Drug delivery system for locally delivering a therapeutic agent and uses thereof - Patents.com

Abstract

Disclosed herein are drug delivery systems and methods for locally delivering therapeutic agents, as well as methods for using such drug delivery systems for the treatment of disease.

Description

[Technical field]

[0001]

[0001] This disclosure relates to drug delivery systems and methods for locally delivering therapeutic agents, and methods for using such drug delivery systems for the treatment of disease. [Background technology]

[0002]

[0002] Most therapeutic agents are delivered to the body systemically by oral / GI absorption or systemic injection. These delivery routes are convenient and suitable for treating systemic diseases. However, more diseases are local disorders. Even if a systemically administered therapeutic agent can effectively treat these disorders, it may target other tissues or binding sites, which may cause side effects or adverse effects. To reduce systemic side effects, a locally administered drug delivery system is desirable. Delivering a therapeutic agent to a desired site is not as easy as taking a drug orally or via injection. Therefore, a long-term, sustained release drug delivery system for locally delivering drugs is essential for such products to be acceptable to physicians and patients. Moreover, the release profile of the therapeutic agent, which maintains an effective concentration at the delivery site after the drug is administered to a subject, can dramatically affect the efficacy of the therapeutic agent. Thus, drug delivery of a therapeutic agent to a specific target tissue or site in the body has been a long-standing challenge in the pharmaceutical industry.

[0003]

[0003] Numerous drug delivery systems have been developed to provide controlled drug delivery with tissue specificity or desired release profile. The most common local drug delivery system is to use biodegradable polymers to control the release rate of therapeutic agents. These drug delivery systems release drugs via both polymer erosion and drug molecule diffusion. This complex release control has imposed great challenges on pharmaceutical manufacturing and quality control.

[0004]

[0004] There are three important characteristics for the success of a localized drug delivery system: the ability to maintain the delivery system at the delivery site, the ability to release the therapeutic agent at a desired rate and profile, and the ability to treat a localized disorder with the therapeutic agent. This disclosure provides a different approach to meet these important characteristics of a localized drug delivery system: the biopolymer maintains the drug delivery system at the delivery site due to its large molecular size; the therapeutic agent is selected from commercially available products or those whose activity has been proven by late stage clinical studies; and the linker covalently bonded to the biopolymer and the therapeutic agent is not chemically stable and upon degradation releases the therapeutic agent at a rate desired for a particular delivery site and a particular disease. Summary of the Invention

[0005] In one aspect, the present disclosure provides a drug delivery system for locally delivering a therapeutic agent at a controlled rate, the system comprising: a biopolymer comprising at least a first binding group BG1 selected from the group consisting of a carboxyl group, an amino group, and combinations thereof; a therapeutic agent comprising at least a second linking group BG2 selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, an amine group, and combinations thereof; A linker capable of covalently linking the biopolymer to a therapeutic agent and retaining the therapeutic agent at the site of administration. Includes; wherein the linker has the structure of formula (I): [ka] (In the formula, U is linked to the biopolymer via BG1 such that at least one amide bond is formed, and U is -N(R 1 )-or [ka] where: [ka] is a nitrogen-containing heteroaryl or nitrogen-containing heterocyclyl optionally containing one or more additional heteroatoms selected from N, O, or S; A is a direct bond, an alkyl, or -(CH 2 CH 2 O) m -, wherein said alkyl is selected from one or more R 2 optionally substituted with a group; B is selected from the group consisting of a direct bond, cycloalkyl, heterocyclyl, aryl, and heteroaryl, where each of cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R 3 optionally substituted with a group; C is a direct bond, an alkyl, or [C(=O)NHCH 2 ] n -, wherein said alkyl is selected from one or more R 4 optionally substituted with a group; V is a direct bond, ester, carbonate, carbamate, -C(=O)NHCH 2 O- or -C(=O)OCH 2 and V is linked to the therapeutic agent via BG2 such that at least one linkage selected from the group consisting of -O-, -OC(=O)-, -NHC(=O)-, -C(=O)NHCH 2 - and -C(=O)OCH 2 - selected from the group consisting of; R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl; R 2 , R 3 , and R 4 each independently represents halogen, hydroxyl, amino, cyano, alkyl, alkoxyl, and C(═O)OR 5 selected from the group consisting of; R 5 is alkyl; m is an integer from 0 to 5; n is an integer from 1 to 4, However, when the biopolymer is chondroitin sulfate, V is not -C(=O)-.

[0006] In some embodiments, the linker in the drug delivery system provided herein has the structure of Formula (Ia)-(Ie): [ka] (In the formula, U and V are as defined above; M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which is selected from one or more R 3 optionally substituted with a group; [ka] Each of is independently alkyl or -C(=O)OCH 3 and optionally substituted with one or more groups selected from: p is an integer ranging from 0 to 10; m and t are independently integers ranging from 1 to 5; q, r, and s are independently integers ranging from 0 to 5; and t is an integer ranging from 1 to 5.

[0007]

[0007] In a further aspect, the present disclosure provides a pharmaceutical composition comprising the drug delivery system provided herein and a pharma- ceutically acceptable excipient.

[0008]

[0008] In another aspect, the present disclosure provides a method for treating a disorder in a subject in need of treatment thereof, the method comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows plasma and synovial time-concentration data for triamcinolone acetonide following a single IA dose of Example 2 (HA, MW 1000 KDa). [Diagram 2]

[0010] FIG. 1 shows plasma and synovial time-concentration data for triamcinolone acetonide following a single IA dose of Example 2 (HA, MW 2000KDa). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010]

[0011] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. Although the present disclosure will be described with the enumerated embodiments, it will be understood that they are not intended to limit the present disclosure to these embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present disclosure as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to implement the present disclosure. The present disclosure is in no way limited to the described methods and materials. In the event that one or more of the incorporated references and similar materials differ or contradict this application, including but not limited to defined terms, term usage, described techniques, etc., the present disclosure shall prevail. All references, patents, patent applications cited in this disclosure are incorporated herein by reference in their entirety.

[0011]

[0012] It is understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms of the same unless the context clearly dictates otherwise.

[0012]

[0013] definition The definition of specific functional groups and chemical terms is described in more detail below.For the purpose of this disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS edition, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein.In addition, the general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, 2nd edition, University Science Books, Sausalito, 2006; Smith and March March's Advanced Organic Chemistry, 6th edition, John Wiley&Sons,Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3rd edition, VCH Publishers,Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4th edition, Cambridge University Press, Cambridge, 2004, the entire contents of each of which are incorporated herein by reference.

[0013]

[0014] At various places in this disclosure, linking substituents are described. It is specifically intended that each linking substituent include both the forward and backward forms of that linking substituent. For example, -NR(CR'R")- includes both -NR(CR'R")- and -(CR'R")NR-. Where a structure clearly requires a linking group, it is understood that the Markush variable recited for that group is the linking group. For example, where a structure requires a linking group and the Markush group definition for that variable recites "alkyl," it is understood that "alkyl" represents a linking alkylene group.

[0014]

[0015] When a bond to a substituent is shown crossing a bond connecting two atoms in a ring, such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom to which such substituent is bonded to the remainder of the compound of a given formula, such substituent may be bonded through any atom in such formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0015]

[0016] Any variable part (e.g., R i When any R occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, a group may have 0 to 2 R i When a group is shown to be substituted with a moiety, the group may contain up to two R i and R at each occurrence is optionally substituted with i are independently i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0016]

[0017] As used herein, "C i~jThe term "carbon atom number" refers to a range of carbon atoms, where i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, where j is greater than i. For example, C 1~6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms. In some embodiments, "C 1~12 The term denotes 1 to 12, in particular 1 to 10, in particular 1 to 8, in particular 1 to 6, in particular 1 to 5, in particular 1 to 4, in particular 1 to 3, or in particular 1 to 2 carbon atoms.

[0017]

[0018] As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated, straight or branched chain hydrocarbon group, which may be optionally substituted, independently, with one or more substituents described below. i~j The term "alkyl" refers to an alkyl having i to j carbon atoms. In some embodiments, an alkyl group contains 1 to 10 carbon atoms. In some embodiments, an alkyl group contains 1 to 9 carbon atoms. In some embodiments, an alkyl group contains 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. 1~10 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1~6Examples of "alkyl" are methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0018]

[0019] As used herein, the term "alkenyl," whether used as part of another term or independently, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon double bond, which may be optionally substituted with one or more substituents, independently, described herein, including groups having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations. In some embodiments, an alkenyl group contains 2-12 carbon atoms. In some embodiments, an alkenyl group contains 2-11 carbon atoms. In some embodiments, an alkenyl group contains 2-11 carbon atoms, 2-10 carbon atoms, 2-9 carbon atoms, 2-8 carbon atoms, 2-7 carbon atoms, 2-6 carbon atoms, 2-5 carbon atoms, 2-4 carbon atoms, 2-3 carbon atoms, and in some embodiments, an alkenyl group contains 2 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethylenyl (or vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2 buten-1-yl, 5-hexenyl, and the like.

[0019]

[0020] As used herein, the term "alkynyl", whether used as part of another term or independently, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond, which may be optionally substituted, independently, with one or more substituents described herein. In some embodiments, an alkenyl group contains 2-12 carbon atoms. In some embodiments, an alkynyl group contains 2-11 carbon atoms. In some embodiments, an alkynyl group contains 2-11 carbon atoms, 2-10 carbon atoms, 2-9 carbon atoms, 2-8 carbon atoms, 2-7 carbon atoms, 2-6 carbon atoms, 2-5 carbon atoms, 2-4 carbon atoms, 2-3 carbon atoms, and in some embodiments, an alkynyl group contains 2 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like.

[0020]

[0021] As used herein, the term "alkoxyl," whether used as part of another term or independently, refers to an alkyl group, as defined above, attached to the parent molecule through an oxygen atom. i~j The term "alkoxy" means the alkyl portion of the alkoxy group has i to j carbon atoms. In some embodiments, the alkoxy group contains 1 to 10 carbon atoms. In some embodiments, the alkoxy group contains 1 to 9 carbon atoms. In some embodiments, the alkoxy group contains 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. "C 1~6 Examples of "alkoxyl" include, but are not limited to, methoxy, ethoxy, propoxy (eg, n-propoxy and isopropoxy), t-butoxy, neopentoxy, n-hexoxy, and the like.

[0021]

[0022] As used herein, the term "amide" refers to -C(=O)NR'-, where R' represents hydrogen, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups.

[0022]

[0023] As used herein, the term "amine" refers to an amine in which one or more hydrogen atoms have been replaced by a substituent, such as N(H) n (R') 3-n where n is 0, 1, or 2, and each R' is independently hydroxyl, nitro, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups, or two R' together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl.

[0023]

[0024] As used herein, the term "amino" refers to -NH 2 Refers to...

[0024]

[0025] As used herein, the term "aryl", whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems having a total of 5-20 ring members, where at least one ring in the system is aromatic, and where each ring in the system contains 3-12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring systems, only one of the rings need be aromatic (e.g., 2,3-dihydroindole), but all of the rings may be aromatic (e.g., quinoline). The second ring may also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. Aryl groups may be substituted at one or more ring positions with the substituents described above.

[0025]

[0026] As used herein, the term "carbamate" refers to -N(R')C(=O)O-, where R' represents hydrogen, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups.

[0026]

[0027] As used herein, the term "carbonate" refers to -OC(=O)O-.

[0027]

[0028] As used herein, the term "carboxyl group" or "carboxyl" refers to --COOH.

[0028]

[0029] As used herein, the term "cycloalkyl", whether used as part of another term or independently, refers to monovalent non-aromatic saturated or partially unsaturated monocyclic and polycyclic ring systems in which all ring atoms are carbon and contain at least three ring-forming carbon atoms. In some embodiments, a cycloalkyl can contain 3-12 ring-forming carbon atoms, 3-10 ring-forming carbon atoms, 3-9 ring-forming carbon atoms, 3-8 ring-forming carbon atoms, 3-7 ring-forming carbon atoms, 3-6 ring-forming carbon atoms, 3-5 ring-forming carbon atoms, 4-12 ring-forming carbon atoms, 4-10 ring-forming carbon atoms, 4-9 ring-forming carbon atoms, 4-8 ring-forming carbon atoms, 4-7 ring-forming carbon atoms, 4-6 ring-forming carbon atoms, 4-5 ring-forming carbon atoms. Cycloalkyl groups can be saturated or partially unsaturated. Cycloalkyl groups can be substituted. In some embodiments, a cycloalkyl group can be a saturated cyclic alkyl group. In some embodiments, a cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double or triple bond in its ring system, hi some embodiments, a cycloalkyl group may be monocyclic or polycyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro[3.6]-decanyl, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, and the like.

[0029]

[0030] As used herein, the term "cyano" refers to --CN.

[0030]

[0031] As used herein, the term "ester" refers to -C(=O)O-.

[0031]

[0032] As used herein, the term "halogen" refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo), and iodine (or iodo).

[0032]

[0033] As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen (including N-oxide).

[0033]

[0034] As used herein, the term "heteroaryl", whether used as part of another term or independently, refers to an aryl group having one or more heteroatoms in addition to carbon atoms. Heteroaryl groups can be monocyclic. Examples of monocyclic heteroaryls include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. Heteroaryl groups also include polycyclic groups in which the aromatic heterocycle is fused to one or more aryl, alicyclic, or heterocyclyl rings, with the group or point of attachment being on the aromatic heterocycle. Examples of polycyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0034]

[0035] As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated carbocyclyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, where one or more ring atoms may be independently optionally substituted with one or more substituents. In some embodiments, the heterocyclyl is a saturated heterocyclyl. In some embodiments, the heterocyclyl is a partially unsaturated heterocyclyl having one or more double bonds in its ring system. In some embodiments, the heterocyclyl may include any oxidized form of carbon, nitrogen, or sulfur, and any quaternized form of a basic nitrogen. "Heterocyclyl" also includes groups in which the heterocyclyl group is fused with a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Heterocyclyl groups may be carbon-linked or nitrogen-linked where possible. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, the group derived from pyrrole can be pyrrol-1-yl (nitrogen-linked) or pyrrol-3-yl (carbon-linked). Furthermore, the group derived from imidazole can be imidazol-1-yl (nitrogen-linked) or imidazol-3-yl (carbon-linked).

[0035]

[0036] In some embodiments, "3- to 12-membered heterocyclyl" refers to a 3- to 12-membered saturated or partially unsaturated, monocyclic or polycyclic heterocyclic ring system having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Fused, spiro, and bridged ring systems may also be included within the scope of this definition. Monocyclic heterocyclyls include, but are not limited to, oxetanyl, 1,1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, piperidinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like. Examples of fused heterocyclyls are phenyl- or pyridinyl-fused rings, such as quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinolidinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, phenyl ... Examples of spiroheterocyclyl include, but are not limited to, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, [1,2,3]triazolo[4,3-a]pyridinyl, and the like. Examples of spiroheterocyclyl include, but are not limited to, spiropyranyl, spirooxazinyl, and the like. Examples of bridged heterocyclyls include, but are not limited to, morphanyl, hexamethylenetetraminyl, 3-aza-bicyclo[3.1.0]hexane, 8-aza-bicyclo[3.2.1]octane, 1-aza-bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane (DABCO), and the like.

[0036]

[0037] As used herein, the term "hydroxyl" refers to --OH.

[0037]

[0038] As used herein, a "linking group" or "BG" refers to a group at a particular location in a first entity (e.g., a biopolymer provided herein, a therapeutic agent) that can react with another group from a second entity (e.g., a linker provided herein) to form a linkage, thereby combining the two entities to form one entity. For example, a carboxyl group contained in one entity can react with an amino group contained in another entity to form an amide linkage that links the two entities together, where the carboxyl group and the amino group can be considered linking groups.

[0038]

[0039] As used herein, the term "linkage" or "linker" refers to a bond or chemical moiety formed from a chemical reaction between functional groups of at least two entities to be linked, thereby forming one molecule or maintaining the association of the entities in sufficiently close proximity. The linker may be incorporated into the resulting linked molecule or structure with or without its reacted functional groups. Such linkages may be covalent or non-covalent. A hydrolytically unstable or degradable linkage means that the linkage is degradable in water or in aqueous solutions, including bodily fluids such as blood. An enzymatically unstable or degradable linkage means that the linkage can be degraded by one or more enzymes. Such degradable linkages include, but are not limited to, ester linkages formed by a carboxylic acid of an entity and an alcohol group of a biologically active agent, such ester groups typically hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of an amine with an aldehyde, phosphate linkages resulting from the reaction of a phosphate group with an alcohol, hydrazone linkages resulting from the reaction of a hydrazide with an aldehyde, acetal linkages resulting from the reaction of an amine group with a carboxyl group.

[0039]

[0040] As used herein, the term "partially unsaturated" refers to a group that contains at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings that have multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0040]

[0041] As used herein, the term "pharmacologically acceptable" indicates that a substance or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the subject being treated therewith.

[0041]

[0042] As used herein, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. "Substituted" or "substituted with" will be understood to include the implicit proviso that such substitution is in accordance with the allowed valence of the substituted atom, and that the substitution results in a stable or chemically feasible compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each of the substitutable positions of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at every position. One of ordinary skill in the art will understand that the substituents themselves may be substituted, where appropriate. Unless specifically described as "unsubstituted", reference to a chemical moiety herein is understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0042]

[0043] As used herein, the terms "therapeutic agent," "drug," "biologically active molecule," "biologically active agent," "active agent," and the like refer to any substance capable of affecting any physical or biochemical property of a biological organism, including, but not limited to, viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, a therapeutic agent includes any substance intended to diagnose, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance the physical or mental well-being of humans or animals.

[0043]

[0044] Drug delivery of therapeutic agents to specific tissues or sites within the body presents various challenges, particularly when it is desired to deliver high doses of poorly water-soluble therapeutic agents locally to specific tissues and when it is desired to avoid high systemic concentrations of the therapeutic agent that can lead to toxic side effects.

[0044]

[0045] Thus, in one aspect, the present disclosure provides a drug delivery system capable of locally delivering a therapeutic agent at a controlled rate, hi some embodiments, the drug delivery system comprises a biopolymer, a therapeutic agent, and a linker capable of covalently linking the biopolymer to the therapeutic agent and retaining the therapeutic agent at the site of administration.

[0045]

[0046] Biopolymers Biopolymers are natural polymers produced by living organisms that contain monomer units that are covalently linked to form larger structures. There are three main classifications of biopolymers: polynucleotides, polypeptides, and polysaccharides, classified according to the monomer units used and the structure of the biopolymer formed. More specifically, polynucleotides, such as RNA and DNA, are long polymers made up of 13 or more nucleotide monomers. Polypeptides or proteins are short polymers of amino acids, some major examples include collagen, actin, and fibrin. Polysaccharides are often polymeric carbohydrate structures linked in linear chains, some examples include cellulose and alginate. Other examples of biopolymers include rubber, suberin, melanin, and lignin.

[0046]

[0047] A variety of biopolymers are useful as polymeric delivery vehicles for delivering therapeutic agents to target cells or tissues. A biopolymer suitable for a particular application is selected based on its ability to target a particular tissue, organ, or cell, and its in vivo stability, i.e., residence time in the circulatory system or in a particular tissue, cell, or organ.

[0047]

[0048] In some embodiments, the biopolymer is selected from biocompatible polymers that include at least a first binding group BG1, which is capable of reacting with a reactive functional group from a second entity (e.g., a linker provided herein) to form a linkage, thereby linking the biopolymer to the second entity (e.g., a linker). The term "biocompatible" as used herein refers to a material that does not have medically unacceptable toxic or deleterious effects on biological functions or is tolerated by the body.

[0048]

[0049] In some embodiments, the biopolymer is selected from biocompatible polymers that at least include a first binding group BG1, where BG1 is selected from the group consisting of hydroxyl, carboxyl, amino, and combinations thereof. BG1 serves as a binding site for conjugation of a linker suitable for linking a therapeutic agent to the biopolymer. BG1 can be present at any site within the backbone of the biopolymer, and thus the link formed between the biopolymer and the linker can be present at any part of the biopolymer.

[0049]

[0050] In some embodiments, the BG1 for reacting with the reactive functional group from the linker can be the same or different. In certain embodiments, the BG1 of the biopolymer is the same. In certain embodiments, the BG1 of the biopolymer is different.

[0050]

[0051] In some embodiments, the biopolymer is a biocompatible polymer containing a carboxyl group as BG1, which can react with a reactive functional group of a suitable linker to form a linkage connecting the carboxyl-containing biopolymer to the linker.

[0051]

[0052] In certain embodiments, the reactive functional group of the linker is an amino or amine, which reacts with a carboxyl group of the biopolymer such that an amide linkage is formed.

[0052]

[0053] In some embodiments, the biopolymer is a biocompatible polymer that contains an amino or amine group as BG1, which can react with a reactive functional group of a suitable linker to form a linkage, thereby generating a biopolymer-linker conjugate.

[0053]

[0054] In certain embodiments, the reactive functional group of the linker is a carboxyl group, which reacts with an amino or amine group of the biopolymer such that an amide linkage is formed.

[0054]

[0055] In some embodiments, the linkage formed from the reaction of the BG1 of the biopolymer with the reactive functional group of the linker is -C(O)N(R 1 )-or [ka] where R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl; [ka] is a nitrogen-containing heteroaryl or nitrogen-containing heterocyclyl optionally containing one or more additional heteroatoms selected from N, O, or S.

[0055]

[0056] In certain embodiments, R 1 is hydrogen.

[0056]

[0057] In certain embodiments, R 1 is alkyl. In certain embodiments, R 1 is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, or C 1~3 In certain embodiments, R 1 is methyl.

[0057]

[0058] In certain embodiments, R 1 is alkenyl. In certain embodiments, R 1 is C 2~6 Alkenyl, C 2~5 Alkenyl, C 2~4 Alkenyl, or C 2~3 alkenyl. In certain embodiments, R 1 is vinyl.

[0058]

[0059] In certain embodiments, R 1 is alkynyl. In certain embodiments, R1 is C 2~6 Alkynyl, C 2~5 Alkynyl, C 2~4 Alkynyl, or C 2~3 In certain embodiments, R 1 is ethynyl.

[0059]

[0060] In certain embodiments, [ka] teeth, [ka] is selected from the group consisting of:

[0060]

[0061] In some embodiments, the biopolymer may be selected from the group consisting of hyaluronic acid (HA), dextran, cellulose, amylose, chitosan, chitin, chondroitin, chondroitin sulfate (CS), gelatin, alginate, carrageenan, gellan, guar gum, pectin, scleroglucan, xanthan, or derivatives thereof.

[0061]

[0062] In some embodiments, the biopolymer can have a number average molecular weight in the range of 400 to 3,000,000 Da, e.g., 1,000 to 3,000,000 Da, 5,000 to 3,000,000 Da, 10,000 to 3,000,000 Da, 20,000 to 3,000,000 Da, 30,000 to 3,000,000 Da, 40,000 to 3,000,000 Da, 50,000 to 3,000,000 Da, or 50,000 to 2,000,000 Da.

[0062]

[0063] In some embodiments, the biopolymer may be selected from the group consisting of HA, chitosan, chondroitin sulfate, or derivatives thereof.

[0063]

[0064] In certain embodiments, the biopolymer is HA. In certain embodiments, the HA can be derived from any source.

[0064]

[0065] In certain embodiments, the biopolymer is CS. In certain embodiments, the HA can be derived from any source.

[0065]

[0066] In certain embodiments, the HA may have a number average molecular weight in the range of 400 to 3,000,000 Da, e.g., 1,000 to 3,000,000 Da, 5,000 to 3,000,000 Da, 10,000 to 3,000,000 Da, 20,000 to 3,000,000 Da, 30,000 to 3,000,000 Da, 40,000 to 3,000,000 Da, 50,000 to 3,000,000 Da, or 50,000 to 2,000,000 Da.

[0066]

[0067] In certain embodiments, the biopolymer is chondroitin sulfate. In certain embodiments, the chondroitin sulfate can be derived from any source.

[0067]

[0068] In certain embodiments, the chondroitin sulfate can have a number average molecular weight in the range of 400 to 3,000,000 Da, e.g., 1,000 to 3,000,000 Da, 5,000 to 3,000,000 Da, 10,000 to 3,000,000 Da, 20,000 to 3,000,000 Da, 30,000 to 3,000,000 Da, 40,000 to 3,000,000 Da, 50,000 to 3,000,000 Da, or 50,000 to 2,000,000 Da.

[0068]

[0069] Therapeutic Agents The present disclosure provides improved delivery systems for the localized delivery of a variety of therapeutic agents.

[0069]

[0070] In some embodiments, the therapeutic agent comprises at least a second linking group, BG2, that is capable of reacting with a reactive functional group and an optional co-reactant from a second entity (e.g., a linker provided herein) to form a linkage, thereby linking the therapeutic agent to the second entity (e.g., a linker).

[0070]

[0071] In some embodiments, the therapeutic agent comprises at least a second linking group BG2 selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, an amine group, and combinations thereof, which serves as a binding site for conjugation of a linker suitable for linking the therapeutic agent to the biopolymer.

[0071]

[0072] In some embodiments, the therapeutic agent comprises a hydroxyl group as BG2, which can react with a reactive functional group of a suitable linker and an optional co-reactant to form a linkage connecting the hydroxyl group-containing therapeutic agent to the linker.

[0072]

[0073] In certain embodiments, a hydroxyl group in the therapeutic agent reacts with a reactive functional group of the linker and an optional co-reactant such that the therapeutic agent is linked to the linker via an ester linkage.

[0073]

[0074] In certain embodiments, a hydroxyl group in the therapeutic agent reacts with a reactive functional group and optional co-reactant of the linker such that the therapeutic agent is linked to the linker via a carbonate linkage.

[0074]

[0075] In certain embodiments, a hydroxyl group in the therapeutic agent reacts with a reactive functional group and optional co-reactant of the linker such that the therapeutic agent is linked to the linker via a carbamate linkage.

[0075]

[0076] In certain embodiments, the hydroxyl group in the therapeutic agent is such that the therapeutic agent is -C(=O)NHCH 2It reacts with a reactive functional group of the linker and an optional co-reactant so as to be linked to the linker via an O-linkage.

[0076]

[0077] In certain embodiments, the hydroxyl group in the therapeutic agent is such that the therapeutic agent is -C(=O)OCH 2 It reacts with a reactive functional group of the linker and an optional co-reactant so as to be linked to the linker via an O-linkage.

[0077]

[0078] In some embodiments, the therapeutic agent comprises a carboxyl group as BG2, which can react with a reactive functional group and optional co-reactant of a suitable linker to form a linkage connecting the carboxyl group-containing therapeutic agent to the linker.

[0078]

[0079] In certain embodiments, a carboxyl group in the therapeutic agent reacts with a reactive functional group and optional co-reactant of the linker such that the therapeutic agent is linked to the linker via an ester linkage.

[0079]

[0080] In certain embodiments, the therapeutic agent comprises an amino or amine group as BG2, which can react with a reactive functional group and optional co-reactant of a suitable linker to form a linkage connecting the amino / amine-containing therapeutic agent to the linker.

[0080]

[0081] In certain embodiments, an amine group in the therapeutic agent reacts with a reactive functional group and optional co-reactant of the linker such that the therapeutic agent is linked to the linker via a direct bond, an amide linkage, a urea linkage, a thiourea linkage, a carbamate linkage, a thiocarbamate linkage, an aza-acetal linkage, a phosphoramidate linkage, or the like.

[0081]

[0082] In some embodiments, the therapeutic agent delivered is an anti-inflammatory agent.

