Drug delivery systems and their use for localized delivery of therapeutic agents

Abstract

Provided 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 to methods for using such drug delivery systems for the treatment of diseases. [Background technology]

[0002]

[0002] Most therapeutic agents are delivered systemically to the body by oral / GI absorption or systemic injection. These delivery routes are convenient and suitable for treating systemic diseases. However, many diseases are localized disorders. While systemically administered therapeutic agents can effectively treat these disorders, they may also target other tissues or binding sites, potentially leading to side effects or adverse effects. To mitigate systemic side effects, locally administered drug delivery systems are desirable. Delivering therapeutic agents to the desired site is not as easy as taking drugs orally or by injection. Therefore, long-lasting, sustained-release drug delivery systems for locally delivering drugs are essential for such products to be accepted by physicians and patients. Furthermore, the release profile of the therapeutic agent to maintain an effective concentration at the delivery site after the drug has been administered to the target can dramatically affect the efficacy of the therapeutic agent. Thus, drug delivery of therapeutic agents to specific target tissues or sites 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 a desired release profile. The most common topical drug delivery systems use biodegradable polymers to control the release rate of therapeutic agents. These drug delivery systems release drugs through both biopolymer erosion and diffusion of drug molecules. This complex release control presents a significant challenge in the manufacturing and quality control of drug products. Therefore, there is a continuing need for drug delivery systems that can deliver therapeutic agents topically to specific tissues while controlling the release of therapeutic agents and mitigating side effects.

[0004]

[0004] For a local drug delivery system to be successful, there are three important characteristics: 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 local disorder with the therapeutic agent. This disclosure provides different methods for satisfying these important characteristics of a local drug delivery system, namely, a biopolymer whose large molecular size allows the drug delivery system to be retained at the delivery site, a therapeutic agent selected from commercially available products or those proven active in late-stage clinical trials, and a linker that covalently bonds to the biopolymer and therapeutic agent, is not chemically stable, and when degraded, releases the therapeutic agent at a desired rate to a specific delivery site and a specific disease. [Overview of the project]

[0005]

[0005] In one aspect, the present disclosure relates to a drug delivery system for locally delivering a therapeutic agent at a controlled rate, A biopolymer comprising at least a first binding group BG1 selected from the group consisting of hydroxyl groups, carboxyl groups, amino groups, and combinations thereof, A therapeutic agent comprising at least a second binding 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 a biopolymer to a therapeutic agent and hold the therapeutic agent at the administration site. Includes, The linker is given by equation (I): [ka] (In the formula, U is linked to the biopolymer via BG1 so that at least one linkage selected from esters or amides is formed, and U is directly linked, -N(R 1 )-, -O-, -C(=O)-, and [ka] selected from the group consisting of, wherein [Chemical formula] is a nitrogen-containing heterocyclyl optionally containing one or more additional heteroatoms selected from N, O, or S, A is selected from a direct bond, alkyl, and -(CH2CH2O) m -, wherein said alkyl is optionally substituted with one or more R a groups, B is selected from the group consisting of a direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, -O-heteroaryl, wherein each of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R b groups, C is selected from a direct bond, -C(=O)-, -C(=O)N(R 2 ), -, -N(R 2 )C(=O)-, -[CH2NHC(=O)] n -, -[NHC(=O)CH2] n -, and -NH(CH2) p C(=O)-, D is selected from a direct bond, alkyl, and aryl, wherein said alkyl is optionally substituted with one or more R c groups, V is connected to the therapeutic agent via BG2 such that at least one linkage selected from the group consisting of amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal, and combinations thereof is formed, and V is a direct bond, -C(=O)-, -N(R 2 )C(=O)-, -N(R 2 )C(S)-, -OC(=O)-, -OC(=S)-, -OC(=O)OCH2-, -C(=O)OCH2-, -N(R 2 )C(=O)OCH2-, -OP(=O)(OPh)-, and -N(R 2Selected from the group consisting of )P(=O)(OPh), R 1 and R 2 These are independently selected from hydrogen, alkyl, alkenyl, and alkynyl, R a , R b , and R c These are halogen, hydroxyl, amino, cyano, nitro, alkyl, alkoxyl, -C(=O)OR e , and are selected independently from =NH, m is an integer between 0 and 4. n is an integer between 1 and 4. The system provides a drug delivery system that includes the structure (where p is an integer from 1 to 4).

[0006]

[0006] In some embodiments, the linker in the drug delivery system provided herein is given by formulas (Ia) to (Im): [ka] [ka] (In the formula, U and V are as defined in

[0005] , M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl, and each of these is one or more R b It is replaced by an optional choice in the base, The structure includes q, r, s, t, u, and v (where q, r, s, t, u, and v are independent integers from 0 to 4).

[0007]

[0007] In some embodiments, the biopolymer in the drug delivery system provided herein is selected from the group consisting of hyaluronic acid, chitosan, chitin, chondroitin, or derivatives thereof.

[0008]

[0008] In some embodiments, the therapeutic agent in the drug delivery system provided herein is selected from the group consisting of anti-inflammatory drugs, Janus kinase (JAK) inhibitors, vascular endothelial growth factor (VEGF) inhibitors, anticancer drugs, and any drug that may have severe systemic toxicity.

[0009]

[0009] In a further embodiment, the present disclosure provides a drug delivery system and a pharmaceutical composition comprising pharmaceutically acceptable excipients provided herein.

[0010]

[0010] In another aspect, the present disclosure provides a method for treating a disorder in a subject requiring treatment of the disorder, the method comprising the step of administering a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein to the subject. [Modes for carrying out the invention]

[0011]

[0011] Details of specific embodiments of the present disclosure will be mentioned next, examples of which are illustrated in the attached structures and formulas. The present disclosure will be described in relation to the listed embodiments, but it will be understood that they are not intended to limit the present disclosure to those embodiments. Rather, the present disclosure is intended to cover all substitutes, modifications and equivalents that may fall 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 may be used in the practice of the present disclosure. The present disclosure is by no means limited to the methods and materials described herein. However, if one or more of the incorporated documents and similar materials differ from or conflict with this application, including defined terms, usage of terms, and described techniques, the present disclosure shall prevail. All documents, patents and patent applications cited herein are incorporated herein by reference in their entirety.

[0012] For clarity, it is understood that certain features of the present disclosure that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the present disclosure that are described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. 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.

[0013]

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

[0014]

[0014] In various places in the present disclosure, linking substituents are described. Each linking substituent is specifically intended to include both the forward and backward forms of the linking substituent. For example, -NR(CR’R”)- includes both -NR(CR’R”)- and -(CR’R”)NR-. When the structure clearly requires a linking group, the Markush variable part listed for that group is understood to be the linking group. For example, when the structure requires a linking group and the definition of the Markush group for its variable part lists "alkyl", "alkyl" is understood to represent a linking alkylene group.

[0015]

[0015] When the bond to a substituent is shown to cross a bond connecting two atoms within a ring, such a substituent can be bonded to any atom within the ring. When a substituent is listed without indicating the atom to which the substituent is bonded to the remainder of a compound of a given formula, such a substituent can be bonded via any atom in such a formula. Combinations of substituents and / or variable parts are permitted, but only when the combination results in a stable compound.

[0016]

[0016] If any variable part (e.g., R i ) appears multiple times in any component of a compound or in a formula, its definition at each occurrence is independent of its definition at all other occurrences. Thus, for example, if a group is shown to be substituted with 0 to 2 R i moieties, the group may optionally be substituted with up to 2 R i moieties, and R i at each occurrence is selected independently of the definition of R i . Combinations of substituents and / or variable parts are also permitted, but only when the combination results in a stable compound.

[0017]

[0017] As used herein, the term "C i~j " indicates a range of carbon atom numbers, where i and j are integers, the range of carbon atom numbers includes the endpoints (i.e., i and j) and each integer point therebetween, and where j is greater than i. For example, C1~6 This 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 "1-12" refers to 1-12, especially 1-10, especially 1-8, especially 1-6, especially 1-5, especially 1-4, especially 1-3, or especially 1-2 carbon atoms.

[0018]

[0018] As used herein, the term “alkyl” means a saturated linear or branched hydrocarbon radical, whether used as part of another term or independently, which may be independently and optionally substituted with one or more substituents as described below. i~j The term "alkyl" refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms. In some embodiments, the alkyl group contains 1 to 9 carbon atoms. In some embodiments, the 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. 1~6 Examples of alkyl groups include 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, and 3,3-dimethyl-2-butyl.

[0019]

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

[0020]

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

[0021]

[0021] As used herein, the term “alkoxyl” refers to an alkyl group, as defined above, bonded to a parent molecule via an oxygen atom, whether used as part of another term or independently. i~j The term "alkoxy" means that 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. 1~6 Examples of "alkoxyl" include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, neopentoxy, and n-hexoxy.

[0022]

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

[0023]

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

[0024]

[0024] As used herein, the term "amino" refers to -NH2.

[0025]

[0025] As used herein, the term “aryl” refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, whether used as part of another term or independently, wherein at least one ring in the system is aromatic, and each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl, which may have one or more substituents. Also, as used herein, the scope of the term “aryl” includes groups in which an aromatic ring is condensed to one or more additional rings. In the case of polycyclic ring systems, only one of the rings needs to be aromatic (e.g., 2,3-dihydroindole), but all rings may be aromatic (e.g., quinoline). The second ring may also be condensed or crosslinked. Examples of polycyclic aryls include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthoimidyl, phenantridinyl, or tetrahydronaphthyl. The aryl group may be substituted with one or more substituents as described above at the ring position.

[0026]

[0026] As used herein, the term "ather-acetal" refers to -N-CH2-O-.

[0027]

[0027] As used herein, the term "carboxyl group" or "carboxyl" refers to -COOH.

[0028]

[0028] As used herein, the term “cycloalkyl” refers to monovalent, non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring systems in which all ring atoms are carbon and which contain at least three ring-forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, and 4 to 5 ring-forming carbon atoms. The cycloalkyl may be saturated or partially unsaturated. The cycloalkyl may be substituted. In some embodiments, the cycloalkyl may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group containing at least one double or triple bond in its ring system. In some embodiments, the cycloalkyl group may be monocyclic or polycyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopenta-1-enyl, 1-cyclopenta-2-enyl, 1-cyclopenta-3-enyl, cyclohexyl, 1-cyclohexa-1-enyl, 1-cyclohexa-2-enyl, 1-cyclohexa-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiropentadienyl, spiro[3,6]-decanyl, bicyclo[1,1,1]pentenyl, and bicyclo[2,2,1]heptenyl.

[0029]

[0029] As used herein, the term "cyano" refers to -CN.

[0030]

[0030] As used herein, the term "ester" refers to -C(=O)O-.

[0031]

[0031] As used herein, the term "carbamate" refers to -NR'(C=O)O-, where R' represents hydrogen, an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, and other suitable organic groups.

[0032]

[0032] As used herein, the term “thiocarbamate” means -NR'(C=S)O-, where R' represents hydrogen, an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, and other suitable organic groups.

[0033]

[0033] As used herein, the term "halogen" refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo), and iodine (or iod).

[0034]

[0034] As used herein, the term “heteroatom” means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen (including N-oxide).

[0035]

[0035] As used herein, the term “heteroaryl” refers to an aryl group having one or more heteroatoms in addition to a carbon atom, whether used as part of another term or independently. 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, pyridadinyl, pyrimidinyl, pyrazinyl, indolidinyl, prinyl, naphthylidinyl, benzofuranyl, and pteridinyl. Heteroaryl groups also include polycyclic groups in which a heteroaromatic ring is fused to one or more aryl rings, alicyclic rings, or heterocyclyl rings, where the radical or bond site is on the heteroaromatic ring. Examples of polycyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranil, benzo[1,3]dioxolyl, dibenzofuranil, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, sinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl.

[0036] As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated carbocyclic 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 optionally be independently substituted with one or more substituents. In some embodiments, heterocyclyl is a saturated heterocyclyl. In some embodiments, heterocyclyl is a partially unsaturated heterocyclyl having one or more double bonds in its ring system. In some embodiments, heterocyclyl may include any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of basic nitrogen. "Heterocyclyl" also includes radicals in which the heterocyclyl radical is fused to a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. The heterocyclyl radical may, where possible, be carbon-linked or nitrogen-linked. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, a group derived from pyrrole may be pyrrol-1-yl (nitrogen-linked) or pyrrol-3-yl (carbon-linked). Further, a group derived from imidazole may be imidazol-1-yl (nitrogen-linked) or imidazol-3-yl (carbon-linked).

[0037]

[0037] In some embodiments, the term “3- to 12-membered heterocyclyl” refers to a 3- to 12-membered saturated or partially unsaturated monocyclic or polycyclic heterocyclic system having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Condensed, spiro, and bridging cyclic systems are also included within this definition. Examples of 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, pyridadinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyridazonyl, pyrrolidinyl, and triazinonyl. Examples of condensed heterocyclyls include, but are not limited to, phenyl condensed rings or pyridinyl condensed rings, such as quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinolidinyl, quinazolinyl, azaindridinyl, pteridinyl, clomenyl, isoclomenyl, indolyl, isoindolyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenadinyl, phenothiazinyl, phenanthiazinyl, imidazo[1,2-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, and [1,2,3]triazolo[4,3-a]pyridinyl groups. Examples of spiroheterocyclils include, but are not limited to, spiropyranil and spirooxazinil. Examples of cross-linked heterocyclils include, but are not limited to, morphanyl, hexamethylenetetraminyl, 3-azabicyclo[3.1.0]hexane, 8-azabicyclo[3.2.1]octane, 1-azabicyclo[2.2.2]octane, and 1,4-diazabicyclo[2.2.2]octane (DABCO).

[0038]

[0038] As used herein, the term "hydroxyl" refers to -OH.

[0039]

[0039] As used herein, the term "nitro" refers to -NO2.

[0040]

[0040] As used herein, the term “urea” means -NR'(C=O)NR”-, where R' and R” independently represent hydrogen, an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, and other suitable organic groups.

[0041]

[0041] As used herein, the term “thiourea” means -NR'H(C=S)NR”-, where R' and R” independently represent hydrogen, an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, and other suitable organic groups.

[0042]

[0042] As used herein, the term "phosphoramide" means -(NR')P(=O)(OR') a (NR) b - refers to R' and R'', where R' and R'' are independently null, hydrogen, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups, and a and b are independently 0, 1, or 2.

[0043]

[0043] As used herein, the terms “binding group” or “BG” refer to a group at a specific position within a first entity (e.g., a biopolymer, therapeutic agent provided herein) that reacts with another group from a second entity (e.g., a linker provided herein) to form a linkage, thereby joining the two entities together to form a single entity. For example, a carboxyl group in one entity may react with an amino group in another entity to form an amide linkage that joins the two entities together, where the carboxyl and amino groups can be considered binding groups.

[0044]

[0044] As used herein, the terms “linking” or “linker” refer to a bond or chemical part formed by a chemical reaction between functional groups of at least two entities being linked, thereby forming a single molecule or maintaining an association of sufficiently closely located entities. Linkers may be incorporated into the resulting linked molecule or structure with or without their reacted functional groups. Such linkings may be covalent or non-covalent. Hydrolytically unstable or degradable linking means that the linking is degradable in water or in aqueous solutions containing bodily fluids such as blood. Enzymatically unstable or degradable linking means that the linking can be degraded by one or more enzymes. Such degradable linkings include, but are not limited to, ester linkings formed by a carboxylic acid of one entity and an alcohol group on a biological activator, such ester groups generally hydrolyzing under physiological conditions to release a biological activator. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages, imine linkages resulting from the reaction of amines and aldehydes, phosphate ester linkages resulting from the reaction of phosphate groups and alcohols, hydrazone linkages resulting from the reaction of hydrazides and aldehydes, acetal linkages resulting from the reaction of aldehydes and alcohols, and amide linkages resulting from the reaction of amine groups and carboxyl groups.

[0045]

[0045] As used herein, the term “partially unsaturated” refers to a radical containing at least one double or triple bond. The term “partially unsaturated” is intended to encompass a ring having multiple unsaturated sites, but not an aromatic (i.e., fully unsaturated) site.

[0046]

[0046] As used herein, the term “pharmaceutically acceptable” means that a substance or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or the subject being treated therewith.

[0047]

[0047] As used herein, the term “substituted” means that one or more hydrogens of the designated moiety are replaced with appropriate substituents, whether preceded by the term “optionally.” “Substituted” or “substituted with” will be understood to include the implicit caveat that such substitution is subject to the acceptable valence of the substituted atom and that the substitution results in a stable or chemically feasible compound that does not spontaneously undergo changes, for example, by rearrangement, cyclization, or elimination. Unless otherwise specified, an “optionally substituted” group may have appropriate substituents at each of its substituted positions, and the substituents may be identical or different at each position if multiple positions in any given structure are substituted with multiple substituents selected from the designated group. Those skilled in the art will understand that substituents may be substituted themselves, where appropriate. Unless otherwise specified, references to chemical moieties herein will be understood to include substituted variants. For example, references to an “aryl” group or moiety implicitly include both substituted and unsubstituted variants.

[0048]

[0048] As used herein, terms such as “therapeutic agent,” “drug,” “biologically active molecule,” “biological activator,” and “activator” refer to any substance that can affect any physical or biochemical properties of a living organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, therapeutic agent includes any substance intended to diagnose, cure, alleviate, treat, or prevent a disease in humans or other animals, or to enhance the physical or mental well-being of a human or animal.

[0049]

[0049] Drug delivery of therapeutic agents to specific tissues or sites within the body presents various challenges, particularly when it is necessary to deliver therapeutic agents with low water solubility to specific tissues at high doses, and when it is necessary to avoid high systemic concentrations of therapeutic agents that could lead to toxic side effects.

[0050]

[0050] Accordingly, the present disclosure provides a drug delivery system capable of delivering a therapeutic agent locally at a controlled rate in one embodiment. In some embodiments, the drug delivery system includes a biopolymer, a therapeutic agent, and a linker that covalently links the biopolymer to the therapeutic agent and can hold the therapeutic agent at the site of administration.

[0051]

[0051] Biopolymer Biopolymers are naturally occurring polymers produced by living organisms, containing monomer units that are covalently bonded to form larger structures. Biopolymers are broadly classified into three types—polynucleotides, polypeptides, and polysaccharides—depending on the monomer units used and the structure of the biopolymer formed. Specifically, polynucleotides, such as RNA and DNA, are long polymers composed of 13 or more nucleotide monomers. Polypeptides, or proteins, are short polymers of amino acids, with collagen, actin, and fibrin being prime examples. Polysaccharides are often linearly linked high-molecular-weight hydrocarbon structures, with some examples including cellulose and alginates. Other examples of biopolymers include rubber, suberin, melanin, and lignin.

[0052]

[0052] Various biopolymers are useful as polymer delivery media for delivering therapeutic agents to target cells or tissues. Biopolymers suitable for a particular application are selected based on their ability to target specific tissues, organs, or cells, and their in vivo stability, i.e., their in vivo residence time in the circulatory system or specific tissues, cells, or organs.

[0053]

[0053] In some embodiments, the biopolymer is selected from biocompatible polymers comprising at least a first binding group BG1 which reacts with reactive functional groups 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, means a substance that does not have medically unacceptable toxicity or adverse effects on biological functions, or is acceptable to the body.

[0054]

[0054] In some embodiments, the biopolymer is selected from biocompatible polymers comprising at least a first binding group BG1, where BG1 is selected from the group consisting of hydroxyl groups, carboxyl groups, amino groups, and combinations thereof. BG1 functions as a binding portion for linker conjugation suitable for linking a therapeutic agent to the biopolymer. BG1 may be located at any site within the biopolymer backbone, and therefore the linkage formed between the biopolymer and the linker may be located at any site within the biopolymer.

[0055]

[0055] In some embodiments, the BG1 for reacting with the reactive functional group from the linker may 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.

[0056]

[0056] In some embodiments, the biopolymer is a biocompatible polymer containing a carboxyl group as BG1, and this carboxyl group can react with the reactive functional group of a suitable linker to form a linkage that connects the carboxyl group-containing biopolymer to the linker.

[0057]

[0057] In certain embodiments, the reactive functional group of the linker is an amino or amine, which reacts with the carboxyl group of the biopolymer so that an amide linkage is formed.

[0058]

[0058] In certain embodiments, the reactive functional group of the linker is hydroxyl, which reacts with the carboxyl group of the biopolymer so that an ester linkage is formed.

[0059]

[0059] In certain embodiments, the reactive functional group of the linker is a halogen, which reacts with the carboxyl group of the biopolymer so that an ester linkage is formed.

[0060]

[0060] In some embodiments, the biopolymer is a biocompatible polymer containing an amino group as BG1, which can react with a reactive functional group of a suitable linker to form a linkage, thereby producing a biopolymer-linker conjugate.

[0061]

[0061] In certain embodiments, the reactive functional group of the linker is a carboxyl group, which reacts with the hydroxyl group of the biopolymer so that an ester linkage is formed.

[0062]

[0062] In some embodiments, the linkage formed from the reaction between the BG1 of the biopolymer and the reactive functional group of the linker is -C(O)N(R 1 )-, [ka] Selected from the group consisting of , and -C(O)O-, where R 1It is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, [ka] This is a nitrogen-containing heterocycline that optionally contains one or more additional heteroatoms selected from N, O, or S.

[0063]

[0063] In a particular embodiment, R 1 It is hydrogen.

[0064]

[0064] In a particular embodiment, [ka] teeth, [ka] It is selected from the group consisting of the following.

[0065]

[0065] In some embodiments, the biopolymer may be selected from the group consisting of hyaluronic acid (HA), dextran, cellulose, amylose, chitosan, chitin, chondroitin, gelatin, alginate, carrageenan, gellan, guar gum, pectin, scleroglucan, and xanthan gum.

[0066]

[0066] In some embodiments, the biopolymer may have a number average molecular weight in the range of 400 to 3,000,000 Da, for example, 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.

[0067]

[0067] In certain embodiments, the biopolymer may be selected from the group consisting of HA, chitosan, chitin, chondroitin, or derivatives thereof.

[0068]

[0068] In certain embodiments, the biopolymer is HA. In certain embodiments, the HA can be derived from any source.

[0069]

[0069] In a particular embodiment, HA may have a number average molecular weight in the range of 400 to 3,000,000 Da, for example, 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.

[0070]

[0070] Therapeutic Agent This disclosure provides an improved delivery system for the local delivery of various therapeutic agents.

[0071]

[0071] In some embodiments, the therapeutic agent includes at least a second binding group BG2 which reacts 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).

[0072]

[0072] In some embodiments, the therapeutic agent comprises at least a second binding 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. BG2 functions as a binding site for linker conjugation suitable for linking the therapeutic agent to a biopolymer.

[0073]

[0073] In some embodiments, the therapeutic agent comprises an amine group as BG2, which can react with the reactive functional group of a suitable linker and an optional co-reactant to form a linkage that connects the amine-containing therapeutic agent to the linker.

[0074]

[0074] In certain embodiments, the amine group in the therapeutic agent reacts with the reactive functional group of the linker and an optional co-reactant such that the therapeutic agent is linked to the linker via direct bonding, amide linkage, urea linkage, thiourea linkage, carbamate linkage, thiocarbamate linkage, aza-acetal linkage, phosphoramidate linkage, etc.

[0075]

[0075] In certain embodiments, the linkage formed from a reaction involving the reactive functional groups of the therapeutic agent BG2 and the linker and an optional co-reactant is -N(R')2-, -C(=O)N(R')-, -C(=O)N(R')2-, -N(R 2 )C(=O)N(R')2-, -N(R 2 )C(=S)N(R')2-, -OC(=O)N(R')2-, -OC(=S)N(R')2-, -OC(=O)OCH2N(R')2-, -N(R 2 )C(=O)OCH2N(R')2-, -OP(=O)(OPh)N(R')2-, and -N(R 2 )P(=O)(OPh)N(R')2- is selected from the group consisting of these, where R 2 R' is selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, and R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, or two R's form a heterocycline together with the nitrogen atom to which they are both bonded.

[0076]

[0076] In a particular embodiment, R 2 It is hydrogen.

[0077]

[0077] In some embodiments, the therapeutic agent contains a carboxyl group as BG2, which can react with the reactive functional group of a suitable linker and an optional co-reactant to form a linkage that connects the carboxyl group-containing therapeutic agent to the linker.

[0078]

[0078] In certain embodiments, the carboxyl groups in the therapeutic agent react with the reactive functional groups of the linker and optional co-reactants so that the therapeutic agent is linked to the linker via ester linkages.

[0079]

[0079] In some embodiments, the therapeutic agent comprises a hydroxyl group as BG2, which can react with the reactive functional group of a suitable linker and an optional co-reactant to form a linkage that connects the hydroxyl-containing therapeutic agent to the linker.

[0080]

[0080] In certain embodiments, the hydroxyl group in the therapeutic agent reacts with the reactive functional group of the linker and an optional co-reactant so that the therapeutic agent is linked to the linker via ester linkage.

[0081]

[0081] In some embodiments, the therapeutic agent to be delivered is selected from the group consisting of nonsteroidal anti-inflammatory drugs (NSAIDs), Janus kinase (JAK) inhibitors, vascular endothelial growth factor (VEGF) inhibitors, anticancer drugs, and any drugs that may have severe systemic toxicity.

[0082]

[0082] In some embodiments, the therapeutic agent to be delivered is an NSAID selected from the group consisting of piroxicam, meloxicam, and diclofenac.

[0083]

[0083] In some embodiments, the therapeutic agent to be delivered is a JAK inhibitor selected from the group consisting of tofacitinib, ruxolitinib, baricitinib, peficitinib, fedratinib, oclacitinib, and upadacitinib.

[0084]

[0084] In some embodiments, the therapeutic agent to be delivered is a VEGF inhibitor selected from the group consisting of axitinib, lapatinib, lenvatinib, pazopanib, nintedanib, sunitinib, and vandetanib.

[0085]

[0085] In some embodiments, the therapeutic agent delivered is tofacitinib.

[0086]

[0086] In some embodiments, the therapeutic agent to be delivered is upadacitinib.

[0087]

[0087] In some embodiments, the therapeutic agent delivered is ruxolitinib.

[0088]

[0088] In some embodiments, the therapeutic agent delivered is baricitinib.

[0089]

[0089] In some embodiments, the therapeutic agent delivered is oclacitinib.

[0090]

[0090] In some embodiments, the therapeutic agent to be delivered is nintedanib.

[0091]

[0091] In some embodiments, the therapeutic agent delivered is sunitinib.

[0092]

[0092] Linker By linking therapeutic agents to biopolymers via appropriate linkers, improved local delivery of therapeutic agents is achieved. By selecting the appropriate linker, the rate of therapeutic agent release from the biopolymer can be controlled, thereby improving the delivery of the therapeutic agent to target cells or tissues.

[0093]

[0093] In some embodiments, multiple linkers may be bound to the therapeutic agent via cleavable links that are cleaved under biological conditions, thereby releasing the therapeutic agent.

[0094]

[0094] A “cleavable linkage” is a relatively unstable linkage that cleaves under physiological conditions. An exemplary releaseable linkage is a hydrolyzable linkage that cleaves (i.e., is hydrolyzed) in reaction with water. The tendency of a linkage to hydrolyze in water may depend not only on the general type of linkage connecting two atoms, but also on the substituents attached to these atoms. Suitable hydrolyzably unstable or weak linkages include, but are not limited to, carboxylic acid esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, oligonucleotides, thioesters, ureas, thioureas, carbamates, thiocarbamates, phosphoramides, and carbonates. Certain functional groups have atoms that can be chemically decomposed by processes other than hydrolysis. Exemplary releaseable linkages of this category include certain carbamates and Fmoc derivatives. Certain molecules containing these types of functionalities when properly linked may be subject to chemical decomposition (release) by the action of a base. In such cases, "cleavage" can occur at high pH values ​​or through the action of biological molecules containing a basic moiety (e.g., histidine). Another exemplary cleavable linkage is an enzymatically cleavable linkage. An "enzymatically cleavable linkage" means a linkage that is cleaved by one or more enzymes.

[0095]

[0095] In some embodiments, the linker is bonded to the biopolymer via a linkage formed from the reactive functional group of the linker and BG1 in the biopolymer, and to the therapeutic agent via a linkage formed from another reactive functional group of the linker and BG2 in the therapeutic agent.

[0096]

[0096] In some embodiments, the linker is given by formula (I): [ka] (In the formula, U is linked to the biopolymer via BG1 of the biopolymer so that at least one linkage selected from esters or amides is formed, and U is directly linked, -N(R 1)-, -O-, -C(=O)-, and [ka] Selected from the group consisting of, here [ka] This is a nitrogen-containing heterocycline optionally comprising one or more additional heteroatoms selected from N, O, or S. A is a direct bond, alkyl, and -(CH2CH2O) m - is selected from, where the alkyl is one or more R a It is replaced by an optional choice in the base, B is selected from the group consisting of direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, where each of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R b It is replaced by an optional choice in the base, C represents direct bonds, -C(=O)-, and -C(=O)N(R). 2 )-,-N(R 2 )C(=O)-,-[CH2NHC(=O)] n -,-[NHC(=O)CH2] n -, and -NH(CH2) p Selected from C(=O)-, D is selected from direct bond, alkyl, and aryl, where the alkyl is one or more R c It is replaced by an optional choice in the base, V is linked to the therapeutic agent via BG2 in the therapeutic agent so as to form at least one linkage selected from the group consisting of amides, ureas, thioureas, carbamates, thiocarbamates, phosphoramidates, aza-acetals, and combinations thereof, and V is directly linked, -C(=O)-, -N(R 2 )C(=O)-, -N(R 2)C(S)-, -OC(=O)-, -OC(=S)-, -OC(=O)OCH2-, -C(=O)OCH2-, -N(R 2 )C(=O)OCH2-, -OP(=O)(OPh)-, and -N(R 2 Selected from the group consisting of )P(=O)(OPh), R 1 and R 2 It is independently selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, R a , R b , and R c These are halogen, hydroxyl, amino, cyano, nitro, alkyl, alkoxyl, -C(=O)OR e , and are selected independently from =NH, R e It is an alkyl, m is an integer between 0 and 4. n is an integer between 1 and 4. The structure includes p (where p is an integer from 1 to 4).

[0097]

[0097] In some embodiments, BG1 is a carboxyl group and U is -N(R) such that an amide linkage is formed and the biopolymer is bonded to the linker. 1 )-is.

[0098]

[0098] In some embodiments, BG1 is a carboxyl group and U is, so that an amide linkage is formed and the biopolymer is bonded to the linker. [ka] That is the case.

