Method for preparing hydrogel microspheres

Abstract

The present invention relates to hydrogel hyaluronic acid (HA) microspheres or pharmaceutically acceptable salts thereof prepared via suspension polymerization. The hydrogel HA microspheres prepared according to the method of the invention can be used as carriers for various substances, such as carriers for various drug parts. The invention also provides a drug conjugate using the hydrogel HA microsphere as a carrier or a pharmaceutically acceptable salt thereof, a method for preparing the drug conjugate, a drug composition containing the drug conjugate, and applications of the drug conjugate and the pharmaceutically acceptable salt.

Description

[0001] The present invention relates to hydrogel hyaluronic acid (HA) microspheres or pharmaceutically acceptable salts thereof prepared via suspension polymerization. The hydrogel HA microspheres prepared according to the method of the present invention can be used as carriers for various substances, for example, carriers for various drug moieties. The present invention also provides a drug conjugate using the hydrogel HA microspheres as a carrier or a pharmaceutically acceptable salt thereof, a method of preparing the drug conjugate, a pharmaceutical composition comprising the drug conjugate, and uses thereof.

[0002] To improve physicochemical or pharmacokinetic properties such as in vivo circulation half-life of a drug, the drug can be coupled to a carrier such as a hydrogel. Typically, hydrogels in drug delivery are used for non-covalent complexation between the drug and the hydrogel, the drug can be embedded in the hydrogel, or covalently embedded via complexation of the drug to the hydrogel.

[0003] Non-covalent means require high efficiency of drug encapsulation to prevent uncontrolled, burst release of the drug upon administration due to disintegration of the drug-hydrogel complex. Limiting diffusion of unbound water-soluble drug molecules requires strong van der Waals interactions, which are often mediated via hydrophobic charged moieties for electrostatic binding. Many conformationally sensitive drugs such as proteins or peptides exhibit functional abnormalities during the complexation process of non-covalent binding drugs and / or during subsequent storage.

[0004] Alternatively, the drug can be covalently conjugated to the hydrogel via a linker moiety in which the linkage between the drug and the linker is stable or via a linker moiety in which the linkage between the drug and the linker moiety is reversible.

[0005] Conventional hydrogels are three-dimensional hydrophilic or amphiphilic polymer networks capable of absorbing large amounts of water. These networks can comprise various polymers which are insoluble due to the presence of covalent and / or physical crosslinking such as ionic, hydrophobic interactions or entanglements.

[0006] Various drug delivery systems comprising drugs covalently linked to hydrogels have been developed. For example, WO 2018 / 175788 Al teaches a hydrogel prodrug composition comprising a crosslinked hyaluronic acid (HA) conjugated to a drug-linker moiety. HA is a biocompatible hydrophilic polysaccharide consisting of D-glucuronic acid and N-acetyl-D-glucosamine. The prodrug can be used for the treatment of ocular diseases requiring repeated intravitreal injections of anti-vascular endothelial growth factor, in particular in the field of neovascular ocular diseases. Specifically, for hydrogel injection applications, it is important to improve the biostability of the hydrogel carrier as well as its injectability through a needle. WO 2018 / 193408 Al describes a drug delivery system consisting of a hyaluronic acid hydrogel linked to a drug moiety via a reversible linker. In this case, the bulk hydrogel can be made injectable via mechanical grinding.

[0007] There remains a need for new drug delivery systems suitable for sustained release of drugs.

[0008] This object is achieved by a process for preparing a hydrogel microsphere comprising crosslinked hyaluronic acid (HA), or a pharmaceutically acceptable salt thereof, wherein the process comprises the steps of:

[0009] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are each different functional moieties from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming a hydrogel HA microsphere;

[0010] (b) optionally, adding a pH adjusting agent to the emulsion of step (a); and

[0011] (c) collecting the hydrogel HA microsphere resulting from step (a) or (b).

[0012] Described herein is a process for preparing HA hydrogel microspheres, or a pharmaceutically acceptable salt thereof, that are readily injectable. Due to the relatively uniform size and shape of the hydrogel microspheres, injectability is improved. The HA hydrogel microspheres can be used as a carrier covalently conjugated to a drug moiety and can provide a valuable tool for local drug delivery.

[0013] In the meaning of the present invention, the terms are used as follows.

[0014] As used herein, the term "about" in combination with a numerical value is used to refer to a range of the stated value plus or minus no more than 20% of the stated value and including the endpoints, in some embodiments plus or minus no more than 15% of the stated value, in some embodiments plus or minus no more than 10% of the stated value, in some embodiments plus or minus no more than 5% of the stated value. For example, the phrase "about 200" is used to refer to a range of 200 + / - 20% and including the endpoints, i.e., a range of 160 to 240 and including the endpoints; in some embodiments, 200 + / - 15%, i.e., a range of 170 to 230 and including the endpoints; in some embodiments, a range of 200 + / - 10% and including the endpoints, i.e., a range of 180 to 220 and including the endpoints; in some embodiments, 200 + / - 5%, i.e., a range of 190 to 210 and including the endpoints.

[0015] As used herein, the term "polysaccharide" also referred to as "glycan", refers to a compound composed of monosaccharide moieties linked via glycosidic linkages. Typically, the term is used for compounds composed of a large number of monosaccharide moieties linked via glycosidic linkages, e.g. ten or more monosaccharide moieties.

[0016] As used herein, the term "glycosaminoglycan" or "mucopolysaccharide" refers to a linear polysaccharide composed of disaccharide units, wherein the repeating disaccharide units are composed of a uronic sugar and an amino sugar, with the exception of the sulfated glycosaminoglycan keratan sulfate, in which case a galactose unit replaces the uronic sugar. Examples of glycosaminoglycan molecules can include a hyaluronic acid, a heparin, a heparan sulfate, a heparosan, a chondroitin sulfate, a dermatan sulfate, or a keratan sulfate molecule.

[0017] As used herein, the term "functionalized hyaluronic acid" refers to any hyaluronic acid derivative that can result from a chemical or enzymatic functionalization or modification of native hyaluronic acid. In particular, the term refers to any hyaluronic acid derivative that can result from a chemical modification or functionalization at a carboxylic acid group.

[0018] As used herein, the term "microsphere" refers to a micrometer-sized particle, which is typically composed of a solid or semi-solid material, and which is generally spherical in shape. Typically, the average diameter of the microspheres of the present application ranges from about 1 μιη to about 1000 μιη, e.g. from about 10 μιη to about 500 μιη or e.g. from about 50 μιη to about 500 μιη, as determined by microscopy, e.g. flow microscopy or laser diffraction or any other suitable method.

[0019] As used herein, the term "dispersed phase" refers to a phase comprising particles or droplets of any size and of any nature distributed or dispersed in a continuous phase. The diameter of the droplets within the dispersed phase can range from about 1 μιη to about 5000 μιη, e.g. from about 10 μιη to about 1000 μιη or e.g. from about 50 μιη to about 500 μιη. In the emulsions of the present application, the average diameter of the droplets in the dispersed phase is typically in the range of 1 μιη to about 1000 μιη, e.g. from 10 μιη to about 500 μιη or e.g. from about 50 μιη to about 500 μιη.

[0020] As used herein, the term "continuous phase" or "continuous phase solution" refers to a fluid phase in which solid or fluid particles or droplets are distributed.

[0021] As used herein, the term "emulsion" refers to a fluid system in which droplets of one liquid are dispersed in another liquid, the one liquid being insoluble or immiscible in the other liquid. If the dispersed phase is an organic substance and the continuous phase is water or an aqueous solution, the emulsion is referred to as an oil-in-water (o / w) emulsion, if the dispersed phase is water or an aqueous solution and the continuous phase is an organic liquid, the emulsion is referred to as a water-in-oil (w / o) emulsion.

[0022] As used herein, the term "suspension polymerization" refers to a polymerization process in which a polymer, such as a hydrogel, is formed as droplets of monomer or monomer-solvent in a continuous phase that is a non-solvent for both the monomer and the polymer formed. As the monomer is converted to polymer, the droplets are converted to viscous, cohesive monomer and / or polymer particles that gradually become spherical solid polymer particles or microspheres. It is understood that, in the context of the present application, the previously mentioned monomer corresponds to the first and second functionalized HA, and the polymer formed corresponds to the HA hydrogel.

[0023] As used herein, the term "drug" refers to a substance used in the treatment, cure, prevention, or diagnosis of a disease or in the enhancement of physical or mental well-being. If a drug is conjugated to another moiety, the portion of the resulting product that is derived from the drug is referred to as a "drug moiety."

[0024] As used herein, the term "drug D-H moiety comprising a primary or secondary amine" refers to a drug moiety comprising at least one primary or secondary amine functional group, which drug can optionally have one or more additional functional groups, including one or more additional primary and / or secondary amine functional groups.

[0025] As used herein, the term "moiety" means a molecular portion that lacks one or more atoms as compared to the corresponding reagent. For example, if a reagent of the formula "H-X-H" is reacted 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 "-" representing a linkage to another moiety. Thus, a drug moiety is released from a reversible bond as a drug.

[0026] It is understood that, if a sequence or chemical structure of a set of atoms is provided that is attached to or interposed in a moiety, the sequence or chemical structure can be attached to the two moieties in either direction, unless otherwise explicitly stated. For example, a moiety "-C(O)N(R x )-" can be attached to two moieties as "-C(O)N(R x )-" or "-N(R x )C(O)-."

[0027] As used herein, the term "protecting group moiety" refers to a moiety that reversibly attaches to a functional group to render it unreactive with, for example, other functional groups. Suitable alcohol (-OH) protecting groups are, for example, acetyl, benzoyl, benzyl, beta-methoxyethoxymethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, trityl, trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyloxy methyl, triisopropylsilyl ether, methyl ether, and ethoxyethyl ether. Suitable carbonyl protecting groups are, for example, acetales and ketals, esters and dithianes. Suitable carboxylic acid protecting groups are, for example, methyl ester, benzyl ester, t-butyl ester, 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-t-butylphenol, silyl ester, ortho ester, and oxazoline. Suitable phosphonic acid protecting groups are, for example, 2-cyanoethyl and methyl.

[0028] As used herein, the term "amine protecting group moiety" refers to a moiety used to reversibly protect an amine functional group during the course of a chemical reaction so that the amine cannot react with, for example, other functional groups.

[0029] As used herein, the term "reducing agent" refers to a compound or element that loses or donates electrons to an electron acceptor (e.g., an oxidizing agent) in an oxidation-reduction chemical reaction.

[0030] As used herein, the term "oxidizing agent" refers to a chemical compound that is capable of oxidizing other compounds.

[0031] As used herein, the term "reagent" means a compound that contains at least one functional group for reaction with another compound or drug's functional group. It is understood that a drug containing a functional group is also a reagent.

[0032] It is recognized by those skilled in the art that the drug conjugate of the present application, or a pharmaceutically acceptable salt thereof, is a prodrug. As used herein, the term "prodrug" refers to a compound which, upon 1 -L 2 - a drug moiety that is reversibly and covalently bound to hyaluronic acid. The prodrug releases the reversibly and covalently bound drug moiety -D or D + In other words, a prodrug is a conjugate comprising a drug moiety that is reversibly and covalently bound to a polymer moiety via at least one -L 1 -L 2 - a drug moiety that is reversibly and covalently bound to hyaluronic acid. The prodrug releases the reversibly and covalently bound drug moiety -D or D

[0033] As used herein, the term "reversible linkage" or "biodegradable linkage" is a linkage that is cleavable in the absence of enzymes under physiological conditions (in aqueous buffer at pH 7.4 and 37°C) with a half-life of between 1 hour and six months, for example between 10 hours and four months, for example between one day and three months, between two days and two months or between three days and one month. However, it will be appreciated that reversible linkages can also be cleavable under other conditions, for example at different pH or at different temperatures, but the test used to determine reversibility is carried out under the above physiological conditions (aqueous buffer, pH 7.4, 37°C). Thus, a "stable linkage" is a linkage with a half-life of more than six months under physiological conditions.

[0034] As used herein, the term "C 1-4 Alkyl" means a straight-chain or branched-chain alkyl moiety having 1 to 4 carbon atoms. If present at the end of a molecule, straight-chain or branched-chain C 1-4 Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl and t-butyl. When two moieties of a molecule are linked by an alkyl group, then such C 1-4 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. C 1-4 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. C 1-4 Each hydrogen of an alkyl carbon can optionally be replaced by a substituent as defined below. Optionally, C 1-4 An alkyl group can be interrupted by one or more moieties as defined below.

[0035] As used herein, the term "C 1-6 Alkyl" means a straight-chain or branched-chain alkyl moiety having 1 to 6 carbon atoms. If present at the end of a molecule, straight-chain and branched-chain C 1-6 Examples of alkyl groups are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When two moieties of a molecule are linked by an alkyl group, then such C 1-6 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. C 1-6 Examples of alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. C 1-6 Each hydrogen of an alkyl carbon can optionally be replaced by a substituent as defined below. Optionally, C 1-6 An alkyl group can be interrupted by one or more moieties as defined below.

[0036] Accordingly, "C1-10 Alkyl", "C 1-20 Alkyl", "C 8-24 Alkyl" or "C 1-50 "Alkyl" means an alkyl chain having 1 to 10, 1 to 20, 8 to 24 or 1 to 50 carbon atoms, respectively, wherein C 1-10 、C 1-20 、C 8-24 or C 1-50 Each hydrogen atom of a carbon may optionally be replaced by a substituent as defined below. Optionally, C 1-10 Alkyl, C 1-20 Alkyl, C 8-24 Alkyl or C 1-50 The alkyl group may be interrupted by one or more moieties as defined below.

[0037] As used herein, the term "C 2-6 "Alkenyl" means a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. If present at the end of the molecule, examples are -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3 and -CH=CH-CH=CH2. When both parts of the molecule are composed of C 2-6 When alkenyl is connected, the C 2-6 An example of an alkenyl group is -CH=CH-. 2-6 Each hydrogen atom of the alkenyl moiety may be optionally replaced by a substituent as defined below. Optionally, C 2-6 An alkenyl group may be interrupted by one or more moieties as defined below.

[0038] Accordingly, the term "C 2-10 Alkenyl", "C 2-20 Alkenyl" or "C 2-50 "Alkenyl" means a straight or branched chain hydrocarbon moiety having 2 to 10, 2 to 20 or 2 to 50 carbon atoms, respectively, containing at least one carbon-carbon double bond. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 Each hydrogen atom of the alkenyl group may be optionally replaced by a substituent as defined below. 2-10 Alkenyl, C 2-20 Alkenyl or C 2-50 An alkenyl group may be interrupted by one or more moieties as defined below.

[0039] As used herein, the term "C 2-6"Alkynyl" means a straight or branched hydrocarbon moiety having 2 to 6 carbon atoms and containing at least one carbon-carbon triple bond. If present at the end of a molecule, examples are -C≡CH, -CH2-C≡CH, CH2-CH2-C≡CH and CH2-C≡C-CH3. When two parts of a molecule are linked by an alkynyl group, an example is -C≡C-. C 2-6 Each hydrogen atom of the alkynyl moiety may be optionally replaced by a substituent as defined below. Optionally, one or more double bonds may be present. Optionally, C 2-6 An alkynyl group may be interrupted by one or more moieties as defined below.

[0040] Accordingly, as used herein, the term "C 2-10 Alkynyl", "C 2-20 Alkynyl" and "C 2-50 "Alkynyl" means a straight or branched chain hydrocarbon moiety having 2 to 10, 2 to 20 or 2 to 50 carbon atoms, respectively, containing at least one carbon-carbon triple bond. 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 Each hydrogen atom of the alkynyl group may be optionally replaced by a substituent as defined below. Optionally, one or more double bonds may be present. Optionally, C 2-10 Alkynyl, C 2-20 Alkynyl or C 2-50 An alkynyl group may be interrupted by one or more moieties as defined below.

[0041] As mentioned above, C 1-4 Alkyl, C 1-6 Alkyl, C 1-10 Alkyl, C 1-20 Alkyl, C 1-50 Alkyl, C 8-24 Alkyl, C 2-6 Alkenyl, C 2-10 Alkenyl, C 2-20 Alkenyl, C 2-50 Alkenyl, C 2-6 Alkynyl, C 2-10 Alkynyl, C 2-20 Alkenyl or C 2-50 The alkynyl group may be optionally interrupted by one or more moieties which, in some embodiments, are selected from

[0042]

[0043] in

[0044] Dashed lines indicate connections to the rest of the moiety or reagent;

[0045] -R and -R aindependently selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl; and said moieties and linkages are optionally further substituted.

[0046] As used herein, the term "C 3-10 "Cycloalkyl" means a cyclic alkyl chain having 3 to 10 carbon atoms, which may be saturated or unsaturated, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. 3-10 Each hydrogen atom of the cycloalkyl group may be replaced by a substituent as defined below. 3-10 "Cycloalkyl" also includes bridged bicyclic rings, such as norbornane or norbornene.

[0047] As used herein, the term "8- to 30-membered carbon polycyclyl" or "8- to 30-membered carbon polycyclic ring" means a cyclic moiety of two or more rings having 8 to 30 ring atoms, wherein two adjacent rings share at least one ring atom, which may contain up to the maximum number of double bonds (fully saturated, partially saturated, or unsaturated aromatic or non-aromatic rings). In some embodiments, an 8- to 30-membered carbon polycyclyl means a cyclic moiety of two, three, four, or five rings. In some embodiments, an 8- to 30-membered carbon polycyclyl means a cyclic moiety of two, three, or four rings.

[0048] As used herein, the term "3- to 10-membered heterocyclyl" or "3- to 10-membered heterocycle" means a ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, which 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 to 4 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and wherein the ring is attached to the rest of the molecule via a carbon or nitrogen atom. Examples of 3- to 10-membered heterocycles include, but are not limited to, aziridine, oxirane, thiirane, aziridine, oxirane, thiirane, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazolin, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepane, The 3- to 10-membered heterocyclic group or each hydrogen atom of the 3- to 10-membered heterocyclic group may be replaced by a substituent as defined below.

[0049] As used herein, the term "8- to 11-membered heterobicyclyl" or "8- to 11-membered heterobicycle" means a heterocyclic moiety having two rings of 8 to 11 ring atoms, wherein at least one ring atom is shared by both rings, and which 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 to 6 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and wherein the ring is attached to the rest of the molecule via a carbon or nitrogen atom. Examples of 8- to 11-membered heterobicyclic rings are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, Purine and pteridine. The term 8- to 11-membered heterobicyclic ring also includes spirocyclic structures of two rings such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-aza-bicyclo[3.2.1]octane. Each hydrogen atom of an 8- to 11-membered heterobicyclic ring or an 8- to 11-membered heterobicyclic ring carbon atom may be replaced by a substituent as defined below.

[0050] Similarly, the term "8- to 30-membered heteropolycyclyl" or "8- to 30-membered heteropolycycle" means a heterocyclic moiety having more than two rings (in some embodiments, three, four, or five rings) of 8 to 30 ring atoms, wherein two adjacent rings share at least one ring atom, and which 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 to 10 ring atoms are replaced by heteroatoms selected from sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and wherein the ring is attached to the rest of the molecule via a carbon or nitrogen atom.

[0051] It is understandable that the structural part The expression "-R x / -R y The pairs, together with the atoms to which they are attached, form C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl or 8- to 11-membered heterobicyclyl" means -R x and -R y The following structure is formed:

[0052]

[0053] Where R is C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, or 8- to 11-membered heterobicyclyl.

[0054] It can also be understood that the structural part The expression "-R x / -R y The atoms to which they are attached, taken together to form a ring -A-"means -R x and -R y The following structure is formed:

[0055]

[0056] As used herein, the term "heteroaromatic N-containing moiety donating a π electron pair" refers to a moiety defined as follows: 1 -, the bond between the drug moiety and the corresponding DH is cleaved to produce the drug DH, wherein the drug moiety -D and, similarly, the corresponding DH comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine or ten) heteroaromatic nitrogen atoms that donate π electron pairs to the aromatic π system. Examples of chemical structures comprising such heteroaromatic nitrogen atoms that donate π electron pairs to the aromatic π system include, but are not limited to, pyrrole, pyrazole, imidazole, isoindazole, indole, indazole, purine, tetrazole, triazole and carbazole. For example, in the following imidazole ring, the heteroaromatic nitrogen that donates π electron pairs to the aromatic π system is marked with a "#":

[0057]

[0058] The heteroaromatic nitrogen atom that donates a π electron pair does not include a heteroaromatic nitrogen atom that donates only one electron (i.e., not a pair of π electrons) to the aromatic π system, such as the nitrogen marked with "§" in the above-mentioned imidazole ring structure. The drug DH can exist in one or more tautomeric forms, for example, in which one hydrogen atom moves between at least two heteroaromatic nitrogen atoms. In all such cases, the linker moiety is covalently and reversibly attached to the heteroaromatic nitrogen that donates a π electron pair to the aromatic π system.

[0059] As used herein, the term "excipient" refers to a diluent, adjuvant, or carrier with which a therapeutic agent, eg, a drug conjugate or pharmaceutical composition, is administered.

[0060] As used herein, the term "free form" of a drug refers to the unmodified pharmacologically active form of the drug, eg, after release from a conjugate.

[0061] As used herein, the term "functional group" means a group of atoms that can react with other groups of atoms. Exemplary functional groups are carboxylic acid, primary amine, secondary amine, tertiary amine, maleimide, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid, phosphonic acid, haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, sulfonamide, sulfuric acid, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine.

[0062] As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine. In some embodiments, the halogen is fluorine or chlorine.

[0063] As used herein, the term "insertion" means that the moiety is inserted between two carbon atoms or, if the insertion is at one end of the moiety, between a carbon or heteroatom and a hydrogen atom, in some embodiments between a carbon and a hydrogen atom.

[0064] As used herein, the term "pharmaceutically acceptable" means a substance that does not cause harm when administered to a patient, preferably means approved by regulatory agencies such as EMA (Europe) and / or FDA (US) and / or any other national regulatory agency for use in animals, preferably in humans.

[0065] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the compounds and is not biologically or otherwise undesirable. In some embodiments, the compounds are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camsylate, chloride / hydrochloride, chlorobenate, citrate, edisylate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, subacetate, tartrate, tosylate, and trifluoroacetate. Mineral acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, ammonium salts and the salts of elements from Groups I to XII of the Periodic Table of Elements. In some embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; basic ion exchange resins; and the like. Certain organic amines include isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine. Pharmaceutically acceptable salts can be synthesized from the parent compound that is basic or acidic in accordance with standard chemical procedures.

[0066] As used herein, the term "peptide" refers to a chain of at least 2 up to and including 50 monomeric parts (also referred to as "amino acid residues") of amino acids linked by peptide (amide) linkages. The amino acid monomers can be selected from proteinogenic and non-proteinogenic amino acids, and can be D-amino acids or L-amino acids. The term "peptide" also includes peptide mimetics, such as peptoids, beta-peptides, cyclic peptides, and depsipeptides, and encompasses such peptide mimetic chains having up to and including 50 monomeric parts.

[0067] As used herein, the term "protein" refers to a chain of more than 50 amino acid monomer portions (may also be referred to as "amino acid residues") linked via peptide linkages, wherein preferably no more than 12000 amino acid monomers are linked via peptide linkages, such as no more than 10000 amino acid monomer portions, no more than 8000 amino acid monomer portions, no more than 5000 amino acid monomer portions, or no more than 2000 amino acid monomer portions.

[0068] As used herein, the term "small molecule drug" refers to an organic compound drug having a molecular weight of less than 1000 Da, such as less than 900 Da or less than 800 Da. It is understood that a nucleobase-based drug moiety (such as an adenine or guanine analog) can also be a type of small molecule drug.

[0069] As used herein, the term "medium molecule drug" refers to a drug that is not a peptide and not a protein and is an organic compound having a molecular weight in the range of 1 kDa to 7.5 kDa, inclusive.

[0070] As used herein, the term "polymer" means a molecule comprising repeating structural units (i.e., monomers) linked by chemical bonds in linear, cyclic, branched, cross-linked, or dendritic fashion, or combinations thereof, which can be of synthetic or biological origin, or a combination of both. The monomers can be the same (in which case the polymer is a homopolymer), or they can be different (in which case the polymer is a heteropolymer). Heteropolymers can also be referred to as "copolymer," including, for example, alternating copolymers, in which different types of monomers alternate; periodic copolymers, in which monomers of different monomer types are arranged in repeating sequences; statistical copolymers, in which different types of monomers are arranged randomly; block copolymers, in which blocks of different homopolymers consisting of only one type of monomer are linked by covalent bonds; and gradient copolymers, in which the composition of different monomers gradually changes along the polymer chain. It is understood that a polymer can also comprise one or more other moieties, such as one or more functional groups. Likewise, it is understood that a peptide or protein is a polymer, even though the side chains of the individual amino acid residues can differ. It is understood that for covalently cross-linked polymers such as hydrogels, no meaningful range of molecular weights can be provided.

[0071] 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 hydrophobic interactions, hydrogen bonds, ionic interactions, and / or covalent chemical cross-linking. Cross-linking provides a network structure and physical integrity.

[0072] As used herein, the term "weight average molecular weight" or "M w " refers to the statistical average molecular weight of all molecules, taking into account the weight of each molecule in determining the contribution to the average molecular weight, in units of g / mol. The higher the molecular weight of a given molecule, the greater the contribution of that molecule to the M wThe weight average molecular weight can be calculated by techniques known in the art that are sensitive to molecular size, such as static light scattering, small angle neutron scattering, X-ray scattering, or sedimentation velocity.

[0073] As used herein, the term "spacer" or "spacer moiety" refers to a moiety suitable for linking two moieties.

[0074] As used herein, 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 termed a "substituent."

[0075] As used herein, the term "substituent" refers, in some embodiments, to a moiety selected from the group consisting of: halogen, -CN, -C(O)OR x1 、-OR x1 、-C(O)R x1 、-C(O)N(R x1 )(R x1a )、-S(O)2N(R x1 )(R x1a )、-S(O)N(R x1 )(R x1a )、-S(O)2R x1 、-S(O)R x1 、-N(R x1 )S(O)2N(R x1a )(R x1b ),-SR x1 、-N(R x1 )(R x1a )、-NO2、-OC(O)R x1 、-N(R x1 )C(O)R x1a 、-N(R x1 )S(O)2R x1a 、-N(R x1 )S(O)R x1a 、-N(R x1 )C(O)OR x1a 、-N(R x1 )C(O)N(R x1a )(R x1b )、-OC(O)N(R x1 )(R x1a ),-T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50Alkynyl is optionally substituted by one or more identical or different -R x2 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-、-S(O)2N(R x3 )-、-S(O)N(R x3 )-、-S(O)2-、-S(O)-、-N(R x3 )S(O)2N(R x3a )-、-S-、-N(R x3 )-、-OC(OR x3 )(R x3a )-、-N(R x3 )C(O)N(R x3a )-and-OC(O)N(R x3 )-;

[0076] -R x1 、-R x1a 、-R x1b Independently selected from: -H, -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R x2 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-、-S(O)2N(R x3 )-、-S(O)N(R x3 )-、-S(O)2-、-S(O)-、-N(R x3 )S(O)2N(R x3a )-、-S-、-N(R x3 )-、-OC(OR x3 )(R x3a )-、-N(R x3 )C(O)N(R x3a)-and -OC(O)N(R x3 )-;

[0077] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R x2 groups, which are the same or different;

[0078] each -R x3 is independently selected from: halo, -CN, oxo (=0), -C(O)OR x3a , -OR x4 , -C(O)R x4a , -C(O)N(R x4b )(R 1-6 ), -S(O)2N(R 1-6 )(R x1 ), -S(O)N(R x1 )(R x1 ), -S(O)2R x1 , -S(O)R x1a , -N(R x1 )S(O)2N(R x1a )(R x1 ), -SR x1a , -N(R x1 )(R x1 ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1 , -N(R x1a )S(O)R x1 , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1a )(R x1 ), -OC(O)N(R x1a )(R x1 ), and C x1a alkyl; wherein C x1 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0079] each -R x1 , -R x1a , -R x1 , -R x1a , -Rx4b independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which can be the same or different.

[0080] In some embodiments, the term "substituent" refers to a moiety selected from halogen, -CN, -C(O)OR x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 )(R x1a ), -S(O)2N(R x1 )(R x1a ), -S(O)N(R x1 )(R x1a ), -S(O)2R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1 )(R x1a ), -SR x1 , -N(R x1 )(R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(R x1 )C(O)OR x1a , -N(R x1 )C(O)N(R x1 )(R x1a ), -OC(O)N(R x1 )(R x1a ), -T 0 , C 1-10 alkyl, C 2-10 alkenyl and C 2-10 alkynyl; wherein -T 0 , C 1-10 alkyl, C 2-10 alkenyl and C 2-10 alkynyl are optionally substituted with one or more -R x2 , which can be the same or different, and wherein C 1-10 alkyl, C 2-10 alkenyl and C 2-10 alkynyl are optionally interrupted with one or more groups selected from -T 0-, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-、-S(O)2N(R x3 )-、-S(O)N(R x3 )-、-S(O)2-、-S(O)-、-N(R x3 )S(O)2N(R x3a )-、-S-、-N(R x3 )-、-OC(OR x3 )(R x3a )-、-N(R x3 )C(O)N(R x3a )-and-OC(O)N(R x3 )-;

[0081] Each-R x1 、-R x1a 、-R x1b 、-R x3 、-R x3a Independently selected from: -H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl;

[0082] Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R x2 replace;

[0083] Each-R x2 Independently selected from: halogen, -CN, oxo (=O), -C(O)OR x4 、-OR x4 、-C(O)R x4 、-C(O)N(R x4 )(R x4a )、-S(O)2N(R x4 )(R x4a )、-S(O)N(R x4 )(R x4a )、-S(O)2R x4 、-S(O)R x4 、-N(R x4 )S(O)2N(R x4a )(R x4b ),-SR x4 、-N(R x4 )(R x4a, -NO2, -OC(O)R x4 , -N(R x4 )C(O)R x4a , -N(R x4 )S(O)2R x4a , -N(R x4 )S(O)R x4a , -N(R x4 )C(O)OR x4a , -N(R x4 )C(O)N(R x4a )(R x4b ), -OC(O)N(R x4 )(R x4a ), and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0084] each -R x4 , -R x4a , -R x4b is independently selected from the group consisting of -H, halogen, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl.

[0085] In some embodiments, the term "substituent" refers to a moiety selected from the group consisting of halogen, -CN, -C(O)OR x1 , -OR x1 , -C(O)R x1 , -C(O)N(R x1 )(R x1a ), -S(O)2N(R x1 )(R x1a ), -S(O)N(R x1 )(R x1a ), -S(O)2R x1 , -S(O)R x1 , -N(R x1 )S(O)2N(R x1a )(R x1b ), -SR x1 , -N(R x1 )(R x1a ), -NO2, -OC(O)R x1 , -N(R x1 )C(O)R x1a , -N(R x1 )S(O)2R x1a , -N(R x1 )S(O)R x1a , -N(Rx1 )C(O)OR x1a 、-N(R x1 )C(O)N(R x1a )(R x1b )、-OC(O)N(R x1 )(R x1a ),-T 0 、C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl; wherein -T 0 、C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl is optionally substituted by one or more identical or different -R x2 substituted, and wherein C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R x3 )-、-S(O)2N(R x3 )-、-S(O)N(R x3 )-、-S(O)2-、-S(O)-、-N(R x3 )S(O)2N(R x3a )-、-S-、-N(R x3 )-、-OC(OR x3 )(R x3a )-、-N(R x3 )C(O)N(R x3a )-and-OC(O)N(R x3 )-;

[0086] Each-R x1 、-R x1a 、-R x1b 、-R x2 、-R x3 、-R x3a Independently selected from: -H, halogen, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl;

[0087] Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R x2Substituted.

[0088] In some embodiments, up to 6 -H atoms in an optionally substituted molecule are independently replaced with a substituent, for example 5 -H atoms are independently replaced with a substituent, 4 -H atoms are independently replaced with a substituent, 3 -H atoms are independently replaced with a substituent, 2 -H atoms are independently replaced with a substituent, or 1 -H atom is replaced with a substituent.

[0089] As used herein, the term "therapeutically effective amount" means an amount that is sufficient to cure, alleviate, or partially arrest the clinical manifestations of a given disease and its complications. The effective amount for a given purpose will depend on the severity of the disease and the weight and general state of the individual.

[0090] As used herein, the term "administered via injection" or "injectability" refers to a combination of factors such as the force applied to the plunger of a syringe containing a drug conjugate described herein swelled in a liquid at a certain concentration (w / v) and at a certain temperature, the needle having a given internal diameter connected to the outlet of the syringe, and the time required to express a certain volume of the drug conjugate through the needle from the syringe.

[0091] As used herein, the term "individual" refers to an animal. Typically, the animal is a mammal. The term "individual" also refers to, for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, and the like. In some embodiments, the individual is a primate. In some embodiments, the individual is a human. In some embodiments, the individual is a non-human primate.

[0092] As used herein, an individual "is in need of" a treatment if the individual would benefit biologically, medically, or in quality of life as a result of the treatment.

[0093] As used herein, the term "water insoluble" means less than 1 g of a compound is soluble in 1 liter of water at 20 °C to form a uniform solution. Thus, the term "water soluble" means 1 g or more of a compound is soluble in 1 liter of water at 20 °C to form a uniform solution.

[0094] As used herein, the term "buffer" or "buffering agent" refers to a chemical compound that maintains the pH of a solution within a desired range.

[0095] As used herein, the term "emulsifier" refers to a chemical compound that absorbs at the newly formed interface between the dispersed phase and the continuous phase solution during emulsion formation, allowing the solutions to mix, and protects the newly formed droplets from immediate re-aggregation, e.g., a surface active ingredient.

