a low molecular weight molecule such as water.

The term "suspension polymerization" as used herein means a heterogeneous and/or biphasic polymerization reaction in which a monomeric reagent is dissolved in a first solvent to form a dispersed phase, and the dispersed phase is emulsified in a second solvent to form a continuous phase. In the present invention, the monomeric reagent is at least one framework reagent and at least one crosslinker reagent. Neither the first solvent nor the monomeric reagent is soluble in the second solvent. The emulsion is prepared by stirring, shaking, exposing to ultrasonic waves or Microsieve^TMUnder emulsification, more preferably by stirring or Microsieve^TMEmulsification is more preferably achieved by stirring. The emulsion is stabilized by a suitable emulsifierAnd (4) determining. The polymerization is initiated by adding a base soluble in the first solvent as an initiator. Suitable commonly known bases suitable for use as initiators may be tertiary bases such as Tetramethylethylenediamine (TMEDA).

The term "immiscible" as used herein refers to the property in which two substances cannot be combined to form a homogeneous mixture.

The term "polyamine" as used herein means comprising more than one amine (-NH-and/or-NH)₂) Such as 2-64 amines, 4-48 amines, 6-32 amines, 8-24 amines, or 10-16 amines. Particularly preferred polyamines comprise from 2 to 32 amines.

As used herein, the term "PEG-based comprising at least X% PEG" with respect to a moiety or agent means that the moiety or agent comprises at least X% (w/w) ethylene glycol units (-CH)₂CH₂O-), wherein the ethylene glycol units may be blocked, arranged alternately or may be distributed randomly within a moiety or agent, preferably all of the ethylene glycol units of said moiety or agent are present as one block; the remaining weight% of the PEG-based moiety or agent is other moieties, especially other moieties selected from the following substituents and bonds:

●C_1-50alkyl radical, C_2-50Alkenyl radical, C_2-50Alkynyl, C_3-10Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl; a naphthyl group; indenyl (indenyl); indanyl (indanyl); and tetralinyl;

● is a bond selected from:

wherein:

the dotted line indicates attachment to a structural moiety or the remainder of the reagent, and

R¹and R^1aIndependent of each otherIs selected from H and C_1-6An alkyl group.

The term "C" as used herein_1-4Alkyl "alone or in combination means a straight or branched alkyl group having 1 to 4 carbon atoms. Straight and branched C if present at the end of the molecule_1-4Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two structural parts of the molecule pass through C_1-4When alkyl groups are attached, then C_1-4An example of an alkyl group is-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅)-、-C(CH₃)₂-、-CH₂-CH₂-CH₂-CH₂-and-CH₂-CH₂-CH₂(CH₃)-。C_1-4Each hydrogen atom of the alkyl group may be replaced by a substituent as defined below.

The term "C" as used herein_1-6Alkyl "alone or in combination means a straight or branched alkyl group having 1 to 6 carbon atoms. Straight and branched C if present at the end of the molecule_1-6Examples of alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 2, 3-dimethylbutyl and 3, 3-dimethylpropyl. When two structural parts of the molecule pass through C_1-6When alkyl groups are attached, then C_1-6An example of an alkyl group is-CH₂-、-CH₂-CH₂-、-CH(CH₃)-、-CH₂-CH₂-CH₂-、-CH(C₂H₅) -and-C (CH)₃)₂-。C_1-6Each hydrogen atom of the alkyl group may be replaced by a substituent as defined below.

Thus, as used herein, the term "C_1-20Alkyl "alone or in combination means a straight or branched alkyl group having 1 to 20 carbon atoms. The term "C_8-18Alkyl radicals "alone or in combinationMeaning a straight-chain or branched alkyl group having 8 to 18 carbon atoms. Thus, as used herein, the term "C_1-50Alkyl "alone or in combination means a straight or branched alkyl group having 1 to 50 carbon atoms. C_1-20Alkyl radical, C_8-18Alkyl and C_1-50Each hydrogen atom of the alkyl group may be replaced with a substituent. In each case, an alkyl group may be present at the end of the molecule or two moieties of the molecule may be linked by an alkyl group.

The term "C" as used herein_2-6Alkenyl "alone or in combination means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond and having from 2 to 6 carbon atoms. If present at the end of the molecule, an example is-CH ═ CH₂、-CH＝CH-CH₃、-CH₂-CH＝CH₂、-CH＝CHCH₂-CH₃and-CH ═ CH₂. When two structural parts of the molecule pass through C_2-6When alkenyl is attached, then C_2-6An example of an alkenyl group is-CH ═ CH-. C_2-6Each hydrogen atom of the alkenyl group may be replaced by a substituent as defined below. Optionally, one or more triple bonds may be present.

Thus, as used herein, the term "C_2-20Alkenyl "alone or in combination means a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms. The term "C_2-50Alkenyl "alone or in combination means a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon double bond and having 2 to 50 carbon atoms. If present at the end of the molecule, an example is-CH ═ CH₂、-CH＝CH-CH₃、-CH₂-CH＝CH₂、-CH＝CHCH₂-CH₃and-CH ═ CH₂. When two moieties of a molecule are linked through an alkenyl group, an example is, for example, -CH ═ CH-. C_2-20Alkenyl or C_2-50Each hydrogen atom of the alkenyl group may be replaced by a substituent as defined below. Optionally, one or more triple bonds may be present.

The term "C" as used herein_2-6Alkynyl "alone or in combination means to encompassAt least one carbon-carbon triple bond and having a straight-chain or branched hydrocarbon residue of 2 to 6 carbon atoms. Examples are-C.ident.CH, -CH if present at the end of the molecule₂-C≡CH、CH₂-CH₂-C ≡ CH and CH₂-C≡C-CH₃. When two moieties of the molecule are linked by an alkynyl group, an example is-C.ident.C-. C_2-6Each hydrogen atom of the alkynyl group may be replaced by a substituent as defined below. Optionally, one or more double bonds may be present.

Thus, as used herein, the term "C_2-20Alkynyl "alone or in combination means a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon triple bond and having 2 to 20 carbon atoms," C_2-50Alkynyl "alone or in combination means a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon triple bond and having 2 to 50 carbon atoms. Examples are-C.ident.CH, -CH if present at the end of the molecule₂-C≡CH、CH₂-CH₂-C ≡ CH and CH₂-C≡C-CH₃. When two moieties of the molecule are linked by an alkynyl group, an example is-C.ident.C-. C_2-20Alkynyl or C_2-50Each hydrogen atom of the alkynyl group may be replaced by a substituent as defined below. Optionally, one or more double bonds may be present.

The term "C" as used herein_3-8Cycloalkyl radicals "or" C_3-8Cycloalkyl ring "means a cycloalkyl chain having 3 to 8 carbon atoms, which may be saturated or unsaturated, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl. Each hydrogen atom of the cycloalkyl carbon may be replaced by a substituent as defined below. The term "C_3-8Cycloalkyl radicals "or" C_3-8Cycloalkyl rings "also include bridged bicyclic rings such as norbornane (norbonane) or norbornene (norbonene). Thus, "C_3-5Cycloalkyl "means a cycloalkyl group having 3 to 5 carbon atoms," C_3-10Cycloalkyl "means cycloalkyl having 3 to 10 carbon atoms.

Thus, as used herein, the term "C_3-10Cycloalkyl "means a carbocyclic ring system having 3 to 10 carbon atoms, which maySaturated or unsaturated, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. The term "C_3-10Cycloalkyl "also includes at least partially saturated monocyclic and bicyclic carbons.

The term "halogen" as used herein means fluorine, chlorine, bromine or iodine. Fluorine or chlorine are particularly preferred.

The term "4-7 membered heterocyclyl" or "4-7 membered heterocyclic ring" as used herein means a ring (fully saturated, partially saturated or unsaturated aromatic or non-aromatic ring) having 4,5,6 or 7 ring atoms and which may contain up to the maximum number of double bonds, wherein at least one to 4 ring atoms are selected from sulfur (including-s (o) -, -s (o)₂-), oxygen and nitrogen (including ═ n (o) -, and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 4-7 membered heterocycles include, but are not limited to, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, and,Azole,Oxazoline, heteroOxazole, isoOxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, and,Oxazolidine, isozymeOxazolidines, thiazolidines, isothiazolidines, thiadiazolidines, cyclic compoundsButanesulfone, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane (diazepane), azepine(azepine) and homopiperazine (homopterazine). Each hydrogen atom of the 4-7 membered heterocyclic group or the 4-7 membered heterocyclic group may be replaced by a substituent as defined below.

The term "8-11 membered heterobicyclic group" or "8-11 membered heterobicyclic" as used herein means a heterocyclic ring system of two rings having 8-11 ring atoms, wherein at least one ring atom is shared by both rings and may contain up to the maximum number of double bonds (fully saturated, partially saturated or unsaturated aromatic or non-aromatic rings), wherein at least one ring atom up to 6 ring atoms are selected from sulfur (including-s (o) -, -s (o))₂-), oxygen and nitrogen (including ═ n (o) -, and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 8-11 membered heterobicyclic rings are indole, indoline (indoline), benzofuran, benzothiophene, benzoAzole, benzisohOxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine(benzazepine), purines and pteridines. The term 8-11 membered heterobicyclic also includes two ring spiro structures, such as 1, 4-dioxa-8-azaspiro [4.5 ]]Decane or bridged heterocycles, e.g. 8-aza-bicyclo [3.2.1]Octane. Each hydrogen atom of an 8-11 membered heterobicyclic group or an 8-11 membered heterobicyclic ring carbon may be replaced by a substituent as defined below.

The term "substituted" means that one or more-H atoms of a molecule or moiety are replaced with a different atom or group of atoms known as a "substituent". Suitable substituents are selected from halogen; CN; COOR⁹；OR⁹；C(O)R⁹；C(O)N(R⁹R^9a)；S(O)₂N(R⁹R^9a)；S(O)N(R⁹R^9a)；S(O)₂R⁹；S(O)R⁹；N(R⁹)S(O)₂N(R^9aR^9b)；SR⁹；N(R⁹R^9a)；NO₂；OC(O)R⁹；N(R⁹)C(O)R^9a；N(R⁹)S(O)₂R^9a；N(R⁹)S(O)R^9a；N(R⁹)C(O)OR^9a；N(R⁹)C(O)N(R^9aR^9b)；OC(O)N(R⁹R^9a)；T；C_1-50An alkyl group; c_2-50An alkenyl group; or C_2-50Alkynyl, wherein T; c_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50Alkynyl is optionally substituted by one or more R, the same or different¹⁰Is substituted, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl groups are optionally interrupted by one or more groups selected from: t, -C (O) O-; -O-; -c (o) -; -C (O) N (R)¹¹)-；-S(O)₂N(R¹¹)-；-S(O)N(R¹¹)-；-S(O)₂-；-S(O)-；-N(R¹¹)S(O)₂N(R^11a)-；-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(R^11a) -; and-OC (O) N (R)¹¹R^11a)；

Wherein:

R⁹、R^9a、R^9bindependently selected from H; t; and C_1-50An alkyl group; c_2-50An alkenyl group; or C_2-50Alkynyl, wherein T; c_1-50An alkyl group; c_2-50Alkenyl radical(ii) a And C_2-50Alkynyl is optionally substituted by one or more R, the same or different¹⁰Is substituted, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl groups are optionally interrupted by one or more groups selected from: t, -C (O) O-; -O-; -c (o) -; -C (O) N (R)¹¹)-；-S(O)₂N(R¹¹)-；-S(O)N(R¹¹)-；-S(O)₂-；-S(O)-；-N(R¹¹)S(O)₂N(R^11a)-；-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(R^11a) -; and-OC (O) N (R)¹¹R^11a)；

T is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cycloalkyl group; 4-7 membered heterocyclyl; or 8-11 membered heterobicyclic group, wherein T is optionally substituted by one or more R which may be the same or different¹⁰Substitution;

R¹⁰is halogen; CN; oxygen (═ O); COOR¹²；OR¹²；C(O)R¹²；C(O)N(R¹²R^12a)；S(O)₂N(R¹²R^12a)；S(O)N(R¹²R^12a)；S(O)₂R¹²；S(O)R¹²；N(R¹²)S(O)₂N(R^12aR^12b)；SR¹²；N(R¹²R^12a)；NO₂；OC(O)R¹²；N(R¹²)C(O)R^12a；N(R¹²)S(O)₂R^12a；N(R¹²)S(O)R^12a；N(R¹²)C(O)OR^12a；N(R¹²)C(O)N(R^12aR^12b)；OC(O)N(R¹²R^12a) (ii) a Or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more, the same or different, halogens;

R¹¹、R^11a、R¹²、R^12a、R^12bindependently selected from H; or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more of the same or different halogens.

In one embodiment, R⁹、R^9a、R^9bMay be independently of one another H.

In one embodiment, R¹⁰Is C_1-6An alkyl group.

In one embodiment, T is phenyl.

Preferably, up to 6-H atoms of the molecule are independently replaced by a substituent, for example 5-H atoms are independently replaced by a substituent, 4-H atoms are 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.

The term "spacer" as used herein means the insertion of one or more atoms between two carbon atoms or at the end of a carbon chain between each carbon atom and a hydrogen atom.

The term "prodrug" as used herein means a compound that undergoes biotransformation before exhibiting its pharmacological effect. Thus, a prodrug may be considered to be a biologically active moiety attached to a specialized non-toxic protecting group that is used in a short-acting manner to alter or eliminate undesirable properties in the parent molecule. This may include enhancing desirable properties in the drug and suppressing undesirable properties.

The term "carrier-linked prodrug" as used herein means a prodrug comprising a temporary linkage of a biologically active moiety to a short-acting carrier group which results in improved physicochemical or pharmacokinetic properties and which is easily removable in vivo, typically by hydrolytic cleavage.

The term "reversible prodrug linker moiety" as used herein means a moiety that is linked at one end to a biologically active moiety D by a reversible bond and at the other end by a permanent bond, in the present invention, by an amine function or a of the backbone moiety^x2And A^y1Reacting to form, thereby linking the biologically active moiety to the hydrogel carrier in the carrier linked prodrug of the invention. "reversible linkage" is a bond which is non-enzymatically hydrolytically degradable, i.e.cleavable, under physiological conditions (aqueous buffer at pH7.4, 37 ℃) with a half-life of 1 hour to 12 months.

In contrast, "permanent linkage" is non-enzymatically hydrolytically degradable under physiological conditions (aqueous buffer at pH7.4, 37 ℃) with a half-life of more than 12 months.

A "biodegradable linkage" is a linkage that is enzymatically and/or non-enzymatically hydrolytically degradable, i.e., cleavable, under physiological conditions (aqueous buffer pH7.4, 37 ℃) with a half-life of 1 hour to 12 months. Preferably, the biodegradable linkages are also non-enzymatically hydrolytically degradable under physiological conditions.

The term "traceless prodrug linker" as used herein means a reversible prodrug linker that releases the free form of the drug upon cleavage. The term "free form" of a drug as used herein means an unmodified pharmacologically active form of the drug.

The term "peptide" as used herein 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 having more than 50 amino acid monomers.

The term "oligonucleotide" as used herein means a short nucleic acid polymer having up to 100 bases.

The term "pharmaceutical composition" as used herein means one or more active ingredients and one or more inert ingredients, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Thus, the pharmaceutical compositions of the present invention include any composition prepared by mixing a prodrug of the present invention linked to a carrier and one or more pharmaceutically acceptable excipients.

The term "excipient" as used herein refers to a diluent, adjuvant, or vehicle with which the therapeutic agent is administered. Such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, 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. When the pharmaceutical composition is administered intravenously, saline and aqueous glucose are preferred excipients. Saline solutions and aqueous dextrose and glycerol solutions are preferred 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, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the pharmaceutical compositions may also contain minor amounts of wetting or emulsifying agents, pH buffering agents, for example, acetates, succinates, TRIS, carbonates, phosphates, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), MES (2- (N-morpholine) ethanesulfonic acid), or may contain detergents such as Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids such as glycine, lysine or histidine. These pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The pharmaceutical compositions may be formulated as suppositories with conventional binders and excipients such as triglycerides. Oral formulations may contain standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. An example of a suitable pharmaceutical excipient is described by e.w. martin in Remington's pharmaceutical sciences. The composition comprises a therapeutically effective amount of the drug or biologically active moiety and an appropriate amount of excipients to provide a form for proper administration to a patient. The formulation should be suitable for the mode of administration.

In general, the term "comprising" or "includes" also includes "consisting of ….

Some of the backbone and crosslinker reagents that can be used as starting materials in process step (a) are commercially available. In addition, the backbone and crosslinker reagents can be prepared according to the methods described in the examples section. Methods for the synthesis of suitable backbone reagents are described in example 1 of WO2011/012715a1, which is incorporated herein by reference. Example 2 of WO2011/012715a1 further provides a method of synthesizing crosslinker reagents having lower molecular weights, which can be modified using standard chemical knowledge to obtain crosslinker reagents suitable for the present invention. Based on these methods, one skilled in the art can apply standard chemical knowledge to arrive at the backbone and crosslinker reagents for use in the present invention.

The mixture of step (a) comprises a first solvent and at least a second solvent. The first solvent is preferably selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol, and water, and a mixture thereof.

At least one backbone agent and at least one cross-linking agent are dissolved in a first solvent, i.e., the dispersed phase of the suspension polymerization. In one embodiment, the backbone reagent and the crosslinker reagent are dissolved separately, i.e. in different containers, using the same or different solvents, preferably the same solvent for both reagents. In another embodiment, the backbone reagent and the crosslinker reagent are dissolved together, i.e., in the same vessel and using the same solvent.

Suitable solvents for the backbone reagent are organic solvents. Preferably, the solvent is selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol, and water, and mixtures thereof. More preferably, the backbone reagent is dissolved in a solvent selected from acetonitrile, dimethylsulfoxide, methanol or a mixture thereof. Most preferably, the backbone reagent is dissolved in dimethylsulfoxide.

In one embodiment, the backbone reagent is dissolved in the solvent at a concentration of 1-300mg/ml, more preferably 5-60mg/ml, most preferably 10-40 mg/ml.

Suitable solvents for the crosslinker agent are organic solvents. Preferably, the solvent is selected from dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, dimethylformamide, acetonitrile, dimethylsulfoxide, propylene carbonate, N-methylpyrrolidone, methanol, ethanol, isopropanol, water or a mixture thereof. More preferably, the crosslinker agent is dissolved in a solvent selected from dimethylformamide, acetonitrile, dimethylsulfoxide, methanol or mixtures thereof. Most preferably, the crosslinker agent is dissolved in dimethyl sulfoxide.

In one embodiment, the crosslinker agent is dissolved in the solvent at a concentration of 5-500mg/ml, more preferably 25-300mg/ml, most preferably 50-200 mg/ml.

The at least one backbone agent and the at least one crosslinker agent are mixed in a weight ratio of 1:99 to 99:1, such as a weight ratio of 2:98 to 90:10, a weight ratio of 3:97 to 88:12, a weight ratio of 3:96 to 85:15, a weight ratio of 2:98 to 90:10 and a weight ratio of 5:95 to 80:20, particularly preferably in a weight ratio of 5:95 to 80:20, wherein the first number relates to the backbone agent and the second number relates to the crosslinker agent.