[0099]

[0099] In a particular embodiment, BG1 is a carboxyl group, and U is [ka] Selected from the group consisting of [ka] That is the case.

[0100]

[0100] In some embodiments, BG1 is a hydroxyl group and U is -C(=O)- so that ester links are formed and the biopolymer is bonded to the linker.

[0101]

[0101] In some embodiments, BG1 is a carboxyl group and U is an -O- or direct bond so that ester links are formed and the biopolymer is bonded to the linker.

[0102]

[0102] In some embodiments, BG1 is an amino group and U is -C(=O)- such that an amide linkage is formed and the biopolymer is bonded to a linker.

[0103]

[0103] In some embodiments, BG2 is an amine group, and V is as follows: (a) direct binding; (b) The therapeutic agent is connected via BG2 to the -N(R) so that a urea linkage is formed and the therapeutic agent is bound to the linker. 2 )C(=O)-, (c) The therapeutic agent is connected via BG2 such that a thiourea linkage is formed and the therapeutic agent is bound to the linker -N(R 2 )C(S)-, (d) -OC(=O)- connected to the therapeutic agent via BG2, such that a carbamate linkage is formed and the therapeutic agent is bound to the linker. (e) -OC(=S)- connected to the therapeutic agent via BG2, such that a thiocarbamate linkage is formed and the therapeutic agent is bound to the linker. (f) -OC(=O)OCH2- connected to the therapeutic agent via BG2, such that an aza-acetal linkage is formed and the therapeutic agent is bound to the linker, (g) -C(=O)OCH2- connected to the therapeutic agent via BG2, such that an aza-acetal linkage is formed. (h) The therapeutic agent is connected via BG2 such that an aza-acetal linkage is formed and the therapeutic agent is bound to the linker -N(R 2 )C(=O)OCH2-, (i) -OP(=O)(OPh)- connected to the therapeutic agent via BG2, such that a phosphoramide linkage is formed and the therapeutic agent is bound to the linker, or (j) A phosphoramidate linkage is formed so that the therapeutic agent is bound to the linker -N(R 2 )P(=O)(OPh)-, (k) -C(=O)- connected to the therapeutic agent via BG2 so that an amide linkage is formed. One of the following will be selected.

[0104]

[0104] In certain embodiments, BG2 is a carboxyl group and V is an -O- or direct bond so that an ester linkage is formed and the therapeutic agent is bonded to the linker.

[0105]

[0105] In certain embodiments, BG2 is a hydroxyl group and V is -C(=O)- such that an ester linkage is formed and the therapeutic agent is bonded to the linker.

[0106]

[0106] In some embodiments, A is a direct bond.

[0107]

[0107] In some embodiments, A is one or more R a It is an alkyl group that is optionally substituted with a base. In certain embodiments, A is one or more R a The base C is replaced by an arbitrary choice. 1~10 It is alkyl. In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl.

[0108]

[0108] In some embodiments, R a is -C(=O)OR e And here Re is alkyl. In certain embodiments, R a is -C(=O)OR e where R e is C 1~8 alkyl, C 1~7 alkyl, C 1~6 alkyl, C 1~5 alkyl, C 1~4 alkyl, or C 1~3 alkyl. In certain embodiments, R a is -C(=O)OCH3.

[0109]

[0109] In some embodiments, A is -(CH2CH2O) m -.

[0110]

[0110] 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.

[0111]

[0111] In some embodiments, B is a direct bond.

[0112]

[0112] In some embodiments, B is alkyl. In certain embodiments, B is C 1~6 alkyl, C 1~5 alkyl, C 1~4 alkyl, C 1~3 alkyl, or C 1~2 alkyl. In certain embodiments, B is ethyl.

[0113]

[0113] In some embodiments, B is cycloalkyl, aryl, or heteroaryl.

[0114]

[0114] In some embodiments, B is cycloalkyl.

[0115]

[0115] In certain embodiments, B is a saturated cycloalkyl. In certain embodiments, B is a partially unsaturated cycloalkyl.

[0116]

[0116] In certain embodiments, B is a 3-8 member cycloalkyl, a 3-7 member cycloalkyl, a 3-6 member cycloalkyl, a 3-5 member cycloalkyl, or a 3-4 member cycloalkyl.

[0117]

[0117] In certain embodiments, B is a saturated 3- to 6-membered cycloalkyl. In certain embodiments, B is a cyclohexyl.

[0118]

[0118] In some embodiments, B is an aryl. In certain embodiments, B is a 5-12 member aryl, a 5-10 member aryl, a 5-8 member aryl, or a 5-6 member aryl.

[0119]

[0119] In certain embodiments, B is phenyl.

[0120]

[0120] In some embodiments, B is a heteroaryl. In certain embodiments, B is a 5-12 member heteroaryl, a 5-10 member heteroaryl, a 5-8 member heteroaryl, or a 5-6 member heteroaryl.

[0121]

[0121] In certain embodiments, B is pyridinyl.

[0122]

[0122] In some embodiments, B is -O-aryl. In certain embodiments, B is -O-phenyl.

[0123]

[0123] In some embodiments, A is a direct bond, and B is selected from the group consisting of direct bonds, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0124]

[0124] In certain embodiments, A is a direct bond, and B is selected from the group consisting of direct bonds, cycloalkyl, aryl, and heteroaryl.

[0125]

[0125] In a particular embodiment, A is a direct bond and B is a direct bond.

[0126]

[0126] In certain embodiments, A is a direct bond and B is a 3-8 member cycloalkyl, a 3-7 member cycloalkyl, a 3-6 member cycloalkyl, a 3-5 member cycloalkyl, or a 3-4 member cycloalkyl. In certain embodiments, A is a direct bond and B is a cyclohexyl.

[0127]

[0127] In certain embodiments, A is a direct bond and B is a 5-12 member aryl, 5-10 member aryl, 5-8 member aryl, or 5-6 member aryl. In certain embodiments, A is a direct bond and B is phenyl.

[0128]

[0128] In certain embodiments, A is a direct bond and B is a 5-12 member heteroaryl, a 5-10 member heteroaryl, a 5-8 member heteroaryl, or a 5-6 member heteroaryl. In certain embodiments, A is a direct bond and B is pyridyl.

[0129]

[0129] In some embodiments, A is an optionally substituted alkyl, and B is selected from the group consisting of directly bonded, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl.

[0130]

[0130] In certain embodiments, A is an optionally substituted alkyl group, and B is selected from the group consisting of direct bonds, aryls, and -O-aryls.

[0131]

[0131] In a particular embodiment, A is one or more R aThe base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is a direct bond, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0132]

[0132] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is an alkyl group, 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 one or more R a The base C is replaced by an arbitrary choice. 1~8 It is an alkyl group, and B is phenyl.

[0133]

[0133] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, and B is -O-aryl. In certain embodiments, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, and B is -O-phenyl.

[0134]

[0134] In some embodiments, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is selected from the group consisting of direct bonds, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0135]

[0135] In a particular embodiment, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is selected from the group consisting of direct bonds, alkyl, aryl, and heteroaryl.

[0136]

[0136] In a particular embodiment, A is -(CH2CH2O) m - where m is an integer from 0 to 4 and B is direct association.

[0137]

[0137] In a particular embodiment, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, C 1~3 Alkyl, or C 1~2 It is alkyl. In certain embodiments, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is ethyl.

[0138]

[0138] In a particular embodiment, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is a 5-12 member aryl, 5-10 member aryl, 5-8 member aryl, or 5-6 member aryl. In certain embodiments, A is -(CH2CH2O) m - where m is an integer from 0 to 4 and B is phenyl.

[0139]

[0139] In a particular embodiment, A is -(CH2CH2O) m - where m is an integer from 0 to 4, and B is a 5-12 member heteroaryl, 5-10 member heteroaryl, 5-8 member heteroaryl, or 5-6 member heteroaryl. In certain embodiments, A is -(CH2CH2O) m - where m is an integer from 0 to 4 and B is pyridinyl.

[0140]

[0140] In some embodiments, C is a direct bond.

[0141]

[0141] In some embodiments, C is -C(=O)-.

[0142]

[0142] In some embodiments, C is -C(=O)N(R 2 )-is.

[0143]

[0143] In some embodiments, C is -N(R 2 )C(=O)-.

[0144]

[0144] In some embodiments, C is -[CH2NHC(=O)] n - is

[0145]

[0145] In some embodiments, C is -[NHC(=O)CH2] n - is

[0146]

[0146] In some embodiments, C is -NH(CH2) p C(=O)-

[0147]

[0147] In some embodiments, A is alkyl, B is selected from the group consisting of directly bonded, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is directly bonded, -C(=O)-, -N(R 2 )C(=O)-,-[CH2NHC(=O)] n -,-[NHC(=O)CH2] n -, and -NH(CH2) p Selected from the group consisting of C(=O)-.

[0148]

[0148] In some embodiments, A is alkyl, B is selected from the group consisting of directly bonded, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is directly bonded, -N(R 2 )C(=O)-, or -[NHC(=O)CH2] n - is

[0149]

[0149] In certain embodiments, A is alkyl, B is selected from the group consisting of direct bond, aryl, and -O-aryl, and C is direct bond or -N(R 2 )C(=O)-.

[0150]

[0150] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is a direct bond, and C is a direct bond or -N(R 2 )C(=O)-, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0151]

[0151] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is a 5-12 membered aryl, 5-10 membered aryl, 5-8 membered aryl, or 5-6 membered aryl, and C is directly bonded or -N(R 2 )C(=O)-, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0152]

[0152] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is phenyl, and C is directly bonded or -N(R 2 )C(=O)-, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0153]

[0153] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is -O-aryl, C is a direct bond, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0154]

[0154] In a particular embodiment, A is one or more R a The base C is replaced by an arbitrary choice. 1~8 It is alkyl, B is -O-phenyl, C is a direct bond, and here R a is -C(=O)OR e And here R e It is an alkyl group.

[0155]

[0155] In some embodiments, A is -(CH2CH2O) m -B is selected from the group consisting of direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and C is direct bond or -N(R 2 )C(=O)-.

[0156]

[0156] In some embodiments, A is -(CH2CH2O) m -where m is 1, 2, 3, or 4, B is selected from the group consisting of direct bonds, alkyl, aryl, and heteroaryl, and C is a direct bond.

[0157]

[0157] In some embodiments, A is -(CH2CH2O) m - where m is 1, 2, 3, or 4, B is a direct bond, and C is a direct bond.

[0158]

[0158] In some embodiments, A is -(CH2CH2O) m - where m is 1, 2, 3, or 4, and B is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, C 1~3 Alkyl, or C 1~2 It is an alkyl group, and C is a direct bond.

[0159]

[0159] In some embodiments, A is -(CH2CH2O) m- where m is 1, 2, 3, or 4, B is an aryl compound, and C is a direct bond. In certain embodiments, A is -(CH2CH2O) m - where m is 1, 2, 3, or 4, B is phenyl, and C is a direct bond.

[0160]

[0160] In some embodiments, A is -(CH2CH2O) m - where m is 1, 2, 3, or 4, B is a heteroaryl, and C is a direct bond. In certain embodiments, A is -(CH2CH2O) m - where m is 1, 2, 3, or 4, B is pyridinyl, and C is a direct bond.

[0161]

[0161] In some embodiments, D is a direct bond.

[0162]

[0162] In some embodiments, D is alkyl. In certain embodiments, D is C 1~6 Alkyl, C 1~5 Alkyl, C 1~4 Alkyl, C 1~3 Alkyl, or C 1~2 It is alkyl.

[0163]

[0163] In some embodiments, D is an aryl. In certain embodiments, D 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, D is a phenyl.

[0164]

[0164] In some embodiments, the linker provided herein is based on formulas (Ia) to (Im): [ka] [ka] (In the formula, U and V are as defined above. M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl, and each of these is one or more R b It is replaced by an optional choice in the base, [ka] Each of these is arbitrarily replaced by -C(=O)OCH3, The structure includes q, r, s, t, u, and v (where q, r, s, t, u, and v are independent integers from 0 to 4).

[0165]

[0165] In some embodiments, M is selected from the group consisting of cyclohexyl, phenyl, pyridinyl, thiazolyl, adamantyl, and 2,5-diaza-bicyclo[2.2.1]heptanyl.

[0166]

[0166] In some embodiments, the linker provided herein is [ka] [ka] [ka] (In the formula, [ka] Each of these contains a structure selected from the group consisting of -C(=O)OCH3 (which is arbitrarily substituted).

[0167]

[0167] Drug Delivery System In one aspect of this disclosure, the therapeutic agent is bound to a biopolymer via a linker, thereby providing a drug delivery system for local delivery of the therapeutic agent to a target site.

[0168]

[0168] The biopolymers of the drug delivery systems provided herein may have one or more therapeutic agents conjugated via linkers. The biopolymers may be conjugated to one or more therapeutic agents via one or more linkers at hydroxyl groups, carboxyl groups, and / or amino groups in the biopolymer backbone.

[0169]

[0169] The drug delivery system of the present disclosure is obtained by conjugation between a biopolymer and a therapeutic agent by a linker, through the formation of a link between the biopolymer and the linker, and a link between the therapeutic agent and the linker.

[0170]

[0170] In some embodiments, the reactive functional group of the linker may first react with BG2 of the therapeutic agent to form a link between the therapeutic agent and the linker, thereby providing a therapeutic agent-linker conjugate. A therapeutic agent-linker conjugate containing another reactive functional group at the end of the linker may then react with BG1 of the biopolymer to form a link between the biopolymer and the linker, thereby providing the drug delivery system of the present disclosure.

[0171]

[0171] In certain embodiments, it is possible to first react a biopolymer BG1 with a reactive functional group of a linker to form a biopolymer-linker conjugate, and then react a therapeutic agent BG2 with another functional group of the linker in the biopolymer-linker conjugate, thereby providing the drug delivery system of the present disclosure.

[0172]

[0172] In some embodiments, the biopolymer selected for the drug delivery system provided herein is HA, and the therapeutic agent selected for the drug delivery system provided herein is tofacitinib.

[0173]

[0173] In certain embodiments, the drug delivery system provided herein is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is selected from the group consisting of the following.

[0174]

[0174] In some embodiments, the therapeutic agent can be conjugated to the biopolymer via a linker at a drug substitution rate (DSR) to the biopolymer, as measured by NMR, 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%, and at least 50%, where the drug substitution rate (DSR) to the biopolymer refers to the ratio of the molar amount of groups substituted with the drug on the biopolymer to the total molar amount of groups that can be substituted with the drug on the biopolymer.

[0175]

[0175] The therapeutic agent may be released from the drug delivery system provided herein by cleavage of the linkage between the linker and the biopolymer or the therapeutic agent. In some embodiments, the release of the therapeutic agent occurs when the linkage between the biopolymer and the linker is cleaved, releasing the therapeutic agent-linker conjugate, which may be considered a prodrug. Subsequent release of the therapeutic agent from the linker may require enzymatic or non-enzymatic cleavage of the linkage between the therapeutic agent and the linker. In some embodiments, the release of the therapeutic agent occurs when the linkage between the therapeutic agent and the linker is cleaved without or before cleavage of the linkage between the biopolymer and the linker. The release of the therapeutic agent may also require an enzymatic or non-enzymatic process.

[0176]

[0176] The release of therapeutic agents may be influenced by various factors, such as the selection of a particular therapeutic agent, linker, and biopolymer, and the administration of the drug delivery system. This disclosure envisions biopolymers with varying molecular weights, binding group BG1, and linking to linkers, linkers with varying reactive functional groups and subunits, and therapeutic agents with various binding group BG2 and linking to linkers.

[0177]

[0177] This disclosure also envisions various local administrations of the drug delivery systems provided herein. In some embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them. In certain embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them by injection. In certain embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them by oral dosage form. In certain embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them by inhalation. In certain embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them by implantation. In certain embodiments, the drug delivery systems provided herein are administered locally to a subject that requires them by topical application. Depending on the particular combination of therapeutic agent, linker, and biopolymer, the release of the therapeutic agent may occur at various locations once administered to the subject. For example, the release of the therapeutic agent may occur at the site of administration.

[0178]

[0178] In some embodiments, administration to a target of the drug delivery system provided herein may result in the release of the therapeutic agent over a period of at least several days to at least several months.

[0179]

[0179] The release of therapeutic agents from 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 rates of therapeutic agents are approximately 0.01% to 20% per day, approximately 0.01% to 15% per day, approximately 0.01% to 10% per day, approximately 0.01% to 9% per day, approximately 0.01% to 8% per day, approximately 0.01% to 7% per day, approximately 0.01% to 6% per day, approximately 0.01% to 5% per day, approximately 0.01% to 4% per day, approximately 0.01% to 3% per day, and approximately 0.01% to 6% per day. It can vary within the range of 2%, approximately 0.01% to 1% per day, approximately 0.01% to 0.5% per day, approximately 0.01% to 0.4% per day, approximately 0.01% to 0.3% per day, approximately 0.01% to 0.2% per day, approximately 0.01% to 0.1% per day, approximately 0.01% to 0.05% per day, approximately 0.01% to 0.04% per day, approximately 0.01% to 0.03% per day, or approximately 0.01% to 0.02% per day.

[0180]

[0180] Pharmaceutical composition In a further embodiment, a pharmaceutical composition comprising the drug delivery system of the present disclosure is provided.

[0181]

[0181] In another embodiment, a pharmaceutical composition comprising the drug delivery system of the present disclosure and at least one pharmaceutically acceptable excipient is provided.

[0182]

[0182] As used herein, the term “pharmaceutical composition” refers to a formulation comprising the drug delivery system of this disclosure in a form suitable for administration to a subject.

[0183]

[0183] As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is generally safe, non-toxic, and useful for preparing a pharmaceutical composition that is not biologically or otherwise undesirable, and includes excipients acceptable for veterinary and human pharmaceutical uses. As used herein, “pharmaceutically acceptable excipient” includes both one and more such excipients. The term “pharmaceutically acceptable excipient” also includes “pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent.”

[0184]

[0184] The pharmaceutical compositions provided herein may be in any form that enables the composition to be administered to subjects including but not limited to humans, and may be formulated to conform to the intended route of administration.

[0185]

[0185] Various routes of administration are intended for the pharmaceutical compositions provided herein, and therefore the pharmaceutical compositions provided herein may be supplied in bulk or in unit dosage forms depending on the intended route of administration. For example, for oral, buccal, and sublingual administration, powders, granules, tablets, pills, capsules, gel caps, and caplets are acceptable solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions are acceptable liquid dosage forms. For injectable administration, gels, solutions, emulsions, and suspensions are acceptable liquid dosage forms, and powders suitable for reconstitution in appropriate solutions are 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 implant administration, solids, semi-solids, and gels may be acceptable dosage forms.

[0186]

[0186] In some embodiments, the pharmaceutical compositions of this disclosure may be in the form of formulations for oral administration.

[0187]

[0187] In some embodiments, the pharmaceutical compositions of this disclosure may be in the form of formulations for administration by injection.

[0188]

[0188] In some embodiments, the pharmaceutical compositions of this disclosure may be in the form of formulations for inhalation administration.

[0189]

[0189] In some embodiments, the pharmaceutical compositions of this disclosure may be in the form of formulations for topical administration.

[0190]

[0190] In certain embodiments, the pharmaceutical compositions provided herein may be formulated in the form of skin patches, which are well known to those skilled in the art.

[0191]

[0191] In addition to these representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are therefore included in this disclosure. Such excipients and carriers are, for example, "Remingtons Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991), and "Remington: The Science and Practice of Pharmacy," Ed. University of the Sciences in Philadelphia, 21 st These are described in Edition, LWW (2005), which are incorporated herein by reference.

[0192]

[0192] In some embodiments, the pharmaceutical compositions of this disclosure may be formulated as single doses. The amount of a single dose compound provided herein varies depending on the target being treated and the specific mode of administration.

[0193]

[0193] In some embodiments, the pharmaceutical compositions of the present disclosure may be formulated to be administered to a subject over a period of time of several days, weeks, months, or even longer.

[0194]

[0194] In a further embodiment, a pharmaceutical composition comprising the drug delivery system of the present disclosure is also provided as a combination therapy of two or more.

[0195]

[0195] Synthesis of a drug delivery system The synthesis of the drug delivery systems provided herein is illustrated in the synthesis schemes of the Examples. The drug delivery systems provided herein can be prepared using any known organic synthesis technique and can be synthesized according to any number of possible synthesis routes; therefore, these schemes are illustrative only and are not intended to limit other possible methods that may be used to prepare the compounds provided herein. Furthermore, the steps in the schemes are for better illustration and may be modified as appropriate. The embodiments of the compounds in the Examples were synthesized for research and potential submission to regulatory authorities.

[0196]

[0196] The reactions for preparing the drug delivery systems of this disclosure can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents can be substantially inactive with the starting materials (reactants), intermediates, or products at the temperature in which the reaction is carried out, which may range from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction can be carried out in one solvent or a mixture of several solvents. Depending on the specific reaction step, a solvent suitable for the specific reaction step can be selected by those skilled in the art.

[0197]

[0197] The preparation of the compounds of this disclosure may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemical properties of protecting groups can be found, for example, in T. Greene and P. GMWuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), P. Kocienski, Protecting Groups, Georg Thieme Verlag, 2003, and Peter GMWuts, Greene's Protective Groups in Organic Synthesis, 5 th These can be found in Edition, Wiley, 2014, and all of them are incorporated herein by reference in their entirety.

[0198]

[0198] The reaction can be monitored by any suitable method known in the art. For example, the formation of the product can be monitored by nuclear magnetic resonance spectroscopy (e.g. 1 H or 13 C) The compounds may be monitored by spectroscopic means such as infrared spectroscopy, spectrophotometric methods (e.g., ultraviolet-visible), and mass spectrometry, or by chromatographic methods such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin-layer chromatography (TLC). Compounds may be purified by a variety of methods, including high-performance liquid chromatography (HPLC) (the entirety of which is incorporated herein by reference in "Preparative LC-MS Purification: Improved Compound Specific Method Optimization" by Karl F. Blom, Brian Glass, Richard Sparks, and Andrew P. Combs J. Combi. Chem. 2004, 6(6), pp. 874-883) and normal-phase silica chromatography.

[0199]

[0199] The known starting materials of this disclosure can be synthesized using or in accordance with methods known in the art, or can be purchased from commercial suppliers. Unless otherwise noted, analytical grade solvents and commercially available reagents were used without further purification.

[0200]

[0200] Unless otherwise specified, all reactions in this disclosure are carried out using dry tubes under positive pressure of nitrogen or argon, or in an anhydrous solvent, and the reaction flasks are typically fitted with rubber septums for the introduction of substrates and reagents via syringes. Glassware is oven-dried and / or heat-dried.

[0201]

[0201] Treatment methods for diseases In a further embodiment, a method is provided for treating a disorder in a subject requiring treatment of the disorder, the method comprising the step of administering a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein to the subject.

[0202]

[0202] 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 may be selected from the group consisting of inflammation, cancer, cardiovascular disease, respiratory disease, vascular endothelial growth factor (VEGF) related diseases, osteoarthritis, neovascular (exudative) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), dermatitis, psoriasis, chronic obstructive pulmonary disease, and asthma.

[0203]

[0203] In this context, the term “therapeutic dose” refers to an amount of a therapeutic agent or a pharmaceutically acceptable salt thereof selected in the drug delivery system provided herein that is effective in providing “therapy” in a subject or in “treating” a disorder, disease, or condition in a subject. [Examples]

[0204]

[0204] For illustrative purposes, the following examples are included. However, it should be understood that these examples are not intended to limit the disclosure but merely to suggest ways of carrying out the disclosure. Those skilled in the art will recognize that the chemical reactions described can be readily adapted to prepare some other compounds of the disclosure, and that alternative methods for preparing the compounds of the disclosure are considered to be within the scope of the disclosure. For example, the synthesis of non-exemplary compounds according to the disclosure can be successfully carried out by modifications obvious to those skilled in the art, for example, by appropriately protecting interfering groups, by utilizing other suitable reagents and construction blocks known in the art other than those described, and / or by making routine modifications to the reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be recognized as applicable to preparing other compounds of the disclosure.

[0205] Example 1 Preparation of a conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0206] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)butyl)carbamate [ka]

[0205] A mixture of tofacitinib (1.5 g, 4.8 mmol, 1 equivalent) and bis(4-nitrophenyl) carbonate (1.61 g, 5.28 mmol, 1.1 equivalents) in dichloromethane (30 mL) was mixed with triethylamine (1.2 g, 12 mmol, 2.5 equivalents) under N2, and the reaction mixture was heated under reflux for 3 hours. Then, tert-butyl(4-aminobutyl) carbamate (0.9 g, 4.82 mmol, 1 equivalent) was added, and the resulting mixture was refluxed for 12 hours. After removing the solvent under reduced pressure, the residue was purified by silica gel chromatography to obtain the title product (2.4 g, yield: 95%); MS (m / z): C 26 H 38 The calculated value of [M+H] for N8O4 is 527.30; the measured value is 527.2.

[0207] Step 2: Preparation of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0206] To a solution of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)butyl)carbamate (2.4 g, 4.56 mmol, 1 equivalent) in ethyl acetate (24 mL), 4 M HCl solution (9.6 mL, 38.4 mmol) was added dropwise under N2 at 0°C. The resulting reaction mixture was stirred at 0°C for 30 minutes, then stirred at room temperature for 2 days. The solvent was removed under reduced pressure, and the resulting solid was stirred in ethyl acetate (24 mL) for 0.5 hours, then filtered to obtain the desired product as the HCl salt (2.1 g, yield: 100%); MS (m / z): C 21 H 30 The calculated [M+H]+ value for N8O2 is 427.25; the measured value is 427.2. 1H-NMR (400 MHz, D2O) δ ppm 8.38 (d, J = 6.7 Hz,1H), 7.82 (d, J = 3.9 Hz 1H), 6.90 (s,1H), 4.72-4.53 (m,1H), 4.16-3.26 (m, 11H), 3.04 (d, J = 6.4 Hz 2H), 2.66-2.46 (m, 1H), 2.03-1.90 (m, 1H), 1.86-1.69 (m, 5H), 1.12 (d, J = 7.1 Hz, 3H).

[0208] Step 3: Preparation of the conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0207] To a solution of sodium hyaluronate (MW50kDa, 0.161g, 0.432 mmol, 1 equivalent) in acetonitrile (22 mL) and H2O (35 mL), 4-methylmorpholine (0.066 g, 0.65 mmol, 1.5 equivalents) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.076 g, 0.432 mmol, 1 equivalent) were added at 0°C. The resulting reaction mixture was stirred at 0°C for 30 minutes, and then stirred at room temperature for 1 hour.

[0209]

[0208] N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.432 mmol, 1 equivalent) was added to the reaction mixture, and the pH of the reaction mixture was then adjusted to 6.5-7 with 4-methylmorpholine. The resulting reaction mixture was stirred at room temperature for 3 days.

[0210]

[0209] NaCl (257 mg, 10 equivalents) in H2O (2 mL) was added to the above reaction mixture and stirred for 0.5 hours. Then, acetone (350 mL) was added dropwise to the mixture, during which time a precipitate formed. The mixture was filtered, and the cake was washed with acetone (10 mL x 3). The wet cake was dissolved in acetonitrile (20 mL) and H2O (40 mL), then dialyzed three times against deionized water using a 3.5 kDa MW cutoff membrane, and then freeze-dried to obtain the title compound (0.15 g, yield: 43.2%, DSR (drug substitution rate) = 17%). 1 H-NMR (400 MHz, D2O / d-DMSO=3:1) δ ppm 8.40-7.90 (m, 0.17H), 7.75-7.20 (m, 0.17H), 6.95-6.25 (m, 0.17H), 4.70-4.20 (m, 2.47H), 4.00-3.23 (m, 11.91H), 2.55-2.30 (m. 0.34H), 1.99 (d, J = 19.7 Hz, 3H), 1.30 (t, J = 6.8 Hz, 0.17H), 1.20-1.10 (m, 0.51H).

[0211]

[0210] Step 3 involved the reaction with sodium hyaluronate (MW500kDa) to obtain the corresponding product (0.16g, yield: 46%, DSR=22%).

[0212]

[0211] Step 3 involved the reaction with sodium hyaluronate (MW2000kDa) to obtain the corresponding product (0.15g, yield: 43.2%, DSR=17%). NMR

[0213] Example 2 Preparation of a conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0214] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)benzyl)carbamate [ka]

[0212] Following Step 1 of Example 1, the title product (1.8 g, yield: 67%) was obtained from tofacitinib (1.5 g, 4.8 mmol, 1 equivalent), bis(4-nitrophenyl) carbonate (1.61 g, 5.28 mmol, 1.1 equivalent), and tert-butyl(4-aminobutyl) carbamate (1.07 g, 4.8 mmol, 1 equivalent); MS (m / z): C 29 H 36 The calculated value of [M+H] for N8O4 is 562.29; the measured value is 562.2.

[0215] Step 2: Preparation of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0213] Following Step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)benzyl)carbamate (1.5 g, 2.675 mmol, 1 equivalent) as the HCl salt (1.33 g, yield: 100%); MS(m / z):C 24 H 28 The calculated [M+H]+ value for N8O2 is 461.23; the measured value is 461.2. 1H-NMR (400 MHz, D2O) δ ppm) δ 8.30 - 8.08 (m, 1H), 7.7-7.3 (m, 5H), 6.80-6.50 (m, 1H), 4.55 (s, 2H), 4.19 (s, 2H), 4.09-3.88 (m, 3H), 3.6-3.19 (m, 5H), 2.44 (br, 1H), 2.0-1.5 (m, 2H), 1.25-1.0 (m, 3H).

[0216] Step 3: Preparation of the conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0214] Following step 3 of Example 1, the title compound (0.18 g, yield: 55%, DSR=38%) was obtained from a reaction mixture of sodium hyaluronate (MW50 kDa, 0.163 g, 0.403 mmol, 1 equivalent) and N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.403 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O / d-DMSO=3:1) δ ppm 9.0-7.0 (m, 2.7 H), 4.75- 4.4(m, 5.5H), 4.4-3.0 (m, 11H), 2.7-2.45 (m, 0.38H), 2.15 (s, 3H), 1.9-1.4 (m, 0.74H), 1.4-0.9 (m, 1.14H).

[0217]

[0215] Following step 3 of Example 1, the corresponding product (0.15 g, yield: 44%, DSR = 21.4%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0218]

[0216] Following step 3 of Example 1, the corresponding product (0.18 g, yield: 53%, DSR = 20.7%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0219] Example 3 Preparation of a conjugate of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenethyl)carbamate with HA [ka]

[0220] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenethyl)carbamate [ka]

[0217] Following Step 1 of Example 1, the title product (2.2 g, yield: 79.7%) was obtained from tofacitinib (1.5 g, 4.8 mmol, 1 equivalent) and tert-butyl(4-aminophenethyl)carbamate (1.14 g, 4.8 mmol, 1 equivalent); MS (m / z): C 30 H 38 The calculated value of [M+H] for N8O4 is 575.30; the measured value is 575.2.