[0096] As used herein, the term "pH adjusting agent" refers to a chemical compound used to change the pH of the droplets within an emulsion, eg, the emulsion of step (a), and to initiate and / or promote the cross-linking reaction between the first and second functionalized HAs.

[0097] As used herein, the term "blocking reagent" refers to a chemical compound used to block unreacted functional groups (eg, -FG1 or -FG2).

[0098] As used herein, the term "flow system" or "continuous flow system" refers to a system in which a process, such as polymer precipitation, is run in a continuous flow stream rather than in batch production.

[0099] As used herein, the term "device for precipitation and separation of polymers" refers to a configuration or apparatus comprising a flow system connected to a collection member.

[0100] As used herein, the term "antisolvent" refers to a solvent in which a polymer (e.g., functionalized HA) is insoluble. The term "insoluble" with respect to a polymer means that less than 1 g of the polymer can be dissolved in 1 liter of the solvent to form a homogeneous solution at room temperature (room temperature can range from 17° C. to 30° C., e.g., 17° C. to 25° C.).

[0101] As used herein, "screen vortex centrifuge" refers to a filter centrifuge that separates solids and liquids from a solid-liquid mixture. In a typical screen vortex centrifuge, the basic principle is to separate the incoming material into two product forms, liquid and solid.

[0102] Generally, the term "comprising" also covers "consisting of.

[0103] The present invention relates to a method for preparing hydrogel microspheres comprising cross-linked hyaluronic acid (HA) or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps:

[0104] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are functional group moieties different from each other, wherein -FG1 on the first functionalized HA is different from the second functionalized HA

[0105] The -FG2 on the surface reacts to form multiple crosslinks, thereby forming hydrogel HA microspheres;

[0106] (b) optionally, adding a pH adjuster to the emulsion of step (a); and

[0107] (c) collecting the hydrogel HA microspheres obtained in step (a) or (b).

[0108] Those skilled in the art will recognize that the inventive process for preparing hydrogel microspheres comprising crosslinked HA or a pharmaceutically acceptable salt thereof can be applied to the preparation of any hydrogel microspheres comprising any crosslinked glycosaminoglycan. In other words, the inventive process can equally be applied to the preparation of hydrogel microspheres comprising crosslinked heparin, heparan sulfate, heparosan, chondroitin sulfate, dermatan sulfate, or keratan sulfate. In particular, the process can be applied to the preparation of hydrogel microspheres comprising crosslinked heparosan.

[0109] The polymerization within the inventive process occurs via suspension polymerization. In step (a), solutions A and B form an emulsion, after a sufficient mixing time has been allowed to proceed, solution A becomes the dispersed phase and solution B becomes the continuous phase. The dispersed phase should be immiscible with the continuous phase. Moreover, in step (a), both functionalized HAs are predominantly or entirely straight HA strands.

[0110] In step (a), the mixing of solutions A and B to form an emulsion can require vigorous agitation, pressure, or other forces, which can be achieved by agitation, for example by agitation with a pitched blade stirrer in combination with a baffle; by shaking, for example by shaking in a container such as a Falcon tube (a closed tube); by using a rotor or stator; by using an ultrasonic device; by using a static mixer, for example by using a packed bead column or a flow disc; by using a membrane with defined pores, for example by using cross-flow membrane emulsification technology in which a liquid form of the material to be formed into microparticles is pushed through a membrane comprising micron-sized pores into a flowing solution of the dispersed phase or into a stirred cell membrane emulsion; by using a tubular membrane with a surface made of a hydrophobic plastic, for example as disclosed in WO 2022 / 198052 A2, which is incorporated herein by reference in its entirety; by using a microfluidic droplet generator; or by spray polymerization in air, for example by using an ultrasonic nebulizer. Suitably, the mixing in step (a) is achieved by agitation, for example by agitation with a pitched blade stirrer in combination with a baffle or by using a microfluidic droplet generator.

[0111] In some embodiments, the inventive process comprises the following steps:

[0112] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are different functional moieties from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres;

[0113] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are different functional moieties from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres;

[0114] (b) adding a pH adjusting agent to the emulsion of step (a); and

[0115] (c) collecting the hydrogel HA microspheres resulting from step (a) or (b).

[0116] In some embodiments, all -FG1 are the same, and all -FG2 are the same.

[0117] Suitably, the first and second functionalized HA of the method of the application are not optionally modified with other functional groups.

[0118] The method of the application optionally further comprises a step of size fractionating the hydrogel microspheres resulting from step (c) to obtain microspheres having a particular particle size distribution.

[0119] Furthermore, the skilled person will recognize that throughout the specification there are optional washing or purification steps between any of the steps of the method of the application.

[0120] In some embodiments, the method of the application comprises the following steps:

[0121] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are different functional moieties from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres;

[0122]

[0123] (b) optionally, adding a pH adjusting agent to the emulsion of step (a);

[0124] (c) collecting the hydrogel HA microspheres resulting from step (a) or (b);

[0125] (d) optionally, size fractionating the hydrogel HA microspheres resulting from step (a), (b) or (c) to obtain microspheres having a particular particle size distribution;

[0126] (e) optionally, washing the microspheres obtained in step (a), (b), (c) or (d);

[0127] (f) optionally, incubating the hydrogel HA microspheres of step (a),

[0128] (b), (c), (d) or (e) in a buffer at a pH ranging from about 7 to about 14, for example from about 8 to about 12, or for example from about 8.5 to about 10;

[0129] ​(g) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), I or (f) with a reducing agent;

[0130] (h) optionally, washing the microspheres obtained in step (f) or (g); and

[0131] (i) optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g) or (h).

[0132] It is understood that step (d) can also be performed after steps (e), (f), (g) or (h).

[0133] In some embodiments, the first and second functionalized HA have a molecular weight independently in the range of about 80 kDa to about 250 kDa, for example about 90 kDa to about 200 kDa or for example about 100 kDa to about 150 kDa. Suitably, the first and second functionalized HA have a molecular weight in the range of 100 kDa to 150 kDa. It is understood that the HA can be polydisperse, comprising polymer chains of varying lengths, and thus the molecular weight is not a single value, i.e., the HA exists as a distribution of chain lengths and molecular weights. For example, if the first or second functionalized HA has a molecular weight of 125 kDa, the HA will comprise polymeric HA strands in the range of about 30 kDa to about 400 kDa.

[0134] In some embodiments, in step (d), the resulting microspheres have a diameter of about 1 μm to about 1000 μm, for example about 50 μm to about 900 μm, for example about 100 μm to about 700 μm, for example about 200 μm to about 500 μm, or for example about 50 μm to about 500 μm, as determined by flow microscopy or other similar methods known in the art. In some embodiments, in step (d), the resulting microspheres have a diameter of 1 μm to 1000 μm, for example 50 μm to 900 μm, for example 100 μm to 700 μm, for example 200 μm to 500 μm, or for example 50 μm to 500 μm, as determined by flow microscopy. In some embodiments, in step (d), the resulting microspheres have a diameter of 50 μm to 500 μm, as determined by flow microscopy. In some embodiments, in step (d), the resulting microspheres have a diameter of 100 μm to 200 μm, as determined by flow microscopy.

[0135] Suitably, in step (d), the resulting microspheres have a d 10 value of < 900 μm, for example a d 90 value of > 100 μm and a d 10 value of < 700 μm, for example a d 90 value of < 900 μm, for example a d 10Value and d≤500μm 90 In some embodiments, in step (d), the resulting microspheres have a d value of ≥ 100 μm. 10 Value and d≤200μm 90 Preferably, for these assays, the microspheres are in succinate buffer.

[0136] As used herein, the parameter d 10 The value represents the point of the size distribution below which 10% of the total volume of material in the sample is contained. Similarly, d 90 The value is the size below which 90% of the volume of the material is contained. It will be appreciated that the expansion of the HA microspheres may be affected by the buffer and / or pH, osmotic pressure and ionic strength in which the microspheres are stored during the assay, and this may therefore have an impact on the d 10 and d 90 Value affects.

[0137] In some embodiments, in step (f), the pH of the buffer is in the range of 8 to 12. In some embodiments, in step (f), the pH of the buffer is in the range of 8.5 to 10. In some embodiments, in step (f), the pH of the buffer is about 9. In some embodiments, in step (f), the pH of the buffer is 9.

[0138] In some embodiments, the methods of the present invention comprise the following steps:

[0139] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are functional group moieties different from each other, and wherein -FG1 on the first functionalized HA is different from -FG1 on the second functionalized HA.

[0140] The -FG2 on the surface reacts to form multiple crosslinks, thereby forming hydrogel HA microspheres;

[0141] (b) adding a pH adjuster to the emulsion of step (a);

[0142] (c) collecting the hydrogel HA microspheres obtained in step (b);

[0143] (d) size fractionating the hydrogel HA microspheres obtained in step (c) to obtain microspheres with a specific particle size distribution;

[0144] (e) optionally, washing the microspheres obtained in step (c) or (d);

[0145] (f) incubating the hydrogel HA microspheres of step (c), (d) or (e) in a buffer having a pH in the range of about 7 to about 14, such as about 8 to about 12, such as about 8.5 to 10;

[0146] (g) incubating the hydrogel HA microspheres of step (c), (d), (e) or (f) with a reducing agent;

[0147] (h) washing the microspheres obtained in step (f) or (g); and

[0148] (i) collecting the hydrogel HA microspheres of step (d), (e), (f), (g) or (h).

[0149] The present application also relates to hydrogel HA microspheres or pharmaceutically acceptable salts thereof obtained by or obtainable by any of the processes of the present application.

[0150] Surprisingly, it was found that the process of the present application allows the use of linear functionalized HA strands with a low degree of substitution in the synthesis of effectively crosslinked hydrogel HA microspheres. It was also observed that due to the size of the microspheres, the injectability of the HA microspheres and of the drug conjugates comprising the hydrogel HA microspheres is significantly improved compared to the injectability of the coacervate gels, such as the coacervate gels disclosed in WO 2018 / 175788 Al. Another advantage of using HA hydrogel microspheres as carriers for drug conjugates is that the microspheres can be washed prior to administration, thus soluble by-products can be easily removed. On the other hand, the coacervate gels disclosed in WO 2018 / 175788 Al cannot be subjected to a washing step, as the gel is obtained by polymerization in a syringe, which is then directly injected into the eye. Furthermore, the coacervate gels disclosed in WO 2018 / 175788 Al are prone to hardening when filled in a syringe, making the process of filling the syringe more challenging. In contrast, the HA hydrogel microspheres of the present application can be stored after synthesis and subsequently easily filled into the final container at a later point in time or further conjugated to the drug moiety in the final container.

[0151] Suitably, solution A of step (a) comprises the first and second functionalized HA and a solvent in which the functionalized HA is soluble.

[0152] In some embodiments, solution A of step (a) comprises the first and second functionalized HA and dimethyl sulfoxide, DMF, DMA or a mixture thereof. In some embodiments, solution A of step (a) comprises the first and second functionalized HA and dimethyl sulfoxide.

[0153] In some embodiments, solution A of step (a) comprises the first and second functionalized HA, dimethyl sulfoxide and water. In some embodiments, solution A of step (a) comprises the first and second functionalized HA, dimethyl sulfoxide and a buffer.

[0154] In some embodiments, solution A of step (a) is an aqueous solution and comprises the first and second functionalized HA and a buffer.

[0155] It will be clear to one skilled in the art that the phrase "buffer" can refer to one buffer or a mixture of two or more buffers.

[0156] Exemplary buffers can be selected from the group consisting of: N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), acetate, 2,2',2"-nitrilotriacetic acid (ADA), adipate, alanine, ammonium, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), arginine, ascorbate, aspartic acid, benzoate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bicarbonate, N,N-bis(2-hydroxyethyl)glycine, bis-(2-hydroxy-ethyl)-amino-tris(hydroxymethyl) 1-Hydroxy-2-aminopropanesulfonic acid (CABS), 1,3-bis(tris(hydroxymethyl)methylamino)propane, borate, 4-(cyclohexylamino)butane-1-sulfonic acid (CABS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), carbonate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), citrate, diethanolamine, 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), edetate, ethanolamine, ethylenediamine, formate, fumarate, gluconate, glutamate, glycine, glycylglycine, guanidine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS) ), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (HEPPS), N-(2-hydroxyethyl)piperazine-N'-(propanesulfonic acid) (HEPPSO), histidine, hydrazine, imidazole, lactate, lysine, malate, maleate, 2-(N-morpholino)ethanesulfonic acid (MES), metaphosphate, methylamine, 4-(4-morpholino)butanesulfonic acid (MOBS), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), pentetate, phosphate, piperazine-N,N'-bis(2-ethanesulfonic acid) )(PIPES), piperazine, piperidine, piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), propionate, pyridine, pyrophosphate, pyruvate, sorbate, succinate, N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid (TABS), ([tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), tartrate, taurine, 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid (TES), tris(hydroxymethyl)methylglycine (tricine), triethanolamine, tromethamine, and α-ketoglutarate.

[0157] The skilled person will appreciate that the corresponding conjugated acids, bases or salts of the buffers and mixtures thereof are also included.

[0158] In some embodiments, the aqueous solution comprises the first and second functionalized HA and a buffer, e.g., a buffer selected from the group consisting of citrate and histidine or mixtures thereof. In some embodiments, the buffer comprises a mixture of citrate and histidine. In some embodiments, the buffer consists of a mixture of citrate and histidine.

[0159] As defined herein, the term "histidine" is intended to encompass both D-histidine and L-histidine and mixtures thereof. In some embodiments, the term "histidine" refers to L-histidine. In some embodiments, the term "histidine" refers to D-histidine. In some embodiments, the term "histidine" refers to a mixture of L-histidine and D-histidine. In some embodiments, the buffer is L-histidine.

[0160] In some embodiments, the buffer consists of a mixture of citrate, L-histidine and sodium chloride.

[0161] In some embodiments, the aqueous solution comprises the first and second functionalized HA, citrate and sodium chloride.

[0162] In some embodiments, the aqueous solution consists of the first and second functionalized HA, citrate and sodium chloride. In some embodiments, the aqueous solution comprises the first and second functionalized HA, histidine and sodium chloride.

[0163] In some embodiments, the aqueous solution comprises the first and second functionalized HA and an emulsifier, and solution B comprises a solvent.

[0164] The buffer can generally be added in an amount of about 0.01 mM to about 500 mM. In some embodiments, the concentration of the buffer is in the range of about 0.5 mM to about 350 mM. In some embodiments, the concentration of the buffer is in the range of about 1 mM to about 250 mM. In some embodiments, the concentration of the buffer is in the range of about 5 mM to 100 mM. In some embodiments, the concentration of the buffer is about 100 mM. In some embodiments, the concentration of the buffer is 100 mM. In some embodiments, the concentration of the buffer is about 5 mM. In some embodiments, the concentration of the buffer is 5 mM.

[0165] Suitably, solution B of step (a) comprises an emulsifier and a solvent.

[0166] The skilled person will appreciate that generally, the phrase "solvent" can refer to one solvent or a mixture of two or more solvents, and the phrase "emulsifier" can refer to one emulsifier or a mixture of two or more emulsifiers.

[0167] Exemplary emulsifiers may be selected from: sorbitan esters, such as sorbitan monolaurate ( 20), sorbitan monooleate ( 80), sorbitan monopalmitate ( 40), Sorbitan monostearate ( 60), Sorbitan sesquioleate ( 83), sorbitan trioleate ( 85) or sorbitan tristearate ( 65); PEG-30 dipolyhydroxystearate (Cithrol TM DPHS); polyglyceryl-3 diisostearate; a mixture of sorbitan oleate and a copolyester of hydroxystearic acid and ethylene glycol (Hypermer TM 1083); Polyoxyethylene sorbitan monooleate (polysorbate 80, 80 and 80R); alcohols such as propanol, butanol, pentanol, hexanol, heptanol or octanol; alkyl and aryl amine salts such as primary, quaternary, secondary or tertiary amine salts; alkyl dimethyl betaine; alkyl ethoxylate sulfate; alkyl phenyl polyoxyethylene ethers such as octylphenol polyether 9, Triton X-100, Igepal TMor Nonidet P40; alkylphosphates, such as monoalkylphosphates or dialkylphosphates; alkylpolyoxyethylenethers, such as lauryl polyether-4, lauryl polyether-9, lauryl polyether-23, cetyl polyether-2, cetyl polyether-10, cetyl polyether-20, cetylstearyl polyether-6, cetylstearyl polyether-20, cetylstearyl polyether-25, stearyl polyether-2, stearyl polyether-10, stearyl polyether-20, oleyl polyether-2, oleyl polyether-10, oleyl polyether-20, decyl polyether-10, or tridecyl polyether-10; alkylsulfates, such as sodium dodecyl sulfate (SDS); alkylxanthates; cholate salts, such as sodium cholate or sodium deoxycholate; cationic lipids, such as hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, dioctadecyldimethylammonium bromide, dioctadecyldimethylammonium chloride, 1,2-diacyl-3-trimethylammoniumpropane, 1,2-diacyl-3-dimethylammoniumpropane, [2,3-bis(octadecyloxy)propyl]trimethylammonium chloride, or [N-(N-dimethylaminoethane)-carbamoyl]cholesterol, dioleoyl); dialkylsulfosuccinates, such as Aerosol OT; ethylenediaminetetra(ethoxylate-block-propoxylate)tetrol, such as Tetronic 304, Tetronic 904, Tetronic 90R4, or Tetronic 1304; fatty acids, such as palmitic acid, oleic acid, lauric acid, myristic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, palmitoleic acid, linoleic acid, linolenic acid, or arachidonic acid, and salts thereof, such as sodium or potassium salts; glycosides, such as octyl glucoside or dodecyl maltoside; linear and branched alkylbenzenesulfonates; poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), such as poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188 F68), poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407, poloxamer 105 benzoate, or poloxamer 182 dibenzoate; polyoxyethylene sorbitan esters, such as polyoxyethylene (40)-sorbitan hexaoleate, polyoxyethylene sorbitan monolaurate (polysorbate 20, 20 and 21), polyoxyethylene sorbitan monopalmitate (polysorbate 40, 40), polyoxyethylene sorbitan monostearate (polysorbate 60, 60 and 61), polyoxyethylene sorbitan trioleate (polysorbate 85, 85) or polyoxyethylene sorbitan tristearate (polysorbate 65, 65); polyvinyl alcohol; polyvinyl pyrrolidone; starch and its derivatives and mixtures thereof.

[0168] In some embodiments, the emulsifier is selected from the group consisting of: sorbitan monolaurate ( 20), sorbitan monooleate ( 80), sorbitan monopalmitate ( 40), Sorbitan monostearate ( 60), Sorbitan sesquioleate ( 83), sorbitan trioleate ( 85) and sorbitan tristearate ( 65).

[0169] In some embodiments, the emulsifier is selected from the group consisting of: sorbitan monooleate ( 80), PEG-30 dipolyhydroxystearate (Cithrol TM DPHS), polyglyceryl-3 diisostearate, and a mixture of a copolyester of sorbitan oleate, hydroxystearic acid and ethylene glycol (Hypermer TM 1083).

[0170] In some embodiments, the emulsifier is selected from the group consisting of sorbitan monooleate, PEG-30 dipolyhydroxystearate, polyglyceryl-3 diisostearate, and a mixture of sorbitan monooleate with a copolyester of hydroxystearic acid and ethylene glycol.

[0171] In some embodiments, the emulsifier is PEG-30 dipolyhydroxystearate. In some embodiments, the emulsifier is polyglyceryl-3 diisostearate. In some embodiments, the emulsifier is a mixture of sorbitan oleate and a copolyester of hydroxystearic acid and ethylene glycol.

[0172] Advantageously, the emulsifier is sorbitan monooleate.

[0173] The emulsifier can be added in an amount of from about 0.01% (w / w) to about 15% (w / w). In some embodiments, the emulsifier is added in an amount of from about 0.01% (w / w) to about 10% (w / w). In some embodiments, the emulsifier is added in an amount of from about 0.1% (w / w) to about 7% (w / w). In some embodiments, the emulsifier is added in an amount of from about 1% (w / w) to about 5% (w / w). In some embodiments, the emulsifier is added in an amount of from about 1.5% (w / w) to about 3.0% (w / w).

[0174] In some embodiments, the emulsifier is added in an amount of about 1.5% (w / w). In some embodiments, the emulsifier is added in an amount of 1.5% (w / w). In some embodiments, the emulsifier is added in an amount of about 0.5% (w / w). In some embodiments, the emulsifier is added in an amount of 0.5% (w / w). In some embodiments, the emulsifier is added in an amount of about 0.25% (w / w). In some embodiments, the emulsifier is added in an amount of 0.25% (w / w).

[0175] In some embodiments, the solvent can be any solvent that is not miscible with the dispersed phase.

[0176] In some embodiments, the solvent is selected from the group consisting of polar solvents, non-polar solvents, fluorocarbons, and ionic liquids.

[0177] In some embodiments, the solvent is selected from the group consisting of: hydrocarbons, such as 3-carene, benzene, camphene, cycloheptane, cyclohexane, decane, dodecane, ethylbenzene, hemellitene, heptane, hexane, isodurene, limonene, mesitylene, m-xylene, n-butylbenzene, n-propylbenzene, nonane, octane, o-xylene, p-cymene, pentadecane, pentane, pinane, pinene, p-menthane, prehnitene, pseudo-camphene, p-xylene, styrene, tetradecane, toluene, tridecane, and undecane; siloxanes, such as cyclomethicone, decamethylcyclopentasiloxane, hexamethyldisiloxane, octamethyltrisiloxane, or liquid polysiloxane (silicone oil), and esters, such as acetyltributylcitrate, castor oil, ethyl laurate, glycerol trioleate, liquid glycerol triester, triacetin, glycerol tributyrate, and triethyl citrate.

[0178] In some embodiments, the solvent is selected from the group consisting of: 3-carene, benzene, camphene, cycloheptane, cyclohexane, decane, dodecane, ethylbenzene, hemellitene, heptane, hexane, isodurene, limonene, mesitylene, m-xylene, n-butylbenzene, n-propylbenzene, nonane, octane, o-xylene, p-cymene, pentadecane, pentane, pinane, pinene, p-menthane, prehnitene, pseudo-camphene, p-xylene, styrene, tetradecane, toluene, tridecane, and undecane.

[0179] In some embodiments, the solvent is selected from the group consisting of acetyltributyl citrate, castor oil, ethyl laurate, glyceryl trioleate, liquid triglyceride, triacetin, glycerol tributyrate, and triethyl citrate.

[0180] In some embodiments, the solvent is heptane or tetradecane. In some embodiments, the solvent is heptane. In some embodiments, the solvent is tetradecane.

[0181] In some embodiments, solution B of step (a) comprises sorbitan monooleate and heptane. In some embodiments, solution B of step (a) consists of sorbitan monooleate and heptane. In some embodiments, solution B of step (a) comprises PEG-30 dipolyhydroxystearate and heptane.

[0182] In some embodiments, solution B of step (a) comprises sorbitan monooleate and tetradecane. In some embodiments, solution B of step (a) consists of sorbitan monooleate and tetradecane. In some embodiments, solution B of step (a) comprises PEG-30 dipolyhydroxystearate and tetradecane.

[0183] In some embodiments, solution B of step (a) comprises Hypermer TM 1083 and heptane. In some embodiments, solution B of step (a) comprises a mixture of sorbitan oleate and a copolyester of hydroxystearic acid and ethylene glycol and heptane.

[0184] In some embodiments, solution B of step (a) comprises polyglyceryl-3 diisostearate and heptane.

[0185] Step (a) can be carried out at a temperature of from about 0 °C to about 150 °C, for example from about 4 °C to about 80 °C, for a sufficient time, for example at least about 10 seconds to at least about 12 hours, for example at least 30 minutes to at least about 12 hours, to allow the functionalised HA to react.

[0186] In some embodiments, the emulsion of step (a) is left at room temperature for about 5 minutes.

[0187] In some embodiments, the emulsion of step (a) is left at room temperature for about 12 hours. It will be appreciated that room temperature can range from 17 °C to 30 °C, for example from 17 °C to 25 °C.

[0188] Suitably, the pH adjusting agent initiates and / or accelerates the cross-linking reaction between the first and second functionalised HAs.

[0189] The pH adjusting agent can be soluble in both solution A and solution B of step (a). This provides the advantage of using controlled reaction conditions to synthesise the hydrogel HA microspheres of the application.

[0190] The pH adjusting agent can be an acid or a base.

[0191] In some embodiments, the base is a non-protic non-nucleophilic amine that is soluble in the dispersed phase and the continuous phase.

[0192] Exemplary bases can be 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, 1,1,4,7,10,10-hexamethyltriethylenetriamine, 1,4,7-trimethyl-1,4,7-triazacyclononane, tris[2-(dimethylamino)ethyl]amine, triethylamine, diisopropylethylamine (DIPEA), trimethylamine, N,N- dimethylethanamine, N,N,N',N'-tetramethyl-1,6-hexanediamine, N,N,N',N",N"- pentamethyldiethylenetriamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5- diazabicyclo[4.3.0]non-5-ene, and hexamethylenetetramine.

[0193] In some embodiments, 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, 1,1,4,7,10,10-hexamethyltriethylenetriamine, 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.

[0194] In some embodiments, the base is N,N,N',N'-tetramethylethylenediamine (TMEDA).

[0195] In some embodiments, prior to step (c), the suspension of step (b) is diluted with a solution comprising a buffering agent, for example a buffering agent having a pH less than or equal to 4. In some embodiments, the solution comprises succinic acid, sodium sulfate, and ethylenediaminetetraacetic acid (EDTA). In some embodiments, the solution comprises succinic acid, ethylenediaminetetraacetic acid (EDTA), and isopropyl alcohol. In some embodiments, the solution comprises sodium chloride.

[0196] In some embodiments, in step (b), the pH adjusting agent is added to the emulsion of step (a), followed by incubation at a temperature ranging from 4 to 50 °C, for example from 10 to 40 °C, for example from 20 to 30 °C. In some embodiments, in step (b), the incubation occurs at about 25 °C, for example at 25 °C.

[0197] In some embodiments, in step (b), the pH adjusting agent increases the pH of the emulsion to about 4. In some embodiments, in step (a), the pH of the emulsion (i.e., before the addition of the pH adjusting agent) is at least 1, for example, about 2. It will be appreciated that in step (a), the pH of the emulsion is determined in the aqueous phase.

[0198] Suitably, in step (d), size fractionation may be performed via: screening, such as wet screening, for example by using a vibrating sieving machine; stirred cell filtration; cross-flow filtration; sedimentation; or centrifugation.

[0199] Advantageously, size fractionation is performed via wet screening.

[0200] In some embodiments, in step (d), the obtained hydrogel HA microspheres are wet-sieved in a solvent in which the dispersed phase particles are swellable.

[0201] In some embodiments, in step (d), the resulting hydrogel HA microspheres are wet sieved in a solvent, such as a solvent comprising a buffer and optionally a water-miscible organic solvent such as a polar solvent. Exemplary solvents can be selected from the group consisting of ethanol, methanol, isopropanol, acetonitrile, dioxane, dimethylformamide, dimethyl sulfoxide, tert-butanol, dimethylacetamide, and N-methylpyrrolidone.

[0202] In some embodiments, in steps (e) and (h), the microspheres are washed with a solution comprising a buffer such as succinic acid. In some embodiments, the solution comprises succinic acid, sodium chloride, ethylenediaminetetraacetic acid, and polyoxyethylene sorbitan monolaurate. In some embodiments, in step (e), the microspheres are washed with a solution comprising succinic acid, sodium chloride, histidine, and pluronic acid. TM The microspheres were washed with a solution of F-68).

[0203] Suitably, in step (f), the hydrogel HA microspheres are incubated in a buffer having a pH above 8, such as borate. In some embodiments, the pH of the buffer is about 9. In some embodiments, the pH of the buffer is 9. In some embodiments, in step (f), the hydrogel HA microspheres are incubated in a buffer having a pH above 8, such as borate. TM F-68).

[0204] The incubation may be performed at a temperature ranging from 4 to 50° C., such as from 10 to 40° C., such as from 20 to 30° C. In some embodiments, in step (f), the incubation is performed at 25° C.

[0205] In some embodiments, the hydrogel HA microspheres of step (f) are treated with a reducing agent.

[0206] Exemplary reducing agents can be selected from the group consisting of 1,3- propanedithiol, 2-mercaptoethanol, 3-mercaptopropionic acid, ascorbic acid, dihydrolipoic acid, dithioerythritol, dithiothreitol, sodium borohydride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and thioglycolic acid.

[0207] In some embodiments, the reducing agent is selected from the group consisting of dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride (TCEP).

[0208] Suitably, the reducing agent is dithiothreitol.

[0209] Chemical modification of HA with functional groups can impart functionality to the HA. One skilled in the art will recognize that one functionalized HA can have more than one reactive functional group, e.g., -FG1or -FG2.

[0210] In some embodiments, -FG1, -FG2, and -FG3are independently selected from the group consisting of:

[0211]

[0212]

[0213]

[0214] wherein the dashed line indicates attachment to the first or second functionalized HA, e.g., to the variable -X'- or -Y'-;

[0215] each -R 08 , -R 08a , and -R 08b is independently selected from the group consisting of: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R 09 , and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010)S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R 010 )C(O)N(R 010a )- and -OC(O)N(R 010 )-;

[0216] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 groups, which are the same or different;

[0217] each -R 09 , -R 010 , and -R 010a is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;

[0218] each -Y 01 is independently selected from -F, -Cl, -Br, and -I;

[0219] each n is independently 1, 2, 3, or 4;

[0220] each -Y 02 and -Y 02a is independently selected from -H and -Br;

[0221] each -Y 03 and -Y 03a is independently selected from -F, -Cl, -Br, -I, -OR, -NR 011 R 011a , and -SR 011 ;

[0222] each -Y 04 is independently selected from -O-, -S-, -NR 011 -, -CR 011 R 011a -; and

[0223] each -R 011 and -R 011a is independently selected from halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 012 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 013 )-、-S(O)2N(R 013 )-、-S(O)N(R 013 )-、-S(O)2-、-S(O)-、-N(R 013 )S(O)2N(R 013a )-、-S-、-N(R 013 )-、-OC(OR 013 )(R 013a )-、-N(R 013 )C(O)N(R 013a )-and-OC(O)N(R 013 )-;

[0224] Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R 012 replace; and

[0225] Each-R 12 、-R 013 and -R 013a Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0226] In some embodiments, -FG1, -FG2, and -FG3 are independently selected from:

[0227]

[0228]

[0229] wherein the dashed line indicates the linkage to the first or second functionalized HA, e.g., to the variable -X'- or -Y'-;

[0230] Each-R 08 、-R08a and -R 08b is independently selected from the group consisting of: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R 09 , which are the same or different, and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010 )S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R 010 )C(O)N(R 010a )-, and -OC(O)N(R 010 )-;

[0231] each T 0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 , which are the same or different;

[0232] each -R 09 , -R 010 , and -R 010a is independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0233] each -Y 01 is independently selected from the group consisting of -F, -Cl, -Br, and -I;

[0234] each n is independently 1, 2, 3, or 4;

[0235] each -Y02 and -Y 02a is independently selected from -H and -Br;

[0236] each -Y 03 and -Y 03a is independently selected from the group consisting of: -F, -Cl, -Br, -I, -OR, -NR 011 R 011a and -SR 011 ;

[0237] each -Y 04 is independently selected from the group consisting of: -O-, -S-, -NR 011 -, -CR 011 R 011a -;

[0238] each -R 011 and -R 011a is independently selected from the group consisting of: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl and C 2-50 alkynyl is optionally substituted with one or more -R 012 which are the same or different, and wherein C 1-50 alkyl, C 2-50 alkenyl and C 2-50 alkynyl is optionally interrupted with one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 013 )-, -S(O)2N(R 013 )-, -S(O)N(R 013 )-, -S(O)2-, -S(O)-, -N(R 013 )S(O)2N(R 013a )-, -S-, -N(R 013 )-, -OC(OR 013 )(R 013a )-, -N(R 013 )C(O)N(R 013a )- and -OC(O)N(R 013 )-;

[0239] each T 0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; wherein each T 0independently optionally substituted with one or more of the same or different -R 012 substituted; and

[0240] each -R 12 , -R 013 , and -R 013a is independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more of the same or different halogen.

[0241] In some embodiments, the pH adjusting agent increases the pH of the emulsion of step (a), and -FG1and -FG2are independently selected from:

[0242]

[0243]

[0244] wherein the dashed line indicates attachment to the first or second functionalized HA, e.g., to the variable -X'- or -Y'-;

[0245] each -R 08 , and -R 08a is independently selected from the group consisting of: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more of the same or different -R 09 , and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010 )S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R 010 )C(O)N(R 010a )-, and -OC(O)N(R 010 )-;

[0246] each T 0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 groups, which are the same or different;

[0247] each -R 09 , -R 010 , and -R 010a is independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;

[0248] each -Y 01 is independently selected from the group consisting of -F, -Cl, -Br, and -I;

[0249] each n is independently 1, 2, 3, or 4;

[0250] each -Y 02 and -Y 02a is independently selected from the group consisting of -H and -Br;

[0251] each -Y 03 and -Y 03a is independently selected from the group consisting of: -F, -Cl, -Br, -I, -OR, -NR 011 R 011a , and -SR 011 ;

[0252] each -R 011 and -R 011a is independently selected from the group consisting of: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R 012 groups, which are the same or different, and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from the group consisting of: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 013 )-, -S(O)2N(R 013 )-, -S(O)N(R013 )-、-S(O)2-、-S(O)-、-N(R 013 )S(O)2N(R 013a )-、-S-、-N(R 013 )-、-OC(OR 013 )(R 013a )-、-N(R 013 )C(O)N(R 013a )-and-OC(O)N(R 013 )-;

[0253] Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R 012 replace;

[0254] Each-R 012 、-R 013 and -R 013a Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different.