[0082]

[0083] In some embodiments, the therapeutic agent delivered is an antiflammatory drug selected from the group consisting of triamcinolone acetonide, meprednisone, prednisolone, hydrocortisone, cortisone, fluocinonide, methylprednisolone, betamethason, and dexamethasone.

[0083]

[0084] Linker Improved localized delivery of therapeutic agents is achieved by linking the therapeutic agent to the biopolymer via a suitable linker. By selecting the appropriate linker, the release rate of the therapeutic agent from the biopolymer can be controlled, thereby improving the delivery of the therapeutic agent to the target cells or tissues.

[0084]

[0085] In some embodiments, multiple linkers are attached to the therapeutic agent via cleavable linkages that are cleaved under biological conditions, thereby allowing release of the therapeutic agent.

[0085]

[0086] A "cleavable linkage" is a relatively unstable linkage that breaks under physiological conditions. Exemplary releasable linkages are hydrolyzable bonds that break (i.e., are hydrolyzed) upon reaction with water. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting the two atoms, but also on the substituents attached to those atoms. Suitable hydrolytically unstable or weak linkages include, but are not limited to, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, ureas, thioureas, carbamates, thiocarbamates, phosphoramidates, and carbonates. Certain functional groups have atoms that can be chemically degraded by processes other than hydrolysis. Exemplary releasable linkages of this class include certain carbamates and Fmoc derivatives. Certain molecules that contain these types of functionalities appropriately attached may undergo chemical degradation (release) upon the action of base. In such cases, "cleavage" may occur at high pH values ​​or through the action of biomolecules that contain a basic moiety (e.g., histidine). Another exemplary cleavable linkage is an enzymatically cleavable linkage. An "enzymatically cleavable linkage" refers to a linkage that is cleaved by one or more enzymes.

[0086]

[0087] In some embodiments, the linker is attached to the biopolymer through a linkage formed from a reactive functional group of the linker and BG1 in the biopolymer, and is attached to the therapeutic agent through a linkage formed from another reactive functional group of the linker and BG2 in the therapeutic agent.

[0087]

[0088] In some embodiments, the linker has the structure of Formula (I): [ka] (In the formula, U is linked to the biopolymer via BG1 such that at least one amide bond is formed, and U is -N(R 1 )-or [ka] where: [ka] is a nitrogen-containing heteroaryl or nitrogen-containing heterocyclyl optionally containing one or more additional heteroatoms selected from N, O, or S; A is a direct bond, an alkyl, or -(CH 2 CH 2 O) m -, wherein said alkyl is selected from one or more R 2 optionally substituted with a group; B is selected from the group consisting of a direct bond, cycloalkyl, heterocyclyl, aryl, and heteroaryl, where each of cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R 3 optionally substituted with a group; C is a direct bond, an alkyl, or [C(=O)NHCH 2 ] n -, wherein said alkyl is selected from one or more R 4 optionally substituted with a group; V is a direct bond, ester, carbonate, carbamate, -C(=O)NHCH 2 O- or -C(=O)OCH 2 and V is linked to the therapeutic agent via BG2 such that at least one linkage selected from the group consisting of -O-, -OC(=O)-, -NHC(=O)-, -C(=O)NHCH 2 - and -C(=O)OCH 2 - selected from the group consisting of; R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl; R 2 , R 3 , and R 4 each independently represents halogen, hydroxyl, amino, cyano, alkyl, alkoxyl, and C(═O)OR5 selected from the group consisting of; R 5 is alkyl; m is an integer from 0 to 5; n is an integer from 1 to 4, With the proviso that when the biopolymer is chondroitin sulfate, V is not -C(=O)-.

[0088]

[0089] In some embodiments, BG1 is a carboxyl group and U is -N(R 1 )-.

[0089]

[0090] In some embodiments, BG1 is a carboxyl group and U is a carboxyl group such that an amide linkage is formed to attach the biopolymer to the linker. [ka] It is.

[0090]

[0091] In certain embodiments, BG1 is a carboxyl group and U is [ka] Selected from the group consisting of [ka] It is.

[0091]

[0092] In some embodiments, BG2 is a hydroxyl group and V is: (a) -C(=O)- linked to a therapeutic agent via BG2 such that an ester linkage is formed; (b) -OC(=O)- linked to a therapeutic agent via BG2 such that a carbonate linkage is formed; (c) -NHC(=O)- linked to a therapeutic agent via BG2 such that a carbamate linkage is formed; (d) -C(=O)NHCH2 -C(=O)NHCH linked to a therapeutic agent via BG2 such that an O-linkage is formed 2 -;or (e) -C(=O)OCH 2 -C(=O)OCH linked to a therapeutic agent via BG2 such that an O-linkage is formed 2 - is selected from one of the following:

[0092]

[0093] In some embodiments, A is a direct bond.

[0093]

[0094] In some embodiments, A is one or more R 2 In certain embodiments, A is an alkyl group optionally substituted with one or more R 2 C optionally substituted with a group 1~10 In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~9 In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~7 It is an alkyl.

[0094]

[0095] In certain embodiments, each R 2 are independently alkyl or -C(=O)OR 5 is selected from.

[0095]

[0096] In certain embodiments, each R 2 is independently 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, or C 1~3 In certain embodiments, R 2 is methyl.

[0096]

[0097] In certain embodiments, each R 2 are independently -C(=O)OR 5 where R 5 is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, or C 1~3 In certain embodiments, R 2 is -C(=O)OCH 3 It is.

[0097]

[0098] In some embodiments, A is -(CH 2 CH 2 O) m -It is.

[0098]

[0099] In certain embodiments, m is an integer from 0 to 4. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4.

[0099]

[0100] In some embodiments, B is a direct bond.

[0100]

[0101] In some embodiments, B is cycloalkyl.

[0101]

[0102] In certain embodiments, B is a 3- to 8-membered cycloalkyl, a 3- to 7-membered cycloalkyl, a 3- to 6-membered cycloalkyl, a 3- to 5-membered cycloalkyl, or a 3- to 4-membered cycloalkyl.

[0102]

[0103] In certain embodiments, B is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, and bicycle[2.2.2]octyl.

[0103]

[0104] In some embodiments, B is aryl. In certain embodiments, B is a 5-12 membered aryl, a 5-10 membered aryl, a 5-8 membered aryl, or a 5-6 membered aryl.

[0104]

[0105] In certain embodiments, B is phenyl.

[0105]

[0106] In certain embodiments, B is heteroaryl. In certain embodiments, B is 5-12 membered heteroaryl, 5-10 membered heteroaryl, 5-8 membered heteroaryl, or 5-6 membered heteroaryl.

[0106]

[0107] In certain embodiments, B is pyridinyl or furyl.

[0107]

[0108] In some embodiments, A is a direct bond and B is selected from the group consisting of a direct bond, cycloalkyl, and aryl.

[0108]

[0109] In certain embodiments, A is a direct bond and B is a direct bond.

[0109]

[0110] In certain embodiments, A is a direct bond and B is a 3-8 membered cycloalkyl, a 3-7 membered cycloalkyl, a 3-6 membered cycloalkyl, a 3-5 membered cycloalkyl, or a 3-4 membered cycloalkyl. In certain embodiments, A is a direct bond and B is cyclobutyl, cyclohexyl, or bicyclo[2.2.2]octyl.

[0110]

[0111] In certain embodiments, A is a direct bond and B is a 5-12 membered aryl, a 5-10 membered aryl, a 5-8 membered aryl, or a 5-6 membered aryl. In certain embodiments, A is a direct bond and B is phenyl.

[0111]

[0112] In certain embodiments, A is a direct bond and B is a 5-12 membered heteroaryl, a 5-10 membered heteroaryl, a 5-8 membered heteroaryl, or a 5-6 membered heteroaryl. In certain embodiments, A is a direct bond and B is pyridyl or furyl.

[0112]

[0113] In some embodiments, A is one or more R 2 and B is an alkyl optionally substituted with a group, and B is selected from the group consisting of a direct bond, aryl, and heteroaryl.

[0113]

[0114] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 alkyl and B is a direct bond, where R 2 is alkyl or -C(=O)OR 5 where R 5 is alkyl.

[0114]

[0115] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 alkyl and B is phenyl.

[0115]

[0116] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 In certain embodiments, A is alkyl and B is heteroaryl. 2 C optionally substituted with a group 1~8 alkyl and B is pyridinyl or furyl.

[0116]

[0117] In some embodiments, A is -(CH 2 CH 2 O) m -, where m is an integer from 0 to 4, and B is a direct bond.

[0117]

[0118] In some embodiments, C is a direct bond.

[0118]

[0119] In some embodiments, C is one or more R 4 In certain embodiments, C is an alkyl group optionally substituted with one or more R 4 C optionally substituted with a group 1~6 In certain embodiments, C is one or more R 4 C optionally substituted with a group 1~5 In certain embodiments, C is one or more R 4 C optionally substituted with a group 1~4 In certain embodiments, C is one or more R 4 C optionally substituted with a group 1~3 In certain embodiments, C is methyl or ethyl.

[0119]

[0120] In certain embodiments, each R 4 are independently alkyl or -C(=O)OR 5 is selected from.

[0120]

[0121] In certain embodiments, each R 4 is independently 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, or C 1~3 In certain embodiments, R 4 is methyl.

[0121]

[0122] In certain embodiments, each R 4 are independently -C(=O)OR 5 where R 5 is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, or C 1~3 In certain embodiments, R 4 is -C(=O)OCH 3 It is.

[0122]

[0123] In some embodiments, C is -[C(=O)NHCH 2 ] n -It is.

[0123]

[0124] In some embodiments, A is a direct bond, B is a direct bond, cycloalkyl, or aryl, and C is a direct bond or one or more R 4 The aryl group is selected from the group consisting of alkyl, optionally substituted with a group.

[0124]

[0125] In certain embodiments, A is a direct bond, B is a direct bond, and C is a direct bond.

[0125]

[0126] In certain embodiments, A is a direct bond, B is cycloalkyl, and C is a direct bond.

[0126]

[0127] In certain embodiments, A is a direct bond, B is aryl, and C is a direct bond or one or more R 4 is an alkyl optionally substituted with a group.

[0127]

[0128] In some embodiments, A is one or more R 2 R is an alkyl group optionally substituted with R, B is selected from the group consisting of a direct bond, aryl, and heteroaryl, and C is a direct bond, one or more R 4 alkyl optionally substituted with a group, or -[C(=O)NHCH 2 ] n - is selected.

[0128]

[0129] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 alkyl, B is a direct bond, and C is a direct bond or -[C(=O)NHCH 2 ] n -It is.

[0129]

[0130] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 A is alkyl, B is phenyl, and C is a direct bond.

[0130]

[0131] In certain embodiments, A is one or more R 2 C optionally substituted with a group 1~8 In certain embodiments, A is alkyl, B is heteroaryl, and C is a direct bond. 2 C optionally substituted with a group 1~8 is alkyl, B is pyridinyl or furyl, and C is a direct bond.

[0131]

[0132] In some embodiments, A is -(CH 2 CH 2 O) m -, B is alkyl, and C is a direct bond.

[0132]

[0133] In some embodiments, A is -(CH 2 CH 2 O) m -, where m is 1, 2, 3, or 4; B is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, C 1~3 Alkyl, or C 1~2 alkyl and C is a direct bond.

[0133]

[0134] In some embodiments, A is -(CH 2 CH 2 O) m -, B is a direct bond, and C is a direct bond.

[0134]

[0135] In some embodiments, the linkers provided herein have the structure of Formula (Ia)-(Ie): [ka] (In the formula, U and V are defined as above; M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which is selected from one or more R 3 optionally substituted with a group; [ka] Each of is independently alkyl or -C(=O)OCH 3 and optionally substituted with one or more groups selected from: p is an integer ranging from 0 to 10; m and t are independently integers ranging from 1 to 5; q, r, and s are independently integers ranging from 0 to 5; and t is an integer ranging from 1 to 5.

[0135]

[0136] In some embodiments, M is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.2]octyl, phenyl, pyridyl, and furyl.

[0136]

[0137] In some embodiments, the linker provided herein is [ka] (In the formula, [ka] Each of is independently alkyl or -C(=O)OCH 3 (optionally substituted with one or more groups selected from).

[0137]

[0138] Drug Delivery Systems In one aspect of the present disclosure, a therapeutic agent is attached to a biopolymer via a linker, thereby providing a drug delivery system for localized delivery of the therapeutic agent to a target site.

[0138]

[0139] The biopolymers of the drug delivery systems provided herein may have one or more therapeutic agents conjugated via a linker. The biopolymer may be conjugated to one or more therapeutic agents via one or more linkers at carboxyl and / or amino groups in the backbone of the biopolymer.

[0139]

[0140] The drug delivery system of the present disclosure is obtained by conjugation between the biopolymer and the therapeutic agent using a linker, via formation of a linkage between the biopolymer and the linker, and a linkage between the therapeutic agent and the linker.

[0140]

[0141] In some embodiments, a reactive functional group of a linker can first react with BG2 of a therapeutic agent to form a linkage between the therapeutic agent and the linker, thereby providing a therapeutic agent-linker conjugate. A therapeutic agent-linker conjugate that includes another reactive functional group at the terminus of the linker can then react with BG1 of a biopolymer to form a linkage between the biopolymer and the linker, thereby providing a drug delivery system of the present disclosure.

[0141]

[0142] In certain embodiments, BG1 of the biopolymer can first be reacted with a reactive functional group of the linker to form a biopolymer-linker conjugate, and then BG2 of the therapeutic agent can be reacted with another functional group of the linker of the biopolymer-linker conjugate, thereby providing the drug delivery system of the present disclosure.

[0142]

[0143] In some embodiments, the biopolymer selected for the drug delivery systems provided herein is hyaluronic acid or chondroitin sulfate, and the therapeutic agent selected for the drug delivery systems provided herein is selected from the group consisting of triamcinolone acetonide, meprednisone, prednisolone, hydrocortisone, cortisone, fluocinonide, methylprednisolone, betamethasone, and dexamethasone.

[0143]

[0144] In certain embodiments, the biopolymer selected for the drug delivery systems provided herein is hyaluronic acid and the therapeutic agent selected for the drug delivery systems provided herein is selected from the group consisting of triamcinolone acetonide, meprednisone, prednisolone, hydrocortisone, cortisone, fluocinonide, methylprednisolone, betamethasone, and dexamethasone.

[0144]

[0145] In certain embodiments, the drug delivery system provided herein comprises: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0145]

[0146] In some embodiments, a therapeutic agent can be conjugated to the biopolymer via a linker with a drug substitution ratio (DSR) for the biopolymer of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% as measured by NMR, where the drug substitution ratio (DSR) for the biopolymer refers to the ratio of the molar amount of groups on the biopolymer that are substituted with the drug to the total molar amount of groups on the biopolymer that can be substituted with the drug.

[0146]

[0147] The therapeutic agent can be released from the drug delivery system provided herein via cleavage of the linkage between the linker and the biopolymer or the therapeutic agent. In some embodiments, release of the therapeutic agent occurs when the linkage between the biopolymer and the linker is cleaved to release the therapeutic agent-linker conjugate, which may be considered a prodrug. Subsequent release of the therapeutic agent from the linker may involve enzymatic or non-enzymatic cleavage of the linkage between the therapeutic agent and the linker. In some embodiments, release of the therapeutic agent occurs when the linkage between the therapeutic agent and the linker is cleaved without or prior to cleavage of the linkage between the biopolymer and the linker. Release of the therapeutic agent may also involve enzymatic or non-enzymatic processes.

[0147]

[0148] The release of the therapeutic agent can be influenced by various factors, such as the selection of the particular therapeutic agent, linker, and biopolymer, administration of the drug delivery system. The present disclosure contemplates biopolymers with various molecular weights, linking groups BG1, linkers; linkers with various reactive functional groups and subunits; and therapeutic agents with various linking groups BG2, linkers.

[0148]

[0149] The present disclosure also contemplates various local administrations of the drug delivery system provided herein. In some embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof. In certain embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof via injection. In certain embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof via oral dosage form. In certain embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof via inhalation. In certain embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof via an implant. In certain embodiments, the drug delivery system provided herein is administered locally to a subject in need thereof via topical application. Depending on the particular therapeutic agent, linker, and biopolymer combination, release of the therapeutic agent may occur at various locations upon administration to a subject. For example, release of the therapeutic agent may occur at the site of administration.

[0149]

[0150] In some embodiments, administration of the drug delivery systems provided herein to a subject can provide release of the therapeutic agent over a period of at least several days to at least several months.

[0150]

[0151] The release of a therapeutic agent from the drug delivery systems provided herein may be characterized by the percentage of therapeutic agent released from the drug delivery system per day. In some embodiments, the release rate of the therapeutic agent is from about 0.01% to about 20% per day, from about 0.01% to about 15% per day, from about 0.01% to about 10% per day, from about 0.01% to about 9% per day, from about 0.01% to about 8% per day, from about 0.01% to about 7% per day, from about 0.01% to about 6% per day, from about 0.01% to about 5% per day, from about 0.01% to about 4% per day, from about 0.01% to about 3% per day, from about 0.01% to about 5% per day, from about 0.01% to about 6% per day, from about 0.01% to about 7% per day, from about 0.01% to about 8% per day, from about 0.01% to about 9% per day, from about 0.01% to about 9% per day, from about 0.01% to about 9% per day, from about 0.01% to about 10% per day, from about 0.01% to about 10% per day, from about 0.01% to about 15 ... It may vary in the range of about 2%, about 0.01% to about 1% per day, about 0.01% to about 0.5% per day, about 0.01% to about 0.4% per day, about 0.01% to about 0.3% per day, about 0.01% to about 0.2% per day, about 0.01% to about 0.1% per day, about 0.01% to about 0.05% per day, about 0.01% to about 0.04% per day, about 0.01% to about 0.03% per day, or about 0.01% to about 0.02% per day.

[0151]

[0152] Pharmaceutical Compositions In a further aspect, a pharmaceutical composition comprising the drug delivery system of the present disclosure is provided.

[0152]

[0153] In another aspect, a pharmaceutical composition is provided comprising the drug delivery system of the present disclosure and at least one pharma- ceutically acceptable excipient.

[0153]

[0154] As used herein, the term "pharmaceutical composition" refers to a formulation that contains the drug delivery system of the present disclosure in a form suitable for administration to a subject.

[0154]

[0155] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and useful for preparing pharmaceutical compositions, including excipients that are acceptable for veterinary and human pharmaceutical use. As used herein, a "pharmaceutically acceptable excipient" includes both one and more than one such excipient. The term "pharmaceutically acceptable excipient" also encompasses "pharmaceutically acceptable carriers" and "pharmaceutically acceptable diluents."

[0155]

[0156] The pharmaceutical compositions provided herein may be in any form that allows for the composition to be administered to a subject, including but not limited to a human, and is formulated to be compatible with the intended route of administration.

[0156]

[0157] Various routes are contemplated for the pharmaceutical compositions provided herein, and therefore the pharmaceutical compositions provided herein may be provided in bulk or unit dosage form depending on the intended route of administration. For example, for oral, buccal, and sublingual administration, powders, granules, tablets, pills, capsules, gelcaps, and caplets may be acceptable solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions may be acceptable liquid dosage forms. For injection administration, gels, solutions, emulsions, and suspensions may be acceptable liquid dosage forms, and powders suitable for reconstitution with a suitable solution may be acceptable solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols may be acceptable dosage forms. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches may be acceptable dosage forms. For vaginal administration, pessaries, tampons, creams, gels, pastes, foams, and sprays may be acceptable dosage forms. For administration by implant, solids, semi-solids and gels can be acceptable dosage forms.

[0157]

[0158] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a formulation for oral administration.

[0158]

[0159] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a formulation for injectable administration.

[0159]

[0160] In some embodiments, a pharmaceutical composition of the present disclosure may be in the form of a formulation for inhaled administration.

[0160]

[0161] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a formulation for topical administration.

[0161]

[0162] In certain embodiments, the pharmaceutical compositions provided herein may be formulated in the form of a skin patch, which are well known to those of ordinary skill in the art.

[0162]

[0163] In addition to the above-mentioned representative dosage forms, pharma- ceutically acceptable excipients and carriers are generally known to those skilled in the art and are therefore included in the present disclosure.Such excipients and carriers are described, for example, in "Remington's Pharmaceutical Sciences" Mack Pub. Co., New Jersey (1991), "Remington: The Science and Practice of Pharmacy", University of the Sciences in Philadelphia, ed., 21st ed., LWW (2005), which are incorporated herein by reference.

[0163]

[0164] In some embodiments, the pharmaceutical compositions of the present disclosure may be formulated as a single dose. The amount of a compound provided herein in a single dose varies depending on the subject being treated and the particular mode of administration.

[0164]

[0165] In some embodiments, the pharmaceutical compositions of the present disclosure may be formulated to be administered to a subject over intervals of days, weeks, months, or even longer.

[0165]

[0166] In a further aspect, pharmaceutical compositions comprising the drug delivery systems of the present disclosure as two or more combination therapies are also provided.

[0166]

[0167] Synthesis of drug delivery systems The synthesis of the drug delivery system provided herein is shown in the synthetic scheme of the examples. The drug delivery system provided herein can be prepared using any known organic synthesis technique and can be synthesized according to any of a number of possible synthetic routes, and therefore these schemes are merely illustrative and are not intended to limit other possible methods that can be used to prepare the compounds provided herein. Furthermore, the steps in the schemes are for better illustration and can be modified accordingly. The compound embodiments in the examples were synthesized for the purpose of research and potential submission to regulatory agencies.

[0167]

[0168] The reactions for preparing the drug delivery system of the present disclosure can be carried out in a suitable solvent, which can be easily selected by those skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, which can range, for example, from the freezing temperature of the solvent to the boiling point of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. Depending on the particular reaction step, a suitable solvent for a particular reaction step can be selected by those skilled in the art.

[0168]

[0169] The preparation of the compounds of the present disclosure may involve the protection and deprotection of various chemical groups.The necessity of protection and deprotection, and the selection of suitable protecting groups can be easily determined by those skilled in the art.The chemistry of protecting groups can be found, for example, in TW Greene and P G M Huts, Protective Groups in Organic Synthesis, 3rd ed., Wiley&Sons,Inc., New York (1999), P. Kocienski, Protecting Groups, Georg Thieme Verlag, 2003, and Peter G M Huts, Greene's Protective Groups in Organic Synthesis, 5th ed., Wiley, 2014, all of which are incorporated herein by reference in their entirety.

[0169]

[0170] The reaction may be monitored according to any suitable method known in the art. For example, the formation of the product may be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g. 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., UV-Vis), mass spectrometry, or chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6(6), pp. 874-883, which is incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0170]

[0171] Known starting materials of this disclosure can be synthesized by or according to methods known in the art or purchased from commercial suppliers. Analytical grade solvents and commercially available reagents were used without further purification unless otherwise noted.

[0172] Unless otherwise stated, all reactions in this disclosure were carried out under a positive pressure of nitrogen or argon or in anhydrous solvents using drying tubes and reaction flasks were typically fitted with rubber septa for the introduction of substrates and reagents via syringe. Glassware was oven-dried and / or heat-dried.

[0171]

[0173] Methods of Treating Disease In a further aspect, there is provided a method of treating a disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein.

[0172]

[0174] The disorder to be treated depends on the selected therapeutic agent in the drug delivery system or pharmaceutical composition provided herein. In some embodiments, the disorder can be an allergic disease, an autoimmune disease, or an inflammatory disease. In certain embodiments, the disorder can be selected from the group consisting of allergic rhinitis, systemic lupus erythematosus, rheumatism, nephrotic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Addison's disease, neurodermatitis, cutaneous pruritus, tendonitis, inflammation, respiratory disease, osteoarthritis, neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), uveitic macular edema (UME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), dermatitis, psoriasis, chronic obstructive pulmonary disease, and asthma.

[0173]

[0175] In this context, the term "therapeutically effective amount" refers to an amount of a selected therapeutic agent, or a pharma- ceutically acceptable salt thereof, in a drug delivery system provided herein effective to provide "therapy" in a subject or to "treat" a disorder, disease, or condition in a subject. EXAMPLES

[0174]

[0176] For illustrative purposes, the following examples are included. However, it should be understood that these examples are not intended to limit the disclosure, but are only intended to suggest a method of carrying out the disclosure. Those skilled in the art will recognize that the described chemical reactions can be easily applied to prepare many other compounds of the disclosure, and alternative methods for preparing compounds of the disclosure are considered to be within the scope of the disclosure. For example, the synthesis of compounds not exemplified in the disclosure can be successfully carried out by modifications obvious to those skilled in the art, such as by appropriately protecting groups that cause interference, by utilizing other suitable reagents and components known in the art other than those described, and / or by routinely changing the reaction conditions. Alternatively, it will be recognized that other reactions disclosed herein or known in the art can be applied to prepare other compounds of the disclosure.

[0175] Example 1 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycinate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycinate [ka]

[0177] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 1086 mg, 2.5 mmol), (tert-butoxycarbonyl)glycine (438 mg, 2.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 527 mg, 2.75 mmol), N,N-dimethylpyridin-4-amine (DMAP, 31 mg, 0.25 mmol) were dissolved in DCM (25 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate=2:1 to 1:1) to give the title compound (1120 mg, yield: 77.3%). MS(m / z):C 31 H 42 FNO 9 [M+H] as + Calculated, 592.67; Found, 536.2 (M+H-56). 1H NMR (400 MHz, chloroform-d) δ 7.21 (d, J = 10.2 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.05 (d, J = 9.4 Hz, 1H), 5.01 - 4.87 (m, 3H), 4.42 (d, J = 8.9 Hz, 1H), 4.11 - 3.97 (m, 2H), 2.63 (tdd, J = 13.9, 6.1, 1.8 Hz, 1H), 2.54 - 2.29 (m, 4H), 2.17 - 2.05 (m, 1H), 1.86 (dt, J = 11.8, 5.2 Hz, 1H), 1.77 - 1.59 (m, 4H), 1.55 (s, 3H), 1.44 (d, J = 9.8 Hz, 12H), 1.21 (s, 3H), 0.93 (s, 3H).

[0176] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycinate hydrochloride [ka]

[0178] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycinate (500 mg, 0.85 mmol) in EtOAc (10 mL) was added slowly 4 M HCl in ethyl acetate (2 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.43 g, yield: 80.6%), which was used without further purification. MS (m / z): C 26 H 34 FNO 7 [M+H] as + Calculated value: 492.56; measured value: 492.3.

[0177] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycinate [ka]

[0179] Hyaluronic acid (201.5 mg, 0.5 mmol carboxylic acid) was dissolved in 40 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 26 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 35 mg, 0.35 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycinate hydrochloride (185 mg, 0.35 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 111 mg, 0.4 mmol) was added, and stirred at room temperature for 72 hours. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200 mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.255 g, yield: 68.3%, DSR=32%); 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.56 (m, 0.32H), 6.56 - 6.45 (m, 0.32H), 6.35 - 6.27 (m, 0.32H), 5.51 - 5.35 (m, 0.32H), 5.19 - 5.00 (m, 0.64H), 4.77 - 4.36 (m, 2.32H), 4.28 - 3.15 (m, 10.64H), 2.90 - 2.45 (m, 1.28H), 2.31 - 2.18 (m, 0.32H), 2.17 - 1.73 (m, 4.28H), 1.69 - 1.47 (m, 1.92H), 1.41 - 1.26 (m, 0.96H), 1.08 - 0.89 (m, 0.96H).