[0221] Step 2: Preparation of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0218] By following step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenethyl)carbamate (1.5 g, 2.61 mmol, 1 equivalent) as the HCl salt (1.33 g, yield: 100%); MS(m / z):C 25 H 30 The calculated [M+H]+ value for N8O2 is 475.25; the measured value is 475.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.31-8.01 (m, 1H), 7.96-7.09 (m, 5H), 6.98-6.54 (m, 1H), 4.50 (dd, 2H), 4.10-3.42 (m, 5H), 3.25 (d, J = 21.8 Hz,5H), 3.02 (t, 2H), 2.43 (d, J = 5.7 Hz, 1H), 1.83 (dd, J = 72.1, 15.3 Hz, 2H), 1.13 (dd, J = 18.5, 13.3 Hz,3H).

[0222] Step 3: Preparation of the conjugate of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenethyl)carbamate with HA. [ka]

[0219] Following step 3 of Example 1, the title compound (0.19 g, yield: 33.4%, DSR=19%) was obtained from sodium hyaluronate (MW50 kDa, 0.157 g, 0.39 mmol, 1 equivalent) and N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.39 mmol, 1 equivalent). 1H-NMR (400 MHz, D2O / DMSO=3:1) δ ppm 8.5-8.25 (m, 0.09H), 7.90-6.72 (m, 1.23H), 4.75-4.25 (m, 1.98H), 4.02-3.17 (m, 12H), 3.10-2.75 (m, 0.76H), 2.54 (br, 0.19H), 2.04 (s, 3H), 1.60-1.25 (m, 0.38H), 1.18-0.98 (m, 0.57H).

[0223]

[0220] In step 3 of Example 1, the reaction with sodium hyaluronate (MW500kDa) yielded the corresponding product (0.147g, yield: 44%, DSR=16%).

[0224]

[0221] Following step 3 of Example 1, the corresponding product (0.175 g, yield: 53%, DSR = 22.9%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0225] Example 4 Preparation of a conjugate of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0226] Step 1: Preparation of tert-butyl(2-(2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)ethoxy)ethoxy)ethyl)carbamate [ka]

[0222] Following Step 1 of Example 1, the title product (3g, yield: 54%) was obtained from tofacitinib (3g, 9.6 mmol, 1 equivalent) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl) carbamate (2.38g, 9.6 mmol, 1 equivalent); MS (m / z): C 28 H 42 The calculated value of [M+H] for N8O6 is 587.32; the measured value is 587.2.

[0227] Step 2: Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0223] Following step 2 of Example 1, the desired product was obtained from tert-butyl(2-(2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)ethoxy)ethoxy)ethyl)carbamate (1.5 g, 2.56 mmol, 1 equivalent) as the HCl salt (1.34 g, yield: 100%); MS(m / z):C 23 H 34 The calculated value of [M+H] for N8O4 is 487.32; the measured value is 487.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.40 (br, 1H), 7.83 (s, 1H), 6.91 (s, 1H), 4.7-4.5 (m, 1H), 4.06-3.92 (m, 3H), 3.83-3.36 (m, 16H), 3.17 (s, 2H), 2.56 (s, 1H), 2.00-1.72 (m, 2H), 1.14 (dd, J = 16.1, 7.0 Hz, 3H).

[0228] Step 3: Preparation of the conjugate of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0224] Following step 3 of Example 1, the title compound (0.2 g, yield: 33%, DSR=36.3%) was obtained from sodium hyaluronate (MW50 kDa, 0.153 g, 0.38 mmol, 1 equivalent) and N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.38 mmol, 1 equivalent). 1 H-NMR (400 MHz, D2O / d-DMSO=3:1) δ ppm 8.52-7.93 (m, 0.37H), 7.82-7.20 (m, 0.33H), 6.99-6.26 (m, 0.39H), 4.75-3.8 (m, 5.09H), 3.8-2.75 (m, 14.93H), 2.5-2.25 (m, 0.36H), 2.05 (s, 3H), 1.82-1.30 (m, 0.73H), 1.2-0.8 (m, 1.09H).

[0229]

[0225] Following step 3 of Example 1, the corresponding product (0.186 g, yield: 56.5%, DSR=32%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0230]

[0226] Following step 3 of Example 1, the corresponding product (0.198 g, yield: 60.2%, DSR = 26.6%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0231] Example 5 Preparation of a conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0232] Step 1: Preparation of tert-butyl(1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methylamino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-yl)carbamate [ka]

[0227] Following Step 1 of Example 1, the title product (1.8 g, yield: 60%) was obtained from tofacitinib (1.5 g, 4.8 mmol, 1 equivalent) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl) carbamate (1.41 g, 4.8 mmol, 1 equivalent); MS (m / z): C 30 H 46 [M+H] of N8O7: Calculated value, 631.35; Measured value, 631.2.

[0233] Step 2: Preparation of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0228] Following step 2 of Example 1, the desired product was obtained from tert-butyl(1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methylamino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-1-oxo-5,8,11-trioxa-2-azatridecane-13-yl)carbamate (1.5 g, 2.38 mmol 1 equivalent) as the HCl salt (1 g, yield: 80%); MS (m / z): C 25 H 38 The calculated value of N8O5's [M+H] is 531.30; the measured value is 531.2. 1 H-NMR (400 MHz, DMSO) δ ppm 9.74 (s, 1H), 8.34 (d, J = 7.0 Hz, 1H), 8.01 (s, 1H), 7.69 (s, 1H), 6.86 (s, 1H), 4.85 (d, J= 3.1 Hz,1H), 4.35-4.0 (m, 10H), 3.7-3.96 (m, 3H), 3.65-3.5 (m, 10H), 2.93 (d, J = 4.3 Hz, 2H), 2.38 (s,1H), 1.89-1.68 (m, 1H), 1.59 (s, 1H), 1.01 (d, J = 4.8 Hz, 3H).

[0234] Step 3: Preparation of the conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0229] Following step 3 of Example 1, the title compound (0.2 g, yield: 62.7%, DSR=24%) was obtained from sodium hyaluronate (MW50 kDa, 0.141 g, 0.35 mmol, 1 equivalent) and N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.35 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O-DMSO=3:1) δ ppm 8.7-8.2 (m, 0.24H), 8.1-7.3 (m, 0.24H), 7.2-6.6 (m, 0.24H), 4.7-4.35 (m, 1H), 4.3-3.1 (m, 17H), 2.7-2.5 (br, 0.24H), 2.14 (br, 3H), 1.93 (br, 0.24H), 1.46 (br, 0.24H), 1.24 (br, 0.72H).

[0235]

[0230] Following step 3 of Example 1, the corresponding product (0.186 g, yield: 58.4%, DSR=26%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0236]

[0231] Following step 3 of Example 1, the corresponding product (0.198 g, yield: 62.2%, DSR=28%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0237] Example 6 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohazide with HA [ka]

[0238] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate [ka]

[0232] Following Step 1 of Example 1, the title product (2.23 g, yield: 75%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and tert-butylhydrazine carboxylate (0.845 g, 6.4 mmol, 1 equivalent); MS (m / z): C 22 H 30 [M+H]+ of N8O4: Calculated value, 471.24; Measured value, 471.2.

[0239] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohdrozide hydrochloride [ka]

[0233] Following step 2 of Example 1, the desired product was obtained from tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate (2 g, 4.25 mmol, 1 equivalent) as the HCl salt (1.5 g, yield: 85%); MS (m / z): C 17 H 22 The calculated [M+H]+ value for N8O2 is 371.19; the measured value is 371.1. 1H-NMR (400 MHz, CD3OD) δ ppm 8.52 (s, 1H), 7.89 (d, J = 3.1 Hz, 1H), 7.13 (d, J = 3.8 Hz, 1H), 4.83 (s, 1H), 4.22-3.32 (m, 9H), 2.55 (br, 1H), 2.05-1.99 (m,1H), 1.90-1.67 (m, 1H), 1.16 (d, J = 7.0 Hz, 3H).

[0240] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide with HA [ka]

[0234] Following step 3 of Example 1, the title compound (0.15 g, yield: 43.2%, DSR=32%) was obtained from sodium hyaluronate (MW50 kDa, 0.202 g, 0.5 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g, 0.5 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O-DMSO=5:1): δ ppm 8.4-7.8 (m, 0.32H), 7.8-7.2 (m, 0.32H), 7.0-6.0 (m, 0.32H), 4.75-4.4 (m, 2.1H), 4.26-3.19 (m, 11.1H), 2.45 (br, 0.32H), 2.05 (br, 3H), 1.85-1.3 (m, 0.65H), 1.18 (br, 0.95H).

[0241]

[0235] Following step 3 of Example 1, the corresponding product (0.18 g, yield: 48.5%, DSR=35%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0242]

[0236] Following step 3 of Example 1, the corresponding product (0.19 g, yield: 51.2%, DSR=30%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0243] Example 7 Preparation of a conjugate of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0244] Step 1: Preparation of tert-butyl(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)ethyl)carbamate [ka]

[0237] Following Step 1 of Example 1, the title product (3 g, yield: 59%) was obtained from tofacitinib (3.2 g, 10.24 mmol, 1 equivalent) and tert-butyl(2-aminoethyl) carbamate (1.64 g, 10.24 mmol, 1 equivalent); MS (m / z): C 24 H 34 The calculated value of [M+H] for N8O4 is 499.27; the measured value is 499.1.

[0245] Step 2: Preparation of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0238] According to step 2 of Example 1, the desired product is obtained from tert-butyl(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)ethyl)carbamate (1 g, 2 mmol, 1 equivalent) as the HCl salt (870 mg, yield: 100%); MS (m / z): C 19 H 26 The calculated [M+H]+ value for N8O2 is 399.22; the measured value is 399.1. 1 H-NMR (400 MHz, CD3OD) δ ppm 8.49 (s, 1H), 7.95 (d, J = 3.6 Hz, 1H), 7.08 (s, 1H), 4.08-3.73 (m, 7.5H), 3.71-3.41 (m, 4.5H), 3.27 (t, 2H), 2.66-2.44 (m, 1H), 2.00-1.67 (m, 2H), 1.16 (d, J = 7.0 Hz, 3H).

[0246] Step 3: Preparation of the conjugate of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0239] Following step 3 of Example 1, the title compound (0.23 g, yield: 66%, DSR=18%) was obtained from sodium hyaluronate (MW50 kDa, 0.186 g, 0.46 mmol, 1 equivalent) and N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.46 mmol, 1 equivalent). 1H-NMR (400 MHz, D2O) δ ppm: 8.23 ​​(m, 0.18H), 7.63 (m, 0.18H), 6.80 (m, 0.18H), 4.64 - 4.24 (m, 2.2H), 4.05 - 2.96 (m, 12.3H), 2.42 (m, 0.18H), 1.98 (s, 3H), 1.80 - 1.67 (m, 0.18H), 1.26 (m, 0.18H), 1.05 (m, 0.54H). 1 H-NMR (400 MHz, D2O) δ ppm: 8.5-7.9 (m, 0.18H), 7.7-7.0 (m, 0.18H), 7.0-5.8 (m, 0.18H), 4.64-4.24 (m, 2.2H), 4.05-2.96 (m, 12.3H), 2.42 (br, 0.18H), 1.98 (s, 3H), 1.80-1.67 (m, 0.18H), 1.26 (br, 0.18H), 1.05 (m, 0.54H).

[0247] Example 8 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0248] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)butyl)(methyl)carbamate [ka]

[0240] Following Step 1 of Example 1, the title product (1 g, yield: 58%) was obtained from tofacitinib (1 g, 3.2 mmol, 1 equivalent) and tert-butyl(4-aminobutyl)(methyl)carbamate (0.65 g, 3.2 mmol, 1 equivalent); MS (m / z): C 27 H 40 The calculated value of [M+H] for N8O4 is 541.32; the measured value is 541.2.

[0249] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0241] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)butyl)(methyl)carbamate (1 g, 1.85 mmol, 1 equivalent) as the HCl salt (0.88 g, yield: 100%); MS(m / z):C 22 H 32 The calculated [M+H]+ value for N8O2 is 441.26; the measured value is 441.1. 1 H-NMR (400 MHz, CD3OD) δ ppm 8.50 (d, J = 4.7 Hz, 1H), 7.94 (d, J = 3.7 Hz, 1H), 7.07 (s, 1H), 4.20 -3.81 (m, 3H), 3.73-3.32 (m, 8H), 3.08 (t, 2H), 2.72 (s, 3H), 2.55 (br, 1H), 1.99 (br, 1H), 1.91-1.67 (m, 5H), 1.16 (d, J = 6.9, 5.2 Hz, 3H).

[0250] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0242] Following step 3 of Example 1, the title compound (0.236 g, yield: 34.4%, DSR=10%) was obtained from sodium hyaluronate (MW50 kDa, 0.169 g, 0.42 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.42 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.4-7.9 (m, 0.1H), 7.75-7.35 (m, 0.1H), 6.95-6.5 (m, 0.1H), 4.7-4.2 (m, 2.7H), 4.0-3.2 (m, 11H), 2.47 (br, 0.1H), 2.02 (br, 3H), 1.29 (br, 0.2H), 1.2-0.95 (m, 0.3H).

[0251]

[0243] Following step 3 of Example 1, the corresponding product (0.18 g, yield: 52.3%, DSR=6%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0252]

[0244] Following step 3 of Example 1, the reaction with sodium hyaluronate (MW2000kDa) yielded the corresponding product KDa (0.17g, yield: 49.4%, DSR=3%).

[0253] Example 9 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbhydrazide with HA [ka]

[0254] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-methylhydrazine-1-carboxylate [ka]

[0245] Following Step 1 of Example 1, the title product (2.2 g, yield: 46%) was obtained from tofacitinib (3.124 g, 10 mmol, 1 equivalent) and tert-butyl 1-methylhydrazine-1-carboxylate (1.45 g, 10 mmol, 1 equivalent); MS (m / z): C 23 H 32 The calculated value of [M+H] for N8O4 is 485.25; the measured value is 485.2.

[0255] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride [ka]

[0246] According to step 2 of Example 1, the desired product was obtained from tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-methylhydrazine-1-carboxylate (1 g, 2.06 mmol, 1 equivalent) as the HCl salt (870 mg, yield: 100%); MS (m / z): C 18H 24 The calculated [M+H]+ value for N8O2 is 385.20; the measured value is 385.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.44 (d, J = 5.0 Hz, 1H), 7.79 (d, J = 3.5 Hz, 1H), 6.98 (s,1H), 4.75 (s, 1H), 4.15-3.79 (m, 4H), 3.72-3.32 (m, 5H), 3.03 (s, 3H), 2.55 (br, 1H), 1.97 (br, 1H), 1.83 (br, 1H), 1.13 (dd, J = 15.1, 7.1 Hz, 3H).

[0256] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbhydrazide with HA [ka]

[0247] Following Step 2 of Example 1, the title compound (0.323 g, yield: 90%, DSR=15%) was obtained from sodium hyaluronate (MW50 kDa, 0.192 g, 0.475 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride (0.2 g, 0.475 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.4-8.2 (m, 0.15H), 7.8-7.5 (m, 0.15H), 7.05-6.75 (m, 0.15H), 4.75-4.2 (m, 2.9H), 4.0-3.2 (m, 11H), 2.47 (br, 0.15H), 2.02 (br, 3H), 1.30 (br, 0.15H), 1.08 (m, 0.45H).

[0257]

[0248] Following step 3 of Example 1, the corresponding product (0.303 g, yield: 83%, DSR=10%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0258]

[0249] Following step 3 of Example 1, the corresponding product (0.327 g, yield: 90.3%, DSR=15%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0259] Example 10 Preparation of a conjugate of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide with HA [ka]

[0260] Step 1: Preparation of tert-butyl1-allyl-2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate [ka]

[0250] Following Step 1 of Example 1, the title product (0.8 g, yield: 25%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and tert-butyl 1-methylhydrazine-1-carboxylate (1.1 g, 6.4 mmol, 1 equivalent); MS (m / z): C 25 H 34 [M+H] of N8O4: Calculated value, 511.27; Measured value, 511.1.

[0261] Step 2: Preparation of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride [ka]

[0251] According to step 2 of Example 1, the desired product was obtained from tert-butyl1-allyl-2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate (0.8 g, 1.57 mmol) as the HCl salt (0.67 g, yield: 96%); MS(m / z):C 20 H 26 The calculated [M+H]+ value for N8O2 is 411.22; the measured value is 411.1. 1 H-NMR (400 MHz, CD3OD) δ ppm 8.50 (d, J = 4.2 Hz, 1H), 7.86 (s, 1H), 7.10 (d, J = 3.8 Hz, 1H), 6.08-5.98 (m, 1H), 5.61-5.52 (m, 2H), 4.34-3.73 (m, 6H), 3.73-3.32 (m, 6H), 2.53 (br, 1H), 2.05-1.9 (m, 1H), 1.87-1.64 (m, 1H), 1.17 (d, J = 7.0 Hz 3H).

[0262] Step 3: Preparation of the conjugate of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide with HA [ka]

[0252] Following step 3 of Example 1, the title compound (0.172 g, yield: 48.6%, DSR=5%) was obtained from sodium hyaluronate (MW50 kDa, 0.182 g, 0.45 mmol, 1 equivalent) and N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride (0.2 g, 0.45 mmol, 1 equivalent); 1H-NMR (400 MHz, D2O) δ ppm 8.50-8.17 (m, 0.05H), 7.85-7.55 (m, 0.05H), 7.05- 6.8 (m, 0.05H), 6.17-5.60 (m, 0.15H), 4.66-4.31 (m, 2H), 4.08-3.29 (m, 10.6H), 2.47 (br, 0.05H), 2.01 (s, 3H), 1.30 (m, 0.1H), 1.19-0.98 (m, 0.15H).

[0263]

[0253] Following step 3 of Example 1, the corresponding product (0.175 g, yield: 49%, DSR=3%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0264]

[0254] Following step 3 of Example 1, the corresponding product (0.186 g, yield: 52.3%, DSR=1%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0265] Example 11 Preparation of a conjugate of N-(5-(aminomethyl)pyridine-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0266] Step 1: Preparation of tert-butyl((6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)pyridine-3-yl)methyl)carbamate [ka]

[0255] Following Step 1 of Example 1, the title product (1 g, yield: 62.5%) was obtained from tofacitinib (0.9 g, 2.88 mmol, 1 equivalent) and tert-butyl((6-aminopyridine-3-yl)methyl)carbamate (0.643 g, 2.88 mmol, 1 equivalent); MS (m / z): C 28 H 35 The calculated value of [M+H] for N9O4 is 526.28; the measured value is 526.1.

[0267] Step 2: Preparation of N-(5-(aminomethyl)pyridine-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0256] According to step 2 of Example 1, the desired product was obtained from tert-butyl((6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)pyridine-3-yl)methyl)carbamate (0.8 g, 1.42 mmol, 1 equivalent) as the HCl salt (0.7 g, yield: 100%); MS(m / z):C 23 H 27 The calculated [M+H]+ value for N9O2 is 462.23; the measured value is 462.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.41-8.31 (m, 2H), 8.1-7.9 (m, 3H), 7.78-7.68 (m, 1H), 6.83 (d, J = 2.4 Hz, 2H), 4.67-4.49 (m, 1H), 4.26 (s, 2H), 4.14-3.82 (m, 5H), 3.66-3.51 (m, 2H), 3.45-3.2 (m, 3H), 2.49 (br, 1H), 2.03-1.85 (m, 1H), 1.77 (m, 1H), 1.13 (dd, J = 24.3, 6.3 Hz, 3H).

[0268] Step 3: Preparation of the conjugate of N-(5-(aminomethyl)pyridine-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0257] Following step 3 of Example 1, the title compound (0.17 g, yield: 50%, DSR=20.5%) was obtained from sodium hyaluronate (MW50 kDa, 0.161 g, 0.4 mmol, 1 equivalent) and N-(5-(aminomethyl)pyridine-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.4 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.6-7.3 (m, 1.12H), 7.2-6.8 (m, 0.11H), 4.7-4.3 (m, 2H), 4.02-3.17 (m, 10.46H), 2.40 (br, 0.21H), 2.00 (s, 3H), 1.61 (br, 0.21H), 1.35-1.2 (m, 0.21H), 1.15-0.85 (m, 0.65H).

[0269]

[0258] Following step 3 of Example 1, the corresponding product (0.177 g, yield: 52.6%, DSR=22.2%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0270] Example 12 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(piperazine-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0271] Step 1: Preparation of tert-butyl 4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenyl)piperazine-1-carboxylate [ka]

[0259] Following Step 1 of Example 1, the title product (0.65 g, yield: 65%) was obtained from tofacitinib (0.52 g, 1.66 mmol, 1 equivalent) and tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (0.46 g, 1.66 mmol, 1 equivalent); MS (m / z): C 32 H 41 The calculated value of [M+H] for N9O4 is 616.33; the measured value is 616.1.

[0272] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(piperazine-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0260] According to step 2 of Example 1, the desired product was obtained from tert-butyl 4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenenyl)piperazine-1-carboxylate (0.6 g, 0.975 mmol, 1 equivalent) as the HCl salt (0.538 mg, yield: 100%); MS (m / z): C 27 H 33 The calculated [M+H]+ value for N9O2 is 516.28; the measured value is 516.1. 1H-NMR (400 MHz, D2O) δ ppm 8.28 (s, 1H), 7.68 (s, 1H), 7.55-7.4 (m, 2H), 7.25-7.10 (m, 2H), 6.78 (s, 1H), 4.55 (s, 1H), 4.12-2.92 (m, 17H), 2.47 (br, 1H), 2.0 -1.85 (m, 1H), 1.85-1.65 (m, 1H), 1.11 (dd, J = 16.6, 7.0 Hz,3H).

[0273] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(4-(piperazine-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0261] Following step 3 of Example 1, the title compound (0.13 g, yield: 41%, DSR=19%) was obtained from sodium hyaluronate (MW50 kDa, 0.145 g, 0.36 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino-N-(4-(piperazine-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.36 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.4-6.8 (m, 1.33H), 4.7-4.4 (m, 2H), 3.9-3.42 (m, 12H), 3.34 (m, 1.42H), 2.47 (br, 0.19H), 2.02 (s, 3H), 1.29 (br, 0.38H), 1.06 (br, 0.57H).

[0274]

[0262] Following step 3 of Example 1, the corresponding product (0.16 g, yield: 49.7%, DSR=20%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0275] Example 13 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(5-(piperazine-1-yl)pyridine-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0276] Step 1: Preparation of tert-butyl 4-(6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)pyridine-3-yl)piperazine-1-carboxylate [ka]

[0263] Following Step 1 of Example 1, the title product (1.3 g, yield: 58.7%) was obtained from tofacitinib (1.12 g, 3.59 mmol, 1 equivalent) and tert-butyl 4-(6-aminopyridine-3-yl)piperazine-1-carboxylate (1 g, 3.59 mmol, 1 equivalent); MS (m / z): C 31 H 40 N 10 O4's [M+H]+ calculated value: 617.32; measured value: 617.2.

[0277] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(5-(piperazine-1-yl)pyridine-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0264] According to step 2 of Example 1, the desired product was obtained from tert-butyl 4-(6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)pyridine-3-yl)piperazine-1-carboxylate (1 g, 1.621 mmol, 1 equivalent) as the HCl salt (0.89 g, yield: 99%); MS (m / z): C 26 H 32 N 10 O2 [M+H]+ Calculated value: 517.28; Measured value: 517.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.33 (d, J = 11.6 Hz, 1H), 8.04 (d, J = 13.2 Hz, 1H), 7.85 (d, J = 19.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 1H), 6.83 (s, 1H), 4.62-4.57 (m,1H), 4.10-3.76 (m, 2H), 3.65-3.21 (m, 15H), 2.47 (m, 1H), 1.94 (m, 1H), 1.77 (m, 1H), 1.12 (dd, J = 24.1, 7.0 Hz, 3H).

[0278] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(5-(piperazine-1-yl)pyridine-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0265] Following step 3 of Example 1, the title compound (0.17 g, yield: 33%, DSR=28%) was obtained from sodium hyaluronate (MW50 kDa, 0.145 g, 0.36 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N-(5-(piperazine-1-yl)pyridine-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.36 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.72-6.98 (m, 1.7H), 4.70-4.3 (m, 2H), 3.80-3.28 (m, 15H), 2.84-2.67 (m, 0.29H), 1.95 (s, 3H), 1.40-1.10 (m, 0.66H), 1.1-0.9 (m, 0.85H).

[0279]

[0266] Following step 3 of Example 1, the corresponding product (0.22 g, yield: 68%, DSR=20%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0280] Example 14 Preparation of a conjugate of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0281] Step 1: Preparation of tert-butyl(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)ethyl)carbamate [ka]

[0267] Following Step 1 of Example 1, the title product (1.3 g, yield: 55.6%) was obtained from tofacitinib (1.24 g, 3.96 mmol, 1 equivalent) and tert-butyl(2-(4-aminophenoxy)ethyl) carbamate (1 g, 3.96 mmol, 1 equivalent); MS (m / z): C 30 H 38 The calculated value of N8O5's [M+H] is 591.30; the measured value is 591.2.

[0282] Step 2: Preparation of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0268] According to step 2 of Example 1, the desired product was obtained from tert-butyl(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)ethyl)carbamate (1 g, 1.69 mmol, 1 equivalent) as the HCl salt (0.892 g, yield: 100%); MS(m / z):C 25 H 30 The calculated value of [M+H] for N8O3 is 491.24; the measured value is 491.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.15 (s, 1H), 7.51 (s, 1H), 7.34 (dd, J = 16.5, 8.8 Hz, 2H), 7.03 (t, 2H), 6.63 (s,1H), 4.45 (s, 1H), 4.38-3.38 (m, 10H), 3.21 (d, J = 15.1 Hz, 2H), 2.39 (s, 1H), 2.0-1.6 (m, 2H), 1.08 (dd, J = 16.9, 6.8 Hz, 3H).

[0283] Step 3: Preparation of the conjugate of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0269] Following step 3 of Example 1, the title compound (0.22 g, yield: 66%, DSR=11%) was obtained from sodium hyaluronate (MW50 kDa, 0.153 g, 0.38 mmol, 1 equivalent) and N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.38 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.0-6.6 (m, 0.77H), 4.53 (br, 1.58H), 4.01-3.19 (m, 12H), 2.77-2.58 (m, 0.11H), 1.95 (s, 3H), 1.28 (br, 0.22H), 1.15-0.95 (m, 0.33H).

[0284]

[0270] Following step 3 of Example 1, the corresponding product (0.24 g, yield: 73%, DSR=10%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0285] Example 15 Preparation of a conjugate of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0286] Step 1: Preparation of tert-butyl (4-(4-nitrophenoxy)butyl)carbamate [ka]

[0271] To a solution of tert-butyl(4-hydroxybutyl) carbamate (3.7 g, 19.5 mmol, 1.1 equivalents) in THF (40 mL), NaH (1.772 g, 44.3 mmol, 2.5 equivalents) was added under N2 at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then 1-fluoro-4-nitrobenzene (2.5 g, 17.72 mmol, 1 equivalent) was added. The resulting mixture was refluxed for 12 hours. After most of the 1-fluoro-4-nitrobenzene had been consumed, the reaction product was quenched with saturated NH4Cl solution in water (100 mL) and extracted with ethyl acetate (40 mL x 2). The combined organic phase was washed with saturated NaCl solution in water (100 mL), dried over anhydrous Na2SO4, and concentrated under high vacuum. The residue was purified by silica gel chromatography to obtain the title product (4 g, yield: 73%); MS (m / z): C 15 H 22 The calculated value of [M+H]+ for N2O5 is 311.15; the measured value is 311.1.

[0287] Step 2: Preparation of tert-butyl(4-(4-aminophenoxy)butyl)carbamate [ka]

[0272] To a solution of tert-butyl(4-(4-nitrophenoxy)butyl)carbamate (2 g, 6.444 mmol, 1 equivalent) in methanol (30 mL), 10% Pd / C (0.2 g) was added, and the reaction mixture was stirred under an H2 balloon at room temperature for 24 hours. After the tert-butyl(4-(4-nitrophenoxy)butyl)carbamate was completely consumed, the reaction mixture was filtered through a Celite pad, and the pad was washed with methanol (10 mL x 2). The combined filtrate was concentrated to obtain the title product (1.8 g, yield: 100%); MS (m / z): C 15 H 24The calculated value of [M+H]+ for N2O3 is 281.18; the measured value is 281.1.

[0288] Step 3: Preparation of tert-butyl(4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)butyl)carbamate [ka]

[0273] Following Step 1 of Example 1, the title product (2.6 g, yield: 59%) was obtained from tofacitinib (2.23 g, 7.133 mmol, 1 equivalent) and tert-butyl (4-(4-aminophenoxy)butyl) carbamate (2 g, 7.133 mmol, 1 equivalent); MS (m / z): C 32 H 42 The calculated value of N8O5's [M+H] is 619.33; the measured value is 619.2.

[0289] Step 4: Preparation of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0274] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)butyl)carbamate (1.3g, 2.1 mmol, 1 equivalent) as the HCl salt (1.1g, yield: 100%); MS(m / z):C 27 H 34 The calculated value of [M+H] for N8O3 is 519.28; the measured value is 519.2. 1H-NMR (400 MHz, D2O) δ ppm 8.14-8.11 (d, J = 10.8 Hz, 1H), 7.43-7.21 (m, 3H), 6.83 (d, J = 8.6 Hz, 2H), 6.70-6.45 (m, 1H), 4.49 (br, 1H), 4.12-3.09 (m, 13H), 2.36 (m, 1H), 1.85-1.55 (m, 6H), 1.04 (d, J = 7.4 Hz, 3H).

[0290] Step 5: Preparation of the conjugate of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0275] Following step 3 of Example 1, the title compound (0.22 g, yield: 66%, DSR=31%) was obtained from sodium hyaluronate (MW50 kDa, 0.153 g, 0.38 mmol, 1 equivalent) and N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.38 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.60-5.22 (m, 2.17H), 4.7-4.2 (m, 3.1H), 4.0-3.25(m, 12H), 3.3-2.9(m, 1.24H), 2.84-2.11 (m, 0.31H), 2.02 (s, 3H), 1.79 (br, 1.24H), 1.30 (br, 0.62H), 1.15-0.5 (m, 0.93H).