[0255] In some embodiments, the pH adjuster lowers the pH of the emulsion of step (a), and -FG1 and -FG2 are independently selected from:

[0256]

[0257] wherein the dashed line indicates the linkage to the first or second functionalized HA, e.g., to the variable -X'- or -Y'-;

[0258] Each-R 08 and -R 08a Independently selected from: halogen, -H, -CN, -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 09 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010 )S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R 010 )C(O)N(R 010a )- and -OC(O)N(R 010 )-;

[0259] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 , which are the same or different; and

[0260] each -R 09 , -R 010 , and -R 010a is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0261] In some embodiments, -FG1is independently selected from:

[0262]

[0263] wherein the dotted line indicates a connection to the first functionalized HA, e.g., to the variable -X’-;

[0264] -Y 01 is independently selected from -F, -Cl, -Br, and -I;

[0265] each -R 08 and -R 08a is independently selected from: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R09 substituted, and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010 )S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R 010 )C(O)N(R 010a )-, and -OC(O)N(R 010 )-;

[0266] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 , which are the same or different; and

[0267] each -R 09 , -R 010 , and -R 010a is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0268] -FG2is independently selected from:

[0269]

[0270] wherein the dotted line indicates a connection to a second functionalization HA, such as to a variable -Y'-;

[0271] each -Y 02 and -Y 02a is independently selected from -H and -Br;

[0272] with the proviso that: -FG1is of formula (y-56), then -FG2is of formula (y-57) or (y-86); if -FG1is of formula (y-1), then -FG2is of formula (y-16) or (y-47); if -FG1is of formula (y-44), then -FG2is of formula (y-16) or (y-47); if -FG1is of formula (y-6), then -FG2is of formula (y-9); if -FG1is of formula (y-49), then -FG2is of formula (y-85); if -FG1is of formula (y-44), then -FG2is of formula (y-47); or if -FG1is of formula (y-39), then -FG2is of formula (y-56).

[0273] In some embodiments, the pH adjusting agent increases the pH of the emulsion of step (a), and -FG1is independently selected from:

[0274]

[0275] wherein the dotted line indicates attachment to the first functionalized HA, e.g., to the variable -X’;

[0276] -Y 01 is independently selected from -F, -CI, -Br, and -I;

[0277] each -R 08 and -R 08a is independently selected from: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R 09 , which are the same or different, and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-, -S(O)2N(R 010 )-, -S(O)N(R 010 )-, -S(O)2-, -S(O)-, -N(R 010 )S(O)2N(R 010a )-, -S-, -N(R 010 )-, -OC(OR 010 )(R 010a )-, -N(R010 )C(O)N(R 010a )- and -OC(O)N(R 010 )-;

[0278] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 09 ; and

[0279] each -R 09 , -R 010 , and -R 010a is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen; and

[0280] -FG2is independently selected from:

[0281]

[0282] wherein the dashed line indicates a connection to the second functionalized HA, e.g., to the variable -Y'-;

[0283] each -Y 02 and -Y 02a is independently selected from -H and -Br;

[0284] with the proviso that: -FG1is of formula (y-56), then -FG2is of formula (y-57) or (y-86); if -FG1is of formula (y-1), then -FG2is of formula (y-16); if -FG1is of formula (y-44), then -FG2is of formula (y-16); or if -FG1is of formula (y-39), then -FG2is of formula (y-56).

[0285] In some embodiments, the pH adjusting agent decreases the pH of the emulsion of step (a), and -FG1is independently selected from:

[0286]

[0287] wherein the dashed line indicates a connection to the first functionalized HA, e.g., to the variable -X'-;

[0288] each -R 08 , -R 08a is independently selected from: halogen, -H, -CN, -T 0 , C 1-50 alkyl, C 2-50 alkenyl, and C2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 09 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-、-S(O)2N(R 010 )-、-S(O)N(R 010 )-、-S(O)2-、-S(O)-、-N(R 010 )S(O)2N(R 010a )-、-S-、-N(R 010 )-、-OC(OR 010 )(R 010a )-、-N(R 010 )C(O)N(R 010a )-and-OC(O)N(R 010 )-;

[0289] Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R 09 replace; and

[0290] Each-R 09 、-R 010 and -R 010a Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0291] -FG2 is independently selected from:

[0292]

[0293] wherein the dashed line indicates the connection to the second functionalized HA, for example, the connection to the variable -Y'-;

[0294] Provided that: if -FG1 has formula (y-6), then -FG2 has formula (y-9); and if -FG1 has formula (y-44), then -FG2 has formula (y-47).

[0295] In some embodiments, -FG1is (y-56), wherein the dashed line indicates attachment to a first functionalized HA, e.g., to a variable -X’; -FG2is (y-57), wherein the dashed line indicates attachment to a second functionalized HA, e.g., to a variable -Y’; the pH adjusting agent increases the pH of the emulsion of step (a) from about 1 to about 9, e.g., from about 1 to about 5.5, or, e.g., from about 2 to about 4, and each -Y 02 and -Y 02a is independently selected from -H and -Br.

[0296] In some embodiments, -FG1is (y-56), wherein the dashed line indicates attachment to a first functionalized HA, e.g., to a variable -X’; -FG2is (y-57), wherein the dashed line indicates attachment to a second functionalized HA, e.g., to a variable -Y’; the pH adjusting agent increases the pH of the emulsion of step (a) from about 1 to about 9, e.g., from about 1 to about 5.5, or, e.g., from about 2 to about 4, and each -Y 02 and -Y 02a is -H.

[0297] In some embodiments, -FG1is (y-56), wherein the dashed line indicates attachment to a first functionalized HA, e.g., to a variable -X’; -FG2is (y-57), wherein the dashed line indicates attachment to a second functionalized HA, e.g., to a variable -Y’; the pH adjusting agent increases the pH of the emulsion of step (a) from 1 to 5.5, and each -Y 02 and -Y 02a is independently selected from -H and -Br.

[0298] In some embodiments, -FG1is (y-56), wherein the dashed line indicates attachment to a first functionalized HA, e.g., to a variable -X’; -FG2is (y-57), wherein the dashed line indicates attachment to a second functionalized HA, e.g., to a variable -Y’; the pH adjusting agent increases the pH of the emulsion of step (a) from 1 to 5.5, and each -Y 02 and -Y 02a is -H.

[0299] In some embodiments, -FG1is (y-56), wherein the dashed line indicates attachment to a first functionalized HA, e.g., to a variable -X’; -FG2is (y-57), wherein the dashed line indicates a connection to the second functionalized HA, e.g., to the variable -Y'-; the pH adjuster increases the pH of the emulsion of step (a) from 2 to 4, and each -Y 02 and -Y 02a is independently selected from the group consisting of -H and -Br.

[0300] In some embodiments, -FG1 is (y-56), wherein the dashed line indicates a connection to the first functionalized HA, e.g., to the variable -X'-; -FG2 is (y-57), wherein the dashed line indicates a connection to the second functionalized HA, e.g., to the variable -Y'-; the pH adjuster increases the pH of the emulsion of step (a) from 2 to 4, and each -Y 02 and -Y 02a is -H.

[0301] In some embodiments, the method of the present application comprises the following steps:

[0302] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are different functional moieties from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein

[0303] The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 units:

[0304]

[0305] The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 units:

[0306]

[0307] wherein

[0308] The unmarked dashed line indicates a connection point to the adjacent unit at the marked dashed line or to a hydrogen atom;

[0309] The marked dashed line indicates a connection point to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0310] Each R a1 is independently selected from the group consisting of -H, C1-10 Alkyl, ammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, alkali metal ions and alkaline earth metal ions;

[0311] Each-R a2 are independently -H or C 1-10 alkyl;

[0312] each -FG1, -FG2 is as defined elsewhere herein;

[0313] Each -X-, -Y- is independently a carbonyl group or absent;

[0314] Each -X'-, -Y'- is independently a spacer moiety or absent;

[0315] (b) optionally, adding a pH adjuster to the emulsion of step (a);

[0316] (c) collecting the hydrogel HA microspheres obtained in step (a) or (b), wherein the hydrogel comprises a plurality of Z 3 unit:

[0317]

[0318] in

[0319] Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms;

[0320] Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group;

[0321] Each-R a2 , -X-, -Y-, -X'- and -Y'- are as defined in step (a);

[0322] Each-L 3 - independently part of the link or not present;

[0323] (d) optionally, size fractionating the hydrogel HA microparticles obtained in step (a), (b) or (c) to obtain microspheres with a specific size distribution;

[0324] (e) optionally, washing the hydrogel HA microspheres obtained in step (a), (b), (c) or (d);

[0325] (f) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), or (e) in a buffer having a pH ranging from about 8 to about 12; (g) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), (e), or (f) with a reducing agent;

[0326] (h) optionally, washing the microspheres obtained in step (f) or (g); and

[0327] (i) optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g), or (h).

[0328] The present application also relates to the above-described method, wherein steps (b), (d), and (e) are not optional, and steps (f), (g), and (h) are absent.

[0329] The present application also relates to the above-described method, wherein steps (b), (d), and (e) are not optional, and steps (f), (g), and (h) are absent, and solution A of step (a) further comprises a buffer, and solution B of step (a) comprises an emulsifier and a solvent, wherein the buffer, emulsifier, and solvent are used as defined elsewhere herein.

[0330] It is understood throughout the specification that -L3- is a linkage resulting from the reaction of -FG1 with -FG2.

[0331] Specific embodiments of -X'- and -Y'- are described elsewhere herein.

[0332] Those skilled in the art will recognize that the hydrogel HA microspheres obtained from any of the above steps can also comprise Z 5 and / or Z 6 units having one or more unreacted -FG1 or -FG2, respectively, i.e., Z 5 and / or Z 6 units of step (a) wherein one or more -FG1 is unreacted with one or more -FG2. It is also understood that the hydrogel can also comprise Z 5 and / or Z 6 units wherein one or more -FG1 and / or -FG2 undergoes hydrolysis or becomes inactive.

[0333] In some embodiments, a majority of the -FG1 or -FG2 moieties do not self-react. As used herein, the term "self-react" with respect to -FG1 or -FG2 means that the -FG1 moieties do not react with another -FG1 moiety, and the -FG2 moieties do not react with another -FG2 moiety.

[0334] According to the chemical structures of the moieties shown below as part of Z 3 units, the hydrogel HA microspheres can be non-degradable or biodegradable:

[0335]

[0336] wherein the unmarked dashed line indicates attachment to -X-, and the dashed line marked with an asterisk indicates attachment to -Y-.

[0337] Suitably, the hydrogel HA microspheres of the present application are biodegradable under physiological conditions.

[0338] In some embodiments, -X- and -Y- are each a carbonyl moiety.

[0339] In some embodiments, the method of the present application comprises the steps of:

[0340] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein

[0341] The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 each of which is:

[0342]

[0343] The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 each of which is:

[0344]

[0345] wherein

[0346] The unlabelled dashed line indicates a point of attachment to an adjacent unit at the labelled dashed line or to a hydrogen atom;

[0347] The labelled dashed line indicates a point of attachment to an adjacent unit at the unlabelled dashed line or to a hydroxyl group;

[0348] Each R a1 is independently selected from the group consisting of: -H, C 1-10 alkyl, an ammonium ion, a tetrabutylammonium ion, a hexadecyltrimethylammonium ion, an alkali metal ion, and an alkaline earth metal ion;

[0349] Each -R a2 is independently -H or C 1-10 alkyl;

[0350] Each -X'-, -Y' is independently a spacer moiety or is absent;

[0351] (b) optionally, adding a pH adjusting agent to the emulsion of step (a);

[0352] (c) collecting the hydrogel HA microspheres resulting from step (a) or (b), wherein the hydrogel comprises a plurality of Z 3 - i units:

[0353]

[0354] wherein

[0355] The unmarked dashed line indicates the point of attachment to the adjacent unit at the dashed line marked with # or to a hydrogen atom;

[0356] The dashed line marked with # indicates the point of attachment to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0357] each -R a2 , -X'- and -Y' are as defined in step (a);

[0358] (d) optionally, size fractionating the hydrogel HA microspheres resulting from step (a), (b) or (c) to obtain microspheres having a particular particle size distribution;

[0359] (e) optionally, washing the hydrogel HA microspheres obtained in step (a), (b), (c) or (d);

[0360] (f) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d) or (e) in a buffer having a pH ranging from about 8 to about 12,

[0361] to provide hydrogel HA microspheres comprising a plurality of Z 3 - i' units:

[0362]

[0363] wherein

[0364] The unmarked dashed line indicates the point of attachment to the adjacent unit at the dashed line marked with # or to a hydrogen atom;

[0365] The dashed line marked with # indicates the point of attachment to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0366] each -R a1 , -R a2 , -X'- and -Y' are as defined in step (a);

[0367] (g) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), (e) or (f) with a reducing agent;

[0368] (h) optionally, washing the microspheres obtained in step (f) or (g); and

[0369] (i) optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g), or (h).

[0370] The present application also relates to the above method, wherein steps (b), (d), and (e) are not optional, and steps (f), (g), and (h) are absent.

[0371] The present application also relates to the above method, wherein steps (b), (d), and (e) are not optional, and steps (f), (g), and (h) are absent, and solution A of step (a) further comprises a buffer, solution B of step (a) comprises an emulsifier and a solvent, and wherein the buffer, emulsifier, and solvent are used as defined elsewhere herein.

[0372] The present application also relates to the above method, wherein steps (b), (d), and (e) are not optional, and steps (f), (g), and (h) are optional.

[0373] Particular embodiments of -X'- and -Y'- are as described elsewhere herein.

[0374] Those skilled in the art will recognize that, in the above step (f), the resulting hydrogel HA microspheres, or pharmaceutically acceptable salts thereof, can comprise a plurality of Z 3 -i' units and / or a plurality of Z 3 -i" units, depending on which of the two carbonyl groups of the thiosuccinimide ring undergoes ring-opening hydrolysis. Z 3 The structure of the -i" unit is shown below:

[0375]

[0376] wherein

[0377] The unmarked dashed line indicates a point of attachment to the adjacent unit at the marked dashed line or to a hydrogen atom;

[0378] The marked dashed line indicates a point of attachment to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0379] each R a1 is independently selected from the group consisting of: -H, C 1-10 alkyl, an ammonium ion, a tetrabutylammonium ion, a hexadecyltrimethylammonium ion, an alkali metal ion, and an alkaline earth metal ion;

[0380] each R a1 , -R a2 is independently -H or C 1-10 alkyl; and

[0381] each -X'- and -Y'- is independently a spacer moiety or a chemical bond.

[0382] In some embodiments, the hydrogel HA microspheres of step (f) comprise a plurality of Z 3 - i' and Z 3 - i" units.

[0383] In some embodiments, the methods of the present application comprise the following steps:

[0384] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein solution A further comprises a buffer, such as citrate and / or histidine, and solution B comprises a solvent, such as heptane or tetradecane, and an emulsifier, such as sorbitan monooleate; wherein,

[0385] The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 each of which is in an -i unit:

[0386]

[0387] The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 each of which is in an -i unit:

[0388]

[0389] wherein

[0390] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0391] The dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0392] each R a1 is H or an alkali metal ion;

[0393] each -R a2 is -H;

[0394] each -X' has formula (x0);

[0395] each -Y' has formula (y0);

[0396] (b) adding a pH adjusting agent to the emulsion of step (a);

[0397] (c) collecting the hydrogel HA microspheres resulting from step (b), wherein the hydrogel comprises a plurality of Z 3- i units:

[0398]

[0399] wherein

[0400] the unmarked dashed line indicates the point of attachment to the adjacent unit at the marked dashed line or to a hydrogen atom;

[0401] the marked dashed line indicates the point of attachment to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0402] each -R a2 , -X'- and -Y' are as defined in step (a);

[0403] (d) size fractionating the hydrogel HA microparticles obtained in step (c) to obtain microspheres having a specific particle size distribution;

[0404] (e) washing the hydrogel HA microspheres obtained in step (d); and

[0405] (f) collecting the hydrogel HA microspheres of step (e).

[0406] It is understood that Z 3 The -i units depict the linkages formed between the first and second functionalized HA. The skilled person understands that HA hydrogels are complex chemical entities and thus there are multiple ways to describe their chemical structure.

[0407] Accordingly, below is shown an alternative way to depict the hydrogel HA microspheres obtained in step (a) or (b), i.e. the hydrogel HA microspheres comprising a plurality of HA strands 1A and a plurality of HA strands 1B, wherein

[0408] each 1A comprises a plurality of linearly linked Z 1 and Z 3 - i(a) units:

[0409]

[0410] each 1B comprises a plurality of linearly linked Z 1 and Z 3 - i(b) units:

[0411]

[0412] wherein

[0413] the unmarked dashed line indicates the point of attachment to the adjacent unit at the marked dashed line or to a hydrogen atom;

[0414] the marked dashed line indicates the point of attachment to the adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0415] Dotted lines marked with * indicate units Z 3 - between -i(a) and Z 3 - between -i(b) such that at least one 1A is crosslinked with at least one 1B; and

[0416] each R a1 , -R a2 , -X'- and -Y' are as defined in step (a).

[0417] It will be appreciated that each 1A will also contain unreacted Z 6 - units which will react with -FG3 as defined elsewhere herein.

[0418] In some embodiments, the method of the present application comprises the steps of:

[0419] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein solution A further comprises citrate and / or histidine, and solution B comprises heptane and sorbitan monooleate or tetradecane and sorbitan monooleate; wherein,

[0420] The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 - each of the -i units:

[0421]

[0422] The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 - each of the -i units:

[0423]

[0424] wherein

[0425] Dotted lines without a mark indicate a point of attachment with an adjacent unit at a dotted line marked with # or with a hydroxyl group;

[0426] Dotted lines marked with # indicate a point of attachment with an adjacent unit at a dotted line without a mark or with a hydroxyl group;

[0427] each R a1 is H or an alkali metal ion;

[0428] each -Ra2 -H;

[0429] each -X' has formula (x4):

[0430]

[0431] wherein the unmarked dashed line indicates a connection to a carbonyl group, the dashed line marked with an asterisk indicates a connection to a sulfur atom, and c0is 7;

[0432] each -Y' has formula (y4):

[0433]

[0434] wherein the unmarked dashed line indicates a connection to a carbonyl group, and the dashed line marked with an asterisk indicates a connection to a nitrogen atom of the maleimide ring;

[0435] (b) adding TMEDA to the emulsion of step (a);

[0436] (c) collecting the hydrogel HA microspheres obtained in step (b), wherein the hydrogel comprises a plurality of Z 3 units:

[0437]

[0438] wherein

[0439] the unmarked dashed line indicates a connection to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0440] the dashed line marked with a # indicates a connection to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0441] each -R a2 , -X' and -Y' are as defined in step (a);

[0442] (d) size fractionating the hydrogel HA microparticles obtained in step (c) to obtain microspheres having a specific particle size distribution;

[0443] (e) washing the hydrogel HA microspheres obtained in step (d); and

[0444] (f) collecting the hydrogel HA microspheres of step (e).

[0445] The present application also relates to the above described method, wherein solution A further comprises a suitable salt, such as NaCl.

[0446] In some embodiments, the method of the present application comprises the following steps:

[0447] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming a hydrogel HA microsphere, wherein solution A further comprises a buffer such as histidine, and solution B comprises a solvent such as heptane or tetradecane and an emulsifier such as PEG-30 dipolyhydroxystearate; wherein,

[0448] The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 each of which is in an -i unit:

[0449]

[0450] The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 each of which is in an -i unit:

[0451]

[0452] wherein

[0453] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0454] The dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0455] each R a1 is H or an alkali metal ion;

[0456] each -R a2 is -H;

[0457] each -X'- and -Y' has formula (y0);

[0458] (b) adding a pH adjusting agent to the emulsion of step (a);

[0459] (c) collecting the hydrogel HA microspheres resulting from step (b), wherein the hydrogel comprises a plurality of Z 3 units:

[0460]

[0461] wherein

[0462] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0463] dashed line labeled with # indicates the point of attachment to the adjacent unit at the unlabeled dashed line or to a hydroxyl group;

[0464] each -R a2 , -X'- and -Y' are as defined in step (a);

[0465] (d) size fractionating the hydrogel HA microparticles obtained in step (c) to obtain microspheres having a specific particle size distribution;

[0466] (e) washing the hydrogel HA microspheres obtained in step (d);

[0467] (f) incubating the hydrogel HA microspheres of step (e) in borate to provide hydrogel HA microspheres comprising a plurality of Z 3 units of -i';

[0468]

[0469] wherein

[0470] the unlabeled dashed line indicates the point of attachment to the adjacent unit at the dashed line labeled with # or to a hydrogen atom;

[0471] the dashed line labeled with # indicates the point of attachment to the adjacent unit at the unlabeled dashed line or to a hydroxyl group;

[0472] each -R a1 , -R a2 , -X'- and -Y' are as defined in step (a);

[0473] (g) incubating the hydrogel HA microspheres of step (f) with DTT;

[0474] (h) washing the microspheres obtained in step (g); and

[0475] (i) collecting the hydrogel HA microspheres of step (h).

[0476] In some embodiments, the methods of the present application comprise the following steps:

[0477] (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein solution A further comprises histidine and solution B comprises heptane or tetradecane and PEG-30 dipolyhydroxystearate; wherein,

[0478] the first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z5 each of the -i units:

[0479]

[0480] the second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 each of the -i units:

[0481]

[0482] wherein

[0483] the unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0484] the dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0485] each R a1 is H or an alkali metal ion;

[0486] each -R a2 is -H;

[0487] each -X'- and -Y'- is of formula (y4):

[0488]

[0489] wherein for -X'- the unmarked dashed line indicates a point of attachment to a carbonyl group and the dashed line marked with an asterisk indicates a point of attachment to a sulfur atom;

[0490] wherein for -Y'- the unmarked dashed line indicates a point of attachment to a carbonyl group and the dashed line marked with an asterisk indicates a point of attachment to a nitrogen atom of a maleimide ring;

[0491] (b) adding TMEDA to the emulsion of step (a);

[0492] (c) collecting the hydrogel HA microspheres resulting from step (b), wherein the hydrogel comprises a plurality of Z 3 each of the -i units:

[0493]

[0494] wherein

[0495] the unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0496] the dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0497] each -R a2, -X'- and -Y'- are as defined in step (a);

[0498] (d) size fractionating the hydrogel HA microparticles obtained in step (c) to obtain microspheres having a specific particle size distribution;

[0499] (e) washing the hydrogel HA microspheres obtained in step (d);

[0500] (f) incubating the hydrogel HA microspheres of step (e) in borate to provide hydrogel HA microspheres comprising a plurality of Z 3 units of -X'- and -Y'- are as defined in step (a);

[0501]

[0502] wherein

[0503] the unlabelled dashed line indicates a point of attachment to an adjacent unit at the labelled dashed line or to a hydrogen atom;

[0504] the labelled dashed line indicates a point of attachment to an adjacent unit at the unlabelled dashed line or to a hydroxyl group;

[0505] each R a1 , -R a2 , -X'- and -Y'- are as defined in step (a);

[0506] (g) incubating the hydrogel HA microspheres of step (f) with DTT;

[0507] (h) washing the microspheres obtained in step (g); and

[0508] (i) collecting the hydrogel HA microspheres of step (h).

[0509] The present application also relates to the above described method, wherein solution A further comprises a salt, such as NaCl.

[0510] It will be appreciated by the skilled person that throughout the description involving units Z 3 , the respective adjacent unit connected to the part of the unit comprising -X'- can be a cross-linking unit as defined in any of the respective Z 3 units, or connected to an unreacted Z 5 unit or Z 1 unit or any other unit present in or producible during polymerisation of the first functionalised HA. Similarly, it will be appreciated that the respective adjacent unit connected to the part of the unit comprising -Y'- can be a cross-linking unit as defined in any of the respective Z 3 units, or connected to an unreacted Z 6 unit or Z 1Any other units present in the unit or second functionalized HA or that can be produced during polymerization. The same principle applies to Z 3 - i, Z 3 - i' and Z 3 - ii" units and their corresponding Z 5 - i and Z 6 - i units.

[0511] The -FG1 functionalization degree of the first functionalized HA can range from about 0.001% to 100%, for example, from about 0.01% to about 90%, for example, from about 0.1% to about 80%, for example, from about 1% to about 70%, for example, from about 1% to about 60%, for example, from about 1% to about 50%, for example, from about 1% to about 40%, for example, from about 1% to about 30%, for example, from about 1% to about 20%, for example, from about 1% to about 15%, for example, from about 1% to about 10%, for example, from about 1% to 7%, for example, from about 2% to about 6%, or for example, from about 3% to about 5%. In some embodiments, the -FG1 functionalization degree of the first functionalized HA is about 5%.

[0512] The -FG2 functionalization degree of the second functionalized HA can range from about 0.001% to 100%, for example, from about 0.01% to about 90%, for example, from about 0.1% to about 80%, for example, from about 1% to about 70%, for example, from about 1% to about 60%, for example, from about 1% to about 50%, for example, from about 1% to about 40%, for example, from about 1% to about 30%, for example, from about 1% to about 20%, for example, from about 1% to about 15%, for example, from about 1% to about 10%, for example, from about 5% to about 15%, for example, from about 6% to about 14%, for example, from about 7% to about 13%, for example, from about 8% to about 12%, for example, from about 10% to about 12%, or for example, from about 9% to about 11%. In some embodiments, the -FG2 functionalization degree of the second functionalized HA is about 10%.

[0513] In some embodiments, the -FG1 functionalization degree of the first functionalized HA is about 5% and the -FG2 functionalization degree of the second functionalized HA is about 10%.

[0514] More particularly, the thiol functional groups are introduced to provide a HA functionalization degree of about 0.001% to 100%, for example, from about 0.01% to about 90%, for example, from about 0.1% to about 80%, for example, from about 1% to about 70%, for example, from about 1% to about 60%, for example, from about 1% to about 50%, for example, from about 1% to about 40%, for example, from about 1% to about 30%, for example, from about 1% to about 20%, for example, from about 1% to about 15%, for example, from about 1% to about 10%, for example, from about 1% to 7%, for example, from about 2% to about 6%, or for example, from about 3% to about 5%.

[0515] Suitably, the thiol functional groups are introduced to provide a HA functionalization degree of about 5%.

[0516] More particularly, maleimide functional groups are introduced to provide a degree of HA functionalization ranging from about 0.001% to 100%, for example, from about 0.01% to about 90%, for example, from about 0.1% to about 80%, for example, from about 1% to about 70%, for example, from about 1% to about 60%, for example, from about 1% to about 50%, for example, from about 1% to about 40%, for example, from about 1% to about 30%, for example, from about 1% to about 20%, for example, from about 1% to about 15%, for example, from about 1% to about 10%, for example, from about 5% to about 15%, for example, from about 6% to about 14%, for example, from about 7% to about 13%, for example, from about 8% to about 12%, for example, from about 10% to about 12%, or for example, from about 9% to about 11%.

[0517] Suitably, maleimide functional groups are introduced to provide a degree of HA functionalization of about 10%.

[0518] Suitably, thiol functional groups are introduced to provide a degree of HA functionalization of about 5%, and maleimide functional groups are introduced to provide a degree of HA functionalization of about 10%.

[0519] As used herein, the term “degree of (HA) functionalization” refers to the relative ratio between the number of functionalized HA disaccharide units contained within a particular HA and the total number of all disaccharide units.

[0520] In some embodiments, each -L 3 is of the formula (x-101):

[0521]

[0522] wherein a dashed line marked with an asterisk indicates a connection to -Y'- and a dashed line marked with no asterisk indicates a connection to -X'-.

[0523] In some embodiments, each -X'- and -Y'- is independently a spacer moiety selected from -T'-, C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl; wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally substituted with one or more -R y1 groups, and wherein C 1-50 alkyl, C 2-50 alkenyl, and C 2-50 alkynyl is optionally interrupted with one or more groups selected from -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-, -S(O)2N(R y2 )-, -S(O)N(R y2)-, -S(O)2-, -S(O)-, -N(R y2 )S(O)2N(R y2a )-, -S-, -N(R y2 )-, -OC(OR y2 )(R y2a )-, -N(R y2 )C(O)N(R y2a )- and -OC(O)N(R y2 )-;

[0524] each -T' is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T' is independently optionally substituted with one or more -R y1 groups, which are the same or different;

[0525] each -R y1 is independently selected from halogen, -CN, oxo (=O), -COOR y3 , -OR y3 , -C(O)R y3 , -C(O)N(R y3 R y3a ), -S(O)2N(R y3 R y3a ), -S(O)N(R y3 R y3a ), -S(O)2R y3 , -S(O)R y3 , -N(R y3 )S(O)2N(R y3a R y3b ), -SR y3 , -N(R y3 R y3a ), -NO2, -OC(O)R y3 , -N(R y3 )C(O)R y3a , -N(R y3 )S(O)2R y3a , -N(R y3 )S(O)R y3a , -N(R y3 )C(O)OR y3a , -N(R y3 )C(O)N(R y3a R y3b ), -OC(O)N(R y3 R y3a ), and C1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0526] Each-R y2 、-R y2a 、-R y3 、-R y3a 、-R y3b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different.

[0527] In some embodiments, each -X'- and -Y'- is independently a spacer moiety selected from: -T'-, C 1-25 Alkyl, C 2-25 Alkenyl and C 2-25 Alkynyl; wherein C 1-25 Alkyl, C 2-25 Alkenyl and C 2-25 Alkynyl is optionally substituted by one or more identical or different -R y1 substituted, and wherein C 1-25 Alkyl, C 2-25 Alkenyl and C 2-25 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-、-S(O)2N(R y2 )-、-S(O)N(R y2 )-、-S(O)2-、-S(O)-、-N(R y2 )S(O)2N(R y2a )-、-S-、-N(R y2 )-、-OC(OR y2 )(R y2a )-、-N(R y2 )C(O)N(R y2a )-and-OC(O)N(R y2 )-;

[0528] Each -T'- is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbon polycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T'- is independently optionally replaced by one or more identical or different -R y1 replace;

[0529] Each-R y1halo, -CN, oxo (=0), -COOR y3 , -OR y3 , -C(O)R y3 , -C(O)N(R y3 R y3a ), -S(O)2N(R y3 R y3a ), -S(O)N(R y3 R y3a ), -S(O)2R y3 , -S(O)R y3 , -N(R y3 )S(O)2N(R y3a R y3b ), -SR y3 , -N(R y3 R y3a ), -NO2, -OC(O)R y3 , -N(R y3 )C(O)R y3a , -N(R y3 )S(O)2R y3a , -N(R y3 )S(O)R y3a , -N(R y3 )C(O)OR y3a , -N(R y3 )C(O)N(R y3a R y3b ), -OC(O)N(R y3 R y3a ) and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0530] each -R y2 , -R y2a , -R y3 , -R y3a , -R y3b is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0531] In some embodiments, each -X'- and -Y'- is independently a spacer moiety selected from -T'-, C 1-18 alkyl, C 2-18 alkenyl and C 2-18 alkynyl; wherein C 1-18 alkyl, C 2-18 alkenyl and C 2-18 alkynyl is optionally substituted with one or more halogen, which are the same or different.Alkynyl is optionally substituted by one or more identical or different -R y1 substituted, and wherein C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-、-S(O)2N(R y2 )-、-S(O)N(R y2 )-、-S(O)2-、-S(O)-、-N(R y2 )S(O)2N(R y2a )-、-S-、-N(R y2 )-、-OC(OR y2 )(R y2a )-、-N(R y2 )C(O)N(R y2a )-and-OC(O)N(R y2 )-;

[0532] Each -T'- is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbon polycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T'- is independently optionally replaced by one or more identical or different -R y1 replace;

[0533] Each-R y1 Independently selected from: halogen, -CN, oxo (=O), -COOR y3 、-OR y3 、-C(O)R y3 、-C(O)N(R y3 R y3a )、-S(O)2N(R y3 R y3a )、-S(O)N(R y3 R y3a )、-S(O)2R y3 、-S(O)R y3 、-N(R y3 )S(O)2N(R y3a R y3b ),-SR y3 、-N(R y3 R y3a )、-NO2、-OC(O)R y3 、-N(R y3 )C(O)R y3a 、-N(Ry3 )S(O)2R y3a 、-N(R y3 )S(O)R y3a 、-N(R y3 )C(O)OR y3a 、-N(R y3 )C(O)N(R y3a R y3b )、-OC(O)N(R y3 R y3a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0534] Each-R y2 、-R y2a 、-R y3 、-R y3a 、-R y3b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0535] In some embodiments, each -X'- and -Y'- is independently a spacer moiety selected from: -T'-, C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl; wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 Alkynyl is optionally substituted by one or more identical or different -R y1 substituted, and wherein C 1-10 Alkyl, C 2-10 Alkenyl and C 2-10 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-、-S(O)2N(R y2 )-、-S(O)N(R y2 )-、-S(O)2-、-S(O)-、-N(R y2 )S(O)2N(R y2a )-、-S-、-N(R y2 )-、-OC(OR y2 )(R y2a )-、-N(R y2 )C(O)N(R y2a )-and-OC(O)N(R y2 )-;

[0536] each -T' is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T' is independently optionally substituted with one or more -R y1 substituents;

[0537] each -R y1 is independently selected from the group consisting of: halogen, -CN, oxo (=0), -COOR y3 , -OR y3 , -C(O)R y3 , -C(O)N(R y3 R y3a ), -S(O)2N(R y3 R y3a ), -S(O)N(R y3 R y3a ), -S(O)2R y3 , -S(O)R y3 , -N(R y3 )S(O)2N(R y3a R y3b ), -SR y3 , -N(R y3 R y3a ), -NO2, -OC(O)R y3 , -N(R y3 )C(O)R y3a , -N(R y3 )S(O)2R y3a , -N(R y3 )S(O)R y3a , -N(R y3 )C(O)OR y3a , -N(R y3 )C(O)N(R y3a R y3b ), -OC(O)N(R y3 R y3a ), and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen; and

[0538] each -R y2 , -R y2a , -R y3 , -R y3a , -R y3b is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen; andThe alkyl group is optionally substituted with one or more halogen groups which may be the same or different.