[0178]

[0180] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.2g, yield: 53.6%, DSR=33%). 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.55 (m, 0.33H), 6.57 - 6.45 (m, 0.33H), 6.35 - 6.25 (m, 0.33H), 5.51 - 5.34 (m, 0.33H), 5.18 - 5.01 (m, 0.66H), 4.77 - 4.36 (m, 2.33H), 4.29 - 3.17 (m, 10.66H), 2.89 - 2.46 (m, 1.32H), 2.30 - 2.19 (m, 0.33H), 2.17 - 1.71 (m, 4.32H), 1.65 - 1.44 (m, 1.98H), 1.38 - 1.23 (m, 0.99H), 1.06 - 0.88 (m, 0.99H).

[0179]

[0181] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.21g, yield: 56.3%, DSR=30%). 1 H NMR (400 MHz, heavy water) δ 7.67 - 7.55 (m, 0.3H), 6.56 - 6.45 (m, 0.3H), 6.36 - 6.27 (m, 0.3H), 5.51 - 5.34 (m, 0.3H), 5.18 - 5.01 (m, 0.6H), 4.78 - 4.35 (m, 2.3H), 4.31 - 3.22 (m, 10.6H), 2.89 - 2.46 (m, 1.2H), 2.33 - 2.19 (m, 0.3H), 2.18 - 1.73 (m, 4.2H), 1.70 - 1.48 (m, 1.8H), 1.40 - 1.26 (m, 0.9H), 1.10 - 0.90 (m, 0.9H).

[0180] Example 2 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0181] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate [ka]

[0182] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 4345 mg, 10 mmol), 4-((tert-butoxycarbonyl)amino)butanoic acid (2642 mg, 13 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2492 mg, 13 mmol), N,N-dimethylpyridin-4-amine (DMAP, 122 mg, 1 mmol) were dissolved in DCM (100 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (6000 mg, yield: 96.9%). MS (m / z): 33 H46 FNO 9 [M+H] as + Calculated value, 620.73; Found value, 564.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.20 (d, J = 10.1 Hz, 1H), 6.35 (dd, J = 10.2, 1.9 Hz, 1H), 6.13 (d, J = 1.8 Hz, 1H), 4.98 (d, J = 4.9 Hz, 1H), 4.90 (s, 2H), 4.68 (s, 1H), 4.42 (d, J = 8.7 Hz, 1H), 3.20 (q, J = 6.7 Hz, 2H), 2.62 (tt, J = 14.2, 7.3 Hz, 1H), 2.54 - 2.35 (m, 5H), 2.17 - 2.06 (m, 1H), 1.92 - 1.82 (m, 3H), 1.74 - 1.58 (m, 5H), 1.55 (s, 3H), 1.44 (d, J = 4.0 Hz, 12H), 1.21 (s, 3H), 0.94 (s, 3H).

[0182] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-aminobutanoate hydrochloride [ka]

[0183] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate (600 mg, 0.97 mmol) in EtOAc (24 mL) was added slowly 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.53 g, yield: 98.3%), which was used without further purification. MS (m / z): C 28 H 38 FNO 7 [M+H] as + Calculated value: 520.61; measured value: 520.1.

[0183] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0184] Hyaluronic acid (201.5 mg, 0.5 mmol carboxylic acid) was dissolved in 40 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 26 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 35 mg, 0.35 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-aminobutanoate hydrochloride (195 mg, 0.35 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 111 mg, 0.4 mmol) was added, and stirred at room temperature for 72 hours. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200 mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.263 g, Yield: 68.6%, DSR=32%); 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.56 (m, 0.32H), 6.59 - 6.47 (m, 0.32H), 6.39 - 6.26 (m, 0.32H), 5.43 - 5.31 (m, 0.32H), 5.20 - 5.01 (m, 0.64H), 4.78 - 4.44 (m, 2.32H), 4.21 - 3.16 (m, 10.64H), 2.91 - 2.51 (m, 1.6H), 2.35 - 2.26 (m, 0.32H), 2.22 - 1.72 (m, 5.56H), 1.70 - 1.47 (m, 1.92H), 1.41 - 1.25 (m, 0.96H), 1.06 - 0.92 (m, 0.96H).

[0184]

[0185] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.196g, yield: 51.1%, DSR=30%). 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.56 (m, 0.3H), 6.58 - 6.48 (m, 0.3H), 6.38 - 6.26 (m, 0.3H), 5.42 - 5.28 (m, 0.3H), 5.18 - 5.00 (m, 0.6H), 4.77 - 4.41 (m, 2.3H), 4.23 - 3.13 (m, 10.6H), 2.87 - 2.51 (m, 1.5H), 2.35 - 2.19 (m, 0.3H), 2.18 - 1.71 (m, 5.4H), 1.69 - 1.43 (m, 1.8H), 1.39 - 1.21 (m, 0.9H), 1.06 - 0.90 (m, 0.9H).

[0185]

[0186] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.191g, yield: 49.8%, DSR=39%). 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.54 (m, 0.39H), 6.58 - 6.47 (m, 0.39H), 6.39 - 6.25 (m, 0.39H), 5.43 - 5.30 (m, 0.39H), 5.19 - 5.00 (m, 0.78H), 4.78 - 4.44 (m, 2.39H), 4.25 - 3.15 (m, 10.78H), 2.89 - 2.50 (m, 1.95H), 2.34 - 2.23 (m, 0.39H), 2.21 - 1.72 (m, 6.12H), 1.70 - 1.46 (m, 2.34 H), 1.41 - 1.23 (m, 1.17H), 1.06 - 0.91 (m, 1.17H).

[0186] Example 3 Preparation of a conjugate of hyaluronan with 4-aminobutyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of tert-butyl (4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12b-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)butyl)carbamate [ka]

[0187] A mixture of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 1304 mg, 3 mmol) and bis(4-nitrophenyl)carbonate (1094 mg, 3.6 mmol) in dichloromethane (27 mL) was 2 Triethylamine (1 mL, 7.2 mmol) was added and the reaction mixture was heated to reflux for 6 h. tert-Butyl (4-hydroxybutyl)carbamate (738 mg, 3.9 mmol) was then added and the resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3 The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (1.4 g, yield: 71.9%); MS (m / z): C 34 H48 FNO 10 [M+H] as + Calculated, 650.75; Found, 594.3 (M+H-56).

[0187] Step 2: Preparation of 4-aminobutyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride [ka]

[0188] To a stirred solution of tert-butyl (4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12b-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)butyl)carbamate (1000 mg, 1.54 mmol) in EtOAc (30 mL) was added slowly 4 M HCl in ethyl acetate (6 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.9 g, yield: 99.8%), which was used without further purification. MS (m / z): C 29 H 40 FNO 8 [M+H] as + Calculated value: 550.64; measured value: 550.3.

[0188] Step 3: Preparation of the conjugate of HA with 4-aminobutyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0189] Hyaluronic acid (101 mg, 0.25 mmol carboxylic acid) was dissolved in 20 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 13 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 18 mg, 0.175 mmol) and 4-aminobutyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride (103 mg, 0.175 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 49mg, 0.175mmol) was added and stirred at room temperature for 72 hours. NaCl (147mg, 2.5mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (100mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.096g, Yield: 48.7%, DSR=27%); 1H NMR (400 MHz, heavy water) δ 7.67 - 7.58 (m, 0.27H), 6.56 - 6.49 (m, 0.27H), 6.34 - 6.27 (m, 0.27H), 5.42 - 5.34 (m, 0.27H), 5.19 - 5.01 (m, 0.54H), 4.80 - 4.45 (m, 2.81H), 4.39 - 3.07 (m, 10.54H), 2.92 - 2.49 (m, 1.35H), 2.32 - 1.68 (m, 3.54H), 1.67 - 1.45 (m, 3.51 H), 1.38 - 1.27 (m, 0.81H), 1.05 - 0.90 (m, 0.81H).

[0189]

[0190] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.068g, yield: 34.5%, DSR=25%). 1 H NMR (400 MHz, heavy water) δ 7.66 - 7.54 (m, 0.25H), 6.55 - 6.47 (m, 0.25H), 6.35 - 6.25 (m, 0.25H), 5.42 - 5.29 (m, 0.25H), 5.18 - 4.97 (m, 0.5H), 4.79 - 4.42 (m, 2.75H), 4.39 - 3.05 (m, 10.5H), 2.89 - 2.48 (m, 1.25H), 2.37 - 1.67 (m, 3.5H), 1.66 - 1.46 (m, 3.25 H), 1.40 - 1.25 (m, 0.75H), 1.07 - 0.90 (m, 0.75H).

[0190]

[0191] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.068g, yield: 34.5%, DSR=15%). 1H NMR (400 MHz, heavy water) δ 7.66 - 7.54 (m, 0.15H), 6.57 - 6.43 (m, 0.15H), 6.34 - 6.25 (m, 0.15H), 5.43 - 5.29 (m, 0.15H), 5.18 - 4.97 (m, 0.3H), 4.79 - 4.43 (m, 2.45H), 4.40 - 3.01 (m, 10.3H), 2.86 - 2.50 (m, 0.75H), 2.37 - 1.66 (m, 3.3H), 1.65 - 1.46 (m, 1.95 H), 1.39 - 1.24 (m, 0.45H), 1.07 - 0.91 (m, 0.45H).

[0191] Example 4 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(4-aminobutyl)carbamate [ka] Step 1: Preparation of tert-butyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)butane-1,4-diyl dicarbamate [ka]

[0192] A mixture of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 1090 mg, 2.5 mmol) and bis(4-nitrophenyl)carbonate (912 mg, 3 mmol) in dichloromethane (80 mL) was 2 Triethylamine (0.85 mL, 6 mmol) was added and the reaction mixture was heated to reflux for 6 h. tert-Butyl (4-aminobutyl)carbamate (612 mg, 3.25 mmol) was then added and the resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3 The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (1.24 g, yield: 76.5%); MS (m / z): C 34 H 49 FN 2 O 9 [M+H] as + Calculated, 649.77; Found, 593.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.23 (d, J = 10.1 Hz, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.14 (t, J = 5.9 Hz, 1H), 4.99 (d, J = 4.8 Hz, 1H), 4.88 (t, J = 13.2 Hz, 2H), 4.69 - 4.58 (m, 1H), 4.41 (dt, J = 10.0, 2.5 Hz, 1H), 3.17 (dt, J = 30.1, 6.1 Hz, 4H), 2.68 - 2.59 (m, 1H), 2.55 - 2.28 (m, 4H), 2.10 (d, J = 4.8 Hz, 2H), 1.93 - 1.75 (m, 2H), 1.70 - 1.53 (m, 9H), 1.44 (d, J = 6.8 Hz, 12H), 1.21 (s, 3H), 0.96 (s, 3H).

[0192] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(4-aminobutyl)carbamate hydrochloride [ka]

[0193] To a stirred solution of tert-butyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)butane-1,4-diyldicarbamate (400 mg, 0.62 mmol) in EtOAc (8 mL) was added slowly 4 M HCl in ethyl acetate (1.6 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.3 g, yield: 82.8%), which was used without further purification. MS (m / z): C 29 H 41 FN 2 O 7 [M+H] as + Calculated value: 549.65; measured value: 549.3.

[0193] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(4-aminobutyl)carbamate [ka]

[0194] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 30 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 35 mg, 0.35 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(4-aminobutyl)carbamate hydrochloride (164 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.151g, Yield: 48%, DSR=27%); 1H NMR (400 MHz, heavy water) δ 7.69 - 7.56 (m, 0.27H), 6.59 - 6.46 (m, 0.27H), 6.36 - 6.25 (m, 0.27H), 5.36 - 5.20 (m, 0.27H), 5.19 - 4.90 (m, 0.54H), 4.76 - 4.42 (m, 2.27H), 4.26 - 3.13 (m, 11.08H), 2.92 - 2.48 (m, 1.08H), 2.39 - 2.22 (m, 0.54H), 2.21 - 1.98 (m, 3.54H), 1.95 - 1.48 (m, 3.24H), 1.42 - 1.26 (m, 0.81H), 1.10 - 0.92 (m, 0.81H).

[0194]

[0195] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.183g, yield: 58.1%, DSR=25%). 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.56 (m, 0.25H), 6.59 - 6.45 (m, 0.25H), 6.37 - 6.23 (m, 0.25H), 5.37 - 5.05 (m, 0.75H), 4.76 - 4.41 (m, 2.25H), 4.25 - 3.09 (m, 11H), 2.91 - 2.46 (m, 1H), 2.34 - 2.20 (m, 0.5H), 2.19 - 1.98 (m, 3.5H), 1.94 - 1.46 (m, 3H), 1.41 - 1.27 (m, 0.75H), 1.09 - 0.92 (m, 0.75H).

[0195]

[0196] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.142g, yield: 45.1%, DSR=15%). 1H NMR (400 MHz, heavy water) δ 7.69 - 7.58 (m, 0.15H), 6.60 - 6.49 (m, 0.15H), 6.40 - 6.26 (m, 0.15H), 5.39 - 5.00 (m, 0.45H), 4.80 - 4.47 (m, 2.15H), 4.20 - 3.12 (m, 10.6H), 2.92 - 2.47 (m, 0.6H), 2.40 - 2.23 (m, 0.3H), 2.21 - 1.97 (m, 3.3H), 1.95 - 1.46 (m, 1.8H), 1.41 - 1.25 (m, 0.45H), 1.08 - 0.93 (m, 0.45H).

[0196] Example 5 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)propanoate [ka]

[0197] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 679 mg, 1.56 mmol), 3-((tert-butoxycarbonyl)amino)propanoic acid (387.5 mg, 1.8 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate=5:1 to 2:1) to give the title compound (650 mg, yield: 68.8%). MS (m / z): 32 H 44 FNO 9 [M+H] as + Calculated value: 606.70; measured value: 606.2. 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.1 Hz, 1H), 6.35 (dd, J = 10.2, 1.9 Hz, 1H), 6.14 (t, J = 1.7 Hz, 1H), 5.17 (s, 1H), 4.99 (dd, J = 11.3, 6.4 Hz, 2H), 4.88 (d, J = 17.6 Hz, 1H), 4.42 (dt, J = 8.4, 2.8 Hz, 1H), 3.46 (t, J = 6.4 Hz, 2H), 2.70 - 2.58 (m, 3H), 2.53 - 2.29 (m, 4H), 2.11 (td, J = 12.6, 5.9 Hz, 1H), 1.87 (dt, J = 11.9, 5.4 Hz, 1H), 1.73 - 1.60 (m, 4H), 1.58 (d, J = 1.5 Hz, 3H), 1.44 (d, J = 4.0 Hz, 12H), 1.22 (s, 3H), 0.94 (s, 3H).

[0197] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate hydrochloride [ka]

[0198] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)propanoate (605 mg, 1 mmol) in EtOAc (25 mL) was added slowly 4 M HCl in ethyl acetate (5 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.54 g, yield: 99.7%), which was used without further purification. MS (m / z): C 27 H 36 FNO 7 [M+H] as + Calculated value: 506.58; measured value: 506.1.

[0198] Step 3: Preparation of the conjugate of HA (hyaluronic acid) with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate [ka]

[0199] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 30 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate hydrochloride (152 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.194g, Yield: 64.1%, DSR=26%); 1H NMR (400 MHz, heavy water) δ 7.67 - 7.51 (m, 0.26H), 6.55 - 6.39 (m, 0.26H), 6.33 - 6.19 (m, 0.26H), 5.40 - 5.23 (m, 0.26H), 5.16 - 4.94 (m, 0.52H), 4.76 - 4.32 (m, 2.26H), 4.18 - 3.12 (m, 10.52H), 2.99 - 2.39 (m, 1.56H), 2.30 - 2.17 (m, 0.26H), 2.16 - 1.67 (m, 4.3H), 1.65 - 1.42 (m, 1.56H), 1.35 - 1.18 (m, 0.78H), 1.04 - 0.86 (m, 0.78H).

[0199]

[0200] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.23g, yield: 76%, DSR=22%). 1 H NMR (400 MHz, heavy water) δ 7.66 - 7.52 (m, 0.22H), 6.55 - 6.39 (m, 0.22H), 6.32 - 6.18 (m, 0.22H), 5.36 - 5.23 (m, 0.22H), 5.16 - 4.95 (m, 0.44H), 4.77 - 4.32 (m, 2.22H), 4.19 - 3.09 (m, 10.44H), 2.94 - 2.40 (m, 1.32H), 2.29 - 2.18 (m, 0.22H), 2.16 - 1.91 (m, 4.1H), 1.90 - 1.42 (m, 1.32H), 1.37 - 1.18 (m, 0.66H), 1.02 - 0.85 (m, 0.66H).

[0200]

[0201] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.099g, yield: 32.7%, DSR=30%). 1H NMR (400 MHz, heavy water) δ 7.66 - 7.51 (m, 0.3H), 6.55 - 6.40 (m, 0.3H), 6.32 - 6.21 (m, 0.3H), 5.39 - 5.25 (m, 0.3H), 5.14 - 4.94 (m, 0.6H), 4.76 - 4.35 (m, 2.3H), 4.21 - 3.14 (m, 10.6H), 2.95 - 2.45 (m, 1.8H), 2.38 - 2.22 (m, 0.3H), 2.18 - 1.68 (m, 4.5H), 1.65 - 1.43 (m, 1.8H), 1.38 - 1.20 (m, 0.9H), 1.05 - 0.87 (m, 0.9H).

[0201] Example 6 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidine-3-carboxylate [ka] Step 1: Preparation of 1-(tert-butyl) 3-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)pyrrolidine-1,3-dicarboxylate [ka]

[0202] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 652 mg, 1.5 mmol), 1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (420 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 375 mg, 1.95 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (910 mg, yield: 96.1%). MS (m / z): C 34 H 46 FNO 9 [M+H] as + Calculated value: 632.74; measured value: 632.2. 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.1 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.05 - 4.80 (m, 3H), 4.42 (s, 1H), 3.70 - 3.33 (m, 4H), 3.19 (q, J = 7.2, 6.8 Hz, 1H), 2.69 - 2.44 (m, 2H), 2.39 (dd, J = 12.9, 5.4 Hz, 2H), 2.30 - 1.99 (m, 4H), 1.91 - 1.82 (m, 1H), 1.70 - 1.58 (m, 4H), 1.55 (s, 3H), 1.46 (s, 9H), 1.43 (s, 3H), 1.22 (s, 3H), 0.93 (s, 3H).

[0202] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidine-3-carboxylate hydrochloride [ka]

[0203] To a stirred solution of 1-(tert-butyl) 3-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)pyrrolidine-1,3-dicarboxylate (600 mg, 0.95 mmol) in EtOAc (24 mL) was added slowly 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.53 g, yield: 97.6%), which was used without further purification. MS (m / z): C 29 H 38 FNO 7 [M+H] as + Calculated value: 532.62; measured value: 532.3.

[0203] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidine-3-carboxylate [ka]

[0204] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 40 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 20 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 35 mg, 0.35 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidine-3-carboxylate hydrochloride (199 mg, 0.35 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 97mg, 0.35mmol) was added and stirred at room temperature for 72 hours. NaCl (439mg, 7.5mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.199g, Yield: 51.4%, DSR=22%); 1H NMR (400 MHz, heavy water) δ 7.68 - 7.48 (m, 0.22H), 6.57 - 6.38 (m, 0.22H), 6.33 - 6.17 (m, 0.22H), 5.47 - 5.23 (m, 0.22H), 5.17 - 4.98 (m, 0.44H), 4.77 - 4.23 (m, 2.22H), 4.19 - 2.99 (m, 11.1H), 2.87 - 2.38 (m, 0.88H), 2.32 -1.68 (m, 4.76H), 1.66 - 1.42 (m, 1.32H), 1.38 - 1.16 (m, 0.66H), 1.03 - 0.82 (m, 0.66H).

[0204]

[0205] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.235g, yield: 60.6%, DSR=7%). 1 H NMR (400 MHz, heavy water) δ 7.71 - 7.62 (m, 0.07H), 6.61 - 6.53 (m, 0.07H), 6.42 - 6.33 (m, 0.07H), 5.47 - 5.37 (m, 0.07H), 5.28 - 5.00 (m, 0.14H), 4.77 - 4.43 (m, 2.07H), 4.26 - 3.02 (m, 10.35H), 2.91 - 2.58 (m, 0.28H), 2.42 -1.77 (m, 3.56H), 1.74 - 1.58(m, 0.42H), 1.44 - 1.35 (m, 0.21H), 1.08 - 0.95 (m, 0.21H).

[0205]

[0206] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.188g, yield: 48.5%, DSR=5%). 1H NMR (400 MHz, heavy water) δ 7.72 - 7.62 (m, 0.05H), 6.62 - 6.52 (m, 0.05H), 6.41 - 6.33 (m, 0.05H), 5.48 - 5.38 (m, 0.05H), 5.26 - 5.01 (m, 0.1H), 4.80 - 4.40 (m, 2.05H), 4.33 - 2.98 (m, 10.25H), 2.89 - 2.55 (m, 0.2H), 2.42 -1.75 (m, 3.4H), 1.73 - 1.55(m, 0.3H), 1.44 - 1.33 (m, 0.15H), 1.10 - 0.97 (m, 0.15H).

[0206] Example 7 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylazetidine-3-carboxylate [ka] Step 1: Preparation of 1-(tert-butyl) 3-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)azetidine-1,3-dicarboxylate [ka]

[0207] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 434.5 mg, 1 mmol), 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid (262 mg, 1.3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 249 mg, 1.3 mmol), N,N-dimethylpyridin-4-amine (DMAP, 13 mg, 0.1 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 2 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (600 mg, yield: 97.2%). MS (m / z): C 33 H 44 FNO 9 [M+H] as + Calculated value: 618.71; measured value: 618.2. 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.1 Hz, 1H), 6.36 (dd, J = 10.2, 1.9 Hz, 1H), 6.14 (t, J = 1.7 Hz, 1H), 5.10 - 4.96 (m, 2H), 4.90 (d, J = 17.6 Hz, 1H), 4.43 (q, J = 3.4 Hz, 1H), 4.24 - 4.09 (m, 4H), 3.48 (tt, J = 8.5, 6.4 Hz, 1H), 2.70 - 2.57 (m, 1H), 2.55 - 2.27 (m, 4H), 2.11 (td, J = 12.5, 5.9 Hz, 1H), 1.87 (dt, J = 11.6, 5.1 Hz, 1H), 1.77 - 1.59 (m, 4H), 1.58 (s, 3H), 1.44 (d, J = 2.6 Hz, 12H), 1.22 (s, 3H), 0.95 (s, 3H).

[0207] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12αa,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylazetidine-3-carboxylate hydrochloride [ka]

[0208] To a stirred solution of 1-(tert-butyl) 3-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)azetidine-1,3-dicarboxylate (300 mg, 0.49 mmol) in EtOAc (12 mL) was added slowly 4 M HCl in ethyl acetate (2.4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.25 g, yield: 92.9%), which was used without further purification. MS (m / z): C 28 H 36 FNO 7 [M+H] as + Calculated value: 518.59; measured value: 518.3.

[0208] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylazetidine-3-carboxylate [ka]

[0209] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 30 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylazetidine-3-carboxylate hydrochloride (155 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.24g, Yield: 78.4%, DSR=15%); 1H NMR (400 MHz, heavy water) δ 7.75 - 7.61 (m, 0.15H), 6.65 - 6.53 (m, 0.15H), 6.43 - 6.32 (m, 0.15H), 5.58 - 5.44 (m, 0.15H), 5.26 - 5.11 (m, 0.3H), 4.81 - 4.41 (m, 2.15H), 4.34 - 3.38 (m, 10.75H), 2.99 - 2.57 (m, 0.3H), 2.45 -1.80 (m, 4.2H), 1.77 - 1.52 (m, 0.9H), 1.48 - 1.37 (m, 0.45H), 1.14 - 1.00 (m, 0.45H).

[0209]

[0210] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.236g, yield: 77%, DSR=19%). 1 H NMR (400 MHz, heavy water) δ 7.73 - 7.62 (m, 0.19H), 6.64 - 6.52 (m, 0.19H), 6.43 - 6.31 (m, 0.19H), 5.58 - 5.42 (m, 0.19H), 5.27 - 5.12 (m, 0.38H), 4.81 - 4.40 (m, 2.19H), 4.30 - 3.19 (m, 10.95H), 2.95 - 2.55 (m, 0.38H), 2.41 -1.75 (m, 4.52H), 1.74 - 1.53 (m, 1.14H), 1.47 - 1.35 (m, 0.57H), 1.12 - 0.97 (m, 0.57H).

[0210]

[0211] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.166g, yield: 54.2%, DSR=20%). 1H NMR (400 MHz, heavy water) δ 7.76 - 7.61 (m, 0.2H), 6.64 - 6.52 (m, 0.2H), 6.43 - 6.32 (m, 0.2H), 5.56 - 5.44 (m, 0.2H), 5.27 - 5.09 (m, 0.4H), 4.80 - 4.43 (m, 2.2H), 4.26 - 3.11 (m, 11H), 2.96 - 2.50 (m, 0.4H), 2.37 -1.75 (m, 4.6H), 1.74 - 1.50 (m, 1.2H), 1.45 - 1.34 (m, 0.6H), 1.11 - 0.95 (m, 0.6H).

[0211] Example 8 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl alaninate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)alaninate [ka]

[0212] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 652 mg, 1.5 mmol), (tert-butoxycarbonyl)alanine (369 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 374 mg, 1.95 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (30 mL). The reaction mixture was stirred at room temperature for 2 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate=5:1 to 2:1) to give the title compound (900 mg, yield: 99.1%). MS(m / z):C 32 H 44 FNO 9 [M+H] as + Calculated value: 606.70; measured value: 606.2. 1 H NMR (400 MHz, chloroform-d) δ 7.22 (dd, J = 10.1, 2.1 Hz, 1H), 6.35 (dd, J = 10.2, 1.9 Hz, 1H), 6.14 (t, J = 1.7 Hz, 1H), 5.13 - 4.78 (m, 4H), 4.42 (d, J = 8.4 Hz, 2H), 2.72 - 2.56 (m, 1H), 2.57 - 2.21 (m, 4H), 2.10 (td, J = 12.5, 5.8 Hz, 1H), 1.92 - 1.81 (m, 1H), 1.79 - 1.58 (m, 4H), 1.55 (m, 3H), 1.51 (dd, J = 11.0, 7.4 Hz, 3H), 1.44 (d, J = 6.6 Hz, 12H), 1.22 (d, J = 3.2 Hz, 3H), 0.93 (d, J = 1.9 Hz, 3H).

[0212] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl alaninate hydrochloride [ka]

[0213] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)alaninate (600 mg, 0.99 mmol) in EtOAc (12 mL) was added slowly 4 M HCl in ethyl acetate (2.4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.51 g, yield: 95.1%), which was used without further purification. MS (m / z): C 27 H 36 FNO 7 [M+H] as + Calculated value: 506.58; measured value: 506.4.

[0213] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl alaninate [ka]

[0214] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 30 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl alaninate hydrochloride (155 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.208g, Yield: 68.7%, DSR=20%); 1 H NMR (400 MHz, heavy water) δ 7.74 - 7.60 (m, 0.2H), 6.64 - 6.53 (m, 0.2H), 6.43 - 6.31 (m, 0.2H), 5.57 - 5.41 (m, 0.2H), 5.22 - 5.05 (m, 0.4H), 4.85 - 4.47 (m, 2.4H), 4.24 - 3.36 (m, 10H), 2.98 - 2.57 (m, 0.8H), 2.42 -1.79 (m, 4.8H), 1.77 - 1.50 (m, 1.2H), 1.45 - 1.31 (m, 0.6H), 1.14 - 0.98 (m, 0.6H).