[0291]

[0276] Following step 3 of Example 1, the corresponding product (0.123 g, yield: 36%, DSR=4%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0292] Example 16 Preparation of a conjugate of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0293] Step 1: Preparation of tert-butyl(2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate [ka]

[0277] To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl) carbamate (3.2 g, 15.59 mmol, 1.1 equivalents) in THF (40 mL), NaH (1.42 g, 35.425 mmol, 2.5 equivalents) was added under N2 at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, then 1-fluoro-4-nitrobenzene (2 g, 14.17 mmol, 1 equivalent) was added, and the resulting mixture was refluxed for 12 hours. After most of the 1-fluoro-4-nitrobenzene had been consumed, the reaction product was quenched with saturated NH4Cl solution in water (100 mL) and extracted with ethyl acetate (50 mL x 2). The combined organic phase was washed with saturated NaCl solution in water (100 mL), dried over anhydrous Na2SO4, and concentrated under high vacuum. The residue was purified by silica gel chromatography to obtain the title product (2 g, yield: 43.3%); MS (m / z): C 15 H 22 The calculated value of [M+H]+ for N2O6 is 327.15; the measured value is 327.1.

[0294] Step 2: Preparation of tert-butyl(2-(2-(4-aminophenoxy)ethoxy)ethyl)carbamate [ka]

[0278] To a solution of tert-butyl(2-(2-(4-nitrophenoxy)ethoxy)ethyl) carbamate (2 g, 6.13 mmol, 1 equivalent) in methanol (40 mL), 10% Pd / C (0.2 g) was added, and the reaction mixture was stirred under an H2 balloon at room temperature for 24 hours. After the tert-butyl(2-(2-(4-nitrophenoxy)ethoxy)ethyl) carbamate was completely consumed, the reaction mixture was filtered through a Celite pad, and the pad was washed with methanol (20 mL x 2). The combined filtrate was concentrated to obtain the title product (1.8 g, yield: 100%); MS (m / z): C 15 H 24 The calculated value of [M+H]+ for N2O4 is 297.17; the measured value is 297.1.

[0295] Step 3: Preparation of tert-butyl(2-(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)ethoxy)ethyl)carbamate [ka]

[0279] Following Step 1 of Example 1, the title product (1.8 g, yield: 57%) was obtained from tofacitinib (1.9 g, 6.07 mmol, 1 equivalent) and tert-butyl (2-(2-(4-aminophenoxy)ethoxy)ethyl) carbamate (1.8 g, 6.07 mmol, 1 equivalent); MS (m / z): C 32 H 42 [M+H]+ of N8O6: Calculated value, 635.32; Measured value, 635.2.

[0296] Step 4: Preparation of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0280] According to step 2 of Example 1, the desired product was obtained from tert-butyl(2-(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)phenoxy)ethoxy)ethyl)carbamate (1 g, 1.575 mmol, 1 equivalent) as the HCl salt (0.89 g, yield: 100%); MS (m / z): C 27 H 34 The calculated value of N8O4's [M+H] is 535.27; the measured value is 535.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.19 (d, J = 9.4 Hz, 1H), 7.52 (d, J = 15.7 Hz, 1H), 7.37 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.72-6.50 (m, 1H), 4.63-4.54 (m, 1H), 4.23 (s, 2H), 4.09-3.81 (m, 7H), 3.74-3.12 (m, 8H), 2.42 (s,1H), 1.77 (m, 2H), 1.08 (d, J = 6.8 Hz, 3H).

[0297] Step 5: Preparation of the conjugate of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0281] Following step 3 of Example 1, the title compound (0.15 g, yield: 32%, DSR=25%) was obtained from sodium hyaluronate (MW50 kDa, 0.141 g, 0.35 mmol, 1 equivalent) and N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.35 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.49-7.25 (m, 0.76H), 7.25-5.75 (m, 1.0H), 4.78-4.02 (m, 3.23H), 4.02-3.22 (m, 12H), 3.2-2.75 (m, 1.23H), 2.5 (br, 0.25H), 2.01 (s, 3H), 1.63-1.16 (m, 0.5H), 1.25-0.5 (m, 0.75H).

[0298]

[0282] Following step 3 of Example 1, the corresponding product (0.132 g, yield: 42%, DSR=28%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0299] Example 17 Preparation of a conjugate between 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropanenitrile and HA [ka]

[0300] Step 1: Preparation of tert-butyl 4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)piperazine-1-carboxylate [ka]

[0283] Following Step 1 of Example 1, the title product (0.9 g, yield: 32%) was obtained from tofacitinib (1.68 g, 5.37 mmol, 1 equivalent) and tert-butylpiperazine-1-carboxylate (1 g, 5.37 mmol, 1 equivalent); MS (m / z): C 26 H 36 [M+H]+ of N8O4: Calculated value, 525.29; Measured value, 525.2.

[0301] Step 2: Preparation of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropannitrile hydrochloride [ka]

[0284] According to step 2 of Example 1, the desired product was obtained from tert-butyl 4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)piperazine-1-carboxylate (0.8 g, 1.525 mmol, 1 equivalent) as the HCl salt (0.7 g, yield: 100%); MS (m / z): C 21 H 28 The calculated [M+H]+ value for N8O2 is 425.23; the measured value is 425.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.41 (d, J = 5.5 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.04 (s, 1H), 4.74-4.57 (m, 1H), 4.17-3.82 (m, 7H), 3.80-3.06 (m, 10H), 2.68-2.51 (m, 1H), 2.06-1.90 (m, 1H), 1.89-1.71 (m, 1H), 1.16 (dd, J = 14.8, 7.1 Hz, 3H).

[0302] Step 3: Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropannitrile and HA. [ka]

[0285] Following step 3 of Example 1, the title compound (0.2 g, yield: 57.3%, DSR=24%) was obtained from sodium hyaluronate (MW50 kDa, 0.175 g, 0.434 mmol, 1 equivalent) and N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.434 mmol, 1 equivalent); 1 1 H-NMR (400 MHz, D2O) δ ppm 8.21 (br, 0.24H), 7.33 (br, 0.24H), 6.91 (br, 0.24H), 4.7-4.3 (m, 2.32H), 4.18-3.12 (m, 14H), 2.46 (br, 0.24H), 2.02 (s, 3H), 1.79 (br, 0.24H), 1.30 (m, 0.24H), 1.18-0.93 (m, 0.72H).

[0303]

[0286] Following step 3 of Example 1, the corresponding product (0.162 g, yield: 46.4%, DSR=28%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0304] Example 18 Preparation of a conjugate between 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropanenitrile and HA [ka]

[0305] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)cyclohexyl)carbamate [ka]

[0287] Following Step 1 of Example 1, the title product (1.785 g, yield: 53.2%) was obtained from tofacitinib (1.895 g, 6.066 mmol, 1 equivalent) and tert-butyl(4-aminocyclohexyl) carbamate (1.3 g, 6.066 mmol, 1 equivalent); MS (m / z): C 28 H 40 The calculated value of [M+H] for N8O4 is 553.32; the measured value is 553.2.

[0306] Step 2: Preparation of N-(4-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0288] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)cyclohexyl)carbamate (1.5 g, 2.71 mmol, 1 equivalent) as the HCl salt (1 g, yield: 77%); MS(m / z):C 23 H 32 The calculated [M+H]+ value for N8O2 is 453.26; the measured value is 453.2. 1H-NMR (400 MHz, D2O) δ ppm 8.6-8.2 (m, 1H), 7.90-7.70 (m, 1H), 6.89 (br, 1H), 4.69-4.59 (m, 1H), 4.14-3.13 (m, 11H), 2.62-2.49 (m, 1H), 2.31-2.11 (m, 1H), 2.05-1.58 (m, 9H), 1.15 (dd, J = 6.6, 5.4 Hz, 3H).

[0307] Step 3: Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropannitrile and HA. [ka]

[0289] Following step 3 of Example 1, the title compound (0.2 g, yield: 55%, DSR=25%) was obtained from sodium hyaluronate (MW50 kDa, 0.178 g, 0.44 mmol, 1 equivalent) and 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropannitrile hydrochloride (0.2 g, 0.44 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.4-7.8 (m, 0.25H), 7.7-7.2 (m, 0.25H), 6.90-6.0 (m, 0.25H), 4.45 (br, 2.5H), 3.77-3.27 (m, 12H), 3.17-3.06 (m, 0.51H), 2.42 (br, 0.25H), 1.96 (s, 3H), 1.85-1.52 (m, 2H), 1.24 (br, 0.5H), 1.1-0.9 (m, 0.75H).

[0308]

[0290] Following step 3 of Example 1, the corresponding product (0.109 g, yield: 30%, DSR=9%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa). Example 19 Preparation of a conjugate of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0309] Step 1: Preparation of tert-butyl(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)cyclohexyl)carbamate [ka]

[0291] Following Step 1 of Example 1, the title product (2 g, yield: 78%) was obtained from tofacitinib (1.46 g, 4.67 mmol, 1 equivalent) and tert-butyl (2-aminocyclohexyl) carbamate (1 g, 4.67 mmol, 1 equivalent); MS (m / z): C 28 H 40 The calculated value of [M+H] for N8O4 is 553.32; the measured value is 553.2.

[0310] Step 2: Preparation of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0292] According to step 2 of Example 1, the desired product was obtained from tert-butyl(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide)cyclohexyl)carbamate (1.3g, 2.352 mmol, 1 equivalent) as the HCl salt (1.15g, yield: 100%); MS(m / z):C 23 H 32 The calculated [M+H]+ value for N8O2 is 453.26; the measured value is 453.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.52-8.36 (m, 1H), 7.90-7.78 (m, 1H), 6.93 (s, 1H), 4.63 (m, 2H), 4.21-3.78 (m, 4H), 3.70-3.25 (m, 6H), 2.55 (br, 1H), 2.14 (m, 1H), 2.04-1.31 (m, 9H), 1.20-1.04 (m, 3H).

[0311] Step 3: Preparation of the conjugate of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0293] According to step 3 of Example 1, the title compound (0.2 g, yield: 59%, DSR=18%) was obtained from sodium hyaluronate (MW50 kDa, 0.161 g, 0.4 mmol, 1 equivalent) and N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.4 mmol, 1 equivalent); 1H-NMR (400 MHz, D2O) δ ppm 8.62-7.85 (m, 0.18H), 7.82-7.41 (m, 0.18H), 6.86-6.70 (m, 0.18H), 4.7-4.2 (m, 2H), 4.23-2.54 (m, 12.16H), 2.42 (br, 0.18H), 2.00 (s, 3H), 1.50-1.2 (m, 0.86H), 1.22-0.87(m, 1.05H).

[0312]

[0294] Following step 3 of Example 1, the corresponding product (0.224 g, yield: 67%, DSR=7%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0313] Example 20 Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbhydrazide with HA [ka]

[0314] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-ethylhydrazine-1-carboxylate [ka]

[0295] Following Step 1 of Example 1, the title product (1.745 g, yield: 43%) was obtained from tofacitinib (2.5 g, 8.114 mmol, 1 equivalent) and tert-butyl 1-ethylhydrazine-1-carboxylate (1.3 g, 8.114 mmol, 1 equivalent); MS (m / z): C 24 H 34 The calculated value of [M+H] for N8O4 is 499.27; the measured value is 499.2.

[0315] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride [ka]

[0296] According to step 2 of Example 1, the desired product was obtained from tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-ethylhydrazine-1-carboxylate (1.4 g, 2.8 mmol, 1 equivalent) as the HCl salt (1 g, yield: 82%); MS (m / z): C 19 H 26 The calculated [M+H]+ value for N8O2 is 399.22; the measured value is 399.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.45 (d, J = 4.8 Hz, 1H), 7.81 (d, J = 3.6 Hz, 1H), 7.00 (s, 1H), 4.76-4.67 (m, 1H), 4.19-3.79 (m, 3H), 3.72-3.31 (m, 8H), 2.56 (br, 1H), 2.06-1.70 (m, 2H), 1.35 (br, 3H), 1.14 (dd, J = 15.2, 7.1 Hz, 3H)

[0316] Step 3: Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbhydrazide with HA [ka]

[0297] Following step 3 of Example 1, the title compound (0.12 g, yield: 34%, DSR=9%) was obtained from sodium hyaluronate (MW50 kDa, 0.186 g, 0.46 mmol, 1 equivalent) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbozide hydrochloride (0.2 g, 0.46 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.4-8.3 (m, 0.09H), 7.69 (d, J = 23.0 Hz, 0.09H), 6.95 (d, J = 10.7 Hz, 0.09H), 4.60-4.41 (m, 2.08H), 3.84 -3.34 (m, 10H), 2.47 (m, 0.09H), 2.01 (s, 3H), 1.82-1.74 (m, 0.19H), 1.31 (br, 0.27H), 1.07 (br, 0.27H).

[0317]

[0298] Following step 3 of Example 1, the corresponding product (0.198 g, yield: 55.3%, DSR=5%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0318] Example 21 Preparation of a conjugate of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysinate with HA [ka]

[0319] Step 1: Preparation of methyl N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-ricinate [ka]

[0299] Following Step 1 of Example 1, the title product (2.2 g, yield: 57.4%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and methyl N6-(tert-butoxycarbonyl)-L-lysinate hydrochloride (1.9 g, 6.4 mmol, 1 equivalent); MS (m / z): C 29 H 42 The calculated value of [M+H] for N8O6 is 599.32; the measured value is 599.2.

[0320] Step 2: Preparation of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-ricinate hydrochloride [ka]

[0300] According to step 2 of Example 1, the desired product was obtained from methyl N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-ricinate (1.5 g, 2.5 mmol, 1 equivalent) as the HCl salt (1.34 g, yield: 100%); MS (m / z): C 24 H 34 The calculated value of [M+H] for N8O4 is 499.27; the measured value is 499.2. 1H-NMR (400 MHz, D2O) δ ppm 8.41 (d, J = 4.4 Hz,1H), 7.79 (dd, J = 9.0, 4.1 Hz, 1H), 6.88 (dd, J = 6.7, 2.3 Hz, 1H), 4.70-4.54 (m, 2H), 4.26-3.80 (m, 8H), 3.67-3.37 (m, 5H), 3.00 (t, J = 7.6 Hz, 3H), 2.67-2.41 (m, 1H), 2.05-1.93 (m, 2H), 1.89-1.62 (m, 4H), 1.62-1.41 (m, 3H), 1.15 (dd, J = 15.9, 7.1 Hz, 3H).

[0321] Step 3: Preparation of the conjugate of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysinate with HA [ka]

[0301] Following step 3 of Example 1, the title compound (0.2 g, yield: 61%, DSR=19%) was obtained from sodium hyaluronate (MW50 kDa, 0.151 g, 0.374 mmol, 1 equivalent) and methyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-ricinate hydrochloride (0.2 g, 0.374 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.25 (m, 0.2H), 7.56 (m, 0.2H), 6.72 (m, 0.2H), 4.72 - 2.85 (m, 15.2H), 2.45 (m, 0.4H), 2.02 (s, 3H), 1.56 (m, 1.2H), 1.31 (m, 0.2H), 1.11 (m, 0.6H). 1H-NMR (400 MHz, D2O) δppm 8.5-7.9 (m, 0.2H), 7.8-7.1 (m, 0.2H), 7.1-6.1 (m, 0.2H), 4.72-2.85 (m, 15.2H), 2.45 (br, 0.4H), 2.02 (s, 3H), 1.8-1.4 (m, 1.2H), 1.31 (br, 0.2H), 1.2-0.95 (m, 0.6H).

[0322]

[0302] Following step 3 of Example 1, the corresponding product (0.2 g, yield: 61%, DSR = 23.2%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0323] Example 22 Preparation of a conjugate between N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide and HA. [ka]

[0324] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)butyl)carbamate [ka]

[0303] To a mixture of tofacitinib (2 g, 6.4 mmol, 1 equivalent) and O-phenylcarbonochloride thioate (2.14 g, 7.04 mmol, 1.1 equivalents) in dichloromethane (40 mL), N,N-dimethylpyridine-4-amine (2.15 g, 17.6 mmol, 2.5 equivalents) was added under N2. The reaction mixture was heated under reflux for 3 hours. Then, tert-butyl(4-aminobutyl)carbamate (1.2 g, 6.4 mmol, 1 equivalent) was added, and the mixture was refluxed for a further 12 hours. After most of the tofacitinib had been consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain the title product (1.16 g, yield: 33.4%); MS (m / z): C 26 H 38 The calculated value of [M+H] for N8O3S is 543.28; the measured value is 543.2.

[0325] Step 2: Preparation of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride [ka]

[0304] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)butyl)carbamate (1.16 g, 2.14 mmol, 1 equivalent) as the HCl salt (1 g, yield: 100%); MS (m / z): C 21 H 30 N8OS [M+H]+ calculated value: 443.23; measured value: 443.2. 1H-NMR (400 MHz, D2O) δ ppm 8.36 (d, J = 36.1 Hz, 2H), 6.92 (s, 1H), 4.63 (d, J = 1.2 Hz, 1H), 4.1-3.75(m, 4H), 3.39 (s, 3H), 3.23 (s, 4H), 2.96 (t, J = 7.5 Hz, 3H), 2.47 (s, 1H), 2.0-1.6 (m, 5H), 1.23-1.06 (m, 4H).

[0326] Step 3: Preparation of the conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbocioamide with HA [ka]

[0305] Following step 3 of Example 1, the title compound (0.18 g, yield: 53%, DSR=20%) was obtained from sodium hyaluronate (MW50 kDa, 0.170 g, 0.42 mmol, 1 equivalent) and N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride (0.2 g, 0.42 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.8-7.4 (m, 0.48H), 7.0-6.0 (m, 0.12H), 4.7-4.25 (m, 2H), 4.18-3.03 (m, 12.8H), 2.47 (br, 0.2H), 2.03 (s, 3H), 1.83 (br, 1H), 1.30 (br, 0.2H), 1.15-1.0 (m, 0.6H).

[0327]

[0306] Following step 3 of Example 1, the corresponding product (0.196 g, yield: 56.8%, DSR=30%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0328]

[0307] Following step 3 of Example 1, the corresponding product (0.203 g, yield: 58.8%, DSR=8%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0329] Example 23 Preparation of a conjugate between N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide and HA. [ka]

[0330] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)benzyl)carbamate [ka]

[0308] Following Step 1 of Example 22, the title product (0.3 g, yield: 5.5%) was obtained from tofacitinib (3 g, 9.6 mmol, 1 equivalent) and tert-butyl(4-aminobenzyl)carbamate (2.134 g, 9.6 mmol, 1 equivalent); MS (m / z): C 29 H 36 The calculated value of [M+H] for N8O3S is 577.26; the measured value is 577.2.

[0331] Step 2: Preparation of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride [ka]

[0309] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)benzyl)carbamate (0.375 g, 0.65 mmol, 1 equivalent) as the HCl salt (0.26 g, yield: 78.8%); MS(m / z):C 24 H 28 N8OS [M+H]+ calculated value: 477.21; measured value: 477.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.43-8.30 (m, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 6.94 (s, 1H), 4.1-3.65 (m, 6H), 3.63-3.25 (m, 6H), 2.47 (s, 1H), 1.93-1.56 (m, 2H), 1.15-1.05 (m, 3H).

[0332] Step 3: Preparation of the conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbocioamide with HA [ka]

[0310] Following step 3 of Example 1, the title compound (0.18 g, yield: 55%, DSR=12%) was obtained from sodium hyaluronate (MW50 kDa, 0.157 g, 0.39 mmol, 1 equivalent) and N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride (0.2 g, 0.39 mmol, 1 equivalent); 1H-NMR (400 MHz, D2O) δ ppm 8.5-6.75 (m, 0.91H), 4.52 (m, 3H), 4.24-2.85 (m, 10.45H), 2.55-2.42 (m, 0.12H), 2.03 (s, 3H), 1.30 (m, 0.24H), 1.13 (m, 0.36H).

[0333]

[0311] Following step 3 of Example 1, the corresponding product (0.166 g, yield: 49.8%, DSR = 24.3%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0334]

[0312] Following step 3 of Example 1, the corresponding product (0.132 g, yield: 39.7%, DSR=14.7%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0335] Example 24 Preparation of a conjugate between N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbocioamide and HA. [ka]

[0336] Step 1: Preparation of tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)phenethyl)carbamate [ka]

[0313] Following Step 1 of Example 22, the title product (0.78 g, yield: 20.6%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and tert-butyl(4-aminophenethyl)carbamate (1.512 g, 6.4 mmol, 1 equivalent); MS (m / z): C 30 H 38 The calculated value of [M+H] for N8O3S is 591.28; the measured value is 591.2.

[0337] Step 2: Preparation of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride [ka]

[0314] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide)phenethyl)carbamate (0.55 g, 0.931 mmol, 1 equivalent) as the HCl salt (0.49 g, yield: 100%); MS(m / z):C 25 H 30 N8OS [M+H]+ Calculated value: 491.23; Measured value: 491.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.43-8.30 (m, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 6.95 (s, 1H), 4.69 (s, 1H), 3.97-3.71 (m, 3H), 3.61-3.37 (m, 4H), 3.23-3.04 (m, 4H), 2.95 (t, 2H), 2.48 (s, 1H), 1.92-1.62 (m, 2H), 1.12 (dd, J = 13.9, 6.0 Hz, 3H).

[0338] Step 3: Preparation of the conjugate of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbocioamide with HA [ka]

[0315] Following step 3 of Example 1, the title compound (0.18 g, yield: 55%, DSR=14%) was obtained from sodium hyaluronate (MW50 kDa, 0.153 g, 0.38 mmol, 1 equivalent) and N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride (0.2 g, 0.38 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.27-6.85 (m, 0.98H), 4.46 (br, 2H), 3.97-3.15 (m, 11.96H), 2.50-2.35 (m, 0.14H), 1.98 (s, 3H), 1.3-1.2 (m, 0.14H), 1.1-0.95 (m, 0.42H).

[0339]

[0316] Following step 3 of Example 1, the corresponding product (0.185 g, yield: 56%, DSR=15%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0340] Example 25 Preparation of a conjugate between N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide and HA [ka]

[0341] Step 1: Preparation of tert-butyl(1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-1-thioxo-5,8,11-trioxa-2-azatridecane-13-yl)carbamate [ka]

[0317] Following Step 1 of Example 22, the title product (1.12 g, yield: 27%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl) carbamate (1.871 g, 6.4 mmol, 1 equivalent); MS (m / z): C 30 H 46 [M+H]+ calculated value for N8O6S: 647.33; measured value: 647.2.

[0342] Step 2: Preparation of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride [ka]

[0318] According to step 2 of Example 1, the desired product was obtained from tert-butyl(1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-1-thioxo-5,8,11-trioxa-2-azatridecane-13-yl)carbamate (1.1 g, 1.7 mmol, 1 equivalent) as the HCl salt (0.84 g, yield: 85%); MS(m / z):C 25 H 38 [M+H]+ calculated value for N8O4S: 547.27; measured value: 547.2. 1H-NMR (400 MHz, D2O) δ ppm 8.5-8.0 (m, 2H), 6.61 (s, 1H), 4.75-4.40 (m, 2H), 4.05-3.5 (m, 18H), 3.40-3.3 (m, 6H), 2.45 (br, 1H), 2.00-1.6 (m, 2H), 1.17-1.04 (m, 3H).

[0343] Step 3: Preparation of the conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide with HA [ka]

[0319] Following step 3 of Example 1, the title compound (0.17 g, yield: 55%, DSR=31%) was obtained from sodium hyaluronate (MW50 kDa, 0.137 g, 0.34 mmol, 1 equivalent) and N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride (0.2 g, 0.34 mmol, 1 equivalent); 1 ¹H-NMR (400 MHz, D2O) δ ppm 8.2-7.6 (m, 0.43H), 7.4-6.8 (m, 0.21H), 6.80-6.0 (m, 0.3H), 4.65-4.1 (m, 4.96H), 4.0-3.0 (m, 15H), 3.25 (m, 3.09H), 2.30 (br, 0.31H), 1.93 (s, 3H), 1.62-1.15 (m, 0.62H), 1.10-0.75 (m, 0.93H). Following step 3 of Example 1, the corresponding product (0.185 g, yield: 58.8%, DSR=25%) was obtained by reaction with sodium hyaluronate (MW500kDa).

[0344]

[0320] Following step 3 of Example 1, the corresponding product (0.231 g, yield: 73.4%, DSR=30%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0345] Example 26 Preparation of a conjugate of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysinate with HA [ka]

[0346] Step 1: Preparation of methyl N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-ricinate [ka]

[0321] Following Step 1 of Example 22, the title product (0.675 g, yield: 17.2%) was obtained from tofacitinib (2 g, 6.4 mmol, 1 equivalent) and methyl N6-(tert-butoxycarbonyl)-L-lysinate hydrochloride (1.9 g, 6.4 mmol, 1 equivalent); MS (m / z): C 29 H 42 [M+H]+ calculated value for N8O5S: 615.30; measured value: 615.2.

[0347] Step 2: Preparation of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-ricinate hydrochloride [ka]

[0322] According to step 2 of Example 1, the desired product was obtained from methyl N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-ricinate (0.625 g, 1.017 mmol, 1 equivalent) as the HCl salt (0.56 g, yield: 100%); MS(m / z):C 24 H 34 The calculated value of [M+H] for N8O3S is 515.25; the measured value is 515.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.45 (s, 1H), 8.33 (s, 1H), 6.92 (s, 1H), 5.09 (t, J = 6.2 Hz, 1H), 4.63 (br, 1H), 3.95- 3.67 (m, 5H), 3.60-3.28 (m, 4H), 3.23 (s, 3H), 2.88 (br, 2H), 2.47 (s, 1H), 2.21-1.96 (m, 2H), 1.92-1.42 (m, 6H), 1.10 (d, J = 6.8 Hz 3H).

[0348] Step 3: Preparation of the conjugate of methyl(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysinate with HA [ka]

[0323] Following step 3 of Example 1, the title compound (0.13 g, yield: 41%, DSR=10%) was obtained from sodium hyaluronate (MW50 kDa, 0.145 g, 0.36 mmol, 1 equivalent) and methyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-ricinate hydrochloride (0.2 g, 0.36 mmol, 1 equivalent); 1H-NMR (400 MHz, D2O) δ ppm 8.75-7.5 (m, 0.27H), 6.87 (br, 0.03H), 5.25 (m, 0.14H), 4.65-4.21 (m, 1.86H), 4.0-2.8 (m, 11.6H), 2.44 (br, 0.1H), 2.00 (s, 3H), 1.7-1.4 (m, 0.62H), 1.28 (t, 0.1H), 1.14-1.0 (m, 0.3H).

[0349]

[0324] Following step 3 of Example 1, the corresponding product (0.117 g, yield: 36.3%, DSR=10%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0350]

[0325] Following step 3 of Example 1, the corresponding product (0.161 g, yield: 50%, DSR=5.7%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0351] Example 27 Preparation of a conjugate of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0352] Step 1: Preparation of 4-(((tert-butoxycarbonyl)amino)methyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0326] Following Step 1 of Example 1, the title product (1.3 g, yield: 53.5%) was obtained from tofacitinib (1.32 g, 4.214 mmol, 1 equivalent) and tert-butyl (4-(hydroxymethyl)benzyl)carbamate (2 g, 4.214 mmol, 1 equivalent); MS (m / z): C 30 H 37 The calculated value of N7O5's [M+H] is 576.29; the measured value is 576.2.

[0353] Step 2: Preparation of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0327] According to step 2 of Example 1, the desired product was obtained from 4-(((tert-butoxycarbonyl)amino)methyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.3 g, 2.25 mmol, 1 equivalent) as the HCl salt (1 g, yield: 87%); MS(m / z):C 25 H 29 The calculated value of [M+H]+ for N7O3 is 476.23; the measured value is 476.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.30 (dd, J = 36.1, 9.5 Hz, 1H), 7.75-7.23 (m, 5H), 6.84 (d, J = 15.6 Hz, 1H), 4.70 (br, 3H), 4.35-3.71 (m, 6H), 3.71-3.23 (m, 5H), 2.50 (d, J = 33.4 Hz, 1H), 2.03-1.62 (m, 2H), 1.17-0.99 (m, 3H).

[0354] Step 3: Preparation of the conjugate of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0328] Following step 3 of Example 1, the title compound (0.2 g, yield: 60%, DSR=22%) was obtained from sodium hyaluronate (MW50 kDa, 0.157 g, 0.39 mmol, 1 equivalent) and 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (0.2 g, 0.39 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.37-7.89 (m, 0.18H), 7.75-7.22 (m, 1.36H), 4.71-4.32 (m, 2.75H), 4.29-3.20 (m, 12.02H), 3.14 (br, 0.34H), 2.32 (br, 0.22H), 2.02 (s, 3H), 1.31 (br, 0.45H), 1.06 (br, 0.65H).

[0355]

[0329] Following step 3 of Example 1, the corresponding product (0.2 g, yield: 60%, DSR = 43.6%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0356]

[0330] Following step 3 of Example 1, the corresponding product (0.175 g, yield: 52.6%, DSR = 35.3%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0357] Example 28 Preparation of a conjugate of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0358] Step 1: Preparation of 4-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0331] Following Step 1 of Example 1, the title product (1.7 g, yield: 28%) was obtained from tofacitinib (3 g, 9.62 mmol, 1 equivalent); MS (m / z): C 32 H 39 [M+H] of N7O7: Calculated value, 634.29; Measured value, 634.2.

[0359] Step 2: Preparation of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0332] According to step 2 of Example 1, the desired product was obtained from 4-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (1 g, 1.578 mmol, 1 equivalent) as the HCl salt (0.9 g, yield: 100%); MS(m / z):C 27 H 31 The calculated value of N7O5's [M+H] is 534.24; the measured value is 534.2. 1 H-NMR (400 MHz, CD3OD) δ ppm 8.55-8.34 (m, 1H), 7.52-7.42 (m, 3H), 7.41-6.74 (m,3H), 5.28 (s, 1H), 4.46-4.21 (m, 1H), 4.09 -3.85 (m, 5H), 3.82 (d, J = 4.2 Hz, 3H), 3.76-3.39 (m, 5H), 3.26-3.03 (m, 1H), 2.55 (br, 1H), 2.0-1.65 (m, 2H), 1.16 (dd, J = 13.2, 5.2 Hz, 3H).