Preferably, the ratio is selected such that the mixture of step (a) comprises a molar excess of amine groups from the backbone reagent compared to the reactive functional end groups of the crosslinker reagent. Thus, the hydrogels produced by the methods of the present invention have free amine groups that can be used to bind other moieties on the hydrogel, such as spacer, affinity ligand, chelator and/or reversible prodrug linker moieties.

The at least one second solvent, i.e. the continuous phase of the suspension polymerization, is preferably an organic solvent, more preferably an organic solvent selected from the group consisting of: linear, branched or cyclic C_5-30An alkane; linear, branched or cyclic C_5-30An olefin; linear, branched or cyclic C_5-30An alkyne; linear or cyclic poly (dimethylsiloxane); aromatic C_6-20A hydrocarbon; and mixtures thereof. Even more preferably, the at least second solvent is selected fromLinear, branched or cyclic C_5-16An alkane; toluene; xylene; trimethylbenzene; hexamethyldisiloxane; or mixtures thereof. Most preferably, the at least second solvent is selected from linear C_7-11Alkanes such as heptane, octane, nonane, decane and undecane.

Preferably, the mixture of step (a) further comprises a detergent. Preferred detergents are Cithrol DPHS, Hypermer 70A, Hypermer B246, Hypermer 1599A, Hypermer 2296 or Hypermer 1083. Most preferred is Cithrol DPHS.

Preferably, the detergent has a concentration of from 0.1g to 100g per 1L of the total mixture (i.e. the dispersed and continuous phases together). More preferably, the detergent has a concentration of 0.5g to 10g per 1L of the total mixture, most preferably the detergent has a concentration of 0.5g to 5g per 1L of the total mixture.

Preferably the mixture of step (a) is an emulsion.

The polymerization of step (b) is initiated by addition of a base. Preferably the base is a non-nucleophilic base soluble in the alkane, more preferably the base is selected from the group consisting of N, N, N ', N ' -Tetramethylethylenediamine (TMEDA), 1, 4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1, 4-diazabicyclo [2.2.2] octane, 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 ' -pentamethyldiethylenetriamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, N, N, N ', N ' -dimethyldiethylenetriamine, N, N, N ' -dimethyldiethylenetriamine, N, N, 1, 8-diazabicyclo [5.4.0, 1, 5-diazabicyclo [4.3.0] non-5-ene and hexamethylenetetramine. Even more preferably, the base is selected from the group consisting of TMEDA, 1, 4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1, 4-diazabicyclo [2.2.2] octane, 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.

The base is added to the mixture of step (a) in an amount of 1 to 500 equivalents per reactive functional end group in the mixture, preferably in an amount of 5 to 50 equivalents, more preferably in an amount of 5 to 25 equivalents, most preferably in an amount of 10 equivalents.

In process step (b), the polymerization of the hydrogels according to the invention is a condensation reaction, which is preferably carried out with continuous stirring of the mixture from step (a). The tip speed (tip speed ═ pi × stirrer rotation speed × stirrer diameter) is preferably 0.2 to 10 meters/second (m/s), more preferably 0.5 to 4m/s, most preferably 1 to 2 m/s.

In a preferred embodiment of step (b), the polymerization is carried out in a cylindrical vessel equipped with baffles. The diameter to height ratio of the vessel may be from 4:1 to 1:2, more preferably the diameter to height ratio of the vessel is from 2:1 to 1: 1.

Preferably, the reaction vessel is equipped with an axial flow stirrer selected from the group consisting of: pitched blade agitators (pitched blade agitator), marine propellers (marine type propeller), or Lightnin A-310. More preferably, the agitator is a pitched blade agitator.

Step (b) may be carried out over a wide temperature range, preferably at a temperature of from-10 ℃ to 100 ℃, more preferably at a temperature of from 0 ℃ to 80 ℃, even more preferably at a temperature of from 10 ℃ to 50 ℃, most preferably at ambient temperature. "ambient temperature" means the temperature present in a typical laboratory environment, preferably means a temperature of 17-25 ℃.

The hydrogel obtained by polymerization is preferably a shaped article, such as a coating, a mesh, a scaffold, a nanoparticle or a microparticle. More preferably, the hydrogel is in the form of particulate beads having a diameter of 1-500 μm, more preferably 10-300 μm, even more preferably 20-150 μm, most preferably 30-130 μm. The above diameters are measured when the hydrogel microparticles are completely hydrated in water.

In one embodiment, the method of making the hydrogel of the present invention further comprises the steps of:

(c) the hydrogel is post-treated.

Step (c) comprises one or more of the following steps:

(c1) the excess liquid is removed from the polymerization reaction,

(c2) the hydrogel is washed to remove the solvent used during polymerization,

(c3) the hydrogel was transferred to a buffer solution,

(c4) the hydrogel is sized/sieved and,

(c5) the hydrogel was transferred to a container and,

(c6) the hydrogel is dried and the dried hydrogel is dried,

(c7) transferring the hydrogel to a special solvent suitable for sterilization, and

(c8) the hydrogel is sterilized, preferably by gamma irradiation.

Preferably, step (c) comprises all of the following steps:

(c1) the excess liquid is removed from the polymerization reaction,

(c2) the hydrogel is washed to remove the solvent used during polymerization,

(c3) the hydrogel was transferred to a buffer solution,

(c4) the hydrogel is sized/sieved and,

(c5) the hydrogel was transferred to a container and,

(c7) transferring the hydrogel to a special solvent suitable for sterilization, and

(c8) the hydrogel is sterilized, preferably by gamma irradiation.

Skeleton reagent

The at least one framing agent has a molecular weight of 1-100kDa, preferably 2-50kDa, more preferably 5-30kDa, even more preferably 5-25kDa, most preferably 5-15 kDa.

Preferably, the backbone reagent is PEG-based comprising at least 10% PEG, more preferably at least 20% PEG, even more preferably at least 30% PEG, most preferably at least 40% PEG.

In one embodiment, the matrix agent is present in the form of an acidic salt thereof, preferably an acid addition salt. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, aspartate, benzoate, benzenesulfonate (besylate), bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate (camsylate), citrate, edisylate (edisylate), ethanesulfonate (esylate), formate, fumarate, glucoheptonate (gluceptate), gluconate, glucuronate, hexafluorophosphate, salicylate (hibenzate), hydrochloride, hydrobromide, hydriodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthenate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate (pamoate), phosphate, hydrogenphosphate, dihydrogenphosphate, sertralite (sacharate), stearate, succinate, tartrate and tosylate. It is particularly preferred that the backbone reagent is present in the form of its hydrochloride salt.

In one embodiment, the at least one scaffold agent is selected from:

a compound of formula (I)

B(–(A⁰)_x1–(SP)_x2–A¹–P–A²–Hyp¹)_x(I)，

Wherein:

b is a branched core, and B is a branched core,

SP is a spacer moiety selected from: c_1-6Alkyl radical, C_2-6Alkenyl and C_2-6An alkynyl group,

p is a PEG-based polymer chain comprising at least 80% PEG, preferably at least 85% PEG, more preferably at least 90% PEG, most preferably at least 95% PEG,

Hyp¹is a compound containing amine (-NH)₂and/or-NH-) or comprises at least two amines (-NH)₂and/or-NH-,

x is an integer of 3 to 16,

x1, x2 independently of one another are 0 or 1, with the proviso that if x2 is 0, then x1 is 0,

A⁰、A¹、A²independently of one another, from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

a compound of formula (II):

Hyp²–A³–P–A⁴–Hyp³(II)，

wherein:

p is as defined above for the compounds of formula (I),

Hyp²、Hyp³independently of one another, contain at least two amines (-NH)₂and/or-NH-), and

A³and A⁴Independently selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

a compound of formula (III):

P¹–A⁵–Hyp⁴(III)，

wherein:

P¹is a PEG-based polymer chain comprising at least 80% PEG, preferably at least 85% PEG, more preferably at least 90% PEG, most preferably at least 95% PEG,

Hyp⁴is a compound containing at least 3 amines (-NH)₂and/or-NH), and

A⁵selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

and

a compound of formula (IV):

T¹–A⁶–Hyp⁵(IV),

wherein:

Hyp⁵is a compound containing at least 3 amines (-NH)₂and/or-NH), and

A⁶selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group; and is

T¹Is selected from C_1-50Alkyl radical, C_2-50Alkenyl or C_2-50Alkynyl, said segments being optionally interrupted by one or more groups selected from:-NH-、-N(C_1-4alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclyl, phenyl or naphthyl.

In the following section, the term "Hyp^x"collectively refers to Hyp¹、Hyp²、Hyp³、Hyp⁴And Hyp⁵。

Preferably, the backbone reagent is a compound of formula (I), (II) or (III), more preferably the backbone reagent is a compound of formula (I) or (III), most preferably the backbone reagent is a compound of formula (I).

In a preferred embodiment, in the compounds of formula (I), x is 4,6 or 8. Preferably, in the compound of formula (I), x is 4 or 8, most preferably, x is 4.

In a preferred embodiment, in the compounds of the formulae (I) to (IV), A⁰、A¹、A²、A³、A⁴、A⁵And A⁶Selected from:

preferably, in the compounds of the formula (I), A⁰Comprises the following steps:

preferably, in the compounds of the formula (I), A¹Comprises the following steps:

preferably, in the compounds of the formula (I), A²Comprises the following steps:

preferably, in the compounds of the formula (II), A³Comprises the following steps:

and A is⁴Comprises the following steps:

preferably, in the compounds of the formula (III), A⁵Comprises the following steps:

preferably, in the compounds of the formula (IV), A⁶Comprises the following steps:

preferably, in the compounds of the formula (IV), T¹Selected from H and C_1-6An alkyl group.

In one embodiment, in the compound of formula (I), the branched core B is selected from the following structures:

wherein:

the dotted line indicates the connection at A⁰Or if x1 and x2 are both 0, then the connection is at A¹In the above-mentioned manner,

t is 1 or 2; it is preferred that t is 1 and,

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.

In a preferred embodiment, B has a structure of formula (a-i), (a-ii), (a-iii), (a-iv), (a-v), (a-vi), (a-vii), (a-viii), (a-ix), (a-x), (a-xiv), (a-xv), or (a-xvi). More preferably, B has a structure of formula (a-iii), (a-iv), (a-v), (a-vi), (a-vii), (a-viii), (a-ix), (a-x) or (a-iv). Most preferably, B has the structure of formula (a-xiv).

A preferred embodiment is B and A⁰Or B and A if x1 and x2 are both 0¹Selected from the following structures:

wherein:

the dashed line indicates a link to SP or P if x1 and x2 are both 0.

More preferably, B and A⁰Or B and A if x1 and x2 are both 0¹Has the structure of formula (b-i), (b-iv), (b-vi) or (b-viii), most preferably has the structure of formula (b-i).

In one embodiment, x1 and x2 of formula (I) are 0.

In one embodiment, the PEG-based polymer chain P has 0.3-40 kDa; for example, a molecular weight of 0.4-35kDa, 0.6-38kDa, 0.8-30kDa, 1-25kDa, 1-15kDa or 1-10 kDa. Most preferably, P has a molecular weight of 1-10 kDa.

In one embodiment, the PEG-based polymer chain P¹Has a molecular weight of 0.3-40 kDa; for example, a molecular weight of 0.4-35kDa, 0.6-38kDa, 0.8-30kDa, 1-25kDa, 1-15kDa or 1-10 kDa. Most preferably, P¹Has a molecular weight of 1-10 kDa.

In one embodiment, in the compound of formula (I) or (II), P has the structure of formula (c-I):

wherein n is from 6 to 900, more preferably n is from 20 to 700, most preferably n is from 20 to 250.

In one embodiment, in the compounds of formula (III), P¹A structure having formula (c-ii):

wherein:

n is 6 to 900, more preferably n is 20 to 700, most preferably n is 20 to 250;

T⁰is selected from C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl optionally interrupted by one or more groups selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O) -, or-S (O)₂-。

In one embodiment, in the compounds of formulae (I) to (IV), the moiety Hyp^xIs a polyamine, preferably comprising moieties of the formulae (d-i), (d-ii), (d-iii) and/or (d-iv) in bound form, if applicable, in R-and/or S-configuration:

wherein:

z1, z2, z3, z4, z5, z6 are independently of one another 1,2, 3,4,5,6, 7 or 8.

More preferably, Hyp^xComprising a bound form, and lysine in R-and/or S-configuration, ornithine, diaminopropionic acid and/or diaminobutyric acid. Most preferably, Hyp^xComprising lysine in bound form and in R-and/or S-configuration.

Hyp^xHas a molecular weight of 40Da to 30kDa, preferably 0.3kDa to 25kDa, more preferably 0.5kDa to 20kDa, even more preferably 1kDa to 20kDa, most preferably 2kDa to 15 kDa.

Hyp^xPreferably selected from:

a moiety of formula (e-i):

wherein:

p1 is an integer from 1 to 5, preferably p1 is 4, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴The above step (1);

a moiety of formula (e-ii):

wherein:

p2, p3 and p4 are identical or different and are each, independently of one another, an integer from 1 to 5, preferably p2, p3 and p4 are 4, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶In the above-mentioned manner,

(iv) a moiety of formula (e-iii):

wherein:

p5-p11 are identical or different and are each, independently of one another, an integer from 1 to 5, preferably p5-p11 are 4, and

if the backbone reagent is of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has formula (IV), the dotted line indicates the linkage at A⁶In the above-mentioned manner,

a moiety of formula (e-iv):

wherein:

p12-p26 are identical or different and are each, independently of one another, an integer from 1 to 5, preferably p12-p26 are 4, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a structure of formula (e-v):

wherein:

p27 and p28 are identical or different and are each, independently of one another, an integer from 1 to 5, preferably p27 and p28 are 4,

q is an integer from 1 to 8, preferably q is 2 or 6, most preferably q is 6, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

(vii) a moiety of formula (e-vi):

wherein:

p29 and p30 are identical or different and are each, independently of one another, an integer from 2 to 5, preferably p29 and p30 are 3, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the skeleton agent hasThe structure of formula (IV) is such that the dotted line indicates the connection at A⁶The above step (1);

a moiety of formula (e-vii):

wherein:

p31-p36 are identical or different and are each, independently of one another, an integer from 2 to 5, preferably p31-p36 are 3, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a moiety of formula (e-viii):

wherein:

p37-p50 are identical or different and are each, independently of one another, an integer from 2 to 5, preferably p37-p50 are 3, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1); and

a moiety of formula (e-ix):

wherein:

p51-p80 are identical or different and are each, independently of one another, an integer from 2 to 5, preferably p51-p80 are 3, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1); and is

Wherein the moieties (e-i) to (e-v) may be in the R-or S-configuration at each chiral centre, preferably all chiral centres of the moieties (e-i) to (e-v) are in the same configuration.

Preferably, Hyp^xHaving a structure of formula (e-i), (e-ii), (e-iii), (e-iv), (e-vi), (e-vii), (e-viii) or (e-ix). More preferably, Hyp^xHaving a structure of formula (e-ii), (e-iii), (e-iv), (e-vii), (e-viii) or (e-ix), even more preferably Hyp^xHaving a structure of formula (e-ii), (e-iii), (e-vii) or (e-viii), most preferably Hyp^xHaving the structure of formula (e-iii).

If Hyp^xComprising lysine in bound form, it is preferred that it comprises D-lysine, since it has surprisingly been found that hydrogels comprising backbone moieties comprising D-lysine-as opposed to those comprising L-lysine-are more stable when administered to a patient.

If the backbone reagent has the structure of formula (I), then it is preferred that the moiety-A²–Hyp¹Is a moiety of the formula:

wherein:

the dotted line indicates the attachment to P; and is

E¹Selected from the formulae (e-i) to (e-ix).

If the skeleton agent has a structure of the formula (II), the preferred structural part Hyp²–A³-a moiety of formula:

wherein:

the dotted line indicates the attachment to P; and is

E¹Selected from the formulae (e-i) to (e-ix);

and preferred moieties-A⁴–Hyp³Is a moiety of the formula:

wherein:

the dotted line indicates the attachment to P; and is

E¹Selected from the formulae (e-i) to (e-ix).

If the backbone reagent has the structure of formula (III), then the preferred moiety-A⁵–Hyp⁴Is a moiety of the formula:

wherein:

the dotted line indicates the connection at P¹The above step (1); and is

E¹Is selected fromFormulae (e-i) to (e-ix).

More preferably, the backbone reagent has the structure of formula (I), and B has the structure of formula (a-xiv).

Even more preferably, the backbone reagent has the structure of formula (I), B has the structure of formula (a-xiv), x1 and x2 are 0, and A is¹is-O-.

Even more preferably, the backbone reagent has the structure of formula (I), B has the structure of formula (a-xiv), A¹is-O-and P has the structure of formula (c-i).

Even more preferably, the backbone reagent is of formula (I), B is of formula (a-xiv), x1 and x2 are 0, A¹is-O-and P has the formula (c-i).

Even more preferably, the backbone reagent is of formula (I), B is of formula (a-xiv), x1 and x2 are 0, A¹is-O-, P has the formula (c-i), A²is-NH- (C ═ O) -and Hyp¹Having formula (e-iii).

Most preferably, the backbone reagent has the formula:

wherein:

n is 10 to 40, preferably 10 to 30, more preferably 10 to 20.

Also preferably, n is 20-30kDa, most preferably n is 28.

SP of formula (I) is selected from C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Spacer moiety of 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-.

Crosslinker reagent

The at least one crosslinker reagent comprises at least 2 carbonyloxy (- (C ═ O) -O-or-O- (C ═ O) -), which are biodegradable bonds. These biodegradable linkages are necessary to make the hydrogel biodegradable. In addition, the at least one crosslinker reagent comprises at least 2 reactive functional end groups that react with the amine of the at least one backbone reagent during the reaction of step (b).

The crosslinker agent has a molecular weight of 6-40kDa, more preferably 6-30kDa, even more preferably 6-20kDa, even more preferably 6-15kDa, most preferably 6-10 kDa.

The crosslinker agent comprises at least 2 reactive functional end groups selected from: active ester groups, active urethane groups, active carbonate groups and active thiocarbonate groups, which during polymerization react with amine groups of the backbone reagent to form amide bonds, i.e. the backbone moiety and the crosslinker moiety are preferably linked by amide bonds.