[0214]

[0215] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.168g, yield: 55.5%, DSR=25%). 1 H NMR (400 MHz, heavy water) δ 7.75 - 7.61 (m, 0.25H), 6.62 - 6.52 (m, 0.25H), 6.42 - 6.32 (m, 0.25H), 5.56 - 5.40 (m, 0.25H), 5.25 - 5.07 (m, 0.5H), 4.84 - 4.46 (m, 2.5H), 4.26 - 3.32 (m, 10H), 2.98 - 2.58 (m, 1H), 2.42 -1.78 (m, 5.25H), 1.77 - 1.51 (m, 1.5H), 1.45 - 1.30 (m, 0.75H), 1.14 - 0.95 (m, 0.75H).

[0215]

[0216] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.202g, yield: 66.7%, DSR=10%). 1 H NMR (400 MHz, heavy water) δ 7.74 - 7.63 (m, 0.1H), 6.63 - 6.52 (m, 0.1H), 6.42 - 6.34 (m, 0.1H), 5.53 - 5.10 (m, 0.3H), 4.82 - 4.47 (m, 2.2H), 4.27 - 3.27 (m, 10H), 2.94 - 2.57 (m, 0.4H), 2.42 -1.78 (m, 3.9H), 1.75 - 1.58 (m, 0.6H), 1.43 - 1.34 (m, 0.3H), 1.11 - 0.98 (m, 0.3H).

[0216] Example 9 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-aminooctanoate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-((tert-butoxycarbonyl)amino)octanoate [ka]

[0217] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 652 mg, 1.5 mmol), 8-((tert-butoxycarbonyl)amino)octanoic acid (506 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 374 mg, 1.95 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (18 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 2:1 to 5:3) to give the title compound (720 mg, yield: 54.7%). MS (m / z): 37 H 54 FNO 9[M+H] as + Calculated, 676.84; Found, 620.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.21 (d, J = 10.1 Hz, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (d, J = 1.9 Hz, 1H), 5.03 - 4.95 (m, 1H), 4.95 - 4.78 (m, 2H), 4.55 (t, J = 5.9 Hz, 1H), 4.42 (d, J = 8.8 Hz, 1H), 3.09 (t, J = 6.9 Hz, 2H), 2.70 - 2.57 (m, 1H), 2.55 - 2.32 (m, 5H), 2.16 - 2.05 (m, 1H), 1.92 - 1.82 (m, 1H), 1.78 - 1.60 (m, 6H), 1.55 (m, 3H), 1.44 (d, J = 5.7 Hz, 14H), 1.39 - 1.29 (m, 7H), 1.22 (s, 3H), 0.95 (s, 3H).

[0217] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-aminooctanoate hydrochloride [ka]

[0218] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-((tert-butoxycarbonyl)amino)octanoate (600 mg, 0.89 mmol) in EtOAc (24 mL) was added slowly 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.533 g, yield: 97.9%), which was used without further purification. MS (m / z): C 32 H 46 FNO 7 [M+H] as + Calculated value: 576.72; measured value: 576.1.

[0218] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-aminooctanoate [ka]

[0219] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 32 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 8-aminooctanoate hydrochloride (172 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.2g, Yield: 62.1%, DSR=8%); 1 H NMR (400 MHz, heavy water) δ 7.65 - 7.56 (m, 0.08H), 6.57 - 6.46 (m, 0.08H), 6.34 - 6.26 (m, 0.08H), 5.35 - 5.25 (m, 0.08H), 5.17 - 5.01 (m, 0.16H), 4.83 - 4.43 (m, 2.08H), 4.28 - 3.07 (m, 10.16H), 2.85 - 2.52 (m, 0.08H), 2.35 -1.69 (m, 4.04H), 1.67 - 1.50 (m, 0.72H), 1.48 - 1.36 (m, 0.72H), 1.05 - 0.92 (m, 0.24H).

[0219]

[0220] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.2g, yield: 62.1%, DSR=12%). 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.58 (m, 0.12H), 6.60 - 6.50 (m, 0.12H), 6.41 - 6.30 (m, 0.12H), 5.41 - 5.28 (m, 0.12H), 5.20 - 5.02 (m, 0.24H), 4.79 - 4.42 (m, 2.12H), 4.31 - 3.08 (m, 10.24H), 2.90 - 2.48 (m, 0.12H), 2.38 -1.74 (m, 4.56H), 1.70 - 1.54 (m, 1.08H), 1.50 - 1.39 (m, 1.08H), 1.08 - 0.94 (m, 0.36H).

[0220]

[0221] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.121g, yield: 37.5%, DSR=22%). 1 H NMR (400 MHz, heavy water) δ 7.66 - 7.53 (m, 0.22H), 6.61 - 6.45 (m, 0.22H), 6.39 - 6.26 (m, 0.22H), 5.35 - 5.03 (m, 0.66H), 4.83 - 4.38 (m, 2.22H), 4.11 - 3.09 (m, 10.44H), 2.82 - 2.41 (m, 0.22H), 2.29 -1.69 (m, 5.86H), 1.67 - 1.49 (m, 1.98H), 1.48 - 1.37 (m, 1.98H), 1.01 - 0.92 (m, 0.66H).

[0221] Example 10 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate [ka] Step 1: Preparation of 1-(tert-butyl) 4-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)piperidine-1,4-dicarboxylate [ka]

[0222] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 652 mg, 1.5 mmol), 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (447 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 374 mg, 1.95 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (18 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (920 mg, yield: 94.9%). MS (m / z): 35 H 48 FNO9 [M+H] as + Calculated value, 646.774; Found value, 590.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.1 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.07 - 4.92 (m, 2H), 4.86 (d, J = 17.7 Hz, 1H), 4.42 (dq, J = 8.9, 2.9 Hz, 1H), 4.01 (d, J = 12.7 Hz, 2H), 2.91 (t, J = 12.3 Hz, 2H), 2.62 (tt, J = 10.1, 4.1 Hz, 2H), 2.55 - 2.27 (m, 4H), 2.10 (td, J = 12.6, 5.9 Hz, 1H), 2.03 - 1.83 (m, 3H), 1.77 - 1.57 (m, 6H), 1.55 (s, 3H), 1.45 (d, J = 10.9 Hz, 12H), 1.22 (s, 3H), 0.94 (s, 3H).

[0222] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6βb-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate hydrochloride [ka]

[0223] To a stirred solution of 1-(tert-butyl) 4-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)piperidine-1,4-dicarboxylate (700 mg, 1.08 mmol) in EtOAc (28 mL) was added slowly 4 M HCl in ethyl acetate (5.6 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.556 g, yield: 93.9%), which was used without further purification. MS (m / z): C 30 H 40 FNO 7 [M+H] as + Calculated value: 546.65; measured value: 546.4.

[0223] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate [ka]

[0224] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 32 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate hydrochloride (163 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.2g, Yield: 63.7%, DSR=27%); 1 H NMR (400 MHz, heavy water) δ 7.66 - 7.54 (m, 0.27H), 6.57 - 6.47 (m, 0.27H), 6.36 - 6.27 (m, 0.27H), 5.45 - 5.33 (m, 0.27H), 5.16 - 4.93 (m, 0.54H), 4.82 - 4.39 (m, 2.27H), 4.28 - 3.21 (m, 10.54H), 3.14 - 2.51 (m, 1.08H), 2.38 -1.73 (m, 4.89H), 1.70 - 1.46 (m, 3.24H), 1.41 - 1.24 (m, 0.81H), 1.08 - 0.92 (m, 0.81H).

[0224]

[0225] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.18g, yield: 57.3%, DSR=16%). 1 H NMR (400 MHz, heavy water) δ 7.67 - 7.53 (m, 0.16H), 6.59 - 6.47 (m, 0.16H), 6.35 - 6.27 (m, 0.16H), 5.47 - 5.31 (m, 0.16H), 5.15 - 4.95 (m, 0.32H), 4.84 - 4.39 (m, 2.16H), 4.26 - 3.19 (m, 10.32H), 3.12 - 2.49 (m, 0.64H), 2.37 -1.73 (m, 4.12H), 1.70 - 1.46 (m, 1.92H), 1.42 - 1.24 (m, 0.48H), 1.07 - 0.91 (m, 0.48H).

[0225]

[0226] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.16g, yield: 51%, DSR=5%). 1 H NMR (400 MHz, heavy water) δ 7.67 - 7.56 (m, 0.05H), 6.57 - 6.49 (m, 0.05H), 6.35 - 6.29 (m, 0.05H), 5.45 - 5.33 (m, 0.05H), 5.18 - 4.98 (m, 0.1H), 4.83 - 4.39 (m, 2.05H), 4.22 - 3.17 (m, 10.1H), 2.95 - 2.39 (m, 0.2H), 2.32 -1.71 (m, 3.35H), 1.68 - 1.50 (m, 0.6H), 1.41 - 1.27 (m, 0.15H), 1.05 - 0.90 (m, 0.15H).

[0226] Example 11 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12βb-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminocyclobutane-1-carboxylate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6b-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylate [ka]

[0227] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 1004 mg, 2.31 mmol), 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylic acid (3649 mg, 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 663 mg, 3.46 mmol), N,N-dimethylpyridin-4-amine (DMAP, 28.3 mg, 0.23 mmol) were dissolved in DCM (30 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (1420 mg, yield: 97.5%). MS (m / z): C34 H 46 FNO 9 [M+H] as + Calculated, 632.74; Found, 576.3 (M+H-56). 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.30 (d, J = 10.2 Hz, 1H), 6.24 (dd, J = 10.1, 1.9 Hz, 1H), 6.02 (t, J = 1.7 Hz, 1H), 5.48 (dt, J = 4.8, 2.1 Hz, 1H), 5.15 (dd, J = 17.9, 11.2 Hz, 1H), 4.87 (t, J = 5.4 Hz, 1H), 4.76 (t, J = 18.2 Hz, 1H), 4.27 - 4.16 (m, 1H), 4.00 - 3.83 (m, 1H), 3.10 - 2.82 (m, 1H), 2.69 - 2.57 (m, 1H), 2.47 - 2.19 (m, 4H), 2.16 - 2.01 (m, 3H), 1.97 - 1.68 (m, 3H), 1.64 - 1.45 (m, 5H), 1.43 - 1.32 (m, 13H), 1.24 (s, 1H), 1.16 (d, J = 4.8 Hz, 3H), 0.84 (d, J = 6.9 Hz, 3H).

[0227] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminocyclobutane-1-carboxylate hydrochloride [ka]

[0228] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6b-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylate (900 mg, 1.42 mmol) in EtOAc (36 mL) was added slowly 4 M HCl (commercially available) in ethyl acetate (7.2 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.7 g, yield: 92.8%), which was used without further purification. MS (m / z): C 29 H 38 FNO 7 [M+H] as + Calculated value: 532.62; measured value: 532.2.

[0228] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12βb-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminocyclobutane-1-carboxylate [ka]

[0229] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 40 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 20 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 35 mg, 0.35 mmol) and 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12βb-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminocyclobutane-1-carboxylate hydrochloride (199 mg, 0.35 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 97mg, 0.35mmol) was added and stirred at room temperature for 72 hours. NaCl (439mg, 7.5mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.18g, Yield: 46.5%, DSR=35%); 1 H NMR (400 MHz, heavy water) δ 7.68 - 7.57 (m, 0.35H), 6.59 - 6.47 (m, 0.35H), 6.36 - 6.29 (m, 0.35H), 5.45 - 5.30 (m, 0.35H), 5.18 - 4.96 (m, 0.7H), 4.81 - 4.33 (m, 2.35H), 4.21 - 3.11 (m, 10.35H), 2.95 - 2.46 (m, 0.7H), 2.33 -1.72 (m, 6.85H), 1.69 - 1.46 (m, 2.8H), 1.39 - 1.20 (m, 1.05H), 1.04 - 0.89 (m, 1.05H).

[0229]

[0230] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.19g, yield: 49%, DSR=34%). 1 H NMR (400 MHz, heavy water) δ 7.68 - 7.58 (m, 0.34H), 6.57 - 6.46 (m, 0.34H), 6.38 - 6.26 (m, 0.34H), 5.41 - 5.30 (m, 0.34H), 5.18 - 4.97 (m, 0.68H), 4.80 - 4.35 (m, 2.34H), 4.19 - 3.15 (m, 10.34H), 2.95 - 2.52 (m, 0.68H), 2.33 -1.72 (m, 6.74H), 1.69 - 1.45 (m, 2.72H), 1.39 - 1.18 (m, 1.02H), 1.07 - 0.90 (m, 1.02H).

[0230]

[0231] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.13g, yield: 33.5%, DSR=25%). 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.56 (m, 0.25H), 6.61 - 6.48 (m, 0.25H), 6.37 - 6.23 (m, 0.25H), 5.42 - 5.30 (m, 0.25H), 5.18 - 4.97 (m, 0.5H), 4.80 - 4.43 (m, 2.25H), 4.22 - 3.07 (m, 10.25H), 2.93 - 2.60 (m, 0.5H), 2.38 -1.92 (m, 5.75H), 1.72 - 1.43 (m, 2H), 1.40 - 1.19 (m, 0.75H), 1.06 - 0.87 (m, 0.75H).

[0231] Example 12 Preparation of a conjugate of hyaluronan with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate [ka] Step 1: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)propanoate [ka]

[0232] (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (fluocinolone acetonide, 453 mg, 1 mmol), 4-((tert-butoxycarbonyl)amino)butanoic acid (246 mg, 1.3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 249 mg, 1.3 mmol), N,N-dimethylpyridin-4-amine (DMAP, 12 mg, 0.1 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (620 mg, yield: 99.5%). MS (m / z): C32 H 43 F 2 NO 9 [M+H] as + Calculated, 624.69; Found, 568.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.15 (dd, J = 10.2, 1.5 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.38 (dd, J = 10.1, 1.9 Hz, 1H), 5.53 - 5.29 (m, 1H), 5.17 (s, 1H), 5.07 - 4.85 (m, 3H), 4.43 (dq, J = 8.8, 2.8 Hz, 1H), 3.46 (q, J = 6.2 Hz, 2H), 2.66 (t, J = 6.1 Hz, 2H), 2.58 - 2.34 (m, 3H), 2.34 - 2.24 (m, 1H), 2.19 (td, J = 12.4, 5.8 Hz, 1H), 1.88 - 1.59 (m, 4H), 1.54(s, 3H), 1.51 - 1.39 (m, 12H), 1.22 (s, 3H), 0.93 (s, 3H).

[0232] Step 2: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate hydrochloride [ka]

[0233] To a stirred solution of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-((tert-butoxycarbonyl)amino)propanoate (400 mg, 0.64 mmol) in EtOAc (16 mL) was added slowly 4 M HCl in ethyl acetate (3.2 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.375 g, yield: 97.9%), which was used without further purification. MS (m / z): C 27 H 35 F 2 NO 7 [M+H] as + Calculated value: 524.57; measured value: 524.3.

[0233] Step 3: Preparation of the conjugate of HA with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate [ka]

[0234] Hyaluronic acid (161 mg, 0.4 mmol carboxylic acid) was dissolved in 32 mL of deionized water in a 100 mL round bottom flask, followed by the dropwise addition of 21 mL of acetonitrile with stirring. Addition of 4-methylmorpholine (NMM, 28 mg, 0.28 mmol) and 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 3-aminopropanoate hydrochloride (157 mg, 0.28 mmol) to the solution caused a transient increase in viscosity. The solution was then cooled to 0°C, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 78mg, 0.28mmol) was added and stirred at room temperature for 72 hours. NaCl (234mg, 4mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200mL) with stirring. The mixture was filtered. The filter cake was collected, washed with absolute alcohol and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.198g, Yield: 64.4%, DSR=30%); 1 H NMR (400 MHz, heavy water) δ 7.67 - 7.54 (m, 0.3H), 6.67 - 6.49 (m, 0.6H), 5.91 - 5.63 (m, 0.3H), 5.44 - 5.32 (m, 0.3H), 5.23 - 5.01 (m, 0.6H), 4.81 - 4.45 (m, 2.3H), 4.21 - 3.31 (m, 10.6H), 3.02 - 2.47 (m, 0.9H), 2.38 -1.74 (m, 4.5H), 1.72 - 1.49 (m, 2.4H), 1.42 - 1.27 (m, 1.5H), 1.09 - 0.90 (m, 1.5H).

[0234] Example 13 Preparation of conjugates of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(((tert-butoxycarbonyl)amino)methyl)benzoate [ka]

[0235] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 679 mg, 1.56 mmol), 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (387.5 mg, 1.8 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (DMAP, 18 mg, 0.15 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (650 mg, yield: 62.4%). MS (m / z): 37 H 46FNO 9 [M+H] as + Calculated value: 668.77; measured value: 668.2. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.97 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 10.2 Hz, 1H), 6.25 (dd, J = 10.1, 1.9 Hz, 1H), 6.03 (t, J = 1.7 Hz, 1H), 5.51 (d, J = 4.1 Hz, 1H), 5.41 (d, J = 18.0 Hz, 1H), 5.01 (d, J = 17.9 Hz, 1H), 4.89 (d, J = 4.6 Hz, 1H), 4.22 (d, J = 6.1 Hz, 3H), 2.73 - 2.60 (m, 1H), 2.58 - 2.42 (m, 2H), 2.39 - 2.30 (m, 1H), 2.15 - 2.06 (m, 1H), 2.03 - 1.92 (m, 2H), 1.82 (t, J = 8.9 Hz, 2H), 1.54 (d, J = 21.4 Hz, 5H), 1.40 (d, J = 8.5 Hz, 12H), 1.23 (s, 3H), 0.90 (s, 3H).

[0235] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate hydrochloride [ka]

[0236] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(((tert-butoxycarbonyl)amino)methyl)benzoate (200 mg, 0.3 mmol) in EtOAc (20 mL) was added slowly 4 M HCl in ethyl acetate (4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.18 g, yield: 99%), which was used without further purification. MS (m / z): C 32 H 38 FNO 7 [M+H] as + Calculated value: 568.65; measured value: 568.3.

[0236] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate [ka]

[0237] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate hydrochloride (120 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.2 g, Yield: 63.4%, DSR=30%); 1H NMR (400 MHz, heavy water) δ 8.24 - 8.12 (m, 0.6H), 7.72 - 7.57 (m, 0.9H), 6.62 - 6.50 (m, 0.3H), 6.40 - 6.30 (m, 0.3H), 5.65 - 5.56 (m, 0.3H), 5.38 - 5.16 (m, 0.9H), 4.78 - 4.43 (m, 2.6H), 4.23 - 3.24 (m, 10.9H), 2.93 - 2.49 (m, 1.2H), 2.45 -1.77 (m, 4.2H), 1.72 - 1.54 (m, 2.4H), 1.49 - 1.38 (m, 0.9H), 1.14 - 0.97 (m, 0.9H).

[0237]

[0238] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.13g, yield: 41.2%, DSR=25%). 1 H NMR (400 MHz, heavy water) δ 8.27 - 8.13 (m, 0.5H), 7.76 - 7.57 (m, 0.75H), 6.64 - 6.50 (m, 0.25H), 6.41 - 6.26 (m, 0.25H), 5.71 - 5.56 (m, 0.25H), 5.39 - 5.13 (m, 0.75H), 4.80 - 4.43 (m, 2.5H), 4.21 - 3.18 (m, 10.75H), 2.99 - 2.54 (m, 1H), 2.44 -1.77 (m, 4H), 1.75 - 1.55 (m, 2H), 1.50 - 1.38 (m, 0.75H), 1.18 - 0.99 (m, 0.75H).

[0238]

[0239] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.13g, yield: 41.2%, DSR=25%). 1H NMR (400 MHz, heavy water) δ 8.26 - 8.12 (m, 0.5H), 7.74 - 7.55 (m, 0.75H), 6.65 - 6.50 (m, 0.25H), 6.40 - 6.27 (m, 0.25H), 5.70 - 5.51 (m, 0.25H), 5.36 - 5.09 (m, 0.75H), 4.84 - 4.41 (m, 2.5H), 4.27 - 3.08 (m, 10.75H), 2.97 - 2.54 (m, 1H), 2.43 -1.78 (m, 4H), 1.75 - 1.56 (m, 2H), 1.50 - 1.37 (m, 0.75H), 1.17 - 0.99 (m, 0.75H).

[0239] Example 14 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(2-aminoethyl)carbamate [ka] Step 1: Preparation of tert-butyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)ethane-1,2-diyl dicarbamate [ka]

[0240] A mixture of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 652 mg, 1.5 mmol) and bis(4-nitrophenyl)carbonate (547 mg, 1.8 mmol) in dichloromethane (80 mL) was 2 Triethylamine (0.42 mL, 3 mmol) was added and the reaction mixture was heated to reflux for 2 h. tert-Butyl (2-aminoethyl)carbamate (312 mg, 1.95 mmol) was then added and the resulting mixture was stirred at room temperature for 2 h. The solution was diluted with DCM and saturated NaHCO 3 The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.65 g, yield: 69.8%); MS (m / z): C 32 H 45 FN 2 O 9 [M+H] as + Calculated, 621.72; Found, 565.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.2 Hz, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 5.56 - 5.32 (m, 1H), 4.99 (d, J = 4.9 Hz, 1H), 4.94 - 4.80 (d, J = 16.9 Hz, 3H), 4.45 - 4.37 (m, 1H), 3.40 - 3.20 (m, 4H), 2.72 - 2.56 (m, 1H), 2.56 - 2.32 (m, 4H), 2.17 - 2.03 (m, 2H), 1.95 - 1.82 (m, 3H), 1.75 (d, J = 14.0 Hz, 1H), 1.70 - 1.59 (m, 3H), 1.45 (s, 9H), 1.43 (s, 3H), 1.21 (s, 3H), 0.96 (s, 3H).

[0240] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(2-aminoethyl)carbamate hydrochloride [ka]

[0241] To a stirred solution of tert-butyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)ethane-1,2-diyldicarbamate (500 mg, 0.8 mmol) in EtOAc (20 mL) was added slowly 4 M HCl in ethyl acetate (4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.386 g, yield: 97.9%), which was used without further purification. MS (m / z): C 27 H 37 FN 2 O 7 [M+H] as + Calculated value: 521.60; measured value: 521.3.

[0241] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(2-aminoethyl)carbamate [ka]

[0242] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(2-aminoethyl)carbamate hydrochloride (111 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (1000 mg, 17 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.2 g, Yield: 65.5%, DSR=18%); 1H NMR (400 MHz, heavy water) δ 7.68 - 7.58 (m, 0.18H), 6.59 - 6.48 (m, 0.18H), 6.37 - 6.29 (m, 0.18H), 5.40 - 5.24 (m, 0.18H), 5.21 - 5.09 (m, 0.18H), 4.82 - 4.40 (m, 2.54H), 4.26 - 3.13 (m, 10.72H), 2.95 - 2.51 (m, 0.72H), 2.37 -1.72 (m, 4.08H), 1.71 - 1.47 (m, 1.08H), 1.40 - 1.30 (m, 0.54H), 1.09 - 0.92 (m, 0.54H).

[0242]

[0243] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.2g, yield: 65.5%, DSR=19%). 1 H NMR (400 MHz, heavy water) δ 7.70 - 7.58 (m, 0.19H), 6.61 - 6.52 (m, 0.19H), 6.41 - 6.30 (m, 0.19H), 5.44 - 5.24 (m, 0.19H), 5.22 - 5.09 (m, 0.19H), 4.81 - 4.40 (m, 2.38H), 4.27 - 3.11 (m, 10.76H), 2.96 - 2.53 (m, 0.76H), 2.41 -1.73 (m, 4.14H), 1.71 - 1.48 (m, 1.14H), 1.44 - 1.31 (m, 0.57H), 1.10 - 0.95 (m, 0.57H).

[0243]

[0244] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.19g, yield: 62.2%, DSR=14%). 1H NMR (400 MHz, heavy water) δ 7.69 - 7.56 (m, 0.14H), 6.61 - 6.49 (m, 0.14H), 6.40 - 6.28 (m, 0.14H), 5.44 - 5.24 (m, 0.14H), 5.22 - 5.07 (m, 0.14H), 4.81 - 4.39 (m, 2.28H), 4.26 - 3.11 (m, 10.56H), 2.92 - 2.49 (m, 0.56H), 2.39 -1.75 (m, 3.84H), 1.72 - 1.48 (m, 0.84H), 1.43 - 1.30 (m, 0.42H), 1.09 - 0.93 (m, 0.42H).

[0244] Example 15 Preparation of a conjugate of hyaluronan with 4-(aminomethyl)benzyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 4-(((tert-butoxycarbonyl)amino)methyl)benzyl 1H-imidazole-1-carboxylate [ka]

[0245] To a solution of tert-butyl (4-(hydroxymethyl)benzyl)carbamate (356 mg, 1.5 mmol) in DCM (10 mL) was added 1,1'-carbonyldiimidazole (CDI, 486 mg, 3 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with saturated NaHCO 3The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.49 g, yield: 98.6%), which was used without further purification.

[0245] Step 2: Preparation of tert-butyl (4-((((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)methyl)benzyl)carbamate [ka]

[0246] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 521 mg, 1.2 mmol) in DMF (10 mL) was 2 4-(((tert-butoxycarbonyl)amino)methyl)benzyl 1H-imidazole-1-carboxylate (477 mg, 1.44 mmol) and K 2 CO 3 (166 mg, 1.2 mmol) was added. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (30 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.42 g, yield: 50.2%); MS (m / z): C 38 H 48 FNO 10 [M+H] as + Calculated value, 698.80; Found value, 642.0 (M+H-56). 1H NMR (400 MHz, chloroform-d) δ 7.35 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 10.1 Hz, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.18 (s, 2H), 5.12 - 4.98 (m, 2H), 4.89 (s, 1H), 4.79 (d, J = 17.8 Hz, 1H), 4.41 (dt, J = 9.1, 2.9 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 2.71 - 2.57 (m, 1H), 2.53 - 2.34 (m, 3H), 2.10 (dt, J = 12.2, 6.2 Hz, 1H), 1.94 - 1.81 (m, 2H), 1.72 - 1.59 (m, 4H), 1.54 (s, 3H), 1.46 (s, 9H), 1.42 (s, 3H), 1.21 (s, 3H), 0.94 (s, 3H).

[0246] Step 3: Preparation of 4-(aminomethyl)benzyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride [ka]

[0247] To a stirred solution of tert-butyl (4-((((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)methyl)benzyl)carbamate (418 mg, 0.6 mmol) in EtOAc (20 mL) was added slowly 4 M HCl (commercially available) in ethyl acetate (4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.3 g, yield: 83.6%), which was used without further purification. MS (m / z): C 33 H 40 FNO 8 [M+H] as + Calculated value: 598.68; measured value: 598.3.

[0247] Step 4: Preparation of the conjugate of HA with 4-(aminomethyl)benzyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0248] Hyaluronic acid (201 mg, carboxylic acid 0.5 mmol) was dissolved in 22 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. The solution was treated with 4-(aminomethyl)benzyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno)-. Addition of [1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (127 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.21 g, Yield: 47.7%, DSR=18%); 1H NMR (400 MHz, heavy water) δ 7.66 - 7.39 (m, 0.9H), 6.59 - 6.46 (m, 0.18H), 6.37 - 6.25 (m, 0.18H), 5.47 - 5.24 (m, 0.54H), 5.16 - 5.01 (m, 0.36H), 4.81 - 4.31 (m, 2.54H), 4.19 - 3.13 (m, 10H), 2.93 - 2.49 (m, 0.72H), 2.33 -1.73 (m, 4.08H), 1.70 - 1.43 (m, 1.08H), 1.36 - 1.28 (m, 0.54H), 1.04 - 0.87 (m, 0.54H).