[0360] Step 3: Preparation of the conjugate of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0333] According to step 3 of Example 1, the title compound (0.2 g, yield: 32%, DSR=20%) was obtained from sodium hyaluronate (MW50 kDa, 0.141 g, 0.35 mmol, 1 equivalent) and 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (0.2 g, 0.35 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 7.87 - 6.43 (m, 1.4 H), 4.51 (m, 2H), 3.70 (m, 14.35H), 2.48 (m, 0.2H), 1.99 (s, 3H), 1.81 - 1.67 (m, 0.2H), 1.29 (m, 0.2H), 1.06 (m, 0.6H).

[0361]

[0334] Following step 3 of Example 1, the corresponding product (0.16 g, yield: 50%, DSR=22%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0362]

[0335] Following step 3 of Example 1, the corresponding product (0.238 g, yield: 74.5%, DSR=22%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0363] Example 29 Preparation of a conjugate of 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka] Step 1: Preparation of 8-((tert-butoxycarbonyl)amino)octyl4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0336] Following Step 1 of Example 1, the title product (0.7 g, yield: 33%) was obtained from tofacitinib (1.15 g, 3.67 mmol, 1 equivalent) and tert-butyl(8-hydroxyoctyl)carbamate (0.9 g, 3.668 mmol, 1 equivalent); MS (m / z): C 30 H 45 The calculated value of N7O5's [M+H] is 584.35; the measured value is 584.3. 1 H-NMR (400 MHz, CDCl3) δ ppm 8.46 (d, J = 8.8 Hz, 1H), 7.46 (dd, J = 10.6, 4.2 Hz,1H), 6.63 (d, J = 4.1 Hz, 1H), 5.13 (s, 1H), 4.65-4.33 (m, 2H), 4.16-3.99 (m, 1H), 3.89-3.74 (m, 1H), 3.69-3.43 (m, 4H), 3.37 (d, J = 18.7 Hz, 2H), 3.10 (d, J = 6.4 Hz, 2H), 2.62-2.41 (m, 1H), 2.02-1.70 (m, 4H), 1.45-1.3 (m, 12H), 1.08 (dd, J = 14.0, 7.1 Hz, 3H).

[0364] Step 2: Preparation of 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0337] According to step 2 of Example 1, the desired product was obtained from 8-((tert-butoxycarbonyl)amino)octyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.7g, 1.2 mmol, 1 equivalent) as the HCl salt (0.6g, yield: 96%); MS(m / z):C 25 H 37 The calculated value of [M+H] for N7O3 is 484.30; the measured value is 484.2.

[0365] Step 3: Preparation of the conjugate of 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0338] Following step 3 of Example 1, the title compound (0.19 g, yield: 54%, DSR=14%) was obtained from sodium hyaluronate (MW50 kDa, 0.153 g, 0.38 mmol, 1 equivalent) and 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (0.2 g, 0.38 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.39 (m, 0.14 H), 7.67 (m, 0.14 H), 6.95 (m, 0.14 H), 4.53 (m, 2H), 3.73 (m, 12H), 2.51 (m, 0.24 H), 2.04 (s, 3H), 1.87 (m, 0.82H), 1.48 (m, 0.92H), 1.11 (m, 0.57H).

[0366]

[0339] Following step 3 of Example 1, the corresponding product (0.191 g, yield: 58.23%, DSR=5.3%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0367] Example 30 Preparation of a conjugate of 4-aminobutyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0368] Step 1: Preparation of 4-(tert-butoxycarbonylamino)butyl4-[[(3S,4S)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0340] Triethylamine (2.7 mL, 19.2 mmol) was added to a stirred mixture of tofacitinib (3000 mg, 9.6 mmol) and bis(4-nitrophenyl) carbonate (3.22 mg, 10.6 mmol) in dichloromethane (60 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 hours. The reaction mixture was then cooled to room temperature. 4-(tert-butoxycarbonylamino)-1-butanol (2000 mg, 10.6 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and washed with saturated NaHCO3 solution, 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 (ethyl acetate:hexane = 1:1) to obtain the title compound (2.64 g, yield: 52.1%). MS(m / z):C 26 H 37 The calculated value of N7O5's [M+H]+ is 528.28; the measured value is 528.2.

[0369] Step 2: Preparation of 4-aminobutyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0341] According to step 2 of Example 1, the title compound was obtained as a white solid (2.59 g, yield: 100%) from 4-(tert-butoxycarbonylamino)butyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (2.955 g, 5.6 mmol). MS(m / z):C 21 H 29 The calculated value of [M+H] for N7O3 is 428.23; the measured value is 428.2. 1 H NMR (400 MHz, D2O) δ ppm 8.39 (d, J = 6.9 Hz, 1H), 7.63 (d, J = 3.8 Hz, 1H), 6.96 (s, 1H), 5.04 (s, 1H), 4.53 (t, J = 6.2 Hz, 2H), 4.01 - 3.70 (m, 4H), 3.67 - 3.54 (m, 1H), 3.41 (d, J = 30.2 Hz, 4H), 3.02 (dd, J = 17.9, 10.6 Hz, 2H), 2.46 (s, 1H), 1.97 - 1.59 (m, 6H), 1.12 - 0.96 (m, 3H).

[0370] Step 3: Preparation of a conjugate of 4-aminobutyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0342] According to step 3 of Example 1, 0.13 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 161.2 mg, 0.399 mmol) and 4-aminobutyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.399 mmol), yield: 36.7%, DSR=12%; 1 H NMR (400 MHz, D2O) δ ppm 8.20 - 8.02 (m, 0.12H), 7.53 - 7.25 (m, 0.12H), 6.82 - 6.62 (m, 0.12H), 4.53 - 4.15 (m, 2.12H), 3.93 - 2.89 (m, 11.56H), 2.02 - 1.44 (m, 3.6H), 1.18 - 1.09 (m, 0.12H), 1.01 - 0.82 (m, 0.36H).

[0371]

[0343] In step 3 of Example 1, the reaction with sodium hyaluronate (MW500kDa) yielded the corresponding product (0.125g, yield: 35.3%, DSR=37%).

[0372]

[0344] Following step 3 of Example 1, the corresponding product (0.134 g, yield: 37.8%, DSR=32%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0373] Example 31 Preparation of a conjugate of (4-aminophenyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0374] Step 1: Preparation of [4-(tert-butoxycarbonylamino)phenyl]4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0345] Following Step 1 of Example 1, the title compound (1.1 g, yield: 40%) was obtained from tofacitinib (1562 mg, 5 mmol) and Tert-butyl(4-hydroxyphenyl)carbamate (1256 mg, 6 mmol). MS(m / z):C 28 H 33 The calculated value of N7O5's [M+H]+ is 548.25; the measured value is 548.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.48 (d, J = 8.0 Hz, 1H), 8.14 - 8.05 (m, 1H), 7.61 (dd, J = 8.0, 4.2 Hz, 1H), 7.42 (m,1H), 7.23 (d, J = 8.9 Hz, 2H), 6.89 - 6.74 (m, 1H), 6.58 (s, 1H), 5.15 (d, J = 3.9 Hz, 1H), 4.11 - 4.01 (m, 1H), 3.88 - 3.78 (m, 1H), 3.67 - 3.45 (m, 4H), 3.43 - 3.31 (m, 3H), 2.59 - 2.44 (m, 1H), 1.94 (tdd, J = 23.2, 8.8, 4.5 Hz, 1H), 1.83 - 1.69 (m, 1H), 1.51 (m, 9H), 1.15 - 1.03 (m, 3H).

[0375] Step 2: Preparation of (4-aminophenyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0346] According to step 2 of Example 1, the title compound was obtained from [4-(tert-butoxycarbonylamino)phenyl]4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.8 g, 1.46 mmol) as a white solid (0.52 g, yield: 73.6%); MS (m / z): C 23 H 25 The calculated value of [M+H] for N7O3 is 448.20; the measured value is 448.3.

[0376] Step 3: Preparation of the conjugate of (4-aminophenyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0347] Following step 3 of Example 1, 0.126 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 116 mg, 0.288 mmol) and (4-aminophenyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (150 mg, 0.288 mmol), yielding 48.3% and a DSR of 10%; 1 H NMR (400 MHz, D2O) δ ppm 8.26 - 8.17 (m, 0.1H), 7.72 - 7.51 (m, 0.2H), 7.38 -7.11 (m,0. 3H), 6.90 -6.78 (m, 0.1H), 4.53 - 4.25 (m, 2.1H), 4.07 - 3.09 (m, 10.9H), 2.49 - 2.34 (m, 0.1H), 2.12 - 1.78 (m, 3.1H), 1.61 - 1.50 (m, 0.1H), 1.26 - 0.92 (m, 0.3H).

[0377]

[0348] In step 3 of Example 1, the corresponding product (0.123 g, yield: 47.1%, DSR: 24%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0378]

[0349] Following step 3 of Example 1, the corresponding product (0.138 g, yield: 52.9%, DSR: 8%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0379] Example 32 Preparation of a conjugate of azetidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0380] Step 1: Preparation of (1-tert-butoxycarbonylazetidine-3-yl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0350] Following Step 1 of Example 1, the title compound (0.48 g, yield: 23.4%) was obtained from tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxyazetidine-1-carboxylate (831.4 mg, 4.8 mmol). MS(m / z):C 25 H 33 The calculated value of N7O5's [M+H] is 512.25; the measured value is 512.1. 1H NMR (400 MHz, CDCl3) δ ppm 8.47 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 11.6, 4.2 Hz, 1H), 6.65 (dd, J = 15.6, 4.0 Hz, 1H), 5.14 (d, J = 3.0 Hz, 1H), 4.38 (dd, J = 10.0, 7.0 Hz, 2H), 4.11 - 3.75 (m, 3H), 3.67 - 3.45 (m, 5H), 3.41 - 3.33 (m, 3H), 2.59 - 2.41 (m, 1H), 2.02 - 1.84 (m, 1H), 1.76 (ddd, J = 23.8, 13.5, 8.5 Hz, 2H), 1.45 (d, J = 6.8 Hz, 9H), 1.11 - 1.02 (m, 3H).

[0381] Step 2: Preparation of azetidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0351] According to step 2 of Example 1, the title compound was obtained as a white solid (0.366 g, yield: 95%) from (1-tert-butoxycarbonylazetidine-3-yl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.44 g, 0.86 mmol). MS(m / z):C 20 H 25 The calculated value of [M+H] for N7O3 is 412.20; the measured value is 412.3.

[0382] Step 3: Preparation of the conjugate of azetidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0352] Following step 3 of Example 1, 0.078 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 83.2 mg, 0.207 mmol) and azetidine-3-yl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (100 mg, 0.207 mmol), yielding 43.4% and a DSR of 12%. 1 H NMR (400 MHz, D2O) δ ppm 8.27 - 8.17 (s, 0.12H), 7.63 - 7.50 (m, 0.12H), 6.92 - 6.78 (m, 0.12H), 4.58 - 4.26 (m, 7.0 Hz, 2.12H), 4.07 - 3.05 (m, 11.56H), 2.45 - 2.35 (m, 0.12H), 2.13 - 1.55 (m, 3.24H), 1.23 (m, 0.12H), 1.11 - 0.89 (m, 0.36H).

[0383] Example 33 Preparation of a conjugate of pyrrolidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0384] Step 1: Preparation of (1-tert-butoxycarbonylpyrrolidine-3-yl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0353] Following Step 1 of Example 1, the title compound (0.94 g, yield: 44.7%) was obtained from tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxypyrrolidine-1-carboxylate (898.8 mg, 4.8 mmol). MS(m / z):C 26 H 35 The calculated value of N7O5's [M+H] is 526.26; the measured value is 526.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.47 (d, J = 6.3 Hz, 1H), 7.38 (dd, J = 11.2, 4.1 Hz, 1H), 6.63 (d, J = 4.1 Hz, 1H), 5.11 (t, J = 11.8 Hz, 1H), 4.08 - 3.77 (m, 2H), 3.73 - 3.47 (m, 8H), 3.36 (d, J = 17.7 Hz, 3H), 2.58 - 2.42 (m, 1H), 2.37 - 2.18 (m, 2H), 1.93 (dddd, J = 23.5, 18.7, 9.0, 4.5 Hz, 1H), 1.82 - 1.72 (m, 2H), 1.47 (s, 9H), 1.08 (dd, J = 14.4, 7.0 Hz, 3H).

[0385] Step 2: Preparation of pyrrolidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0354] According to step 2 of Example 1, the title compound was obtained from (1-tert-butoxycarbonylpyrrolidine-3-yl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.6 g, 1.14 mmol) as a white solid (0.526 g, yield: 100%). MS(m / z):C 21 H 27The calculated value of [M+H] for N7O3 is 426.21; the measured value is 426.3.

[0386] Step 3: Preparation of the conjugate of pyrrolidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0355] Following step 3 of Example 1, 0.178 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 161.6 mg, 0.401 mmol) and pyrrolidine-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.401 mmol), yielding 50% and a DSR of 20%; 1 H NMR (400 MHz, D2O) δ ppm 8.34 - 8.18 (m, 0.2H), 7.66 - 7.38 (m, 0.2H), 6.87 -6.68 (m, 0.2H), 4.58 - 4.26 (m, 2.2H), 4.07 - 3.07 (m, 12.6H), 2.53 - 2.19 (m, 0.6H), 2.10 - 1.60 (m, 3.4H), 1.29 - 1.18 (m, 0.2H), 1.15 - 0.86 (m, 0.6H).

[0387]

[0356] Following step 3 of Example 1, the corresponding product (0.185 g, yield: 52.2%, DSR: 33%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0388] Example 34 Preparation of a conjugate of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0389] Step 1: Preparation of 3-(tert-butoxycarbonylamino)propyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0357] Following Step 1 of Example 1, the title compound (1.03 g, yield: 50%) was obtained from tofacitinib (1249.5 mg, 4 mmol) and 3-(Boc-amino)-1-propanol (841.1 mg, 4.8 mmol). MS(m / z):C 25 H 35 The calculated value of N7O5's [M+H] is 514.26; the measured value is 514.2. 1 H NMR (400 MHz, CDCl3) δ ppm 8.53 (d, J = 10.4 Hz, 1H), 7.53 (dd, J = 10.8, 4.0 Hz, 1H), 7.15 (s, 1H), 6.67 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 30.4 Hz, 1H), 4.57 (dd, J = 14.1, 8.5 Hz, 2H), 4.06 - 3.78 (m, 2H), 3.67 -3.48 (m, 4H), 3.47 - 3.41 (m, 2H), 3.41- 3.33 (m, 3H), 2.56 - 2.36 (m, 1H), 2.09 - 2.01 (m, 1H), 1.93 (tdd, J = 22.0, 8.8, 4.5 Hz, 1H), 1.83 - 1.65 (m, 2H), 1.45 (s, 9H), 1.07 (dd, J = 16.9, 7.1 Hz, 3H).

[0390] Step 2: Preparation of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0358] According to step 2 of Example 1, the title compound was obtained as a white solid (0.9 g, 100%) from 3-(tert-butoxycarbonylamino)propyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.03 g, 2 mmol). MS(m / z):C 20 H 27 [M+H]+ of N7O3: Calculated value, 414.21; Measured value, 414.3.

[0391] Step 3: Preparation of the conjugate of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0359] According to step 3 of Example 1, 0.162 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 165.6 mg, 0.411 mmol) and 3-aminopropyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.411 mmol), yielding 45.2% and a DSR of 15%. 1H NMR (400 MHz, D2O) δ ppm 8.34 - 8.20 (m, 0.15H), 7.63 - 7.50 (m, 0.15H), 6.91 - 6.78 (m, 0.15H), 4.49 - 4.31 (m, 2.15H), 4.06 - 3.07 (m, 11.95H), 2.44 - 2.35 (m, 0.15H), 2.16 - 1.56 (m, 3.3H), 1.21 (t, J = 7.0 Hz, 0.15H), 1.05 - 0.90 (m, 0.45H).

[0392]

[0360] Following step 3 of Example 1, the corresponding product (0.161 g, yield: 44.9%, DSR: 17%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0393] Example 35 Preparation of a conjugate of (4-aminocyclohexyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0394] Step 1: Preparation of [4-(tert-butoxycarbonylamino)cyclohexyl]4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0361] Following Step 1 of Example 1, the title compound (0.851 g, yield: 45%) was obtained from tofacitinib (1249.5 mg, 4 mmol) and tert-butyl(4-hydroxycyclohexyl)carbamate (1033.4 mg, 4.8 mmol). MS(m / z):C 28 H 39The calculated value of N7O5's [M+H] is 554.30; the measured value is 554.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.46 (d, J = 5.8 Hz, 1H), 7.42 (dd, J = 9.5, 4.2 Hz, 1H), 6.61 (d, J = 4.1 Hz, 1H), 5.19 - 5.04 (m, 1H), 5.03 - 4.89 (m, 1H), 4.45 (s, 1H), 4.08 - 3.76 (m, 2H), 3.63 - 3.46 (m, 4H), 3.36 (d, J = 16.4 Hz, 3H), 2.49 (ddd, J = 24.3, 11.9, 6.4 Hz, 1H), 2.26 - 2.06 (m, 4H), 1.93 (dddd, J = 18.5, 13.7, 8.9, 4.4 Hz, 1H), 1.82 - 1.59 (m, 4H), 1.45 (s, 9H), 1.34 (td, J = 13.3, 2.7 Hz, 2H), 1.15 - 0.99 (m, 3H).

[0395] Step 2: Preparation of (4-aminocyclohexyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0362] According to step 2 of Example 1, the title compound was obtained as a white solid (0.55 g, yield: 100%) from [4-(tert-butoxycarbonylamino)cyclohexyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.62 g, 1.12 mmol). MS(m / z):C 23 H 31 The calculated value of [M+H] for N7O3 is 454.24; the measured value is 454.3.

[0396] Step 3: Preparation of the conjugate of (4-aminocyclohexyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0363] Following step 3 of Example 1, 0.173 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 153 mg, 0.38 mmol) and (4-aminocyclohexyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.38 mmol), yielding 49.9% and a DSR of 11%. 1 H NMR (400 MHz, D2O) δ ppm 8.24 - 8.02 (m, 0.11H), 7.57 - 7.37 (m, 0.11H), 6.80 - 6.64 (m, 0.11H), 4.96 - 4.86 (m, 0.11H), 4.57 - 4.27 (m, 2H), 4.07 - 3.04 (m, 10.99H), 2.46 - 2.30 (m, 0.11H), 2.27 - 1.76 (m, 3.77H), 1.74 - 1.33 (m, 0.66H), 1.22 (d, J = 6.9 Hz, 0.11H), 1.09 - 0.85 (m, 0.33H).

[0397]

[0364] Following step 3 of Example 1, the corresponding product (0.162 g, yield: 46.7%, DSR: 27%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0398] Example 36 Preparation of a conjugate of 4-piperidyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0399] Step 1: Preparation of (1-tert-butoxycarbonyl-4-piperidyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0365] Following Step 1 of Example 1, the title compound (0.537 g, yield: 24.9%) was obtained from tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (966 mg, 4.8 mmol). MS(m / z):C 27 H 37 The calculated value of N7O5's [M+H] is 540.28; the measured value is 540.2.

[0400] Step 2: Preparation of 4-piperidyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0366] According to step 2 of Example 1, the title compound was obtained from (1-tert-butoxycarbonyl-4-piperidyl)4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.47 g, 0.87 mmol) as a white solid (0.25 g, yield: 58%). MS(m / z):C 22 H 29 The calculated value of N7O3's [M+H] is 440.23; the measured value is 440.3.

[0401] Step 3: Preparation of the conjugate of 4-piperidyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0367] Following step 3 of Example 1, 0.161 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 169.7 mg, 0.421 mmol) and 4-piperidyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.421 mmol), yielding 44.4% and a DSR of 11%. 1 H NMR (400 MHz, D2O) δ ppm 8.27 - 8.04 (m, 0.11H), 7.61 - 7.38 (m,0.1 1H), 6.84 - 6.68 (m, 0.11H), 4.61 - 4.28 (m, 2.11H), 4.12 - 3.05 (m, 11.43H), 2.41 - 2.30 (m, 0.11H), 2.19 - 1.58 (m, 3.66H), 1.20 (t, J = 6.9 Hz, 0.11H), 1.08 - 0.85 (m, 0.33H).

[0402] Example 37 Preparation of a conjugate of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0403] Step 1: Preparation of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0368] Triethylamine (1.1 mL, 8 mmol) was added to a stirred mixture of tofacitinib (1250 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1460 mg, 4.8 mmol) in dichloromethane (40 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 hours. The reaction mixture was then cooled to room temperature. Tert-butyl(2-(2-hydroxyethoxy)ethyl) carbamate (985.2 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and washed with saturated NaHCO3 solution, water and saturated brine solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the title compound (1.3 g, yield: 59.8%). MS(m / z):C 26 H 37 The calculated value of [M+H] for N7O6 is 544.28; the measured value is 544.2.

[0404] Step 2: Preparation of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride [ka]

[0369] According to step 2 of Example 1, the title compound was obtained as a white solid (1.1 g, yield: 95.9%) from 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.3 g, 2.39 mmol). MS(m / z):C 21 H29 [M+H] of N7O4: Calculated value, 444.22; Measured value, 444.2.

[0405] Step 3: Preparation of the conjugate of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0370] Following step 3 of Example 1, 0.175 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 156.4 mg, 0.388 mmol) and 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.388 mmol), yielding 50% and a DSR of 15%; 1 H NMR (400 MHz, D2O) δ ppm 8.23 ​​- 8.09 (m, 0.15H), 7.58 - 7.39 (m, 0.15H), 6.86 - 6.65 (m, 0.15H), 4.58 - 4.25 (m, 2.15H), 4.02 - 2.94 (m, 12.55H), 2.43 - 2.28 (m, 0.15H), 2.16 - 1.56 (m, 3.3H), 1.10 - 0.89 (m, 0.45H).

[0406]

[0371] Following step 3 of Example 1, the corresponding product (0.182 g, yield: 52%, DSR: 17%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0407] Example 38 Preparation of the methyl(2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate conjugate [ka]

[0408] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate [ka]

[0372] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1323 mg, 3 mmol), (S)-methyl6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1336 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 863 mg, 4.5 mmol), and 1-hydroxybenzotriazole (HOBt, 608 mg, 4.5 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (2.08 mL, 15 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:150-1:80) to obtain the title compound (1.726 g, yield: 83.3%). MS(m / z):C 33 H 49 [M+H]+ calculated value of N9O7: 684.37; measured value: 684.1. 1H NMR (400 MHz, CDCl3) δ ppm9.89 (dd, J = 13.5, 7.5 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 7.72 (dd, J = 11.9, 4.0 Hz, 1H), 6.75 - 6.50 (m, 2H), 5.14 (s, 1H), 4.75 - 4.48 (m, 2H), 4.12 - 3.76 (m, 2H), 3.73 - 3.32 (m, 9H), 3.11 (d, J = 5.4 Hz, 2H), 2.58 - 2.42 (m, 1H), 2.41 - 2.25 (m, 2H), 2.09 - 1.65 (m, 6H), 1.54 - 1.32 (m, 15H), 1.10 (dd, J = 12.7, 7.1 Hz, 3H).

[0409] Step 2: Preparation of methyl(2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate hydrochloride [ka]

[0373] According to step 2 of Example 1, the title compound was obtained from methyl(2S)-6-(tert-butoxycarbonylamino)-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate (1.5 g, 2.137 mmol) as a white solid (1.426 g, yield: 99.6%). MS(m / z):C 28 H 41 The calculated value of N9O5's [M+H] is 584.32; the measured value is 584.1.

[0410] Step 3: Preparation of the methyl(2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate conjugate. [ka]

[0374] Following step 3 of Example 1, 0.13 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 123 mg, 0.305 mmol) and methyl(2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate hydrochloride (200 mg, 0.36 mmol), yield: 45%, DSR: 5%; 1 H NMR (400 MHz, D2O) δ ppm 8.29 - 8.16 (m, 0.05H), 7.64 - 7.51 (m, 0.05H), 6.82 - 6.65 (m, 0.05H), 4.57 - 4.22 (m, 2.05H), 3.99 - 2.89 (m, 10.75H), 2.42 - 2.29 (m, 0.1H), 2.14 - 1.55 (m, 3.3H), 1.47 - 1.35 (m, 0.3H), 1.24 - 1.11 (m, 0.15H).

[0411] Example 39 Preparation of a conjugate of N-[4-(2-aminoethylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0412] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid [ka]

[0375] Triethylamine (33.9 g, 336.3 mmol) was added to a stirred mixture of tofacitinib (30 g, 96.1 mmol) and bis(4-nitrophenyl) carbonate (43.3 g, 142.4 mmol) in dichloromethane (300 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 16 hours. The reaction mixture was then cooled to 0-10 °C. 300 mL aqueous solutions of 4-aminobutyric acid (19.8 g, 192.2 mmol), triethylamine (19.4 g, 192.2 mmol), and tetrabutylammonium bromide (0.3 g) were added. The reaction mixture was stirred at 0-10 °C for 16 hours, the pH of the reaction mixture was adjusted to 3-4 with 20% aqueous citric acid solution, and the organic layer was washed with 10% aqueous citric acid solution, 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 (methanol / dichloromethane = 1:100-1:50) to obtain the title compound (10.3 g, 24%). MS(m / z):C 21 H 27 [M+H] of N7O4: Calculated value, 442.2; Measured value, 442.3.

[0413] Step 2: Preparation of tert-butyl N-[2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]ethyl]carbamate [ka]

[0376] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1323 mg, 3 mmol), N-Boc-ethylenediamine (721 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 863 mg, 4.5 mmol), and 1-hydroxybenzotriazole (HOBt, 607.5 mg, 4.5 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (1.25 mL, 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:100~1:50) to obtain the title compound (1.58 g, yield: 90%). MS(m / z):C 28 H 41 The calculated value of N9O5's [M+H] is 584.32; the measured value is 584.2. 1 H NMR (400 MHz, CDCl3) δ ppm 10.02 - 9.67 (m, 1H), 8.29 (d, J = 8.9 Hz, 1H), 7.72 (dd, J = 10.5, 4.0 Hz, 1H), 6.58 (dd, J = 14.2, 10.2 Hz, 2H), 5.32 (d, J = 17.1 Hz, 1H), 5.13 (s, 1H), 4.20 - 3.70 (m, 2H), 3.69 - 3.44 (m, 6H), 3.39 - 3.14 (m, 7H), 2.60 - 2.40 (m, 1H), 2.26 (dt, J = 15.3, 7.4 Hz, 2H), 2.11 - 1.83 (m, 3H), 1.83 - 1.70 (m, 1H), 1.41 (d, J = 28.2 Hz, 9H), 1.09 (dd, J = 12.6, 7.1 Hz, 3H).

[0414] Step 3: Preparation of N-[4-(2-aminoethylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0377] According to step 2 of Example 1, the title compound was obtained as a white solid (1.04 g, yield: 100%) from tert-butyl N-[2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]ethyl]carbamate (1.18 g, 2 mmol). MS(m / z):C 23 H 33 The calculated value of [M+H] for N9O3 is 484.27; the measured value is 484.3.

[0415] Step 4: Preparation of the conjugate of N-[4-(2-aminoethylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0378] Following step 3 of Example 1, 0.174 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 145 mg, 0.36 mmol) and N-[4-(2-aminoethylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.36 mmol), yielding 51.4% and a DSR of 30%; 1H NMR (400 MHz, D2O) δ ppm 8.24 - 8.13 (m, 0.2H), 7.61 - 7.47 (m, 0.2H), 6.79 - 6.61 (m, 0.2H), 4.58 - 4.16 (m, 2.2H), 4.10 - 2.77 (m, 13H), 2.42 - 2.18 (m, 0.6H), 2.04 - 1.55 (m, 4H), 1.06 - 0.85 (m, 0.6H).

[0416]

[0379] In step 3 of Example 1, the reaction with sodium hyaluronate (MW500kDa) yielded the corresponding product (0.186g, yield: 54.9%, DSR: 39%).

[0417]

[0380] Following step 3 of Example 1, the corresponding product (0.189 g, yield: 55.8%, DSR: 30%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0418] Example 40 Preparation of a conjugate of N-[4-(4-aminobutylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0419] Step 1: Preparation of tert-butyl N-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]butyl]carbamate [ka]

[0381] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (886 mg, 2 mmol), N-Boc-1,4-butanediamine (564 mg, 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 575.1 mg, 3 mmol), and 1-hydroxybenzotriazole (HOBt, 405 mg, 3 mmol) were dissolved in dichloromethane (30 mL), and triethylamine (0.84 mL, 6 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:100~1:50) to obtain the title compound (0.52 g, yield: 42.5%). MS(m / z):C 30 H 45 The calculated value of N9O5's [M+H] is 612.35; the measured value is 612.1. 1 H NMR (400 MHz, d-DMSO) δ ppm 9.62 (s, 1H), 8.29 (t, J = 14.4 Hz, 1H), 7.77 (s, 1H), 7.61 (d, J = 22.9 Hz, 1H), 6.77 (d, J = 28.6 Hz, 2H), 4.86 (s, 1H), 4.22 - 3.61 (m, 5H), 3.43 - 3.33 (m, 3H), 3.27 (s, 3H), 2.94 (d, J = 25.8 Hz, 2H), 2.86 (s, 2H), 2.37 (s, 1H), 2.13 (t, J = 7.0 Hz, 2H), 1.91 - 1.39 (m, 5H), 1.33 (s, 12H), 0.97 (t, J = 14.9 Hz, 3H).

[0420] Step 2: Preparation of N-[4-(4-aminobutylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0382] According to step 2 of Example 1, the title compound was obtained as a white solid (0.32 g, yield: 83%) from tert-butyl N-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]butyl]carbamate (0.5 g, 0.818 mmol). MS(m / z):C 25 H 37 The calculated value of N9O3's [M+H] is 512.30; the measured value is 512.3.

[0421] Step 3: Preparation of the conjugate of N-[4-(4-aminobutylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0383] Following step 3 of Example 1, 0.162 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 147.2 mg, 0.365 mmol) and N-[4-(4-aminobutylamino)-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.365 mmol), yielding 47.5% and a DSR of 33%; 1H NMR (400 MHz, D2O) δ ppm 8.26 -7.86 (m Hz, 0.33H), 7.64 - 7.18 (m, 0.33H), 6.81 - 6.32 (m, 0.33H), 4.52 - 4.28 (m, 2.33H), 4.06 - 2.81 (m, 14.95H), 2.41- 2.21 (m, 0.99H), 1.99- 1.63 (m, 4.32H), 1.47 - 1.26 (m, 1.32H), 1.11-0.91 (m, 0.99H).