In a preferred embodiment, the crosslinker agent is a compound of formula (V-I):

wherein:

each D¹、D²、D³And D⁴Are identical or different and are each, independently of one another, selected from the group consisting of-O-, -NR⁵-, -S-and-CR⁶R^6a-；

Each R is¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁶And R^6aAre identical OR different and are each, independently of one another, selected from the group consisting of-H, -OR⁷、-NR⁷R^7a、-SR⁷And C_1-6An alkyl group; optionally each pair of R¹/R²、R³/R⁴、R^1a/R^2aAnd R^3a/R^4aMay independently form a chemical bond and/or each pair R¹/R^1a、R²/R^2a、R³/R^3a、R⁴/R^4a、R⁶/R^6a、R¹/R²、R³/R⁴、R^1a/R^2aAnd R^3a/R^4aIndependently of one another, together with the atoms to which they are attached form C_3-8Cycloalkyl either forms ring a or is linked together with the atoms to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclic or adamantyl group;

each R is⁵Independently selected from-H and C_1-6An alkyl group; optionally, each pair of R¹/R⁵、R²/R⁵、R³/R⁵、R⁴/R⁵And R⁵/R⁶May independently form a chemical bond and/or be joined together with the atoms to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclic group;

each R is⁷、R^7aIndependently selected from H and C_1-6An alkyl group;

a is selected from indenyl, indanyl and tetrahydronaphthyl;

P²comprises the following steps:

m is 120-;

r1, r2, r7, r8 are independently 0 or 1;

r3, r6 are independently 0,1, 2,3 or 4;

r4, r5 are independently 1,2, 3,4,5,6, 7, 8, 9 or 10;

s1, s2 are independently 1,2, 3,4,5 or 6;

Y¹、Y²are identical or different and are each, independently of one another, selected from the formulae (f-i) to (f-vi):

wherein:

the dashed lines indicate attachment to the rest of the molecule,

b is 1,2, 3 or 4,

X^His Cl, Br, I or F.

Preferably, the crosslinker agent is a compound of formula (V-II):

wherein:

D¹、D²、D³and D⁴Are identical or different and are each, independently of one another, selected from the group consisting of-O-, -NR⁵-, -S-and-CR⁵R^5a-；

R¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵And R^5aAre identical or different and are each, independently of one another, selected from H and C_1-6An alkyl group; optionally, one or more pairs of R¹/R^1a、R²/R^2a、R³/R^3a、R⁴/R^4a、R¹/R²、R³/R⁴、R^1a/R^2aAnd R^3a/R^4aForm chemical bonds or are linked together with the atoms to which they are attached to form C_3-8Cycloalkyl either forms ring a or is linked together with the atoms to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclic or adamantyl group;

a is selected from phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl;

P²comprises the following steps:

m is 120-;

r1, r2, r7, r8 are independently 0 or 1;

r3, r6 are independently 0,1, 2,3 or 4;

r4, r5 are independently 1,2, 3,4,5,6, 7, 8, 9 or 10;

s1, s2 are independently 1,2, 3,4,5 or 6;

Y¹、Y²are identical or different and are each, independently of one another, selected from the formulae (f-i) to (f-vi):

wherein:

the dashed lines indicate attachment to the rest of the molecule,

b is 1,2, 3 or 4

X^HIs Cl, Br, I or F.

It should be understood that the structural components:

representing at least 2 reactive functional end groups.

Y of the formulae (V-I) and (V-II) is preferred¹And Y²A junction having the formula (f-i), (f-ii) or (f-v)And (5) forming. More preferably, Y¹And Y²Having the structure of formula (f-i) or (f-ii), most preferably, Y¹And Y²Has a structure of formula (f-i).

Preferably, the moieties Y of the formulae (V-I) and (V-II)¹And Y²All having the same structure. More preferably, the moiety Y¹And Y²All have the structure of formula (f-i).

Preferably, r1 for formulae (V-I) and (V-II) is 0.

Preferably, r1 and s1 of both formulae (V-I) and (V-II) are 0.

Preferably, one or more pairs R of the formulae (V-I) and (V-II)¹/R^1a、R²/R^2a、R³/R^3a、R⁴/R^4a、R¹/R²、R³/R⁴、R^1a/R^2aAnd R^3a/R^4aForm chemical bonds or are linked together with the atoms to which they are attached to form C_3-8Cycloalkyl or form ring a.

Preferably, one or more pairs R of the formulae (V-I) and (V-II)¹/R²、R^1a/R^2a、R³/R⁴、R^3a/R^4aTaken together with the atoms to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclic group.

Preferably, the crosslinker reagents of formulae (V-I) and (V-II) are symmetrical, i.e., moieties:

having a structure portion:

the same structure.

In a preferred embodiment, s1, s2, r1 and r8 of formulae (V-I) and (V-II) are 0.

In another preferred embodiment, s1, s2, r1 and r8 of formulae (V-I) and (V-II) are 0 and r4 and r5 of formulae (V-I) and (V-II) are 1.

Preferred crosslinker reagents have the formulae (V-1) to (V-54):

wherein:

if applicable, each crosslinker agent may be in the form of a racemic mixture thereof; and is

m、Y¹And Y²As defined above.

Even more preferred crosslinker reagents are of formulae (Va-1) to (Va-54):

wherein:

if applicable, each crosslinker agent may be in the form of a racemic mixture thereof; and is

m、Y¹And Y²As defined above.

It has surprisingly been found that the use of a crosslinker agent having a branch (i.e. a residue other than H) on the carbon alpha to the carbonyloxy group results in the formation of a hydrogel which is more resistant to enzymatic degradation, for example by esterase degradation.

Similarly, it was surprisingly found that the fewer atoms present between (C ═ O) of the carbonyloxy group and (C ═ O) of the adjacent active ester, active carbamate, active carbonate or active thiocarbamate, the more resistant the resulting hydrogel is to degradation, for example by esterase.

Thus, crosslinker reagents V-11 to V-54, V-1, V-2, Va-11 to Va-54, Va-1 and Va-2 are preferred crosslinker reagents. The crosslinker reagents Va-11 to Va-54, Va-1 and Va-2 are more preferred crosslinker reagents. Most preferred is crosslinker reagent Va-14.

In another embodiment, the crosslinker reagent V-1, V-2, V-5, V-6, V-7, V-8, V-9, V-10, V-11, V-12, V-13, V-14, V-15, V-16, V-17, 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, V-40, V-41, V-42, V-43, V-9, V-21, V, V-44, V-45, V-46, V-47, V-48, V-49, V-50, V-51, V-52, V-53 and V-54 are preferred cross-linker reagents. More preferably, the at least one crosslinker agent has the 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 V-46, and most preferably, the at least one crosslinker agent has the formula V-5, V-6, V-9 or V-14.

In another embodiment, the crosslinker reagent 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-32, Va, Va-44, Va-45, Va-46, Va-47, Va-48, Va-49, Va-50, Va-51, Va-52, Va-53 and Va-54 are even more preferred cross-linking agent reagents. More preferably, the at least one cross-linking agent reagent has 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, the at least one cross-linking agent reagent has formula Va-5, Va-6, Va-9 or Va-14.

The preferred embodiments of the compounds of formulae (V-I) and (V-II) as described above thus apply to the preferred compounds of formulae (V-1) to (V-54), and more preferably to the compounds of formulae (Va-1) to (Va-54).

In another aspect, the present invention relates to a hydrogel obtainable by a process as defined above.

The hydrogel contains 0.01-1 mmol/g primary amine group (-NH)₂) More preferably, 0.02 to 0.5 mmoles per gram primary amine group, most preferably 0.05 to 0.3 mmoles per gram primary amine group. The term "X mmoles/gram primary amine group" means that 1 gram of dry hydrogel contains X mmoles primary amine groups. The measurement of the amine content of the hydrogels was performed according to Gude et al (Letters in PeptideSecience, 2002, 9 (4): 203-206, which is incorporated herein by reference in its entirety), and is also described in detail in the examples section.

Preferably, the term "dry" as used herein means having a residual water content of at most 10%, preferably less than 5%, more preferably less than 2% (measured according to Karl Fischer). The preferred method of drying is lyophilization.

Hydrogel-spacer conjugates and methods of making

In another aspect, the present invention relates to a method of making a hydrogel-spacer conjugate, the method comprising the steps of:

(d) reacting the hydrogel from step (b) or (c) with a spacer reagent of formula (VI) in the presence of a solvent to obtain a hydrogel-spacer conjugate:

A^x1-S⁰-A^x2(VI)，

wherein:

S⁰is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl, said segment being optionally substitutedOne or more radical spacers selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S, -C (O) -, -C (O) NH, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclic group, phenyl group and naphthyl group;

A^x1is a functional group that reacts with an amine group of the hydrogel; and is

A^x2Is a functional group.

Preferably, A^x1Selected from the group consisting of activated carboxylic acids; cl- (C ═ O) -; NHS- (C ═ O) -, where NHS is N-hydroxysuccinimide; ClSO₂-；R¹(C ═ O) -; i-; br-; cl-; SCN-; and CN-, wherein R¹Selected from H, C_1-6Alkyl, alkenyl, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl.

Most preferably, A^x1Is an activated carboxylic acid.

Suitable activating agents for obtaining the activated carboxylic acids are, for example, N' -dicyclohexyl-carbodiimide (DCC), 1-ethyl-3-carbodiimide (EDC), benzotriazol-1-yl-oxytripyrrolidinylHexafluorophosphate (PyBOP), bromotripyrrolidinonylHexafluorophosphate (PyBrOP), 1-cyano-2-ethoxy-2-oxoethyleneaminooxy) dimethylamino-morpholinyl-carbonHexafluorophosphate (COMU), 1-Hydroxybenzotriazole (HOBT), 1-hydroxy-7-azabenzotriazole (HOAT), O- (6-chlorobenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (O- (6-chlorobenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, HCTU), 1-H-benzotriazol(HBTU), (O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU), and O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate (TBTU).

Preferably, A^x2Selected from-maleimide, -SH, -NH₂、-SeH、-N₃、-C≡CH、-CR¹＝CR^1aR^1b、-OH、-(CH＝X⁰)–R¹、-(C＝O)–S–R¹、-(C＝O)-H、-NH-NH₂、-O-NH₂、-Ar–X⁰、-Ar–Sn(R¹)(R^1a)(R^1b)、-Ar–B(OH)(OH)，

Having an optional protecting group;

wherein:

X⁰is-OH, -NR¹R^1a-SH and-SeH,

ar is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl, and

R¹、R^1a、R^1bindependently of each other selected from H, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl.

More preferably, A^x2Is selected from-NH₂Maleimide and thiol, most preferably A^x2Is maleimide. Also preferred is a thiol (-SH) group.

Suitable reaction conditions are described in the examples section and are known to those skilled in the art.

Process step (d) can be carried out in the presence of a base. Suitable bases include conventional inorganic or organic bases. These preferably include alkaline earth metal or alkali metal cyanides, hydroxides, amides, alkoxides, acetates, carbonates or bicarbonates, for example sodium hydride, sodium amide, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium acetate, potassium acetate, calcium acetate, ammonium acetate, sodium carbonate, potassium bicarbonate, sodium bicarbonate or ammonium carbonate, and tertiary amines such as trimethylamine, triethylamine, tributylamine, N-dimethylaniline, N-dimethylbenzylamine, pyridine, N-methylpiperidine, N-methylmorpholine, N-dimethylaminopyridine, Diazabicyclooctane (DABCO), Diazabicyclononene (DBN), N-Diisopropylethylamine (DIPEA), Diazabicycloundecene (DBU) or collidine (colidine).

Process step (d) may be carried out in the presence of a solvent. Suitable solvents for carrying out process step (d) of the present invention include organic solvents. These preferably include water and aliphatic, alicyclic or aromatic hydrocarbons, such as petroleum ether, hexane, heptane, cyclohexane, methylcyclohexane, benzene, toluene, xylene or decalin; halogenated hydrocarbons such as chlorobenzene, dichlorobenzene, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or trichloroethane; alcohols, such as methanol, ethanol, n-or iso-propanol, n-or iso-butanol, sec-or tert-butanol, ethylene glycol, propane-1, 2-diol, ethoxyethanol, methoxyethanol, diethylene glycol monomethyl ether, dimethyl ether, diethylene glycol; acetonitrile, N-methyl-2-pyrrolidone (NMP), Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 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, e.g. diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl tert-amyl ether, diAn alkane, tetrahydrofuran, 1, 2-dimethoxyethane, 1, 2-diethoxyethane or anisole;or mixtures thereof. Preferably, the solvent is selected from water, acetonitrile or N-methyl-2-pyrrolidone.

In another aspect, the present invention relates to a hydrogel-spacer conjugate obtainable by the above method.

Method for preparing prodrug connected with carrier

In another aspect, the present invention relates to a method of making a prodrug linked to a carrier, the method comprising the steps of:

(e) reacting the hydrogel of step (b) or (c) or the hydrogel-spacer conjugate of step (d) with a prodrug linker-biologically active moiety reagent of formula (VII) in the presence of a solvent to give a carrier-linked prodrug:

A^y1-L-D (VII)，

wherein:

A^y1a functional group A for reaction with an amine of the hydrogel of step (b) or (c) or for reaction with the hydrogel-spacer conjugate of step (d)^x2The functional group of the reaction is a functional group,

l is a prodrug linker;

d is a bioactive moiety.

Process step (e) may be carried out in the presence of a solvent. Suitable solvents for carrying out step (e) of the process of the present invention include organic solvents. These preferably include water and aliphatic, alicyclic or aromatic hydrocarbons, such as petroleum ether, hexane, heptane, cyclohexane, methylcyclohexane, benzene, toluene, xylene or decalin; halogenated hydrocarbons such as chlorobenzene, dichlorobenzene, dichloromethane, chloroform, carbon tetrachloride, dichloroethane or trichloroethane; alcohols, such as methanol, ethanol, n-or iso-propanol, n-or iso-butanol, sec-or tert-butanol, ethylene glycol, propane-1, 2-diol, ethoxyethanol, methoxyethanol, diethylene glycol monomethyl ether, dimethyl ether, diethylene glycol; acetonitrile, N-methyl-2-pyrrolidone (NMP), Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-dimethylAcetamide, nitromethane, nitrobenzene, Hexamethylphosphoramide (HMPT), 1, 3-dimethyl-2-imidazolidinone (DMI), 1, 3-dimethyl-3, 4,5, 6-tetrahydro-2 (1H) -pyrimidindinone (DMPU), ethyl acetate, acetone, butanone; ethers, e.g. diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl tert-amyl ether, diAn alkane, tetrahydrofuran, 1, 2-dimethoxyethane, 1, 2-diethoxyethane or anisole; or mixtures thereof. Preferably, the solvent is selected from water, acetonitrile or N-methyl-2-pyrrolidone.

If the prodrug linker-bioactive moiety reagent of formula (VII) reacts with the amine of the hydrogel of step (b) or (c), then A^y1Is maleimide or OH-, preferably maleimide.

If a of the prodrug linker-bioactive moiety reagent of formula (VII) is coupled to the hydrogel-spacer conjugate of step (d)^x2Reaction, then A^y1Is determined by the relation with A^y1Reaction of A^x2The structure of (1). Preferred A^x2/A^y1The pairs are selected from the following:

wherein:

X⁰is-OH, -NR¹R^1a-SH and-SeH;

R¹、R^1a、R^1bindependently of each other selected from H, C_1-6Alkyl, aryl, heteroaryl, and heteroaryl,C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl; and is

Ar is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl.

More preferably, A^y1Selected from-SH or-maleimide, most preferably A^y1is-SH, therefore, preferred is A^x2Is-maleimide or-SH, most preferably A^x2Is-maleimide.

In a particularly preferred embodiment, A^x2Is thiol and A of the formula (VII)^y1Has formula (VIIa):

T–PG⁰–S- (VIIa)，

wherein:

t is H or a tag moiety;

PG⁰is a sulfur activating moiety; and is

S is sulfur.

In one embodiment, PG of formula (VIIa)⁰Selected from:

wherein:

the dotted line marked with an asterisk indicates attachment to T of formula (VIIa), the unlabeled line indicates attachment to the sulfur of formula (VIIa);

ar is a group moiety, which is optionally further substituted;

R⁰¹、R⁰³、R⁰⁴independently of one another, is a bond, or is C_1-50An alkyl group; c_2-50An alkenyl group; or C_2-50Alkynyl, wherein C_1-50Alkyl radical；C_2-50An alkenyl group; and C_2-50Alkynyl is optionally substituted by one or more R, the same or different³Is substituted, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl groups are optionally interrupted by one or more groups selected from: -Q-, -C (O) O-; -O-; -c (o) -; -C (O) N (R)⁴)-；-S(O)₂N(R⁴)-；-S(O)N(R⁴)-；-S(O)₂-；-S(O)-；-N(R⁴)S(O)₂N(R^4a)-；-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(R^4a) -; and-OC (O) N (R)⁴R^4a)；

R⁰²is-H; c_1-50An alkyl group; c_2-50An alkenyl group; or C_2-50Alkynyl, wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50Alkynyl is optionally substituted by one or more R, the same or different³Is substituted, and wherein C_1-50An alkyl group; c_2-50An alkenyl group; and C_2-50The alkynyl groups are optionally interrupted by one or more groups selected from: -Q-, -C (O) O-; -O-; -c (o) -; -C (O) N (R)⁴)-；-S(O)₂N(R⁴)-；-S(O)N(R⁴)-；-S(O)₂-；-S(O)-；-N(R⁴)S(O)₂N(R^4a)-；-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(R^4a) -; and-OC (O) N (R)⁴R^4a)；

Q is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cycloalkyl group; 4-7 membered heterocyclyl; and 8-11 membered heterobicyclic group, wherein T is optionally substituted by one or more R which may be the same or different³Substitution;

R³is halogen; -CN; oxygen (═ O); -COOR⁵；-OR⁵；-C(O)R⁵；-C(O)N(R⁵R^5a)；

-S(O)₂N(R⁵R^5a)；-S(O)N(R⁵R^5a)；-S(O)₂R⁵；-S(O)R⁵；-N(R⁵)S(O)₂N(R^5aR^5b)；-SR⁵；-N(R⁵R^5a)；-NO₂；-OC(O)R⁵；-N(R⁵)C(O)R^5a；-N(R⁵)S(O)₂R^5a；-N(R⁵)S(O)R^5a；-N(R⁵)C(O)OR^5a；-N(R⁵)C(O)N(R^5aR^5b)；-OC(O)N(R⁵R^5a) (ii) a Or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more, the same or different, halogens; and is

R⁴、R^4a、R⁵、R^5a、R^5bIndependently selected from-H; or C_1-6Alkyl radical, wherein C_1-6Alkyl is optionally substituted with one or more of the same or different halogens.

Preferably, R⁰¹Is C_1-6An alkyl group. Even more preferably R⁰¹Is selected from-CH₂-、-CH₂-CH₂-and-CH₂-CH₂-CH₂-。

Preferably, R⁰²Selected from H and C_1-6An alkyl group.

Preferably, R⁰³Is C_1-6An alkyl group, a carboxyl group,

preferably, R⁰⁴Is C_1-6An alkyl group, a carboxyl group,

preferably, Ar is selected from:

wherein:

the dotted line marked with an asterisk indicates the attachment to T of formula (VIIa), not markedThe dotted line represents PG attached to formula (VIIa)⁰On the remainder of (c);

w is independently O, S or N;

w' is N; and is

Wherein Ar is optionally substituted with one or more substituents independently selected from NO₂Cl and F.

More preferably, PG of formula (VIIa)⁰Selected from:

wherein:

the dotted line marked with an asterisk indicates attachment to T of formula (VIIa), the unlabeled line indicates attachment to the sulfur of formula (VIIa); and is

Ar、R⁰¹、R⁰²、R⁰³And R⁰⁴As used above.

More preferably, PG of formula (VIIa)⁰Comprises the following steps:

wherein:

the dotted line marked with an asterisk indicates attachment to T of formula (VIIa), and the non-marked dotted line indicates attachment to the sulfur of formula (VIIa).