[0539] In some embodiments, each -X'- and -Y'- is independently a spacer moiety selected from: -T'-, C 1-5 Alkyl, C 2-5 Alkenyl and C 2-5 Alkynyl; wherein C 1-5 Alkyl, C 2-5 Alkenyl and C 2-5 Alkynyl is optionally substituted by one or more identical or different -R y1 substituted, and wherein C 1-5 Alkyl, C 2-5 Alkenyl and C 2-5 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-、-S(O)2N(R y2 )-、-S(O)N(R y2 )-、-S(O)2-、-S(O)-、-N(R y2 )S(O)2N(R y2a )-、-S-、-N(R y2 )-、-OC(OR y2 )(R y2a )-、-N(R y2 )C(O)N(R y2a )-and-OC(O)N(R y2 )-;

[0540] Each -T'- is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbon polycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each -T'- is independently optionally replaced by one or more identical or different -R y1 replace;

[0541] Each-R y1 Independently selected from: halogen, -CN, oxo (=O), -COOR y3 、-OR y3 、-C(O)R y3 、-C(O)N(R y3 R y3a )、-S(O)2N(R y3 R y3a )、-S(O)N(R y3 R y3a )、-S(O)2R y3 、-S(O)Ry3 、-N(R y3 )S(O)2N(R y3a R y3b ),-SR y3 、-N(R y3 R y3a )、-NO2、-OC(O)R y3 、-N(R y3 )C(O)R y3a 、-N(R y3 )S(O)2R y3a 、-N(R y3 )S(O)R y3a 、-N(R y3 )C(O)OR y3a 、-N(R y3 )C(O)N(R y3a R y3b )、-OC(O)N(R y3 R y3a ) and C 1-4 Alkyl; among which C 1-4 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0542] Each-R y2 、-R y2a 、-R y3 、-R y3a 、-R y3b Independently selected from -H and C 1-4 Alkyl; among which C 1-4 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different.

[0543] In some embodiments, -X'- has the formula (x0):

[0544]

[0545] in

[0546] Unmarked dashed lines indicate connections to -X-, and dashed lines marked with an asterisk indicate connections to -FG1 or -L 3 -connection;

[0547] v0 is selected from 0 and 1;

[0548] -X 1 -、-X 4 - independently C 1-10 Alkyl, the C 1-10 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -T-, -N(R y1 )-, -C(O)O- and -C(O)N(Ry1 )-group; and said C 1-10 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T, -N(R y1 )- and -C(O)N(R y2 R y2a ) is substituted with a group;

[0549] Where -R y1 、-R y2 、-R y2a Independently selected from H and C 1-4 alkyl;

[0550] -X 2 -selected from -N(R 1 )-, -O-, -S- and -Se-;

[0551] =X 3 Selected from =O, =N(R 1 ) and =S;

[0552] -X 5 -It's C 1-20 Alkyl, the C 1-20 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -C(O)O-, -T-, -N(R y1 )- and -N(R y1 )C(O)-; and said C 1-20 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T, -N(R y1 )- and -C(O)N(R y2 R y2a ) is substituted with a group;

[0553] -R 1 Independently selected from -H, C 1-5 Alkyl and -T;

[0554] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 2 replace;

[0555] -R 2 Selected from: halogen, -CN, oxo, -C(O)OR 3 、-OR 3 、-C(O)R 3 、-C(O)N(R 3 )(R 3a )、-S(O)2N(R 3)(R 3a )、-S(O)N(R 3 )(R 3a )、-S(O)2R 3 、-S(O)R 3 、-N(R 3 )S(O)2N(R 3a )(R 3b ),-SR 3 、-N(R 3 )(R 3a )、-NO2、-OC(O)R 3 、-N(R 3 )C(O)R 3a 、-N(R 3 )S(O)2R 3a 、-N(R 3 )S(O)R 3a 、-N(R 3 )C(O)OR 3a 、-N(R 3 )C(O)N(R 3a )(R 3b )、-OC(O)N(R 3 )(R 3a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0556] Where -R 3 、-R 3a and -R 3b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0557] In some embodiments, v0 of formula (x0) is 0. In some embodiments, v0 of formula (x0) is 1.

[0558] In some embodiments, -X'- has the formula (x1):

[0559]

[0560] in

[0561] Unmarked dashed lines indicate connections to -X-, and dashed lines marked with an asterisk indicate connections to -FG1 or -L 3 -connection;

[0562] b0 is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0563] -X 1 -、-X 4 - independently is C 1-5 alkyl, said C 1-5 alkyl is optionally interrupted with one or more groups independently selected from -0-, -T-, -N(R y1 )- and -C(O)N(R y1 )-; and said C 1-5 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a ); wherein -R y1 , -R y2 , -R y2a are independently selected from -H and C 1-4 alkyl;

[0564] -X 2 - is selected from -N(R 1 )-, -O-, -S- and -Se-;

[0565] =X 3 is selected from =O, =N(R 1 ) and =S;

[0566] -X 6 - is C 1-10 alkyl, said C 1-10 alkyl is optionally interrupted with one or more groups independently selected from -O-, -C(O)O-, -T-, -N(R y1 )- and -N(R y1 )C(O)-; and said C 1-10 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a );

[0567] -R 1 is independently selected from -H, C 1-5 alkyl and -T;

[0568] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 2 groups, which are the same or different;

[0569] -R 2 is selected from: halogen, -CN, oxo, -C(O)OR3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 )(R 3a ), -S(O)2N(R 3 )(R 3a ), -S(O)N(R 3 )(R 3a ), -S(O)2R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a )(R 3b ), -SR 3 , -N(R 3 )(R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a )(R 3b ), -OC(O)N(R 3 )(R 3a ), and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0570] wherein -R 3 , -R 3a , and -R 3b are independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0571] In some embodiments, -X'- has formula (x2):

[0572]

[0573] wherein

[0574] the unlabelled dotted line indicates the connection to -X-, and the dotted line labelled with an asterisk indicates the connection to -FG1or -L 3

[0575] ​b0 is selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0576] -X 1 -, -X 4 -, -X 7 -, -X 8 - is independently C 1-5 alkyl, said C 1-5 alkyl is optionally interrupted with one or more groups independently selected from -0-, -T-, -N(R y1 )-, and -C(O)N(R y1 )-; and said C 1-5 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -NH(R y1 ), and -C(O)N(R y2 R y2a ); wherein -R y1 , -R y2 , -R y2a are independently selected from H and C 1-4 alkyl;

[0577] -R 1 , -R 5 , -R 10 are independently selected from -H, C 1-5 alkyl, and -T;

[0578] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 6 groups, which are the same or different;

[0579] -R 6 is selected from: halogen, -CN, oxo, -C(O)OR 7 , -OR 7 , -C(O)R 7 , -C(O)N(R 7 )(R 7a ), -S(O)2N(R 7 )(R 7a ), -S(O)N(R 7 )(R 7a ), -S(O)2R 7 , -S(O)R 7 , -N(R 7 )S(O)2N(R 7a )(R 7b ), -SR 7 , -N(R 7)(R 7a ), -NO2, -OC(O)R 7 , 7 )C(O)R 7a , 7 )S(O)2R 7a , 7 )S(O)R 7a , 7 )C(O)OR 7a , 7 )C(O)N(R 7a )(R 7b ), 7 )C(O)N(R 7a )(R 1-6 ) and C 1-6 alkyl; wherein the C 7 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0580] wherein -R 7a , -R 7b and -R 1-6 are independently selected from -H and C 1-6 alkyl; wherein the C 3 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0581] In some embodiments, -X'- has formula (x3):

[0582]

[0583] wherein

[0584] the unlabelled dotted line indicates the connection to -X-, and the dotted line labelled with an asterisk indicates the connection to -FG1or -L 1 ;

[0585] b0 is selected from the group consisting of: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

[0586] -X 4 -, -X 1-5 - is independently C 1-5 alkyl, which C y1 alkyl is optionally interrupted with one or more groups independently selected from -O-, -T-, -N(R y1 )- and -C(O)N(R 1-5 ); and which C y1 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -NH(R y2 ); and y2a) is substituted with a group; wherein -R y1 、-R y2 、-R y2a Independently selected from H and C 1-4 alkyl;

[0587] -R 1 、-R 5 、-R 10 Independently selected from -H, C 1-5 Alkyl and -T;

[0588] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 6 replace;

[0589] -R 6 Selected from: halogen, -CN, oxo, -C(O)OR 7 、-OR 7 、-C(O)R 7 、-C(O)N(R 7 )(R 7a )、-S(O)2N(R 7 )(R 7a )、-S(O)N(R 7 )(R 7a )、-S(O)2R 7 、-S(O)R 7 、-N(R 7 )S(O)2N(R 7a )(R 7b ),-SR 7 、-N(R 7 )(R 7a )、-NO2、-OC(O)R 7 、-N(R 7 )C(O)R 7a 、-N(R 7 )S(O)2R 7a 、-N(R 7 )S(O)R 7a 、-N(R 7 )C(O)OR 7a 、-N(R 7 )C(O)N(R 7a )(R 7b )、-OC(O)N(R 7 )(R 7a ) and C 1-6 Alkyl; among which C 1-6alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0590] wherein -R 7 , -R 7a , and -R 7b are independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0591] In some embodiments, b0 of formula (xl), (x2), or (x3) is 1. In some embodiments, b0 of formula (xl), (x2), or (x3) is 2. In some embodiments, b0 of formula (xl), (x2), or (x3) is 3. In some embodiments, b0 of formula (xl), (x2), or (x3) is 4. In some embodiments, b0 of formula (xl), (x2), or (x3) is 5. In some embodiments, b0 of formula (xl), (x2), or (x3) is 6. In some embodiments, b0 of formula (xl), (x2), or (x3) is 7. In some embodiments, b0 of formula (xl), (x2), or (x3) is 8. In some embodiments, b0 of formula (xl), (x2), or (x3) is 9. In some embodiments, b0 of formula (xl), (x2), or (x3) is 10.

[0592] In some embodiments, -X'- has formula (x4):

[0593]

[0594] wherein

[0595] the unlabelled dashed line indicates the connection to -X-, and the dashed line labelled with an asterisk indicates the connection to -FG1or -L 3 ; and

[0596] c0 is selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0597] In some embodiments, c0 of formula (x4) is 1. In some embodiments, c0 of formula (x4) is 2. In some embodiments, c0 of formula (x4) is 3. In some embodiments, c0 of formula (x4) is 4. In some embodiments, c0 of formula (x4) is 5. In some embodiments, c0 of formula (x4) is 6. In some embodiments, c0 of formula (x4) is 7. In some embodiments, c0 of formula (x4) is 8. In some embodiments, c0 of formula (x4) is 9. In some embodiments, c0 of formula (x4) is 10.

[0598] In some embodiments, -Y'- has the formula (y0):

[0599]

[0600] in

[0601] Unmarked dashed lines indicate connections to -Y-, and dashed lines marked with asterisks indicate connections to -FG2, -L 3 -、-L 4 -or-L 5 -connection;

[0602] -Y 1 -、-Y 4 - independently C 1-10 Alkyl, the C 1-10 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -T-, -N(R y1 )-, -C(O)O- and -C(O)N(R y1 )-group; and said C 1-10 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a ) is substituted with a group; wherein -R y1 、-R y2 、-R y2a Independently selected from H and C 1-4 alkyl;

[0603] -Y 2 -selected from -N(R 2 )-, -O-, -S- and -Se-;

[0604] =Y 3 Selected from =O, =N(R 2 ) and =S;

[0605] -R 1 、-R 2 Independently selected from -H, C 1-5 Alkyl and -T;

[0606] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 3 replace;

[0607] -R 3 Selected from: halogen, -CN, oxo, -C(O)OR 4 、-OR4 、-C(O)R 4 、-C(O)N(R 4 )(R 4a )、-S(O)2N(R 4 )(R 4a )、-S(O)N(R 4 )(R 4a )、-S(O)2R 4 、-S(O)R 4 、-N(R 4 )S(O)2N(R 4a )(R 4b ),-SR 4 、-N(R 4 )(R 4a )、-NO2、-OC(O)R 4 、-N(R 4 )C(O)R 4a 、-N(R 4 )S(O)2R 4a 、-N(R 4 )S(O)R 4a 、-N(R 4 )C(O)OR 4a 、-N(R 4 )C(O)N(R 4a )(R 4b )、-OC(O)N(R 4 )(R 4a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0608] Where -R 4 、-R 4a and -R 4b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

[0609] In some embodiments, -X'- has the formula (y0), wherein the unlabeled dashed line indicates linkage to -X- and the dashed line labeled with an asterisk indicates linkage to -FG1.

[0610] In some embodiments, -Y of formula (y0) 2 - is -N(R 2 In some embodiments, -Y of formula (y0) 2 - is -O-. In some embodiments, -Y of formula (y0) 2- is -S-. In some embodiments, -Y of formula (y0) 2 -for-Se-.

[0611] In some embodiments, =Y of formula (y0) 3 =O. In some embodiments, =Y of formula (y0) 3 =N(R 2 In some embodiments, =Y of formula (y0) 3 =S.

[0612] In some embodiments, -Y'- has the formula (y1):

[0613]

[0614] in

[0615] Unmarked dashed lines indicate connections to -Y-, and dashed lines marked with asterisks indicate connections to -FG2, -L 3 -、-L 4 -or-L 5 -connection;

[0616] -Y 1 -、-Y 4 - independently C 1-10 Alkyl, the C 1-10 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -T-, -N(R y1 )-, -C(O)O- and -C(O)N(R y1 )-group; and said C 1-10 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a ) is substituted with a group; wherein -R y1 、-R y2 、-R y2a Independently selected from H and C 1-4 alkyl;

[0617] -Y 2 -selected from -N(R 2 )-, -O-, -S- and -Se-;

[0618] -R 1 、-R 2 Independently selected from -H, C 1-5 Alkyl and -T;

[0619] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 3 substituted;

[0620] -R 3 selected from: halo, -CN, oxo, -C(O)OR 4 , -OR 4 , -C(O)R 4 , -C(O)N(R 4 )(R 4a ), -S(O)2N(R 4 )(R 4a ), -S(O)N(R 4 )(R 4a ), -S(O)2R 4 , -S(O)R 4 , -N(R 4 )S(O)2N(R 4a )(R 4b ), -SR 4 , -N(R 4 )(R 4a ), -NO2, -OC(O)R 4 , -N(R 4 )C(O)R 4a , -N(R 4 )S(O)2R 4a , -N(R 4 )S(O)R 4a , -N(R 4 )C(O)OR 4a , -N(R 4 )C(O)N(R 4a )(R 4b ), -OC(O)N(R 4 )(R 4a ), and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0621] wherein -R 4 , -R 4a , and -R 4b are independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0622] In some embodiments, -X'- has formula (y1), wherein the unstarred dashed line indicates the connection to -X-, and the starred dashed line indicates the connection to -FG1.

[0623] In some embodiments, -Y'- has formula (y2):

[0624]

[0625] wherein

[0626] the unmarked dashed line indicates the connection to -Y-, and the dashed line marked with an asterisk indicates the connection to -FG2, -L 3 , -L 4 , or -L 5 ;

[0627] -R 1 , -R 5 are independently selected from the group consisting of: -H, methyl, ethyl, propyl, and isopropyl; and

[0628] a1, a2 are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, preferably from 1, 2, 3, 4, 5, 6, 7, and 8, more preferably from 1, 2, 3, 4, 5, and 6, or even more preferably from 1, 2, and 3.

[0629] In some embodiments, -X'- has formula (y2), wherein the unmarked dashed line indicates the connection to -X-, and the dashed line marked with an asterisk indicates the connection to -FG1.

[0630] In some embodiments, -Y'- has formula (y3):

[0631]

[0632] wherein

[0633] the unmarked dashed line indicates the connection to -Y-, and the dashed line marked with an asterisk indicates the connection to -FG2, -L 3 , -L 4 , or -L 5 ; and

[0634] -R 1 , -R 5 are independently selected from the group consisting of: -H, methyl, ethyl, propyl, and isopropyl.

[0635] In some embodiments, -X'- has formula (y3), wherein the unmarked dashed line indicates the connection to -X-, and the dashed line marked with an asterisk indicates the connection to -FG1 or -L 3 .

[0636] In some embodiments, -Y'- has formula (y4):

[0637]

[0638] wherein

[0639] the unmarked dashed line indicates the attachment to -Y- and the dashed line marked with an asterisk indicates the attachment to -FG2, -L 3 , -L 4 or -L 5 .

[0640] In some embodiments, -X- has formula (y4), wherein the unmarked dashed line indicates the attachment to -X- and the dashed line marked with an asterisk indicates the attachment to -FG1 or -L 3 .

[0641] The hydrogel HA microspheres of the present application or a pharmaceutically acceptable salt thereof can advantageously be used as a drug carrier, as they prolong the half-life of a drug and exhibit physiological tolerability. Alternatively, they can be used for encapsulating drugs or other substances. Moreover, as hyaluronic acid exhibits inertness when exposed to tissue, the hydrogel HA microspheres can have medical uses and be used as dilators for Schlemm’s canal during viscocanalostomy. Other uses include medical cosmetic fillers, such as dermal fillers (for filling tissue space under the epidermis); biodegradable implants; injectable materials for regenerating articular cartilage; building artificial structures that can be used to build vascularized tissue; tissue engineering / enhancing or regenerative materials; grafting materials or injectable bone graft compositions, or as stem cell microsphere gel composites for subcutaneous injection.

[0642] The present application also relates to a method of preparing a drug conjugate or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps:

[0643] (a) providing the hydrogel HA microspheres or a pharmaceutically acceptable salt thereof obtained by any one of the methods of the present application, wherein the hydrogel comprises a plurality of Z 5 and / or Z 6 units having one or more unreacted -FG1 or -FG2;

[0644] (b) providing a mono-conjugation reagent D-L 1 -L 2 -FG3, a bi-conjugation reagent FG3-L 2 -L 1 -D-L 1 -L 2 -FG3 or a tri-conjugation reagent of formula (t):

[0645]

[0646] wherein -D is independently a drug moiety covalently and reversibly conjugated to -L 1 -

[0647] each -L 1 independently is a reversible linker moiety;

[0648] each -L 2 independently is a spacer moiety or a chemical bond;

[0649] each -FG3independently is a functional group that reacts with -FG1on Z 5 or -FG2on Z 6 ;

[0650] (c) mixing the hydrogel HA microspheres of step (a) with the mono-, di- or triconjugated reagent of step (b);

[0651] (d) optionally mixing the drug conjugate or a pharmaceutically salt thereof of step (c) with a blocking reagent; and

[0652] (e) collecting the drug conjugate or a pharmaceutically acceptable salt thereof of step (c) or (d).

[0653] The method of preparing a drug conjugate or a pharmaceutically acceptable salt thereof can further comprise the optional step of washing or purifying the drug conjugate obtained in step (c) or (d).

[0654] The present application also relates to a drug conjugate or a pharmaceutically acceptable salt thereof obtainable by the method of the present application.

[0655] The person skilled in the art will recognize that the drug conjugate or a pharmaceutically acceptable salt thereof of the present application can also comprise one or more unreacted -FG1or -FG2. In order to avoid unwanted reactions of said groups with other functional groups on the drug moiety or compounds that can be found at the site where the drug conjugate is administered, a blocking reagent is used.

[0656] Exemplary blocking reagents can be selected from the group consisting of:

[0657]

[0658] More specifically, in order to avoid unwanted reactions of unreacted maleimides on the HA hydrogel microspheres with nucleophilic compounds such as thiols, in particular glutathione, or functional groups such as amines on the drug moiety, said unreacted maleimides are reacted with a blocking reagent. Advantageously, the blocking reagent of formula (r01) inhibits the retro-Michael reaction and exchange reaction of the formed thiosuccinimide in the presence of other thiol containing compounds at physiological pH and temperature. This is particularly beneficial for the administration of the drug conjugate or a pharmaceutically acceptable salt thereof to tissues or organs where glutathione naturally occurs, such as the eye.

[0659] In some embodiments, the reagent of step (b) is a mono-conjugate reagent D-L 1 -L 2 -FG3. In some embodiments, the reagent of step (b) is a bis-conjugate reagent FG3-L 2 -L 1 -D-L 1 -L 2 -FG3. In some embodiments, the reagent of step (b) is a tris-conjugate reagent of formula (t).

[0660] mono-conjugate reagent D-L 1 -L 2 -FG3 can be obtained by any method known in the art. Subsequently, the resulting drug-reversible prodrug linker conjugate is labeled with a purification tag to form a mixture for purification. The mono-conjugate reagent D-L 1 -L 2 -FG3 is purified from the mixture by chromatographic separation, then the purification tag is removed from the labeled D-L 1 -L 2 -FG3 to form purified D-L 1 -L 2 -FG3.

[0661] As used herein, a "purification tag" refers to a moiety that, when conjugated to a second moiety, imparts one or more physical and / or chemical properties that are not present in the second moiety without the tag moiety, the different physical and / or chemical properties allowing for purification of such conjugates.

[0662] Purification tags within the scope of the present disclosure are described in WO 2015 / 052155 Al, which is incorporated by reference herein in its entirety. Thus, in some embodiments, the purification tag comprises a moiety of formula (ax):

[0663]

[0664] wherein the dotted line indicates the linkage to D-L 1 -L 2 -FG3;

[0665] -R 1 , -R 1a , -R 1b , -R 2 , -R 2a , -R 2b , -R 3 , -R 3a , -R 3b , -R 4 , -R4a , -R 4b each R is independently H or methyl;

[0666] each m is independently 1, 2, 3, 4, 5, 6, 7 or 8;

[0667] each n is independently 1, 2, 3, 4, 5, 6, 7 or 8;

[0668] each x is independently 1, 2, 3, 4, 5, 6, 7 or 8; and

[0669] each y is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0670] In some embodiments, -FG3has formula (y-56) or (y-57):

[0671]

[0672] wherein the dotted line indicates the attachment to -L 2 ; and

[0673] -Y 02 and -Y 02a are independently selected from -H and -Br.

[0674] In some embodiments, the reagent of step (b) is a mono-conjugating reagent D-L 1 -L 2 -FG3:

[0675]

[0676] As used herein throughout the specification, for convenience, the vast majority of structures are not depicted with stereochemistry, thus representing all possible stereoisomers.

[0677] In some embodiments, the reagent of step (b) is a mono-conjugating reagent D-L 1 -L 2 -FG3:

[0678]

[0679] The present application also relates to the use of the hydrogel microspheres of the present application or a pharmaceutically acceptable salt thereof as a carrier in a drug conjugate or a pharmaceutically acceptable salt thereof.

[0680] Another aspect of the present application relates to a drug conjugate comprising hydrogel HA microspheres or a pharmaceutically acceptable salt thereof, obtainable by the method of the present application.

[0681] The present application also relates to a drug conjugate or a pharmaceutically acceptable salt thereof comprising HA hydrogel microspheres comprising crosslinked HA chains covalently and reversibly conjugated to a plurality of drug moieties, the drug conjugate comprising a plurality of the following units:

[0682]

[0683] wherein

[0684] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked # or to a hydrogen atom;

[0685] The dashed line marked # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0686] wherein each -D is independently a drug moiety covalently and reversibly conjugated to -L 1 ;

[0687] each -L 4 is independently a linker moiety; and

[0688] each R a1 , -R a2 , -X-, -Y-, -X'-, 1 -Y'-, 2 -L 3 , and -L a1 is as defined elsewhere herein.

[0689] The skilled person understands that the drug conjugate or a pharmaceutically acceptable salt thereof of the present application can comprise small amounts of other HA units that can arise from various intramolecular reactions, for example, HA units in which -D acts as a crosslinking agent between two functionalized HA strands. In addition, the drug conjugate or a pharmaceutically acceptable salt thereof can comprise small amounts of HA units in which degradation or other modifications occur.

[0690] In some embodiments, the drug conjugate or a pharmaceutically acceptable salt thereof comprises HA hydrogel microspheres comprising crosslinked HA chains covalently and reversibly conjugated to a plurality of drug moieties, the drug conjugate comprising a plurality of each of the following units:

[0691]

[0692]

[0693] wherein the unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked # or to a hydrogen atom; the dashed line marked # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group; and each R a1 , -R a2、-X'-、-Y'-、-D、-L 1 -and-L 2 - used as defined elsewhere herein.

[0694] In some embodiments, each -L 3 -、-L 4 -or-L 5 -Independently selected from:

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701] For -L 3 -, one dotted line indicates the connection to -X'-, and the other dotted line indicates the connection to -Y'-; for -L 4 -, a dotted line indicates the difference between -L 2 -, another dotted line indicates the connection to -Y'-; and for -L 5 -, one dotted line indicates the connection to -Y'-, and the other dotted line indicates the connection to -BA;

[0702] -Y- is selected from -O-, -S-, -NR 05 -、-CR 05 R 05a -;

[0703] Each-R 04 、-R 04a 、-R 04b 、-R 04c 、-R 05 and -R 05a Independently selected from: halogen, -H, -CN, -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 06 substituted, and wherein C 1-50 Alkyl, C 2-50alkenyl and C 2-50 alkynyl is optionally interrupted with one or more groups selected from -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 07 )-, -S(O)2N(R 07 )-, -S(O)N(R 07 )-, -S(O)2-, -S(O)-, -N(R 07 )S(O)2N(R 07a )-, -S-, -N(R 07 )-, -OC(OR 07 )(R 07a )-, -N(R 07 )C(O)N(R 07a )- and -OC(O)N(R 07 )-;

[0704] each T 0 is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 is independently optionally substituted with one or more -R 06 , which are the same or different; and

[0705] each -R 06 , -R 07 , and -R 07a is independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

[0706] In some embodiments, all moieties -L 3 - have the same structure. In some embodiments, all moieties -L 4 - have the same structure. In some embodiments, all moieties -L 5 - have the same structure. In some embodiments, all moieties -L 3 - have the same structure, all moieties -L 4 - have the same structure and all moieties -L 5 - have the same structure, the structure between different moieties can be the same or different. In some embodiments, all moieties -L 3 -, all moieties -L 4 - and all moieties -L 5 - have the same chemical structure.

[0707] In some embodiments, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises HA hydrogel microspheres comprising crosslinked HA chains covalently and reversibly conjugated to a plurality of drug moieties, or a pharmaceutically acceptable salt thereof, the drug conjugate comprising a plurality of each of the following units:

[0708]

[0709] wherein

[0710] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0711] The dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0712] each R a1 , -R a2 , -X-, -Y-, -X'-, -Y'-, -D, -L 1 , -L 2 , -L 3 , -L 4 - are used as defined elsewhere herein;

[0713] each -L 5 is independently a linking moiety; and

[0714] each -BA is a capping agent.

[0715] The drug conjugate of the present application, or a pharmaceutically acceptable salt thereof, can comprise a range of about 50% to about 98% of Z 1 , a range of about 0.1% to about 20% of Z 2 , a range of about 0.1% to about 20% of Z 3 , and a range of about 0.1% to about 10% of Z 4 .

[0716] In some embodiments, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises a range of about 75% to about 98% of Z 1 , a range of about 0.1% to about 10% of Z 2 , a range of about 0.1% to about 10% of Z 3 , and a range of about 0.1% to about 5% of Z 4 .

[0717] In some embodiments, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises a range of about 78% to about 96% of Z 1 , a range of about 2% to about 10% of Z 2 , a range of about 1% to about 7% of Z 3 , and a range of about 0.5% to about 5% of Z 4 .

[0718] In some embodiments, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises Z in the range of about 90.5% to about 94.8% 1 , Z in the range of about 2.7% to about 6.4% 2 , Z in the range of about 1.1% to about 2.1% 3 , and Z in the range of about 0.8% to about 1.9% 4 .

[0719] Suitably, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises about 92.9% Z 1 , about 4.3% Z 2 , about 1.5% Z 3 , and about 1.3% Z 4 .

[0720] It can be appreciated that the percentages provided above are calculated based on the total number of units present in the drug conjugate.

[0721] Exemplary capping agents can be selected from:

[0722]

[0723] wherein the dotted line indicates the point of attachment to -L 5 -.

[0724] In some embodiments, the capping agent has formula (b01), (b02), or (b04). In some embodiments, the capping agent has formula (b01). In some embodiments, the capping agent has formula (b02). In some embodiments, the capping agent has formula (b03). In some embodiments, the capping agent has formula (b04). In some embodiments, the capping agent has formula (b05).

[0725] Suitably, the drug conjugate, or a pharmaceutically acceptable salt thereof, comprises HA hydrogel microspheres comprising crosslinked HA chains covalently and reversibly conjugated to a plurality of drug moieties, or a pharmaceutically acceptable salt thereof, wherein the drug conjugate comprises a plurality of each of the following units:

[0726]

[0727]

[0728] wherein

[0729] The unmarked dotted line indicates the point of attachment to the adjacent unit at the dotted line marked with # or to a hydrogen atom;

[0730] The dotted line marked with # indicates the point of attachment to the adjacent unit at the unmarked dotted line or to a hydroxyl group;

[0731] each R a1 is independently selected from the group consisting of -H, C 1-10 alkyl, an ammonium ion, a tetrabutylammonium ion, a cetyltrimethylammonium ion, an alkali metal ion, and an alkaline earth metal ion;

[0732] each -R a2 is independently -H or C 1-10 alkyl;

[0733] each -X'-, -Y' is independently a spacer moiety or absent;

[0734] wherein each -D is independently a drug moiety covalently and reversibly conjugated to -L 1 ;

[0735] each -L 1 is independently a reversible linker moiety;

[0736] each -L 2 is independently a spacer moiety or absent; and

[0737] wherein the drug conjugate comprises a range of about 50% to about 98% of Z 1 , a range of about 0.1% to about 20% of Z 2 -i, a range of about 0.1% to about 20% of Z 3 -i and a range of about 0.1% to about 10% of Z 4 -i.

[0738] In particular, the above drug conjugate comprises a range of about 86% to about 96% of Z 1 , a range of about 0.1% to about 12.9% of Z 2 -i, a range of about 0.34% to about 3.57% of Z 3 -i and a range of about 0.1% to about 9.5% of Z 4 -i.