[0248]

[0249] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.19g, yield: 43.1%, DSR=18%). 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.38 (m, 0.9H), 6.56 - 6.45 (m, 0.18H), 6.35 - 6.24 (m, 0.18H), 5.46 - 5.25 (m, 0.54H), 5.17 - 4.99 (m, 0.36H), 4.80 - 4.35 (m, 2.54H), 4.20 - 3.06 (m, 10H), 2.91 - 2.50 (m, 0.72H), 2.35 -1.72 (m, 4.08H), 1.70 - 1.44 (m, 1.08H), 1.36 - 1.27 (m, 0.54H), 1.04 - 0.89 (m, 0.54H).

[0249]

[0250] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.2g, yield: 45.4%, DSR=15%). 1H NMR (400 MHz, heavy water) δ 7.70 - 7.43 (m, 0.75H), 6.63 - 6.50 (m, 0.15H), 6.41 - 6.32 (m, 0.15H), 5.50 - 5.31 (m, 0.45H), 5.22 - 5.03 (m, 0.3H), 4.82 - 4.40 (m, 2.45H), 4.25 - 3.09 (m, 10H), 2.94 - 2.52 (m, 0.6H), 2.38 -1.76 (m, 3.9H), 1.72 - 1.51 (m, 0.9H), 1.42 - 1.32 (m, 0.45H), 1.07 - 0.90 (m, 0.45H).

[0250] Example 16 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidin-3-yl carbonate [ka] Step 1: Preparation of 1-(tert-butoxycarbonyl)pyrrolidin-3-yl 1H-imidazole-1-carboxylate [ka]

[0251] To a solution of tert-butyl 3-hydroxypyrrolidine-1-carboxylate (281 mg, 1.5 mmol) in DCM (10 mL) was added 1,1'-carbonyldiimidazole (CDI, 486 mg, 3 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.35 g, yield: 83%), which was used without further purification. MS (m / z): C 13 H 19 N 3 O 4 [M+H] as + Calculated value: 282.31; measured value: 282.1.

[0251] Step 2: Preparation of tert-butyl 3-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)pyrrolidine-1-carboxylate [ka]

[0252] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 564 mg, 1.3 mmol) in DMF (10 mL) was 2 1-(tert-butoxycarbonyl)pyrrolidin-3-yl 1H-imidazole-1-carboxylate (365 mg, 1.3 mmol) and K 2 CO 3 (179 mg, 1.3 mmol) was added. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (30 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.7 g, yield: 50.2%); MS (m / z): C 34 H 46 FNO 10 [M+H] as +Calculated, 648.74; Found, 592.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.24 - 7.18 (m, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 5.21 (t, J = 4.1 Hz, 1H), 5.13 (d, J = 17.8 Hz, 1H), 5.00 (dd, J = 5.2, 2.2 Hz, 1H), 4.90 - 4.69 (m, 1H), 4.48 - 4.35 (m, 1H), 3.53 (d, J = 29.5 Hz, 4H), 2.70 - 2.30 (m, 5H), 2.26 - 2.06 (m, 3H), 1.87 (dt, J = 11.8, 5.4 Hz, 1H), 1.76 - 1.52 (m, 7H), 1.47 (s, 9H), 1.43 (s, 3H), 1.21 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H).

[0252] Step 3: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidin-3-yl carbonate hydrochloride [ka]

[0253] To a stirred solution of tert-butyl 3-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)pyrrolidine-1-carboxylate (421 mg, 0.65 mmol) in EtOAc (20 mL) was added slowly 4 M HCl (commercially available) in ethyl acetate (4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.3 g, yield: 79%), which was used without further purification. MS (m / z): C 29 H 38 FNO 8 [M+H] as + Calculated value: 548.62; measured value: 548.2.

[0253] Step 4: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidin-3-yl carbonate [ka]

[0254] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpyrrolidin-3-yl carbonate hydrochloride (117 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.19 g, Yield: 61%, DSR=9%); 1H NMR (400 MHz, heavy water) δ 7.66 - 7.57 (m, 0.09H), 6.57 - 6.49 (m, 0.09H), 6.38 - 6.29 (m, 0.09H), 5.54 - 5.30 (m, 0.27H), 5.18 - 5.03 (m, 0.09H), 4.82 - 4.38 (m, 2.09H), 4.23 - 3.20 (m, 10.36H), 2.96 - 2.50 (m, 0.36H), 2.45 -1.72 (m, 3.36H), 1.70 - 1.46 (m, 0.9H), 1.40 - 1.27 (m, 0.27H), 1.06 - 0.89 (m, 0.27H).

[0254]

[0255] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.15g, yield: 48.2%, DSR=10%). 1 H NMR (400 MHz, heavy water) δ 7.65 - 7.56 (m, 0.1H), 6.57 - 6.48 (m, 0.1H), 6.38 - 6.28 (m, 0.1H), 5.52 - 5.30 (m, 0.3H), 5.22 - 5.04 (m, 0.1H), 4.81 - 4.37 (m, 2.1H), 4.27 - 3.10 (m, 10.4H), 2.93 - 2.51 (m, 0.4H), 2.43 -1.72 (m, 3.4H), 1.69 - 1.46 (m, 1H), 1.39 - 1.27 (m, 0.3H), 1.06 - 0.88 (m, 0.3H).

[0255]

[0256] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.16g, yield: 51.4%, DSR=6%). 1H NMR (400 MHz, heavy water) δ 7.69 - 7.61 (m, 0.06H), 6.61 - 6.51 (m, 0.06H), 6.40 - 6.31 (m, 0.06H), 5.52 - 5.33 (m, 0.18H), 5.24 - 5.06 (m, 0.06H), 4.82 - 4.40 (m, 2.06H), 4.25 - 3.14 (m, 10.24H), 2.94 - 2.59 (m, 0.24H), 2.52 -1.73 (m, 3.24H), 1.71 - 1.50 (m, 0.6H), 1.41 - 1.32 (m, 0.18H), 1.06 - 0.92 (m, 0.18H).

[0256] Example 17 Preparation of a conjugate of hyaluronan with 2-aminoethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 2-((tert-butoxycarbonyl)amino)ethyl 1H-imidazole-1-carboxylate [ka]

[0257] To a solution of tert-butyl (2-hydroxyethyl)carbamate (1600 mg, 10 mmol) in DCM (100 mL) was added 1,1'-carbonyldiimidazole (CDI, 3200 mg, 20 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (2.5 g, yield: 98%), which was used without further purification. MS (m / z): C 11 H 17 N 3 O 4 [M+H] as + Calculated value: 256.27; measured value: 256.2.

[0257] Step 2: Preparation of tert-butyl (2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)ethyl)carbamate [ka]

[0258] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 434 mg, 1 mmol) in DMF (10 mL) was 2 2-((tert-butoxycarbonyl)amino)ethyl 1H-imidazole-1-carboxylate (505 mg, 2 mmol) and K 2 CO 3 (138 mg, 1 mmol) was added. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (30 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.26 g, yield: 41.8%); MS (m / z): C 32 H 44 FNO 10 [M+H] as +Calculated, 622.70; Found, 566.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.21 (d, J = 10.2 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (d, J = 1.8 Hz, 1H), 5.11 - 4.99 (m, 2H), 4.92 (s, 1H), 4.81 (d, J = 17.8 Hz, 1H), 4.43 (dq, J = 8.9, 2.7 Hz, 1H), 4.24 (q, J = 5.8 Hz, 2H), 3.54 - 3.37 (m, 2H), 2.71 - 2.28 (m, 5H), 2.10 (td, J = 12.5, 5.8 Hz, 1H), 1.87 (dt, J = 12.0, 5.3 Hz, 1H), 1.74 - 1.60 (m, 4H), 1.55 (s, 3H), 1.45 (s, 9H), 1.43 (s, 3H), 1.21 (s, 3H), 0.95 (s, 3H).

[0258] Step 3: Preparation of 2-aminoethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride [ka]

[0259] To a stirred solution of tert-butyl (2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)ethyl)carbamate (249 mg, 0.4 mmol) in EtOAc (15 mL) was added slowly 4 M HCl in ethyl acetate (3 mL) in an ice bath. The reaction mixture was stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.22 g, yield: 98.6%), which was used without further purification. MS (m / z): C 27 H 36 FNO 8 [M+H] as + Calculated value: 522.58; measured value: 522.2.

[0259] Step 3: Preparation of the conjugate of HA with 2-aminoethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0260] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-aminoethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride (112 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (4.3 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.18 g, Yield: 59%, DSR=14%); 1H NMR (400 MHz, heavy water) δ 7.68 - 7.59 (m, 0.14H), 6.58 - 6.50 (m, 0.14H), 6.37 - 6.28 (m, 0.14H), 5.42 - 5.29 (m, 0.14H), 5.24 - 5.09 (m, 0.28H), 4.80 - 4.34 (m, 2.42H), 4.26 - 3.20 (m, 10.28H), 2.96 - 2.51 (m, 0.7H), 2.35 -1.73 (m, 3.28H), 1.70 - 1.49 (m, 1.26H), 1.43 - 1.31 (m, 0.42H), 1.08 - 0.94 (m, 0.42H).

[0260]

[0261] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.18g, yield: 59%, DSR=16%). 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.59 (m, 0.16H), 6.60 - 6.51 (m, 0.16H), 6.40 - 6.30 (m, 0.16H), 5.50 - 5.32 (m, 0.16H), 5.24 - 5.01 (m, 0.32H), 4.81 - 4.38 (m, 2.48H), 4.23 - 3.15 (m, 10.32H), 2.94 - 2.49 (m, 0.8H), 2.37 -1.74 (m, 3.32H), 1.71 - 1.50 (m, 1.44H), 1.43 - 1.32 (m, 0.48H), 1.09 - 0.93 (m, 0.48H).

[0261]

[0262] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.19g, yield: 62.2%, DSR=14%). 1H NMR (400 MHz, heavy water) δ 7.70 - 7.59 (m, 0.14H), 6.60 - 6.50 (m, 0.14H), 6.40 - 6.32 (m, 0.14H), 5.52 - 5.32 (m, 0.14H), 5.24 - 5.02 (m, 0.28H), 4.82 - 4.34 (m, 2.42H), 4.27 - 3.13 (m, 10.28H), 3.02 - 2.49 (m, 0.7H), 2.38 -1.75 (m, 3.28H), 1.73 - 1.48 (m, 1.26H), 1.42 - 1.32 (m, 0.42H), 1.10 - 0.95 (m, 0.42H).

[0262] Example 18 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-((tert-butoxycarbonyl)amino)pentanoate [ka]

[0263] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 435 mg, 1 mmol), 5-((tert-butoxycarbonyl)amino)pentanoic acid (282 mg, 1.3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 249 mg, 1.3 mmol), N,N-dimethylpyridin-4-amine (DMAP, 12 mg, 0.1 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 24 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate=5:1 to 2:1) to give the title compound (600 mg, yield: 94.7%). MS(m / z):C 34 H 48 FNO 9 [M+H] as + Calculated, 634.75; Found, 578.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.21 (d, J = 10.1 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 4.98 (d, J = 4.9 Hz, 1H), 4.90 (d, J = 2.4 Hz, 2H), 4.63 (t, J = 5.8 Hz, 1H), 4.42 (d, J = 8.7 Hz, 1H), 3.14 (q, J = 6.7 Hz, 2H), 2.69 - 2.58 (m, 1H), 2.55 - 2.43 (m, 3H), 2.43 - 2.32 (m, 3H), 2.28 - 2.18 (m, 1H), 2.11 (td, J = 12.5, 5.9 Hz, 1H), 1.87 (dt, J = 11.9, 5.3 Hz, 1H), 1.80 - 1.53 (m, 10H),1.49 - 1.39 (m, 12H), 1.21 (s, 3H), 0.94 (s, 3H).

[0263] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate hydrochloride [ka]

[0264] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-((tert-butoxycarbonyl)amino)pentanoate (600 mg, 0.95 mmol) in EtOAc (24 mL) was added slowly 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.51 g, yield: 94.2%), which was used without further purification. MS (m / z): C 29 H 40 FNO 7 [M+H] as + Calculated value: 534.64; measured value: 534.3.

[0264] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate [ka]

[0265] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate hydrochloride (114 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (4.3 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.17 g, Yield: 55.1%, DSR=10%); 1H NMR (400 MHz, heavy water) δ 7.65 - 7.56 (m, 0.1H), 6.56 - 6.47 (m, 0.1H), 6.36 - 6.27 (m, 0.1H), 5.37 - 5.26 (m, 0.1H), 5.19 - 5.01 (m, 0.2H), 4.80 - 4.38 (m, 2.1H), 4.29 - 3.09 (m, 10.2H), 2.89 - 2.46 (m, 0.5H), 2.37 -1.67 (m, 3.3H), 1.66 - 1.49 (m, 1.4H), 1.40 - 1.30 (m, 0.3H), 1.05 - 0.90 (m, 0.3H).

[0265]

[0266] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.19g, yield: 61.6%, DSR=16%). 1 H NMR (400 MHz, heavy water) δ 7.67 - 7.57 (m, 0.16H), 6.57 - 6.49 (m, 0.16H), 6.37 - 6.28 (m, 0.16H), 5.40 - 5.28 (m, 0.16H), 5.20 - 5.02 (m, 0.32H), 4.79 - 4.40 (m, 2.16H), 4.29 - 3.08 (m, 10.32H), 2.94 - 2.51 (m, 0.8H), 2.39 -1.68 (m, 3.48H), 1.66 - 1.50 (m, 2.24H), 1.42 - 1.30 (m, 0.48H), 1.06 - 0.93 (m, 0.48H).

[0266]

[0267] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.17g, yield: 55.1%, DSR=8%). 1H NMR (400 MHz, heavy water) δ 7.67 - 7.55 (m, 0.08H), 6.56 - 6.46 (m, 0.08H), 6.36 - 6.26 (m, 0.08H), 5.38 - 5.27 (m, 0.08H), 5.20 - 5.01 (m, 0.16H), 4.79 - 4.36 (m, 2.08H), 4.27 - 3.04 (m, 10.16H), 2.89 - 2.43 (m, 0.4H), 2.35 -1.68 (m, 3.24H), 1.67 - 1.45 (m, 1.12H), 1.40 - 1.27 (m, 0.24H), 1.05 - 0.90 (m, 0.24H).

[0267] Example 19 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycylglycylglycinate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycylglycylglycinate [ka]

[0268] 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 435 mg, 1 mmol), (tert-butoxycarbonyl)glycylglycylglycine (376 mg, 1.3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 249 mg, 1.3 mmol), N,N-dimethylpyridin-4-amine (DMAP, 12 mg, 0.1 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 4 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate=5:1 to 2:1) to give the title compound (690 mg, yield: 97.8%). MS(m / z):C 35 H 48 FN 3 O 11 [M+H] as + Calculated value: 706.78; measured value: 706.2. 1 H NMR (400 MHz, chloroform-d) δ 7.57 - 7.37 (m, 2H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.61 (s, 1H), 5.06 - 4.93 (m, 3H), 4.43 (dt, J = 9.3, 2.9 Hz, 1H), 4.39 - 4.19 (m, 2H), 3.96 (ddt, J = 41.3, 22.8, 10.7 Hz, 4H), 3.79 - 3.65 (m, 1H), 2.63 (td, J = 13.6, 5.8 Hz, 1H), 2.54 - 2.34 (m, 2H), 2.29 (dt, J = 14.0, 3.4 Hz, 1H), 2.15 - 2.01 (m, 2H), 1.87 (dd, J = 12.5, 5.8 Hz, 1H), 1.77 - 1.53 (m, 7H), 1.44 (d, J = 9.4 Hz, 12H), 1.20 (s, 3H), 0.89 (s, 3H).

[0268] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycylglycylglycinate hydrochloride [ka]

[0269] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycylglycylglycinate (600 mg, 0.85 mmol) in EtOAc (24 mL) was added slowly in an ice bath 4 M HCl in ethyl acetate (4.8 mL). The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.5 g, yield: 91.7%), which was used without further purification. MS (m / z): C 30 H 40 FN 3 O 9 [M+H] as + Calculated value: 606.66; measured value: 606.3.

[0269] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl glycylglycylglycinate [ka]

[0270] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylglycylglycylglycinate hydrochloride (128 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) to the solution temporarily increased the viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (4.3 mL) was added. NaCl (1000 mg, 17 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.18 g, Yield: 55.9%, DSR=13%); 1H NMR (400 MHz, heavy water) δ 7.66 - 7.56 (m, 0.13H), 6.56 - 6.45 (m, 0.13H), 6.35 - 6.27 (m, 0.13H), 5.43 - 5.25 (m, 0.13H), 5.22 - 4.98 (m, 0.39H), 4.81 - 4.41 (m, 2.13H), 4.35 - 3.27 (m, 10.65H), 2.90 - 2.51 (m, 0.52H), 2.36 -1.71 (m, 3.39H), 1.69 - 1.46 (m, 1.17H), 1.39 - 1.30 (m, 0.39H), 1.07 - 0.90 (m, 0.39H).

[0270]

[0271] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.2g, yield: 62.1%, DSR=16%). 1 H NMR (400 MHz, heavy water) δ 7.69 - 7.58 (m, 0.16H), 6.60 - 6.49 (m, 0.16H), 6.39 - 6.29 (m, 0.16H), 5.50 - 5.28 (m, 0.16H), 5.24 - 5.08 (m, 0.48H), 4.80 - 4.48 (m, 2.16H), 4.39 - 3.20 (m, 10.8H), 2.98 - 2.55 (m, 0.64H), 2.40 -1.73 (m, 3.48H), 1.71 - 1.49 (m, 1.44H), 1.42 - 1.31 (m, 0.48H), 1.09 - 0.93 (m, 0.48H).

[0271]

[0272] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.2g, yield: 62.1%, DSR=13%). 1H NMR (400 MHz, heavy water) δ 7.70 - 7.59 (m, 0.13H), 6.61 - 6.50 (m, 0.13H), 6.41 - 6.31 (m, 0.13H), 5.51 - 5.28 (m, 0.13H), 5.25 - 5.08 (m, 0.39H), 4.82 - 4.44 (m, 2.13H), 4.37 - 3.15 (m, 10.65H), 2.96 - 2.54 (m, 0.52H), 2.40 -1.75 (m, 3.39H), 1.72 - 1.48 (m, 1.17H), 1.43 - 1.33 (m, 0.39H), 1.10 - 0.92 (m, 0.39H).

[0272] Example 20 Preparation of a conjugate of hyaluronan with 1-aminopropan-2-yl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 1-((tert-butoxycarbonyl)amino)propan-2-yl 1H-imidazole-1-carboxylate [ka]

[0273] To a solution of tert-butyl (2-hydroxypropyl)carbamate (525 mg, 3 mmol) in DCM (30 mL) was added 1,1'-carbonyldiimidazole (CDI, 972 mg, 6 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.8 g, yield: 98.7%), which was used without further purification. MS (m / z): C 12 H 19 N 3 O 4 [M+H] as + Calculated value: 270.14; measured value: 270.2.

[0273] Step 2: Preparation of tert-butyl (2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)propyl)carbamate [ka]

[0274] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (triamcinolone acetonide, 1041 mg, 2.4 mmol) in DMF (30 mL) was 2 1-((tert-butoxycarbonyl)amino)propan-2-yl 1H-imidazole-1-carboxylate (800 mg, 3 mmol) and K 2 CO 3 (414 mg, 13 mmol) was added. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (90 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.7 g, yield: 45.9%); MS (m / z): C 33 H 46 FNO 10 [M+H] as +Calculated value, 636.31; Found value, 580.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.26 (s, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 5.10 - 4.97 (m, 2H), 4.94 - 4.78 (m, 3H), 4.47 - 4.37 (m, 1H), 3.49 - 3.17 (m, 2H), 2.63 (td, J = 13.7, 5.9 Hz, 1H), 2.53 - 2.30 (m, 4H), 2.17 - 2.07 (m, 2H), 1.86 (dt, J = 11.9, 5.4 Hz, 1H), 1.77 - 1.58 (m, 6H), 1.55 (d, J = 1.8 Hz, 3H), 1.49 - 1.38 (m, 9H), 1.31 (dd, J = 7.9, 6.4 Hz, 3H), 1.20 (d, J = 4.0 Hz, 3H), 0.96 (d, J = 7.1 Hz, 3H).

[0274] Step 3: Preparation of 1-aminopropan-2-yl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride [ka]

[0275] To a stirred solution of tert-butyl (2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)propyl)carbamate (699 mg, 1.1 mmol) in EtOAc (20 mL) was added slowly 4 M HCl in ethyl acetate (4 mL) in an ice bath. The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.62 g, yield: 98.6%), which was used without further purification. MS (m / z): C 28 H 38 FNO 8 [M+H] as + Calculated value: 536.26; measured value: 536.3.

[0275] Step 4: Preparation of the conjugate of HA with 1-aminopropan-2-yl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0276] Hyaluronic acid (201 mg, carboxylic acid 0.5 mmol) was dissolved in 22 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 20 mL of 1,4-dioxane with stirring. The solution was treated with 1-aminopropan-2-yl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indenoyl 1-aminopropan-2-yl ester ... Addition of [1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (114 mg, 0.2 mmol), N-hydroxysuccinimide (NHS, 46 mg, 0.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 38 mg, 0.2 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (200 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.22 g, Yield: 71.4%, DSR=17%); 1H NMR (400 MHz, heavy water) δ 7.71 - 7.61 (m, 0.17H), 6.64 - 6.50 (m, 0.17H), 6.43 - 6.31 (m, 0.17H), 5.49 - 5.33 (m, 0.17H), 5.26 - 4.98 (m, 0.68H), 4.80 - 4.44 (m, 2.17H), 4.25 - 3.09 (m, 10.34H), 2.96 - 2.50 (m, 0.68H), 2.41 -1.73 (m, 3.34H), 1.72 - 1.53 (m, 1.7H), 1.48 - 1.34 (m, 1.02H), 1.10 - 0.91 (m, 0.51H).

[0276]

[0277] Using this procedure, sodium hyaluronate (MW 500KDa) was reacted to give the corresponding product (0.19g, yield: 61.6%, DSR=14%). 1 H NMR (400 MHz, heavy water) δ 7.71 - 7.60 (m, 0.14H), 6.64 - 6.51 (m, 0.14H), 6.43 - 6.32 (m, 0.14H), 5.52 - 5.32 (m, 0.14H), 5.26 - 4.98 (m, 0.56H), 4.81 - 4.43 (m, 2.14H), 4.26 - 3.20 (m, 10.28H), 3.04 - 2.53 (m, 0.56H), 2.40 -1.74 (m, 3.28H), 1.73 - 1.52 (m, 1.4H), 1.50 - 1.33 (m, 0.84H), 1.10 - 0.93 (m, 0.42H).

[0277]

[0278] Using this procedure, sodium hyaluronate (MW 2000KDa) was reacted to give the corresponding product (0.16g, yield: 51.9%, DSR=14%). 1H NMR (400 MHz, heavy water) δ 7.72 - 7.59 (m, 0.14H), 6.66 - 6.53 (m, 0.14H), 6.42 - 6.30 (m, 0.14H), 5.51 - 5.32 (m, 0.14H), 5.25 - 4.95 (m, 0.56H), 4.80 - 4.40 (m, 2.14H), 4.32 - 3.08 (m, 10.28H), 2.96 - 2.52 (m, 0.56H), 2.28 -1.74 (m, 3.28H), 1.73 - 1.52 (m, 1.4H), 1.51 - 1.34 (m, 0.84H), 1.11 - 0.90 (m, 0.42H).

[0278] Example 21 Preparation of a conjugate of hyaluronan with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate [ka] Step 1: Preparation of 1-(tert-butyl) 4-(2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)piperidine-1,4-dicarboxylate [ka]

[0279] (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (679 mg, 1.5 mmol), 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (447 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (431 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (18 mg, 0.15 mmol) were dissolved in DCM (30 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / EtOAc=2:1 to 1:1) to give the title compound (940 mg, yield: 94%). MS (m / z): 35 H 47 F 2 NO 9 [M+H] as + Calculated value, 664.32; Found value, 608.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.14 (dd, J = 10.1, 1.5 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 6.38 (dd, J = 10.2, 1.9 Hz, 1H), 5.53 - 5.28 (m, 1H), 5.04 - 4.84 (m, 3H), 4.42 (dd, J = 7.4, 4.3 Hz, 1H), 4.01 (d, J = 12.4 Hz, 2H), 2.98 - 2.85 (m, 2H), 2.66 - 2.58 (m, 1H), 2.57 - 2.35 (m, 3H), 2.29 (dd, J = 7.1, 5.0 Hz, 1H), 2.19 (td, J = 12.5, 6.0 Hz, 1H), 2.02 - 1.88 (m, 2H), 1.83 - 1.59 (m, 6H), 1.54 (s, 3H), 1.50 - 1.40 (m, 12H), 1.23 (s, 3H), 0.93 (s, 3H).

[0279] Step 2: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate hydrochloride [ka]

[0280] To a stirred solution of 1-(tert-butyl) 4-(2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)piperidine-1,4-dicarboxylate (800 mg, 1.2 mmol) in EtOAc (32 mL) was added slowly 4 M HCl in ethyl acetate (6.4 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 19 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.6 g, yield: 88%), which was used without further purification. MS (m / z): C 30 H 39 F 2 NO 7 [M+H] as + Calculated value: 564.27; measured value: 564.3.

[0280] Step 3: Preparation of the conjugate of HA with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate [ka]

[0281] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethylpiperidine-4-carboxylate hydrochloride (120 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (270 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.204 g, Yield: 65%, DSR=6%);1 H NMR (400 MHz, heavy water) δ 7.55 - 7.45 (m, 0.06H), 6.55 - 6.43 (m, 0.12H), 579 - 5.57 (m, 0.06H), 5.36 - 5.27 (m, 0.06H), 5.18 - 4.98 (m, 0.12H), 4.72 - 4.27 (m, 2.06H), 4.22 - 3.15 (m, 10.12H), 3.11 - 2.66(m, 0.18H), 2.55 - 2.41 (m, 0.24H), 2.35 -1.65 (m, 3.48H), 1.64 - 1.54 (m, 0.18H), 1.53 - 1.46 (m, 0.18H), 1.35 - 1.27 (m, 0.18H), 1.00 - 0.89 (m, 0.18H).

[0281] Example 22 Preparation of conjugates of hyaluronan with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate [ka] Step 1: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(((tert-butoxycarbonyl)amino)methyl)benzoate [ka]

[0282] (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (679 mg, 1.5 mmol), 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid (490 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (18 mg, 0.15 mmol) were dissolved in DCM (30 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 5:1 to 2:1) to give the title compound (1000 mg, yield: 97%). MS (m / z): 37 H 45 F 2 NO 9 [M+H] as + Calculated value, 686.31; Found value, 630.2 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 8.09 - 8.00 (m, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.13 (dd, J = 10.1, 1.4 Hz, 1H), 6.45 (s, 1H), 6.38 (dd, J = 10.1, 1.9 Hz, 1H), 5.45 (ddd, J = 11.6, 6.6, 1.9 Hz, 1H), 5.13 (d, J = 3.3 Hz, 2H), 5.07 - 4.92 (m, 2H),4.50 - 4.29 (m, 3H), 2.61 - 2.40 (m, 2H), 2.40 - 2.16 (m, 3H), 1.90 - 1.61 (m, 4H), 1.54 (s, 3H), 1.51 - 1.42 (m, 12H), 1.29 (s, 3H), 1.00 (s, 3H).

[0282] Step 2: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate hydrochloride [ka]

[0283] To a stirred solution of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(((tert-butoxycarbonyl)amino)methyl)benzoate (800 mg, 1.17 mmol) in EtOAc (32 mL) was added slowly 4 M HCl in ethyl acetate (6.4 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 40 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.54 g, yield: 78%), which was used without further purification. MS (m / z): C 32 H 37 F 2 NO 7 [M+H] as + Calculated value: 586.25; measured value: 586.4.