Most preferably, PG of formula (VIIa)⁰Comprises the following steps:

wherein:

the dotted line marked with an asterisk indicates attachment to T of formula (VIIa), and the non-marked dotted line indicates attachment to the sulfur of formula (VIIa).

In a preferred embodiment, T of formula (VIIa) is H.

In another embodiment, T of formula (VIIa) comprises a polymeric moiety. Preferably, T of formula (VIIa) comprises a polymer selected from: 2-methacryloyl-oxyethylphosphorylcholine, poly (acrylic acid), poly (acrylate), poly (acrylamide), poly (alkoxy) polymer, poly (amide), poly (amidoamine), poly (amino acid), poly (anhydride), poly (asparagine), poly (butyric acid), poly (glycolic acid), polybutylene terephthalate, poly (caprolactone), poly (carbonate), poly (cyanoacrylate), poly (dimethylacrylamide), poly (ester), poly (ethylene), poly (alkylene glycol), poly (ethylene oxide), poly (ethyl phosphate), poly (ethyl phosphate), poly (ethylene glycol, propylene glycol, polyethylene glycolOxazoline), poly (glycolic acid), poly (hydroxyethyl acrylate), poly (hydroxyethyl-Oxazoline), poly (hydroxy methacrylate), poly (hydroxy propyl methacrylamide), poly (hydroxy propyl methacrylate)Oxazoline), poly (iminocarbonate), poly (lactic acid), poly (lactic-co-glycolic acid), poly (methacrylamide), poly (methacrylate), poly (methyl methacrylate)Oxazolines, poly (organophosphazenes), poly (orthoesters), poly(s)Oxazolines, poly (propylene glycol), poly (siloxanes), poly (urethanes), poly (vinyl alcohol), poly (vinylamine), poly (vinyl methyl ether), poly (vinyl pyrrolidone), siloxanes, cellulose, carboxymethylMethylcellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid and derivatives, functionalized hyaluronic acid, mannan, pectin, rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch and other carbohydrate-based polymers, xylan and copolymers thereof.

If T of formula (VIIa) is a polymeric moiety, then preferably T has a molecular weight of at least 1kDa, preferably at least 3kDa, most preferably at least 5 kDa. If T of formula (VIIa) is a polymeric moiety, it preferably has a molecular weight of at most 1000kDa, such as at most 800kDa, at most 500kDa, at most 250kDa, at most 200kDa or at most 100 kDa.

In another embodiment, T of formula (VIIa) comprises an affinity ligand. Preferably T of formula (VIIa) comprises, more preferably T is an affinity ligand moiety selected from the group consisting of: 4-aminobenzamidine, 3- (2' -aminobenzhydryloxy) tropane, -aminocaproyl-p-chlorobenzamide, 1-amino-4- [3- (4, 6-dichlorotriazin-2-ylamino) -4-sulfophenylamino]Anthraquinone-2-sulfonic acid, 2- (2 '-amino-4' -methylphenylsulfanyl) -N, N-dimethylbenzylamine dihydrochloride, angiopoietin-1, aplimo (aptamers), arotinoid, avermectin, biotin, calmodulin, cocaethylene, cetospolone B (cytosponone B), N-dihexyl-2- (4-fluorophenyl) indole-3-acetamide, N-dipropyl-2- (4-chlorophenyl) -6, 8-dichloro-imidazole [1,2-a ]]Pyridine-3-acetamide, 5-fluoro-2 '-deoxyuridine 5' - (p-aminophenyl) monophosphate, S-hexyl-L-glutathione, (S, S) -4-phenyl- α - (4-phenyl)Oxazolidin-2-ylidene) -2-Oxazoline-2-acetonitrile, Pro-Leu-Gly hydroxamate, 2- (4- (2- (trifluoromethyl) phenyl) piperidine-1-carboxamide) benzoic acid, trimethyl (m-aminophenyl) ammonium chloride, urocortin III (urocortin III), cofactors such as adenosine triphosphate, s-adenosylmethionine, ascorbic acid, cobalamin, and the like,Coenzyme A, coenzyme B, coenzyme M, coenzyme Q, coenzyme F420, cytidine triphosphate, flavin mononucleotide, flavin adenine dinucleotide, glutathione, heme, lipoamide, menaquinone, methylfuran, methylcobalamin, molybdopterin, NAD +, NADP +, nucleotide sugar, adenosine-5' -phosphosulfate, pyridoxal phosphate, polyhistidine, pyrroloquinoline quinone, riboflavin, streptavidin, tetrahydrobiopterin, tetrahydromethanepterin, tetrahydrofolic acid, Biotin Carboxyl Carrier Protein (BCCP), 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, strep-tag, thioredoxin, Triazines and antibody fragments.

If T of formula (VIIa) saturates the affinity ligand, then preferably the affinity ligand is polyhistidine.

In another embodiment, T of formula (VIIa) comprises a charged moiety. Preferably, T of formula (VIIa) comprises at least one positive and/or negative charge. It should be understood that the positive and negative charge numbers of T are unequal to ensure that T is a charged molecule.

Preferably, T of formula (VIIa) comprises at least one positive or negative charge, such as at least 2 positive or negative charges, at least 3 positive or negative charges, at least 4 positive or negative charges, at least 5 positive or negative charges, at least 6 positive or negative charges, at least 7 positive or negative charges, at least 8 positive or negative charges, at least 9 positive or negative charges, at least 10 positive or negative charges, at least 11 positive or negative charges, at least 12 positive or negative charges, at least 13 positive or negative charges, at least 14 positive or negative charges, or at least 15 positive or negative charges.

More preferably, T of formula (VIIa) comprises at least one positive charge, such as 1 positive charge, 2 positive charges, 3 positive charges, 4 positive charges, 5 positive charges, 6 positive charges, 7 positive charges, 8 positive charges, 9 positive charges, 10 positive charges, 11 positive charges, 12 positive charges, 13 positive charges, 14 positive charges, or 15 positive charges. More preferably, T of formula (VIIa) comprises 1 positive charge, 2 positive charges, 3 positive charges, 4 positive charges, 5 positive charges, 6 positive charges, 7 positive charges, or 8 positive charges. More preferably, T of formula (VIIa) comprises 2 positive charges, 3 positive charges, 4 positive charges, 5 positive charges, or 6 positive charges.

Preferably, at least one positive charge of T of formula (VIIa) is substituted by ammonium orProvided is a method.

Preferably, T of formula (VIIa) comprises a polyamide comprising at least one quaternary ammonium residue and/or at least one protonated ammonium residue, optionally comprising other functional groups. Preferably, such optional additional functional groups are 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 4 quaternary ammonium residues and/or 4 protonated ammonium residues.

Preferably, T of formula (VIIa) comprises a polyamine in bound form. More preferably, T of formula (VIIa) comprises a polyamine in a bound form selected from: ethylenediamine, 1, 3-diaminopropane, hexamethylenediamine, cadaverine, putrescine, spermine, spermidine, norspermidine (norsperamine), and tetraethylmethylenediamine.

Even more preferably, T of formula (VIIa) comprises a moiety of formula (a):

wherein:

dotted line indicates a connection to PG⁰The above step (1);

R¹、R^1a、R^1b、R²、R^2a、R^2b、R³、R^3a、R^3b、R⁴、R^4a、R^4bindependently of one another, H or methyl;

each m is independently 1,2, 3,4,5,6, 7 or 8;

each n is independently 1,2, 3,4,5,6, 7 or 8;

each x is independently 1,2, 3,4,5,6, 7 or 8;

each y is independently 0,1, 2,3, 4,5,6, 7 or 8; and is

SP is a spacer moiety.

Preferably, the moiety of formula (a) is symmetrical, i.e. the moiety:

and structural part (c):

are the same.

In one embodiment, R of formula (a)¹、R^1a、R^1bAre both methyl groups.

In another embodiment, R of formula (a)¹Is H, and R of formula (a)^1aAnd R^1bAre both methyl groups.

In one embodiment, R of formula (a)²、R^2a、R^2bAre both methyl groups.

In another embodiment, R of formula (a)²Is H and R of formula (a)^2aAnd R^2bAre both methyl groups.

In one embodiment, R of formula (a)³、R^3a、R^3bAre both methyl groups.

In another embodiment of formula (a)R³Is H, and R of formula (a)^3aAnd R^3bAre both methyl groups.

In one embodiment, R of formula (a)⁴、R^4a、R^4bAre both methyl groups.

In another embodiment, R of formula (a)⁴Is H, and R of formula (a)^4aAnd R^4bAre both methyl groups.

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, most preferably, m of formula (a) is 4.

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, most preferably, n of formula (a) is 3.

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, most preferably, x of formula (a) is 1.

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, most preferably, y of formula (a) is 1.

In a preferred embodiment, R¹、R^1a、R^1b、R²、R^2a、R^2b、R³、R^3a、R^3b、R⁴、R^4a、R^4bIs methyl; m is 4; n is 3; y is 1 and x is 1.

In another preferred embodiment, R¹、R²、R³、R⁴Is H; r^1a、R^1b、R^2a、R^2b、R^3a、R^3b、R^4a、R^4bIs methyl, m is 4; n is 3; y is 1 and x is 1.

A preferred counterion for T of formula (I) is Cl^-、TFA^-And SO₄ ^-。

If A is^y1Having the formula (VIIa) and T being a tag moiety, T may be used to tag each D from x moieties A^y1L is linked and where x is a positive integer, e.g., 1,2, 3,4,5,6, 7, 8, 9 or 10, of a prodrug linker-biologically active moiety reagent mixture of formula (VII) having the indicated number of moieties A per moiety D^y1-L-a prodrug linker of formula (VII) -a biologically active moiety reagent. T can thus be used to isolate a mono-conjugate of the prodrug linker-biologically active moiety reagent of formula (VII).

The method of isolating the prodrug linker-biologically active substance mono-conjugate depends on the labeling moiety T of formula (VIIa).

If T of formula (VIIa) is a polymeric moiety having a molecular weight of at least 10% (w/w) of D of formula (VII), the separation step is preferably size exclusion chromatography.

If T of formula (VIIa) comprises an affinity ligand, the separation step is preferably affinity chromatography.

If T of formula (VIIa) comprises a charged moiety, the separation step is preferably ion exchange chromatography.

In a preferred embodiment, T of formula (VIIa) is a charged moiety and the separation step is preferably ion exchange chromatography.

Preferably, process step (e) comprises the steps of: reacting the hydrogel-spacer conjugate of step (d) with a prodrug-bioactive moiety reagent of formula (VII).

Suitable reaction conditions are described in the examples section and are known to those skilled in the art.

Thus, the process for preparing a prodrug linked to a carrier preferably comprises the steps of:

(d) reacting the hydrogel from step (b) or (c) with a spacer reagent of formula (VI) in the presence of a solvent to obtain a hydrogel-spacer conjugate:

A^x1-S⁰-A^x2(VI)，

wherein:

S⁰is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl, said segments being optionally interrupted by one or more groups selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S, -C (O) -, -C (O) NH, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclic group, phenyl group and naphthyl group;

A^x1is a functional group that reacts with an amine group of the hydrogel; and is

A^x2Is a functional group; and

(e) reacting the hydrogel-spacer conjugate of step (d) with a prodrug linker-biologically active moiety reagent of formula (VII) in the presence of a solvent to obtain a carrier-linked prodrug:

A^y1-L-D (VII)，

wherein:

A^y1functional group A as a conjugate with the hydrogel-spacer of step (d)^x2A reactive functional group;

l is a prodrug linker;

d is a bioactive moiety.

Preferred A^x2And A^y1The combinations of (a) and (b) are as described above.

The prodrug linker reagent or prodrug linker moiety that is part of the prodrug linker-biologically active moiety reagent may have the structure of any prodrug linker moiety known in the art.

Preferably, the prodrug linker reagent or prodrug linker moiety that is part of the prodrug linker-biologically active moiety reagent is a traceless prodrug linker reagent or moiety.

Preferred prodrug linkers are disclosed and are available as described in WO 2005/099768 a 2. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-i) or (h-ii):

wherein:

A^y1is a functional group as defined in step (e),

d is a biologically active moiety linked to L via an amine group of the corresponding drug;

x is a spacer moiety such as R₅-Y₆，

Y₁、Y₂Independently O, S or NR₆，

Y₃、Y₅Independently of each other is O or S,

Y₄is O, NR₆Or C (R)₇)(R₈)-，

Y₆Is O, S, NR₆Succinimide, maleimide, unsaturated carbon-carbon bond, or any heteroatom containing a free electron pair, or absent,

R₂、R₃independently of one another, from: a hydrogel, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl, cyano, nitro, halogen, carboxy, carboxyalkyl, alkylcarbonyl, or carboxyamidoalkyl;

R₄selected from hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroarylA radical, substituted or unsubstituted linear, branched or cyclic alkoxy, substituted or unsubstituted linear, branched or cyclic heteroalkoxy, aryloxy or heteroaryloxy, cyano, halogen,

R₅selected from substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl,

R₆selected from the group consisting of hydrogels, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl,

R₇、R₈independently selected from hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl, carboxyalkyl, alkylcarbonyl, carboxyamidoalkyl, cyano or halogen,

w is selected from the group consisting of substituted or unsubstituted linear, branched or cyclic alkyl, aryl, substituted or unsubstituted linear, branched or cyclic heteroalkyl, substituted or unsubstituted heteroaryl,

nu is a nucleophile which is a nucleic acid,

n is zero or a positive integer, and

ar is a polysubstituted aromatic hydrocarbon or a polysubstituted aromatic heterocyclic ring.

It is understood that in the formulae (h-i) and (h-ii), L corresponds to the link A^y1And structural moieties of D.

Preferably, R of the formulae (h-i) and (h-ii)₂、R₃、R4、R₅、R₆、R₇And R₈Independently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl.

Preferably, Y of the formulae (h-i) and (h-ii)₆Is C_1-20Alkyl radical, C_2-20Alkenyl or C_2-20Alkynyl.

Preferably, the Nu of formulae (h-i) and (h-ii) is selected from the following nucleophiles: primary, secondary and tertiary amino groups, thiol groups, carboxylic acids, hydroxylamines, hydrazines and nitrogen-containing heteroaryl groups.

Preferably, W of the formulae (h-i) and (h-ii) is- (CR)₉R₁₀)_b-, wherein R₉And R₁₀Independently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl, and wherein b is 1,2, 3,4 or 5.

Preferably, n of formulae (h-i) and (h-ii) is 0,1 or 2, more preferably, n is 0 or 1, most preferably n is 0.

Preferably, Ar of formulae (h-i) and (h-ii) is selected from:

other preferred prodrug linkers are disclosed and are available as described in WO 2006/136586 a 2. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-iii), (h-iv) or (h-v):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to L via an amine group of the corresponding drug forming an amide bond;

x is a spacer moiety such as R₁₃-Y1；

Y1 is O, S, NR₆Succinimide, maleimide, unsaturated carbon-carbon bonds or containing free electron pairsAny heteroatom, either absent,

R₁₃selected from substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl;

R₂and R₃Independently selected from hydrogen, acyl, or a protecting group for hydroxyl;

R₄-R₁₂independently selected from hydrogen, substituted or unsubstituted linear, branched or cyclic alkyl or heteroalkyl, aryl, substituted or unsubstituted heteroaryl, cyano, nitro, halogen, carboxy, carboxamide.

It will be appreciated that in formulae (h-iii), (h-iv) and (h-v), L corresponds to the link A^y1And structural moieties of D.

Preferably, R of the formulae (h-iii), (h-iv) and (h-v)₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁And R₁₂Independently selected from H, C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl.

Preferably, in formulae (h-iii), (h-iv) and (h-v), Y1 is C_1-20Alkyl radical, C_2-20Alkenyl or C_2-20Alkynyl.

Another preferred prodrug linker is disclosed and is obtainable as described in WO 2009/095479 a 2. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-vi):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule by an aromatic amine of the corresponding drug through formation of an amide bond;

x is C (R)⁴R^4a)；N(R⁴)；O；C(R⁴R^4a)-C(R⁵R^5a)；C(R⁵R^5a)-C(R⁴R^4a)；C(R⁴R^4a)-N(R⁶)；N(R⁶)-C(R⁴R^4a)；C(R⁴R^4a) -O; or O-C (R)⁴R^4a)；

X¹Is C; or S (O);

X²is C (R)⁷,R^7a) (ii) a Or C (R)⁷,R^7a)-C(R⁸,R^8a)；

R¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵、R^5a、R⁶、R⁷、R^7a、R⁸、R^8aIndependently selected from H; and C_1-4An alkyl group;

optionally, one or more pairs of R^1a/R^4a、R^1a/R^5a、R^4a/R^5a、R^4a/R^5a、R^7a/R^8aForming a chemical bond;

optionally, one or more pairs of R¹/R^1a、R²/R^2a、R⁴/R^4a、R⁵/R^5a、R⁷/R^7a、R⁸/R^8aAre linked together with the atom to which they are attached to form C_3-8A cycloalkyl group; or a 4-7 membered heterocyclic group;

optionally, one or more pairs of R¹/R⁴、R¹/R⁵、R¹/R⁶、R⁴/R⁵、R⁷/R⁸、R²/R³Together with the atoms to which they are attached form a ringA；

Optionally, R³/R^3aTaken together with the nitrogen atom to which they are attached to form a 4-7 membered heterocyclic ring;

a is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cycloalkyl group; 4-7 membered heterocyclyl; and 8-11 membered heterobicyclic group;

provided that R is¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵、R^5a、R⁶、R⁷、R^7a、R⁸Or R^8aWith one hydrogen in A^y1And (4) replacing.

It is understood that in formula (h-vi), L corresponds to the link A^y1And structural moieties of D.

Another preferred prodrug linker is disclosed and is obtainable as described in WO 2011/012721 a1 and WO 2011/012722a 1. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-vii):

wherein:

d is a biologically active moiety linked to the rest of the molecule by an aromatic amine of the corresponding drug through formation of an amide bond;

A^y1is a functional group as defined in step (e);

X¹is C (R)¹R^1a) Or a cyclic fragment selected from: c_3-8Cycloalkyl, 4-7 membered heterocyclyl, phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl or 8-11 membered heterobicyclic group,

wherein:

if X is¹Is a cyclic fragment, then said cyclic fragment is incorporated into L by means of 2 adjacent ring atoms¹And X adjacent to the carbon atom of the amide bond¹Is also a carbon atom;

X²is a chemical bond or is selected from C (R)³R^3a),、N(R³)、O、C(R³R^3a)-C(R⁴R^4a)、C(R³R^3a)-N(R⁴)、N(R³)-C(R⁴R^4a)、C(R³R^3a) -O or O-C (R)³R^3a)，

Wherein:

if X is¹Is a cyclic fragment, then X²Is a chemical bond, C (R)³R^3a)、N(R³) Or O;

optionally, if X¹Is a cyclic fragment and X²Is C (R)³R^3a) Then L is¹Inner X¹Fragment and X²The order of the fragments can be varied, and the cyclic fragments are incorporated into L by means of 2 adjacent ring atoms¹Performing the following steps;

R¹、R³and R⁴Independently selected from H, C_1-4Alkyl and-N (R)⁵R^5a)；

R^1a、R²、R^3a、R^4aAnd R^5aIndependently selected from H and C_1-4An alkyl group;

R⁵is C (O) R⁶；

R⁶Is C_1-4An alkyl group;

optionally, a pair of R^1a/R^4a、R^3a/R^4aOr R^1a/R^3aForming a chemical bond;

provided that R is¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵、R^5aOr R⁶A hydrogen of^y1And (4) replacing.