[0739] Advantageously, the drug conjugate or pharmaceutically acceptable salt thereof comprises HA hydrogel microspheres comprising crosslinked HA chains covalently and reversibly conjugated to a plurality of drug moieties or pharmaceutically acceptable salts thereof, wherein the drug conjugate comprises a plurality of each of the following units:

[0740]

[0741]

[0742] wherein

[0743] the unlabelled dashed line indicates a point of attachment to the adjacent unit at the dashed line marked with # or to a hydrogen atom;

[0744] Dashed lines marked with # indicate the point of attachment to an adjacent unit at an unlabeled dashed line or to a hydroxyl group; and

[0745] Each R a1 is -H or alkali metal ion;

[0746] Each-R a2 is -H;

[0747] Each -X'- has (x0):

[0748]

[0749] in

[0750] Unlabeled dashed lines indicate attachment to the carbonyl group, and dashed lines marked with an asterisk indicate attachment to the sulfur atom;

[0751] v0 is selected from 0 and 1;

[0752] -X 1 -、-X 4 - independently C 1-10 Alkyl, the C 1-10 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -T-, -N(R y1 )-, -C(O)O- and -C(O)N(R y1 )-group; and said C 1-10 The alkyl chain is optionally substituted with one or more independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a ) group substitution;

[0753] Where -R y1 、-R y2 、-R y2a Independently selected from -H and C 1-4 alkyl;

[0754] -X 2 -selected from -N(R 1 )-, -O-, -S- and -Se-;

[0755] =X 3 Selected from =O, =N(R 1 ) and =S;

[0756] -X 5 -It's C 1-20 Alkyl, the C 1-20 The alkyl group is optionally interrupted by one or more groups independently selected from -O-, -C(O)O-, -T-, -N(R y1)-and -N(R y1 )C(O)-; and said C 1-20 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -NH(R y1 ) and -C(O)N(R y2 R y2a );

[0757] -R 1 is independently selected from -H, C 1-5 alkyl and -T;

[0758] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more R 2 groups, which are the same or different;

[0759] -R 2 is selected from: halo, -CN, oxo, -C(O)OR 3 , -OR 3 , -C(O)R 3 , -C(O)N(R 3 )(R 3a ), -S(O)2N(R 3 )(R 3a ), -S(O)N(R 3 )(R 3a ), -S(O)2R 3 , -S(O)R 3 , -N(R 3 )S(O)2N(R 3a )(R 3b ), -SR 3 , -N(R 3 )(R 3a ), -NO2, -OC(O)R 3 , -N(R 3 )C(O)R 3a , -N(R 3 )S(O)2R 3a , -N(R 3 )S(O)R 3a , -N(R 3 )C(O)OR 3a , -N(R 3 )C(O)N(R 3a )(R 3b ), -OC(O)N(R 3 )(R 3a ), and C 1-6alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and

[0760] wherein -R 3 , -R 3a , and -R 3b are independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0761] each -Y' has the formula (y0):

[0762]

[0763] wherein

[0764] the unlabelled dotted line indicates the attachment to the carbonyl group, and the dotted line labelled with an asterisk indicates the attachment to the nitrogen atom of the thiosuccinimide ring;

[0765] -Y 1 -, -Y 4 - is independently C 1-10 alkyl, which C 1-10 alkyl is optionally interrupted with one or more groups independently selected from the group consisting of -0-, -T-, -N(R y1 )-, -C(O)O-, and -C(O)N(R y1 )-; and which C 1-10 alkyl chain is optionally substituted with one or more groups independently selected from the group consisting of -OH, -T, -NH(R y1 ), and -C(O)N(R y2 R y2a ); wherein -R y1 , -R y2 , -R y2a are independently selected from the group consisting of H and C 1-4 alkyl;

[0766] -Y 2 - is selected from the group consisting of -N(R 2 )-, -O-, -S-, and -Se-;

[0767] =Y 3 is selected from the group consisting of =O, =N(R 2 ), and =S;

[0768] -R 1 , -R 2 are independently selected from the group consisting of -H, C 1-5 alkyl, and -T;

[0769] wherein each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 3 replace;

[0770] -R 3 Selected from: halogen, -CN, oxo, -C(O)OR 4 、-OR 4 、-C(O)R 4 、-C(O)N(R 4 )(R 4a )、-S(O)2N(R 4 )(R 4a )、-S(O)N(R 4 )(R 4a )、-S(O)2R 4 、-S(O)R 4 、-N(R 4 )S(O)2N(R 4a )(R 4b ),-SR 4 、-N(R 4 )(R 4a )、-NO2、-OC(O)R 4 、-N(R 4 )C(O)R 4a 、-N(R 4 )S(O)2R 4a 、-N(R 4 )S(O)R 4a 、-N(R 4 )C(O)OR 4a 、-N(R 4 )C(O)N(R 4a )(R 4b )、-OC(O)N(R 4 )(R 4a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and

[0771] Where -R 4 、-R 4a and -R 4b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0772] Each -D is independently covalently and reversibly conjugated to -L 1 The drug part;

[0773] Each-L 1 - is a linker moiety of formula (I):

[0774]

[0775] in

[0776] The dashed line indicates the connection to the nitrogen of -D via formation of an amide bond;

[0777] -X- is -C(R 4 R 4a )-;-N(R 4 )-;-O-;-C(R 4 R 4a )-C(R 5 R 5a )-;-C(R 5 R 5a )-C(R 4 R 4a )-;-C(R 4 R 4a )-N(R 6 )-;-N(R 6 )-C(R 4 R 4a )-;-C(R 4 R 4a )-O-;-OC(R 4 R 4a )-;or-C(R 7 R 7a )-;

[0778] X 1 is C; or S(O);

[0779] -X 2 -for-C(R 8 R 8a )-;or-C(R 8 R 8a )-C(R 9 R 9a )-;

[0780] =X 3 =O; =S; or =N-CN;

[0781] -R 1 、-R 1a 、-R 2 、-R 2a 、-R 4 、-R 4a 、-R 5 、-R 5a 、-R 6, -R 8 , -R 8a , -R 9 , -R 9a are independently selected from -H and C 1-6 alkyl;

[0782] -R 3 , -R 3a are independently selected from -H and C 1-6 alkyl, provided that in the case where one or both of -R 3 , -R 3a are not -H, they are attached to N to which they are attached via an sp 3 hybridized carbon atom;

[0783] -R 7 is -N(R 10 R 10a ); or -NR 10 -(C=0)-R 11 ;

[0784] -R 7a , -R 10 , -R 10a , -R 11 are independently -H; or C 1-10 alkyl;

[0785] Optionally, one or more of -R 1a -R 4a , -R 1a -R 5a , -R 1a -R 7a , -R 4a -R 5a , -R 8a -R 9a form a chemical bond;

[0786] Optionally, one or more of -R 1 -R 1a , -R 2 -R 2a , -R 4 -R 4a , -R 5 -R 5a , -R 8 -R 8a , -R 9 -R 9a , taken together with the atoms to which they are attached, form a C 3-10 cycloalkyl; or a 3- to 10-membered heterocyclyl;

[0787] Optionally, one or more of -R1 -R 4 -R 1 -R 5 -R 1 -R 6 -R 1 -R 7a -R 4 -R 5 -R 4 -R 6 -R 8 -R 9 -R 2 -R 3 one or more pairs of -R

[0788] -R 3 -R 3a together with the nitrogen atom to which they are attached form a 3- to 10-membered heterocycle;

[0789] ring A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and

[0790] each -L 1 - is substituted with -L 2 -; provided that the hydrogen marked with an asterisk in formula (I) is not replaced with a substituent; or

[0791] each -L 1 - is a linker moiety of formula (XI):

[0792]

[0793] wherein

[0794] the dotted line indicates the attachment to the nitrogen of the primary or secondary amine of -D;

[0795] v is selected from 0 or 1 ;

[0796] -X 1 - is selected from the group consisting of -C(R 8 )(R 8a )-, -N(R 9 )- and -O-;

[0797] =X 2 is selected from =O and =N(R 10 );

[0798] -X 3 is selected from -O, -S and -Se;

[0799] each p is independently selected from 0 or 1, provided that at most one p is 0;

[0800] -R 6 , -R 6a , -R 10 is independently selected from the group consisting of: -H, -C(R 11 )(R 11a )(R 11b ), and -T;

[0801] -R 9 is selected from the group consisting of: -C(R 11 )(R 11a )(R 11b ), and -T;

[0802] -R 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 4 , -R 4a , -R 5 , -R 5a , -R 7 , -R 8 -R 8a , -R 11 , -R 11a , and -R 11b are independently selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with one or more -R 13 , which are the same or different; and wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally interrupted with one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R 14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14 )C(O)N(R 14a )-, and -OC(O)N(R 14 )-;

[0803] -R 12 , -R 12a , and -R 12b are independently selected from the group consisting of -H, -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally substituted with one or more -R 13 , which are the same or different; and wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl is optionally interrupted with one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R 14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14)C(O)N(R 14a )- and -OC(O)N(R 14 )-;

[0804] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 13 groups, which are the same or different;

[0805] -R 13 is selected from: halo, -CN, oxo, -C(O)OR 15 , -OR 15 , -C(O)R 15 , -C(O)N(R 15 )(R 15a ), -S(O)2N(R 15 )(R 15a ), -S(O)N(R 15 )(R 15a ), -S(O)2R 15 , -S(O)R 15 , -N(R 15 )S(O)2N(R 15a )(R 15b ), -SR 15 , -N(R 15 )(R 15a ), -NO2, -OC(O)R 15 , -N(R 15 )C(O)R 15a , -N(R 15 )S(O)2R 15a , -N(R 15 )S(O)R 15a , -N(R 15 )C(O)OR 15a , -N(R 15 )C(O)N(R 15a )(R 15b ), -OC(O)N(R 15 )(R 15a ), and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0806] wherein -R 14 , -R 14a , -R 15 , -R 15a , and -R 15bindependently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[0807] optionally, one or more pairs of -R 1 -R 1a , -R 2 -R 2a , -R 3 -R 3a , -R 4 -R 4a , -R 5 -R 5a , or -R 8 -R 8a form, together with the atoms to which they are attached, a C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, or 8- to 11-membered heterobicyclyl;

[0808] optionally, one or more pairs of -R 1 -R 2 , -R 1 -R 8 , -R 1 -R 9 , -R 2 -R 9 , or -R 2 -R 10 form, together with the atoms to which they are attached, a ring-A-;

[0809] wherein -A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl;

[0810] optionally, one or more pairs of -R 3 -R 6 , -R 4 -R 6 , -R 5 -R 6 , -R 6 -R 6a , or -R 6 -R 7 form, together with the atoms to which they are attached, a ring-A'-;

[0811] wherein -A'- is selected from the group consisting of 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl; and

[0812] -L 1 substituted with -L 2- substituted, with the proviso that the hydrogen marked with an asterisk in formula (XI) is not replaced by a substituent;

[0813] each -L 2 - is a spacer moiety, and

[0814] wherein the drug conjugate comprises Z in the range of about 50% to about 98% 1 , Z in the range of about 0.1% to about 20% 2 - i. Z in the range of about 0.1% to about 20% 3 - i and Z in the range of about 0.1% to about 10% 4 - i.

[0815] The unit percentages in the HA microspheres or drug conjugates and pharmaceutically acceptable salts thereof provided throughout this application can be determined by any method known to one of skill in the art and based on the information contained in the following paragraphs. Amine-functionalized HA can be obtained by coupling a diamine to one or more carboxyl groups of HA having a Mw of about 100 kDa to about 150 kDa. Prior to functionalization, the HA can be optionally purified according to methods known in the art. The degree of substitution of the amine-functionalized HA obtained can be in the range of about 3% to about 15%, for example, about 3% to about 6% or, for example, about 9% to 15%. The degree of substitution can be determined by any method known in the art, for example, by an ortho-phenylenediamine (OPA) assay based on the reaction of OPA with primary amines or by an enzymatic assay. Maleimide-functionalized HA can be obtained by further converting the terminal amino groups on the amine-functionalized HA to N-substituted amides having a spacer moiety (-L 2″ -) containing a maleimide group at the terminus. The degree of substitution of the maleimide-functionalized HA obtained can be in the range of about 9% to about 15%. The degree of substitution can be determined by any method known in the art, for example, a reverse Ellman’s assay (i.e., by reacting the free maleimide groups with a known excess of 2-mercaptoethanol at neutral pH and then determining the remaining thiol by Ellman’s assay) or by an enzymatic assay. Thiol-functionalized HA can be obtained by further converting the terminal amino groups on the amine-functionalized HA to N-substituted amides having a spacer moiety (-L 2″ -) containing a thiol group at the terminus. The degree of substitution of the thiol-functionalized HA obtained can be in the range of about 3% to about 6%. The degree of substitution can be determined by any method known in the art, for example, by Ellman’s assay or by an enzymatic assay. Varying the degree of maleimide and thiol functionalization allows for different crosslinking densities and different drug loadings in the hydrogel conjugates described throughout this application. Moreover, the methods of preparing thiol and maleimide-functionalized HA are described in WO 2018 / 175788 Al, which is hereby incorporated by reference in its entirety.

[0816] The obtained maleimide-functionalized HA and thiol-functionalized HA are reacted with each other in a ratio (w / w) that can be from about 10: 1 to 0.6: 1, for example, from about 8: 1 to 1.1: 1, for example, from about 5: 1 to 1.2: 1, or for example, from about 3: 1 to 1.5: 1, advantageously about 2.15: 1, to provide HA microspheres. It is assumed that the reaction goes to completion, i.e., all thiol functional groups have reacted. Thus, the amount of unreacted maleimide on the HA microspheres can be determined, for example, by the reverse Ellman’s assay or enzymatic assay. Based on the non-swollen, completely dried (lyophilized) hydrogel, i.e., the above calculation does not take into account water or swelling behavior, the determined amount of maleimide on the resulting hydrogel HA microspheres can be from 80 to 250 pmol / g. To obtain the drug conjugate or pharmaceutically acceptable salt thereof, the hydrogel HA microspheres are mixed with the monoconjugate D-L 1 -L 2 -FG3 (wherein -D, -L 1 -, -L 2 - and -FG3 are used as described elsewhere herein) in a molar ratio that can be from about 0.5 (maleimide functional groups): 1 (monoconjugate) to about 3 (maleimide functional groups): 1 (monoconjugate), advantageously 1.2 (maleimide functional groups): 1 (monoconjugate). An excess of capping reagent (as described elsewhere herein) is then added. When the drug is a protein, there are multiple ways to determine the protein content. The protein content is determined by quantitative amino acid analysis. The drug conjugate or pharmaceutically acceptable salt thereof is incubated with 6 M HC1 / TFA in a microwave apparatus at 190 °C for about 30 minutes, which breaks the protein down into individual amino acids. After hydrolysis, the amino acids are derivatized with carbamidate 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate to yield stable, UV-active urea compounds. Subsequently, the derivatives are separated by reverse phase chromatography and quantified by UV detection. The concentration of the amino acid derivatives can then be converted to the protein concentration. Another method to determine the protein loading is to utilize enzymatic hydrolysis (e.g., hyaluronidase or chondroitinase) to perform HA hydrolysis or HA crosslinker hydrolysis at elevated pH / temperature followed by protein quantification at 280 nm.

[0817] In some embodiments, one alternative method to depict the drug conjugate or pharmaceutically acceptable salt thereof of the present application is shown below, i.e., as a drug conjugate comprising a plurality of HA strand 2A and a plurality of HA strand 2B, wherein:

[0818] each 2A comprises a plurality of linearly linked Z 1 , Z 3 (a), Z 2 , and Z 4 units:

[0819]

[0820]

[0821] each 2B comprises a plurality of Z 1 and Z 3 (b) units:

[0822]

[0823] wherein

[0824] unlabeled dashed lines indicate points of attachment to adjacent units at labeled dashed lines or to a hydrogen atom;

[0825] labeled dashed lines indicate points of attachment to adjacent units at unlabeled dashed lines or to a hydroxyl group;

[0826] dashed lines labeled with an * represent cross-linking points between units Z 3 (a) and Z 3 (b), such that at least one 2A is cross-linked to at least one 2B;

[0827] each -D, R a1 , -R a2 , -X-, -Y-, -X'-, 1 -, -L 2 -, -L 4 -, -L 5 -, -BA are as defined elsewhere herein; and

[0828] each -L 3a - is and each -L 3b - is

[0829] In some embodiments, the drug conjugate of the application, or a pharmaceutically acceptable salt thereof, is shown below, i.e., as a drug conjugate comprising a plurality of HA strands 2A and a plurality of HA strands 2B, wherein:

[0830] each 2A comprises a plurality of Z 1 , Z 3 -i(a), Z 2 -i, and Z 4 -i units:

[0831]

[0832]

[0833] each 2B comprises a plurality of Z 1 and Z 3- i(b) units:

[0834]

[0835] wherein

[0836] The unmarked dashed line indicates a point of attachment to an adjacent unit at the dashed line marked with a # or to a hydrogen atom;

[0837] The dashed line marked with a # indicates a point of attachment to an adjacent unit at the unmarked dashed line or to a hydroxyl group;

[0838] The dashed line marked with a * represents a unit Z 3 - i(a) and Z 3 - i(b) are crosslinked such that at least one 2A is crosslinked to at least one 2B;

[0839] each R a1 is H or an alkali metal ion;

[0840] each -R a2 is -H;

[0841] each -D is as defined elsewhere herein;

[0842] each -X' - has the formula (x4):

[0843]

[0844] wherein the unmarked dashed line indicates a point of attachment to a carbonyl group, the dashed line marked with an asterisk indicates a point of attachment to a sulfur atom, and c0 is 7;

[0845] each -Y' - has the formula (y4):

[0846]

[0847] wherein the unmarked dashed line indicates a point of attachment to a carbonyl group, and the dashed line marked with an asterisk indicates a point of attachment to a nitrogen atom of the maleimide ring;

[0848] each -L 2 - L 1 - has the formula (s1) or (s2):

[0849]

[0850] wherein the dashed line indicates a point of attachment to a sulfur atom, and the dashed line marked with an asterisk indicates a point of attachment to -D.

[0851] It is emphasized that in the above embodiments, the -D moiety is only suitably attached to the 2A chain, i.e. not to the 2B chain. Similarly, the capping agent is also only attached to the 2A chain.

[0852] Generally, throughout the application, it is understood that in the functionalized HA (HA strand) and in 1A, 1B, 2A and 2B, the disaccharide units are connected to each other via beta-1,4 and beta-1,3 glycosidic bonds.

[0853] The present application also relates to a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof, obtainable or obtained by the method of the present application.

[0854] Another aspect of the present application is a pharmaceutical composition comprising a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof of the present application and at least one pharmaceutically acceptable excipient.

[0855] The present application also relates to a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present application for use as a medicament.

[0856] The present application also relates to a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present application for use as a medicament.

[0857] The present application also relates to a pharmaceutical conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present application for use in a method of treatment of a disease treatable with D-H or D or a pharmaceutically acceptable salt thereof, for example an ocular disease.

[0858] Exemplary eye conditions are selected from: age-related macular degeneration (AMD), macular degeneration, macular edema, diabetic macular edema (DME), retinopathy, diabetic retinopathy (DR), other ischemia-related retinopathies, retinopathy of prematurity (ROP), retinal vein occlusion (RVO), CNV, corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, uveitic macular edema, branch retinal vein occlusion (BRVO), central retinal vein occlusion (CRVO), submacular hemorrhage, polypoidal choroidal vasculopathy (PCV), retinal microaneurysms, retinal arterial occlusion (RAO), branch retinal arterial occlusion (BRAO), central retinal arterial occlusion (CRAO), subfoveal hemorrhage, subretinal hemorrhage, radiation retinopathy, exudative retinal detachment, Eales disease, neovascular macular disease) and Sjogren's disease.

[0859] The ocular disorder can also be selected from the group consisting of age-related macular degeneration (AMD), macular degeneration, macular oedema, diabetic macular oedema (DME), retinopathy, diabetic retinopathy (DR), other ischaemia-related retinopathies, retinopathy of prematurity (ROP), retinal vein occlusion (RVO), CNV, corneal neovascularisation, diseases associated with corneal neovascularisation, retinal neovascularisation, diseases associated with retinal / choroidal neovascularisation, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, familial exudative vitreoretinopathy (FEVR), Coats' disease, Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular capillary telangiectasia, iridal neovascularisation, intraocular neovascularisation, retinal degeneration, cystoid macular oedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis, Leber congenital amaurosis, uveitis, choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury and Sjogren's disease.

[0860] Suitably, the ocular disease is selected from AMD, DME, DR or RVO. Preferably, the ocular disease is AMD.

[0861] A further aspect of the application is a method of preventing or treating a disease which can be prevented or treated with D-H or D, such as an ocular disease, the method comprising the step of administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical conjugate of the application, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.

[0862] In some embodiments, all -D moieties of the pharmaceutical conjugate, or pharmaceutically acceptable salt thereof, are the same, i.e. have the same chemical structure. In this case, all -D moieties of the pharmaceutical conjugate are derived from the same type of drug molecule.

[0863] ​In some embodiments, the drug conjugates of the present invention or pharmaceutically acceptable salts thereof contain different -D moieties, i.e., -D moieties with different chemical structures. These different structures are derived from different types of drug molecules. In some embodiments, the drug conjugates of the present invention contain two different types of -D moieties. In some embodiments, the drug conjugates of the present invention contain three different types of -D moieties. In some embodiments, the drug conjugates of the present invention contain four different types of -D moieties. In some embodiments, the drug conjugates of the present invention contain five different types of -D moieties.

[0864] If the drug conjugate of the present invention contains more than one type of -D, all -D moieties may be conjugated to the same type of -L. 1 - combined with, or can be used with different types of -L 1 - combined, i.e., the first type of -D can be combined with the first type of -L 1 -Combined, the second type of -D can be combined with the second type of -L 1 -binding, etc. In some embodiments, different types of -L are used 1 - may allow for different release kinetics of different types of -D, such as a faster release of a first type of -D, a moderate release of a second type of -D, and a slow release of a third type of -D. Thus, in some embodiments, the drug conjugates of the present invention comprise one type of -L 1 -. In some embodiments, the drug conjugates of the present invention comprise two types of -L 1 In some embodiments, the drug conjugates of the present invention comprise three types of -L 1 In some embodiments, the drug conjugates of the present invention comprise four types of -L 1 -.

[0865] In some embodiments, the drug conjugates of the present invention comprise one type of -D and one type of -L 1 In some embodiments, the drug conjugates of the present invention comprise two types of -D and two types of -L 1 In some embodiments, the drug conjugates of the present invention comprise three types of -D and three types of -L 1 In some embodiments, the drug conjugates of the present invention comprise four types of -D and four types of -L 1 -.

[0866] In some embodiments, all -L 1 - moieties have the same structure. In some embodiments, the drug conjugate comprises two or more different types of -L 1-parts, such as two, three, four or five different types of -L 1 -part. The two or more different types of -L 1 - moieties can be combined with -D of the same or different types. Use different types of -L 1 - allowing the release of the same or different types of drugs DH from the conjugate of the present invention with different release half-lives, for example, a first group of drugs - L having a short release half-life 1 -Part with a second group with a long half-life -L 1 -Partial combination.

[0867] In some embodiments, -D is selected from the group consisting of: a small molecule drug moiety, a medium size drug moiety, a peptide drug moiety, and a protein drug moiety.

[0868] In some embodiments, -D is a small molecule drug moiety.

[0869] In some embodiments, -D is a peptide drug moiety. In some embodiments, -D is a protein drug moiety. In some embodiments, such a protein moiety is a monoclonal or polyclonal antibody or a fragment or fusion thereof.

[0870] In some embodiments, -L 1 - a cysteine ​​residue linked to -D. In some embodiments, -L 1 - is linked to a histidine residue of -D. In some embodiments, -L 1 - is linked to a lysine residue of -D. In some embodiments, -L 1 - a tryptophan residue linked to -D. In some embodiments, -L 1 - is linked to a serine residue of -D. In some embodiments, -L 1 - is linked to a threonine residue of -D. In some embodiments, -L 1 - is linked to a tyrosine residue of -D. In some embodiments, -L 1 - is linked to an aspartic acid residue of -D. In some embodiments, -L 1 -a glutamic acid residue linked to -D. In some embodiments, -L 1 -Arginine residue linked to -D.

[0871] In some embodiments, at least one -L 1 -moiety is linked to the amino acid residue of -D and one or more additional -L 1 The -moiety is linked to the modifying moiety present in -D.

[0872] -L 1- can be connected to -D via any type of linkage, provided that the linkage is reversible.

[0873] In some embodiments, -L 1 - is connected to -D via a linkage selected from amide, ester, carbamate, acetal, acetal amine, imine, oxime, hydrazone, disulfide, and acylguanidine. In some embodiments, -L 1 - is connected to -D via a linkage selected from amide, ester, carbamate, and acylguanidine. It will be appreciated that such linkages can themselves be irreversible, but reversibility can be -L 1 - the role of certain atomic groups or moieties present in -L

[0874] In some embodiments, -L 1 - is connected to -D via an ester linkage. In some embodiments, -L 1 - is connected to -D via a carbamate linkage. In some embodiments, -L 1 - is connected to -D via an acylguanidine linkage. In some embodiments, -L 1 - is connected to -D via an amide linkage.

[0875] In some embodiments, -L 1 - is connected to -D via the nitrogen of a side chain amine function of a lysine residue of -D. Suitably, -L 1 - is connected to -D via the nitrogen of an amine function, for example the nitrogen of a side chain amine function of a lysine residue of -D, and the linkage formed between -D and -L 1 - is an amide.

[0876] In some embodiments, -L 1 - has a structure as disclosed in WO 2009 / 095479 A2, which is incorporated herein by reference in its entirety. Thus, in some embodiments, -L 1 - moiety has the formula (I):

[0877]

[0878] wherein

[0879] the dotted line indicates attachment to the nitrogen of -D by formation of an amide bond;

[0880] - X- is -C(R 4 R 4a )-; -N(R 4 )-; -O-; -C(R 4 R 4a )-C(R 5 R 5a )-; -C(R 5 R5a )-C(R 4 R 4a )-; -C(R 4 R 4a )-N(R 6 )-; -N(R 6 )-C(R 4 R 4a )-; -C(R 4 R 4a )-O-; -O-C(R 4 R 4a )-; or -C(R 7 R 7a )-;

[0881] X 1 is C; or S(O);

[0882] -X 2 - is -C(R 8 R 8a )-; or -C(R 8 R 8a )-C(R 9 R 9a )-;

[0883] = X 3 is =O; =S; or =N-CN;

[0884] -R 1 , -R 1a , -R 2 , -R 2a , -R 4 , -R 4a , -R 5 , -R 5a , -R 6 , -R 8 , -R 8a , -R 9 , -R 9a are independently selected from -H and C 1-6 alkyl;

[0885] -R 3 , -R 3a are independently selected from -H and C 1-6 alkyl, provided that in the case that one or both of -R 3 , -R 3a are not -H, they are attached to N to which they are attached via an sp 3 heteroatom carbon;

[0886] -R 7 is -N(R 10 R10a ); or -NR 10 -(C=O)-R 11 ;

[0887] -R 7a 、-R 10 、-R 10a 、-R 11 are independently -H; or C 1-10 alkyl;

[0888] Optionally, -R 1a / -R 4a 、-R 1a / -R 5a 、-R 1a / -R 7a 、-R 4a / -R 5a 、-R 8a / -R 9a One or more pairs of them form chemical bonds;

[0889] Optionally, -R 1 / -R 1a 、-R 2 / -R 2a 、-R 4 / -R 4a 、-R 5 / -R 5a 、-R 8 / -R 8a 、-R 9 / -R 9a One or more pairs of atoms in the C 3-10 Cycloalkyl; or 3- to 10-membered heterocyclic group;

[0890] Optionally, -R 1 / -R 4 、-R 1 / -R 5 、-R 1 / -R 6 、-R 1 / -R 7a 、-R 4 / -R 5 、-R 4 / -R 6 、-R 8 / -R 9 、-R 2 / -R 3 One or more pairs of them, together with the atoms to which they are attached, combine to form ring A;

[0891] Optionally, -R 3 / -R3a Together with the nitrogen atom to which they are attached, they form a 3- to 10-membered heterocyclic ring;

[0892] Ring A is selected from: phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and

[0893] Each-L 1 -被-L 2 -substituted and optionally further substituted, provided that hydrogens marked with an asterisk in formula (I) are not replaced by substituents.

[0894] In some embodiments, -L 1 - has formula (I), wherein the dashed line indicates the connection to the nitrogen of the amine of the lysine side chain of -D. In some embodiments, -L 1 - has formula (I), wherein the dashed line indicates the connection to the nitrogen of the amine at the N-terminus of -D. In some embodiments, -L of formula (I) 1 - has not been further replaced.

[0895] It is understood that if -R 3 / -R 3a Together with the nitrogen atom to which they are attached, they form a 3- to 10-membered heterocyclic ring, and only those in which the atom directly attached to the nitrogen is sp 3 In other words, -R 3 / -R 3a Together with the nitrogen atom to which they are attached, these 3- to 10-membered heterocyclic rings have the following structure:

[0896]

[0897] in

[0898] Dashed line indicates -L 1 - the rest of the connection;

[0899] The ring contains 3 to 10 atoms including at least one nitrogen; and

[0900] R # and R ## Indicates sp 3 Hybridized carbon atoms.

[0901] It is also understood that the 3- to 10-membered heterocyclic ring may be further substituted.

[0902] -R of formula (I) 3 / -R 3a Illustrative embodiments of suitable 3- to 10-membered heterocyclic rings formed together with the nitrogen atom to which they are attached are as follows:

[0903]

[0904] in

[0905] Dashed lines indicate connections to the rest of the molecule; and

[0906] -R is selected from -H and C 1-6 alkyl.

[0907] -L of formula (I) 1 - may be optionally further substituted. Generally, any substituent may be used as long as the cleavage principle is not affected, that is, the hydrogens marked with an asterisk in formula (I) are not replaced and Part of the nitrogen is still part of the primary, secondary or tertiary amine, that is, -R 3 and -R 3a Independently of each other -H or via sp 3 The hybridized carbon atom is connected to -N<.

[0908] In some embodiments, -R 1 or -R 1a L 2 In some embodiments, -R 2 or -R 2a L 2 In some embodiments, -R 3 or -R 3a L 2 In some embodiments, -R 4 L 2 In some embodiments, -R 5 or -R 5a L 2 In some embodiments, -R 6 L 2 In some embodiments, -R 7 or -R 7a L 2 In some embodiments, -R 8 or -R 8a L 2 In some embodiments, -R 9 or -R 9a L 2 -replace.

[0909] Suitably, -R 11-L 2 - substituted.

[0910] In some embodiments, -L 1 - has a structure as disclosed in WO 2018 / 193408 Al, which is incorporated herein by reference in its entirety. Thus, in some embodiments, -L 1 - is partially of formula (II):

[0911]

[0912] wherein

[0913] the dotted line indicates the connection to -D, which is a primary amine, a secondary amine, or a ring nitrogen atom of a nitrogen heteroaromatic ring;

[0914] -R 1 is -H or C1-C4 alkyl;

[0915] -R 1a is -H or C1-C4 alkyl, or -CR 1 R 1a in combination form a C3-C6 cycloalk-1,1 -diyl;

[0916] -R 2 is independently at each occurrence selected from C1-C4 alkyl or oxo, or two -R 2 groups in combination form a fused C3-C6 cycloalkyl or a spiro C3-C6 cycloalk-1,1 -diyl;

[0917] a is 0, 1, 2, 3, or 4;

[0918] -R 3 is -H or C1-C4 alkyl;

[0919] -R 3a is -H or C1-C4 alkyl, or -CR 3 R 3a in combination form a C3-C6 cycloalk-1,1 -diyl;

[0920] -Y is -C(O)R 4 , -C(O)OR 4 , -C(O)NHR 4 , -C(O)NR 5 R 6 , -SiR 5 R 6 R 7 or -CR 12 R 12a OR 13 ;

[0921] -R12 is -H or C1-C4alkyl;

[0922] -R 12a is -H or C1-C4alkyl, or -CR 12 R 12a forms a C3-C6cycloalk-1,1-diyl in combination;

[0923] -R 13 is C1-C4alkyl; or -CHR 12 OR 13 forms a 5-, 6-, or 7-membered cyclic ether in combination;

[0924] -R 4 is C1-C8alkyl or C3-C7cycloalkyl, wherein cycloalkyl is optionally substituted with 0, 1, or 2 independently selected C1-C4alkyl groups, and wherein alkyl is optionally substituted with C1-C4alkoxy;

[0925] -R 5 and -R 6 are each independently selected from C1-C4alkyl and C3-C6cycloalkyl;

[0926] -R 7 is C1-C8alkyl, C3-C7cycloalkyl, C1-C8alkoxy, C3-C7cycloalkoxy, heterocycloalkyloxy, or -(OCHR 3 CH2) b O-C1-C4alkyl, wherein heterocycloalkyloxy is a 4- to 7-membered saturated heterocyclic ring having one ring heteroatom selected from N, O, and S and optionally substituted with 0, 1, or 2 independently selected C1-C4alkyl groups;

[0927] b is an integer ranging from 1 to 10;

[0928] -Z is -CH-L 2 - or -N-L 2 -; and

[0929] wherein each -L 1 - is optionally further substituted.

[0930] In some embodiments, -L 1 - has formula (IIa):

[0931]

[0932] wherein

[0933] the dashed line marked with an asterisk indicates attachment to the nitrogen atom of -D by formation of an amide bond;

[0934] the unmarked dashed line indicates attachment to -L2 - is connected; and

[0935] - R 4 is -CH3, -CH2-O-CH3, -CH2CH3or -CH(CH3)2.

[0936] In some embodiments, -L 1 - has the formula (IIa) and -L 2 - has the formula (IIa'):

[0937]

[0938] wherein

[0939] the unlabelled dashed line indicates the connection to -L 1 - is connected; and

[0940] the dashed line marked with an asterisk indicates the connection to -L 4 -.

[0941] In some embodiments, -L 1 - has the structure as disclosed in WO2016 / 020373A1, which is incorporated herein by reference in its entirety. Thus, in some embodiments, -L 1 - is a moiety having the formula (III):

[0942]

[0943] wherein

[0944] the dashed line indicates the connection to a primary or secondary amine or hydroxyl group of -D by forming an amide or ester linkage, respectively;

[0945] - R 1 , -R 1a , -R 2 , -R 2a , -R 3 and -R 3a are independently of each other selected from the group consisting of: -H, -C(R 8 R 8a R 8b ), -C(=O)R 8 , -C≡N, -C(=NR 8 )R 8a , -CR 8 (=CR 8a R 8b ), -C≡CR 8 and -T;

[0946] - R 4 , -R 5 and -R 5aare independently selected from: -H, -C(R 9 R 9a R 9b ) and -T;

[0947] a1 and a2 are independently 0 or 1;

[0948] Each-R 6 、-R 6a 、-R 7 、-R 7a 、-R 8 、-R 8a 、-R 8b 、-R 9 、-R 9a 、-R 9b Independently selected from: -H, halogen, -CN, -COOR 10 、-OR 10 、-C(O)R 10 、-C(O)N(R 10 R 10a )、-S(O)2N(R 10 R 10a )、-S(O)N(R 10 R 10a )、-S(O)2R 10 、-S(O)R 10 、-N(R 10 )S(O)2N(R 10a R 10b ),-SR 10 、-N(R 10 R 10a )、-NO2、-OC(O)R 10 、-N(R 10 )C(O)R 10a 、-N(R 10 )S(O)2R 10a 、-N(R 10 )S(O)R 10a 、-N(R 10 )C(O)OR 10a 、-N(R 10 )C(O)N(R 10a R 10b )、-OC(O)N(R 10 R 10a ), -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl; wherein -T, C 1-20 Alkyl, C 2-20 Alkenyl and C 2-20alkenyl and C 11 substituted, and wherein C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally interrupted by one or more groups selected from: -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-;

[0949] each -R 10 , -R 10a , -R 10b is independently selected from: -H, -T, C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl; wherein -T, C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally substituted by one or more groups, which can be the same or different, -R 11 , and wherein C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl is optionally interrupted by one or more groups selected from: -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 12 )-, -S(O)2N(R 12 )-, -S(O)N(R 12 )-, -S(O)2-, -S(O)-, -N(R 12 )S(O)2N(R 12a )-, -S-, -N(R 12 )-, -OC(OR 12 )(R 12a )-, -N(R 12 )C(O)N(R 12a )- and -OC(O)N(R 12 )-;

[0950] Each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally replaced by one or more identical or different -R 11 replace;

[0951] Each-R 11 are independently selected from halogen, -CN, oxo (=O), -COOR 13 、-OR 13 、-C(O)R 13 、-C(O)N(R 13 R 13a )、-S(O)2N(R 13 R 13a )、-S(O)N(R 13 R 13a )、-S(O)2R 13 、-S(O)R 13 、-N(R 13 )S(O)2N(R 13a R 13b ),-SR 13 、-N(R 13 R 13a )、-NO2、-OC(O)R 13 、-N(R 13 )C(O)R 13a 、-N(R 13 )S(O)2R 13a 、-N(R 13 )S(O)R 13a 、-N(R 13 )C(O)OR 13a 、-N(R 13 )C(O)N(R 13a R 13b )、-OC(O)N(R 13 R 13a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0952] Each-R 12 、-R 12a 、-R 13 、-R 13a 、-R 13b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different;

[0953] Optionally, -R 1 / -R 1a 、-R 2 / -R 2a 、-R 3 / -R 3a 、-R 6 / -R 6a 、-R 7 / -R 7a One or more pairs of atoms in the C 3-10 a cycloalkyl group or a 3- to 10-membered heterocyclic group;

[0954] Optionally, -R 1 / -R 2 、-R 1 / -R 3 、-R 1 / -R 4 、-R 1 / -R 5 、-R 1 / -R 6 、-R 1 / -R 7 、-R 2 / -R 3 、-R 2 / -R 4 、-R 2 / -R 5 、-R 2 / -R 6 、-R 2 / -R 7 、-R 3 / -R 4 、-R 3 / -R 5 、-R 3 / -R 6 、-R 3 / -R 7 、-R 4 / -R 5 、-R 4 / -R 6 、-R 4 / -R 7 、-R 5 / -R 6 、-R 5 / -R 7 、-R 6 / -R 7 One or more pairs of them, together with the atoms to which they are attached, combine to form ring A;

[0955] A is selected from: phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl;

[0956] wherein -L 1 - is optionally further substituted. 2 - is optionally further substituted. 1 - is optionally further substituted.