[0283] Step 3: Preparation of the conjugate of HA with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate [ka]

[0284] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-(aminomethyl)benzoate hydrochloride (124 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (270 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid (Sodium hyaluronate MW 50KDa 0.218 g, Yield: 68%, DSR=35%);1 H NMR (400 MHz, heavy water) δ 8.20 - 8.04 (m, 0.7H), 7.67 - 7.41 (m, 1.05H), 6.57 - 6.38 (m, 0.7H), 5.81 - 5.42 (m, 1.05H), 5.31 - 5.02 (m, 0.7H), 4.76 - 4.31 (m, 2.7H), 4.14 - 3.02 (m, 10H), 2.59- 1.66 (m, 5.8H), 1.65 - 1.43 (m, 2.1H), 1.40 - 1.27 (m, 1.05H), 1.07 - 0.87 (m, 1.05H).

[0284] Example 23 Preparation of a conjugate of hyaluronan with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-aminohexanoate [ka] Step 1: Preparation of 2-((2S,6αS,6βR,7S,8αaS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-((tert-butoxycarbonyl)amino)hexanoate [ka]

[0285] (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (679 mg, 1.5 mmol), 6-((tert-butoxycarbonyl)amino)hexanoic acid (451 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (18 mg, 0.15 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / ethyl acetate = 2:1 to 1:1) to give the title compound (922 mg, yield: 92%). MS (m / z): C 35 H 49 F 2 NO 9 [M+H] as + Calculated value, 666.34; Found value, 566.3 (M+H-100). 1 H NMR (400 MHz, chloroform-d) δ 7.15 (dd, J = 10.1, 1.4 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 6.37 (dd, J = 10.2, 1.9 Hz, 1H), 5.33 (ddd, J = 11.6, 6.6, 1.9 Hz, 1H), 4.99 (d, J = 4.8 Hz, 1H), 4.90 (s, 2H), 4.62 (s, 1H), 4.42 (dt, J = 8.5, 2.9 Hz, 1H), 3.12 (q, J = 6.8 Hz, 2H), 2.67 (s, 1H), 2.58 - 2.34 (m, 4H), 2.33 - 2.25 (m, 1H), 2.24 - 2.13 (m, 1H), 1.84 - 1.74 (m, 2H), 1.73 - 1.59 (m, 4H), 1.57 - 1.49 (m, 5H), 1.48 - 1.36 (m, 14H), 1.22 (s, 3H), 0.94 (s, 3H).

[0285] Step 2: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-aminohexanoate hydrochloride [ka]

[0286] To a stirred solution of 2-((2S,6αS,6βR,7S,8αaS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-((tert-butoxycarbonyl)amino)hexanoate (800 mg, 1.2 mmol) in EtOAc (32 mL) was added slowly 4 M HCl in ethyl acetate (6.4 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.669 g, yield: 98%), which was used without further purification. MS (m / z): C 30 H 41 F 2 NO 7 [M+H] as + Calculated value: 566.29; measured value: 566.3.

[0286] Step 3: Preparation of the conjugate of HA with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-aminohexanoate [ka]

[0287] Hyaluronic acid (101 mg, 0.25 mmol carboxylic acid) was dissolved in 11 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 11 mL of 1,4-dioxane with stirring. Addition of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 6-aminohexanoate hydrochloride (150 mg, 0.25 mmol), N-hydroxysuccinimide (58 mg, 0.5 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (48 mg, 0.25 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (140 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.09 g, Yield: 37%, DSR=21%);1 H NMR (400 MHz, heavy water) δ 7.56 - 7.38 (m, 0.21H), 6.55 - 6.34 (m, 0.42H), 5.78- 5.53 (m, 0.21H), 5.33 - 5.18 (m, 0.42H), 5.13 - 4.90 (m, 0.42H), 4.70 - 4.26 (m, 2.21H), 4.19 - 2.99 (m, 10.42H), 2.60 - 2.35 (m, 1.05H), 2.28 - 1.92 (m, 3.21H), 1.90 - 1.32 (m, 3.15H), 1.31 - 1.24 (m, 0.63H), 1.00 - 0.82 (m, 0.63H).

[0287] Example 24 Preparation of a conjugate of hyaluronan with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate [ka] Step 1: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-((tert-butoxycarbonyl)amino)pentanoate [ka]

[0288] (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (679 mg, 1.5 mmol), 5-((tert-butoxycarbonyl)amino)pentanoic acid (424 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (18 mg, 0.15 mmol) were dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / EtOAc=2:1 to 1:1) to give the title compound (950 mg, yield: 97%). MS (m / z): 34 H 47 F 2 NO 9 [M+H] as + Calculated, 652.32; Found, 596.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.15 (dd, J = 10.1, 1.5 Hz, 1H), 6.43 (d, J = 2.1 Hz, 1H), 6.37 (dd, J = 10.2, 1.9 Hz, 1H), 5.50 - 5.31 (m, 1H), 4.99 (d, J = 4.8 Hz, 1H), 4.90 (d, J = 6.5 Hz, 2H), 4.65 (s, 1H), 4.42 (dt, J = 8.7, 2.8 Hz, 1H), 3.14 (q, J = 6.8 Hz, 2H), 2.69 (s, 1H), 2.58 - 2.40 (m, 3H), 2.40 - 2.33 (m, 1H), 2.33 - 2.25 (m, 1H), 2.24 - 2.13 (m, 1H), 1.86 - 1.66 (m, 6H), 1.61 - 1.51 (m, 5H), 1.49 - 1.39 (m, 12H), 1.22 (s, 3H), 0.94 (s, 3H).

[0288] Step 2: Preparation of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate hydrochloride [ka]

[0289] To a stirred solution of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-((tert-butoxycarbonyl)amino)pentanoate (800 mg, 1.23 mmol) in EtOAc (32 mL) was added slowly 4 M HCl in ethyl acetate (6.4 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 18 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.67 g, yield: 98%), which was used without further purification. MS (m / z): C 29 H 39 F 2 NO 7 [M+H] as + Calculated value: 552.27; measured value: 552.3.

[0289] Step 3: Preparation of the conjugate of HA with 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate [ka]

[0290] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 5-aminopentanoate hydrochloride (118 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (270 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.19 g, Yield: 61%, DSR=22%);1 H NMR (400 MHz, heavy water) δ 7.59 - 7.45 (m, 0.22H), 6.59 - 6.41 (m, 0.44H), 5.82- 5.55 (m, 0.22H), 5.36 - 5.23 (m, 0.22H), 5.16 - 4.93 (m, 0.66H), 4.73 - 4.34 (m, 2H), 4.19 - 3.10 (m, 10.44H), 2.68 - 2.36 (m, 0.66H), 2.32 - 1.92 (m, 3.66H), 1.91 - 1.56 (m, 2.42H), 1.55 - 1.42 (m, 0.66H), 1.36 - 1.25 (m, 0.66H), 1.02 - 0.85 (m, 0.66H).

[0290] Example 25 Preparation of a conjugate of hyaluronan with 5-aminopentyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 5-((tert-butoxycarbonyl)amino)pentyl 1H-imidazole-1-carboxylate [ka]

[0291] To a solution of tert-butyl (5-hydroxypentyl)carbamate (407 mg, 2 mmol) in DCM (30 mL) was added 1,1'-carbonyldiimidazole (648 mg, 4 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.58 g, yield: 97%), which was used without further purification. MS (m / z): C 14 H 23 N 3 O 4 [M+H] as + Calculated value: 298.17; measured value: 298.4.

[0291] Step 2: Preparation of tert-butyl (5-(((2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)pentyl)carbamate [ka]

[0292] A solution of (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (905 mg, 2 mmol) in DMF (20 mL) was added to N 2 5-((tert-butoxycarbonyl)amino)pentyl 1H-imidazole-1-carboxylate (594 mg, 2 mmol) and K 2 CO 3 (276 mg, 2 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (90 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.8 g, yield: 58%); MS (m / z): C 35 H 49 F 2 NO 10[M+H] as + Calculated value, 682.33; Found value, 626.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.19 - 7.11 (m, 1H), 6.44 (d, J = 2.2 Hz, 1H), 6.37 (dd, J = 10.1, 1.9 Hz, 1H), 5.50 - 5.28 (m, 1H), 5.04 - 4.94 (m, 2H), 4.84 (d, J = 17.9 Hz, 1H), 4.60 (s, 1H), 4.42 (dq, J = 8.8, 2.7 Hz, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.12 (q, J = 6.7 Hz, 2H), 2.68 (s, 1H), 2.59 - 2.41 (m, 1H), 2.40 - 2.25 (m, 2H), 2.23 - 2.12 (m, 1H), 1.84 - 1.59 (m, 6H), 1.58 - 1.49 (m, 5H), 1.48 - 1.39 (m, 14H), 1.21 (s, 3H), 0.95 (s, 3H).

[0292] Step 3: Preparation of 5-aminopentyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride [ka]

[0293] To a stirred solution of tert-butyl (5-(((2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)pentyl)carbamate (600 mg, 0.88 mmol) in EtOAc (24 mL) was slowly added 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 20 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.5 g, yield: 97%), which was used without further purification. MS (m / z): C 30 H 41 F 2 NO 8 [M+H] as + Calculated value: 582.28; measured value: 582.3.

[0293] Step 4: Preparation of the conjugate of HA with 5-aminopentyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0294] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 5-aminopentyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (124 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (1 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (270 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.19 g, Yield: 59%, DSR=22%); 1H NMR (400 MHz, heavy water) δ 7.58 - 7.43 (m, 0.22H), 6.59 - 6.40 (m, 0.44H), 5.82- 5.56 (m, 0.22H), 5.39 - 5.25 (m, 0.22H), 5.17 - 4.92 (m, 0.44H), 4.72 - 4.39 (m, 2.66H), 4.37 - 3.02 (m, 10.44H), 2.61 - 2.37 (m, 0.66H), 2.30 - 1.92 (m, 3.22H), 1.91 - 1.38 (m, 3.52H), 1.37 - 1.24 (m, 0.66H), 1.02 - 0.83 (m, 0.66H).

[0294] Example 26 Preparation of a conjugate of hyaluronan with 6-aminohexyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate [ka]

[0295] To a solution of 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate (435 mg, 2 mmol) in DCM (20 mL) was added 1,1'-carbonyldiimidazole (648 mg, 4 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.61 g, yield: 97%), which was used without further purification. MS (m / z): C 15 H 25 N 3 O 4 [M+H] as + Calculated value: 312.18; measured value: 312.2.

[0295] Step 2: Preparation of tert-butyl (6-(((2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)hexyl)carbamate [ka]

[0296] A solution of (6α,9α,11β,16β)-6,9-difluoro-11,21-dihydroxy-16,17-[(1-methylethylidene)bis(oxy)]pregna-1,4-diene-3,20-dione (311 mg, 1 mmol) in DMF (8 mL) was added to N 2 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate (452 ​​mg, 1 mmol) and K 2 CO 3(138 mg, 1 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (24 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (20 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.45 g, yield: 64%); MS (m / z): C 36 H 51 F 2 NO 10 [M+H] as + Calculated, 696.35; Found, 640.4 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.19 - 7.10 (m, 1H), 6.44 (d, J = 2.1 Hz, 1H), 6.37 (dd, J = 10.1, 1.9 Hz, 1H), 5.50 - 5.31 (m, 1H), 5.05 - 4.94 (m, 2H), 4.83 (d, J = 17.8 Hz, 1H), 4.57 (t, J = 5.9 Hz, 1H), 4.48 - 4.36 (m, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.11 (q, J = 6.6 Hz, 2H), 2.65 - 2.42 (m, 2H), 2.37 (d, J = 13.9 Hz, 1H), 2.34 - 2.25 (m, 1H), 2.24 - 2.13 (m, 1H), 1.88 - 1.58 (m, 8H), 1.54 (s, 3H), 1.52 - 1.31 (m, 16H), 1.21 (s, 3H), 0.96 (s, 3H).

[0296] Step 3: Preparation of 6-aminohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0297] To a stirred solution of tert-butyl (6-(((2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)hexyl)carbamate (430 mg, 0.62 mmol) in EtOAc (17.2 mL) was slowly added 4 M HCl in ethyl acetate (3.4 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 21 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.39 g, yield: 99%), which was used without further purification. MS (m / z): C 31 H 43 F 2 NO 8 [M+H] as + Calculated value: 596.30; measured value: 596.3.

[0297] Step 4: Preparation of the conjugate of HA with 6-aminohexyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0298] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 6-aminohexyl (2-((2S,6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-2,6β-difluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride (126 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (1 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.19 g, Yield: 59%, DSR=15%); 1H NMR (400 MHz, heavy water) δ 7.59 - 7.47 (m, 0.15H), 6.59 - 6.43 (m, 0.3H), 5.79- 5.58 (m, 0.15H), 5.39 - 5.25 (m, 0.15H), 5.16 - 4.91 (m, 0.3H), 4.71 - 4.37 (m, 2.45H), 4.36 - 2.88 (m, 10.3H), 2.59 - 2.39 (m, 0.45H), 2.32 - 1.66 (m, 3.15H), 1.65 - 1.34 (m, 2.7H), 1.33 - 1.26 (m, 0.45H), 1.02 - 0.86 (m, 0.45H).

[0298] Example 27 Preparation of a conjugate of hyaluronan with 6-aminohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate [ka]

[0299] To a solution of 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate (435 mg, 2 mmol) in DCM (20 mL) was added 1,1'-carbonyldiimidazole (648 mg, 4 mmol). The resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.61 g, yield: 97%), which was used without further purification. MS (m / z): C 15 H 25 N 3 O 4 [M+H] as + Calculated value: 312.18; measured value: 312.2.

[0299] Step 2: Preparation of tert-butyl (6-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)hexyl)carbamate [ka]

[0300] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (435 mg, 1 mmol) in DMF (8 mL) was 2 6-((tert-butoxycarbonyl)amino)hexyl 1H-imidazole-1-carboxylate (311 mg, 1 mmol) and K 2 CO 3(414 mg, 13 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water (25 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (20 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.35 g, yield: 51%); MS (m / z): C 36 H 52 FNO 10 [M+H] as + Calculated value, 678.36; Found value, 622.4 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.21 (d, J = 10.1 Hz, 1H), 6.34 (dd, J = 10.2, 1.9 Hz, 1H), 6.14 (t, J = 1.7 Hz, 1H), 5.05 - 4.95 (m, 2H), 4.83 (d, J = 17.8 Hz, 1H), 4.55 (s, 1H), 4.42 (d, J = 8.8 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 3.11 (q, J = 6.7 Hz, 2H), 2.72 - 2.59 (m, 1H), 2.55 - 2.34 (m, 3H), 2.21 (s, 1H), 2.16 - 2.06 (m, 5.9 Hz, 1H), 1.92 - 1.82 (m, 1H), 1.75 - 1.59 (m, 8H), 1.55 (s, 3H), 1.52 - 1.32 (m, 16H), 1.21 (s, 3H), 0.96 (s, 3H).

[0300] Step 3: Preparation of 6-aminohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0301] To a stirred solution of tert-butyl (6-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)hexyl)carbamate (330 mg, 0.48 mmol) in EtOAc (13.2 mL) was added 4 M HCl in ethyl acetate (2.6 mL) slowly in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 21 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.28 g, yield: 94%), which was used without further purification. MS (m / z): C 31 H 44 FNO 8 [M+H] as + Calculated value: 578.31; measured value: 578.3.

[0301] Step 4: Preparation of the conjugate of HA with 6-aminohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0302] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. Addition of 6-aminohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride (123 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1.7 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.21 g, Yield: 66%, DSR=15%); 1H NMR (400 MHz, heavy water) δ 7.62 - 7.51 (m, 0.15H), 6.53 - 6.42 (m, 0.15H), 6.33- 6.22 (m, 0.15H), 5.38 - 5.24 (m, 0.15H), 5.15 - 4.92 (m, 0.3H), 4.74 - 4.21 (m, 2.45H), 4.20 - 3.00 (m, 10.3H), 2.87 - 2.45 (m, 0.6H), 2.34 - 1.66 (m, 3H), 1.65 - 1.35 (m, 3H), 1.34 - 1.27 (m, 0.45H), 1.02 - 0.86 (m, 0.45H).

[0302] Example 28 Preparation of conjugates of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-aminoethoxy)acetate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetate [ka]

[0303] A mixture of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (652 mg, 1.5 mmol), 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetic acid (428 mg, 1.95 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (432 mg, 2.25 mmol), N,N-dimethylpyridin-4-amine (18 mg, 0.15 mmol) was dissolved in DCM (20 mL). The reaction mixture was stirred at room temperature for 3 h. The solution was diluted with DCM and washed with water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (PE / EtOAc=2:1 to 1:1) to give the title compound (900 mg, yield: 95%). MS(m / z): C 33 H 46 FNO 10 , [M+H] as 636.31 + Calculated value; Found value, 580.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.22 (d, J = 10.3 Hz, 1H), 6.35 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 5.17 - 4.87 (m, 3H), 4.53 - 4.37 (m, 1H), 4.26 (d, J = 4.3 Hz, 2H), 3.65 (t, J = 5.0 Hz, 2H), 3.36 (s, 2H), 2.72 - 2.57 (m, 1H), 2.54 - 2.26 (m, 3H), 2.17 - 2.06 (m, 1H), 1.96 - 1.81 (m, 1H), 1.79 - 1.59 (m, 6H), 1.56 (s, 3H), 1.49 - 1.40 (m, 12H), 1.22 (s, 3H), 0.95 (s, 3H).

[0303] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-aminoethoxy)acetate [ka]

[0304] To a stirred solution of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetate (600 mg, 0.94 mmol) in EtOAc (24 mL) was added slowly 4 M HCl in ethyl acetate (4.8 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 21 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.46 g, yield: 91%), which was used without further purification. MS (m / z): C 28 H 38 FNO 8 [M+H] as + Calculated value: 536.26; measured value: 536.1.

[0304] Step 3: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-aminoethoxy)acetate [ka]

[0305] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22 mL of 1,4-dioxane with stirring. The addition of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2-(2-aminoethoxy)acetate hydrochloride (114 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (1.3 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50KDa 0.199 g, Yield: 64%, DSR=15%); 1H NMR (400 MHz, heavy water) δ 7.65 - 7.55 (m, 0.15H), 6.55 - 6.44 (m, 0.15H), 6.34 - 6.21 (m, 0.15H), 5.45 - 5.32 (m, 0.15H), 5.17 - 5.01 (m, 0.3H), 4.71 - 4.37 (m, 2.45H), 4.23 - 3.03 (m, 10.6H), 2.89 - 2.45 (m, 0.6H), 2.32 - 1.69 (m, 3.9H), 1.66 - 1.46 (m, 0.9H), 1.38 - 1.29 (m, 0.45H), 1.03 - 0.89 (m, 0.45H).

[0305] Example 29 Preparation of a conjugate of hyaluronan with 4-(2-aminoethyl)phenyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of tert-butyl (4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenethyl)carbamate [ka]

[0306] A mixture of tert-butyl(4-hydroxyphenethyl)carbamate (711 mg, 3 mmol) and bis(4-nitrophenyl)carbonate (1094 mg, 3.6 mmol) in dichloromethane (30 mL) was diluted with N 2 Triethylamine (758 mg, 7.5 mmol) was added and the reaction mixture was heated to reflux for 7 h. 9α-Fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (1042 mg, 2.4 mmol) was then added and the resulting mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and saturated NaHCO 3 The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.65 g, yield: 38%); MS (m / z): C 38 H 48 FNO 10 [M+H] as + Calculated value, 698.33; Found value, 642.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.25 - 7.10 (m, 5H), 6.34 (dd, J = 10.1, 2.0 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 5.13 (d, J = 17.7 Hz, 1H), 5.02 (d, J = 4.9 Hz, 1H), 4.90 (d, J = 17.7 Hz, 1H), 4.57 (s, 1H), 4.47 - 4.35 (m, 1H), 3.36 (d, J = 7.5 Hz, 2H), 2.80 (t, J = 7.0 Hz, 2H), 2.63 (td, J = 13.6, 5.9 Hz, 1H), 2.55 - 2.30 (m, 3H), 2.18 - 2.02 (m, 2H), 1.92 - 1.82 (m, 1H), 1.75 - 1.60 (m, 4H), 1.53 (s, 3H), 1.48 - 1.39 (m, 12H), 1.23 (s, 3H), 0.95 (s, 3H).

[0306] Step 2: Preparation of 4-(2-aminoethyl)phenyl(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate [ka]

[0307] To a stirred solution of tert-butyl (4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenethyl)carbamate (488 mg, 0.7 mmol) in EtOAc (20 mL) was added slowly 4 M HCl in ethyl acetate (5 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.4 g, yield: 95%), which was used without further purification. MS (m / z): C 33 H 40 FNO 8 [M+H] as + Calculated value: 598.27; measured value: 598.3.

[0307] Step 3: Preparation of the conjugate of HA with 4-(2-aminoethyl)phenyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0308] Hyaluronic acid (201 mg, 0.5 mmol carboxylic acid) was dissolved in 22.5 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 22.5 mL of 1,4-dioxane with stirring. The addition of 4-(2-aminoethyl)phenyl(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (127 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) to the solution caused a transient increase in viscosity. The pH of the reaction mixture was adjusted with 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3The solution (7.5 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (4.3 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.19 g, Yield: 59%, DSR=20%); 1 H NMR (400 MHz, heavy water) δ 7.64 - 7.52 (m, 0.2H), 7.51 - 7.36 (m, 0.2H), 7.35 - 7.14 (m, 0.4H), 6.98 - 6.85 (m, 0.2H), 6.55 - 6.42 (m, 0.2H), 6.35 - 6.21 (m, 0.2H), 5.53 - 5.41 (m, 0.2H), 5.19 - 5.04 (m, 0.4H), 4.70 - 4.32 (m, 2.2H), 4.25 - 3.03 (m, 10.4H), 3.01 - 2.40 (m, 1.2H), 2.33 - 1.66 (m, 4.2H), 1.65 - 1.41 (m, 1.2H), 1.36 - 1.20 (m, 0.6H), 1.03 - 0.87 (m, 0.6H).

[0308] Example 30 Preparation of a conjugate of hyaluronan with (1S,2S)-2-aminocyclohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of (1S,2S)-2-((tert-butoxycarbonyl)amino)cyclohexyl 1H-imidazole-1-carboxylate [ka]

[0309] To a solution of tert-butyl ((1S,2S)-2-hydroxycyclohexyl)carbamate (215 mg, 1 mmol) in DCM (10 mL) was added 1,1'-carbonyldiimidazole (324 mg, 2 mmol). The resulting mixture was stirred at room temperature for 19 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a white solid (0.3 g, yield: 48%), which was used without further purification. MS (m / z): C 15 H 23 N 3 O 4 [M+H] as + Calculated value, 310.17; measured value, 310.2.

[0309] Step 2: Preparation of tert-butyl ((1S,2S)-2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)cyclohexyl)carbamate [ka]

[0310] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (435 mg, 1 mmol) in DMF (10 mL) was 2 Below, (1S,2S)-2-((tert-butoxycarbonyl)amino)cyclohexyl 1H-imidazole-1-carboxylate (309 mg, 1 mmol) and K 2 CO3 (138 mg, 1 mmol) was added and the resulting mixture was stirred at room temperature for 19 h. The reaction mixture was poured into water (40 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.3 g, yield: 44%); MS (m / z): C 36 H 50 FNO 10 [M+H] as + Calculated value, 676.34; Found value, 620.4 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.24 (d, J = 10.3 Hz, 1H), 6.33 (dd, J = 10.1, 1.9 Hz, 1H), 6.12 (d, J = 1.8 Hz, 1H), 5.09 - 4.95 (m, 2H), 4.76 (d, J = 17.7 Hz, 1H), 4.64 (s, 1H), 4.51 - 4.38 (m, 2H), 3.59 (s, 1H), 2.81 - 2.75 (m, 1H), 2.69 - 2.57 (m, 1H), 2.54 - 2.29 (m, 3H), 2.15 - 2.01 (m, 3H), 1.91 - 1.74 (s, 2H), 1.72 - 1.51 (m, 8H), 1.48 - 1.40 (m, 12H), 1.38 - 1.28 (m, 4H), 1.20 (s, 3H), 0.94 (s, 3H).

[0310] Step 3: Preparation of (1S,2S)-2-aminocyclohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0311] To a stirred solution of tert-butyl ((1S,2S)-2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)cyclohexyl)carbamate (300 mg, 0.44 mmol) in EtOAc (10 mL) was added slowly 4 M HCl in ethyl acetate (2 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 21 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.26 g, yield: 96%), which was used without further purification. MS (m / z): C 31 H 42 FNO 8 [M+H] as + Calculated value: 576.29; measured value: 576.2.

[0311] Step 4: Preparation of (1S,2S)-2-aminocyclohexyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0312] Hyaluronic acid (202 mg, carboxylic acid 0.5 mmol) was dissolved in 22.5 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 22.5 mL of 1,4-dioxane with stirring. The solution was treated with (1S,2S)-2-aminocyclohexyl(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4 Addition of [1,2-d][1,3]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (122 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (7.5 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (4.3 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (225 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.195 g, Yield: 61%, DSR=11%); 1H NMR (400 MHz, heavy water) δ 7.67 - 7.53 (m, 0.11H), 6.58 - 6.43 (m, 0.11H), 6.33 - 6.21 (m, 0.11H), 5.39 - 5.24 (m, 0.11H), 5.15 - 4.91 (m, 0.22H), 4.74 - 4.33 (m, 2.22H), 4.32 - 3.01 (m, 10.11H), 2.95 - 2.41 (m, 0.55H), 2.40 - 1.66 (m, 3.99H), 1.65 - 1.47 (m, 0.66H), 1.46 - 1.25 (m, 0.77H), 1.03 - 0.87 (m, 0.33H).

[0312] Example 31 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl (7-azaspiro[3.5]non-2-yl) carbonate [ka] Step 1: Preparation of tert-butyl 2-((1H-imidazole-1-carbonyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate [ka]

[0313] To a solution of tert-butyl 2-hydroxy-7-azaspiro[3.5]nonane-7-carboxylate (362 mg, 1.5 mmol) in DCM (10 mL) was added 1,1'-carbonyldiimidazole (486 mg, 3 mmol). The resulting mixture was stirred at room temperature for 18 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.5 g, yield: 99%), which was used without further purification. MS (m / z): C 17 H 25 N 3 O 4 [M+H] as + Calculated value: 336.18; measured value: 336.3.

[0313] Step 2: Preparation of tert-butyl 2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate [ka]

[0314] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (652 mg, 1.5 mmol) in DMF (15 mL) was 2 tert-Butyl 2-((1H-imidazole-1-carbonyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate (503 mg, 1.5 mmol) and K 2 CO 3(207 mg, 1.5 mmol) was added and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was poured into water (45 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.27 g, yield: 25%); MS (m / z): C 38 H 52 FNO 10 [M+H] as + Calculated value, 702.36; Measured value, 646.3 (M+H-56)). 1 H NMR (400 MHz, chloroform-d) δ 7.19 (d, J = 10.2 Hz, 1H), 6.34 (dd, J = 10.2, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.13 - 4.92 (m, 3H), 4.77 (d, J = 17.8 Hz, 1H), 4.42 (dt, J = 9.2, 2.9 Hz, 1H), 3.44 - 3.22 (m, 4H), 2.71 - 2.56 (m, 1H), 2.55 - 2.27 (m, 5H), 2.15 - 2.04 (m, 2H), 2.01 - 1.93 (m, 2H), 1.91 - 1.83 (m, 1H), 1.73 - 1.66 (m, 4H), 1.65 - 1.51 (m, 7H), 1.45 (s, 9H), 1.43 (s, 3H), 1.21 (s, 3H), 0.94 (s, 3H).