[0422]

[0384] In step 3 of Example 1, the corresponding product (0.243 g, yield: 71.3%, DSR: 18%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0423]

[0385] Following step 3 of Example 1, the corresponding product (0.168 g, yield: 49.3%, DSR: 20%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0424] Example 41 Preparation of a conjugate of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0425] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid [ka]

[0386] Following Step 1 of Example 1, the title compound (9.5 g, yield: 62.5%) was obtained from tofacitinib (10 g, 32 mmol) and 4-aminobenzoic acid (8.7 g, 64 mmol). MS(m / z):C 24 H 25 N7O4's [M+H] + Calculated value: 476.1; Measured value: 476.3.

[0426] Step 2: Preparation of tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate [ka]

[0387] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol), N-Boc-1,4-butanediamine (564 mg, 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 575.1 mg, 3 mmol), and 1-hydroxybenzotriazole (HOBt, 405 mg, 3 mmol) were dissolved in dichloromethane (24 mL), and triethylamine (0.84 mL, 6 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (ethyl acetate:hexane = 10:100 to 50:100) to obtain the title compound (0.89 g, yield: 69%). MS(m / z):C 33 H 43 [M+H] of N9O5: Calculated value, 646.33; Measured value, 646.1. 1H NMR (400 MHz, d-DMSO) δ ppm 12.21 (s, 1H), 8.42 (s, 2H), 7.89 (d, J = 7.6 Hz, 2H), 7.73 (d, J = 7.8 Hz, 3H), 6.84 (d, J = 55.3 Hz, 2H), 4.89 (s, 1H), 4.18 - 3.60 (m, 4H), 3.47 - 3.32 (m, 2H), 3.23 (s, 5H), 2.92 (s, 2H), 2.42 - 2.33 (m, 1H), 1.90 - 1.17 (m, 15H), 1.01 (d, J = 5.2 Hz, 3H).

[0427] Step 3: Preparation of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0388] According to step 2 of Example 1, the title compound was obtained from tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.87 g, 1.35 mmol) as a white solid (1 g, yield: 83%). MS(m / z):C 28 H 35 The calculated value of [M+H] for N9O3 is 546.28; the measured value is 546.3.

[0428] Step 4: Preparation of the conjugate of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0389] Following step 3 of Example 1, 0.26 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 138.6 mg, 0.344 mmol) and N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.344 mmol), yielding 93.9% and a DSR of 23%. 1 H NMR (400 MHz, D2O) δ ppm 8.32 - 8.12 (m, 0.23H), 7.88 - 7.23 (m, 1.15H), 6.80 - 6.64 (m, 0.23H), 4.54 - 4.30 (m, 2.23H), 3.99 - 2.81 (m, 12.99H), 2.06 - 1.37 (m, 4.61H), 1.04 - 0.87 (m, 0.69H).

[0429]

[0390] In step 3 of Example 1, the corresponding product (0.145 g, yield: 52.4%, DSR: 15%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0430]

[0391] Following step 3 of Example 1, the corresponding product (0.104 g, yield: 37.5%, DSR: 33%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0431] Example 42 Preparation of a conjugate of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0432] Step 1: Preparation of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid [ka]

[0392] Triethylamine (97.2 g, 960.3 mmol) was added to a stirred mixture of tofacitinib (100 g, 320.1 mmol) and bis(4-nitrophenyl) carbonate (146 g, 480.1 mmol) in dichloromethane (2000 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 16 hours. The reaction mixture was then cooled to 0-10 °C. 300 mL aqueous solutions of glycine (48.1 g, 640.2 mmol), triethylamine (32 g, 316.8 mmol), and tetrabutylammonium bromide (10.3 g) were added. The reaction mixture was stirred at 0-10 °C for 16 hours, the pH of the reaction mixture was adjusted to 3-4 with acetic acid, and the organic layer was washed with 0.5 N hydrochloric acid solution, 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 (methanol / dichloromethane = 1:50~1:10) to obtain the title compound (89.8 g, yield: 67.8%). MS(m / z):C 19 H 23 [M+H] of N7O4: Calculated value, 414.1; Measured value, 414.5.

[0433] Step 2: Preparation of tert-butyl N-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]butyl]carbamate [ka]

[0393] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg, 1.5 mmol), N-Boc-1,4-butanediamine (423 mg, 2.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 431.3 mg, 2.25 mmol), and 1-hydroxybenzotriazole (HOBt, 303.7 mg, 2.25 mmol) were dissolved in dichloromethane (10 mL), and triethylamine (0.62 mL, 4.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:100~1:40) to obtain the title compound (0.65 g, yield: 74%). MS(m / z):C 28 H 41 The calculated value of N9O5's [M+H] is 584.32; the measured value is 584.2. 1 H NMR (400 MHz, d-DMSO) δ ppm 9.92 (t, J = 5.2 Hz, 1H), 8.32 (d, J = 5.8 Hz, 1H), 8.07 (t, J = 5.4 Hz, 1H), 7.63 (d, J = 4.0 Hz, 1H), 6.79 (d, J = 26.3 Hz, 2H), 4.86 (s, 1H), 4.16 - 3.89 (m, 4H), 3.86 - 3.54 (m, 2H), 3.43 - 3.33 (m, 2H), 3.28 (s, 3H), 3.03 (t, J = 19.0 Hz, 2H), 2.88 (d, J = 5.7 Hz, 2H), 2.38 (s, 1H), 1.72 (dt, J = 78.7, 27.2 Hz, 3H), 1.45 - 1.18 (m, 12H), 1.00 (d, J = 7.1 Hz, 3H).

[0434] Step 3: Preparation of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0394] According to step 2 of Example 1, the title compound was obtained as a white solid (0.26 g, yield: 98%) from tert-butyl N-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]butyl]carbamate (0.3 g, 0.51 mmol). MS(m / z):C 23 H 33 The calculated value of [M+H] for N9O3 is 484.27; the measured value is 484.3. 1 H NMR (400 MHz, d-DMSO) δ ppm 9.90 (s, 1H), 8.34 (d, J = 6.7 Hz, 1H), 8.21 (s, 1H), 7.91 (s, 2H), 7.66 (s, 1H), 6.84 (s, 1H), 4.85 (s, 1H), 4.13 - 3.66 (m, 7H), 3.39 (s, 1H), 3.28 (s, 1H), 3.10 (d, J = 5.3 Hz, 2H), 2.70 (d, J = 37.4 Hz, 2H), 2.34 (d, J = 27.1 Hz, 2H), 1.77 (d, J = 41.2 Hz, 1H), 1.55 - 1.39 (m, 4H), 1.15 (t, J = 7.1 Hz, 2H), 1.00 (d, J = 6.5 Hz, 3H).

[0435] Step 4: Preparation of the conjugate of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0395] Following step 3 of Example 1, 0.102 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 101 mg, 0.25 mmol) and N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (130 mg, 0.25 mmol), yielding 45% and a DSR of 40%; 1 H NMR (400 MHz, D2O) δ ppm 8.26 - 7.85 (m, 0.4H), 7.63 - 7.18 (m, 0.4H), 6.83 - 6.31 (m, 0.4H), 4.58 - 4.28 (m, 2.4H), 4.15 - 2.90 (m, 16H), 2.43 - 2.10 (m, 0.8H), 2.06 - 1.69 (m, 4.4H), 1.59 - 1.37 (m, 1.2H), 1.19 - 0.87 (m, 0.8H).

[0436]

[0396] In step 3 of Example 1, the corresponding product (0.092 g, yield: 40.6%, DSR: 26%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0437]

[0397] Following step 3 of Example 1, the corresponding product (0.143 g, yield: 63%, DSR: 21%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0438] Example 43 Preparation of a conjugate of N-[2-(2-aminoethylamino)-2-oxoethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0439] Step 1: Preparation of tert-butyl N-[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]ethyl]carbamate [ka]

[0398] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (1239 mg, 3 mmol), N-Boc-ethylenediamine (721 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 863 mg, 4.5 mmol), and 1-hydroxybenzotriazole (HOBt, 607.5 mg, 4.5 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (1.25 mL, 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:100~1:60) to obtain the title compound (1.1 g, yield: 66%). MS(m / z):C 26 H 37 The calculated value of N9O5's [M+H] is 556.29; the measured value is 556.2. 1H NMR (400 MHz, CDCl3) δ ppm 10.37 - 10.14 (m, 1H), 8.31 (d, J = 6.3 Hz, 1H), 7.76 - 7.54 (m, 1H), 7.03 (t, J = 18.4 Hz, 1H), 6.59 (d, J = 3.9 Hz, 1H), 5.13 (s, 1H), 5.01 (s, 1H), 4.26 - 4.11 (m, 2H), 4.09 - 3.72 (m, 2H), 3.67 - 3.46 (m, 4H), 3.42 - 3.33 (m, 5H), 3.28 (dd, J = 10.8, 5.5 Hz, 2H), 2.50 (ddd, J = 18.5, 12.3, 6.0 Hz, 1H), 2.09 - 1.84 (m, 1H), 1.83 - 1.72 (m, 1H), 1.54 - 1.30 (m, 9H), 1.09 (dd, J = 11.8, 7.1 Hz, 3H).

[0440] Step 2: Preparation of N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0399] According to step 2 of Example 1, the title compound was obtained as a white solid (0.78 g, yield: 99%) from tert-butyl N-[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]ethyl]carbamate (0.89 g, 1.6 mmol). MS(m / z):C 21 H 29 The calculated value of [M+H] for N9O3 is 456.23; the measured value is 456.2.

[0441] Step 3: Preparation of the conjugate of N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0400] According to step 3 of Example 1, 0.165 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 152.7 mg, 0.379 mmol) and N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.379 mmol), yield: 47.7%, DSR: 40%; 1 H NMR (400 MHz, D2O) δ ppm 8.26 - 7.82 (m, 0.4H), 7.60 - 7.26 (m, 0.4H), 6.77 - 6.26 (m, 0.4H), 4.60 - 4.21 (m, 2.4H), 4.08 - 2.99 (m, 16H), 2.42 - 2.18 (m, 0.4H), 2.04 - 1.55 (m, 3.8H), 1.06 - 0.85 (m, 1.2H).

[0442]

[0401] In step 3 of Example 1, the corresponding product (0.173 g, yield: 50%, DSR: 28%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0443]

[0402] Following step 3 of Example 1, the corresponding product (0.18 g, yield: 52%, DSR: 40%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0444] Example 44 Preparation of a conjugate of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0445] Step 1: Preparation of tert-butyl N-[2-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]ethyl]carbamate [ka]

[0403] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (1652 mg, 4 mmol), tert-butyl 4-aminophenethylcarbamate (1418 mg, 6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1151 mg, 6 mmol), and 1-hydroxybenzotriazole (HOBt, 810 mg, 6 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (1.665 mL, 12 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:150~1:100) to obtain the title compound (1.77 g, yield: 70%). MS(m / z):C 32 H 41 [M+H] of N9O5: Calculated value, 632.32; Measured value, 632.2. 1H NMR (400 MHz, CDCl3) δ ppm 10.59 - 10.19 (m, 1H), 8.30 (dd, J = 17.1, 7.1 Hz, 2H), 7.67 (dd, J = 13.2, 4.0 Hz, 1H), 7.49 (t, J = 11.3 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 6.61 (t, J = 4.7 Hz, 1H), 5.13 (s, 1H), 4.54 (s, 1H), 4.38 - 4.23 (m, 2H), 4.13 - 3.72 (m, 2H), 3.68 - 3.44 (m, 4H), 3.42 - 3.24 (m, 5H), 2.75 (t, J = 6.8 Hz, 2H), 2.50 (ddd, J = 19.1, 11.9, 5.6 Hz, 1H), 2.08 - 1.83 (m, 1H), 1.83 - 1.64 (m, 1H), 1.43 (s, 9H), 1.09 (t, J = 8.2 Hz, 3H).

[0446] Step 2: Preparation of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0404] According to step 2 of Example 1, the title compound was obtained as a white solid (1.3 g, yield: 97.7%) from tert-butyl N-[2-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]ethyl]carbamate (1.48 g, 2.345 mmol). MS(m / z):C 27 H 33 The calculated value of [M+H] for N9O3 is 532.27; the measured value is 532.2.

[0447] Step 3: Preparation of the conjugate of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0405] Following step 3 of Example 1, 0.136 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 133.4 mg, 0.331 mmol) and N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.331 mmol), yielding 41.5% and a DSR of 20%; 1 H NMR (400 MHz, D2O) δ ppm 8.33 - 8.06 (m, 0.2H), 7.68 - 7.50 (m, 0.2H), 7.48 - 7.13 (m, 0.8H), 6.85 - 6.67 (m, 0.2H), 4.59 - 4.23 (m, 2.2H), 4.07 - 2.84 (m, 12.8H), 2.46 - 2.22 (m, 0.4H), 2.05 - 1.70 (m, 3.2H),1.21 - 1.25 (mz, 0.2H), 1.07 - 0.90 (m, 0.6H).

[0448]

[0406] In step 3 of Example 1, the corresponding product (0.158 g, yield: 48.2%, DSR: 22%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0449]

[0407] Following step 3 of Example 1, the corresponding product (0.146 g, yield: 44.5%, DSR: 16%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0450] Example 45 Preparation of a conjugate of methyl(2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate with HA [ka]

[0451] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate [ka]

[0408] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (1239 mg, 3 mmol), (S)-methyl6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1336 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 863 mg, 4.5 mmol), and 1-hydroxybenzotriazole (HOBt, 608 mg, 4.5 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (2.1 mL, 15 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:150-1:80) to obtain the title compound (1.638 g, yield: 83.3%). MS(m / z):C 31 H 45 [M+H] of N9O7: Calculated value, 656.34; Measured value, 656.1. 1H NMR (400 MHz, CDCl3) δ ppm 10.36 (dd, J = 12.6, 7.1 Hz, 1H), 8.33 (d, J = 9.1 Hz, 1H), 7.72 (dd, J = 12.9, 4.0 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.61 (t, J = 4.2 Hz, 1H), 5.14 (s, 1H), 4.63 (dt, J = 33.6, 16.8 Hz, 2H), 4.19 (t, J = 14.2 Hz, 2H), 4.11 - 3.76 (m, 2H), 3.73 (s, 3H), 3.66 - 3.57 (m, 1H), 3.55 - 3.39 (m, 3H), 3.36 (s, 2H), 3.17 - 2.98 (m, 2H), 2.59 - 2.41 (m, 1H), 2.03 - 1.84 (m, 2H), 1.83 - 1.68 (m, 2H), 1.53 - 1.39 (m, 11H), 1.39 - 1.26 (m, 3H), 1.09 (dd, J = 12.3, 7.1 Hz, 3H).

[0452] Step 2: Preparation of methyl(2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate hydrochloride [ka]

[0409] Following Step 2 of Example 1, the title compound was obtained from methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate (1.4 g, 2.137 mmol) as a white solid (1.26 g, yield: 99.6%), which was used without further purification. MS(m / z):C 26 H 37The calculated value of N9O5's [M+H] is 556.29; the measured value is 556.2.

[0453] Step 3: Preparation of the conjugate of methyl(2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate with HA. [ka]

[0410] Following step 3 of Example 1, 0.131 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 128.2 mg, 0.318 mmol) and methyl(2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate hydrochloride (200 mg, 0.318 mmol), yielding 40.6% and a DSR of 30%. 1 H NMR (400 MHz, D2O) δ ppm 8.27 - 7.99 (m, 0.3H), 7.64 - 7.33 (m, 0.3H), 6.82 - 6.51 (m, 0.3H), 4.58 - 4.30 (d, J = 28.8 Hz, 2.3H), 4.10 - 2.99 (m, 14.5H), 2.41 - 2.27 (m, 0.3H), 2.09 - 1.58 (m, 4.8H), 1.53 - 1.29 (m, 0.9H), 1.25 - 0.93 (m, 0.9H).

[0454]

[0411] Following step 3 of Example 1, the corresponding product (0.134 g, yield: 41.5%, DSR: 20%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0455] Example 46 Preparation of a conjugate of N-[4-[4-(2-aminoethyl)anilino]-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0456] Step 1: Preparation of tert-butyl N-[2-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]phenyl]ethyl]carbamate [ka]

[0412] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1764 mg, 4 mmol), tert-butyl 4-aminophenethylcarbamate (1418 mg, 6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1151 mg, 6 mmol), and 1-hydroxybenzotriazole (HOBt, 810 mg, 6 mmol) were dissolved in dichloromethane (40 mL), and triethylamine (1.66 mL, 12 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:150~1:100) to obtain the title compound (2.02 g, yield: 76.6%). MS(m / z):C 34 H 45 The calculated value of N9O5's [M+H] is 660.35; the measured value is 660.3. 1H NMR (400 MHz, CDCl3) δ ppm 10.00 (dd, J = 14.1, 8.0 Hz, 1H), 8.48 (d, J = 26.6 Hz, 1H), 8.28 (d, J = 9.5 Hz, 1H), 7.73 (dd, J = 10.8, 4.0 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 6.60 (t, J = 4.6 Hz, 1H), 5.14 (s, 1H), 4.56 (s, 1H), 4.12 - 3.71 (m, 2H), 3.62 (t, J = 9.0 Hz, 3H), 3.48 (s, 3H), 3.35 (d, J = 8.6 Hz, 5H), 2.76 (t, J = 6.8 Hz, 2H), 2.60 - 2.35 (m, 3H), 2.14 - 2.04 (m, 2H), 2.02 - 1.84 (m, 1H), 1.84 - 1.68 (m, 1H), 1.43 (s, 9H), 1.10 (dd, J = 12.0, 7.1 Hz, 3H).

[0457] Step 2: Preparation of N-[4-[4-(2-aminoethyl)anilino]-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0413] According to step 2 of Example 1, the title compound was obtained from tert-butyl N-[2-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]phenyl]ethyl]carbamate (1.73 g, 2.625 mmol) as a white solid (1.56 g, yield: 99.8%). MS(m / z):C 29 H 37 The calculated value of N9O3's [M+H] is 560.30; the measured value is 560.1.

[0458] Step 3: Preparation of the conjugate of N-[4-[4-(2-aminoethyl)anilino]-4-oxobutyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0414] Following step 3 of Example 1, 0.154 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 127.3 mg, 0.316 mmol) and N-[4-[4-(2-aminoethyl)anilino]-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.316 mmol), yielding 47.8% and a DSR of 33%. 1 H NMR (400 MHz, D2O) δ ppm 8.10 - 7.85 (m, 0.33H), 7.54 - 7.32 (m, 0.33H), 7.24 - 6.81 (m, 1.32H), 6.68 - 6.42 (m, 0.33H), 4.59 - 4.21 (m, 2.33H), 4.04 - 2.92 (m, 14.29H), 2.73 - 2.63 (m, 0.66H), 2.50 - 2.23 (m, 0.99H), 2.11 - 1.56 (m, 3.99H), 1.30 - 1.16 (m, 0.33H), 1.13 - 0.87 (m, 0.99H).

[0459]

[0415] In step 3 of Example 1, the corresponding product (0.136 g, yield: 42.2%, DSR: 18%) was obtained by the reaction with sodium hyaluronate (MW500 kDa).

[0460]

[0416] Following step 3 of Example 1, the corresponding product (0.158 g, yield: 49.1%, DSR: 17%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0461] Example 47 Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidylmethyl-amino]-N-[2-oxo-2-(4-piperazine-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0462] Step 1: Preparation of tert-butyl 4-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]piperazine-1-carboxylate [ka]

[0417] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg, 1.5 mmol), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (624 mg, 2.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 431.3 mg, 2.25 mmol), and 1-hydroxybenzotriazole (HOBt, 304 mg, 2.25 mmol) were dissolved in dichloromethane (20 mL), and triethylamine (0.624 mL, 4.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:100-1:50) to obtain the title compound (0.55 g, yield: 54.5%). MS(m / z):C 34 H 44 N 10 O5's [M+H]+ calculated value: 673.34; measured value: 673.3. MS(m / z):C 34 H 44 N 10 O5's [M+H]+ calculated value: 673.34; measured value: 673.3. 1H NMR (400 MHz, CDCl3) δ ppm 10.45 (dd, J = 13.6, 7.8 Hz, 1H), 8.33 (d, J = 9.1 Hz, 1H), 8.07 (d, J = 10.2 Hz, 1H), 7.69 (dd, J = 12.1, 4.0 Hz, 1H), 7.43 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.62 (t, J = 4.3 Hz, 1H), 5.14 (s, 1H), 4.30 (d, J = 5.8 Hz, 2H), 4.11 - 3.74 (m, 2H), 3.68 - 3.43 (m, 8H), 3.38 (d, J = 20.1 Hz, 3H), 3.10 (dd, J = 28.1, 23.2 Hz, 4H), 2.60 - 2.39 (m, 1H), 2.01 - 1.84 (m, 1H), 1.83 - 1.66 (m, 1H), 1.48 (s, 9H), 1.09 (t, J = 8.5 Hz, 3H).

[0463] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-(4-piperazine-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0418] According to step 2 of Example 1, the title compound was obtained as a white solid (0.43 g, yield: 99%) from tert-butyl 4-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]piperazine-1-carboxylate (0.48 g, 0.714 mmol). MS(m / z):C 29 H 36 N 10 O 31 [M+H]+calculated value, 573.29; measured value, 573.2.

[0464] Step 3: Preparation of the conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidylmethyl-amino]-N-[2-oxo-2-(4-piperazine-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0419] Following step 3 of Example 1, 0.121 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 99.54 mg, 0.247 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-(4-piperazine-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg, 0.247 mmol), yielding 49% and a DSR of 25%; 1 H NMR (400 MHz, D2O) δ ppm 8.27 - 8.09 (m, 0.25H), 7.62 - 6.96 (m, 0.25H), 6.79 - 6.64 (m, 0.25H), 4.57 - 4.24 (m, 2.15H), 4.09 - 2.77 (m, 14.25H), 2.41 - 2.31 (m, 0.25H), 2.16 - 1.45 (m, 4.25H), 1.24 - 1.12 (m, 0.25H), 1.07 - 0.78 (m, 0.75H).

[0465]

[0420] Following step 3 of Example 1, the corresponding product (0.107 g, yield: 43.6%, DSR: 35%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0466] Example 48 Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0467] Step 1: Preparation of tert-butyl 4-[6-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]-3-pyridyl]piperazine-1-carboxylate [ka]

[0421] 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg, 1.5 mmol), tert-butyl 4-(6-aminopyridine-3-yl)piperazine-1-carboxylate (626 mg, 2.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 431.3 mg, 2.25 mmol), and 1-hydroxybenzotriazole (HOBt, 304 mg, 2.25 mmol) were dissolved in dichloromethane (20 mL), and triethylamine (0.624 mL, 4.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:100-1:50) to obtain the title compound (0.4 g, yield: 39.6%). MS(m / z):C 33 H 43 N 11 O5's [M+H]+ calculated value: 674.34; measured value: 674.3.1 H NMR (400 MHz, CDCl3) δ ppm 10.54 - 10.40 (m, 1H), 8.55 (d, J = 20.7 Hz, 1H), 8.34 (d, J = 14.9 Hz, 1H), 8.12 (t, J = 8.1 Hz, 1H), 7.93 (d, J = 2.8 Hz, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.33 - 7.28 (m, 1H), 6.62 (d, J = 3.7 Hz, 1H), 5.15 (s, 1H), 4.33 (d, J = 17.0 Hz, 2H), 4.14 - 3.77 (m, 2H), 3.73 - 3.45 (m, 8H), 3.39 (d, J = 18.0 Hz, 3H), 3.18 - 2.99 (m, 4H), 2.52 (dd, J = 16.9, 10.4 Hz, 1H), 2.01 - 1.85 (m, 1H), 1.83 - 1.69 (m, 1H), 1.48 (s, 9H), 1.10 (dd, J = 12.7, 7.1 Hz, 3H).

[0468] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0422] According to step 2 of Example 1, the title compound was obtained as a white solid (0.32 g, yield: 98%) from tert-butyl 4-[6-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]-3-pyridyl]piperazine-1-carboxylate (0.36 g, 0.535 mmol). MS(m / z):C 28 H 35 N 11O3's [M+H]+ calculated value: 574.29; measured value: 574.3.

[0469] Step 3: Preparation of the conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0423] Following step 3 of Example 1, 0.126 g of the title compound was obtained from sodium hyaluronate (MW50 kDa, 99.14 mg, 0.246 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg, 0.246 mmol), yielding 52.5% and a DSR of 22%; 1 H NMR (400 MHz, D2O) δ ppm 8.26 - 8.07 (m, 0.22H), 8.01 - 7.82 (m, 0.22H), 7.71 - 7.40 (m, 0.44H), 6.81 - 6.57 (m, 0.22H), 4.64 - 4.28 (m, 2.22H), 4.20 - 2.73 (m, 13.74H), 2.42 - 2.32 (m, 0.22H), 2.20 - 1.54 (m, 3.44H), 1.09 - 0.78 (m, 0.66H).

[0470]

[0424] Following step 3 of Example 1, the corresponding product (0.116 g, yield: 48.3%, DSR: 26%) was obtained by the reaction with sodium hyaluronate (MW2000 kDa).

[0471] Example 49 Preparation of a conjugate of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid with HA [ka]

[0472] Step 1: Preparation of (2,5-dioxopyrrolidine-1-yl)2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetate [ka]

[0425] A stirring mixture of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (826 mg, 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg, 2.4 mmol) in dichloromethane (20 mL) was slowly added in an ice bath. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered and then concentrated under reduced pressure to obtain the title compound (1 g, yield: 98%), which was used without further purification. MS(m / z):C 23 H 26 [M+H] of N8O6: Calculated value, 511.19; Measured value, 511.1.

[0473] Step 2: Preparation of the conjugate of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid with HA [ka]

[0426] Conversion of sodium hyaluronic acid to TBA hyaluronic acid. Amberlite 732, a strongly acidic ion exchange resin, was added to an aqueous solution of sodium hyaluronate, and the mixture was stirred at room temperature for 8 hours. The solution was filtered, and the filtrate was then neutralized with an aqueous solution of tetrabutylammonium hydroxide (TBA-OH). The resulting aqueous solution was immediately frozen and freeze-dried to obtain the TBA salt of hyaluronic acid (HA-TBA).

[0474]

[0427] Triethylamine (0.12 mL, 0.875 mmol) was slowly added at 0-15°C to a stirred mixture of hyaluronic acid TBA salt (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL). Then, (2,5-dioxopyrrolidine-1-yl)2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetate (255 mg, 0.5 mmol) was added to the reaction mixture at 0-15°C in anhydrous DMSO (20 mL). The reaction mixture was stirred at room temperature for 16 hours. Then, a 2.5 wt% sodium chloride solution (7 mL) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (250 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa MW cutoff) and lyophilization. Sodium hyaluronate MW 10 kDa MW 0.17 g, yield: 43.9%, DSR: 20%; 1 H NMR (400 MHz, D2O) δ ppm 8.26 - 8.12 (m, 0.2H), 7.62 - 7.46 (m, 0.2H), 6.80 - 6.67 (m, 0.2H), 4.54 - 4.18 (m, 2.2H), 4.09 - 2.94 (m, 12.2H), 2.41 - 2.38 (m, 0.2H), 2.12 - 1.58 (m, 3.4H), 1.09 - 0.87 (m, 0.6H).

[0475]

[0428] This step resulted in the reaction of hyaluronic acid TBA (MW50kDa) to obtain the corresponding product (0.245g, yield: 613.3%, DSR: 6%).

[0476]

[0429] This step resulted in the reaction of hyaluronic acid TBA (MW500kDa) to obtain the corresponding product (0.274g, yield: 70.8%, DSR: 23%).

[0477]

[0430] This step resulted in the reaction of hyaluronic acid TBA (MW2000kDa) to obtain the corresponding product (0.237g, yield: 61.2%, DSR: 24%).

[0478] Example 50 Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid with HA [ka]

[0479] Step 1: Preparation of (2,5-dioxopyrrolidine-1-yl)4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoate [ka]

[0431] A stirring mixture of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (882 mg, 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg, 2.4 mmol) in dichloromethane (25 mL) was slowly added in an ice bath with a solution of N,N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (15 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered and then concentrated under reduced pressure to obtain the title compound (1 g, yield: 93%), which was used without further purification. MS(m / z):C 25 H 30 The calculated value of N8O6's [M+H] is 538.22; the measured value is 538.1.

[0480] Step 2: Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid with HA [ka]

[0432] Triethylamine (0.12 mL, 0.875 mmol) was slowly added at 0-15°C to a stirred mixture of hyaluronic acid TBA salt (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL). Then, (2,5-dioxopyrrolidine-1-yl)4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoate (269 mg, 0.5 mmol) in anhydrous DMSO (20 mL) was added to the reaction mixture at 0-15°C. The reaction mixture was stirred at room temperature for 16 hours. Then, 7 mL of 2.5 wt% sodium chloride solution was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (250 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa MW cutoff) and lyophilization. Sodium hyaluronate MW 10 kDa, 0.166 g, yield: 41.3%, DSR: 10%; 1 ¹H NMR (400 MHz, D2O) δ ppm: 8.26 - 8.09 (m, 0.1H), 7.61 - 7.44 (m, 0.1H), 6.78 - 6.65 (m, 0.1H), 4.60 - 4.22 (m, 2.1H), 4.06 - 2.94 (m, 11.1H), 2.55 - 2.31 (m, 0.3H), 2.15 - 1.57 (m, 3.4H), 1.11 - 0.86 (m, 0.3H). Sodium hyaluronate MW 50kDa, 0.181g, yield: 45.1%, DSR: 14%;

[0481]

[0433] This step involves the reaction of hyaluronic acid TBA (MW500kDa) to obtain the corresponding product (0.263g, yield: 65.5%, DSR: 5%).

[0482]

[0434] This step resulted in the reaction of hyaluronic acid TBA (MW2000kDa) to obtain the corresponding product (0.17g, yield: 42.3%, DSR: 7%).

[0483] Example 51 Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid with HA [ka]

[0484] Step 1: Preparation of (2,5-dioxopyrrolidine-1-yl)4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoate [ka]

[0435] A stirring mixture of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg, 2.4 mmol) in dichloromethane (40 mL) was slowly added in an ice bath to a solution of N,N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered and then concentrated under reduced pressure to obtain the title compound (1.1 g, yield: 96%), which was used without further purification. MS(m / z):C 28 H 28 The calculated value of [M+H] for N8O6 is 573.21; the measured value is 573.1.