[0957] -L 1 The optional further substituents of -L

[0958] -L 1 - is not further substituted.

[0959] -L 1 - has a structure as disclosed in EP1536334B1, WO2009 / 009712A1, WO2008 / 034122A1, WO2009 / 143412A2, WO201 1 / 082368A2 and US8618124B2, which are hereby incorporated by reference in their entirety.

[0960] -L 1 - has a structure as disclosed in US8946405B2 and US8754190B2, which are hereby incorporated by reference in their entirety. Thus, in some embodiments, -L 1 - has formula (IV):

[0961]

[0962] wherein

[0963] the dotted line indicates the attachment to -D via a functional group of -D selected from the group consisting of -OH, -SH and -NH2;

[0964] m is 0 or 1 ;

[0965] -R 1 and -R 2 at least one or both are independently of each other selected from the group consisting of: -CN, -N02, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(0)R 3 , -S(0)R 3 , -S(0)2R 3 and -SR 4 ,

[0966] -R 1 and -R 2one and only one of which is selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;

[0967] -R 3 selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 9 and -N(R 9 )2;

[0968] -R 4 selected from the group consisting of: optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0969] each -R 5 is independently selected from the group consisting of: -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0970] -R 9 is selected from the group consisting of: -H and optionally substituted alkyl;

[0971] -Y- is absent, and -X- is -O- or -S-; or

[0972] -Y- is -N(Q)CH2-, and -X- is -O-;

[0973] Q is selected from the group consisting of: optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

[0974] optionally, -R 1 and -R 2 may together form a 3- to 8-membered ring; and

[0975] optionally, two -R 9 together with the nitrogen to which they are attached form a heterocyclic ring;

[0976] wherein -L 1 - is substituted with -L 2 - and wherein -L 1 - is optionally further substituted.

[0977] In the context of Formula (IV) only, the terms used have the following meanings:

[0978] As used herein, the term "alkyl" includes straight chain, branched chain, or cyclic saturated hydrocarbon radicals having from 1 to 8 carbon atoms, or in some embodiments, from 1 to 6 or 1 to 4 carbon atoms.

[0979] The term "alkoxy" includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0980] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond.

[0981] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond.

[0982] The term "aryl" includes aromatic hydrocarbon radicals having from 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthryl. The term "heteroaryl" includes aromatic rings containing from 3 to 15 carbons, preferably aromatic rings containing from 3 to 7 carbons, containing at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

[0983] In some instances, an alkenyl, alkynyl, aryl, or heteroaryl moiety can be coupled to the remainder of the molecule via an alkylene linkage. In these cases, the substituent will be referred to as an alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating the alkylene moiety between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which it is coupled.

[0984] The term "halogen" includes bromo, fluoro, chloro, and iodo.

[0985] The term "heterocycle" refers to a 4- to 8-membered aromatic or non-aromatic ring containing from 3 to 7 carbon atoms and at least one N, O, or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, and the exemplary groups provided above for the term "heteroaryl."

[0986] When a ring system is optionally substituted, suitable substituents are selected from alkyl, alkenyl, alkynyl, or another ring, each optionally further substituted. Optional substituents on any group, including the groups described above, include halo, nitro, cyano, -OR, -SR, -NR2, -OCOR, -NRCOR, -COOR, -CONR2, -SOR, -SO2R, -SONR2, -SO2NR2, where each R is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, or two R groups together with the atom to which they are attached form a ring.

[0987] In some embodiments, -L 1- having a structure as disclosed in WO2013 / 036857A1, which is incorporated herein in its entirety by reference. Thus, in some embodiments, -L 1 - having the formula (V):

[0988]

[0989] wherein

[0990] dashed lines indicate attachment via the amine functionality of -D to -D;

[0991] -R 1 is selected from: optionally substituted C1-C6 linear, branched, or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; alkoxy; and -NR 5 2;

[0992] -R 2 is selected from: -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0993] -R 3 is selected from: -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0994] -R 4 is selected from: -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

[0995] each -R 5 is independently selected from: -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; or when two -R 5 may be taken together to be a cyclic alkyl or cyclic heteroalkyl;

[0996] wherein -L 1 - is optionally further substituted, and wherein -L 2 - is optionally further substituted, and wherein -L 1 - is optionally further substituted.

[0997] In the context of formula (V) only, the terms used have the following meanings:

[0998] “Alkyl”, “alkenyl”, and “alkynyl” include straight chain, branched chain, or cyclic hydrocarbon groups having from 1 to 8 carbons or 1 to 6 carbons or 1 to 4 carbons, where alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon-carbon double bonds, and alkynyl includes one or more carbon-carbon triple bonds. Unless otherwise specified, such groups contain 1-6 C atoms.

[0999] "Aryl" includes aromatic hydrocarbon groups having 6-18 carbons, preferably 6-10 carbons, including, for example, phenyl, naphthyl, and anthracene groups. "Heteroaryl" includes aromatic rings containing 3 to 15 carbons, preferably aromatic rings containing 3 to 7 carbons, containing at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, indenyl, and the like.

[1000] The term "substituted" means that an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group contains one or more substituents in place of one or more hydrogen atoms. The substituents can typically be selected from the group consisting of: halogen, including F, Cl, Br, and I; lower alkyl, including straight chain, branched, and cyclic; lower haloalkyl, including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy, including straight chain, branched, and cyclic; SH; lower alkylthio, including straight chain, branched, and cyclic; amino, alkylamino, dialkylamino; silyl, including alkylsilyl, alkoxy silyl, and arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic acid ester, carboxylic acid amide, aminocarbonyl; aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketone; sulfone; sulfonamide; aryl, including phenyl, naphthyl, and anthracene; heteroaryl, including 5-membered heteroaryl groups, including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole, 6-membered heteroaryl groups, including pyridine, pyrimidine, pyrazine, and fused heteroaryl groups, including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole, and benzisothiazole.

[1001] In some embodiments, -L 1 - has a structure as disclosed in US7585837B2, which is incorporated by reference in its entirety. Thus, in some embodiments, -L 1 - has a formula (VI):

[1002]

[1003] wherein

[1004] the dotted line indicates the attachment via the amine functionality of -D to -D;

[1005] R 1 and R 2 are independently selected from the group consisting of: hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkylaryl, arylalkyl, halogen, nitro, -SO3H, -SO2NHR 5 , amino, ammonium, carboxyl, PO3H2, and OPO3H2;

[1006] R 3 , R 4 and R5 independently selected from the group consisting of: hydrogen, alkyl, and aryl;

[1007] wherein -L 1 - is optionally further substituted. 2 - is optionally further substituted. 1 - is optionally further substituted.

[1008] Suitable substituents for formula (VI) are alkyl (e.g., C 1-6 alkyl), alkenyl (e.g., C 2-6 alkenyl), alkynyl (e.g., C 2-6 alkynyl), aryl (e.g., phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (e.g., aromatic 4- to 7-membered heterocycle), or a halogen moiety.

[1009] The terms used have the following meanings only in the context of formula (VI):

[1010] The terms "alkyl," "alkoxy," "alkoxyalkyl," "aryl," "alkaryl," and "aralkyl" mean alkyl groups having 1-8, preferably 1-4, carbon atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, and aryl groups having 6-10 carbon atoms, such as phenyl and naphthyl. The term "halogen" includes bromine, fluorine, chlorine, and iodine.

[1011] In some embodiments, -L 1 - has a structure as disclosed in WO 2002 / 089789 Al, which is incorporated by reference in its entirety. Thus, in some embodiments, -L 1 - has formula (VII):

[1012]

[1013] wherein

[1014] the dotted line indicates the attachment via the amine functionality of -D to -D;

[1015] Y1and Y2are independently O, S, or NR 7 ;

[1016] R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of: hydrogen, C 1-6 alkyl, C 3-12 branched alkyl, C 3-8 cycloalkyl, C 1-6 substituted alkyl, C 3-8 substituted cycloalkyl, aryl, substituted aryl, aralkyl, C 1-6heteroalkyl, substituted C 1-6 heteroalkyl, C 1-6 alkoxy, phenoxy, and C 1-6 heteroalkoxy;

[1017] Ar is a moiety that forms a poly-substituted aromatic hydrocarbon or a poly- substituted heterocyclyl when included in Formula (VII);

[1018] X is a bond or a moiety that is actively transported into a target cell, a hydrophobic moiety, or a combination thereof,

[1019] y is 0 or 1;

[1020] wherein -L 1 - is optionally further substituted. 2 - is optionally further substituted. 1 - is optionally further substituted.

[1021] In the context of Formula (VII) only, the terms used have the following meanings:

[1022] The term "alkyl" is understood to include, for example, straight chain, branched chain, substituted C 1-12 alkyl, including alkoxy, C 3-8 cycloalkyl, or substituted cycloalkyl, and the like.

[1023] The term "substituted" is understood to include the addition of or replacement of one or more atoms within a functional group or compound with one or more different atoms.

[1024] Substituted alkyl includes carboxyalkyl, aminoalkyl, dialkylamino, hydroxyalkyl, and mercaptoalkyl; substituted cycloalkyl includes, for example, the moiety 4-chlorocyclohexyl; aryl includes, for example, the moiety naphthyl; substituted aryl includes, for example, the moiety 3-bromo-phenyl; aralkyl includes, for example, the moiety tolyl; heteroalkyl includes, for example, the moiety ethylthiophene; substituted heteroalkyl includes, for example, the moiety 3-methoxythiophene; alkoxy includes, for example, the moiety methoxy; and phenoxy includes, for example, the moiety 3-nitrophenoxy. Halo- is understood to include fluoro, chloro, iodo, and bromo.

[1025] In some embodiments, -L 1 comprises the substructure of Formula (VIII):

[1026]

[1027] wherein

[1028] the dashed line marked with an asterisk indicates attachment to the nitrogen of -D through formation of an amide bond;

[1029] the unmarked dashed line indicates attachment to -L 1the remainder of -L

[1030] wherein -L 1 - is substituted with -L 2 - is substituted with -L 1 - is optionally further substituted.

[1031] In some embodiments, -L 1 - has formula (VIII), wherein the dashed line marked with an asterisk indicates the attachment to the nitrogen of the amine of the lysine side chain of -D. In some embodiments, -L 1 - has formula (VIII), wherein the dashed line marked with an asterisk indicates the attachment to the nitrogen of the amine of the N-terminus of -D.

[1032] In some embodiments, -L 1 - is substituted with one -L 2 - moiety. In some embodiments, -L 1 - is not further substituted.

[1033] In some embodiments, -L 1 - comprises the substructure of formula (IX):

[1034]

[1035] wherein

[1036] the dashed line marked with an asterisk indicates the attachment to the nitrogen of -D via formation of a carbamate bond;

[1037] the unmarked dashed line indicates the attachment to the remainder of -L 1 -; and

[1038] wherein -L 1 - is substituted with -L 2 - is substituted with -L 1 - is optionally further substituted.

[1039] In some embodiments, -L 1 - has formula (IX), wherein the dashed line marked with an asterisk indicates the attachment to the nitrogen of the amine of the lysine side chain of -D.

[1040] In some embodiments, -L 1 - has formula (IX), wherein the dashed line marked with an asterisk indicates the attachment to the nitrogen of the amine of the N-terminus of -D.

[1041] In some embodiments, -L 1 - is not further substituted.

[1042] In some embodiments, -L 1- having the formula (IX-a):

[1043]

[1044] in

[1045] The dashed line marked with an asterisk indicates the connection to the nitrogen of -D, and the unmarked dashed line indicates the connection to the nitrogen of -L. 2 -connection;

[1046] n is 0, 1, 2, 3 or 4;

[1047] =Y1 is selected from =O and =S;

[1048] -Y 2 - is selected from -O- and -S-;

[1049] -Y3- is selected from -O- and -S-;

[1050] -Y4- is selected from -O-, -NR 5 -and-C(R 6 R 6a )-;

[1051] =Y5 is selected from =O and =S;

[1052] -R 3 、-R 5 、-R 6 、-R 6a are independently selected from the group consisting of: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl;

[1053] -R 4 Selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl;

[1054] -W-Selected from: C 1-20 Alkyl, optionally interrupted by one or more groups selected from: C 3-10 Cycloalkyl, 8- to 30-membered carbon polycyclic group, 3- to 10-membered heterocyclic group, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 )-;

[1055] -Nu is a nucleophilic group selected from the following: -N(R7 R 7a ), -N(R 7 OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH,

[1056]

[1057] -Ar- is selected from:

[1058]

[1059] wherein

[1060] the dotted line indicates the connection to the rest of -L 1 -;

[1061] -Z 1 - is selected from -O-, -S- and -N(R 7 )-, and

[1062] -Z 2 - is -N(R 7 )-; and

[1063] -R 7 , -R 7a , -R 7b are independently of each other selected from -H, C 1-6 alkyl, C 2-6 alkenyl and C 2-6 alkynyl;

[1064] wherein -L 1 - is optionally further substituted.

[1065] In some embodiments, -L 1 - has formula (IX-a), wherein the dotted line marked with an asterisk indicates the connection to the nitrogen of the amine of the lysine side chain of -D.

[1066] In some embodiments, -L 1 - has formula (IX-a), wherein the dotted line marked with an asterisk indicates the connection to the nitrogen of the amine of the N-terminus of -D.

[1067] In some embodiments, -L 1 - of formula (IX-a) is not further substituted.

[1068] In some embodiments, -L 1 - has formula (IX-b):

[1069]

[1070] in

[1071] The dashed line marked with an asterisk indicates the connection to the nitrogen of -D, and the unmarked dashed line indicates the connection to the nitrogen of -L. 2 -connection;

[1072] n is 0, 1, 2, 3 or 4;

[1073] =Y1 is selected from =O and =S;

[1074] -Y 2 - is selected from -O- and -S-;

[1075] -Y3- is selected from -O- and -S-;

[1076] -Y4- is selected from -O-, -NR 5 -and-C(R 6 R 6a )-;

[1077] =Y5 is selected from =O and =S;

[1078] -R 2 、-R 3 、-R 5 、-R 6 、-R 6a are independently selected from the group consisting of: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl;

[1079] -R 4 Selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl;

[1080] -W-Selected from: C 1-20 Alkyl, optionally interrupted by one or more groups selected from: C 3-10 Cycloalkyl, 8- to 30-membered carbon polycyclic group, 3- to 10-membered heterocyclic group, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 )-;

[1081] -Nu is a nucleophilic group selected from the following: -N(R 7 R 7a )、-N(R 7OH), -N(R 7 )-N(R 7a R 7b ), -S(R 7 ), -COOH,

[1082]

[1083] -Ar- is selected from:

[1084]

[1085] wherein

[1086] the dotted line indicates the attachment to the rest of -L 1 -;

[1087] -Z 1 - is selected from: -O-, -S- and -N(R 7 )-, and

[1088] -Z 2 - is -N(R 7 )-; and

[1089] -R 7 , -R 7a , -R 7b are independently of each other selected from: -H, C 1-6 alkyl, C 2-6 alkenyl and C 2-6 alkynyl;

[1090] wherein -L 1 - is optionally further substituted.

[1091] In some embodiments, -L 1 - has formula (IX-b), wherein the dotted line marked with an asterisk indicates the attachment to the nitrogen of the amine of the lysine side chain of -D.

[1092] In some embodiments, -L 1 - has formula (IX-b), wherein the dotted line marked with an asterisk indicates the attachment to the nitrogen of the amine of the N-terminus of -D.

[1093] In some embodiments, -L 1 - of formula (IX-b) is not further substituted.

[1094] In some embodiments, =Y 1 - of formulae (IX-a) and (IX-b) is =0.

[1095] In some embodiments, -Y 2 - of formulae (IX-a) and (IX-b) is -O-.

[1096] In some embodiments, -Y of Formula (IX-a) and (IX-b) 3 - is -O-.

[1097] In some embodiments, -Y of Formula (IX-a) and (IX-b) 4 -for-NR 5 -.

[1098] In some embodiments, =Y of Formula (IX-a) and (IX-b) 5 =O.

[1099] In some embodiments, n of Formula (IX-a) and (IX-b) is 0 or 1. In some embodiments, n of Formula (IX-a) and (IX-b) is 0. In some embodiments, n of Formula (IX-a) and (IX-b) is 1.

[1100] In some embodiments, -R of formula (IX-b) 2 is selected from the group consisting of: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. 2 is selected from the group consisting of: -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R 2 In some embodiments, -R of formula (IX-b) is selected from -H, methyl and ethyl. 2 is -H.

[1101] In some embodiments, -R of Formula (IX-a) and (IX-b) 3 is selected from the group consisting of: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In some embodiments, -R 3 is selected from the group consisting of: -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R 3 In some embodiments, -R of formula (IX-a) and (IX-b) is selected from -H, methyl and ethyl. 3 is -H.

[1102] In some embodiments, each -R 4 is independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R 4 is selected from the group consisting of: methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R 4Selected from methyl and ethyl.

[1103] In some embodiments, -R of Formula (IX-a) and (IX-b) 5 is selected from the group consisting of: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In some embodiments, -R 5 is selected from the group consisting of: -H, methyl, ethyl, n-propyl and isopropyl. In some embodiments, -R 5 In some embodiments, -R 5 It is a methyl group.

[1104] In some embodiments, -R of Formula (IX-a) and (IX-b) 6 and -R 6a is independently selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In some embodiments, -R 6 and -R 6a In some embodiments, -R of formula (IX-a) and (IX-b) is independently selected from: -H, methyl, ethyl, n-propyl and isopropyl. 6 and -R 6a In some embodiments, -R of formula (IX-a) and (IX-b) is independently selected from -H, methyl and ethyl. 6 and -R 6a Both are -H.

[1105] In some embodiments, Ar of formula (IX-a) and (IX-b) is phenyl. In some embodiments, Ar of formula (IX-a) and (IX-b) is

[1106]

[1107] Therein, the dashed lines indicate the connections to the remainder of the moieties of formulae (IX-a) and (IX-b).

[1108] In some embodiments, W of formula (IX-a) and (IX-b) is C 1-20 Alkyl, optionally interrupted by C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S- and -N(R 7 In some embodiments, W of formula (IX-a) and (IX-b) is C 1-10 Alkyl, optionally interrupted by C 3-10 Cycloalkyl, -C(O)-, -C(O)N(R7 )-, -O-, -S-, and -N(R 7 )-. In some embodiments, W of formula (IX-a) and (IX-b) is C 1-6 alkyl, optionally interrupted with C 3-10 cycloalkyl, -C(O)-, -C(O)N(R 7 )-, -O-, -S-, and -N(R 7 )-. In some embodiments, W of formula (IX-a) and (IX-b) is

[1109]

[1110] wherein

[1111] dashed lines indicate the connection to the rest of the moiety of formula (IX-a) or (IX-b), respectively.

[1112] In some embodiments, -Nu of formula (IX-a) and (IX-b) is -N(R 7 R 7a ).

[1113] In some embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from the group consisting of -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, and t-butyl. In some embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from the group consisting of -H, methyl, ethyl, n-propyl, and i-propyl. In some embodiments, -R 7 , -R 7a , and -R 7b are each independently selected from the group consisting of methyl or ethyl. In some embodiments, -R 7 , -R 7a , and -R 7b are each -H.

[1114] In some embodiments, -L 1 has formula (IX-c):

[1115]

[1116] wherein

[1117] the dashed line marked with an asterisk indicates the connection to the nitrogen of -D;

[1118] Unmarked dashed lines indicate the same as -L 2 - connection; and

[1119] s1 is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[1120] In some embodiments, -L 1 - has formula (IX-c), wherein the dashed line marked with an asterisk indicates the connection to the amine nitrogen of the lysine side chain of -D.

[1121] In some embodiments, -L 1 - has formula (IX-c), wherein the dashed line marked with an asterisk indicates the connection to the amine nitrogen at the N-terminus of -D.

[1122] In some embodiments, s1 of Formula (IX-c) is an integer selected from 1, 2, 3, 4, and 5. In some embodiments, s1 of Formula (IX-c) is 1. In some embodiments, s1 of Formula (IX-c) is 2. In some embodiments, s1 of Formula (IX-c) is 3. In some embodiments, s1 of Formula (IX-c) is 4. In some embodiments, s1 of Formula (IX-c) is 5.

[1123] In some embodiments, -L 1 -having the formula (IX-d):

[1124]

[1125] in

[1126] The dashed line marked with an asterisk indicates the connection to the nitrogen of -D; and

[1127] Unmarked dashed lines indicate the same as -L 2 -connection.

[1128] In some embodiments, -L 1 - has the formula (IX-d), wherein the dashed line marked with an asterisk indicates the connection to the amine nitrogen of the lysine side chain of -D.

[1129] In some embodiments, -L 1 - has the formula (IX-d), wherein the dashed line marked with an asterisk indicates the connection to the amine nitrogen at the N-terminus of -D.

[1130] In some embodiments, -L 1 - has a structure as disclosed in WO2020 / 206358A1, which is incorporated herein by reference in its entirety. Thus, in some embodiments, -L 1 - a moiety having the formula (X):

[1131]

[1132] wherein

[1133] the unlabelled dashed line indicates the attachment to -D;

[1134] the dashed line marked with an asterisk indicates the attachment to -L 2 ;

[1135] n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

[1136] -R 1 and -R 2 are independently an electron withdrawing group, alkyl, or -H, and wherein at least one of -R 1 or -R 2 is an electron withdrawing group;

[1137] each -R 4 is independently C1-C3 alkyl, or the two -R 4 together with the carbon atom to which they are attached form a 3- to 6-membered ring; and

[1138] -Y- is absent when -D is a drug moiety attached via an amine, or -Y- is -N(R 6 )CH2- when -D is a drug moiety attached via a phenol, alcohol, thiol, thiol, imidazole, or non-basic amine; wherein -R 6 is optionally substituted C1-C6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[1139] In some embodiments, n of formula (X) is an integer selected from 1, 2, 3, 4, 5, and 6. In some embodiments, n of formula (X) is an integer selected from 1, 2, and 3. In some embodiments, n of formula (X) is an integer from 0, 1, 2, and 3. In some embodiments, n of formula (X) is 1. In some embodiments, n of formula (X) is 2. In some embodiments, n of formula (X) is 3.

[1140] In some embodiments, the electron withdrawing group of -R 1 and -R 2 of formula (X) is selected from: -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR 3 , -SOR 3 , or -SO2R 3 , wherein -R 3 is -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 8 , or -NR8 2, where each -R 8 is independently -H or optionally substituted alkyl, or two -R 8 groups together with the nitrogen to which they are attached form a heterocyclic ring; or -SR 9 , where -R 9 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[1141] In some embodiments, the -R 1 group of formula (X) is -H, -F, -Cl, -Br, or -I. 2 In some embodiments, the electron withdrawing group of -R 1 and -R 2 is -CN. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is -NO2. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted aryl comprising 6 to 10 carbons. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted heteroaryl comprising 3 to 7 carbons and comprising at least one N, O, or S atom. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, or indenyl. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted alkenyl comprising 2 to 20 carbon atoms. In some embodiments, the electron withdrawing group of -R 1 and -R 2 is an optionally substituted alkynyl comprising 2 to 20 carbon atoms. In some embodiments, the electron withdrawing group of -R 3 and -R 3 is -COR 3 , -SOR 3 , or -SO2R 8 , where -R 8 is -H, optionally substituted alkyl comprising 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 8or -NR 8 2, wherein each -R 8 is independently -H or optionally substituted alkyl comprising 1 to 20 carbon atoms, or two -R 8 groups together with the nitrogen to which they are attached form a heterocycle. In some embodiments, -R 1 of formula (X) is -H, -F, -Cl, -Br, -I, -CN, -OR 2 , -SR 9 , -NR 9 2R or -N3.

[1142] In some embodiments, at least one of -R 1 and -R 2 of formula (X) is -CN, -SOR 3 , or -SO2R 3 . In some embodiments, at least one of -R 1 and -R 2 of formula (X) is -CN or -SO2R 3 . In some embodiments, at least one of -R 1 and -R 2 of formula (X) is -CN or -SO2R 3 , wherein -R 3 is optionally substituted alkyl, optionally substituted aryl, or -NR 8 2. In some embodiments, at least one of -R 1 and -R 2 of formula (X) is -CN, -SO2N(CH3)2, -SO2CH3, phenyl substituted with -SO2, phenyl substituted with -SO2 and -Cl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[1143] In some embodiments, each -R 4 of formula (X) is independently C1-C3 alkyl. In some embodiments, both -R 4 are methyl.

[1144] In some embodiments, -Y- of formula (X) is absent. In some embodiments, -Y- of formula (X) is -N(R 6 )CH2-.

[1145] In some embodiments, -L 1 - has formula (X), wherein n is 1, -R1 is -CN, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is -SO2N(CH3)2, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is SO2CH3, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is -SO2N(CH2CH2)2CHCH3, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is phenyl substituted with -SO2, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is phenyl substituted with -SO2 and -Cl, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is -SO2N(CH2CH2)2O, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is -SO2CH(CH3)2, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1 is -SO2N(CH3)(CH2CH3), -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 has the formula (X), wherein n is 1, -R 1-SO2N(CH2CH2OCH3)2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 1, -R 1 -phenyl substituted with -SO2and -CH3, -R 2 -H, and -R 4 -CH3.

[1146] In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -CN, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2N(CH3)2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2CH3, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2N(CH2CH2)2CHCH3, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -phenyl substituted with -SO2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -phenyl substituted with -SO2and -Cl, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2N(CH2CH2)2O, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1-SO2CH(CH3)2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2N(CH3)(CH2CH3), -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -SO2N(CH2CH2OCH3)2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 2, -R 1 -phenyl substituted with -SO2 and -CH3, -R 2 -H, and -R 4 -CH3.

[1147] In some embodiments, -L 1 -Formula (X) where n is 3, -R 1 -CN, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 3, -R 1 -SO2N(CH3)2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 3, -R 1 -SO2CH3, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 3, -R 1 -SO2N(CH2CH2)2CHCH3, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 3, -R 1 -phenyl substituted with -SO2, -R 2 -H, and -R 4 -CH3. In some embodiments, -L 1 -Formula (X) where n is 3, -R 1is -SO2N(CH2CH2)2O, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 - has the formula (X) wherein n is 3, -R 1 is -SO2N(CH2CH2)2O, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 - has the formula (X) wherein n is 3, -R 1 is -SO2CH(CH3)2, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 - has the formula (X) wherein n is 3, -R 1 is -SO2N(CH3)(CH2CH3), -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 - has the formula (X) wherein n is 3, -R 1 is -SO2N(CH2CH2OCH3)2, -R 2 is -H, and -R 4 is -CH3. In some embodiments, -L 1 - has the formula (X) wherein n is 3, -R 1 is -SO2N(CH2CH2OCH3)2, -R 2 is -H, and -R 4 is -CH3.

[1148] The terms used have the following meanings, only in the context of formula (X):

[1149] The term "alkyl" refers to a straight chain, branched, or cyclic saturated hydrocarbon group having from 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In some embodiments, the alkyl group is straight chain or branched. Examples of straight chain or branched alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. In some embodiments, the alkyl group is cyclic. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, and cyclohexyl.

[1150] The term "alkoxy" refers to an alkyl group bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, and cyclobutoxy.

[1151] The term "alkenyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon double bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[1152] The term "alkynyl" refers to a non-aromatic unsaturated hydrocarbon having a carbon-carbon triple bond and 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[1153] The term "aryl" refers to an aromatic hydrocarbon group having 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthryl. The term "heteroaryl" refers to an aromatic ring containing 3 to 15 carbons containing at least one N, O, or S atom, preferably an aromatic ring containing 3 to 7 carbons containing at least one N, O, or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indolyl, and indenyl.

[1154] In some embodiments, an alkenyl, alkynyl, aryl, or heteroaryl moiety can be coupled to the remainder of the molecule via an alkyl linkage. In these cases, the substituent will be referred to as an alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating the alkylene moiety between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is coupled.

[1155] The term "halogen" or "halo" refers to bromo, fluoro, chloro, and iodo.

[1156] The term "heterocycle" or "heterocyclyl" refers to a 3- to 15-membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, and the exemplary groups provided above for the term "heteroaryl." In some embodiments, the heterocycle or heterocyclyl is non-aromatic. In some embodiments, the heterocycle or heterocyclyl is aromatic.

[1157] The term "optionally substituted" means that a group can be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents which can be the same or different. Examples of substituents include alkyl, alkenyl, alkynyl, halo, -CN, -OR aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aaC(O)R bb , -NR aa C(O)OR bb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)2NR aa R bb , -P(O)(OR aa )(OR bb ), heterocyclyl, heteroaryl, or aryl, wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl is independently optionally substituted with -R cc , wherein each of -R aa and -R bb is independently -H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or -R aa and -R bb together with the nitrogen atom to which they are attached form a heterocyclyl group, which is optionally substituted with alkyl, alkenyl, alkynyl, halo, hydroxyl, alkoxy, or -CN, and wherein: each -R cc is independently alkyl, alkenyl, alkynyl, halo, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[1158] In some embodiments, -L 1 has a structure as disclosed in WO 2021 / 136808 A1, which is incorporated by reference in its entirety. Thus, in some embodiments, -L 1 has a moiety having formula (XI):

[1159]

[1160] wherein

[1161] the dotted line indicates attachment to the nitrogen of a primary or secondary amine of -D;

[1162] v is selected from 0 or 1;

[1163] -X 1 is selected from -C(R 8 )(R 8a )-, -N(R 9 )-, and -O-;

[1164] = X 2 selected from =0 and =N(R 10 );

[1165] -X 3 selected from -0-, -S-, and -Se;

[1166] each p is independently selected from 0 or 1, provided that at most one p is 0;

[1167] -R 6 , -R 6a , -R 10 are independently selected from -H, -C(R 11 )(R 11a )(R 11b ), and -T;

[1168] -R 9 is selected from -C(R 11 )(R 11a )(R 11b ), and -T;

[1169] -R 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 4 , -R 4a , -R 5 , -R 5a , -R 7 , -R 8 -R 8a , -R 11 , -R 11a , and -R 11b are independently selected from -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein the C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally substituted with one or more -R 13 , which are the same or different; and wherein the C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally interrupted by one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R 14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14 )C(O)N(R 14a )-, and -OC(O)N(R 14 )-;

[1170] -R 12 , -R 12a , -R 12b are independently selected from the group consisting of -H, -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein the -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally substituted with one or more -R 13 , which are the same or different; and wherein the C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally interrupted by one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14 )C(O)N(R 14a )- and -OC(O)N(R 14 )-;

[1171] wherein each T is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 13 groups, which are the same or different;

[1172] -R 13 is selected from: halogen, -CN, oxo, -C(O)OR 15 , -OR 15 , -C(O)R 15 , -C(O)N(R 15 )(R 15a ), -S(O)2N(R 15 )(R 15a ), -S(O)N(R 15 )(R 15a ), -S(O)2R 15 , -S(O)R 15 , -N(R 15 )S(O)2N(R 15a )(R 15b ), -SR 15 , -N(R 15 )(R 15a ), -NO2, -OC(O)R 15 , -N(R 15 )C(O)R 15a , -N(R 15 )S(O)2R 15a , -N(R 15 )S(O)R 15a , -N(R 15 )C(O)OR 15a , -N(R 15 )C(O)N(R 15a )(R 15b ), -OC(O)N(R 15 )(R 15a ), and C 1-6alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[1173] wherein -R 14 , -R 14a , -R 15 , -R 15a and -R 15b are independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;

[1174] optionally, one or more pairs of -R 1 -R 1a , -R 2 -R 2a , -R 3 -R 3a , -R 4 -R 4a , -R 5 -R 5a or -R 8 -R 8a are combined to form, together with the atoms to which they are attached, a C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl or 8- to 11-membered heterobicyclyl;

[1175] optionally, one or more pairs of -R 1 -R 2 , -R 1 -R 8 , -R 1 -R 9 , -R 2 -R 9 or -R 2 -R 10 are combined to form, together with the atoms to which they are attached, a ring-A-;

[1176] wherein -A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl and 8- to 11-membered heterobicyclyl;

[1177] optionally, one or more pairs of -R 3 -R 6 , -R 4 -R 6 , -R 5 -R 6 , -R 6 -R 6a or -R 6 -R 7one or more of the pairs -A'- together with the atoms to which they are attached form a ring;

[1178] wherein -A'- is selected from a 3- to 10-membered heterocyclyl and an 8- to 11-membered heterobicyclyl; and

[1179] -L 1 - substituted and optionally further substituted, with the proviso that the hydrogen marked with an asterisk in formula (XI) is not replaced by a substituent. 2 - substituted and optionally further substituted, with the proviso that the hydrogen marked with an asterisk in formula (XI) is not replaced by a substituent.