[0314] Step 3: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl (7-azaspiro[3.5]non-2-yl) carbonate [ka]

[0315] To a stirred solution of tert-butyl 2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)-7-azaspiro[3.5]nonane-7-carboxylate (270 mg, 0.38 mmol) in EtOAc (10.8 mL) was added slowly 4 M HCl in ethyl acetate (2.2 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 21 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.23 g, yield: 95%), which was used without further purification. MS (m / z): C 33 H 44 FNO 8 [M+H] as + Calculated value, 602.31; measured value, 602.4.

[0315] Step 4: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(7-azaspiro[3.5]non-2-yl)carbonate [ka]

[0316] Hyaluronic acid (161 mg, carboxylic acid 0.4 mmol) was dissolved in 17.8 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 17.8 mL of 1,4-dioxane with stirring. The solution was treated with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxane. The addition of (sol-8β-yl)-2-oxoethyl (7-azaspiro[3.5]non-2-yl) carbonate hydrochloride (102 mg, 0.16 mmol), N-hydroxysuccinimide (37 mg, 0.32 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (31 mg, 0.16 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (1 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (250 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.129 g, Yield: 50%, DSR=6%); 1H NMR (400 MHz, heavy water) δ 7.63 - 7.52 (m, 0.06H), 6.51 - 6.44 (m, 0.06H), 6.31 - 6.24 (m, 0.06H), 5.37 - 5.26 (m, 0.06H), 5.15 - 4.94 (m, 0.12H), 4.74 - 4.31 (m, 2.06H), 4.29 - 3.02 (m, 10.24H), 2.88 - 2.42 (m, 0.36H), 2.32 - 1.64 (m, 3.3H), 1.63 - 1.46 (m, 0.84H), 1.34 - 1.24 (m, 0.18H), 1.02 - 0.88 (m, 0.18H).

[0316] Example 32 Preparation of a conjugate of hyaluronan with 2-(2-aminoethoxy)ethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 1H-imidazole-1-carboxylate [ka]

[0317] To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (308 mg, 1.5 mmol) in DCM (10 mL) was added 1,1'-carbonyldiimidazole (486 mg, 3 mmol). The resulting mixture was stirred at room temperature for 18 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to give the title compound as a yellow oil (0.44 g, yield: 98%), which was used without further purification. MS (m / z): C 13 H 21 N 3 O 5 [M+H] as + Calculated value: 300.15; measured value: 300.2.

[0317] Step 2: Preparation of tert-butyl (2-(2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)ethoxy)ethyl)carbamate [ka]

[0318] A solution of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (652 mg, 1.5 mmol) in DMF (15 mL) was 2 2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 1H-imidazole-1-carboxylate (449 mg, 1.5 mmol) and K 2 CO 3(207 mg, 1.5 mmol) was added and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was poured into water (45 mL) at room temperature, filtered, and the filter cake was dissolved in DCM (40 mL). The solution was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.36 g, yield: 36%); MS (m / z): C 34 H 48 FNO 11 [M+H] as + Calculated value, 666.32; Found value, 610.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.20 (d, J = 10.1 Hz, 1H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.8 Hz, 1H), 4.92 (dd, J = 71.2, 11.0 Hz, 4H), 4.38 (d, J = 30.1 Hz, 2H), 3.72 (s, 2H), 3.56 (dt, J = 5.5, 3.8 Hz, 2H), 3.33 (s, 2H), 2.70 - 2.57 (m, 1H), 2.55 - 2.34 (m, 3H), 2.19 - 2.07 (m, 1H), 1.93 - 1.78 (m, 1H), 1.73 - 1.59 (m, 6H), 1.56 (s, 3H), 1.46 (s, 9H), 1.43 (s, 3H), 1.21 (s, 3H), 0.97 (s, 3H).

[0318] Step 3: Preparation of 2-(2-aminoethoxy)ethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0319] To a stirred solution of tert-butyl (2-(2-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)ethoxy)ethyl)carbamate (360 mg, 0.54 mmol) in EtOAc (14.4 mL) was slowly added 4 M HCl in ethyl acetate (2.9 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 20 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.3 g, yield: 92%), which was used without further purification. MS (m / z): C 29 H 40 FNO 9 [M+H] as + Calculated value: 566.27; measured value: 566.4.

[0319] Step 4: Preparation of the conjugate of HA with 2-(2-aminoethoxy)ethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka]

[0320] Hyaluronic acid (161 mg, carboxylic acid 0.4 mmol) was dissolved in 17.8 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 17.8 mL of 1,4-dioxane with stirring. The solution was treated with 2-(2-aminoethoxy)ethyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5] Addition of indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate hydrochloride (96 mg, 0.16 mmol), N-hydroxysuccinimide (37 mg, 0.32 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (31 mg, 0.16 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, followed by the addition of 1M HCl solution (1 mL). NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by the dropwise addition of absolute alcohol (250 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.13 g, Yield: 51%, DSR=8%); 1H NMR (400 MHz, heavy water) δ 7.63 - 7.50 (m, 0.08H), 6.52 - 6.43 (m, 0.08H), 6.33 - 6.21 (m, 0.08H), 5.40 - 5.28 (m, 0.08H), 5.17 - 4.94 (m, 0.16H), 4.73 - 4.34 (m, 2.16H), 4.19 - 2.98 (m, 10.48H), 2.85 - 2.42 (m, 0.32H), 2.33 - 1.65 (m, 3.16H), 1.64 - 1.42 (m, 0.88H), 1.35 - 1.24 (m, 0.24H), 1.02 - 0.87 (m, 0.24H).

[0320] Example 33 Preparation of conjugates of hyaluronan with methyl (S)-2-amino-3-(4-((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate [ka] Step 1: Preparation of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate [ka]

[0321] A mixture of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione (694 mg, 1.6 mmol) and bis(4-nitrophenyl)carbonate (730 mg, 2.4 mmol) in dichloromethane (20 mL) was 2 Triethylamine (0.7 mL, 5 mmol) was added and the reaction mixture was heated to reflux for 18 h. Methyl (tert-butoxycarbonyl)-L-tyrosinate (590 mg, 2 mmol) was then added and the resulting mixture was stirred at room temperature for 7 h. The solution was diluted with DCM and saturated NaHCO 3 The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the title product (0.4 g, yield: 33%); MS (m / z): C 40 H 50 FNO 12 [M+H] as + Calculated value, 756.33; Found value, 700.3 (M+H-56). 1 H NMR (400 MHz, chloroform-d) δ 7.23 - 7.09 (m, 5H), 6.34 (dd, J = 10.1, 1.9 Hz, 1H), 6.13 (t, J = 1.7 Hz, 1H), 5.11 (d, J = 17.6 Hz, 1H), 5.02 (d, J = 4.9 Hz, 2H), 4.91 (d, J = 17.6 Hz, 1H), 4.63 - 4.52 (m, 1H), 4.45 - 4.35 (m, 1H), 3.73 (s, 3H), 3.20 - 2.95 (m, 2H), 2.69 - 2.56 (m, 1H), 2.55 - 2.30 (m, 3H), 2.20 - 2.07 (m, 2H), 1.93 - 1.82 (m, 1H), 1.75 - 1.56 (m, 4H), 1.54 (s, 3H), 1.47 - 1.36 (m, 12H), 1.23 (s, 3H), 0.95 (s, 3H).

[0321] Step 2: Preparation of methyl (S)-2-amino-3-(4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate [ka]

[0322] To a stirred solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate (378 mg, 0.5 mmol) in EtOAc (15 mL) was slowly added 4 M HCl in ethyl acetate (3 mL) in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 20 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.34 g, yield: 98%), which was used without further purification. MS (m / z): C 35 H 42 FNO 10 [M+H] as + Calculated value: 656.28; measured value: 656.3.

[0322] Step 3: Preparation of the conjugate of HA with methyl (S)-2-amino-3-(4-((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate [ka]

[0323] Hyaluronic acid (201 mg, carboxylic acid 0.5 mmol) was dissolved in 22.5 mL of deionized water in a 100 mL round-bottom flask, followed by the dropwise addition of 22.5 mL of 1,4-dioxane with stirring. The solution was treated with methyl(S)-2-amino-3-(4-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[ The addition of 1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)phenyl)propanoate hydrochloride (138 mg, 0.2 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg, 0.2 mmol) caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3The solution (7.5 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (4.3 mL) was added. NaCl (293 mg, 5 mmol) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.19 g, Yield: 57%, DSR=30%); 1 H NMR (400 MHz, heavy water) δ 7.61 - 7.50 (m, 0.3H), 7.47 - 7.14 (m, 1.2H), 6.51 - 6.42 (m, 0.3H), 6.32 - 6.20 (m, 0.3H), 5.51 - 5.34 (m, 0.3H), 5.17 - 5.02 (m, 0.9H), 4.72 - 4.35 (m, 2.3H), 4.18 - 2.98 (m, 11.5H), 2.82 - 2.39 (m, 1.2H), 2.30 - 1.64 (m, 4.8H), 1.63 - 1.41 (m, 1.8H), 1.34 - 1.23 (m, 0.9H), 1.02 - 0.82 (m, 0.9H).

[0323] Example 34 Preparation of conjugates of HA with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0324] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,10,12,13,14,15,16,17-decahydro-3H-cyclopenta[a]phenanthrene-3,11(6H)-dione instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.274 g, yield: 50.4%). MS (m / z): C 30 H 41 NO 8 [M+H] as + Calculated value, 544.28; Found value, 566.2 [M+Na] + .

[0324] Step 2: Preparation of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0325] 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy The title compound was prepared in a similar manner to Example 2, step 2 by using -6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate instead, and obtained as a white solid (0.21 g, yield: 98%). MS (m / z): C 25 H 33 NO 6 [M+H] as + Calculated value: 444.23; measured value: 444.2. 1 H NMR (400 MHz, heavy water) δ 7.88 (d, J = 10.2 Hz, 1H), 6.32 (dd, J = 10.2, 2.0 Hz, 1H), 6.24 (t, J = 1.6 Hz, 1H), 5.15 (d, J = 18.1 Hz, 1H), 5.03 (d, J = 18.1 Hz, 1H), 3.12 (t, J = 7.8 Hz, 2H), 2.94 (d, J = 12.1 Hz, 1H), 2.72 - 2.64 (m, 3H), 2.58 - 2.48 (m, 1H), 2.46 - 2.15 (m, 5H), 2.14 - 1.90 (m, 3H), 1.83 (ddd, J = 15.1, 9.5, 5.6 Hz, 1H), 1.58 (qd, J = 12.1, 5.7 Hz, 1H), 1.48 (s, 3H), 1.42 - 1.20 (m, 2H), 0.69 (s, 3H).

[0325] Step 3: Preparation of the conjugate of HA with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0326] Hyaluronic acid (227 mg, 0.564 mmol carboxylic acid) was dissolved in 25 mL of deionized water in a 100 mL round bottom flask followed by the dropwise addition of 25 mL of 1,4-dioxane with stirring. The solution was treated with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride (100 mg, 0.226 mmol), N-hydroxysuccinimide (452 ​​mg, 0.451 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (45 mg, 0.226 mmol), which caused a transient increase in viscosity. The pH of the reaction mixture was adjusted to 5% NaHCO 3 The pH was adjusted to 6-6.5 with 5% NaHCO. The reaction mixture was then stirred at room temperature for 24 hours. 3 The solution (2 mL) was added to the reaction mixture and stirred for 1 hour, after which 1M HCl solution (1 mL) was added. NaCl (340 mg) was then added to the reaction mixture, which was stirred for 1 hour, followed by dropwise addition of absolute alcohol (300 mL) with stirring at -10°C. The mixture was filtered. The filter cake was collected, washed with absolute alcohol, and dried in vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 500KDa 0.255 g, Yield: 83.9%, DSR=17%). 1 H NMR (400 MHz, D 2O) δ 7.89 (d, J = 9.8 Hz, 0.17H), 6.32 (d, J = 10.1 Hz, 0.17H), 6.23 (s, 0.17H), 5.20 - 4.91 (m, 0.34H), 4.68 - 4.33 (m, 2H), 4.27 - 3.05 (m, 10H), 2.95 - 2.83 (m, 0.34H), 2.72 - 2.14 (m, 1.7H), 2.13 - 1.67 (m, 3.68H), 1.66 - 1.26 (m, 1.02H), 0.69 (s, 0.51H).

[0326]

[0327] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.251g, yield: 82.6%, DSR=20%). 1 H NMR (400 MHz, D 2 O) δ 7.89 (d, J = 9.7 Hz, 0.2H), 6.31 (d, J = 10.0 Hz, 0.2H), 6.20 (s, 0.2H), 5.07 (dd, J = 46.2, 18.0 Hz, 0.4H), 4.68 - 4.33 (m, 2H), 4.22 - 3.10 (m, 10H), 2.93 (d, J = 11.5 Hz, 0.4H), 2.73 - 2.13 (m, 2H), 2.12 - 1.65 (m, 3.8H), 1.64 - 1.24 (m, 1.2H), 0.68 (s, 0.6H).

[0327] Example 35 Preparation of the conjugate of CS with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0328] The title compound was prepared similarly to Example 34, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.238 g, yield: 70%, DSR=32%). 1 H NMR (400 MHz, D 2 O) δ 7.89 (d, J = 9.6 Hz, 0.32H), 6.31 (d, J = 10.1 Hz, 0.32H), 6.23 (s, 0.32H), 5.10 (dd, J = 34.0, 21.8 Hz, 0.64H), 4.69 - 4.38 (m, 2H), 4.31 - 3.13 (m, 10H), 3.04 - 2.82 (m, 0.64H), 2.77 - 2.14 (m, 3.2H), 2.13 - 1.75 (m, 4.28H), 1.70 - 1.15 (m, 1.92H), 0.68 (d, J = 8.5 Hz, 0.96H).

[0328] Example 36 Preparation of conjugates of HA with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0329] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.283 g, yield: 78.6%). MS (m / z): C 30 H 43 NO 8 [M+H] as + Calculated value, 546.30; Found value, 568.2 [M+Na] + .

[0329] Step 2: Preparation of 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0330] 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydro The title compound was prepared in a similar manner to Example 2, step 2 by using 4-((tert-butoxycarbonyl)amino)-2-oxoethyl instead of 4-((tert-butoxycarbonyl)amino)-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl) as a white solid (0.22 g, yield: 99%). MS (m / z): C 25 H 35 NO 6 [M+H] as + Calculated value: 446.25; measured value: 446.2. 1 H NMR (400 MHz, heavy water) δ 7.60 (d, J = 10.0 Hz, 1H), 6.39 (dd, J = 10.0, 1.9 Hz, 1H), 6.16 (s, 1H), 5.19 (d, J = 18.0 Hz, 1H), 5.06 (d, J = 18.0 Hz, 1H), 4.54 (d, J = 3.5 Hz, 1H), 3.12 (q, J = 6.3, 4.9 Hz, 2H), 2.72 - 2.55 (m, 4H), 2.46 (d, J = 12.2 Hz, 1H), 2.21 (d, J = 10.5 Hz, 1H), 2.10 - 1.96 (m, 3H), 1.83 (d, J = 13.5 Hz, 2H), 1.73 - 1.52 (m, 3H), 1.46 (s, 3H), 1.38 - 1.29 (m, 1H), 1.17 - 1.12 (m, 2H), 0.90 (s, 3H).

[0330] Step 3: Preparation of the conjugate of HA with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0331] 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate was reacted with 2-((8S,9S,10R,13S,14S, The title compound was prepared similarly to Example 34, step 3 by using 17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.204g, yield: 67%, DSR=18%) 1 H NMR (400 MHz, D 2 O) δ 7.61 (d, J = 10.0 Hz, 0.18H), 6.39 (d, J = 10.6 Hz, 0.18H), 6.17 (s, 0.18H), 5.20 - 4.96 (m, 0.36H), 4.69 - 4.35 (m, 2.18H), 4.06 - 2.97 (m, 10.36H), 2.76 - 2.41 (m, 0.9H), 2.25 - 1.82 (m, 4.26H), 1.73 - 1.40 (m, 1.08H), 1.26 - 1.10 (m, 0.36H), 0.91 (s, 0.54H).

[0331]

[0332] Using this procedure, sodium hyaluronate (MW 50 KDa) was reacted to give the corresponding product (0.224 g, yield: 73.6%, DSR=23%). 1 H NMR (400 MHz, D 2 O) δ 7.61 (d, J = 10.1 Hz, 0.23H), 6.39 (d, J = 10.1 Hz, 0.23H), 6.16 (s, 0.23H), 5.21 - 4.95 (m, 0.46H), 4.68 - 4.43 (m, 2.23H), 4.15 - 3.13 (m, 10.46H), 2.72 - 2.39 (m, 1.15H), 2.31 - 1.77 (m, 4.61H), 1.72 - 1.43 (m, 1.38H), 1.26 - 1.10 (m, 0.46H), 0.91 (s, 0.69H).

[0332] Example 37 Preparation of the conjugate of CS with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0333] The title compound was prepared similarly to Example 36, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.232 g, yield: 68.5%, DSR=34%). 1 H NMR (400 MHz, D 2O) δ 7.68 - 7.57 (m, 0.34H), 6.41 - 6.34 (m, 0.34H), 6.16 (s, 0.34H), 5.24 - 4.95 (m, 0.68H), 4.68 - 4.43 (m, 2.34H), 4.27 - 3.18 (m, 10.68H), 2.70 - 2.41 (m, 1.7H), 2.24 - 1.82 (m, 5.38H), 1.71 - 1.41 (m, 2.04H), 1.22 - 1.15 (m, 0.68H), 0.91 (s, 1.02H).

[0333] Example 38 Preparation of conjugates of HA with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0334] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,2,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.327 g, yield: 59.7%). MS (m / z): C 30 H 45 NO 8 [M+H] as + Calculated value, 548.31; Found value, 570.2 [M+Na] + .

[0334] Step 2: Preparation of 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0335] 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7- The title compound was prepared in the same manner as in Example 2, step 2, by using instead of hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate, and obtained as a white solid (0.28 g, yield: 97%). MS (m / z): C 25 H 37 NO 6 [M+H] as + Calculated value: 448.26; measured value: 448.2. 1 H NMR (400 MHz, D 2 O) δ 5.81 (s, 1H), 5.20 (d, J = 18.0 Hz, 1H), 5.07 (d, J = 18.0 Hz, 1H), 4.53 (d, J = 2.8 Hz, 1H), 3.19 - 3.08 (m, 2H), 2.78 - 2.53 (m, 5H), 2.47 - 2.30 (m, 2H), 2.22 (dt, J = 13.2, 4.6 Hz, 1H), 2.19 - 1.81 (m, 8H), 1.80 - 1.70 (m, 1H), 1.62 (ddd, J = 13.9, 8.5, 5.5 Hz, 1H), 1.56 - 1.37 (m, 4H), 1.21 - 1.10 (m, 2H), 0.88 (s, 3H).

[0335] Step 3: Preparation of the conjugate of HA with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0336] 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate was reacted with 2-((8S,9S,10R,13S,1 The title compound was prepared similarly to Example 34, step 3 by using 4S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.237g, yield: 77.7%, DSR=28%). 1 H NMR (400 MHz, D 2 O) δ 5.79 (s, 0.28H), 5.26 - 4.95 (m, 0.56H), 4.71 - 4.33 (m, 2.28H), 4.12 - 3.06 (m, 10H), 2.95 - 2.83 (m, 0.56H), 2.70 - 2.28 (m, 1.96H), 2.26 - 1.56 (m, 6.08H), 1.54 - 1.35 (m, 1.12H), 1.23 - 1.09 (m, 0.56H), 0.87 (s, 0.84H).

[0336]

[0337] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.196g, yield: 64.3%, DSR=11%). 1H NMR (400 MHz, D 2 O) δ 5.79 (s, 0.11H), 5.11 (dd, J = 51.2, 17.7 Hz, 0.22H), 4.66 - 4.24 (m, 2.11H), 4.17 - 3.02 (m, 10H), 2.97 - 2.80 (m, 0.22H), 2.72 - 2.27 (m, 0.77H), 2.26 - 1.56 (m, 4.21H), 1.54 - 1.37 (m, 0.44H), 1.23 - 1.11(m, 0.22H), 0.87 (s, 0.33H).

[0337] Example 39 Preparation of the conjugate of CS with 2-((8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0338] The title compound was prepared similarly to Example 38, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.275 g, yield: 81.1%, DSR=30%). 1 H NMR (400 MHz, D 2 O) δ 5.80 (s, 0.3H), 5.13 (dd, J = 47.6, 17.8 Hz, 0.6H), 4.68 - 4.38 (m, 2.3H), 4.30 - 3.15 (m, 10H), 2.97 - 2.88 (m, 0.6H), 2.68 - 2.29 (m, 2.1H), 2.28 - 1.58 (m, 6.3H), 1.52 - 1.33 (m, 1.2H), 1.29 - 1.06 (m, 0.6H), 0.87 (s, 0.9H).

[0338] Example 40 Preparation of conjugates of HA with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0339] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-1,6,7,8,9,10,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3,11(2H)-dione instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.68 g, yield: 89.7%). MS (m / z): C 30 H 43 NO 8 [M+H] as + Calculated value, 546.30; Found value, 568.2 [M+Na] + .

[0339] Step 2: Preparation of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0340] 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydro The title compound was prepared in a similar manner to Example 2, step 2 by using tert-butoxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate instead of tert-butoxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate as a white solid (0.48 g, yield: 98%). MS (m / z): C 25 H 35 NO 6 [M+H] as + Calculated value: 446.25; measured value: 446.2. 1 H NMR (400 MHz, D 2 O) δ 5.85 (s, 1H), 5.16 (d, J = 18.1 Hz, 1H), 5.03 (d, J = 18.1 Hz, 1H), 3.19 - 3.07 (m, 2H), 2.97 (d, J = 12.2 Hz, 1H), 2.70 - 2.51 (m, 6H), 2.43 - 2.25 (m, 5H), 2.22 - 1.94 (m, 5H), 1.87 - 1.66 (m, 2H), 1.56 (ddd, J = 18.5, 12.2, 6.1 Hz, 1H), 1.51 - 1.32 (m, 4H), 0.66 (s, 3H).

[0340] Step 3: Preparation of the conjugate of HA with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0341] 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate The title compound was prepared similarly to Example 34, step 3 by using 1,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.243g, yield: 79.8%, DSR=12%). 1 H NMR (400 MHz, D 2 O) δ 5.84 (s, 0.12H), 5.19 - 4.98 (m, 0.24H), 4.64 - 4.35 (m, 2H), 4.09 - 2.98 (m, 10.24H), 2.92 - 2.85 (m, 0.12H), 2.73 - 2.27 (m, 1.32H), 2.26 - 1.49 (m, 3.96H), 1.48 - 1.25 (m, 0.48H), 0.64 (s, 0.36H).

[0341]

[0342] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.262g, yield: 86.1%, DSR=8%). 1 H NMR (400 MHz, D 2O) δ 5.85 (s, 0.08H), 5.18 - 4.95 (m, 0.16H), 4.66 - 4.29 (m, 2H), 4.23 - 3.13 (m, 10.16H), 3.00 - 2.93 (m, 0.08H), 2.74 - 2.28 (m, 0.88H), 2.27 - 1.46 (m, 3.64H), 1.45 - 1.25 (m, 0.32H), 0.65 (s, 0.24H).

[0342] Example 41 Preparation of the conjugate of CS with 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0343] The title compound was prepared similarly to Example 40, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.235 g, yield: 69.4%, DSR=16%). 1 H NMR (400 MHz, D 2 O) δ 5.77 (s, 0.16H), 5.13 - 4.89 (m, 0.32H), 4.65 - 4.33 (m, 2H), 4.21 - 3.02 (m, 10.32H), 2.96 - 2.88 (m, 0.16H), 2.65 - 2.22 (m, 1.76H), 2.19 - 1.41 (m, 4.28H), 1.40 - 1.25 (m, 0.64H), 0.58 (s, 0.48H). Example 42 Preparation of conjugates of HA with 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0344] The title compound was prepared in a similar manner to Example 2, step 1, by using (6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-6,10,13-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.612 g, yield: 81.9%). MS (m / z): C 31 H 45 NO 8 [M+H] as + Calculated value, 560.31; Found value, 582.2 [M+Na] + .

[0343] Step 2: Preparation of 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0345] 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxyethyl The title compound was prepared in a similar manner to Example 2, step 2 by using 1,2-dihydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate instead of 1,2-dihydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate as a white solid (0.52 g, yield: 97.8%). MS (m / z): C 26 H 37 NO 6 [M+H] as + Calculated value: 460.26; measured value: 460.2. 1H NMR (400 MHz, DMSO) δ 7.57 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 10.1 Hz, 1H), 6.14 - 6.01 (m, 1H), 5.24 - 5.11 (m, 1H), 5.03 (d, J = 17.9 Hz, 1H), 4.54 - 4.38 (m, 1H), 3.16 - 3.04 (m, 2H), 2.78 - 2.54 (m, 4H), 2.28 - 1.92 (m, 5H), 1.86 - 1.38 (m, 8H), 1.13 (d, J = 5.6 Hz, 3H), 1.02 - 0.71 (m, 5H).

[0344] Step 3: Preparation of the conjugate of HA with 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0346] 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate was reacted with 2-((8S,9S,10R,13S,1 The title compound was prepared in the same manner as in Example 34, step 3, by using 4S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.28g, yield: 91%, DSR=22%). 1 H NMR (400 MHz, D 2O) δ 7.67 - 7.55 (m, 0.22H), 6.46 - 6.32 (m, 0.22H), 6.14 - 6.08 (m, 0.22H), 5.21 - 5.00 (m, 0.44H), 4.65 - 4.24 (m, 2.22H), 4.10 - 3.05 (m, 10.44H), 2.85 - 2.44 (m, 0.88H), 2.34 - 1.75 (m, 4.54H), 1.71 - 1.39 (m, 1.32H), 1.21 - 1.09 (m, 0.66H), 0.98 - 0.81 (m, 1.1H).

[0345]

[0347] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.277g, yield: 90%, DSR=26%). 1 H NMR (400 MHz, D 2 O) δ 7.67 - 7.56 (m, 0.26H), 6.44 - 6.35 (m, 0.26H), 6.15 - 6.08 (m, 0.26H), 5.17 - 4.97 (m, 0.52H), 4.65 - 4.40 (m, 2.26H), 4.16 - 3.12 (m, 10.52H), 2.86 - 2.39 (m, 1.04H), 2.35 - 1.72 (m, 4.82H), 1.71 - 1.38 (m, 1.56H), 1.20 - 1.08 (m, 0.78H), 0.98 - 0.81 (s, 1.3H).

[0346] Example 43 Preparation of the conjugate of CS with 2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0348] The title compound was prepared similarly to Example 42, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.27 g, yield: 79%, DSR=32%). 1 H NMR (400 MHz, D 2 O) δ 7.66 - 7.54 (m, 0.32H), 6.45 - 6.34 (m, 0.32H), 6.15 - 6.06 (m, 0.32H), 5.22 - 4.96 (m, 0.64H), 4.67 - 4.41 (m, 2.32H), 4.32 - 3.13 (m, 10.64H), 2.83 - 2.41 (m, 1.28H), 2.33 - 1.70 (m, 5.24H), 1.68 - 1.32 (m, 1.92H), 1.19 - 1.06 (m, 0.96H), 0.98 - 0.80 (m, 1.6H).