[0485] Step 2: Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid with HA. [ka]

[0436] Triethylamine (0.12 mL, 0.875 mmol) was slowly added at 0-15°C to a stirred mixture of hyaluronic acid TBA salt (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL). Then, (2,5-dioxopyrrolidine-1-yl)4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoate (286 mg, 0.5 mmol) in anhydrous DMSO (20 mL) was added to the reaction mixture at 0-15°C. The reaction mixture was stirred at room temperature for 16 hours. Then, 7 mL of 2.5 wt% sodium chloride solution was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (250 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa MW cutoff) and lyophilization. Sodium hyaluronate MW 10 kDa, 0.177: 42.3%, DSR: 10%; 1 ¹H NMR (400 MHz, D2O) δ ppm: 8.09 - 7.92 (m, 0.2H), 7.86 - 7.69 (m, 0.2H), 7.54 - 7.32 (m, 0.3H), 6.83 - 6.74 (m, 0.1H), 4.58 - 4.22 (m, 2.1H), 4.05 - 3.05 (m, 10.9H), 2.39 - 2.27 (m, 0.1H), 2.12 - 1.65 (m, 3.2H), 1.11 - 0.92 (m, 0.3H). Sodium hyaluronate MW 50kDa, 0.2g, yield: 47.8%, DSR: 6%;

[0486]

[0437] This step, by reaction with hyaluronic acid TBA (MW500kDa), yields the corresponding product (0.187g, yield: 44.7%, DSR: 5%).

[0487]

[0438] This step resulted in the reaction of hyaluronic acid TBA (MW2000kDa) to obtain the corresponding product (0.1g, yield: 23.9%, DSR: 2%).

[0488] Example 52 Preparation of a conjugate of O-(4-aminophenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate with HA [ka]

[0489] Step 1: Preparation of O-(4-((tert-butoxycarbonyl)amino)phenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate [ka]

[0439] A mixture of tert-butyl(4-hydroxyphenyl) carbamate (2 g, 9.56 mmol, 1 equivalent) and N,N-dimethylpyridine-4-amine (2.92 g, 23.9 mmol, 2.5 equivalents) in dichloromethane (40 mL, 20 V) was mixed with thiophosgene (1.21 g, 10.5 mmol, 1.1 equivalents) under N2, and the reaction mixture was stirred at room temperature for 0.5 hours. Then, tofacitinib (2.99 g, 9.56 mmol, 1 equivalent) was added, and the resulting mixture was stirred at room temperature for a further 12 hours. After most of the tofacitinib had been consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain the title product (1.827 g, yield: 34%); MS (m / z): C 28 H 33 [M+H]+ calculated value for N7O4S: 564.23; measured value: 564.1.

[0490] Step 2: Preparation of O-(4-aminophenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride [ka]

[0440] According to step 2 of Example 1, the desired product was obtained from O-(4-((tert-butoxycarbonyl)amino)phenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate (1.5 g, 2.661 mmol, 1 equivalent) as the HCl salt (1.33 g, yield: 100%); MS(m / z):C 23 H 25 The calculated [M+H]+ value for N7O2S is 64.18; the measured value is 464.1. 1 H-NMR (400 MHz, D2O) δ ppm 8.45-8.30 (m, 1H), 7.66 -7.44 (m, 2H), 7.44-7.22 (m, 2H), 7.09-6.74 (m, 2H), 4.73 (s, 1H), 4.06-3.94 (m, 3H), 3.71-3.17 (m, 6H), 2.56 (s, 1H), 2.03-1.70 (m, 2H), 1.13 (dd, J = 14.8, 6.8 Hz, 3H).

[0491] Step 3: Preparation of the conjugate of O-(4-aminophenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate with HA [ka]

[0441] According to step 3 of Example 1, the title compound (0.15 g, yield: 45%, DSR=14.3%) was obtained from sodium hyaluronate (MW50 kDa, 0.161 g, 0.4 mmol, 1 equivalent) and O-(4-aminophenyl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride (0.2 g, 0.4 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 7.7-7.55 (m, 0.5H), 7.35-7.2 (m, 0.5H), 4.6-4.45 (m, 1.43H), 3.85-3.4 (m, 12H), 2.46-2.43 (m, 0.14H), 2.00 (s, 3.0H), 1.9-1.45 (m, 2.86H), 1.3-1.25 (m, 0.43H).

[0492]

[0442] This procedure yielded the corresponding product (0.175 g, yield: 52%, DSR=4.6%) by reaction with sodium hyaluronate (MW2000 kDa).

[0493] Example 53 Preparation of a conjugate between O-(piperidine-4-yl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbocioate and HA. [ka]

[0494] Step 1: Preparation of tert-butyl 4-((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothio)oxy)piperidine-1-carboxylate [ka]

[0443] A mixture of tert-butyl 4-hydroxypiperidine-1-carboxylate (2 g, 9.94 mmol, 1 equivalent) and N,N-dimethylpyridine-4-amine (3.03 g, 9.94 mmol, 2.5 equivalents) in dichloromethane (40 mL, 20 V) was mixed with thiophosgene (1.26 g, 10.934 mmol, 1.1 equivalents) under N2, and the reaction mixture was stirred at room temperature for 0.5 hours. Then, tofacitinib (3.1 g, 9.94 mmol, 1 equivalent) was added, and the resulting mixture was stirred at room temperature for a further 12 hours. After most of the tofacitinib had been consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain the title product (1.2 g, yield: 22%); MS (m / z): C 27 H 37 [M+H]+ calculated value for N7O4S: 556.26; measured value: 556.2.

[0495] Step 2: Preparation of O-(piperidine-4-yl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride [ka]

[0444] According to step 2 of Example 1, the desired product was obtained from tert-butyl 4-((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothio)oxy)piperidine-1-carboxylate (1 g, 1.8 mmol, 1 equivalent) as the HCl salt (0.885 g, yield: 100%); MS (m / z): C 22 H 29 The calculated [M+H]+ value for N7O2S is 456.21; the measured value is 456.1. 1H-NMR (400 MHz, D2O) δ ppm 8.49 (d, J = 9.7 Hz, 1H), 8.00 (s, 1H), 7.03 (s, 1H), 5.93 (br, 1H), 5.03 (s, 1H), 4.15-4.0 (m, 1H), 3.79 -3.31 (m, 11H), 3.29-3.05 (m, 1H), 2.52 (br, 1H), 2.45-2.28(m, 4H), 2.0-1.7(m, 3H), 1.11 (dd, J = 17.1, 7.1 Hz, 3H).

[0496] Step 3: Preparation of the conjugate of O-(piperidine-4-yl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate with HA. [ka]

[0445] Following step 3 of Example 1, the title compound (0.17 g, yield: 34%, DSR=12.6%) was obtained from sodium hyaluronate (MW50 kDa, 0.164 g, 0.406 mmol, 1 equivalent) and O-(piperidine-4-yl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride (0.2 g, 0.406 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm δ 8.25-7.0 (m, 0.22H), 7.0-6.5 (m, 0.08H), 5.95-5.49(m, 0.08H), 4.45-4.25 (m, 1.26H), 3.8-2.9(m, 12.63H), 2.33 (br, 0.13H), 1.88 (s, 3H), 1.81-1.72 (m, 0.51H), 1.23-1.07 (m, 0.25H), 0.99-0.82 (m, 0.38H).

[0497]

[0446] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.192g, yield: 56.6%, DSR=4.6%).

[0498] Example 54 Preparation of a conjugate of 4-aminobutyl((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl) carbonate with HA [ka]

[0499] Step 1: Preparation of 3-((3R,4R)-4-methyl-3-(methyl(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropannitrile [ka]

[0447] To a mixture of tofacitinib (9 g, 28.81 mmol, 1 equivalent) in dichloromethane (180 mL, 20 V), DIPethyl acetate (3.745 g, 28.81 mmol, 1 equivalent) was added under N2 conditions, and the reaction mixture was stirred at room temperature for 0.5 hours. Then, (2-(chloromethoxy)ethyl)trimethylsilane (4.8 g, 28.81 mmol, 1 equivalent) was added, and the resulting mixture was stirred overnight at room temperature. After most of the tofacitinib had been consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to obtain the title product (9 g, yield: 71%); MS (m / z): C 22 H 34 The calculated [M+H]+ value for N6O2Si is 443.25; the measured value is 443.2.

[0500] Step 2: Preparation of 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)(methyl)amino)-4-methylpiperidine-1-yl)-3-oxopropannitrile [ka]

[0448] To a mixture of 3-((3R,4R)-4-methyl-3-(methyl(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino)piperidine-1-yl)-3-oxopropanenitrile (5 g, 11.3 mmol, 1 equivalent) in dichloromethane (100 mL, 20 V), TFA (6.44 g, 56.5 mmol, 5 equivalents) was added dropwise under N2 at 0°C. The resulting reaction mixture was stirred at 0°C for 30 minutes, then warmed to room temperature, and subsequently stirred at this temperature for 24 hours. After most of the SM had been consumed, NaHCO3 (saturated) was added to the solution to adjust the pH to 8 at 0°C. The mixture was then poured into a separatory funnel and separated. The organic phase was washed with saturated NaCl, dried over Na2SO4, and then concentrated to obtain the title product (3.5 g, yield: 90%); MS (m / z): C 17 H 22 The calculated [M+H]+ value for N6O2 is 343.18; the measured value is 343.1.

[0501] Step 3: Preparation of tert-butyl(4-((((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methoxy)carbonyl)oxy)butyl)carbamate [ka]

[0449] Following Step 1 of Example 1, the title product (0.85 g, yield: 52%) was obtained from 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)(methyl)amino)-4-methylpiperidine-1-yl)-3-oxopropanenitrile (1 g, 2.92 mmol, 1 equivalent) and tert-butyl(4-hydroxybutyl)carbamate (1 g, 2.92 mmol, 1 equivalent); MS(m / z):C 27 H 39 The calculated value of N7O6's [M+H] is 558.30; the measured value is 558.2.

[0502] Step 4: Preparation of 4-aminobutyl((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl)carbonate hydrochloride [ka]

[0450] According to step 2 of Example 1, the desired product was obtained from tert-butyl(4-((((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methoxy)carbonyl)oxy)butyl)carbamate (0.85 g, 1.524 mmol, 1 equivalent) as the HCl salt (0.753 mg, yield: 100%); MS(m / z):C 22 H 31 The calculated value of N7O4's [M+H] is 458.24; the measured value is 458.1. 1 H-NMR (400 MHz, CD3OD) δ ppm 8.41 (s, 1H), 7.64 (d, J = 3.7 Hz, 1H), 7.01 (d, J = 3.4 Hz, 1H), 6.28 (s, 2H), 4.69 (s, 1H), 4.21-3.40 (m, 11H), 2.95 (t, J = 6.8 Hz, 1H), 2.56 (br, 1H), 2.05 (br, 1H), 1.87 -1.66 (m, 5H), 1.23-1.14 (m, 3H).

[0503] Step 5: Preparation of the conjugate of 4-aminobutyl((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl) carbonate with HA. [ka]

[0451] According to step 3 of Example 1, the title compound (0.18 g, yield: 56%, DSR=21%) was obtained from sodium hyaluronate (MW50 kDa, 0.161 g, 0.4 mmol, 1 equivalent) and 4-aminobutyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl) carbonate hydrochloride (0.2 g, 0.4 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.2-7.9 (m, 0.21H), 7.3-7.0 (m, 0.21H), 6.8-6.3 (m, 0.21H), 6.1-5.1(m, 0.42H), 4.45 (d, J = 27.3 Hz, 2.84H), 3.8-3.3 (m, 12H), 2.40 (br, 0.21H), 1.97 (s, 3H), 1.65-1.45 (m, 0.94H), 1.25 (t, J = 6.9 Hz, 0.21H), 1.05-0.75 (m, 0.7H).

[0504]

[0452] This step resulted in the reaction of hyaluronic acid TBA (MW2000kDa) to obtain the corresponding product (0.172g, yield: 47.6%, DSR=14%).

[0505] Example 55 Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl(4-aminobutyl)carbamate and HA. [ka]

[0506] Step 1: Preparation of tert-butyl((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl)butan-1,4-diyldicarbamate [ka]

[0453] Following Step 1 of Example 1, the title product (0.878 g, yield: 90%) was obtained from 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl)(methyl)amino)-4-methylpiperidine-1-yl)-3-oxopropanenitrile (0.6 g, 1.75 mmol, 1 equivalent), bis(4-nitrophenyl) carbonate (0.587 g, 1.928 mmol, 1.1 equivalent), and tert-butyl(4-aminobutyl) carbamate (0.33 g, 1.75 mmol, 1 equivalent); m / z(ESI): 557.31[M+H] + MS(m / z):C 27 H 40 The calculated value of N8O5's [M+H] is 557.31; the measured value is 557.2.

[0507] Step 2: Preparation of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl(4-aminobutyl)carbamate hydrochloride [ka]

[0454] According to step 2 of Example 1, the desired product was obtained from tert-butyl((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl)butan-1,4-diyldicarbamate (0.8 g, 1.44 mmol, 1 equivalent) as the HCl salt (0.7 g, yield: 100%); MS(m / z):C 22 H 32 The calculated value of [M+H] for N8O3 is 457.25; the measured value is 457.2. 1 H-NMR (400 MHz, D2O) δ ppm 8.33 (t, J = 6.7 Hz, 1H), 7.52 (dd, J = 17.0, 3.7 Hz, 1H), 6.88 (d, J = 9.3 Hz, 1H), 6.17 (d, J = 24.8 Hz, 2H), 4.58 (s, 1H), 4.10-3.81 (m, 3H), 3.68-3.16 (m, 5H), 3.16-2.88 (m, 5H), 2.56 (br, 1H), 2.0-1.97 (m, 1H), 1.83-1.72 (m, 1H), 1.67-1.49 (m, 4H), 1.13 (dd, J = 14.9, 7.0 Hz, 3H).

[0508] Step 3: Preparation of the conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl(4-aminobutyl)carbamate with HA. [ka]

[0455] Following step 3 of Example 1, the title compound (0.17 g, yield: 50%, DSR=6%) was obtained from sodium hyaluronate (MW50 kDa, 0.161 g, 0.4 mmol, 1 equivalent) and 4-aminobutyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidine-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-yl)methyl(4-aminobutyl)carbamate hydrochloride (0.2 g, 0.4 mmol, 1 equivalent); 1 H-NMR (400 MHz, D2O) δ ppm 8.15-7.95 (m, 0.06H), 7.27 (br, 0.06H), 6.68 (br, 0.06H), 6.1-5.9 (m, 0.12H), 4.40 (d, 2H), 3.93-3.11 (m, 10.8H), 2.37 (br, 0.06H), 2.01 -1.79 (s, 3H), 1.6-1.5 (m, 0.36H), 1.0-0.9 (m, 0.18H).

[0509]

[0456] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.150g, yield: 44.9%, DSR=8%).

[0510] Example 56 Preparation of a conjugate of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA [ka]

[0511] Step 1: Preparation of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide [ka]

[0457] Triethylamine (0.558 mL, 8 mmol) was added to a stirred mixture of tofacitinib (1.245 g, 4 mmol) in toluene (50 mL). The reaction mixture was heated to 50°C. Then, 2-chloroethyl isocyanate (1688 mg, 16 mmol) was added to the reaction mixture. The reaction mixture was stirred at this temperature for 4 hours. Then, the reaction mixture was filtered and concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:100~1:20) to obtain the title compound (1.2 g, 71.9%). MS(m / z):C 19 H 24 The calculated [M+H]+ value for ClN7O2 is 418.16; the measured value is 418.1.

[0512] Step 2: Preparation of the conjugate of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide with HA. [ka]

[0458] To a stirred mixture of hyaluronic acid TBA salt (HA-TBA, 331 mg, 0.5 mmol, sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL), a solution of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide (209 mg, 0.5 mmol) in anhydrous DMSO (2 mL) was slowly added. The reaction mixture was stirred at room temperature for 7 days, followed by dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 40 mL of deionized water. The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa MW cutoff) and lyophilization. Sodium hyaluronate MW 50kDa, 0.177g, yield: 42.3%, DSR: 10%; 1H NMR (400 MHz, d-DMSO) δ ppm 8.29 - 8.17 (m, 0.1H), 7.68 - 7.54 (m, 0.1H), 6.82 - 6.68 (m,0. 1H), 4.63 -4.28 (m, 2.1H), 4.23 - 3.17 (m, 11.3H), 2.44 - 2.33 (m, 0.1H), 2.14 -1.58 (m, 3.2H), 1.47 - 1.36 (m, 0.1H), 1.28- 1.17 (m, 0.1H), 1.11 - 0.89 (m, 0.3H).

[0513] Example 57 Preparation of a conjugate of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA [ka]

[0514] Step 1: Preparation of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate [ka]

[0459] To a stirred mixture of tofacitinib (156 mg, 0.5 mmol) and K2CO3 (138 mg, 1 mmol) in anhydrous DMF (2 mL), 2-chloroethyl chloroformate (0.1 mL, 1 mmol) was added. The reaction mixture was stirred at room temperature for 30 minutes. The solution was diluted with dichloromethane 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 (methanol / dichloromethane = 1:100~1:50) to obtain the title compound (0.08 g, 40%). MS(m / z):C 19 H 23The calculated [M+H]+ value for ClN6O3 is 419.15; the measured value is 419.3.

[0515] Step 2: Preparation of the conjugate of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate with HA. [ka]

[0460] A stirring mixture of hyaluronic acid TBA salt (HA-TBA, MW500kDa; 331mg, 0.5 mmol, sodium hyaluronate MW50kDa) in anhydrous DMSO (20mL) was slowly added to a solution of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (209mg, 0.5 mmol) in anhydrous DMSO (2mL). The reaction mixture was stirred at room temperature for 7 days, followed by dropwise addition of acetone (200mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 40mL of deionized water. The title compound was obtained by thorough dialysis of the solution against deionized water (3.5kDa MW cutoff) and lyophilization. 0.28g, yield: 73.5%, DSR: 25%; 1 H NMR (400 MHz, D2O) δ ppm 8.21 - 7.99 (m, 0.25H), 7.57 - 7.10 (m, 0.25H), 6.83 - 6.57 (m, 0.25H), 4.54 - 4.31 (m, 2.25H), 4.09 - 2.73 (m, 12.75H), 2.39 - 2.23 (m, 0.25H), 2.02 - 1.50 (m, 3.75H),1.32 - 1.21 (m, 0.25H), 1.00- 0.79 (m, 0.75H).

[0516]

[0461] This step resulted in the reaction of HA-TBA (sodium hyaluronate MW2000kDa) to obtain the corresponding product (0.18g, yield: 47.2%, DSR=20%).

[0517] Example 58 Preparation of a conjugate between 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile and HA [ka]

[0518] Step 1: Preparation of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile [ka]

[0462] To a stirred mixture of tofacitinib (156 mg, 0.5 mmol) and K2CO3 (138 mg, 1 mmol) in anhydrous DMF (2 mL), 2-chloromethoxyethyl chloride (142 mg, 1.1 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The solution was diluted with dichloromethane and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:100~1:50) to obtain the title compound (0.11 g, 55%). MS(m / z):C 19 H 25 The calculated [M+H]+ value for ClN6O2 is 405.17; the measured value is 405.3.

[0519] Step 2: Preparation of a conjugate of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile and HA. [ka]

[0463] A stirring mixture of hyaluronic acid TBA salt (HA-TBA, 331 mg, 0.5 mmol, sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL) was slowly added to a solution of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile (202 mg, 0.5 mmol) in anhydrous DMSO (2 mL). The reaction mixture was stirred at room temperature for 4 days, followed by dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 40 mL of deionized water. Thorough dialysis of the solution against deionized water (3.5 kDa MW cutoff) and lyophilization yielded 0.28 g of the title compound, with a yield of 74.9% and a DSR of 10%. 1 H NMR (400 MHz, d-DMSO) δ ppm 8.28 - 8.14 (m, 0.1H), 7.67 - 7.56 (m, 0.1H), 6.82 - 6.68 (m,0. 1H), 4.66 - 4.28 (m, 2.1H), 4.23 - 3.17 (m, 11.3H), 2.44 - 2.33 (m, 0.1H), 2.19 - 1.55 (m, 3.2H), 1.47 - 1.36 (m, 0.1H), 1.28- 1.17 (m, 0.1H), 1.11 - 0.89 (m, 0.3H).

[0520]

[0464] This step resulted in the reaction of HA-TBA (sodium hyaluronate MW2000kDa) to obtain the corresponding product (0.175g, yield: 46.8%, DSR=35%).

[0521] Example 59 Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate [ka]

[0522] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate [ka]

[0465] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol), (S)-methyl6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (890 mg, 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 575 mg, 3 mmol), and 1-hydroxybenzotriazole (HOBt, 405 mg, 3 mmol) were dissolved in DCM (40 mL), and triethylamine (1.39 mL, 10 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with DCM and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / DCM = 1:150~1:80) to obtain the title compound (1.1 g, yield: 76.7%). MS(m / z):C 36 H 47 [M+H] of N9O7: Calculated value, 718.35; Measured value, 718.3. 1H NMR (400 MHz, CDCl3) δ ppm 12.29 (d, J = 30.5 Hz, 1H), 8.39 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.6 Hz, 2H), 7.78 (dd, J = 10.8, 6.4 Hz, 3H), 6.85-6.70 (m, 1H), 6.66 (d, J = 3.7 Hz, 1H), 5.11 (d, J = 23.2 Hz, 1H), 4.89-4.74 (m, 1H), 4.62 (s, 1H), 4.09 (dd, J = 13.0, 3.8 Hz, 1H), 3.95-3.70 (m, 4H), 3.66-3.45 (m, 4H), 3.39 (d, J = 14.1 Hz, 3H), 3.13 (d, J = 6.0 Hz, 2H), 2.60-2.43 (m, 1H), 2.06-1.91 (m, 2H), 1.91-1.75 (m, 2H), 1.59-1.49 (m, 2H), 1.40 (d, J = 19.4 Hz, 11H), 1.27 (d, J = 12.0 Hz, 1H), 1.10 (t, J = 7.0 Hz, 3H).

[0523] Step 2: Preparation of methyl(2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate hydrochloride [ka]

[0466] A stirred solution of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate (1 g, 1.39 mmol) in ethyl acetate (20 mL) was slowly added in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.9 g, yield: 99%), which was used without further purification. MS(m / z):C 31 H 39 The calculated value of N9O5's [M+H] is 618.30; the measured value is 618.1.

[0524] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate. [ka]

[0467] Sodium hyaluronate (117 mg, 0.29 mmol carboxylic acid, MW 50 kDa) was dissolved in 23.4 mL of deionized water in a 100 mL round-bottom flask, and then 16 mL of acetonitrile was added dropwise while stirring. 4-methylmorpholine (NMM, 59 mg, 0.58 mmol) was added to the solution to temporarily increase its viscosity. The solution was then cooled to 0°C, and 2-chloro-4,6-dimethoxy-1,3,5-triazine (51 mg, 0.29 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The solution was mixed with methyl(2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate hydrochloride (200 mg, 0.29 mmol) and stirred at room temperature for 72 hours. Then, NaCl (170 mg, 2.9 mmol) was added to the reaction mixture and stirred for 1 hour, followed by dropwise addition of acetone (290 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa Mw cutoff) and lyophilization. 0.119g, yield: 41.9%, DSR: 20%; 1 H NMR (400 MHz, D2O) δ ppm 8.35-8.23 (m, 0.2H), 7.89-7.40 (m, 1H), 6.83-6.71 (m, 0.2H), 4.61-4.16 (m, 2.2H), 4.13-2.47 (m, 13.4H), 2.09-1.29 (m, 4.6H), 1.23-1.16 (m, 0.2H), 1.06-0.81 (m, 0.6H).

[0525]

[0468] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.119g, yield: 41.9%, DSR=21%).

[0526] Example 60 Preparation of a conjugate between HA (sodium hyaluronate) and N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide [ka]

[0527] Step 1: Preparation of 3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid [ka]

[0469] Triethylamine (1.12 mL, 8 mmol) was added to a stirred mixture of tofacitinib (1248 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1459 mg, 4.8 mmol) in DCM (40 mL). The reaction mixture was heated to 45°C and stirred at this temperature for 5 hours. The reaction mixture was then cooled to room temperature. 3-aminobenzoic acid (658 mg, 4.8 mmol) was added. The reaction mixture was stirred at 45°C for 16 hours. The reaction mixture was filtered. The filtrate was washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol = 20:1) to obtain the title compound (1.8 g, yield: 94.7%). MS(m / z):C 24 H 25 [M+H] of N7O4: Calculated value, 476.19; Measured value, 476.1.

[0528] Step 2: Preparation of tert-butyl N-[4-[[3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate [ka]

[0470] 3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (1188 mg, 2.5 mmol), N-Boc-1,4-butanediamine (705 mg, 3.75 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 719 mg, 3.75 mmol), and 1-hydroxybenzotriazole (HOBt, 507 mg, 3.75 mmol) were dissolved in DCM (40 mL), and triethylamine (1.04 mL, 7.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with DCM and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / DCM = 1:150~1:80) to obtain the title compound (1 g, yield: 62%). MS(m / z):C 33 H 43 [M+H]+ of N9O5: Calculated value, 646.33; Measured value, 646.3.

[0529] Step 3: Preparation of N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0471] To a stirred solution of tert-butyl N-[4-[[3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.6 g, 0.93 mmol) in ethyl acetate (12 mL), 2.4 mL of 4 M HCl (commercial) (in an ice bath) was slowly added. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.54 g, yield: 99%), which was used without further purification. MS(m / z):C 28 H 35 The calculated value of [M+H] for N9O3 is 546.28; the measured value is 546.3.

[0530] Step 4: Preparation of a conjugate between HA (sodium hyaluronate) and N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide [ka]

[0472] Sodium hyaluronate (186 mg, 0.462 mmol carboxylic acid, MW 50 kDa) was dissolved in 37.2 mL of deionized water in a 100 mL round-bottom flask, and then 24.2 mL of acetonitrile was added dropwise while stirring. N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.324 mmol) and 4-methylmorpholine (NMM, 32 mg, 0.324 mmol) were added to the solution at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 128 mg, 0.462 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (270 mg, 4.62 mmol) was added to the reaction mixture and stirred for 1 hour, followed by dropwise addition of acetone (250 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). The title compound was obtained by thorough dialysis of the solution with deionized water (3.5 kDa Mw cutoff) and lyophilization. 0.209 g, yield: 49.9%, DSR: 22%; 1 H NMR (400 MHz, D2O) δ ppm 8.06-7.86 (m, 0.22H), 7.56-7.41 (m, 1.1H), 6.56-6.42 (m, 0.22H), 4.63-4.18 (m, 2.22H), 4.05-2.37 (m, 12.86H), 2.13-1.70 (m, 3.66H), 1.69-1.07 (m, 1.54H).

[0531]

[0473] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.214g, yield: 51.1%, DSR=18%).

[0532] Example 61 Preparation of a conjugate between HA (sodium hyaluronate) and N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide [ka]

[0533] Step 1: Preparation of tert-butyl N-[2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]ethyl]carbamate [ka]

[0474] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (1425 mg, 3 mmol), N-Boc-ethylenediamine (721 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 863 mg, 4.5 mmol), and 1-hydroxybenzotriazole (HOBt, 607.5 mg, 4.5 mmol) were dissolved in DCM (40 mL), and triethylamine (1.25 mL, 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solution was diluted with DCM and washed with water and saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / DCM = 1:150~1:80) to obtain the title compound (1.23 g, yield: 66.4%). MS(m / z):C 31 H 39 The calculated value of N9O5's [M+H] is 618.30; the measured value is 618.2. 1H NMR (400 MHz, CDCl3) δ ppm 12.26 (d, J = 23.3 Hz, 1H), 8.38 (d, J = 9.3 Hz, 1H), 7.81 (dd, J = 39.5, 8.6 Hz, 5H), 7.24 (s, 1H), 6.65 (d, J = 4.0 Hz, 1H), 5.11 (d, J = 33.0 Hz, 2H), 4.13-3.74 (m, 2H), 3.68-3.11 (m, 11H), 2.62-2.45 (m, 1H), 2.08-1.72 (m, 2H), 1.44 (s, 9H), 1.10 (t, J = 8.6 Hz, 3H).

[0534] Step 2: Preparation of N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride [ka]

[0475] To a stirred solution of tert-butyl N-[2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]ethyl]carbamate (0.6 g, 0.97 mmol) in ethyl acetate (6 mL), 2.4 mL of 4 M HCl (commercial) (in an ice bath) was slowly added. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.5 g, yield: 99%), which was used without further purification. MS(m / z):C 26 H 31 The calculated value of [M+H]+ for N9O3 is 518.25; the measured value is 518.2.

[0535] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide [ka]

[0476] Sodium hyaluronate (208 mg, 0.516 mmol carboxylic acid, MW 50 kDa) was dissolved in 41.6 mL of deionized water in a 100 mL round-bottom flask, and then 27 mL of acetonitrile was added dropwise while stirring. N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.361 mmol) and 4-methylmorpholine (NMM, 36.5 mg, 0.361 mmol) were added to the solution at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 142.8 mg, 0.516 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (303 mg, 5.16 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (250 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). The title compound was obtained by thorough dialysis of the solution against deionized water (3.5 kDa Mw cutoff) and lyophilization. 0.25 g, yield: 55.1%, DSR: 22%; 1H NMR (400 MHz, D2O) δ ppm 8.36-8.18 (m, 0.22H), 7.92-6.84 (m, 1.1H), 6.68-6.55 (m, 0.22H), 4.61-4.22 (m, 2.22H), 4.13-2.65 (m, 12.86H), 2.24-1.52 (m, 3.66H), 1.30-1.09 (m, 0.66H). This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.24g, yield: 52.9%, DSR=26%).

[0536] Example 62 Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0537] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0477] Triethylamine (1.25 mL, 9 mmol) was added to a stirred mixture of trans-N-methyl-1-[4-[methyl(7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino]cyclohexyl]methanesulfonamide (1012 mg, 3 mmol) and bis(4-nitrophenyl) carbonate (1094 mg, 3.6 mmol) in DCM (40 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 hours. The reaction mixture was then cooled to room temperature. (S)-methyl6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1069 mg, 3.6 mmol) was added. The reaction mixture was stirred at 45 °C for 16 hours. The reaction mixture was filtered. The filtrate was washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=100:1) to obtain the title compound (0.66 g, yield: 35.3%). MS(m / z):C 28 H 45 [M+H]+ calculated value for N7O7S: 624.31; measured value: 624.2. 1 H NMR (400 MHz, CDCl3) δ ppm 10.38 (d, J = 7.6 Hz, 1H), 8.36 (s, 1H), 7.67 (d, J = 4.1 Hz, 1H), 6.59 (d, J = 4.0 Hz, 1H), 4.78 - 4.69 (m, 2H), 4.58 (s, 1H), 4.22 - 4.05 (m, 1H), 3.81 (s, 3H), 3.24 (s, 3H), 3.14 (d, J = 5.4 Hz, 2H), 3.01 (dd, J = 15.2, 5.9 Hz, 2H), 2.86 (d, J = 5.3 Hz, 3H), 2.21 (d, J = 11.6 Hz, 2H), 2.07 - 1.87 (m, 5H), 1.71 (dd, J = 23.8, 11.5 Hz, 2H), 1.59 - 1.48 (m, 4H), 1.47 - 1.32 (m, 11H).