[1180] In some embodiments, the dashed line in formula (XI) indicates attachment to a nitrogen of a primary amine of -D. In some embodiments, the dashed line in formula (XI) indicates attachment to a nitrogen of a secondary amine of -D.

[1181] In some embodiments, -X 3 is -O. In some embodiments, -X 3 is -S. In some embodiments, -X 3 is -Se.

[1182] In some embodiments, -R 6 is -H. In some embodiments, -R 6 is -C(R 11 )(R 11a )(R 11b ). In some embodiments, -R 6 is -T.

[1183] In some embodiments, -R 6a is -H. In some embodiments, -R 6a is -C(R 11 )(R 11a )(R 11b ). In some embodiments, -R 6a is -T.

[1184] In some embodiments, -R 6 and -R 6a are both -H.

[1185] In some embodiments, v in formula (XI) is 0. In some embodiments, v in formula (XI) is 1.

[1186] In some embodiments, -X 1 - is -C(R 8 )(R 8a )-. In some embodiments, -X1 -N(R 9 )-. In some embodiments, -X 1 - is -O-.

[1187] In some embodiments, =X 2 - is =O. In some embodiments, =X 2 - is =N(R 10 ).

[1188] In some embodiments, -R 9 - is -C(R 11 )(R 11a )(R 11b ). In some embodiments, -R 9 - is -T.

[1189] In some embodiments, -R 10 - is -H. In some embodiments, -R 10 - is -C(R 11 )(R 11a )(R 11b ). In some embodiments, -R 10 - is -T.

[1190] In some embodiments, -R 1 - is selected from: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 1 is -H. In some embodiments, -R 1 is halogen. In some embodiments, -R 1 is -T. In some embodiments, -R 1 is C 1-6 alkyl. In some embodiments, -R 1 is C 2-6 alkenyl. In some embodiments, -R 1 is C 2-6 alkynyl. In some embodiments, -R 1 is selected from the group consisting of: -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1191] In some embodiments, -R 1a is selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 1aIn some embodiments, -R 1a In some embodiments, -R 1a In some embodiments, -R 1a C 1-6 In some embodiments, -R 1a C 2-6 In some embodiments, -R 1a C 2-6 In some embodiments, -R 1a Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1192] In some embodiments, -R 2 Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 2 In some embodiments, -R 2 In some embodiments, -R 2 In some embodiments, -R2 C 1-6 In some embodiments, -R 2 C 2-6 In some embodiments, -R 2 C 2-6 In some embodiments, -R 2 Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1193] In some embodiments, -R 2a Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 2a In some embodiments, -R 2a In some embodiments, -R 2a In some embodiments, -R 2a C 1-6 In some embodiments, -R 2a C 2-6alkenyl. In some embodiments, -R is -H. In some embodiments, -R is halogen. In some embodiments, -R is -T. In some embodiments, -R is C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 2a C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 2-6 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 2a selected from: -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1194] C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 selected from: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R is -H. In some embodiments, -R is halogen. In some embodiments, -R is -T. In some embodiments, -R is C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 In some embodiments, -R is -H. In some embodiments, -R is halogen. In some embodiments, -R is -T. In some embodiments, -R is C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 In some embodiments, -R is -H. In some embodiments, -R is halogen. In some embodiments, -R is -T. In some embodiments, -R is C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 In some embodiments, -R is -H. In some embodiments, -R is halogen. In some embodiments, -R is -T. In some embodiments, -R is C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 1-6 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 2-6 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 2-6 C1-C6alkyl. In some embodiments, -R is C1-C6alkenyl. In some embodiments, -R is C1-C6alkynyl. 3selected from the group consisting of: -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1195] In some embodiments, -R 3a is selected from the group consisting of: -H, -halo, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 3a of Formula (XI) is -H. In some embodiments, -R 3a of Formula (XI) is -halo. In some embodiments, -R 3a of Formula (XI) is -T. In some embodiments, -R 3a of Formula (XI) is C 1-6 alkyl. In some embodiments, -R 3a of Formula (XI) is C 2-6 alkenyl. In some embodiments, -R 3a of Formula (XI) is C 2-6 alkynyl. In some embodiments, -R 3a of Formula (XI) is selected from the group consisting of: -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1196] In some embodiments, -R 4 is selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 4 of Formula (XI) is -H. In some embodiments, -R 4 of Formula (XI) is halogen. In some embodiments, -R 4 of Formula (XI) is -T. In some embodiments, -R 4 of Formula (XI) is C 1-6 alkyl. In some embodiments, -R 4 of Formula (XI) is C 2-6 alkenyl. In some embodiments, -R 4 of Formula (XI) is C 2-6 alkynyl. In some embodiments, -R 4 of Formula (XI) is selected from the group consisting of: -H, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1197] In some embodiments, -R 4a of Formula (XI) is selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 4a In some embodiments, -R 4a In some embodiments, -R 4a In some embodiments, -R 4a C 1-6 In some embodiments, -R 4a C 2-6 In some embodiments, -R 4a C 2-6 In some embodiments, -R 4a Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1198] In some embodiments, -R 5 Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12)(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 5 In some embodiments, -R 5 In some embodiments, -R 5 In some embodiments, -R 5 C 1-6 In some embodiments, -R 5 C 2-6 In some embodiments, -R 5 C 2-6 In some embodiments, -R 5 Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1199] In some embodiments, -R 5a Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 5a of formula (XI) is -H. In some embodiments, -R 5a of formula (XI) is halogen. In some embodiments, -R 5a of formula (XI) is -T. In some embodiments, -R 5a of formula (XI) is C 1-6 alkyl. In some embodiments, -R 5a of formula (XI) is C 2-6 alkenyl. In some embodiments, -R 5a of formula (XI) is C 2-6 alkynyl. In some embodiments, -R 5a of formula (XI) is selected from the group consisting of: -H, methyl, ethyl, n- propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2- dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1200] In some embodiments, -R 7 of formula (XI) is selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 7 In some embodiments, -R 7 In some embodiments, -R 7 In some embodiments, -R 7 C 1-6 In some embodiments, -R 7 C 2-6 In some embodiments, -R 7 C 2-6 In some embodiments, -R 7 Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1201] In some embodiments, -R 8 Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl. In some embodiments, -R 8 of Formula (XI) is -H. In some embodiments, -R 8 of Formula (XI) is halogen. In some embodiments, -R 8 of Formula (XI) is -T. In some embodiments, -R 8 of Formula (XI) is C 1-6 alkyl. In some embodiments, -R 8 of Formula (XI) is C 2-6 alkenyl. In some embodiments, -R 8 of Formula (XI) is C 2-6 alkynyl. In some embodiments, -R 8 of Formula (XI) is selected from the group consisting of: -H, methyl, ethyl, n- propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2- dimethylpropyl, 3-methylbutyl, 1-methylbutyl, and 1-ethylpropyl.

[1202] In some embodiments, -R 8a of Formula (XI) is selected from the group consisting of: -H, halogen, -CN, -C(O)OR 12 , -OR 12 , -C(O)R 12 , -C(O)N(R 12 )(R 12a ), -S(O)2N(R 12 )(R 12a ), -S(O)N(R 12 )(R 12a ), -S(O)2R 12 , -S(O)R 12 , -N(R 12 )S(O)2N(R 12a )(R 12b ), -SR 12 , -NO2, -N(R 12 )C(O)OR 12a , -N(R 12 )C(O)N(R 12a )(R 12b ), -OC(O)N(R 12 )(R 12a ), -T, C 1-6 alkyl, C 2-6 alkenyl, and C2-6 In some embodiments, -R 8a In some embodiments, -R 8a In some embodiments, -R 8a In some embodiments, -R 8a C 1-6 In some embodiments, -R 8a C 2-6 In some embodiments, -R 8a C 2-6 In some embodiments, -R 8a Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1203] In some embodiments, -R 11 Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 11 In some embodiments, -R 11In some embodiments, -R 11 In some embodiments, -R 11 C 1-6 In some embodiments, -R 11 C 2-6 In some embodiments, -R 11 C 2-6 In some embodiments, -R 11 Selected from: -H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

[1204] In some embodiments, -R 11a Selected from: -H, halogen, -CN, -C(O)OR 12 、-OR 12 、-C(O)R 12 、-C(O)N(R 12 )(R 12a )、-S(O)2N(R 12 )(R 12a )、-S(O)N(R 12 )(R 12a )、-S(O)2R 12 、-S(O)R 12 、-N(R 12 )S(O)2N(R 12a )(R 12b ),-SR 12 、-NO2、-N(R 12 )C(O)OR 12a 、-N(R 12 )C(O)N(R 12a )(R 12b )、-OC(O)N(R 12 )(R 12a ), -T, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 In some embodiments, -R 11a In some embodiments, -R 11a In some embodiments, -R 11a In some embodiments, -R 11a C 1-6alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 11a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 11a alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkynyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 11a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C

[1205] alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 1-6 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 1-6 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12 alkynyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkynyl. In some embodiments, -R is selected from the group consisting of -H, -T, C

[1206] alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 1-6 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 1-6 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12a alkynyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkynyl. In some embodiments, -R is selected from the group consisting of -H, -T, C

[1207] alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 12b alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 1-6 alkyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C 2-6 alkenyl. In some embodiments, -R is selected from the group consisting of -H, -T, C12b -H. In some embodiments, -R 12b -T. In some embodiments, -R 12b -C. In some embodiments, -R 1-6 alkyl. In some embodiments, -R 12b -C. In some embodiments, -R 2-6 alkenyl. In some embodiments, -R 12b -C. In some embodiments, -R 2-6 alkynyl.

[1208] In some embodiments, T of formula (XI) is selected from the group consisting of: phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl. In some embodiments, T of formula (XI) is phenyl. In some embodiments, T of formula (XI) is naphthyl. In some embodiments, T of formula (XI) is indenyl. In some embodiments, T of formula (XI) is indanyl. In some embodiments, T of formula (XI) is tetrahydronaphthyl. In some embodiments, T of formula (XI) is tetrahydronaphthyl. In some embodiments, T of formula (XI) is C 3-10 cycloalkyl. In some embodiments, T of formula (XI) is 3- to 10-membered heterocyclyl. In some embodiments, T of formula (XI) is 8- to 11-membered heterobicyclyl.

[1209] In some embodiments, T of formula (XI) is substituted with one or more -R 13 groups of formula (XI). In some embodiments, T of formula (XI) is substituted with one -R 13 group of formula (XI). In some embodiments, T of formula (XI) is not substituted with -R 13 groups of formula (XI).

[1210] In some embodiments, T of formula (XI) is substituted with one -R 13 group of formula (XI). In some embodiments, T of formula (XI) is not substituted with -R 13 groups of formula (XI).

[1211] In some embodiments, -R 13 of formula (XI) is selected from the group consisting of: halogen, -CN, oxo, -C(O)OR 15 , -OR 15 , -C(O)R 15 , -C(O)N(R 15 )(R 15a ), -S(O)2N(R 15 )(R 15a ), -S(O)N(R 15 )(R 15a ), -S(O)2R 15 , -S(O)R 15 , -N(R 15)S(O)2N(R 15a )(R 15b ),-SR 15 、-N(R 15 )(R 15a )、-NO2、-OC(O)R 15 、-N(R 15 )C(O)R 15a 、-N(R 15 )S(O)2R 15a 、-N(R 15 )S(O)R 15a 、-N(R 15 )C(O)OR 15a 、-N(R 15 )C(O)N(R 15a )(R 15b )、-OC(O)N(R 15 )(R 15a ) and C 1-6 In some embodiments, -R 13 In some embodiments, -R 13 In some embodiments, -R 13 In some embodiments, -R 13 -C(O)OR 15 In some embodiments, -R 13 For-OR 15 In some embodiments, -R 13 -C(O)R 15 In some embodiments, -R 13 -C(O)N(R 15 )(R 15a In some embodiments, -R 13 -S(O)2N(R 15 )(R 15a In some embodiments, -R 13 -S(O)N(R 15 )(R 15a In some embodiments, -R 13 is -S(O)2R 15 In some embodiments, -R 13 -S(O)R 15 In some embodiments, -R 13 -N(R 15)S(O)2N(R 15a )(R 15b ) In some embodiments, -R 13 of Formula (XI) is -SR 15 In some embodiments, -R 13 of Formula (XI) is -N(R 15 )(R 15a ) In some embodiments, -R 13 of Formula (XI) is -NO2. In some embodiments, -R 13 of Formula (XI) is -OC(O)R 15 In some embodiments, -R 13 of Formula (XI) is -N(R 15 )C(O)R 15a In some embodiments, -R 13 of Formula (XI) is -N(R 15 )S(O)2R 15a In some embodiments, -R 13 of Formula (XI) is -N(R 15 )S(O)R 15a In some embodiments, -R 13 of Formula (XI) is -N(R 15 )C(O)OR 15a In some embodiments, -R 13 of Formula (XI) is -N(R 15 )C(O)N(R 15a )(R 15b ) In some embodiments, -R 13 of Formula (XI) is -OC(O)N(R 15 )(R 15a ) In some embodiments, -R 13 of Formula (XI) is C 1-6 alkyl.

[1212] In some embodiments, -R 14 of Formula (XI) is selected from -H and C 1-6 alkyl. In some embodiments, -R 14 of Formula (XI) is -H. In some embodiments, -R 14 of Formula (XI) is C 1-6 alkyl.

[1213] In some embodiments, -R 14a of Formula (XI) is selected from -H and C 1-6 alkyl. In some embodiments, -R 14a-H. In some embodiments, -R 14a is C 1-6 alkyl. In some embodiments, -R

[1214] -H. In some embodiments, -R 15 is selected from -H and C 1-6 alkyl. In some embodiments, -R 15 -H. In some embodiments, -R 15 is C 1-6 alkyl. In some embodiments, -R

[1215] -H. In some embodiments, -R 15a is selected from -H and C 1-6 alkyl. In some embodiments, -R 15a -H. In some embodiments, -R 15a is C 1-6 alkyl. In some embodiments, -R

[1216] -H. In some embodiments, -R 15b is selected from -H and C 1-6 alkyl. In some embodiments, -R 15b -H. In some embodiments, -R 15b is C 1-6 alkyl. In some embodiments, -R

[1217] -H. In some embodiments, -R 1 and -R 1a , together with the atom to which they are attached, combine to form a C 3-10 cycloalkyl. In some embodiments, -R 1 and -R 1a , together with the atom to which they are attached, combine to form a 3- to 10-membered heterocyclyl. In some embodiments, -R 1 and -R 1a , together with the atom to which they are attached, combine to form an 8- to 11-membered heterobicyclyl.

[1218] -H. In some embodiments, -R 2 and -R 2a , together with the atom to which they are attached, combine to form a C 3-10 cycloalkyl. In some embodiments, -R 2 and -R 2a , together with the atom to which they are attached, combine to form a 3- to 10-membered heterocyclyl. In some embodiments, -R 2 and -R 2awith the atom to which they are attached form an 8- to 11-membered heterobicyclyl.

[1219] In some embodiments, -R of formula (XI) is 3 and -R 3a with the atom to which they are attached form a C 3-10 cycloalkyl. In some embodiments, -R of formula (XI) is 3 and -R 3a with the atom to which they are attached form a 3- to 10-membered heterocyclyl. In some embodiments, -R of formula (XI) is 3 and -R 3a with the atom to which they are attached form an 8- to 11-membered heterobicyclyl.

[1220] In some embodiments, -R of formula (XI) is 4 and -R 4a with the atom to which they are attached form a C 3-10 cycloalkyl. In some embodiments, -R of formula (XI) is 4 and -R 4a with the atom to which they are attached form a 3- to 10-membered heterocyclyl. In some embodiments, -R of formula (XI) is 4 and -R 4a with the atom to which they are attached form an 8- to 11-membered heterobicyclyl.

[1221] In some embodiments, -R of formula (XI) is 5 and -R 5a with the atom to which they are attached form a C 3-10 cycloalkyl. In some embodiments, -R of formula (XI) is 5 and -R 5a with the atom to which they are attached form a 3- to 10-membered heterocyclyl. In some embodiments, -R of formula (XI) is 5 and -R 5a with the atom to which they are attached form an 8- to 11-membered heterobicyclyl.

[1222] In some embodiments, -R of formula (XI) is 8 and -R 8a with the atom to which they are attached form a C 3-10 cycloalkyl. In some embodiments, -R of formula (XI) is 8 and -R 8a with the atom to which they are attached form a 3- to 10-membered heterocyclyl. In some embodiments, -R of formula (XI) is 8 and -R 8atogether with the atoms to which they are attached form an 8- to 11-membered heterobicyclyl.

[1223] In some embodiments, -R 1 and -R 2 together with the atoms to which they are attached form a ring -A- of Formula (XI).

[1224] In some embodiments, -R 1 and -R 8 together with the atoms to which they are attached form a ring -A- of Formula (XI).

[1225] In some embodiments, -R 1 and -R 9 together with the atoms to which they are attached form a ring -A- of Formula (XI).

[1226] In some embodiments, -R 2 and -R 9 together with the atoms to which they are attached form a ring -A- of Formula (XI).

[1227] In some embodiments, -R 2 and -R 10 together with the atoms to which they are attached form a ring -A- of Formula (XI).

[1228] In some embodiments, -A- of Formula (XI) is phenyl. In some embodiments, -A- of Formula (XI) is naphthyl. In some embodiments, -A- of Formula (XI) is indenyl. In some embodiments, -A- of Formula (XI) is indanyl. In some embodiments, -A- of Formula (XI) is tetrahydronaphthyl. In some embodiments, -A- of Formula (XI) is C 3-10 cycloalkyl. In some embodiments, -A- of Formula (XI) is 3- to 10-membered heterocyclyl. In some embodiments, -A- of Formula (XI) is 8- to 11-membered heterobicyclyl.

[1229] In some embodiments, -R 3 and -R 6 together with the atoms to which they are attached form a ring -A'- of Formula (XI).

[1230] In some embodiments, -R 4 and -R 6 together with the atoms to which they are attached form a ring -A'- of Formula (XI).

[1231] In some embodiments, -R5 and -R 6 together with the atoms to which they are attached form a ring -A'- of Formula (XI).

[1232] In some embodiments, -R 6 and -R 6a together with the atoms to which they are attached form a ring -A'- of Formula (XI).

[1233] In some embodiments, -R 6 and -R 7 together with the atoms to which they are attached form a ring -A'- of Formula (XI).

[1234] In some embodiments, -A'- of Formula (XI) is a 3- to 10-membered heterocyclyl. In some embodiments, -A'- of Formula (XI) is an 8- to 11-membered heterobicyclyl.

[1235] In some embodiments, -L 1 is of Formula (XIa):

[1236]

[1237] wherein

[1238] the dotted line indicates attachment to the nitrogen of a primary or secondary amine of -D;

[1239] -R 1 , -R 1a , -R 2 , -R 2a , -R 3 , -R 3a , -R 5 , -R 5a , -R 6 and -R 6a are used as defined in Formula (XI); and

[1240] -L 1 - is substituted with -L 2 - is substituted and optionally further substituted, with the proviso that the hydrogen marked with an asterisk in Formula (XIa) is not replaced with a substituent.

[1241] In some embodiments, the dotted line in Formula (XIa) indicates attachment to the nitrogen of a primary amine of -D. In some embodiments, the dotted line in Formula (XIa) indicates attachment to the nitrogen of a secondary amine of -D.

[1242] In some embodiments, -R 1 is -H. In some embodiments, -R 1a is -H. In some embodiments, -R2 In some embodiments, -R 2a In some embodiments, -R 3 In some embodiments, -R 3a In some embodiments, -R 5 In some embodiments, -R 5a In some embodiments, -R 6 In some embodiments, -R 6a is -H.

[1243] In some embodiments, -L of Formula (XIa) 1 - has not been further replaced.

[1244] In some embodiments, -R 1 is -H, the -H is replaced by -L 2 In some embodiments, -R 1a is -H, the -H is replaced by -L 2 In some embodiments, -R 2 is -H, the -H is replaced by -L 2 In some embodiments, -R 2a is -H, the -H is replaced by -L 2 In some embodiments, -R 3 is -H, the -H is replaced by -L 2 In some embodiments, -R 3a is -H, the -H is replaced by -L 2 In some embodiments, -R 5 is -H, the -H is replaced by -L 2 In some embodiments, -R 5a is -H, the -H is replaced by -L 2 In some embodiments, -R 6 is -H, the -H is replaced by -L 2 In some embodiments, -R 6a is -H, the -H is replaced by -L 2 -replace.

[1245] In some embodiments, -L 1 - having the formula (XIb):

[1246]

[1247] in

[1248] The dashed line indicates the connection to the nitrogen of the primary or secondary amine of -D; and

[1249] -L 1 -被-L 2 -substituted and optionally further substituted, provided that the hydrogens marked with an asterisk in formula (XIb) are not replaced by substituents.

[1250] In some embodiments, the dashed line in Formula (XIb) indicates attachment to the nitrogen of the primary amine of -D. In some embodiments, the dashed line in Formula (XIb) indicates attachment to the nitrogen of the secondary amine of -D.

[1251] In some embodiments, -L of formula (XIb) 1 - has not been further replaced.

[1252] In some embodiments, -L 1 - having the formula (XIc):

[1253]

[1254] in

[1255] The unlabeled dashed line indicates the connection to the nitrogen of the primary or secondary amine of -D, and

[1256] The dotted line marked with # indicates the same as -L 2 -connection.

[1257] In some embodiments, the unlabeled dashed line in Formula (XIc) indicates attachment to the nitrogen of the primary amine of -D. In some embodiments, the unlabeled dashed line in Formula (XIc) indicates attachment to the nitrogen of the secondary amine of -D.

[1258] In some embodiments, -L 1 - has a structure as disclosed in WO 2020 / 254603 A1, which is incorporated herein by reference in its entirety. Thus, in some embodiments, -L 1 -part has formula (XII):

[1259]

[1260] in

[1261] The dashed line indicates the connection to the heteroaromatic N of -D donating a π electron pair;

[1262] n is an integer selected from 0, 1, 2, 3 and 4;

[1263] =X 1 Selected from =O, =S and =N(R 4 );

[1264] -X 2 - is selected from -O-, -S-, -N(R5 )- and -C(R 6 )(R 6a )-;

[1265] -X 3 - selected from: -C(R 10 )(R 10a )-, -C(R 11 )(R 11a )-C(R 12 )(R 12a )-, -O- and -C(O)-;

[1266] -R 1 , -R 1a , -R 6 , -R 6a , -R 10 , -R 10a , -R 11 , -R 11a , -R 12 , -R 12a and -R 2 and -R 2a are each independently selected from: -H, -C(O)OH, halogen, -CN, -OH, C 1-6 alkyl, C 2-6 alkenyl and C 2-6 alkynyl; wherein C 1-6 alkyl, C 2-6 alkenyl and C 2-6 alkynyl are optionally substituted with one or more -R 13 which are the same or different; and wherein C 1-6 alkyl, C 2-6 alkenyl and C 2-6 alkynyl are optionally interrupted with one or more groups selected from: -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R 14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14 )C(O)N(R 14a )- and -OC(O)N(R 14 )-;

[1267] -R3 , -R 4 , -R 5 , -R 7 , -R 8 , and -R 9 are independently selected from the group consisting of: -H, -T, -CN, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally substituted with one or more -R 13 groups, which are the same or different; and wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally interrupted with one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 14 )-, -S(O)2N(R 14 )-, -S(O)N(R 14 )-, -S(O)2-, -S(O)-, -N(R 14 )S(O)2N(R 14a )-, -S-, -N(R 14 )-, -OC(OR 14 )(R 14a )-, -N(R 14 )C(O)N(R 14a )-, and -OC(O)N(R 14 )-;

[1268] each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R 13 groups, which are the same or different;

[1269] wherein -R 13 is selected from the group consisting of: -H, -NO2, -OCH3, -CN, -N(R 14 )(R 14a ), -OH, -C(O)OH, and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;

[1270] wherein -R 14 and -R 14a are independently selected from -H and C 1-6 alkyl; wherein C 1-6alkyl is optionally substituted with one or more halogen, which are the same or different;

[1271] optionally, -R 1 optionally, -R 1a optionally, -R 2 optionally, -R 2a optionally, two adjacent -R 2 optionally, -R 6 optionally, -R 6a optionally, -R 10 optionally, -R 10a optionally, -R 11 optionally, -R 11a and -R 12 optionally, -R 12a one or more pairs of -R 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, or 8- to 11-membered heterobicyclyl;

[1272] optionally, -R 1 optionally, -R 2 optionally, -R 1 optionally, -R 5 optionally, -R 1 optionally, -R 6 optionally, -R 1 optionally, -R 9 optionally, -R 1 optionally, -R 10 optionally, -R 3 optionally, -R 6a optionally, -R 4 optionally, -R 5 optionally, -R 4 optionally, -R 6 optionally, -R 5 optionally, -R 10 and -R 6 optionally, -R 10 one or more pairs of -R

[1273] wherein -A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl;

[1274] optionally, -R 1 and adjacent -R 2 form a carbon-carbon double bond, provided that n is selected from 1, 2, 3, and 4;

[1275] optionally, two adjacent -R 2 form a carbon-carbon double bond, provided that n is selected from 2, 3, and 4;

[1276] provided that if -X 2 - is -N(R5 )-, then -X 3 - is selected from: and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 5, 6, or 7 atoms, and if present, a carbon-carbon double bond formed between -R 1 and -R 2 or between two adjacent -R 2 groups is in the cis configuration; and each -L 1 - is substituted with -L 2 - and optionally further substituted.

[1277] It is understood that the "N" in the phrase "heteroaromatic N donating a pi electron pair" refers to a nitrogen.

[1278] It is understood that two adjacent -R 2 groups in formula (XII) can only be present if n is at least 2.

[1279] It is understood that the expression "distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk" refers to the total number of atoms in the shortest distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk, and also includes the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk. For example, in the following structure, n is 1, and the distance between the nitrogen marked with an asterisk and the carbon marked with an asterisk is 5:

[1280]

[1281] In the following structure, n is 2, -R 1 and -R 1a form a cyclohexyl group, and the distance between the nitrogen marked with an asterisk and the carbon marked with an asterisk is 6:

[1282]

[1283] In some embodiments, =X 1 of formula (XII) is =0. In some embodiments, =X 1 of formula (XII) is =S. In some embodiments, =X 1 of formula (XII) is =N(R 4 ).

[1284] In some embodiments, -X 2 - of formula (XII) is -0-. In some embodiments, -X 2 - of formula (XII) is -S-. In some embodiments, -X 2 - of formula (XII) is -N(R 5 )-. In some embodiments, -X 2 - of formula (XII) is -C(R 6)(R 6a )-.

[1285] In some embodiments, -X 3 - is

[1286] In some embodiments, -X 3 - is

[1287] In some embodiments, -X 3 - is

[1288] In some embodiments, -X 3 - is -C(R 10 )(R 10a )-. In some embodiments, -X 3 - is -C(R 11 )(R 11a )-C(R 12 )(R 12a )-. In some embodiments, -X 3 - is -O-. In some embodiments, -X 3 - is -C(O)-.

[1289] In some embodiments, -X 2 - is -N(R 5 )-, -X 3 - is and the distance between the starred nitrogen atom and the starred carbon atom in Formula (XII) is 5 atoms.

[1290] In some embodiments, -X 2 - is -N(R 5 )-, -X 3 - is and the distance between the starred nitrogen atom and the starred carbon atom in Formula (XII) is 6 atoms.

[1291] In some embodiments, -X 2 - is -N(R 5 )-, -X 3 - is and the distance between the starred nitrogen atom and the starred carbon atom in Formula (XII) is 7 atoms.

[1292] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 5 atoms.

[1293] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 6 atoms.

[1294] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 7 atoms.

[1295] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 5 atoms.

[1296] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 6 atoms.

[1297] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 7 atoms.

[1298] In some embodiments, -X in formula (XII) is -N(R 2 - is -N(R 5 )-, -X in formula (XII) is -N(R 3 - is -N(R And the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 5 atoms.

[1299] In some embodiments, -X of formula (XII) 2 - is -N(R 5 )-, -X of formula (XII) 3 -for And the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 6 atoms.

[1300] In some embodiments, -X of formula (XII) 2 - is -N(R 5 )-, -X of formula (XII) 3 -for And the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XII) is 7 atoms.

[1301] In some embodiments, =X of formula (XII) 1 =O, -X in formula (XII) 2 -for-C(R 6 )(R 6a )-, -X of formula (XII) 3 -for And -R 3 Contains no amines.

[1302] In some embodiments, -R 1 、-R 1a 、-R 6 、-R 6a 、-R 10 、-R 10a 、-R 11 、-R 11a 、-R 12 、-R 12a and formula (XII)-R 2 and -R 2a Each of the following is independently selected from: -H, -C(O)OH, halogen, -CN, -OH, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Alkynyl.

[1303] In some embodiments, -L 1 - has a structure as disclosed in WO 2020 / 254602 A1, which is incorporated herein by reference in its entirety. 1 - is suitable for drug D that, when bound to -L 1 - contains electron-donating heteroaromatic N+ partially or fully quaternary ammonium cation and becomes -D upon ligation + partially or fully quaternary ammonium cation and becomes -D upon ligation. Thus, in some embodiments, -L 1 - has formula (XII):

[1304]

[1305] wherein

[1306] the dashed line indicates attachment to -D + of N + ; and

[1307] t is selected from 0, 1, 2, 3, 4, 5, and 6;

[1308] -A- is a ring selected from monocyclic or bicyclic aryl and heteroaryl, provided that -A- is attached to -Y and -C(R 1 )(R 1a )- via a carbon atom; wherein said monocyclic or bicyclic aryl and heteroaryl are optionally substituted with one or more -R 2 , which are the same or different;

[1309] -R 1 , -R 1a , and each -R 2 is independently selected from the group consisting of: -H, -C(O)OH, -halo, -NO2, -CN, -OH, C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl; wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally substituted with one or more -R 3 , which are the same or different; and wherein C 1-6 alkyl, C 2-6 alkenyl, and C 2-6 alkynyl are optionally interrupted with one or more groups selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 4 )-, -S(O)2N(R 4 )-, -S(O)N(R 4 )-, -S(O)2-, -S(O)-, -N(R 4 )S(O)2N(R 4a )-, -S-, -N(R 4 )-, -OC(OR 4 )(R 4a )-, -N(R 4 )C(O)N(R 4a )-, and -OC(O)N(R 4 )-;

[1310] each -T- is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, wherein each -T- is independently optionally substituted with one or more -R 3 substituents, which are the same or different;

[1311] wherein -R 3 is selected from: -H, -NO2, -OCH3, -CN, -N(R 4 )(R 4a ), -OH, -C(O)OH, and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;

[1312] wherein -R 4 and -R 4a are independently selected from -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more halogens, which are the same or different;

[1313] -Y is selected from:

[1314]

[1315] and a peptidyl moiety;

[1316] wherein

[1317] the dashed line marked with an asterisk indicates the attachment to -A-.

[1318] -Nu is a nucleophilic group;

[1319] -Y 1 - is selected from -O-, -C(R 10 )(R 10a )-, -N(R 11 )-, and -S-;

[1320] =Y 2 is selected from =O, =S, and =N(R 12 );

[1321] -Y 3 - is selected from -O-, -S-, and -N(R 13 )-;

[1322] -E- is selected from C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, and -Q-; wherein C 1-6 alkyl, C2-6 alkenyl, C 2-6 alkynyl is optionally substituted with one or more identical or different -R 14 ;

[1323] -R 5 , -R 6 , each -R 7 , -R 8 , -R 9 , -R 10 , -R 10a , -R 11 , -R 12 , and -R 13 are independently selected from the group consisting of C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, and -Q; wherein C 1-20 alkyl, C 2-20 alkenyl, and C 2-20 alkynyl is optionally substituted with one or more identical or different -R 14 ; and wherein C 1-20 alkyl, C 2-20 alkenyl, and C 2-20 alkynyl is optionally interrupted with one or more groups selected from -Q-, -C(O)O-, -O-, -C(O)-, -C(O)N(R 15 )-, -S(O)2N(R 15 )-, -S(O)N(R 15 )-, -S(O)2-, -S(O)-, -N(R 15 )S(O)2N(R 15a )-, -S-, -N(R 15 )-, -OC(OR 15 )R 15a -, -N(R 15 )C(O)N(R 15a )-, and -OC(O)N(R 15 )-; each Q is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl, wherein each Q is independently optionally substituted with one or more identical or different -R 14 ;

[1324] wherein -R 14 , -R 15 , and -R 15a are independently selected from the group consisting of -H and C 1-6 alkyl; wherein C 1-6 alkyl is optionally substituted with one or more identical or different halogens; and

[1325] each -L 1 - is optionally further substituted. 2 - is optionally further substituted.