It is understood that in formula (h-vii), L corresponds to the link A^y1And structural moieties of D.

Another preferred prodrug linker is disclosed and is obtainable as described in WO 2011/089214 a 1. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-viii):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule via an aromatic hydroxyl (-OH) group of the corresponding drug by formation of a carbamate linkage;

R¹is selected from C_1-4An alkyl group; a heteroalkyl group; c_3-8A cycloalkyl group; and is

R²、R^2a、R³And R^3aIndependently selected from hydrogen, substituted or unsubstituted linear, branched or cyclic C_1-4Alkyl or heteroalkyl;

each d is independently 2,3 or 4;

provided that R is¹、R²、R^2a、R³Or R^3aA hydrogen of^y1And (4) replacing.

It is understood that in formula (h-viii), L corresponds to the link A^y1And structural moieties of D.

Another preferred prodrug linker is disclosed and is obtainable as described in WO 2011/089216 a 1. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-ix):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule via an aliphatic amine of the corresponding drug by formation of an amide bond;

X₁selected from O, S or CH-R^1a；

R¹And R^1aIndependently selected from H, OH, CH₃；

R²、R^2a、R⁴And R^4aIndependently selected from H and C_1-4An alkyl group, a carboxyl group,

R³and R^3aIndependently selected from H, C_1-4Alkyl and R⁵；

R⁵Selected from:

wherein:

the dashed lines indicate connections to the rest of the structural part,

provided that R is¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4aAnd R⁵A hydrogen of^y1And (4) replacing.

It is understood that in formula (h-ix), L corresponds to the link A^y1And structural moieties of D.

Preferably, R of formula (h-ix)³Is H, and R of formula (H-ix)^3aIs R⁵。

Preferably, R of formula (h-ix)⁴/R^4aIs H.

Optionally, one or more pairs R of formula (h-ix)³/R^3a、R⁴/R^4a、R³/R⁴May independently form one or more groups selected from C_3-8A cyclic fragment of a cycloalkyl, 4-7 membered heterocyclyl or 8-11 membered heterobicyclic group.

Optionally, R of formula (h-ix)³、R^3a、R⁴And R^4aIs further covered with C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, phenyl, 4-7 membered heterocycle or halogen.

Another preferred prodrug linker is disclosed and is obtainable as described in WO 2011/089215 a 1. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-x):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule by an aromatic amine of the corresponding drug through formation of an amide bond;

R¹、R^1a、R²、R³、R^3a、R⁴and R^4aIndependently selected from H and C_1-4An alkyl group;

optionally, R¹、R^1a、R²、R³、R^3a、R⁴And R^4aAny two of which may independently form one or more loop fragments selected from: c_3-8Cycloalkyl, 4-7 membered heterocyclyl, phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, or 8-11 membered heterobicyclic group;

optionally R¹、R^1a、R²、R³、R^3a、R⁴And R^4aFurther substituted with a substituent selected from: c_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, or 8-11 membered heterobicyclic group;

provided that R is¹、R^1a、R²、R³、R^3a、R⁴And R^4aA hydrogen of^y1And (4) replacing.

It is understood that in formula (h-x), L corresponds to the link A^y1And structural moieties of D.

Another preferred prodrug linker is disclosed and is obtainable as described in PCT/EP 2012/065748. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-xi):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule via the carboxylic acid group (- (C ═ O) -OH) of the corresponding drug by formation of a carboxylic ester bond;

R¹selected from unsubstituted alkyl groups; a substituted alkyl group; unsubstituted phenyl; a substituted phenyl group; unsubstituted naphthyl; substituted naphthyl; unsubstituted indenyl; a substituted indenyl group; an unsubstituted indanyl group; substituted indanyl; unsubstituted tetralinyl; substituted tetralinyl; unsubstituted C_3-10A cycloalkyl group; substituted C_3-10A cycloalkyl group; unsubstituted 4-7 membered heterocyclyl; substituted 4-7 membered heterocyclyl; unsubstituted 8-11 membered heterobicyclic group; and substituted 8-11 membered heterobicyclic group;

R²selected from the group consisting of H, unsubstituted alkyl and substituted alkyl,

R³and R⁴Independently selected from the group consisting of H, unsubstituted alkyl, and substituted alkyl;

e is 0 or 1;

optionally, R¹And R³Taken together with the atoms to which they are attached to form ring a;

a is selected from C_3-10A cycloalkyl group; 4-7 membered aliphatic heterocyclic group; and 8-11 membered aliphatic heterobicyclic group, wherein A is unsubstituted or substituted;

q is selected from C_1-50Alkyl radical, C_2-50Alkenyl or C_2-50Alkynyl, said segments being optionally interrupted by one or more groups selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclyl, phenyl or naphthyl.

It is understood that in formula (h-xi), L corresponds to the link A^y1And structural moieties of D.

Another preferred prodrug linker is disclosed and may be as described in EP 12165516. Thus, preferred prodrug linkers-biologically active moiety agents A^y1-L-D has the structure of formula (h-xii):

wherein:

A^y1is a functional group as defined in step (e);

d is a biologically active moiety linked to the rest of the molecule via the hydroxyl group of the corresponding drug by formation of an ester or carbamate linkage;

y is-C (R)¹)(R^1a) -; or-N (R)¹)-；

X is-C (R)⁴)(R^4a)-；-N(R⁴)-；-O-；-C(R⁴)(R^4a)-C(R⁵)(R^5a)-；-C(R⁴)(R^4a)-N(R⁶)-；-N(R⁶)-C(R⁴)(R^4a)-；-C(R⁴)(R^4a)-O-；-O-C(R⁴)(R^4a)-；-C(O)-N(R⁶) -; or-N (R)⁶)-C(O)-；

X¹Is composed ofOr

X²is-C (R)⁷)(R^7a) -; or-C (R)⁷)(R^7a)-C(R⁸)(R^8a)-；

X³Is ═ O; (ii) S; or ═ N-CN;

R¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵、R^5a、R⁶、R⁷、R^7a、R⁸、R^8aindependently selected from H; c_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-20Heteroalkyl group and Y₁-T; and independently no 0,1 or more pairs R^1a/R^4a、R^1a/R^5a、R^4a/R^5a、R^7a/R^8aAnd the corresponding carbon atoms to which they are attached form a cis double bond;

Y¹is a chemical bond or C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6An alkynyl group;

t is selected from phenyl; a naphthyl group; an indenyl group; indanyl; tetrahydronaphthyl; c_3-10A cycloalkyl group; 4-7 membered heterocyclyl; or 8-11 membered heterobicyclic group, wherein T is optionally substituted by one or more R which may be the same or different⁹Substitution;

R⁹is halogen; -CN; oxygen (═ O); -C (O) OH; -OH; -S (O)₂NH₂；-S(O)NH₂；-S(O)₂OH；-S(O)OH；-SH；-NH₂；-NO₂；C_1-6Alkyl, or C_1-10A heteroalkyl group;

optionally, one or more pairs of R¹/R^1a、R¹/R⁴、R¹/R⁶、R¹/R⁵、R²/R^2a、R²/R³、R⁴/R^4a、R⁴/R⁵、R⁵/R^5a、R⁷/R^7a、R⁷/R⁸、R⁸/R^8aTaken together with the atoms to which they are attached to form a ring T;

optionally, R³/R^3aTaken together with the nitrogen atom to which they are attached to form a 4-7 membered heterocyclic ring;

provided that R is¹、R^1a、R²、R^2a、R³、R^3a、R⁴、R^4a、R⁵、R^5a、R⁶、R⁷、R^7a、R⁸Or R^8aA hydrogen of^y1And (4) replacing.

It is understood that in formula (h-xii), L corresponds to the link A^y1And structural moieties of D.

In a preferred embodiment, the prodrug linker-biologically active moiety reagent A^y1-L-D has the formula (h-i) or (h-ii).

In another preferred embodiment, the prodrug linker-biologically active moiety agent A^y1-L-D has the formula (h-vi).

Any drug containing at least one functional group can be conjugated to the prodrug linker reagent to give the prodrug linker-biologically active moiety reagent A^y1-L-D. Such drugs are selected from the group consisting of polypeptides, proteins and oligonucleotides. Preferably, the drug is a protein.

Preferably, D has a molecular weight of 2-500kDa, more preferably 5-250kDa, more preferably 5-100kDa, most preferably 10-60 kDa.

Preferably, the drug is a protein drug that modulates the activity of one or more biological targets selected from the group consisting of basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor α (TGFa), transforming growth factor β (TGF β), platelet-derived growth factor (PDGF), angiogenin, platelet-derived endothelial 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, heparin binding cytokine, placental growth factor, pleiotrophin, PTN, growth factor (progranulin), polytriactin, tumor necrosis factor- α (TNF-alpha), tumor angiogenesis inhibitor (beta angiostatin), angiostatin (XVIII), heparin-derived angiostatin (hIV), heparin-induced protein I/VEGF), heparin fragment (CDI/IP-derived interferon fragment), heparin fragment (CDI), heparin fragment (CDIV), fragment II), fragment of human interferon (CDIV), fragment of human fibronectin/I), fragment (CDIV), fragment of human interferon (CDIV), fragment of human heparitin, fragment (CDIV), fragment of human interferon (CDIV), fragment II), fragment of human interferon (CDIV), fragment II), fragment of human interferon (CDIV), fragment of TNF-derived human interferon (CDIV), fragment of human interferon (CDIV), and fragment of TNF-derivedEnzyme inhibitors (TIMP), 2-methoxyestradiol, placental ribonuclease inhibitors, plasminogen activator inhibitors, platelet factor-4 (PF4), prolactin 16kD fragment, proliferation protein-related protein (PRP), retinoids, tetrahydrocortisol-S, thrombin-sensitizing protein-I (TSP-I), angiostatin (vasostatin) and angiostatin (calreticulin fragment), prostaglandins, growth hormones, insulin-like growth factor-I (IGF-I), sphingosine-1-phosphate (sphingosine-1-phosphate), factor D, RTP801, complement inhibitors including C1, C3 and C5, α₂Adrenergic agonists, mTOR, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), lens epithelial-derived growth factor (LEDGF), rod-derived cone activity factor (RdCVF), pigment epithelial cell-derived factor (PEDF).

If the drug is a protein, it is preferably selected from the group consisting of ACTH, adenosine deaminase, argasidase, albumin, alpha-1 antitrypsin (AAT), alpha-1 protease inhibitor (API), arabinosidase, alteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal and fragments or fusions), antithrombin III, antitrypsin, aprotinin, asparaginase, biphenanine, bone morphogenetic protein (bone-morphogenic protein), calcitonin (salmon), collagenase, DNase, endorphin, enfuvirtide (enfuvirtide), enkephalin, erythropoietin, factor VIIa, factor VIII, factor VIIIa, factor IX, plasmin, fusion protein, follicle stimulating hormone, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon-like peptides such as GLP-1, glucagon-like peptides, Glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), chorionic gonadotropin (hCG), hemoglobin, hepatitis B vaccine, hirudin, hyaluronidase, Edynonidase (iduronidase), immunoglobulin, influenza vaccine, interleukins (1 α,1 β,2, 3,4, 6, 10, 11, 12), IL-1 receptor antagonists (rhIL-1ra), insulin, interferons (α 2a, α 2b, α 2c, β 1a, β 1b, γ 1a, γ 1b), Keratinocyte Growth Factor (KGF), lactase, leuprolide (lerolide), levothyroxine, luteinizing hormone, Lyme disease (lyme) vaccine, natriuretic peptide, pancreatic lipase, papain, parathyroid hormone, PDGF, pepsin, phospholipase (PLAP), platelet activating factor acetylhydrolase (platactrivatidylactin), PAF-AH), prolactin, protein C, octreotide (octreotide), secretin, sertraline (sermorelin), superoxide dismutase (SOD), somatotropin (growth hormone), somatostatin, streptokinase, sucrase, tetanus toxin fragment, galactosidase, thrombin, thymosin, thyroid stimulating hormone, thyrotropin, transforming growth factor, Tumor Necrosis Factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), transferrin, TSH, urate oxidase, and urokinase.

If the drug is an antibody, it may be a monoclonal or polyclonal antibody or fragment or fusion thereof. Preferred antibody fragments are selected from the group consisting of Fab (fragment, antigen binding), f (ab)2 fragments, Fc (fragment, crystallizable), pFc' fragments, Fv (fragment, variable), scFv (single chain variable fragment), di-scFv/diabody, bispecific T-cell engagers (engage), CDRs (complementarity determining regions), single domain antibodies (sdABs/nanobody), heavy chain (α, γ, μ) or heavy chain fragments, light chain (λ, κ) or light chain fragments, VH fragments (variable regions of heavy chains), VL fragments (variable regions of light chains), VHH fragments, and VNAR fragments.

If the drug is an affinity scaffold protein, it is preferably selected from the group consisting of shark-derived affinity scaffold proteins, Kunitz domain-derived affinity scaffold proteins, citizen (centryrin) -derived affinity scaffold proteins, ubiquitin-derived affinity scaffold proteins, lipocalin-derived affinity scaffold proteins, ankyrin-derived affinity scaffold proteins, non-sahedides (disulfide-rich affinity scaffold proteins), fibronectin-derived affinity scaffold proteins, camelid-derived antibody fragments (camelid-derived antibody fragments) and affinity scaffold proteins, camelid-derived antibody fragments (llama-derived antibody fragments) and affinity scaffold proteins, transferrin-derived affinity scaffold proteins, and pumpkin-type (squash-type) protease inhibitors with cysteine knot-scaffold-derived affinity scaffold proteins.

In another aspect, the present invention relates to carrier-linked prodrugs obtainable by the process of the present invention for preparing carrier-linked prodrugs.

Such carrier-linked prodrugs release the drug molecule with a half-life of 1 hour to 12 months, e.g., 6 hours to 12 months, 12 hours to 11 months, 1 day to 10 months, 3 days to 9 months, 6 days to 9 months, 1 week to 9 months, 2 weeks to 9 months, 3 weeks to 8 months, 4 weeks to 8 months, 6 weeks to 7 months, 8 weeks to 7 months, 10 weeks to 6 months, 12 weeks to 6 months, or 16 weeks to 5 months.

Another aspect of the invention is a pharmaceutical composition comprising a prodrug of the linking carrier of the invention or a pharmaceutical salt thereof together with a pharmaceutically acceptable excipient.

A further aspect of the invention is a prodrug of a linking carrier of the invention for use as a medicament or a pharmaceutical composition comprising a prodrug of a linking carrier of the invention

Yet another aspect of this invention is a method of treating, controlling, delaying or preventing a mammalian patient, preferably a human, in need of treatment for one or more conditions, which comprises administering to said patient a therapeutically effective amount of a linking carrier prodrug of the present invention or a pharmaceutical composition comprising a linking carrier prodrug of the present invention or a pharmaceutically acceptable salt thereof.

Examples

Materials and methods

Materials:

amino 4-arm PEG5000 was obtained from JenKem Technology, Beijing, p.r. Cithrol^TMDPHS is available from Croda International Pic, Cowick Hall, United Kingdom.

Cis-1, 4-cyclohexanedicarboxylic acid was obtained from TCI EUROPE N.V., Boerenveldseweg 6-Haven 1063,2070Zwijndrecht, Belgium.

Isopropylmalonic acid was obtained from ABCR GmbH & Co.KG,76187Karlsruhe, Germany.

Monobenzyl glutarate was obtained from IRIS Biotec GmbH,95615Marktredwitz, Germany.

N- (3-maleimidopropyl) -21-amino-4, 7,10,13,16, 19-hexaoxa-heneicosanoic acid pentafluorophenyl ester (maleimide-NH-PEG 6-PFE) and N- (3-maleimidopropyl) -39-amino-4, 7,10,13,16,19,22,25,28,31,34, 37-dodecaoxa-nonadecanoic acid pentafluorophenyl ester (maleimide-NH-PEG 12-PFE) were obtained from Biomatrik inc, jiang, p.r.china.

Oxyma pure and Fmoc-L-Asp (OtBu) -OH were purchased from Merck Biosciences GmbH, Schwalbach/Ts, Germany.

4-Nitrophenyl carbonate (5-methyl-2-oxo-1, 3-dioxol-4-yl) -methyl ester was purchased from Chemzon scientific Inc., Lachine, QC, Canada.

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

The method comprises the following steps:

RP-HPLC was performed on 100 × 20mm or 100 × 40mm C18 ReProSil-Pur 300 ODS-35. mu. columns (Dr. Maisch, Ammerbuch, Germany) connected to Waters 600 or 2535HPLC systems and Waters 2487 or 2489 absorbance detectors, respectively₂0.1% TFA in O) and solution B (0.1% TFA in acetonitrile). The HPLC fractions containing the product were combined and lyophilized.

Flash chromatography purification was performed on an Isolera One system from Biotage AB, Sweden using a Biotage KP-Sil silica cartridge and n-heptane, ethyl acetate and methanol as eluents. The product was detected at 254 nm. For products that do not show absorption above 240nm, the fraction is sieved through LC/MS.

Analysis ultra high performance lc (uplc) was performed on a Waters Acquity system from Thermo Scientific equipped with a Waters BEH 300C 18 column (2.1 × 50mm, 1.7 μm particle size), connected to an LTQ Orbitrap Discovery mass spectrometer.

HPLC-electrospray ionization mass spectrometry (HPLC-ESI-MS) the mass spectrum of the sample was analyzed by chromatography on a column equipped with an Acquity PDA detector, with a Waters acquisition UPLC BEH 300C 18 RP column (2.1 × 50mm,1.7 μm, flow rate: 0.25 mL/min; solvent A: UP-H₂0+ 0.04% TFA, solvent B: UP-acetonitrile + 0.05% TFA) on a Thermo LTQ Orbitrap Discovery high resolution/high precision mass spectrometer connected Waters acquisition UPLC.

The MS spectra of the PEG product show a series of (CH) due to the polydispersity of the PEG starting material₂CH₂O)_nA structural portion. For easier explanation, only one single representative m/z signal is given in the examples.

Example 1

Synthesis of backbone reagents 1a, 1g and 1 h:

the backbone reagent 1a was synthesized as described in example 1 of WO2011/012715A1, except that Boc-DLys (Boc) -OH was used instead of Boc-LLys (Boc) -OH.

MS：m/z 888.50＝[M+10H⁺]¹⁰⁺(calculated value 888.54)

Backbone reagent 1g was synthesized from amino 4-arm PEG50001b according to the following protocol:

for the synthesis of compound 1b, amino 4-arm PEG5000(MW about 5350g/mol, 10.7g, 2.00 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (4.73g, 12.0 mmol) were dissolved in 43mL of DCM (anhydrous) and DIPEA (3.10g, 24.0 mmol, 4.18mL) was added at room temperature. After 10 minutes, 1, 9-bis-boc-1, 5, 9-triazacyclononane (5.30g, 16.0 mmol) was added and the mixture was stirred for 15 minutes. Additional 1, 9-bis-boc-1, 5, 9-triazacyclononane (0.33g, 1.0 mmol) was then added. After complete dissolution, the reaction mixture was filtered and the solvent was evaporated at room temperature.