[0538] Step 2: Preparation of methyl(2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride [ka]

[0478] A stirred solution of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (0.61 g, 0.97 mmol) in ethyl acetate (12 mL) was slowly added with 4 M HCl (commercial) (2.4 mL) in an ice bath. The reaction mixture was cooled to 0°C and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.54 g, yield: 99%), which was used without further purification. MS(m / z):C 23 H 37 [M+H]+ calculated value for N7O5S: 524.25; measured value: 524.2.

[0539] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate. [ka]

[0479] Sodium hyaluronate (101.6 mg, 0.252 mmol carboxylic acid, MW 50 kDa) was dissolved in 21 mL of deionized water in a 100 mL round-bottom flask, and then 13.2 mL of acetonitrile was added dropwise while stirring. 4-methylmorpholine (NMM, 51 mg, 0.504 mmol) was added to the solution to temporarily increase its viscosity. The solution was then cooled to 0°C, and 2-chloro-4,6-dimethoxy-1,3,5-triazine (44.3 mg, 0.252 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The solution was mixed with methyl(2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride (150 mg, 0.252 mmol) and stirred at room temperature for 72 hours. Then, NaCl (148 mg, 2.53 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (148 mg, 2.53 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried in vacuum to obtain the title compound as a white solid. 0.118g, yield: 52.9%, DSR: 16%; 1 H NMR (400 MHz, D2O) δ ppm 8.29 - 8.17 (m, 0.16H), 7.66 - 7.54 (m, 0.16H), 6.84 - 6.70 (m, 0.16H), 4.59 - 4.15 (m, 2.16H), 4.11 - 2.37 (m, 12.24H), 2.11 -1.73 (m, 4.44H), 1.71 - 1.11 (m, 0.96H).

[0540]

[0480] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.105g, yield: 47.1%, DSR=20%).

[0541] Example 63 Preparation of a conjugate between HA (sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide [ka]

[0542] Step 1: Preparation of tert-butyl N-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate [ka]

[0481] A mixture of trans-N-methyl-1-[4-[methyl(7H-pyrrolo[2,3-d]pyrimidine-4-yl)amino]cyclohexyl]methanesulfonamide (1350 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1337.6 mg, 4.4 mmol) in DMSO (14 mL) was stirred at room temperature for 4 hours. Then, tert-butylhydrazine carboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filtered cake was washed with a saturated NaHCO3 solution and water. Then, the filtered cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to obtain the title compound (1.67 g, yield: 84.3%). MS(m / z):C 21 H 33 The calculated value of N7O5S's [M+H] is 496.22; the measured value is 496.1. 1H NMR (400 MHz, CDCl3) δ ppm 11.38 (s, 1H), 8.30 (s, 1H), 7.63 (d, J = 4.1 Hz, 1H), 6.56 (dd, J = 25.8, 3.5 Hz, 2H), 4.75 (s, 1H), 4.36 (q, J = 5.1 Hz, 1H), 3.22 (d, J = 11.1 Hz, 3H), 2.97 (d, J = 6.3 Hz, 2H), 2.83 (d, J = 5.3 Hz, 3H), 2.20 (t, J = 14.4 Hz, 2H), 2.04 - 1.94 (m, 1H), 1.87 (d, J = 10.8 Hz, 2H), 1.74 - 1.62 (m, 2H), 1.51 (s, 9H), 1.43 - 1.32 (m, 2H).

[0543] Step 2: Preparation of 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride [ka] To a stirred solution of tert-butyl N-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate (0.6 g, 1.21 mmol) in ethyl acetate (12 mL), 2.4 mL of 4 M HCl (commercially available) in ethyl acetate was slowly added in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.52 g, yield: 99%), which was used without further purification. MS(m / z):C 16 H 25 The calculated value of [M+H] for N7O3S is 396.17; the measured value is 396.1.

[0544] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide [ka]

[0483] Sodium hyaluronate (246 mg, 0.61 mmol carboxylic acid, MW 50 kDa) was dissolved in 49.2 mL of deionized water in a 100 mL round-bottom flask, and then 32 mL of acetonitrile was added dropwise while stirring. 4-methylmorpholine (NMM, 43 mg, 0.427 mmol) was added to the solution to temporarily increase its viscosity. 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride (200 mg, 0.427 mmol) and 4-methylmorpholine (NMM, 43 mg, 0.427 mmol) were added to the solution at room temperature to temporarily increase its viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 169 mg, 0.61 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (357 mg, 6.1 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (400 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (357 mg, 6.1 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried in vacuum to obtain the title compound as a white solid. 0.29 g, yield: 62.8%, DSR: 23%; 1H NMR (400 MHz, D2O) δ ppm 8.26 - 8.09 (m, 0.23H), 7.63 - 7.52 (m, 0.23H), 6.85 - 6.67 (m, 0.23H), 4.70 - 4.28 (m, 2.23H), 4.20 - 2.53 (m, 11.84H), 2.23 - 1.40 (mz, 4.61H), 1.34 - 1.06 (m, 0.46H).

[0484] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.305g, yield: 66.1%, DSR=31%).

[0545] Example 64 Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0546] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0485] Triethylamine (1.25 mL, 9 mmol) was added to a stirred mixture of 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)pyrazole-1-yl]azetidine-3-yl]acetonitrile (1114 mg, 3 mmol) and bis(4-nitrophenyl) carbonate (1094 mg, 3.6 mmol) in DCM (40 mL). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 hours. The reaction mixture was then cooled to room temperature. (S)-methyl 6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1069 mg, 3.6 mmol) was added. The reaction mixture was stirred at 45 °C for 16 hours. 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. The crude residue was purified by column chromatography (DCM:methanol=100:1) to obtain the title compound (1.3g, yield: 65.9%). MS(m / z):C 29 H 39 [M+H]+ calculated value for N9O7S: 658.26; measured value: 658.1. 1 H NMR (400 MHz, CDCl3) δ ppm 9.93 (d, J = 7.6 Hz, 1H), 8.95 (s, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 8.04 (d, J = 4.0 Hz, 1H), 6.85 (d, J = 4.1 Hz, 1H), 5.45-5.34 (m, 1H), 4.76-4.71 (m, 1H), 4.66 (d, J = 9.4 Hz, 2H), 4.63-4.56 (m, 1H), 4.52-4.42 (m, 1H), 4.28 (d, J = 9.6 Hz, 2H), 3.84 (s, 3H), 3.44 (s, 2H), 3.20-3.07 (m, 2H), 2.10-1.94 (m, 2H), 1.88-1.76 (m, 1H), 1.61-1.35 (m, 12H), 1.27 (s, 3H).

[0547] Step 2: Preparation of methyl(2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride [ka]

[0486] A stirred solution of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (1.23 g, 1.87 mmol) in ethyl acetate (25 mL) was slowly added in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (1.1 g, yield: 99%), which was used without further purification. MS(m / z):C 24 H 31 [M+H]+ calculated value for N9O5S: 558.21; measured value: 558.1.

[0548] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate. [ka]

[0487] Sodium hyaluronate (175 mg, 0.434 mmol carboxylic acid, MW 50 kDa) was dissolved in 35 mL of deionized water in a 100 mL round-bottom flask, and then 23 mL of acetonitrile was added dropwise while stirring. Methyl(2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride (200 mg, 0.317 mmol) and 4-methylmorpholine (NMM, 32 mg, 0.317 mmol) were added to the solution at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 120 mg, 0.434 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (254 mg, 4.34 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (300 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (254 mg, 4.34 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried under vacuum to obtain the title compound as a white solid. 0.2 g, yield: 50.1%, DSR: 20%; 1 H NMR (400 MHz, D2O) δ ppm 8.72 - 8.57 (m, 0.2H), 8.41 - 8.16 (m, 0.2H), 7.96 - 7.82 (m, 0.2H), 7.45 - 7.29 (m, 0.2H), 7.02 - 6.87 (m, 0.2H), 4.56 - 4.02 (m, 3.4H), 3.97 - 2.84 (m, 11.4H), 2.03 - 1.81 (m, 3.6H), 1.43 (m, 1.2H).

[0549]

[0488] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.154g, yield: 38.6%, DSR=30%).

[0550] Example 65 Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbozide [ka]

[0551] Step 1: Preparation of tert-butyl N-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate [ka]

[0489] A mixture of 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)pyrazole-1-yl]azetidine-3-yl]acetonitrile (1486 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1337.6 mg, 4.4 mmol) in DMSO (20 mL) was stirred at room temperature for 7 hours. Then, tert-butylhydrazine carboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filtered cake was washed with a saturated NaHCO3 solution and water. Then, the filtered cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=100:1) to obtain the title compound (0.758 g, yield: 35.8%). MS(m / z):C 22 H 27[M+H]+ calculated value for N9O5S: 530.18; measured value: 530.1. 1 H NMR (400 MHz, CDCl3) δ ppm 10.93 (d, J = 1.8 Hz, 1H), 8.87 (s, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 7.95 (dd, J = 28.4, 4.1 Hz, 1H), 6.82 (t, J = 17.0 Hz, 1H), 6.70 (s, 1H), 4.64 (d, J = 9.3 Hz, 2H), 4.23 (t, J = 23.2 Hz, 2H), 3.42 (s, 2H), 3.09 (q, J = 7.4 Hz, 2H), 1.52 (s, 9H), 1.42 (t, J = 7.4 Hz, 3H).

[0552] Step 2: Preparation of 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbozide trifluoroacetate [ka]

[0490] Trifluoroacetic acid (2.4 mL) was slowly added to a stirred mixture of tert-butyl N-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonylazetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate (0.6 g, 1.13 mmol) in DCM (9.6 mL) using an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure to obtain the title compound as a white solid (0.61 g, yield: 99%), which was used without further purification. MS(m / z):C 17 H 19 [M+H]+ calculated value for N9O3S: 430.13; measured value: 430.0.

[0553] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbozide. [ka]

[0491] Sodium hyaluronate (212 mg, 0.526 mmol carboxylic acid, MW 50 kDa) was dissolved in 42.4 mL of deionized water in a 100 mL round-bottom flask, and then 28 mL of acetonitrile was added dropwise while stirring. To the solution, 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidine-3-yl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (200 mg, 0.368 mmol) and 4-methylmorpholine (NMM, 37 mg, 0.368 mmol) were added at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 146 mg, 0.526 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (308 mg, 5.26 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (300 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (308 mg, 5.26 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried under vacuum to obtain the title compound as a white solid. 0.2 g, yield: 48.1%, DSR: 40%; 1H NMR (400 MHz, D2O) δ ppm 8.89-8.663 (m, 0.4H), 8.49-8.17 (m, 0.4H), 8.04-7.84 (m, 0.4H), 7.05-6.91 (m, 0.4H), 4.43 (d, J = 34.6 Hz, 3.6H), 3.46 (dd, J = 86.0, 63.7 Hz, 11.6H), 1.93 (s, 3H), 1.26 (d, J = 43.7 Hz, 1.2H).

[0554]

[0492] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.154g, yield: 37%, DSR=40%).

[0555] Example 66 Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0556] Step 1: Preparation of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate [ka]

[0493] A mixture of (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)pyrazole-1-yl]propanenitrile (1486 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1141 mg, 3.754 mmol) in DMSO (12 mL) was stirred at room temperature for 7 hours. Then, (S)-methyl 6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1448 mg, 4.88 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filtered cake was washed with a saturated NaHCO3 solution and water. Then, the filtered cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=150:1) to obtain the title compound (0.828 g, yield: 34.9%). MS(m / z):C 30 H 40 The calculated value of N8O5's [M+H] is 593.31; the measured value is 593.2. 1 H NMR (400 MHz, CDCl3) δ ppm 9.95 (d, J = 7.5 Hz, 1H), 8.91 (s, 1H), 8.32 (d, J = 11.9 Hz, 2H), 7.99 (d, J = 4.0 Hz, 1H), 6.82 (d, J = 4.0 Hz, 1H), 4.76 (dd, J = 12.8, 7.5 Hz, 1H), 4.60 (s, 1H), 4.28 (td, J = 9.9, 3.8 Hz, 1H), 3.81 (s, 3H), 3.22-3.05 (m, 3H), 2.97 (dd, J = 17.0, 3.8 Hz, 1H), 2.61 (dt, J = 16.5, 8.4 Hz, 1H), 2.13-1.88 (m, 3H), 1.65-1.50 (m, 7H), 1.44-1.35 (m, 9H), 1.27 (d, J = 12.8 Hz, 4H).

[0557] Step 2: Preparation of methyl(2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride [ka]

[0494] A stirred solution of methyl(2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (0.6 g, 1.01 mmol) in ethyl acetate (12 mL) was slowly added with 4 M HCl (commercial) (2.4 mL) in ethyl acetate over an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.53 g, yield: 99%), which was used without further purification. MS(m / z):C 25 H 32 The calculated value of [M+H] for N8O3 is 493.25; the measured value is 493.2.

[0558] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and methyl(2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate. [ka]

[0495] Sodium hyaluronate (204 mg, 0.506 mmol carboxylic acid, MW 50 kDa) was dissolved in 40.8 mL of deionized water in a 100 mL round-bottom flask, and then 26.5 mL of acetonitrile was added dropwise while stirring. Methyl(2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride (200 mg, 0.354 mmol) and 4-methylmorpholine (NMM, 36 mg, 0.354 mmol) were added to the solution at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 140 mg, 0.506 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (207 mg, 3.54 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (300 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (207 mg, 3.54 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried under vacuum to obtain the title compound as a white solid. 0.195 g, yield: 45.1%, DSR: 27%; 1 H NMR (400 MHz, D2O) δ ppm 8.59-8.45 (m, 0.27H), 8.33-7.88 (m, 0.81H), 6.91-6.80 (m, 0.27H), 4.57-4.02 (m, 2.54H), 3.97-2.72 (m, 11.89H), 2.05-1.72 (m, 3.81H), 1.70-0.99 (m, 2.97H).

[0559]

[0496] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.197g, yield: 45.5%, DSR=16%).

[0560] Example 67 Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbozide [ka]

[0561] Step 1: Preparation of tert-butyl N-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate [ka]

[0497] A mixture of 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)pyrazole-1-yl]azetidine-3-yl]acetonitrile (1617 mg, 4 mmol), triethylamine (1.67 mL, 12 mmol), and bis(4-nitrophenyl) carbonate (1337.6 mg, 4.4 mmol) in DMSO (20 mL) was stirred at room temperature for 7 hours. Then, tert-butylhydrazine carboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filtered cake was washed with a saturated NaHCO3 solution and water. Then, the filtered cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=100:1) to obtain the title compound (1.02 g, yield: 54.9%). MS(m / z):C 23 H 28 The calculated value of [M+H] for N8O3 is 465.22; the measured value is 465.2. 1H NMR (400 MHz, CDCl3) δ ppm 10.97 (d, J = 1.9 Hz, 1H), 8.86 (s, 1H), 8.31 (d, J = 15.2 Hz, 2H), 7.97 (d, J = 4.0 Hz, 1H), 6.87-6.84 (m, 1H), 6.62 (s, 1H), 4.27 (td, J = 10.0, 3.9 Hz, 1H), 3.14 (dd, J = 17.0, 8.6 Hz, 1H), 2.97 (dd, J = 17.0, 3.9 Hz, 1H), 2.61 (dt, J = 17.0, 8.6 Hz, 1H), 2.04-1.92 (m, 1H), 1.79-1.62 (m, 5H), 1.54 (d, J = 13.3 Hz, 9H), 1.35-1.19 (m, 3H).

[0562] Step 2: Preparation of 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohdrozide hydrochloride [ka]

[0498] To a stirred solution of tert-butyl N-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate (0.75 g, 1.61 mmol) in ethyl acetate (15 mL), 3 mL of 4 M HCl (commercial) in ethyl acetate was slowly added in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with ethyl acetate and concentrated under reduced pressure to obtain the title compound as a white solid (0.557 g, yield: 95%), which was used without further purification. MS(m / z):C 18 H 20 N8O's [M+H] + Calculated value: 365.17; Measured value: 365.1.

[0563] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohdrozide [ka]

[0499] Sodium hyaluronate (263.5 mg, 0.654 mmol carboxylic acid, MW50 kDa) was dissolved in 53 mL of deionized water in a 100 mL round-bottom flask, and then 34 mL of acetonitrile was added dropwise while stirring. To the solution, 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazole-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (200 mg, 0.458 mmol) and 4-methylmorpholine (NMM, 46 mg, 0.458 mmol) were added at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 181 mg, 0.654 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (382 mg, 6.54 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (300 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (382 mg, 6.54 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried in vacuum to obtain the title compound as a white solid. 0.316 g, yield: 66.5%, DSR: 33%; 1H NMR (400 MHz, D2O) δ ppm 8.88 - 8.65 (m, 0.33H), 8.45 - 7.28 (m, 0.99H), 7.07 - 6.82 (m, 0.33H), 4.68 - 4.27 (m, 2.33H), 4.21 - 2.62 (m, 10.66H), 2.47 - 2.23 (m, 0.33H), 2.15 - 1.32 (m, 4.32H), 1.31 - 1.02 (m, 0.99H).

[0564]

[0500] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.308g, yield: 64.8%, DSR=21%).

[0565] Example 68 Preparation of a conjugate between HA (sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-aminoacetate [ka]

[0566] Step 1: Preparation of [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-(tert-butoxycarbonylamino)acetate [ka]

[0501] A mixture of 3-[(3R,4R)-3-[[7-(hydroxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile (600 mg, 1.754 mmol), 2-(tert-butoxycarbonylamino)acetic acid (1.23 mg, 7.02 mmol), and triphenylphosphine (1840 mg, 7.02 mmol) in THF (120 mL) was stirred at 0°C to 10°C for 10 minutes. Then, diisopropyl azodicarboxylic acid (1420 mg, 7.02 mmol) was added dropwise. The reaction mixture was stirred at this temperature for 16 hours. The reaction mixture was poured into a saturated NaHCO3 solution in an ice bath. The mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (Â:hexane = 1:2) to obtain the title compound (400 mg, yield: 45.7%). MS(m / z):C 24 H 33 The calculated value of N7O5's [M+H] is 500.25; the measured value is 500.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.31 (t, J = 16.1 Hz, 1H), 7.15 (dd, J = 13.4, 3.7 Hz, 1H), 6.56 (t, J = 3.9 Hz, 1H), 6.23 (d, J = 2.8 Hz, 2H), 5.14 (s, 1H), 4.96 (s, 1H), 4.10 - 3.98 (m, 1H), 3.97 - 3.76 (m, 3H), 3.68 - 3.45 (m, 4H), 3.42 - 3.30 (m, 3H), 2.57 - 2.43 (m, 1H), 2.01 - 1.85 (m, 1H), 1.82 - 1.69 (m, 1H), 1.51 - 1.32 (m, 9H), 1.27 - 1.24 (m, 2H), 1.13 - 1.09 (m, 1H).

[0567] Step 2: Preparation of [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-aminoacetate trifluoroacetate [ka]

[0502] To a stirred mixture of [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-(tert-butoxycarbonylamino) acetate (0.35 g, 0.7 mmol) in DCM (10 mL), trifluoroacetic acid (2 mL) was slowly added in an ice bath. The reaction mixture was cooled to room temperature and then stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure to obtain the title compound as a white solid (0.27 g, yield: 96.7%), which was used without further purification. MS(m / z):C 19 H 25 [M+H] of N7O3: Calculated value, 400.20; Measured value, 400.1.

[0568] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-aminoacetate. [ka]

[0503] Sodium hyaluronate (178 mg, 0.443 mmol carboxylic acid, MW 50 kDa) was dissolved in 53 mL of deionized water in a 100 mL round-bottom flask. To the solution, [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl 2-aminoacetate trifluoroacetate (150 mg, 0.443 mmol) and 4-methylmorpholine (NMM, 44.8 mg, 0.443 mmol) were added at room temperature to temporarily increase the viscosity. The reaction mixture was stirred at this temperature for 2 hours. To the solution, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 122 mg, 0.443 mmol) was added and the mixture was stirred at room temperature for 72 hours. Then, NaCl (259 mg, 4.43 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (300 mL) while stirring. The mixture was filtered. The filtered cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Then, NaCl (259 mg, 4.43 mmol) was added to the reaction mixture and stirred for 1 hour, followed by the dropwise addition of acetone (200 mL) while stirring. The mixture was filtered. The filtered cake was collected, washed with acetone, and dried under vacuum to obtain the title compound as a white solid. 0.15 g, yield: 44.5%, DSR: 21%; 1 H NMR (400 MHz, D2O) δ ppm 8.24 - 8.11 (m, 0.21H), 7.40 - 7.27 (m, 0.21H), 6.78 - 6.67 (m, 0.21H), 6.31 - 6.07 (m, 0.42H), 4.59 - 4.17 (m, 2.21H), 4.11 - 2.95 (m, 12.31H), 2.49 - 2.35 (m, 0.21H), 2.00 - 1.61 (m, 3.42H), 1.27 - 1.16 (m, 0.21H), 1.07 - 0.90 (m, 0.42H).

[0569]

[0504] This step, by reaction with sodium hyaluronate (MW2000kDa), yielded the corresponding product (0.108g, yield: 45.7%, DSR=19%).

[0570] Example 69 Preparation of a conjugate between HA (sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl-4-aminobutanoate [ka]

[0571] Step 1: Preparation of [5-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-yl]methyl-4-(tert-butoxycarbonylamino)butanoate [ka]

[0505] A mixture of 3-[(3R,4R)-3-[[7-(hydroxymethyl)pyrrolo[2,3-d]pyrimidine-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile (300 mg, 0.877 mmol), 4-(tert-butoxycarbonylamino)butanoic acid (713 mg, 3.51 mmol), and triphenylphosphine (920 mg, 3.51 mmol) in THF (60 mL) was stirred at 0°C to 10°C for 10 minutes. Then, diisopropyl azodicarboxylic acid (709.5 mg, 3.51 mmol) was added dropwise. The reaction mixture was stirred at this temperature for 16 hours. The reaction mixture was poured into a saturated NaHCO3 solution in an ice bath. The mixture was diluted with ethyl acetate and washed with saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (Â:hexane = 1:2) to obtain the title compound (240 mg, yield: 51.9%). MS(m / z):C 26 H37 The calculated value of N7O5's [M+H]+ is 528.28; the measured value is 528.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.34 (d, J = 7.5 Hz, 1H), 7.16 (dd, J = 13.8, 3.7 Hz, 1H), 6.55 (t, J = 4.1 Hz, 1H), 6.17 (d, J = 3.4 Hz, 2H), 5.14 (s, 1H), 4.61 (s, 1H), 4.09 - 3.99 (m, 1H), 3.89 - 3.56 (m, 3H), 3.54 - 3.31 (m, 5H), 3.12 (d, J = 6.1 Hz, 2H), 2.50 (d, J = 26.3 Hz, 1H), 2.37 (t, J = 7.3 Hz, 2H), 2.01 - 1.86 (m, 1H), 1.78 (dd, J = 14.2, 7.1 Hz, 2H), 1.50 - 1.30 (m, 9H), 1.31 -1.21 (m, 3H), 1.13 - 1.09 (m, 1H).

[0572] Step 2: Preparation of [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amin...

Claims

1. A drug delivery system for locally delivering therapeutic agents at a controlled rate, A biopolymer comprising at least a first binding group BG1, wherein the biopolymer is hyaluronic acid. A therapeutic agent comprising at least a second binding group BG2, wherein the therapeutic agent is tofacitinib, oclacitinib, baricitinib, ruxolitinib, nintedanib, or sunitinib, and A linker that covalently links a biopolymer to a therapeutic agent, allowing the therapeutic agent to be held at the administration site. Includes, Here, the linker is given by equation (I): 【Chemistry 1】 (In the formula, U is connected to the biopolymer via BG1, where, BG1 is a carboxyl group and U is -N(R) such that an amide linkage is formed. １ )or, 【Chemistry 2】 It is; BG1 is a carboxyl group and U is -O- or a direct bond so that an ester linkage is formed; or In order for an ester linkage to be formed, BG1 is a hydroxyl group and U is -C(=O)-, A is directly bonded, alkyl, and -(CH ２ CH ２ O) ｍ - is selected from, where the alkyl is one or more R ａ It is replaced by an optional choice in the base, B is selected from the group consisting of direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, where each of alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R ｂ It is replaced by an optional choice in the base, C is selected from direct bond, -C(=O)-, -C(=O)N(R ２ ), -N(R ２ ), -C(=O)-, -[CH ２ NHC(=O)] ｎ -, -[NHC(=O)CH ２ ｎ -, and -NH(CH ２ ) ｐ C(=O)-, and is selected from​ D is selected from direct bond, alkyl, and aryl, where the alkyl is one or more R ｃ It is replaced by an optional choice in the base, BG2 is an amine group, V is connected to the therapeutic agent via BG2 and is selected from one of the following (a) to (i): (a) Connected to the therapeutic agent via BG2 so that a urea linkage is formed -N(R ２ ) C (= O) -, (b) The therapeutic agent is connected via BG2 such that a thiourea linkage is formed -N(R ２ ) C(S)-, (c) Connected to the therapeutic agent via BG2 such that a thiocarbamate linkage is formed -OC(=S)-, (d) -OC(=O)OCH connected to the therapeutic agent via BG2 so that an ather-acetal linkage is formed. ２ - (e) -C(=O)OCH connected to the therapeutic agent via BG2 so that an ather-acetal linkage is formed. ２ - (f) The therapeutic agent is connected via BG2 such that an ather-acetal linkage is formed -N(R ２ )C(=O)OCH ２ - (g) A phosphoramidate linkage is formed, connected to the therapeutic agent via BG2 -OP(=O)(OPh)-, or (h) Connected to the therapeutic agent via BG2 such that a phosphoramidate linkage is formed -N(R ２ )P(=O)(OPh)-, R １ and R ２ It is independently selected from the group consisting of hydrogen, alkyl, alkenyl, and alkynyl, R ａ , R ｂ , and R ｃ These are halogen, hydroxyl, amino, cyano, nitro, alkyl, alkoxyl, -C(=O)OR ｅ , and are selected independently of NH, R ｅ It is an alkyl, m is an integer between 0 and 4. n is an integer from 1 to 4, and also, The drug delivery system comprising the structure (where p is an integer from 1 to 4).

2. A drug delivery system according to claim 1, U, 【Transformation 3】 When U is, 【Chemistry 4】 A drug delivery system selected from the group consisting of the following:

3. A drug delivery system according to claim 1, A is a direct bond, and B is selected from the group consisting of direct bonds, cycloalkyl, heterocyclyl, aryl, and heteroaryl; A is alkyl, and B is selected from the group consisting of directly bonded, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl; or, A is - (CH ２ CH ２ O) ｍ - and B is selected from the group consisting of direct bonds, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; The aforementioned drug delivery system.

4. A drug delivery system according to claim 3, A is a direct bond, and B is selected from the group consisting of direct bonds, cycloalkyl, aryl, and heteroaryl; A is alkyl, and B is selected from the group consisting of direct bond, aryl, and -O-aryl; or, A is - (CH ２ CH ２ O) ｍ - and B is selected from the group consisting of direct bonds, alkyl, aryl, and heteroaryl; The aforementioned drug delivery system.

5. A drug delivery system according to claim 1, A is alkyl, B is selected from the group consisting of direct bond, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is direct bond, -C(=O)-, -N(R ２ )C(=O)-,-[CH ２ NHC (=O) ｎ -, -[NHC(=O)CH ２ ] ｎ -, and -NH(CH ２ ) ｐ Selected from the group consisting of C(=O)-; or, A is - (CH ２ CH ２ O) ｍ - and B is selected from the group consisting of direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and C is direct bond or -N(R ２ )C (=O)-; The aforementioned drug delivery system.

6. A drug delivery system according to claim 5, A is alkyl, B is selected from the group consisting of direct bond, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is direct bond, -N(R ２ )C(=O)-, or-[NHC(=O)CH ２ ] ｎ - is; The aforementioned drug delivery system.

7. The linker states: Equations (Ia) to (Im): 【Chemistry 5-1】 【Chemistry 5-2】 (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, and each of these is one or more R ｂ It is replaced by an optional choice in the base, 【Transformation 6】 Each of these is -C(=O)OCH ３ It is replaced by an optional selection, The drug delivery system according to claim 1, comprising the structure (where q, r, s, t, u, and v are independently integers from 0 to 5).

8. A drug delivery system according to claim 7, M is selected from the group consisting of cyclohexyl, phenyl, pyridinyl, thiazolyl, and adamantyl, and each of these is one or more R ｂ It is replaced by an optional choice in the base. The aforementioned drug delivery system.

9. Linker, 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 (In the formula, 【Transformation 8】 Each of these is -C(=O)OCH ３ The drug delivery system according to claim 7, comprising a structure selected from the group consisting of (which is optionally substituted).

10. A drug delivery system according to any one of claims 1 to 9, wherein the therapeutic agent is tofacitinib. 【Request Item 11】 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 【Chemistry 9-4】 【Chemistry 9-5】 【Chemistry 9-6】 【Chemistry 9-7】 【Chemistry 9-8】 【Chemistry 9-9】 [Chemistry 9-10] 【Chemistry 9-11】 【Chemistry 9-12】 【Chemistry 9-13】 [Chemistry 9-14] A drug delivery system selected from the group consisting of the following.

12. A drug delivery system according to any one of claims 1 to 11, which is administered locally to a subject requiring a drug delivery system.

13. The drug delivery system according to claim 12, which is administered topically to a subject requiring a drug delivery system by injection, oral dosage form, inhalation, implantation, or topical application.

14. A pharmaceutical composition comprising a drug delivery system according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient.

15. A drug delivery system for a therapeutically effective amount according to any one of claims 1 to 13 or a pharmaceutical composition according to claim 14, for use in a method of treating a disorder in a subject requiring treatment of the disorder.

16. The drug delivery system or pharmaceutical composition according to claim 15, wherein the disorder is selected from the group consisting of inflammation, cancer, cardiovascular disease, respiratory disease, vascular endothelial growth factor (VEGF)-related diseases, osteoarthritis, neovascular (exudative) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), dermatitis, psoriasis, chronic obstructive pulmonary disease, and asthma.

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