[1326] It will be appreciated that, in some embodiments, -D + may comprise both an electron-donating heteroaromatic N + and a quaternary ammonium cation, and similarly, the corresponding D can comprise both an electron-donating heteroaromatic N and a tertiary amine. It will also be appreciated that, if D is bound to -L 1 - then -D + and -L 1 - form a quaternary ammonium cation, wherein a counter anion can be present. Examples of counter anions include, but are not limited to, chloride, bromide, acetate, bicarbonate, sulfate, bisulfate, nitrate, carbonate, alkylsulfonate, arylsulfonate, and phosphate.

[1327] -L 1 of formula (XIII) is optionally further substituted as described elsewhere herein.

[1328] In some embodiments, -L 1 of formula (XIII) is not further substituted.

[1329] Such drug moieties -D + comprise at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) electron-donating heteroaromatic N + or quaternary ammonium cation, and similarly, the corresponding released drug D comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) electron-donating heteroaromatic N or tertiary amine. Examples of chemical structures that include a heteroaromatic nitrogen atom (i.e., N + or N) that donates one electron to the aromatic pi system include, but are not limited to, pyridine, pyridazine, pyrimidine, quinoline, quinazoline, quinoxaline, pyrazole, imidazole, isoindazole, indazole, purine, tetrazole, triazole, and triazine. For example, in the following imidazole ring, the heteroaromatic nitrogen that donates one electron to the aromatic pi system is marked with a “§”:

[1330]

[1331] Such electron-donating heteroaromatic nitrogen atoms do not include heteroaromatic nitrogen atoms that donate a pair of electrons (i.e., not one electron) to the aromatic pi system, such as the nitrogen marked with a “#” in the above imidazole ring structure. Drug D can exist in one or more tautomeric forms, such as where one hydrogen atom is moved between at least two heteroaromatic nitrogen atoms. In all such cases, the linker moiety is covalently and reversibly attached at the electron-donating heteroaromatic nitrogen to the aromatic pi system.

[1332] As used herein, the term "monocyclic or bicyclic aryl" means an aromatic hydrocarbon ring system which can be monocyclic or bicyclic, wherein a monocyclic aryl ring consists of at least 5 ring carbon atoms and can comprise up to 10 ring carbon atoms, and wherein a bicyclic aryl ring consists of at least 8 ring carbon atoms and can comprise up to 12 ring carbon atoms. Each hydrogen atom of a monocyclic or bicyclic aryl group can be replaced by a substituent as defined below.

[1333] As used herein, the term "monocyclic or bicyclic heteroaryl" means a monocyclic aromatic ring system which can comprise 2 to 6 ring carbon atoms and 1 to 3 ring heteroatoms, or a bicyclic aromatic ring system which can comprise 3 to 9 ring carbon atoms and 1 to 5 ring heteroatoms, for example selected from nitrogen, oxygen, and sulfur. Examples of monocyclic or bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzothiophenyl, furanyl, imidazolyl, indolyl, azaindolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, tetrazinyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, quinazolinyl, quinoxalinyl, triazolyl, thiazolyl, and thiophenyl. Each hydrogen atom of a monocyclic or bicyclic heteroaryl group can be replaced by a substituent as defined below.

[1334] As used herein, the term "nucleophilic group" or "nucleophile" refers to a reagent or functional group that forms a bond with a reactive ligand (i.e., an electrophile or functional group) by donating two bonding electrons.

[1335] In some embodiments, t of formula (XIII) is 0. In some embodiments, t of formula (XIII) is 1. In some embodiments, t of formula (XIII) is 2. In some embodiments, t of formula (XIII) is 3. In some embodiments, t of formula (XIII) is 4. In some embodiments, t of formula (XIII) is 5. In some embodiments, t of formula (XIII) is 6.

[1336] In some embodiments, -A- of formula (XIII) is a ring selected from monocyclic or bicyclic aryl and heteroaryl, provided that -A- is connected to -Y and -C(R 1 )(R 1a )- via a carbon atom. In some embodiments, -A- of formula (XIII) is substituted with one or more -R 2 of formula (XIII). In some embodiments, -A- of form...

Claims

1. A method for preparing hydrogel microspheres comprising cross-linked hyaluronic acid (HA) or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps: (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and optionally other functional groups and a second functionalized HA modified with one or more -FG2 and optionally other functional groups, wherein -FG1 and -FG2 are each a functional group moiety different from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres; (b) optionally, adding a pH adjuster to the emulsion of step (a); and (c) collecting the hydrogel HA microspheres obtained in step (a) or (b).

2. The method of claim 1, wherein the solution A of step (a) further comprises a buffer.

3. The method according to claim 1 or 2, wherein the solution B of step (a) comprises an emulsifier and a solvent.

4. The method of any one of claims 1 to 3, wherein the method comprises the following steps: (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and a second functionalized HA modified with one or more -FG2, wherein -FG1 and -FG2 are functional group moieties different from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres; (b) optionally, adding a pH adjuster to the emulsion of step (a); (c) collecting the hydrogel HA microspheres obtained in step (b); (d) optionally, size-fractionating the hydrogel HA microspheres obtained in step (c) to obtain microspheres with a specific size distribution; (e) optionally, washing the microspheres obtained in step (c) or (d); (f) optionally, incubating the hydrogel HA microspheres of step (c), (d), or (e) in a buffer having a pH ranging from about 8 to about 12; (g) optionally, incubating the hydrogel HA microspheres of step (c), (d), (e) or (f) with a reducing agent; (h) optionally, washing the microspheres obtained in step (f) or (g); and (i) Optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g) or (h).

5. The method of any one of claims 1 to 4, wherein -FG1 is independently selected from: Wherein the dotted line indicates the connection to the first functionalized HA; -Y 01 independently selected from -F, -Cl, -Br and -I; Each-R 08 and -R 08a Independently selected from: halogen, -H, -CN, -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 09 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-、-S(O)2N(R 010 )-、-S(O)N(R 010 )-、-S(O)2-、-S(O)-、-N(R 010 )S(O)2N(R 010a )-、-S-、-N(R 010 )-、-OC(OR 010 )(R 010a )-、-N(R 010 )C(O)N(R 010a )-and-OC(O)N(R 010 )-; Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R 09 replace; and Each-R 09 、-R 010 and -R 010a Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and -FG2 is independently selected from: Wherein the dotted line indicates the connection to the second functionalized HA; Each-Y 02 and -Y 02a independently selected from -H and -Br; The conditions are: if -FG1 has formula (y-56), then -FG2 has formula (y-57) or (y-86); if -FG1 has formula (y-1), then -FG2 has formula (y-16) or (y-47); if -FG1 has formula (y-44), then -FG2 has formula (y-16) or (y-47); if -FG1 has formula (y-6), then -FG2 has formula (y-9); if -FG1 has formula (y-49), then -FG2 has formula (y-85); if -FG1 has formula (y-44), then -FG2 has formula (y-47); or if -FG1 has formula (y-39), then -FG2 has formula (y-56).

6. The method of any one of claims 1 to 5, wherein the pH adjuster increases the pH of the emulsion of step (a), and -FG1 is independently selected from: Wherein the dotted line indicates the connection to the first functionalized HA; -Y 01 independently selected from -F, -Cl, -Br and -I; Each-R 08 and -R 08a Independently selected from: halogen, -H, -CN, -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T 0 、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R 09 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T 0 -, -C(O)O-, -O-, -C(O)-, -C(O)N(R 010 )-、-S(O)2N(R 010 )-、-S(O)N(R 010 )-、-S(O)2-、-S(O)-、-N(R 010 )S(O)2N(R 010a )-、-S-、-N(R 010 )-、-OC(OR 010 )(R 010a )-、-N(R 010 )C(O)N(R 010a )-and-OC(O)N(R 010 )-; Each T 0 independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 Cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T 0 are optionally replaced by one or more identical or different -R 09 replace; and Each-R 09 、-R 010 and -R 010a Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and -FG2 is independently selected from: Wherein the dotted line indicates the connection to the second functionalized HA; Each-Y 02 and -Y 02a independently selected from -H and -Br; The conditions are: if -FG1 has formula (y-56), then -FG2 has formula (y-57) or (y-86); if -FG1 has formula (y-1), then -FG2 has formula (y-16); if -FG1 has formula (y-44), then -FG2 has formula (y-16); or if -FG1 has formula (y-39), then -FG2 has formula (y-56).

7. The method of any one of claims 1 to 6, wherein -FG1 is Wherein the dotted line indicates the connection with the first functionalized HA, -FG2 is wherein the dotted line represents the connection to the second functionalized HA, the pH adjusting agent increases the pH of the emulsion of step (a) from about 1 to about 9, preferably from about 1 to about 5.5, more preferably from about 2 to about 4, and wherein each -Y 02 and -Y 02a are independently selected from -H and -Br.

8. The method of any one of claims 1 to 7, wherein the method comprises the following steps: (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more -FG1 and a second functionalized HA modified with one or more -FG2, wherein -FG1 and -FG2 are functional group moieties different from each other, and wherein -FG1 on the first functionalized HA reacts with -FG2 on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 Each of the units: The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 Each of the units: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each R a1 Independently selected from: -H, C 1-10 Alkyl, ammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, alkali metal ions and alkaline earth metal ions; Each-R a2 are independently -H or C 1-10 alkyl; Each -FG1 and -FG2 is independently a functional group moiety; Each -X-, -Y- is independently a carbonyl group or absent; Each -X'-, -Y'- is independently a spacer moiety or absent; (b) adding a pH adjuster to the emulsion of step (a); (c) collecting the hydrogel HA microspheres obtained in step (a) or (b), wherein the hydrogel comprises a plurality of Z 3 unit: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each-R a2 , -X-, -Y-, -X'- and -Y'- are as defined in step (a); Each-L 3 - independently part of the link or not present; (d) optionally, size fractionating the hydrogel HA microparticles obtained in step (a), (b) or (c) to obtain microspheres with a specific size distribution; (e) optionally, washing the hydrogel HA microspheres obtained in step (a), (b), (c) or (d); (f) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), or (e) in a buffer having a pH ranging from about 8 to about 12; (g) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), (e) or (f) with a reducing agent; (h) optionally, washing the microspheres obtained in step (f) or (g); and (i) Optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g) or (h).

9. The method of any one of claims 1 to 8, wherein the method comprises the following steps: (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 -i Each of the units: The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 -i Each of the units: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each R a1 Independently selected from: -H, C 1-10 Alkyl, ammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, alkali metal ions and alkaline earth metal ions; Each-R a2 are independently -H or C 1-10 alkyl; Each -X'-, -Y'- is independently a spacer moiety or absent; (b) adding a pH adjuster to the emulsion of step (a); (c) collecting the hydrogel HA microspheres obtained in step (a) or (b), wherein the hydrogel comprises a plurality of Z 3 -i unit: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each-R a2 , -X'- and -Y'- are as defined in step (a); (d) optionally, size fractionating the hydrogel HA microparticles obtained in step (a), (b) or (c) to obtain microspheres with a specific size distribution; (e) optionally, washing the hydrogel HA microspheres obtained in step (a), (b), (c) or (d); (f) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), or (e) in a buffer having a pH ranging from about 8 to about 12, To provide multiple Z 3 -i' unit hydrogel HA microspheres: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each R a1 、-R a2 , -X'- and -Y'- are as defined in step (a); (g) optionally, incubating the hydrogel HA microspheres of step (a), (b), (c), (d), (e) or (f) with a reducing agent; (h) optionally, washing the microspheres obtained in step (f) or (g); and (i) Optionally, collecting the hydrogel HA microspheres of step (d), (e), (f), (g) or (h).

10. The process of claim 8 or 9, wherein steps (d) and (e) are not optional and steps (f) to (h) are absent.

11. The method of any one of claims 1 to 10, wherein the pH adjuster 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, 1,1,4,7,10,10-hexamethyltriethylenetetramine, 1,4,7-trimethyl-1,4,7-triazacyclononane, Tris[2-(dimethylamino)ethyl]amine, triethylamine, diisopropylethylamine (DIPEA), trimethylamine, N,N-dimethylethylamine, N,N,N',N'-tetramethyl-1,6-hexanediamine, N,N,N',N",N"-pentamethyldiethylenetriamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]nonane-5-ene and hexamethylenetetramine.

12. The method of any one of claims 1 to 11, wherein each -X'- and -Y'- is independently a spacer moiety selected from the group consisting of: -T'-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R y1 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y2 )-、-S(O)2N(R y2 )-、-S(O)N(R y2 )-、-S(O)2-、-S(O)-、-N(R y2 )S(O)2N(R y2a )-、-S-、-N(R y2 )-、-OC(OR y2 )(R y2a )-、-N(R y2 )C(O)N(R y2a )-and-OC(O)N(R y2 )-; Each T' is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic and 8- to 30-membered heteropolycyclic; wherein each T' is independently optionally replaced by one or more identical or different -R y1 replace; Each-R y1 Independently selected from: halogen, -CN, oxo (=O), -COOR y3 、-OR y3 、-C(O)R y3 、-C(O)N(R y3 R y3a )、-S(O)2N(R y3 R y3a )、-S(O)N(R y3 R y3a )、-S(O)2R y3 、-S(O)R y3 、-N(R y3 )S(O)2N(R y3a R y3b ),-SR y3 、-N(R y3 R y3a )、-NO2、-OC(O)R y3 、-N(R y3 )C(O)R y3a 、-N(R y3 )S(O)2R y3a 、-N(R y3 )S(O)R y3a 、-N(R y3 )C(O)OR y3a 、-N(R y3 )C(O)N(R y3a R y3b )、-OC(O)N(R y3 R y3a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and Each-R y2 、-R y2a 、-R y3 、-R y3a 、-R y3b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

13. The method of any one of claims 1 to 12, wherein the method comprises the following steps: (a) mixing solution A with solution B to form an emulsion, wherein solution A comprises a first functionalized HA modified with one or more thiol functional groups and a second functionalized HA modified with one or more maleimide functional groups, wherein the thiol functional groups on the first functionalized HA react with the maleimide functional groups on the second functionalized HA to form a plurality of crosslinks, thereby forming hydrogel HA microspheres, wherein, The first functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 -i Each of the units: The second functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 6 -i Each of the units: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each R a1 is H or alkali metal ion; Each-R a2 is -H; Each -X'- has the formula (x4): wherein the unmarked dashed line indicates the connection to the carbonyl group, the dashed line marked with an asterisk indicates the connection to the sulfur atom, and c0 is 7; Each -Y'- has the formula (y4): wherein the unlabeled dashed line indicates the connection to the carbonyl group, and the dashed line marked with an asterisk indicates the connection to the nitrogen atom of the maleimide ring; (b) adding TMEDA to the emulsion of step (a); (c) collecting the hydrogel HA microspheres obtained in step (a) or (b), wherein the hydrogel comprises a plurality of Z 3 -i unit: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each-R a2 , -X'- and -Y'- are as defined in step (a); (d) size fractionating the hydrogel HA microparticles obtained in step (c) to obtain microspheres with a specific particle size distribution; (e) washing the hydrogel HA microspheres obtained in step (d); and (f) Collecting the hydrogel HA microspheres from step (e).

14. The method of any one of claims 1 to 13, wherein the molecular weight of the first and second functionalized HAs independently ranges from about 80 kDa to about 250 kDa, such as from about 90 kDa to about 200 kDa, or such as from about 100 kDa to about 150 kDa.

15. The method of any one of claims 9 to 14, wherein Z 5 The degree of thiol functionalization in -i is about 5% and Z 6 The degree of maleimide functionalization in -i is about 10%.

16. The method of any one of claims 1 to 15, wherein in step (a), solution A further comprises citrate and / or histidine, preferably at a pH of about 2.

17. The method of any one of claims 1 to 16, wherein in step (a), solution B comprises heptane and sorbitan monooleate, or tetradecane and sorbitan monooleate.

18. The method of any one of claims 1 to 17, wherein in step (b), the pH adjusting agent is TMEDA and the pH of the emulsion of step (a) is increased, preferably to a pH of about 4.

19. The process of any one of claims 1 to 18, wherein in step (d), size fractionation is performed via wet screening.

20. Hydrogel HA microspheres or pharmaceutically acceptable salts thereof, obtainable by the method according to any one of claims 1 to 19.

21. Use of the hydrogel HA microspheres or pharmaceutically acceptable salts thereof according to claim 20 as a carrier in a drug conjugate or a pharmaceutically acceptable salt thereof.

22. A drug conjugate or a pharmaceutically acceptable salt thereof, comprising the hydrogel HA microspheres according to claim 21.

23. A drug conjugate or a pharmaceutically acceptable salt thereof, comprising hyaluronic acid (HA) hydrogel microspheres comprising cross-linked HA chains or a pharmaceutically acceptable salt thereof, comprising cross-linked HA chains covalently and reversibly conjugated to a plurality of drug moieties, wherein the drug conjugate comprises a plurality of each of the following units: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; Each R a1 Independently selected from: -H, C 1-10 Alkyl, ammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, alkali metal ions and alkaline earth metal ions; Each-R a2 are independently -H or C 1-10 alkyl; Each -X-, -Y- is independently a carbonyl group or absent; Each -X'-, -Y'- is independently a spacer moiety or absent; wherein each -D is independently covalently and reversibly conjugated to -L 1 - the drug portion; Each-L 1 - are independently reversible linker moieties; Each-L 2 - is independently a spacer moiety or absent; Each-L 3 -、-L 4 -、-L 5 - independently part of the link or does not exist; and Each -BA is independently a blocking agent.

24. The drug conjugate of claim 23 or a pharmaceutically acceptable salt thereof, wherein the drug conjugate comprises Z in the range of about 50% to about 98%. 1 , Z in the range of about 0.1% to about 20% 2 , Z in the range of about 0.1% to about 20% 3 and Z in the range of about 0.1% to about 10% 4 .

25. The drug conjugate according to claim 23 or 24, or a pharmaceutically acceptable salt thereof, wherein -BA is selected from: The dotted line indicates the 5 -connection.

26. The drug conjugate according to any one of claims 23 to 25, or a pharmaceutically acceptable salt thereof, wherein the drug conjugate comprises a plurality of each of the following units: Each R a1 Independently selected from: -H, C 1-10 Alkyl, ammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, alkali metal ions and alkaline earth metal ions; Each-R a2 are independently -H or C 1-10 alkyl; Each -X'-, -Y'- is independently a spacer moiety or absent; wherein each -D is independently covalently and reversibly conjugated to -L 1 - the drug portion; Each-L 1 - are independently reversible linker moieties; Each-L 2 - is independently a spacer moiety or absent; and wherein the drug conjugate comprises Z in the range of about 50% to about 98% 1 , Z in the range of about 0.1% to about 20% 2 -i. Z in the range of about 0.1% to about 20% 3 -i and Z in the range of about 0.1% to about 10% 4 -i.

27. The drug conjugate of any one of claims 23 to 26, or a pharmaceutically acceptable salt thereof, wherein -D is selected from a small molecule drug moiety, a medium-sized drug moiety, a peptide drug moiety, and a protein drug moiety.

28. The drug conjugate according to any one of claims 23 to 27, or a pharmaceutically acceptable salt thereof, wherein -D is a protein drug moiety.

29. The drug conjugate according to any one of claims 23 to 28, or a pharmaceutically acceptable salt thereof, wherein -D is a monoclonal or polyclonal antibody or a fragment or fusion thereof.

30. The drug conjugate according to any one of claims 23 to 29, or a pharmaceutically acceptable salt thereof, wherein each R a1 is H or an alkali metal ion and -R a2 is -H.

31. The drug conjugate according to any one of claims 23 to 30, or a pharmaceutically acceptable salt thereof, wherein each -L 2 - is selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y1 )-、-S(O)2N(R y1 )-、-S(O)N(R y1 )-、-S(O)2-、-S(O)-、-N(R y1 )S(O)2N(R y1a )-、-S-、-N(R y1 )-、-OC(OR y1 )(R y1a )-、-N(R y1 )C(O)N(R y1a )-、-OC(O)N(R y1 )-、C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T'-, C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R y2 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y3 )-、-S(O)2N(R y3 )-、-S(O)N(R y3 )-、-S(O)2-、-S(O)-、-N(R y3 )S(O)2N(R y3a )-、-S-、-N(R y3 )-、-OC(OR y3 )(R y3a )-、-N(R y3 )C(O)N(R y3a )-and-OC(O)N(R y3 )-; -R y1 and -R y1a Independently selected from: -H, -T', C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl; wherein -T', C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 Alkynyl is optionally substituted by one or more identical or different -R y2 substituted, and wherein C 1-50 Alkyl, C 2-50 Alkenyl and C 2-50 The alkynyl group is optionally interrupted by one or more groups selected from: -T'-, -C(O)O-, -O-, -C(O)-, -C(O)N(R y4 )-、-S(O)2N(R y4 )-、-S(O)N(R y4 )-、-S(O)2-、-S(O)-、-N(R y4 )S(O)2N(R y4a )-、-S-、-N(R y4 )-、-OC(OR y4 )(R y4a )-、-N(R y4 )C(O)N(R y4a )-and-OC(O)N(R y4 )-; Each T' is independently selected from phenyl, naphthyl, indenyl, indanyl, tetrahydronaphthyl, C 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbocyclic and 8- to 30-membered heteropolycyclic; wherein each T' is independently optionally replaced by one or more identical or different -R y2 replace; Each-R y2 Independently selected from: halogen, -CN, oxo (=O), -C(O)OR y5 、-OR y5 、-C(O)R y5 、-C(O)N(R y5 )(R y5a )、-S(O)2N(R y5 )(R y5a )、-S(O)N(R y5 )(R y5a )、-S(O)2R y5 、-S(O)R y5 、-N(R y5 )S(O)2N(R y5 )(R y5a ),-SR y5 、-N(R y5 )(R y5a )、-NO2、-OC(O)R y5 、-N(R y5 )C(O)R y5a 、-N(R y5 )S(O)2R y5a 、-N(R y5 )S(O)R y5a 、-N(R y5 )C(O)OR y5a 、-N(R y5 )C(O)N(R y5 )(R y5a )、-OC(O)N(R y5 )(R y5a ) and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more halogen groups which may be the same or different; and Each-R y3 、-R y3a 、-R y4 、-R y4a 、-R y5 、-R y5a and -R y5b Independently selected from -H and C 1-6 Alkyl; among which C 1-6 The alkyl group is optionally substituted with one or more same or different halogens.

32. The drug conjugate according to any one of claims 23 to 31 or a pharmaceutically acceptable salt thereof, wherein each -L 1 - having formula (I): in The dashed line indicates the connection to the nitrogen of -D via formation of an amide bond; -X- is -C(R 4 R 4a )-; -N(R 4 )-; -O-; -C(R 4 R 4a )-C(R 5 R 5a )-; -C(R 5 R 5a )-C(R 4 R 4a )-; -C(R 4 R 4a )-N(R 6 )-; -N(R 6 )-C(R 4 R 4a )-; -C(R 4 R 4a )-O-; -O-C(R 4 R 4a )-; or -C(R 7 R 7a )-; X 1 is C; or S(O); -X 2 -for-C(R 8 R 8a )-;or-C(R 8 R 8a )-C(R 9 R 9a )-; =X 3 =O; =S; or =N-CN; -R 1 、-R 1a 、-R 2 、-R 2a 、-R 4 、-R 4a 、-R 5 、-R 5a 、-R 6 、-R 8 、-R 8a 、-R 9 、-R 9a Independently selected from -H and C 1-6 alkyl; -R 3 、-R 3a Independently selected from -H and C 1-6 Alkyl, provided that, in -R 3 、-R 3a When one or both of them are not -H, they are connected to N, wherein they are connected to N via sp 3 A hybridized carbon atom is connected to the N; -R 7 -N(R 10 R 10a ); or -NR 10 -(C=O)-R 11 ; -R 7a 、-R 10 、-R 10a 、-R 11 are independently -H; or C 1-10 alkyl; Optionally, -R 1a / -R 4a 、-R 1a / -R 5a 、-R 1a / -R 7a 、-R 4a / -R 5a 、-R 8a / -R 9a One or more pairs of them form chemical bonds; Optionally, -R 1 / -R 1a 、-R 2 / -R 2a 、-R 4 / -R 4a 、-R 5 / -R 5a 、-R 8 / -R 8a 、-R 9 / -R 9a One or more pairs of atoms in the C 3-10 Cycloalkyl; or 3- to 10-membered heterocyclic group; Optionally, -R 1 / -R 4 、-R 1 / -R 5 、-R 1 / -R 6 、-R 1 / -R 7a 、-R 4 / -R 5 、-R 4 / -R 6 、-R 8 / -R 9 、-R 2 / -R 3 One or more pairs of them, together with the atoms to which they are attached, combine to form ring A; Optionally, -R 3 / -R 3a Together with the nitrogen atom to which they are attached, they form a 3- to 10-membered heterocyclic ring; Ring A is selected from: phenyl; naphthyl; indenyl; indanyl; tetrahydronaphthyl; C 3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and each - L 1 -substituted by - L 2 - substituted, provided that the hydrogen marked with an asterisk in formula (I) is not replaced by a substituent.

33. A pharmaceutical composition comprising the drug conjugate according to any one of claims 23 to 32 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

34. The drug conjugate according to any one of claims 23 to 32 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 33, for use as a medicament.

35. The drug conjugate according to any one of claims 23 to 32 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 33, for use in a method for treating a disease treatable with DH or a pharmaceutically acceptable salt thereof.

36. A method for preparing a drug conjugate or a pharmaceutically acceptable salt thereof, wherein the method comprises the following steps: (a) providing hydrogel HA microspheres or pharmaceutically acceptable salts thereof obtained by the method of any one of claims 1 to 22, wherein the hydrogel comprises one or more unreacted -FG1 or -FG2; (b) Providing a single conjugation reagent DL 1 -L 2 -FG3, double conjugated reagent FG3-L 2 -L 1 -DL 1 -L 2 -FG3 or a tri-conjugation reagent of formula (t): wherein each -D is independently covalently and reversibly conjugated to -L 1 - the drug portion; Each-L 1 - are independently reversible linker moieties; Each-L 2 - is independently a spacer moiety or absent; Each -FG3 is independently a functional group that reacts with one -FG1 or -FG2; (c) mixing the hydrogel HA microspheres of step (a) with the single conjugation, double conjugation or triple conjugation reagent of step (b); (d) mixing the drug conjugate of step (c) or a pharmaceutically acceptable salt thereof with a blocking agent; and (e) collecting the drug conjugate or a pharmaceutically acceptable salt thereof of step (c) or (d).

37. A drug conjugate obtainable by the method of claim 36 or a pharmaceutically acceptable salt thereof.

38. A method for precipitating a polymer in a flow system, wherein the method comprises the steps of: (a') flowing a first solution comprising a polymer through a first channel and flowing a second solution comprising an antisolvent through a second channel, optionally simultaneously; (b') combining the first solution and the second solution of step (a'); (c') passing the combined mixture of step (b') into at least one precipitation unit; and (d') precipitating the polymer; Where more than one precipitation unit is present, the mixture containing the precipitated polymer flowing out of one precipitation unit is combined with the second solution containing an antisolvent or optionally combined with another solution containing an antisolvent before flowing into another precipitation unit.

39. A method for separating a polymer in an apparatus for precipitating and separating a polymer, wherein the method comprises the following steps: (a') flowing a first solution comprising a polymer through a first channel and flowing a second solution comprising an antisolvent through a second channel, optionally simultaneously; (b') combining the first solution and the second solution of step (a'); (c') passing the combined mixture of step (b') into at least one precipitation unit; (d') precipitating the polymer; and (e') separating the precipitate of step (d'), Where more than one precipitation unit is present, the mixture containing the precipitated polymer flowing out of one precipitation unit is combined with the second solution containing an antisolvent or optionally combined with another solution containing an antisolvent before flowing into another precipitation unit.

40. The method of claim 38 or 39, wherein there is a precipitation unit.

41. The process of claim 38 or 39, wherein there are two precipitation units and the mixture comprising the precipitated polymer flowing out of the first precipitation unit is combined with the second solution comprising the antisolvent and then flowed into the second precipitation unit.

42. The method of any one of claims 38 to 41, wherein the polymer is selected from the group consisting of: a polysaccharide such as hyaluronic acid, hyaluronic acid and derivatives or functionalized hyaluronic acid, heparin, heparan sulfate, heparin precursors, chondroitin sulfate, dermatan sulfate, keratan sulfate, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, polydextrose, or dextrin; a polyether such as polyethylene glycol or polypropylene glycol; a polyester such as polyhydroxybutyrate, polyglycolic acid, polybutylene terephthalate, polycaprolactone, polylactic acid, or poly(lactic-co-glycolic acid); a protein such as gelatin or collagen; a polyolefin such as poly(2-methacryloyl-oxyethylphosphorylcholine), polyacrylic acid, Polyacrylates, polyacrylamides, polycyanoacrylates, polydimethylacrylamides, polyethylene, polyhydroxyethyl acrylate, poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), poly(hydroxypropyl methacrylate), polyvinyl alcohol, polyvinylamine, poly(vinyl methyl ether) or polyvinylpyrrolidone; polyoxazolines, for example poly(methyloxazoline) or poly(ethyloxazoline); polyamides; poly(amidoamines); polyamino acids; polyanhydrides; polyasparagine; polycarbonates; poly(alkylene phosphates), for example poly(ethylene phosphate); poly(iminocarbonates); poly(methacrylamides); poly(organophosphazenes); polyorthoesters; polysiloxanes and polyurethanes.

43. The method of any one of claims 38-42, wherein the polymer is selected from the group consisting of: a polysaccharide, such as hyaluronic acid, hyaluronic acid and derivatives or functionalized hyaluronic acid, heparin, heparan sulfate, a heparin precursor, chondroitin sulfate, dermatan sulfate, keratan sulfate, cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, polydextrose, or dextrin; a polyether, such as polyethylene glycol; and a protein, such as gelatin or collagen.

44. The method of any one of claims 38-43, wherein the polymer is functionalized HA.

45. The method of any one of claims 38-44, wherein the first solution comprises functionalized HA, wherein the functionalized HA comprises a plurality of the following linearly linked Z 1 and Z 5 -i Each of the units: or multiple of the following linearly connected Z 1 and Z 6 -i Each of the units: or multiple of the following linearly connected Z 1 and Z 7 -i Each of the units: in Unlabeled dashed lines indicate points of connection to adjacent units at the dashed lines marked with # or to hydrogen atoms; Dashed lines marked with # indicate the point of connection to the adjacent unit at the unmarked dashed line or to the hydroxyl group; -X'- has the formula (x4): wherein the unmarked dashed line indicates the connection to the carbonyl group, the dashed line marked with an asterisk indicates the connection to the sulfur atom, and c0 is 7; -Y'- has the formula (y4): wherein the unlabeled dashed line indicates the connection to the carbonyl group, and the dashed line marked with an asterisk indicates the connection to the nitrogen atom of the maleimide ring; Each R a1 is H or an alkali metal; Each-R a2 is -H; and The second solution is ethanol.

46. ​​A flow system for precipitating a polymer, comprising: - a container containing a first solution comprising the polymer; - at least one storage container containing a second solution comprising an antisolvent; at least one combining unit for combining the first solution and the second solution or for combining the mixture emerging from the precipitation unit with the second solution comprising an antisolvent or optionally with another solution comprising an antisolvent; - at least one precipitation unit for precipitating the polymer; wherein the container, the at least one storage container, the at least one merging unit and the at least one settling unit are connected via connecting channels to provide a continuous flow path, wherein - the first solution flows from the container to the merging unit through the first channel; - a second solution flows from the at least one storage unit to the merging unit through a second channel; - the combined mixture passes through at least one of said precipitation units, and Where more than one precipitation unit is present, the mixture containing the precipitated polymer flowing out of one precipitation unit is combined with the second solution containing an antisolvent or optionally combined with another solution containing an antisolvent before flowing into another precipitation unit.

47. The flow system of claim 46, wherein the container and the at least one storage container are further connected to valves and / or pumps to control the flow rate of the mixture comprising the polymer and the antisolvent.

48. The flow system of claim 46 or 47, further comprising a storage container for storing a buffer, wherein the storage container is connected to the outflow port of the container via a channel and a valve.

49. An apparatus for precipitating and isolating a polymer, wherein the apparatus comprises: 1) A flow system for precipitating a polymer, comprising: - a container containing a first solution comprising the polymer; - at least one storage container containing a second solution comprising an antisolvent; at least one combining unit for combining the first solution and the second solution or for combining the mixture emerging from the precipitation unit with the second solution comprising an antisolvent or optionally with another solution comprising an antisolvent; - at least one precipitation unit for precipitating the polymer; wherein the container, the at least one storage container, the at least one merging unit and the at least one settling unit are connected via connecting channels to provide a continuous flow path, wherein - the first solution flows from the container to the merging unit through the first channel; - a second solution flows from the at least one storage unit to the merging unit through a second channel; - the combined mixture passes through at least one of said precipitation units, and wherein if there is more than one precipitation unit, the mixture containing the precipitated polymer flowing out of one precipitation unit is combined with the second solution containing the antisolvent or optionally combined with another solution containing the antisolvent before flowing into another precipitation unit; and II) A collecting member for separating the precipitated polymer.

50. The flow system of any one of claims 38 to 48 or the device of claim 49, wherein the merging unit is a Y-type, T-type, X-type, arrow-type mixer or a static mixer.

51. The flow system of any one of claims 38 to 48 or the device of claim 49, wherein the merging unit is a Y-type or T-type mixer.

52. The flow system of any one of claims 38 to 48 or the apparatus of claim 49, wherein the precipitation unit is a coiled reactor or a tubular reactor.

53. The device of claim 49, wherein the collection component comprises a centrifuge.

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