The residue was dissolved in 40mL iPrOH and diluted with 320mL MTBE. The product was allowed to settle at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 200mL of cold MTBE (0 ℃). The product was dried under vacuum overnight.

Yield: 11.1g (83%) of a white solid 1 b.

MS：m/z 1112.86＝[M+6H]⁶⁺(calculated value 1113.04).

For the synthesis of compound 1c, boc-protected compound 1b (11.1g, 1.66 mmol) was dissolved in 40mL of 3m hci in MeOH and stirred at 45 ℃ for 20 min, then at 55 ℃ for 10 min. For precipitation, 10mL of LMEOH and 200mL of MTBE were added and the mixture was stored at-20 ℃ for 16 hours. The precipitate was collected by filtration through a glass filter Por.3 and washed with 200mL of cold MTBE (0 ℃). The product was dried under vacuum overnight.

Yield: 9.14g (89%) of white powder 1c (HCl salt).

MS：m/z 979.45＝[M+6H]⁶⁺(calculated value 979.55).

For the synthesis of compound 1d, compound 1c (9.06g, 1.47 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (6.95g, 17.6 mmol) were dissolved in 50mL DCM (anhydrous) and DIPEA (4.56g, 35.3 mmol, 6.15mL) was added at room temperature. After 10 minutes, 1, 9-bis-boc-1, 5, 9-triazacyclononane (7.80g, 23.5 mmol) was added and the mixture was stirred for 15 minutes. Additional 1, 9-bis-boc-1, 5, 9-triazacyclononane (0.49g, 1.5 mmol) was then added. After complete dissolution, the solvent was evaporated at room temperature.

The residue was dissolved in 35mL iPrOH at 40 ℃ and diluted with 200mL MTBE. The product was allowed to settle at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 200mL of cold MTBE (0 ℃). The product was dried under vacuum overnight to give 1d as a white solid.

Yield: 11.6g (90%) of a white solid 1 d.

MS：m/z 1248.08＝[M+7H]⁷⁺(calculated value 1248.27).

For the synthesis of compound 1e, boc-protected compound 1d (11.4g, 1.31 mmol) was dissolved in 40mL of 3m hci in MeOH and stirred at 45 ℃ for 20 min, then at 55 ℃ for 10 min. For precipitation, 10mL of LMEOH and 200mL of MTBE were added and the mixture was stored at-20 ℃ for 16 hours. The precipitate was collected by filtration through a glass filter Por.3 and washed with 200mL of cold MTBE (0 ℃). The product was dried under vacuum overnight to give 1e as a white powder.

Yield: 7.60g (75%) of 1e (HCl salt) as a white powder.

MS：m/z 891.96＝[M+8H]⁸⁺(calculated value 892.13).

For the synthesis of compound 1f, compound 1e (7.56g, 0.980 mmol, HCl salt) and bis (pentafluorophenyl) carbonate (9.27g, 23.0 mmol) were dissolved in 250mL DCM (anhydrous) and DIPEA (6.08g, 47.0 mmol, 8.19mL) was added at 35 ℃. After 10 minutes, 1, 9-bis-boc-1, 5, 9-triazacyclononane (5.30g, 16.0 mmol) was added and the mixture was stirred for 15 minutes. Additional 1, 9-bis-boc-1, 5, 9-triazacyclononane (0.33g, 1.0 mmol) was added. After complete dissolution, the solvent was evaporated at room temperature.

The residue was dissolved in 250mL iPrOH at 60 ℃ and diluted with 1350mL MTBE. The product was allowed to settle at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 400mL of cold MTBE (0 ℃). The product was dried under vacuum overnight to give 1f as a glassy solid.

Yield: 11.1g (83%) of a glassy solid 1 f.

MS：m/z 1312.01＝[M+10H]¹⁰⁺(calculated value 1312.21).

For the synthesis of 1g of backbone reagent, boc-protected compound 1f (7.84g, 0.610 mmol) was dissolved in 16mL MeOH at 37 deg.C, and 4M HCl (4 deg.C) was added at room temperature in two portions55mL of a pre-cooled solution in an alkane. The mixture was stirred for 20 minutes without cooling. After 20 minutes, 110mL of 3M HCl in MeOH was added. The solution was distributed into 24 Falcon tubes (50mL) and precipitated by adding 40mL of cold MTBE (-20 ℃ C.) to each Falcon tube. After centrifugation at 3214rcf for 1 min, the supernatant was decanted, and the glassy solid was dissolved in 5mL MeOH per Falcon tube and precipitated by again adding 40mL cold MTBE (-20 ℃) to each Falcon tube. The supernatant was decanted and the remaining solid was dried under vacuum overnight.

Yield: 5.74g (87%) of a white glassy solid 1g (HCl salt).

MS：m/z 965.46＝[M+10H]¹⁰⁺(calculated value 965.45).

Backbone reagent 1h was synthesized as described in the examples of WO2011/012715A1 for Compound 1e, except that Boc-DLys (Boc) -OH was used instead of Boc-LLys (Boc) -OH.

MS：m/z 848.52＝[M+8H⁺]⁸⁺(calculated value 848.57).

Example 2

Synthesis of crosslinker reagents 2d, 2g, rac-2k, rac-2o, 2s, 2v, rac-2y, 2ac, 2ag and 2ak

Crosslinker reagent 2e was prepared from monobenzyl azelate and PEG10000 according to the following scheme:

for the synthesis of monobenzyl azelate 2a, a mixture of azelaic acid (37.6g, 200 mmol), benzyl alcohol (21.6g, 200 mmol), p-toluenesulfonic acid (0.80g, 4.2 mmol) and 240mL of toluene was refluxed in a dean Stark apparatus for 7 hours. After cooling, the solvent was evaporated and 300mL saturated NaHCO was added₃Aqueous solution the mixture was extracted with 3 × 200mL MTBE the combined organic phases were taken over Na₂SO₄The product was purified on 2 × 340g silica using ethyl acetate/heptane (10:90 → 25:75) as eluent, the eluent was evaporated and the residue was dried under vacuum overnight.

Yield: 25.8g (46%) of a colorless oil 2 a.

MS：m/z 279.16＝[M+H]⁺(calculated value 279.16).

For the synthesis of compound 2b, monobenzyl azelate 2a (3.90g, 14.0 mmol) and PEG10000 (40.0g, 4.00 mmol) were dissolved in 64mL dichloromethane and cooled with an ice bath. A solution of DCC (2.89g, 14.0 mmol) and DMAP (0.024g, 0.020 mmol) in 32mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 65mL of dichloromethane and diluted with 308mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 250mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 40.8g (97%) of white powder 2 b.

MS：m/z 835.50＝[M+14H]¹⁴⁺(calculated value 835.56).

For the synthesis of compound 2c, compound 2b (40.6g, 3.86 mmol) was dissolved in methyl acetate (250mL) and 203mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 37.2g (93%) of a glassy solid 2 c.

MS：m/z 882.53＝[M+13H]¹³⁺(calculated value 882.51).

For the synthesis of compound 2d, compound 2c (32.0g, 3.10 mmol) and TSTU (3.73g, 12.4 mmol) were dissolved in 150mL dichloromethane at room temperature. DIPEA (1.60g, 12.4 mmol) was then added and the mixture was stirred for 1 hour. The resulting suspension was filtered and the filtrate was diluted with 170mL of dichloromethane and washed with 140mL of 750g water/197 g NaCl/3g NaOH solution. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 200mL of toluene, diluted with 180mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 100mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 28.8g (88%) of white powder 2 d.

MS：m/z 795.47＝[M+15H]¹⁵⁺(calculated value 795.54).

Crosslinker reagent 2g was prepared from monobenzyl azelate and PEG6000 according to the following protocol.

For the synthesis of compound 2e, monobenzyl azelate 2a (6.50g, 23.3 mmol) and PEG6000 (40.0g, 6.67 mmol) were dissolved in 140mL of dichloromethane and cooled with an ice bath. A solution of DCC (4.81g, 23.3 mmol) and DMAP (0.040g, 0.33 mmol) in 40mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 70mL of dichloromethane and diluted with 300mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 500mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 41.2g (95%) of white powder 2 e.

MS：m/z 833.75＝[M+8H]⁸⁺(calculated value 833.74).

For the synthesis of compound 2f, compound 2e (41.2g, 6.32 mmol) was dissolved in methyl acetate (238mL) and ethanol (40mL), then 400mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 38.4g (96%) of a glassy solid 2 f.

MS：m/z 750.46＝[M+9H]⁹⁺(calculated value 750.56).

For the synthesis of compound 2g, compound 2f (38.2g, 6.02 mmol) and TSTU (7.25g, mmol) were dissolved in 130mL of dichloromethane at room temperature. DIPEA (3.11g, 24.1 mmol) was then added and the mixture was stirred for 1 hour. The resulting suspension is filtered, the filtrate is diluted with 100mL of dichloromethane and washed with 200mL of 750g of water/197 g of NaCl/3g of NaOH solution. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 210mL of toluene, diluted with 430mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 450mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 35.8g (91%) of white powder 2 g.

MS：m/z 857.51＝[M+8H]⁸⁺(calculated value 857.51).

Crosslinker reagent 2k was prepared from monobenzyl isopropyl malonate and PEG10000 according to the following protocol:

for the synthesis of monobenzyl isopropylmalonate rac-2h, isopropylmalonic acid (35.0g, 239 mmol), benzyl alcohol (23.3g, 216 mmol) and DMAP (1.46g, 12.0 mmol) were dissolved in 100mL acetonitrile, the mixture was cooled to 0 ℃ with an ice bath, a solution of DCC (49.4g, 239 mmol) in 150mL acetonitrile was added over 15 minutes at 0 ℃ the ice bath was removed and the reaction mixture was stirred at room temperature overnight, then the solid was filtered off, the filtrate was evaporated under vacuum at 40 ℃ and the residue was dissolved in 300mL MTBE, the solution was purified with 2 × 300mL saturated NaHCO₃Extracting the aqueous solution, and then mixingThe combined aqueous phases were acidified with 6N hydrochloric acid to pH 1-3, the resulting emulsion was extracted with 2 × 300mL MTBE and the solvent was evaporated, the combined organic phases were washed with 200mL saturated aqueous NaCl and over MgSO₄And (5) drying. The product was purified on 340g silica using ethyl acetate/heptane (10:90 → 20:80) as eluent. The eluent was evaporated and the residue was dried under vacuum overnight.

Yield: 9.62g (17%) of colorless oil rac-2 h.

MS：m/z 237.11＝[M+H]⁺(calculated value 237.11).

For the synthesis of compound rac-2i, monobenzyl isopropylmalonate rac-2h (945mg, 4.00 mmol) and PEG10000(10.0g, 4.00 mmol) were dissolved in 20mL dichloromethane and cooled with an ice bath. A solution of DCC (825mg, 4.00 mmol) and DMAP (6mg, 0.05 mmol) in 10mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 20mL of dichloromethane and diluted with 150mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 500mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 9.63g (92%) of rac-2i as a white powder.

MS：m/z 742.50＝[M+16H]¹⁶⁺(calculated value 742.51).

For the synthesis of compound rac-2j, compound rac-2i (3.38g, 0.323 mmol) was dissolved in methyl acetate (100mL) and 105mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 3.25g (98%) of rac-2j, a glassy solid.

MS：m/z 731.25＝[M+16H]¹⁶⁺(calculated value 731.25).

For the synthesis of compound rac-2k, compound rac-2j (3.10g, 0.302 mmol) and TSTU (0.364g, 1.21 mmol) were dissolved in 15mL of dichloromethane at room temperature, DIPEA (0.156g, 1.21 mmol) was then added and the mixture was stirred for 45 min, the resulting suspension was filtered and the filtrate was washed with 2 × 10mL of 0.5M phosphate buffer pH 6.5 the organic phase was MgSO 2₄Dried and the solvent evaporated under vacuum. The residue was dissolved in 20mL of toluene, diluted with 10mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 250mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 2.66g (84%) of rac-2k as a white powder.

MS：m/z 743.37＝[M+16H]¹⁶⁺(calculated value 743.38).

The crosslinker reagent rac-2o was prepared from cis-1, 4-cyclohexanedicarboxylic acid and PEG10000 according to the following protocol:

for the synthesis of monobenzyl cis-1, 4-cyclohexanedicarboxylate rac-2l, cis-1, 4-cyclohexanedicarboxylic acid (20.0g, 116 mmol), benzyl alcohol (11.3g, 105 mmol) and DMAP (710mg, 5.81 mmol) were dissolved in 200mL THF the mixture was cooled to 0 ℃ with an ice bath, a solution of DCC (49.4g, 239 mmol) in 100mL THF was added over 15 minutes at 0 ℃ the ice bath was removed and the reaction mixture was stirred at room temperature overnight, then the solid was filtered off, the filtrate was evaporated at 40 ℃ and the residue was dissolved in 300mL MTBE, the solution was taken up with 2 × 300mL saturated NaHCO 2₃The aqueous solution is extracted, the combined aqueous phases are then acidified with 6N hydrochloric acid to pH 1-3, the emulsion obtained is extracted with 2 × 300mL MTBE and the solvent is evaporated, the combined organic phases are washed with 200mL saturated aqueous NaCl and over MgSO₄And (5) drying. The product is prepared in340g of silica are purified using ethyl acetate/heptane (10:90 → 20:80) as eluent. The eluent was evaporated and the colourless oily residue crystallised during drying under vacuum overnight.

Yield: 4.82g (16%) of rac-2l as colorless crystals.

MS：m/z 263.13＝[M+H]⁺(calculated value 263.13).

For the synthesis of compound rac-2m, mono-benzyl cis-1, 4-cyclohexanedicarboxylate rac-2l (2.10g, 8.00 mmol) and PEG10000 (20.0g, 10.0 mmol) were dissolved in 50mL of dichloromethane and cooled with an ice bath. A solution of DCC (1.65g, 8.00 mmol) and DMAP (0.012g, 0.10 mmol) in 25mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 55mL of dichloromethane and diluted with 300mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 250mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 18.2g (87%) of rac-2m as a white powder.

MS：m/z 745.76＝[M+16H]¹⁶⁺(calculated value 745.77).

For the synthesis of compound rac-2n, compound rac-2m (9.00g, 0.857 mmol) was dissolved in methyl acetate (100mL) and 157mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 8.83g (100%) of rac-2n as a glassy solid.

MS：m/z 734.50＝[M+16H]¹⁶⁺(calculated value 734.50).

For the synthesis of compound rac-2o, the following will be usedThe compounds rac-2n (8.92g, 0.864 mmol) and TSTU (1.04g, 3.64 mmol) were dissolved in 35mL dichloromethane at room temperature then DIPEA (0.447g, 3.46 mmol) was added and the mixture stirred for 45 min the resulting suspension was filtered and the filtrate was washed with 2 × 10mL 0.5M phosphate buffer pH 6.5 the organic phase was washed over MgSO 10₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 50mL of toluene, diluted with 25mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 400mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 7.62g (84%) of rac-2o as a white powder.

MS：m/z 702.60＝[M+16H]¹⁶⁺(calculated value 702.59).

Crosslinker reagent 2s was prepared from monobenzyl suberate and PEG10000 according to the following scheme:

monobenzyl suberate 2p was synthesized from suberic acid and benzyl alcohol according to monobenzyl azelate 2 a.

Compound 2q was synthesized from monobenzyl suberate 2p and PEG10000 according to PEG derivative 2 b.

Compound 2r was synthesized from compound 2q according to PEG derivative 2 c.

For the synthesis of compound 2s, compound 2r (18.0g, 1.74 mmol) and p-nitrophenyl carbonate (2.12g, 6.76 mmol) were dissolved in 70mL acetonitrile at room temperature. DIPEA (0.90g, 6.76 mmol) in 1.0mL of dichloromethane was then added and the mixture was stirred for 17 hours. The mixture was diluted with 270mL MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 200mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 17.6g (96%) of pale yellow powder for 2 s.

MS：m/z 822.20＝[M+14H]¹⁴⁺(calculated value 822.25).

Crosslinker reagent 2v was prepared from monobenzyl suberate 2p and PEG6000 according to the following scheme:

compound 2t was synthesized from monobenzyl suberate 2p and PEG6000 according to PEG derivative 2 b.

Compound 2u was synthesized from compound 2t according to PEG derivative 2 c.

For the synthesis of compound 2v, compound 2u (2.40g, 0.38 mmol) and p-nitrophenyl carbonate (4.61g, 1.52 mmol) were dissolved in 6mL acetonitrile at room temperature. DIPEA (0.20g, 1.52 mmol) in 0.14mL of dichloromethane was then added and the mixture was stirred for 1 hour. The mixture was diluted with 26mL MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 100mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 2.37g (95%) of pale yellow powder 2 v.

MS：m/z 774.34＝[M+9H]⁹⁺(calculated value 774.34).

Crosslinker reagent 2y was prepared from monobenzyl isopropyl malonate and PEG8000 according to the following scheme:

for the synthesis of compound rac-2w, monobenzyl isopropylmalonate rac-2h (2.25g, 9.50 mmol) and PEG8000 (19.0g, 2.38 mmol) were dissolved in 100mL of dichloromethane and cooled with an ice bath. A solution of DCC (1.96g, 9.50 mmol) and DMAP (14mg, 0.12 mmol) in 10mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 40mL of dichloromethane and diluted with 270mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 500mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 18.5g (92%) of rac-2w as a white powder.

MS：m/z 737.43＝[M+13H]¹³⁺(calculated value 737.42).

For the synthesis of compound rac-2X, compound rac-2w (18.4g, 2.18 mmol) was dissolved in methyl acetate (160mL) and 254mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 17.7g (98%) of rac-2X as a glassy solid.

MS：m/z 723.51＝[M+13H]¹³⁺(calculated value 723.55).

For the synthesis of compound rac-2y, compound rac-2x (13.6g, 1.65 mmol) and TSTU (1.96g, 6.60 mmol) were dissolved in 60mL dichloromethane at room temperature. DIPEA (852mg, 6.60 mmol) was then added and the mixture was stirred for 45 min. The resulting suspension was filtered, the filtrate diluted with 70mL ethyl acetate and washed with 70mL 0.5M phosphate buffer pH 6.5. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum. The residue was dissolved in 80mL of toluene and the remaining solid was filtered off and washed with 20mL of toluene. The combined toluene fractions were diluted with 35mL of MTBE at room temperature and stored overnight at-20 ℃. The precipitate was collected by filtration through a glass filter Por.3 and washed with 600mL of cold MTBE (-20 deg.C)) And (6) washing. The product was dried under vacuum overnight.

Yield: 12.1g (87%) of rac-2y as a white powder.

MS：m/z 738.51＝[M+13H]¹³⁺(calculated value 738.49).