[0347] Example 44 Preparation of conjugates of HA with 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0349] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.61 g, yield: 82.8%). MS (m / z): C 31 H 44 FNO 8 [M+H] as + Calculated value, 578.31; Found value, 600.2 [M+Na] + .

[0348] Step 2: Preparation of 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0350] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro The title compound was prepared in the same manner as in Example 2, step 2, by using 4-((tert-butoxycarbonyl)amino)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)-4-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl) instead of 4-((tert-butoxycarbonyl)amino)butanoate, and obtained as a white solid (0.52 g, yield: 98.9%). MS (m / z): C 26 H 36 FNO 6 [M+H] as + Calculated value: 478.25; measured value: 478.2. 1 H NMR (400 MHz, D 2 O) δ 7.55 (d, J = 10.1 Hz, 1H), 6.45 (dd, J = 10.2, 2.0 Hz, 1H), 6.26 (s, 1H), 5.10 (s, 2H), 4.48 - 4.30 (m, 1H), 3.12 (t, J = 7.7 Hz, 2H), 2.90 - 2.43 (m, 5H), 2.29 - 1.91 (m, 7H), 1.58 (s, 4H), 1.34 - 1.17 (m, 2H), 1.12 (d, J = 7.3 Hz, 3H), 1.02 (s, 3H).

[0349] Step 3: Preparation of the conjugate of HA with 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0351] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate was reacted with 2-((8S,9S,10R,1 The title compound was prepared similarly to Example 34, step 3 by using 3S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.251g, yield: 80.5%, DSR=22%). 1 H NMR (400 MHz, D 2 O) δ 7.56 (d, J = 9.8 Hz, 0.22H), 6.45 (d, J = 9.8 Hz, 0.22H), 6.25 (s, 0.22H), 5.08 (s, 0.44H), 4.66 - 4.29 (m, 2.22H), 4.17 - 3.03 (m, 10.44H), 2.95 - 2.35 (m, 1.1H), 2.31 - 1.73 (m, 4.54H), 1.72 - 1.44 (m, 0.88H), 1.33 - 1.27 (m, 0.44H), 1.11 (d, J = 7.0 Hz, 0.66H), 1.03 (s, 0.66H).

[0350]

[0352] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.262g, yield: 84.1%, DSR=15%). 1 H NMR (400 MHz, D 2 O) δ 7.57 (d, J = 9.9 Hz, 0.15H), 6.45 (d, J = 10.0 Hz, 0.15H), 6.25 (s, 0.15H), 5.13 (s, 0.3H), 4.65 - 4.21 (m, 2.15H), 4.19 - 3.06 (m, 10.3H), 3.01 - 2.43 (m, 0.75H), 2.37 - 1.74 (m, 4.05H), 1.72 - 1.44 (m, 0.6H), 1.27 - 1.19 (m, 0.3H), 1.12 (d, J = 7.1 Hz, 0.45H), 1.03 (s, 0.45H).

[0351] Example 45 Preparation of conjugates of CS with 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0353] The title compound was prepared similarly to Example 44, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.223 g, yield: 64.5%, DSR=25%). 1 H NMR (400 MHz, D 2O) δ 7.57 (d, J = 9.8 Hz, 0.25H), 6.45 (d, J = 9.9 Hz, 0.25H), 6.25 (s, 0.25H), 5.08 (s, 0.5H), 4.68 - 4.35 (m, 2.25H), 4.32 - 3.16 (m, 10.5H), 3.00 - 2.42 (m, 1.25H), 2.38 - 1.80 (m, 4.75H), 1.74 - 1.47 (m, 1H), 1.32 - 1.17 (m, 0.5H), 1.12 (d, J = 6.5 Hz, 0.75H), 1.02 (s, 0.75H).

[0352] Example 46 Preparation of conjugates of HA with 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka] Step 1: Preparation of 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate

[0354] The title compound was prepared in a similar manner to Example 2, step 1, by using (8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one instead of 9α-fluoro-11β,16α,17,21-tetrahydroxy-1,4-pregnadiene-3,20-dione, and obtained as a white solid (0.37 g, yield: 94.2%). MS (m / z): C 31 H 44 FNO 8 [M+H] as + Calculated value, 578.31; Found value, 600.2 [M+Na] + .

[0353] Step 2: Preparation of 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate hydrochloride

[0355] 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)butanoate was reacted with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro The title compound was prepared in the same manner as in Example 2, step 2, by using 4-((tert-butoxycarbonyl)amino)-2-oxoethyl 4-((tert-butoxycarbonyl)amino)-4-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl) instead of 4-((tert-butoxycarbonyl)amino)butanoate, and obtained as a white solid (0.29 g, yield: 95.7%). MS (m / z): C 26 H 36 FNO 6 [M+H] as + Calculated value: 478.25; measured value: 478.2. 1 H NMR (400 MHz, D 2 O) δ 7.56 (d, J = 10.1 Hz, 1H), 6.46 (dd, J = 10.1, 1.4 Hz, 1H), 6.26 (s, 1H), 5.17 - 5.02 (m, 2H), 4.43 (d, J = 9.6 Hz, 1H), 3.19 - 3.04 (m, 2H), 2.82 - 2.42 (m, 5H), 2.37 - 1.96 (m, 5H), 1.95 - 1.71 (m, 2H), 1.70 - 1.44 (m, 4H), 1.29 (d, J = 6.6 Hz, 2H), 1.01 (s, 3H), 0.91 (d, J = 7.2 Hz, 3H).

[0354] Step 3: Preparation of the conjugate of HA with 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate

[0356] 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate was reacted with 2-((8S,9S,10R,1 The title compound was prepared similarly to Example 34, step 3 by using 3S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead, and obtained as a white solid. (Sodium hyaluronate MW 500KDa 0.238g, yield: 76.3%, DSR=22%). 1 H NMR (400 MHz, D 2 O) δ 7.57 (d, J = 9.8 Hz, 0.22H), 6.45 (d, J = 10.1 Hz, 0.22H), 6.25 (s, 0.22H), 5.09 (dd, J = 32.2, 17.9 Hz, 0.44H), 4.65 - 4.21 (m, 2.22H), 4.20 - 2.82 (m, 10.44H), 2.58 (m, 1.1H), 2.37 - 1.67 (m, 4.54H), 1.64 - 1.40 (m, 0.88H), 1.35 - 1.21 (m, 0.44H), 1.01 (s, 0.66H), 0.90 (d, J = 7.0Hz, 0.66H).

[0355]

[0357] Using this procedure, sodium hyaluronate (MW 50KDa) was reacted to give the corresponding product (0.224g, yield: 71.9%, DSR=12%). 1 H NMR (400 MHz, D 2 O) δ 7.57 (d, J = 10.0 Hz, 0.12H), 6.45 (d, J = 9.8 Hz, 0.12H), 6.25 (s, 0.12H), 5.08 (q, J = 18.0 Hz, 0.24H), 4.68 - 4.25 (m, 2.12H), 4.22 - 2.86 (m, 10.24H), 2.85 - 2.39 (m, 0.6H), 2.38 - 1.68 (m, 3.84H), 1.65 - 1.41 (m, 0.48H), 1.37 - 1.24 (m, 0.24H), 1.01 (s, 0.36H), 0.90 (d, J = 7.0Hz, 0.36H).

[0356] Example 47 Preparation of conjugates of CS with 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate [ka]

[0358] The title compound was prepared similarly to Example 46, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.243 g, yield: 70.3%, DSR=29%). 1 H NMR (400 MHz, D 2O) δ 7.58 (d, J = 9.9 Hz, 0.29H), 6.45 (d, J = 9.6 Hz, 0.29H), 6.25 (s, 0.29H), 5.09 (dd, J = 33.2, 22.1 Hz, 0.58H), 4.68 - 4.34 (m, 2.29H), 4.32 - 2.84 (m,10.58H), 2.83 - 2.36 (m, 1.45H), 2.35 - 1.66 (m, 5.03H), 1.65 - 1.38 (m, 1.16H), 1.34 - 1.12 (m, 0.58H), 1.01 (s, 0.87H), 0.90 (d, J = 6.5 Hz, 0.87H).

[0357] Example 48 Preparation of conjugates of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 7-azaspiro[3.5]nonane-2-carboxylate [ka] Step 1: Preparation of 7-(tert-butyl)2-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) 7-azaspiro[3.5]nonane-2,7-dicarboxylate

[0359] The title compound was prepared similarly to Example 1, step 1, by using 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid instead of (tert-butoxycarbonyl)glycine, and obtained as a white solid (0.525 g, yield: 97%). MS (m / z): C 38 H 52 FNO 9 [M+H] as + Calculated value, 686.36; Found value, 708.3 [M+Na] + . 1 H NMR (400 MHz, DMSO) δ 7.32 (d, J = 10.1 Hz, 1H), 6.24 (dd, J = 10.1, 1.8 Hz, 1H), 6.06 - 5.88 (m, 2H), 5.53 (d, J = 4.0 Hz, 1H), 5.15 (d, J = 17.9 Hz, 1H), 4.86 (d, J = 4.7 Hz, 1H), 4.76 (d, J = 17.9 Hz, 1H), 4.20 (s, 1H), 3.08 - 2.90 (m, 4H), 2.69 - 2.57 (m, 1H), 2.49 - 2.29 (m, 4.3 Hz, 2H), 2.16 - 1.79 (m, 7H), 1.77 - 1.64 (m, 3H), 1.63 - 1.47 (m, 5H), 1.46 - 1.28 (m, 15H), 1.19 - 1.08 (m, 3H), 0.90 - 0.75 (m, 3H).

[0358] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 7-azaspiro[3.5]nonane-2-carboxylate hydrochloride

[0360] 7-(tert-Butyl)2-(2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)7-azaspiro[3.5]nonane-2,7-dicarboxylate was reacted with 2-((6αS,6βR,7S,8αS, The title compound was prepared in the same manner as in Example 1, step 1, by using instead of 8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycinate, and obtained as a white solid (0.28 g, yield: 97%). MS(m / z):C 33 H 44 FNO 7 [M+H] as + Calculated value: 586.31; measured value: 586.3.

[0359] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 7-azaspiro[3.5]nonane-2-carboxylate

[0361] 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 7-azaspiro[3.5]nonane-2-carbo The title compound was prepared in a similar manner to Example 34, step 3 by using 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate instead of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate, as a white solid. (Sodium hyaluronate MW 500KDa 0.358g, yield: 48.7%, DSR=9%). 1 H NMR (400 MHz, D 2 O) δ 7.58 - 7.51 (m, 0.09H), 6.49 - 6.41 (m, 0.09H), 6.26 (s, 0.09H), 5.36 - 4.85 (m, 0.36H), 4.70 - 4.29 (m, 2.09H), 4.28 - 3.05 (m, 10.36H), 3.04 - 2.32 (m, 0.27H), 2.31 - 1.65 (m, 4.08H), 1.64 - 1.43 (m, 0.81H), 1.28 (s, 0.27H), 0.93 (s, 0.27H).

[0360] Example 49 Preparation of the conjugate of CS with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 7-azaspiro[3.5]nonane-2-carboxylate [ka]

[0362] The title compound was prepared similarly to Example 48, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.18 g, yield: 62.9%, DSR=20%). 1 H NMR (400 MHz, D 2 O) δ 7.63 - 7.50 (m, 0.2H), 6.47 - 6.40 (m, 0.2H), 6.33 - 6.23 (m, 0.2H), 5.21 - 4.87 (m, 0.8H), 4.69 - 4.39 (m, 2.2H), 4.38 - 3.02 (m, 10.8H), 3.00 - 2.30 (m, 0.6H), 2.29 - 1.67 (m, 5.4H), 1.66 - 1.44 (m, 1.8H), 1.29 (s, 0.6H), 0.94 (s, 0.6H).

[0361] Example 50 Preparation of a conjugate of hyaluronan with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 14-amino-3,6,9,12-tetraoxatetradecanoate [ka] Step 1: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecan-19-oate

[0363] The title compound was prepared similarly to Example 1, step 1 by using 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-oic acid instead of (tert-butoxycarbonyl)glycine, and obtained as a white solid (1.1 g, yield: 95.9%). MS (m / z): C 39 H 58 FNO 13 [M+H] as + Calculated value, 768.39; Found value, 790.3 [M+Na] + .

[0362] Step 2: Preparation of 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 14-amino-3,6,9,12-tetraoxatetradecanoate hydrochloride

[0364] 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecane-19-oate was synthesized by the method described above. The title compound was prepared in the same manner as in Example 1, step 1, by using instead of αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl(tert-butoxycarbonyl)glycinate, and obtained as a white solid (0.6 g, yield: 98.6%). MS(m / z):C 34 H 50 FNO 11 [M+H] as + Calculated value: 668.34; measured value: 668.3. 1H NMR (400 MHz, DMSO) δ 7.91 (s, 4H), 7.34 (d, J = 10.1 Hz, 1H), 6.24 (dd, J = 10.1, 1.7 Hz, 1H), 6.02 (s, 1H), 5.58 (d, J = 4.5 Hz, 1H), 5.23 (d, J = 17.9 Hz, 1H), 5.00 - 4.73 (m, 2H), 4.35 - 4.20 (m, 3H), 3.73 - 3.58 (m, 14H), 3.09 - 2.92 (m, 2H), 2.64 (td, J = 13.6, 5.8Hz, 1H), 2.47 - 2.30 (m, 2H), 2.05 (d, J = 13.5 Hz, 1H), 2.01 - 1.88 (m, 1H), 1.87 - 1.78 (m, 1H), 1.72 (d, J = 13.2 Hz, 1H), 1.66 - 1.45 (m, 5H), 1.42 - 1.29 (m, 4H), 1.21 - 1.11 (m, 3H), 0.85 (d, J = 8.4 Hz, 3H).

[0363] Step 3: Preparation of the conjugate of HA with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 14-amino-3,6,9,12-tetraoxatetradecanoate

[0365] 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 14-amino-3,6,9,12-tetraoxate The title compound was prepared similarly to Example 34, step 3 by using tradecanoate instead of 2-((8S,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3,11-dioxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 4-aminobutanoate, to give a white solid. (Sodium hyaluronate MW 500KDa 0.235g, yield: 93.6%, DSR=18%). 1 H NMR (400 MHz, D 2 O) δ 7.62 - 7.52 (m, 0.18H), 6.53 - 6.43 (m, 0.18H), 6.27 (s, 0.18H), 5.42 - 5.27 (m, 0.18H), 5.19 - 4.90 (m, 0.36H), 4.72 - 4.31 (m, 2.54H), 4.30 - 3.05 (m, 12.88H), 3.04 - 2.28 (m, 0.54H), 2.27 - 1.65 (m, 3.72H), 1.64 -1.44 (m, 1.62H), 1.26 (s, 0.54H), 0.93 (s, 0.54H).

[0364] Example 51 Preparation of the conjugate of CS with 2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl 14-amino-3,6,9,12-tetraoxatetradecanoate [ka]

[0366] The title compound was prepared similarly to Example 50, step 3 by using CS (chondroitin sulfate) instead of HA (hyaluronic acid) and obtained as a white solid (0.268 g, yield: 97.8%, DSR=38%). 1 H NMR (400 MHz, D 2 O) δ 7.62 - 7.52 (m, 0.38H), 6.53 - 6.42 (m, 0.38H), 6.26 (s, 0.38H), 5.41 - 5.28 (m, 0.38H), 5.20 - 4.91 (m, 0.76H), 4.75 - 4.37 (m, 3.14H), 4.35 - 3.00 (m, 16.08H), 2.98 - 2.28 (m, 1.14H), 2.27 - 1.67 (m, 4.52H), 1.66 -1.44 (m, 3.42H), 1.26 (s, 1.14H), 0.92 (s, 1.14H).

[0365] Example 52 Preparation of a conjugate of hyaluronan with 2-aminopropyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate [ka] Step 1: Preparation of 2-((tert-butoxycarbonyl)amino)propyl 1H-imidazole-1-carboxylate

[0367] The title compound was prepared similarly to Example 20, step 1, by using tert-butyl (1-hydroxypropan-2-yl)carbamate instead of tert-butyl (2-hydroxypropyl)carbamate, and obtained as a white solid (0.716 g, yield: 88.7%). MS (m / z): C 12 H 19 N 3 O 4 [M+H] as + Calculated value: 270.34; measured value: 270.2.

[0366] Step 2: Preparation of tert-butyl (1-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',β1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)propan-2-yl)carbamate

[0368] The title compound was prepared in a similar manner to Example 20, step 2, by using 2-((tert-butoxycarbonyl)amino)propyl 1H-imidazole-1-carboxylate instead of 1-((tert-butoxycarbonyl)amino)propan-2-yl 1H-imidazole-1-carboxylate, and obtained as a white solid (0.58 g, yield: 39.7%). 33 H 46 FNO 10 [M+H] as + Calculated value, 636.31; Found value, 658.3 [M+Na] + .

[0367] Step 3: Preparation of 2-aminopropyl (2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl) carbonate hydrochloride

[0369] tert-Butyl (1-(((2-((6αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',β1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethoxy)carbonyl)oxy)propan-2-yl)carbamate to 1-aminopropan-2-yl(2-((6 The title compound was prepared in the same manner as in Example 20, step 3, by using αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate instead of αS,6βR,7S,8αS,8βS,11αR,12αS,12βS)-6β-fluoro-7-hydroxy-6α,8α,10,10-tetramethyl-4-oxo-1,2,4,6α,6β,7,8,8α,11α,12,12α,12β-dodecahydro-8βH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8β-yl)-2-oxoethyl)carbonate, and obtained as a white solid (0.5 g, yield: 98%). MS (m / z): C 28 H 38 FNO 8 [M+H] as + Calculated value: 536.26; measured value: 536.1. 1H NMR (400 MHz, DMSO) δ 8.19 (s, 2H), 7.38 (d, J = 10.1 Hz, 1H), 6.30 (dd, J = 10.1, 1.4 Hz, 1H), 6.08 (s, 1H), 5.61 (s, 1H), 5.30 (dd, J = 18.0, 3.4 Hz, 1H), 4.94 (d, J = 4.5 Hz, 1H), 4.87 (dd, J = 18.1, 2.3 Hz, 1H), 4.29 (dt, J = 17.4, 9.6 Hz, 3H), 3.59 (d, J = 4.6 Hz, 1H), 2.76 - 2.64 (m, 1H), 2.62 - 2.45 (m, 1H), 2.40 (d, J = 9.6 Hz, 1H), 2.14 - 1.95 (m, 2H), 1.92 - 1....

Claims

1. 1. A drug delivery system for locally delivering a therapeutic agent at a controlled rate, comprising: a biopolymer comprising at least a first binding group BG1 selected from the group consisting of a carboxyl group, an amino group, and combinations thereof; a therapeutic agent comprising at least a second linking group BG2 selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, an amine group, and combinations thereof; A linker that can covalently link the biopolymer to the therapeutic agent and retain the therapeutic agent at the site of administration. Including; wherein the linker has the structure of formula (I): 【Chemical Formula 1】 (In the formula, U is linked to the biopolymer via BG1 such that at least one amide bond is formed, and U is —N(R １ )-or 【Chemistry 2】 is selected from, where: 【Chemistry 3】 is a nitrogen-containing heteroaryl or nitrogen-containing heterocyclyl optionally containing one or more additional heteroatoms selected from N, O, or S; A is a direct bond, alkyl, and —(CH ２ CH ２ O) ｍ -, wherein said alkyl is selected from one or more R ２ optionally substituted with a group; B is selected from the group consisting of a direct bond, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from one or more R ３ optionally substituted with a group; C is a direct bond, an alkyl, or [C(=O)NHCH ２ ] ｎ -, wherein said alkyl is selected from one or more R ４ optionally substituted with a group; V is a direct bond, an ester, a carbonate, a carbamate, or —C(═O)NHCH ２ O-, or -C(=O)OCH ２ and V is linked to the therapeutic agent via BG2 such that at least one bond selected from the group consisting of -O-, -OC(=O)-, -NHC(=O)-, -C(=O)NHCH ２ -, and -C(=O)OCH ２ - selected from the group consisting of; R １ is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl; R ２ , R ３ , and R ４ each independently represents a halogen, hydroxyl, amino, cyano, alkyl, alkoxyl, and C(═O)OR ５ selected from the group consisting of: R ５ is alkyl; m is an integer from 0 to 5; n is an integer from 1 to 4, With the proviso that when the biopolymer is chondroitin sulfate, V is not —C(═O)—.

2. BG1 is a carboxyl group and U is -N(R １ 2. The drug delivery system of claim 1, wherein

3. BG1 is a carboxyl group and U is 【Chemistry 4】 The drug delivery system of claim 1, wherein

4. U, 【Chemistry 5】 selected from the group consisting of 【Chemistry 6】 The drug delivery system of claim 3, wherein

5. R １ The drug delivery system of claim 2 , wherein is hydrogen.

6. BG2 is a hydroxyl group and V is: (a) —C(═O)— linked to a therapeutic agent via BG2 such that an ester linkage is formed; (b) —OC(═O)— linked to a therapeutic agent via BG2 such that a carbonate linkage is formed; (c) —NHC(═O)— linked to a therapeutic agent via BG2 such that a carbamate linkage is formed; (d) -C(=O)NHCH ２ -C(=O)NHCH linked to a therapeutic agent via BG2 such that an O-linkage is formed. ２ -;or (e)-C(=O)OCH ２ -C(=O)OCH linked to a therapeutic agent via BG2 such that an O-linkage is formed. ２ - The drug delivery system of claim 1 , wherein the drug delivery system is selected from one of the following:

7. The drug delivery system of claim 1 , wherein A is a direct bond.

8. A is one or more R ２ 2. The drug delivery system of claim 1, wherein R is an alkyl optionally substituted with a group.

9. Each R ２ are independently alkyl or —C(═O)OR ５ The drug delivery system of claim 8, wherein the drug delivery system is selected from the group consisting of:

10. A is -(CH ２ CH ２ O) ｍ The drug delivery system of claim 1, wherein

11. The drug delivery system of claim 1 , wherein B is a direct bond.

12. 10. The drug delivery system of claim 1, wherein B is cycloalkyl.

13. 13. The drug delivery system of claim 12, wherein B is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, and bicyclo[2.2.2]octyl.

14. 10. The drug delivery system of claim 1, wherein B is aryl or heteroaryl.

15. 15. The drug delivery system of claim 14, wherein B is selected from the group consisting of phenyl, pyridyl, and furyl.

16. 2. The drug delivery system of claim 1, wherein A is a direct bond and B is selected from the group consisting of a direct bond, a cycloalkyl, and an aryl.

17. A is one or more R ２ 2. The drug delivery system of claim 1, wherein B is alkyl optionally substituted with a group, and B is selected from the group consisting of a direct bond, aryl, and heteroaryl.

18. A is -(CH ２ CH ２ O) ｍ - and B is a direct bond.

19. The drug delivery system of claim 1 , wherein C is a direct bond.

20. C is one or more R ４ 2. The drug delivery system of claim 1, wherein R is an alkyl optionally substituted with a group.

21. Each R ４ are independently alkyl or —C(═O)OR ５ 21. The drug delivery system of claim 20, wherein the drug delivery system is selected from:

22. C is -[C(=O)NHCH ２ ] ｎ The drug delivery system of claim 1, wherein

23. A is a direct bond, B is a direct bond or cycloalkyl, and C is a direct bond or one or more R ４ 2. The drug delivery system of claim 1, wherein the alkyl group is selected from the group consisting of alkyl optionally substituted with a group.

24. A is one or more R ２ and C is an alkyl optionally substituted with a group selected from the group consisting of a direct bond, aryl, and heteroaryl; and C is a direct bond, one or more R ４ alkyl optionally substituted with a group, or —[C(═O)NHCH ２ ] ｎ The drug delivery system of claim 1, wherein the drug delivery system is selected from the group consisting of:

25. A is -(CH ２ CH ２ O) ｍ -, B is a direct bond, and C is one or more R ４ 2. The drug delivery system of claim 1, wherein R is an alkyl optionally substituted with a group.

26. The linker has a structure of formula (Ia)-(Ie): 【Chemistry 7】 (In the formula, U and V are as defined in claim 1; M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which is selected from one or more R ３ optionally substituted with a group; 【Chemistry 8】 each independently represents alkyl or —C(═O)OCH ３ optionally substituted with one or more groups selected from p is an integer ranging from 0 to 10; m and t are independently integers ranging from 1 to 5; q, r, and s are independently integers ranging from 0 to 5; t is an integer ranging from 1 to 5.

27. 27. The drug delivery system of claim 26, wherein M is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.2]octyl, phenyl, pyridyl, and furyl.

28. The linker 【Chemistry 9】 (In the formula, 【Chemistry 10】 each independently represents alkyl or —C(═O)OCH ３ 27. The drug delivery system of claim 26, comprising a structure selected from the group consisting of:

29. 2. The drug delivery system of claim 1, wherein the biopolymer is selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, or derivatives thereof.

30. 30. The drug delivery system of claim 29, wherein the biopolymer is hyaluronic acid.

31. 30. The drug delivery system of claim 29, wherein the biopolymer is chondroitin sulfate.

32. 10. The drug delivery system of claim 1, wherein the therapeutic agent is selected from the group consisting of triamcinolone acetonide, meprednisone, prednisolone, hydrocortisone, cortisone, fluocinonide, methylprednisolone, betamethasone, and dexamethasone.

33. 33. The drug delivery system of claim 32, wherein the biopolymer is hyaluronic acid.

34. [Chemical 11-1] 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 【Chemistry 11-5】 【Chemistry 11-6】 【Chemistry 11-7】 【Chemistry 11-8】 10. The drug delivery system of claim 1, selected from the group consisting of:

35. 10. The drug delivery system of claim 1, wherein the drug delivery system is administered locally to a subject in need thereof.

36. 36. The drug delivery system of claim 35, wherein the drug delivery system is administered locally via injection to a subject in need thereof.

37. 36. The drug delivery system of claim 35, wherein the drug delivery system is administered locally via an oral dosage form to a subject in need thereof.

38. 36. The drug delivery system of claim 35, wherein the drug delivery system is administered locally via inhalation to a subject in need thereof.

39. 36. The drug delivery system of claim 35, wherein the drug delivery system is administered locally via an implant to a subject in need thereof.

40. 36. The drug delivery system of claim 35, wherein the drug delivery system is administered locally via topical application to a subject in need thereof.

41. A pharmaceutical composition comprising the drug delivery system of claim 1 and a pharmaceutically acceptable excipient.

42. 42. A drug delivery system according to any one of claims 1 to 40 or a pharmaceutical composition according to claim 41 for use in a method for treating a disorder in a subject in need thereof, said method comprising administering a therapeutically effective amount of said drug delivery system or pharmaceutical composition to a subject.

43. 43. The drug delivery system or pharmaceutical composition of claim 42, wherein the disorder is an allergic disease, an autoimmune disease, or an inflammatory disease.

44. 44. The drug delivery system or pharmaceutical composition of claim 43, wherein the disorder is selected from the group consisting of allergic rhinitis, systemic lupus erythematosus, rheumatism, nephrotic syndrome, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Addison's disease, neurodermatitis, cutaneous pruritus, tendonitis, inflammation, respiratory disease, osteoarthritis, neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), uveitis macular edema (UME), dermatitis, psoriasis, chronic obstructive pulmonary disease, and asthma.

45. 44. The drug delivery system or pharmaceutical composition of claim 43, wherein the disorder is selected from the group consisting of tendonitis, osteoarthritis, neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), uveitis macular edema (UME), dermatitis, psoriasis, chronic obstructive pulmonary disease, and asthma.

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