Crosslinker reagent 2ac was prepared from monobenzyl sebacate and PEG10000 according to the following scheme:

for the synthesis of monobenzyl sebacate 2z, a mixture of sebacic acid (20.2g, 100 mmol), benzyl alcohol (10.8g, 100 mmol), p-toluenesulfonic acid (0.40g, 2.1 mmol) and 120mL of toluene was refluxed in a dean Stark apparatus for 19 hours. After cooling, the solvent was evaporated and 150mL of saturated NaHCO was added₃Aqueous solution the mixture was extracted with 3 × 100mM MTBE the combined organic phases were purified over Na₂SO₄Dried and the solvent evaporated. The product was purified on 340g silica using ethyl acetate/heptane (10:90 → 25:75) as eluent. The eluent was evaporated and the residue was dried under vacuum overnight.

Yield: 13.4g (46%) of colorless oil 2 z.

MS：m/z 293.16＝[M+H]⁺(calculated value 293.16).

For the synthesis of compound 2aa, monobenzyl sebacate 2z (1.02g, 3.50 mmol) and PEG10000(10.0g, 1.00 mmol) were dissolved in 40mL dichloromethane and cooled with an ice bath. A solution of DCC (722mg, 3.5 mmol) and DMAP (6.1mg, 0.05 mmol) in 12mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 12mL of dichloromethane and diluted with 45mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 250mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 9.92g (94%) of white powder 2 aa.

MS：m/z 793.48＝[M+15H]¹⁵⁺(calculated value 793.56).

For the synthesis of compound 2ab, compound 2aa (9.83g, 0.93 mmol) was dissolved in methyl acetate (100mL) and 123mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 9.14g (95%) of glassy solid 2 ab.

MS：m/z 840.51＝[M+14H]¹⁴⁺(calculated value 840.44).

For the synthesis of compound 2ac, compound 2ab (9.00g, 0.87 mmol) and TSTU (1.05g, 3.47 mmol) were dissolved in 30mL of dichloromethane at room temperature, then DIPEA (449mg, 3.47 mmol) was added and the mixture was stirred for 1 hour. The resulting suspension was filtered and diluted with 30mL of dichloromethane, washed with 60mL of 750g water/197 g NaCl/3g NaOH solution. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 45mL of toluene, diluted with 35mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 350mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 8.29g (90%) of white powder 2 ac.

MS：m/z 794.47＝[M+15H]¹⁵⁺(calculated value 794.48).

Crosslinker reagent 2ag was prepared from monobenzyl undecanedioate and PEG10000 according to the following scheme:

for the synthesis of monobenzyl undecanedioate 2ad, a mixture of undecanedioic acid (21.6g, 100 mmol), benzyl alcohol (10.8g, 100 mmol), p-toluenesulfonic acid (0.40g, 2.1 mmol) and 120mL of toluene was refluxed in a dean Stark apparatus for 19 hours. After cooling, the solvent was evaporated and 150mL of saturated NaHCO was added₃Aqueous solution the mixture was extracted with 3 × 100mL MTBE 3 combined organic phases were extracted over Na₂SO₄Dried and the solvent evaporated. The product was purified on 340g silica using ethyl acetate/heptane (10:90 → 25:75) as eluent. The eluent was evaporated and the residue was dried under vacuum overnight.

Yield: 14.8g (48%) of colorless oil 2 ad.

MS：m/z 307.19＝[M+H]⁺(calculated value 307.19).

For the synthesis of compound 2ae, monobenzyl undecanedioate 2ad (1.07g, 3.50 mmol) and PEG10000(10.0g, 1.00 mmol) were dissolved in 40mL of dichloromethane and cooled with an ice bath. A solution of DCC (722mg, 3.5 mmol) and DMAP (6.1mg, 0.05 mmol) in 12mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 13mL of dichloromethane and diluted with 43mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 250mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 10.0g (95%) of 2ae as a white powder.

MS：m/z 792.48＝[M+15H]¹⁵⁺(calculated value 792.49).

For the synthesis of compound 2af, compound 2ae (9.83g, 0.93 mmol) was dissolved in methyl acetate (100mL) and 103mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 8.90g (92%) of 2af glassy solid.

MS：m/z 780.47＝[M+15H]¹⁵⁺(calculated value 780.47).

For the synthesis of compound 2ag, compound 2af (8.75g, 0.84 mmol) and TSTU (1.01g, 3.37 mmol) were dissolved in 30mL of dichloromethane at room temperature. DIPEA (435mg, 3.37 mmol) was then added and the mixture was stirred for 1 hour. The resulting suspension was filtered and the filtrate was diluted with 30mL of dichloromethane and washed with 60mL of 750g water/197 g NaCl/3g NaOH solution. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 38mL of toluene, diluted with 30mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 350mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 7.51g (84%) of 2ag as a white powder.

MS：m/z 793.48＝[M+14H]¹⁴⁺(calculated value 793.38).

Crosslinker reagent 2ak was prepared from monobenzylglutarate and PEG10000 according to the following protocol:

for the synthesis of compound 2ai, monobenzyl glutarate 2ah (1.95g, 8.78 mmol) and PEG10000 (25.1g, 2.51 mmol) were dissolved in 70mL dichloromethane and cooled with an ice bath. A solution of DCC (1.81g, 8.78 mmol) and DMAP (15.3mg, 0.13 mmol) in 18mL 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 ℃ and the solid was filtered off. The solvent was evaporated under vacuum.

The residue was dissolved in 70mL of dichloromethane and diluted with 360mL of MTBE at room temperature. The mixture was stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 500mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 24.9g (95%) of white powder 2 ai.

MS：m/z 740.76＝[M+16H]¹⁶⁺(calculated value 740.76).

For the synthesis of compound 2aj, compound 2ai (24.9g, 2.39 mmol) was dissolved in methyl acetate (220mL) and 302mg of palladium on charcoal was added. The mixture was stirred at room temperature overnight under an atmosphere of hydrogen at ambient pressure. The reaction mixture was filtered through a pad of celite, the filtrate was evaporated and dried under vacuum overnight.

Yield: 24.3g (99%) of a glassy solid 2 aj.

MS：m/z 729.50＝[M+16H]¹⁶⁺(calculated 729.49).

For the synthesis of compound 2ak, compound 2aj (23.9g, 2.33 mmol) and TSTU (2.81g, 9.33 mmol) were dissolved in 100mL dichloromethane at room temperature. DIPEA (1.21g, 9.33 mmol) was then added and the mixture was stirred for 1 hour. The resulting suspension was filtered and the filtrate was diluted with 20mL of dichloromethane and washed with 120mL of 750g water/197 g NaCl/3g NaOH solution. The organic phase is over MgSO₄Dried and the solvent evaporated under vacuum.

The residue was dissolved in 125mL of toluene, diluted with 100mL of MTBE at room temperature and stored at-20 ℃ overnight. The precipitate was collected by filtration through a glass filter Por.3 and washed with 650mL of cold MTBE (-20 ℃). The product was dried under vacuum overnight.

Yield: 22.5g (92%) of white powder 2 ak.

MS：m/z 741.63＝[M+16H]¹⁶⁺(calculated value 741.63).

Example 3

Preparation of hydrogel beads 3a, 3b, 3c and 3d containing free amino groups

218mg Cithrol in a cylindrical 250mL reactor with bottom outlet, diameter 60mm, equipped with baffles^TMAn emulsion of DPHS in 100mL of undecane was stirred at 580rpm at ambient temperature with an isojet stirrer 50mm in diameter. A solution of 250mg 1a and 2205mg 2d in 22.1g DMSO was added and stirred at room temperature for 10 minutes to form a suspension. 1.1mL of TMEDA was added to conduct polymerization. The mixture was stirred for 16 hours. 1.7mL of acetic acid was added, followed by 100mL of 15 wt% aqueous sodium chloride solution after 10 minutes. After 10 minutes, the stirrer was stopped and the phases were allowed to separate. After 2 hours, the aqueous phase containing the hydrogel was drained off.

For bead size classification, the water-hydrogel suspension was diluted with 40mL of ethanol and wet sieved using a Retsch AS200 controlled sieving machine on 125, 100, 75, 63, 50, 40 and 32 μm steel sieves for 15 minutes. The screening amplitude is 1.5mm, and the water flow is 300 mL/min. The bead fractions remaining on the 63 and 75 μm sieves were collected and washed 3 times with 0.1% AcOH, 10 times with ethanol and dried at 0.1 mbar for 16 hours to give 670mg of 3a as a white powder.

The amino group content of the hydrogel was determined to be 0.145 mmol/g by conjugation of fmoc-amino acids to free amino groups of the hydrogel followed by fmoc-determination.

3b is prepared as described for 3a, except that a stirrer speed of 560rpm is used, 350mg 1a, 2548mg2g, 26.1g DMSO, 257mg Cithrol^TMDPHS, 1.5mL TMEDA and 2.4mL acetic acid gave 550mg of 3b as a white powder, free amino groups 0.120 mmol/g.

3c are prepared as described for 3a, except that 5 is appliedStirrer speed of 60rpm, 250mg of 1a, 3019mg of rac-2k, 32.7g of DMSO, 290mg of Cithrol^TMDPHS, 1.1mL of TMEDA and 1.7mL of acetic acid gave 770mg of 3c as a white powder with 0.126 mmol/g of free amino groups.

3d was prepared as described for 3a, except that 250mg 1a, 2258mg rac-2o, 22.6g DMSO, 222mg Cithrol were used^TMDPHS, 1.1mL of TMEDA and 1.7mL of acetic acid gave 186mg of 3d as a white powder, free amino groups 0.153 mmol/g.

3e was prepared as described for 3a, except that 250mg 1a, 2168mg rac-2y, 21.8g DMSO, 215mg Cithrol were used^TMDPHS, 1.1mL TMEDA, 1.7mL acetic acid gave 710mg of 3e as a white powder with 0.154 mmol/g free amino groups.

3f was prepared as described for 3a except that 290mg of the backbone reagent described in example 1 of WO2011/012715A1 as 1g, 2281mg 2v, 15.8g DMSO, 290mg Cithrol were used with a stirrer speed of 400rpm^TMDPHS, 2.2mL TMEDA, 3.3mL acetic acid, were sieved over 250, 180, 125, 90 and 63 μm steel sieves and the product was collected on 180 μm sieves. Work-up gave 820mg of 3f as a pale yellow powder, free amino 0.108 mmol/g.

3g were prepared as described for 3a, except that 300mg of the backbone reagent described in example 1 of WO2011/012715A1 as 1g, 2520mg 2s, 32.4g DMSO, 300mg Cithrol were used, using a stirrer speed of 500rpm^TMDPHS, 2.2mL TMEDA, 3.4mL acetic acid gave 770mg as a pale yellow powder of 3g, free amino 0.175 mmol/g.

3h was prepared as described for 3a, except that 200mg 1a, 1995mg 2ac, 19.8g DMSO, 195mg Cithrol were used^TMDPHS, 0.9mL TMEDA, 1.4mL acetic acid gave 650mg of 3h as a white powder with 0.131 mmol/g free amino groups.

3i was prepared as described for 3a, except 150mg 1a, 1591mg 2ag, 15.7g DMSO, 154mg Cithrol was used^TMDPHS, 0.7mL TMEDA, 1.0mL acetic acid gave 300mg of 3i as a white powder, free amino 0.123 mmol/g.

3j are prepared as described for 3a, except that a stirrer speed of 570rpm is applied, using 360mg 1h, 2567mg2ak, 26.3g DMSO, 257mg Cithrol^TMDPHS, 2.2mL TMEDA, 3.4mL acetic acid gave 744mg of 3j as a white powder with 0.097 mmol/g free amino groups.

Example 4

Synthesis of linker reagent 4c

Linker reagent 4c was synthesized according to the following scheme:

4a Synthesis:

Fmoc-L-Asp (OtBu) -OH (1.00g, 2.43 mmol) with DCC (0.70g, 3.33 mmol) was dissolved in DCM (25 mL.) Oxyma pure (0.51g, 3.58 mmol) and collidine (0.50mL, 3.58 mmol) were added in one portion and a solution of N-Boc-ethylenediamine (0.41g, 2.56 mmol) in DCM (15mL) was added slowly, after stirring the mixture at room temperature for 90 minutes, the precipitate formed was filtered off and the filtrate was washed with aqueous HCl (0.1M, 50mL), the aqueous layer was extracted with DCM (2 × 20mL) and the combined organic portions were extracted with saturated aqueous NaHCO₃(3 × 25mL) and brine (1 × 50mL) and washed over Na₂SO₄Dried, filtered and concentrated under vacuum. The crude solid was purified by flash chromatography. The intermediate N-boc-N' - (N-fmoc-4-tert-butyl-L-aspartyl) -ethylenediamine (0.98g, 1.77 mmol, 73%) was obtained as a white solid

MS：m/z 554.29＝[M+H]⁺(calculated value: 554.29)

N-boc-N' - (N-fmoc-4-tert-butyl-L-aspartyl) -ethylenediamine (0.98g, 1.77 mmol) was dissolved in THF (15mL), DBU (0.31mL) was added and the solution was stirred at room temperature for 12 minutes. The reaction was quenched with AcOH (0.5ml), concentrated under vacuum and the residue purified by flash chromatography to give 4a as a white solid (0.61g, 1.77 mmol, 73% over 2 steps).

MS：m/z 332.38＝[M+H]⁺(calculated value 332.22).

4b Synthesis:

6-Acetylthiohexanoic acid (0.37g, 1.95 mmol) was dissolved in DCM (19.5mL) and Oxyma pure (0.35g, 2.48 mmol) and DCC (0.40g, 1.95 mmol) were added in one portion. The solution was stirred at room temperature for 30 min, filtered and the filtrate added to a solution of 4a (0.61g, 1.77 mmol) in DCM (10.5 mL). DIPEA (0.46mL, 2.66 mmol) was added to the solution and the reaction was stirred at room temperature for 2 hours. The solution is taken up in aqueous H₂SO₄(0.1M, 2 × 30mL), saturated aqueous NaHCO₃(2 × 20mL) and brine (1 × 20 mL.) the organic layer was washed over Na₂SO₄Dried, filtered and concentrated under vacuum. The crude material was purified by flash chromatography to give N-boc-N' - (N-6-acetylthiohexyl-4-tert-butyl-L-aspartyl) -ethylenediamine (0.65g, 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-aspartyl) -ethylenediamine (0.60g, 1.18 mmol) was dissolved in TFA (5mL) and TES (0.13mL) and water (0.13mL) were added. The mixture was stirred at room temperature for 30 minutes. In N₂TFA was removed from the stream and the crude 4b was dissolved in H2O/ACN 1:1 and purified by RP-HPLC.

Yield: 0.39g, 0.85 mmol (TFA salt), 72%.

MS：m/z 348.25＝[M+H]⁺(calculated value 348.16).

4c Synthesis

4b (TFA salt, 0.38g, 0.80 mmol) was dissolved in DMF (5mL) and 4-nitrophenyl carbonate (5-methyl-2-oxo-1, 3-dioxol-4-yl) -methyl ester (0.26g, 0.88 mmol) and DIPEA (0.28mL, 1.6 mmol) were added. The resulting suspension was diluted with DCM (5mL) and stirred at room temperature for 3 hours. More DIPEA (0.28ml1.6 mmol) was added and stirring was continued for 2 hours. The DCM was concentrated in vacuo and the residue was diluted with H2O/ACN 3:1 and purified by RP-HPLC to give N- (5-methyl-2-oxo-1, 3-dioxol-4-yl) -methyl-oxycarbonyl-N' - (N-6-acetylthiohexyl-L-aspartyl) -ethylenediamine (0.31g, 0.62 mmol, 77%) as a colourless oil.

MS：m/z 504.16＝[M+H]⁺(calculated value 504.17).

N- (5-methyl-2-oxo-1, 3-dioxol-4-yl) -methyloxycarbonyl-N' - (N-6-acetylthiohexyl-L-aspartyl) -ethylenediamine (150mg, 0.30 mmol) was dissolved in DCM (17.5mL) and NHS (41mg, 0.36 mmol), DCC (74mg, 0.36 mmol) and DMAP (4mg, 0.03 mmol) were added in one portion. The reaction was stirred at room temperature for 1 hour and the resulting suspension was filtered. The precipitate was washed with a small amount of DCM and the combined filtrates were concentrated in vacuo. Purification of 4c by RP-HPLC gave a colourless oil (144mg, 0.24 mmol, 80%).

MS：m/z 601.18＝[M+H]⁺(calculated value 601.18).

Example 5

Preparation of Maleimide-functionalized hydrogel beads 5a

259.3mg of dry hydrogel beads 3a were incubated for 15 minutes in 10mL of 1% n-propylamine in NMP, then washed twice with 1% n-propylamine in NMP and twice with 2% DIPEA in NMP. 171mg of maleimide-NH-PEG 12-PFE was dissolved in 1mL of NMP and added to the washed hydrogel beads 3 a. The hydrogel suspension was incubated at room temperature for 2 hours. The resulting maleimide-functionalized hydrogel beads 5a were treated with NMP, 20mM succinate each time,1mM Na₂EDTA, 0.01% Tween20 at pH 3.0, water, and 0.1% acetic acid, 0.01% Tween20 were washed 5 times.

Preparation of Maleimide-functionalized hydrogel beads 5b

117.7mg of dry hydrogel beads 3a were swollen in 5mL of NMP, washed 5 times with NMP and 5 times with 2% DIPEA in NMP. 5 equivalents (56mg) of maleimide-NH-PEG 6-PFE (based on the amine content of the hydrogel beads) were dissolved in 0.5mL NMP and added to the washed hydrogel beads 5 b. The hydrogel suspension was incubated at room temperature for 2.5 hours. The resulting maleimide-functionalized hydrogel beads were washed 5 times with each NMP, followed by 0.1% acetic acid, 0.01% Tween 20.

Example 6

Synthesis of short-acting Lucentis-linker-hydrogel prodrug 6c

4.6mg of Lucentis (described as Lucentis-NH in the following protocol)₂) (460. mu.L of 10mg/mLLucentis 10mM histidine solution, 10 wt% α -trehalose, 0.01% Tween20, pH 5.5) was exchanged with 10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride buffer, pH7.4, and the Lucentis concentration was adjusted to 16.4mg/mL 6mg linker reagent 4c was dissolved in 100. mu.L DMSO to give a concentration of 100 mM.1 molar equivalents of linker reagent 4c relative to the amount of Lucentis was added to the Lucentis solution

The pH of the reaction mixture was adjusted to pH 6.5 by adding 1M sodium citrate at pH 5.0 and Na was added₂EDTA to a final concentration of 5 mM. To remove the protecting group of 6a, 0.5M NH was added₂OH (dissolved in 10mM sodium citrate, 140mM sodium chloride, 5mM Na)₂EDTA, pH 6.5) to 45mMFinal concentration, and the deprotection reaction was incubated at room temperature for 4 hours to give the Lucentis-linker mono-conjugate 6 b. A mixture of Lucentis and Lucentis-linker monoconjugate 6b was mixed with 10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride, 5mM Na, pH 6.5₂EDTA, 0.01% Tween20 buffer exchange, and the total concentration of the two Lucentis species was adjusted to 11.8 mg/mL. The Lucentis-linker mono-conjugate 6b content in the mixture was determined by ESI-MS to be 20%.

10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride, 5mM Na at pH 6.5₂EDTA, 4mg of Lucentis/Lucentis-linker mono-conjugate 6b mixture in 0.01% Tween20 was added to 1mg of maleimide functionalized hydrogel beads 5a and incubated overnight at room temperature to give a short acting Lucentis-linker-hydrogel prodrug 6 c.

Example 7

In vitro release kinetics-in vitro half-life determination

Lucentis-linker-hydrogel prodrug 6c (containing about 1mg Lucentis) was treated with 60mM sodium phosphate, 3mM Na, pH7.4₂EDTA, 0.01% Tween20 were washed 5 times and finally suspended in 1mL of the above buffer. The suspension was incubated at 37 ℃. The suspension was buffer exchanged after different time intervals and analyzed by HPLC-SEC at 220 nm. Peaks corresponding to released Lucentis were pooled and the total amount of released Lucentis was plotted against total incubation time. Curve fitting software was applied to determine the first order cleavage rate.

Example 8

Preparation of hydrogel beads 8a and 8b containing free amino groups

8a as for3a except that 400mg of [ PEG1250-LLys is used₂-LLys₄(NH₂)₈]₄、5082mg 2s、33.5g DMSO、400mg Cithrol^TMDPHS, 2.3mL TMEDA and 4.8mL acetic acid gave 1460mg 8a as a white powder, free amino 0.057 mmol/g.

8b are prepared as described for 3a, except that 290mg [ PEG1250-LLys is used₂-LLys₄(NH₂)₈]₄、2281mg2v、26.1g DMSO、257mg Cithrol^TMDPHS, 1.7mL TMEDA and 3.5mL acetic acid gave 820mg of 8b as a white powder with 0.108 mmol/g free amino groups.

8c were prepared as described for 3a, except that 250mg [ PEG1250-LLys was used₂-LLys₄(NH₂)₈]₄、2168mg rac-2y、21.8g DMSO、215mg Cithrol^TMDPHS, 1.1mL TMEDA and 1.7mL acetic acid gave 810mg of 8c as a white powder, free amino groups 0.154 mmol/g.

Example 9

Preparation of Biotin conjugated hydrogels 9a and 9b

11mg of 8a was transferred to a syringe equipped with a glass filter. Hydrogel beads 8a were incubated with NMP and H each time₂O/ACN/TFA (1/1/0.002; v/v/v) were washed 3 times each. 1.14mg of biotinimidohexanoic acid N-hydroxysuccinimide ester (2.5. mu. mol) was dissolved in 250. mu. L H₂O/ACN/TFA (1/1/0.002; v/v/v) was added to the washed hydrogel beads 8a and incubated at room temperature for 5 minutes with stirring (15 rpm). 50 μ L of 0.5M sodium phosphate pH7.4 was added and the reaction mixture was incubated at room temperature for 60 minutes with stirring (15 rpm). The biotin-conjugated hydrogel 9a thus obtained was treated with H each time₂O/ACN/TFA (1/1/0.002; v/v/v) and 5 washes with 10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride, 10% ACN, pH7.4, respectively.

9b was prepared according to 9a except that 11.3mg of hydrogel beads 8b and 2.2mg of N-hydroxysuccinimide biotinimidohexanoate (4.8. mu. mol) were used.

Example 10

Preparation of streptavidin-biotin hydrogels 10a and 10b

6mg of streptavidin was dissolved in 10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride, 10% ACN at pH7.4, added to the biotin-conjugated hydrogel 9a (prepared from 11mg 8 a) and incubated at room temperature for 60 minutes with stirring (15rpm), giving a streptavidin-biotin hydrogel 10 a. 10a was washed 2 times with 850. mu.L 10mM sodium phosphate, 2.7mM potassium chloride, 140mM sodium chloride, 10% ACN, pH7.4, and the wash fractions were combined. The amount of streptavidin bound to the biotin conjugated hydrogel was indirectly quantified by photometric determination at a streptavidin concentration of 280nm in the bound wash fraction and found to be 2.6 mg.

10b was prepared according to 10a except that biotin-conjugated hydrogel 9b (prepared from 11.3mg 8 b) and 8.1mg streptavidin were used. The amount of streptavidin bound to the biotin-conjugated hydrogel 9b was found to be 0.5 mg.

Example 11

Closed hydrogel bead 11

Hydrogel beads were synthesized according to the procedure described in example 3c and functionalized with maleimide groups according to the procedure described in example 5 b. Thereafter, 4mL of the 10mg/mL hydrogel suspension was transferred to a 20mL syringe equipped with a frit. The solvent was removed and the hydrogel was washed 10 times with 5mL 10mM histidine/10 wt% α, α -trehalose/0.01% Tween20/pH 5.5. The solvent was removed and 5mL of 1mM β -mercaptoethanol in 10mM histidine/10 wt% α, α -trehalose/0.01% Tween20/pH 5.5 was introduced into the syringe. The resulting suspension was incubated at ambient temperature for 5 minutes with gentle shaking. The solvent was drained and the hydrogel was treated with 5mL of 1mM β -mercaptoethanol in 10mM histidine/10 wt% α, α -trehalose/0.01% Tween20/pH 5.5 for an additional 9 times. The solvent was drained each time. The hydrogel beads were then washed 10 times with 5mL each time of 10mM histidine/10 wt% α, α -trehalose/0.01% Tween20/pH 5.5, draining off the solvent each time. The hydrogel beads were then washed 10 times with 5mL PBS-T/pH 7.4 each time, draining the solvent each time. Finally fresh PBS-T/pH 7.4 was introduced into the syringe and the suspension was transferred to a Falcon tube to give 11.

Abbreviations:

ac acetyl group

ACN acetonitrile

AcOH acetic acid

Asp aspartic acid

Boc tert-butoxycarbonyl

DBU 1, 8-diazabicyclo (5.4.0) undec-7-ene

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DIPEA diisopropylethylamine

DMAP dimethylaminopyridine

DMF dimethyl formamide

DMSO dimethyl sulfoxide

Fmoc fluorenylmethoxycarbonyl

HPLC high performance liquid chromatography

iPrOH Isopropanol

Maleimide-NH-PEG 6-PFE

N- (3-Maleimidopropyl) -21-amino-4, 7,10,13,16, 19-hexaoxa-heneicosanoic acid pentafluorophenyl ester

Maleimide-NH-PEG 12-PFE

N- (3-Maleimidopropyl) -39-amino-4, 7,10,13,16,19,22,25,28,31,34, 37-dodecaoxa-nonadecanoic acid pentafluorophenyl ester

MeOAc methyl acetate

MeOH methanol

MS mass spectrometry

MTBE methyl tert-butyl ether

NHS N-hydroxysuccinimide

Oxyma Pure 2-cyano-2- (hydroxyimino) acetic acid ethyl ester

PEG polyethylene glycol

RP-HPLC reversed-phase high performance liquid chromatography

RT Room temperature

tBu tert-butyl

TAN 1,5, 9-triazacyclononane

TES Triethylsilane

TFA trifluoroacetic acid

THF tetrahydrofuran

TMEDA N, N, N ', N' -tetramethylethylenediamine

TSTU O- (N-succinimidyl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate

Claims (30)

1. A method of making a prodrug linked to a carrier comprising the steps of:

(a) providing a mixture comprising:

(a-i) at least one framing agent, wherein the at least one framing agent has a molecular weight of 1-100kDa and comprises at least 3 amines (-NH)₂and/or-NH-);

(a-ii) at least one crosslinker agent, wherein the at least one crosslinker agent has a molecular weight of 6-40kDa, wherein the at least one crosslinker agent comprises:

(i) at least 2 carbonyloxy (- (C ═ O) -O-or-O- (C ═ O) -), and additionally

(ii) At least 2 reactive functional end groups selected from: an active ester group, an active urethane group, an active carbonate group and an active thiocarbonate group,

and is PEG-based comprising at least 70% PEG; and

(a-iii) a first solvent and at least a second solvent, said second solvent being immiscible in the first solvent,

wherein the weight ratio of the at least one framing agent to the at least one cross-linking agent is 1:99 to 99: 1;

(b) polymerizing the mixture of step (a) in suspension polymerization to form a hydrogel;

(c) optionally post-treating the hydrogel;

(d) optionally, reacting the hydrogel of step (b) or (c) with a spacer reagent of formula (VI) in the presence of a solvent to obtain a hydrogel-spacer conjugate:

A^x1-S⁰-A^x2(VI)，

wherein:

S⁰is selected from C_1-50Alkyl radical, C_2-50Alkenyl and C_2-50Alkynyl, said segments being optionally interrupted by one or more groups selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S, -C (O) -, -C (O) NH, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclic group, phenyl group and naphthyl group;

A^x1is a functional group that reacts with an amine group of the hydrogel; and is

A^x2Is a functional group; and

(e) reacting the hydrogel of step (b) or (c) or the hydrogel-spacer conjugate of step (d) with a prodrug linker-biologically active moiety reagent of formula (VII) in the presence of a solvent to give a carrier-linked prodrug:

A^y1-L-D (VII)，

wherein:

A^y1for reaction with an amine of the hydrogel of step (b) or (c) or for reaction with the hydrogel-spacer conjugate of step (d)Functional group A of^x2A reactive functional group;

l is a prodrug linker;

d is a bioactive moiety.

2. The method of claim 1, wherein the mixture of step (a) further comprises a detergent.

3. The method of claim 2, wherein the detergent is Cithrol DPHS, Hypermer 70A, HypermerB246, Hypermer 1599A, Hypermer 2296, or Hypermer 1083.

4. The process according to claim 1, wherein the polymerization in step (b) is initiated by addition of a base.

5. The process according to claim 4, wherein the base is selected from the group consisting of N, N, N ', N ' -Tetramethylethylenediamine (TMEDA), 1, 4-dimethylpiperazine, 4-methylmorpholine, 4-ethylmorpholine, 1, 4-diazabicyclo [2.2.2] octane, 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 "-pentamethyldiethylenetriamine, 1, 8-diazabicyclo [5.4.0] undec-7-ene, 1, 5-diazabicyclo [4.3.0] non-5-ene and hexamethylenetetramine.

6. The process according to claim 1 or 2, wherein the mixture of step (a) is an emulsion.

7. The method according to claim 1, wherein the at least one framing agent is selected from the group consisting of:

a compound of formula (I):

B(–(A⁰)_x1–(SP)_x2–A¹–P–A²–Hyp¹)_x(I)，

wherein:

b is a branched core, and B is a branched core,

SP is a spacer moiety selected from: c_1-6Alkyl radical, C_2-6Alkenyl and C_2-6An alkynyl group,

p is a polymer chain comprising at least 80% PEG,

Hyp¹is a compound containing amine (-NH)₂and/or-NH-) or contain at least 2 amines (-NH)₂and/or-NH-,

x is an integer of 3 to 16,

x1, x2 independently of one another are 0 or 1, with the proviso that if x2 is 0, then x1 is 0,

A⁰、A¹、A²independently of one another, from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

a compound of formula (II):

Hyp²–A³–P–A⁴–Hyp³(II)

wherein:

p is as defined above for the compounds of formula (I),

Hyp²、Hyp³independently of one another, contain at least two amines (-NH)₂and/or-NH-), and

A³and A⁴Independently selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

a compound of formula (III):

P¹–A⁵–Hyp⁴(III)

wherein:

P¹to comprise at least80% of the polymer chains of PEG,

Hyp⁴is a compound containing at least 3 amines (-NH)₂and/or-NH), and

A⁵selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group;

and

a compound of formula (IV):

T¹–A⁶–Hyp⁵(IV)

wherein:

Hyp⁵is a compound containing at least 3 amines (-NH)₂and/or-NH), and

A⁶selected from:

wherein R is¹And R^1aIndependently of one another, selected from H and C_1-6An alkyl group; and is

T¹Is selected from C_1-50Alkyl radical, C_2-50Alkenyl or C_2-50Alkynyl, said segments being optionally interrupted by one or more groups selected from: -NH-, -N (C)_1-4Alkyl) -, -O-, -S-, -C (O) NH-, -C (O) N (C)_1-4Alkyl) -, -O-C (O) -, -S (O)₂-, 4-7 membered heterocyclyl, phenyl or naphthyl.

8. The method according to claim 7, wherein Hyp¹、Hyp²、Hyp³、Hyp⁴And Hyp⁵Selected from:

a moiety of formula (e-i):

wherein:

p1 is an integer of 1 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴In the above-mentioned manner,

a moiety of formula (e-ii):

wherein:

p2, p3 and p4 are identical or different and are each, independently of one another, an integer from 1 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

(iv) a moiety of formula (e-iii):

wherein:

p5-p11 are identical or different and are each, independently of one another, an integer from 1 to 5, and

if the backbone reagent is of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a moiety of formula (e-iv):

wherein:

p12-p26 are identical or different and are each, independently of one another, an integer from 1 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a moiety of formula (e-v):

wherein:

p27 and p28 are the same or different and are each, independently of one another, an integer from 1 to 5,

q is an integer of 1 to 8, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

(vii) a moiety of formula (e-vi):

wherein:

p29 and p30 are identical or different and are each, independently of one another, an integer from 2 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the formula(II) in which the dotted line indicates the connection at A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a moiety of formula (e-vii):

wherein:

p31-p36 are identical or different and are each, independently of one another, an integer from 2 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1);

a moiety of formula (e-viii):

wherein:

p37-p50 are identical or different and are each, independently of one another, an integer from 2 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1); and

a moiety of formula (e-ix):

wherein:

p51-p80 are identical or different and are each, independently of one another, an integer from 2 to 5, and

if the backbone reagent has the structure of formula (I), the dotted line indicates the linkage at A²If the backbone reagent has the structure of formula (II), the dotted line indicates the linkage to A³Or A⁴If the backbone reagent has the structure of formula (III), the dotted line indicates the linkage at A⁵If the backbone reagent has the structure of formula (IV), the dotted line indicates the linkage at A⁶The above step (1); and

wherein the moieties (e-i) to (e-v) may be in the R-or S-configuration at the respective chiral center.

9. The method according to claim 7 or 8, wherein the backbone reagent is a compound of formula (I).

10. The process according to claim 7, wherein the branched core B is selected from the following structures:

wherein the dotted line indicates the connection at A⁰Or if x1 and x2 are both 0, then the connection is at A¹In the above-mentioned manner,

t is 1 or 2 and the ratio is,

v is 1,2, 3,4,5,6, 7, 8, 9, 10, 11, 12, 13 or 14.

11. The method according to claim 10, wherein B has the formula (a-xiv).

12. The method according to claim 7 or 8, wherein A is⁰Comprises the following steps:

13. the method according to claim 7 or 8, wherein x1 and x2 are 0.

14. The method according to claim 7 or 8, wherein P has the structure of formula (c-i):

wherein n is 6-900.

15. A method according to claim 7 or 8, wherein the moiety-A²–Hyp¹Is a moiety of the formula:

wherein:

the dotted line indicates the attachment to P; and is

E¹Selected from the formulae (e-i) to (e-ix).

16. The method of claim 1, wherein the backbone reagent has the formula:

wherein:

n is 10 to 40.

17. The method of claim 16, wherein n is 20 to 30.

18. A method according to claim 1 or 2, wherein the skeleton agent is present in the form of its acid salt.

19. A process according to claim 1 or 2, wherein the crosslinker agent is a compound of formula (V-II):

wherein:

D¹、D²、D³and D⁴Are identical or different and are each independently selected from O, NR⁵S and CR⁵R^5a；

R¹、R^1a、R²、R²、R³、R^3a、R⁴、R^4a、R⁵And R^5aAre identical or different and are each, independently of one another, selected from H and C_1-6An alkyl group; optionally, one or more pairs of R¹/R^1a、R²/R^2a、R³/R^3a、R⁴/R^4a、R¹/R²、R³/R⁴、R^1a/R^2aAnd R^3a/R^4aForm chemical bonds or are linked together with the atoms to which they are attached to form C_3-8Cycloalkyl is either linked together to form a ring a or together with the atoms to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclic group or adamantyl;

a is selected from phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl;

P²comprises the following steps:

m is 120-920;

r1, r2, r7, r8 are independently 0 or 1;

r3, r6 are independently 0,1, 2,3 or 4;

r4, r5 are independently 1,2, 3,4,5,6, 7, 8, 9 or 10;

s1, s2 are independently 1,2, 3,4,5 or 6;

Y¹、Y²are identical or different and are each, independently of one another, selected from the formulae (f-i) to (f-vi):

wherein:

the dashed lines indicate attachment to the rest of the molecule,

b is 1,2, 3 or 4,

X^His Cl, Br, I or F.

20. The method according to claim 1 or 2, wherein the crosslinker reagent has the formulae (V-1) to (V-54):

wherein:

if applicable, each crosslinker agent may be in the form of a racemic mixture thereof; and is

m is 120-920;

Y¹、Y²are identical or different and are each, independently of one another, selected from the formulae (f-i) to (f-vi):

wherein:

the dashed lines indicate attachment to the rest of the molecule,

b is 1,2, 3 or 4,

X^His Cl, Br, J or F.

21. The method according to claim 1, wherein the hydrogel obtained by polymerization is a shaped article.

22. A method according to claim 21, wherein the hydrogel is in the form of particulate beads having a diameter of 1 to 500 μm.

23. The method according to claim 1, wherein A is^x1Selected from the group consisting of activated carboxylic acids; cl- (C ═ O) -; NHS- (C ═ O) -, where NHS is N-hydroxysuccinimide; ClSO₂-；R¹(C ═ O) -; i-; br-; cl-; SCN-; and CN-,

wherein:

R¹selected from H, C_1-6Alkyl, alkenyl, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl.

24. The method according to claim 1 or 23, wherein a is^x2Selected from-maleimide, -SH, -NH₂、-SeH、-N₃、-C≡CH、-CR¹＝CR^1aR^1b、-OH、-(CH＝X⁰)–R¹、-(C＝O)–S–R¹、-(C＝O)-H、-NH-NH₂、-O-NH₂、-Ar–X⁰、-Ar–Sn(R¹)(R^1a)(R^1b)、-Ar–B(OH)(OH)，

Having an optional protecting group;

wherein:

X⁰is-OH, -NR¹R^1a-SH and-SeH,

ar is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl, and

R¹、R^1a、R^1bindependently of each other selected from H, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl.

25. The method according to claim 1, wherein A is^x2/A^y1Selected from the following:

wherein:

X⁰is-OH, -NR¹R^1a-SH and-SeH;

R¹、R^1a、R^1bindependently of each other selected from H, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 4-7 membered heterocyclyl, 8-11 membered heterobicyclic, phenyl, naphthyl, indenyl, indanyl and tetrahydronaphthyl; and is

Ar is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, and tetrahydronaphthyl.

26. The method according to claim 1, wherein A is^x2Is a thiol group, and A^y1Has formula (VIIa):

T–PG⁰–S-(VIIa)，

wherein:

t is H or a tag moiety;

PG⁰is a sulfur activating moiety; and is

S is sulfur.

27. The method according to claim 1, wherein D has a molecular weight of 2-500 kDa.

28. A carrier-linked prodrug obtainable by the process for the preparation of a carrier-linked prodrug according to claim 1.

29. A pharmaceutical composition comprising the carrier-linked prodrug of claim 28 or a pharmaceutical salt thereof and a pharmaceutically acceptable excipient.

30. A carrier-linked prodrug according to claim 28 or a pharmaceutical composition according to claim 29 for use as a medicament.