Drug delivery system for local delivery of therapeutic agents and uses thereof

Abstract

Provided herein are drug delivery systems and methods for the local delivery of therapeutic agents and methods for treating disease using such drug delivery systems.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202180049488.5, application date July 13, 2021, and invention name “Drug delivery system for local delivery of therapeutic agents and its use”. The original application is a national phase application with international application number PCT / CN2021 / 105899, which claims priority to the international application with application number PCT / CN2020 / 102115 and application date July 15, 2020. Technical Field

[0002] The present disclosure relates to drug delivery systems and methods for the local delivery of therapeutic agents and methods for treating disease using such drug delivery systems. Background Art

[0003] Most therapeutic agents are delivered to the body systemically through oral / GI absorption or systemic injection. These delivery routes are convenient and suitable for treating systemic diseases. However, many diseases are local conditions. Although the therapeutic agents applied systemically can effectively treat these conditions, these therapeutic agents can also target other tissues or binding sites that may cause side effects or adverse reactions. In order to reduce systemic side effects, a drug delivery system for local administration is desirable. It is not as easy as taking drugs orally or by injection to deliver the therapeutic agent to the desired site. Therefore, a long-term, sustained-release drug delivery system for local delivery of drugs is a prerequisite for doctors and patients to accept such products. In addition, after administering the drug to the subject, the release curve of the therapeutic agent maintaining an effective concentration at the delivery site may significantly affect the effectiveness of the therapeutic agent. Therefore, the drug delivery of therapeutic agents at specific target tissues or sites in the body represents a long-term challenge in the pharmaceutical industry.

[0004] Many drug delivery systems have been developed to provide controlled drug delivery with tissue-specific or desired release profiles. The most common local drug delivery system uses biodegradable polymers to control the rate of release of therapeutic agents. These drug delivery systems release drugs by both biopolymer erosion and drug molecule diffusion. This complex release control has brought great challenges to pharmaceutical product manufacturing and quality control. Therefore, it is still necessary to develop a drug delivery system that can locally deliver therapeutic agents to specific tissues when the therapeutic agent release is controlled and side effects are reduced.

[0005] There are three key properties of a successful topical drug delivery system: the ability to maintain the delivery system at the delivery site; the ability to release the therapeutic agent at a desired rate and profile; and the ability to treat a localized condition with the therapeutic agent. The present disclosure provides a different approach to achieving these key properties for topical drug delivery systems. Specifically, a biopolymer, which maintains the drug delivery system at the delivery site due to its large molecular size; a therapeutic agent, which is selected from a marketed product or has demonstrated activity in late-stage clinical studies; and a linker covalently bound to the biopolymer and the therapeutic agent, which is chemically labile and, upon degradation, releases the therapeutic agent at a desired rate to a specific delivery site and specific condition. Summary of the Invention

[0006] In one aspect, the present disclosure provides a drug delivery system for locally delivering a therapeutic agent at a controlled rate, the drug delivery system comprising:

[0007] A biopolymer comprising at least a first binding group BG1, wherein the first binding group BG1 is selected from the group consisting of: a hydroxyl group, a carboxyl group, an amino group, and a combination thereof;

[0008] a therapeutic agent, the 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; and

[0009] a linker that covalently attaches the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration;

[0010] Wherein the linker comprises a structure of formula (I):

[0011]

[0012] in

[0013] U is linked to the biopolymer via BG1 such that at least one bond selected from ester or amide is formed, and U is selected from the group consisting of: a direct bond, -N(R 1 )-, -O-, -C(=O)- and in is a nitrogen-containing heterocyclic group, which optionally includes one or more additional heteroatoms selected from N, O or S;

[0014] A is selected from a direct bond, an alkyl group and -(CH2CH2O) m -, wherein the alkyl group is optionally replaced by one or more R a group substitution;

[0015] B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, an -O-cycloalkyl group, an -O-heterocyclyl group, an -O-aryl group, an -O-heteroaryl group, wherein each of the alkyl group, the cycloalkyl group, the heterocyclyl group, the aryl group and the heteroaryl group is optionally replaced by one or more R b group substitution;

[0016] 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)-;

[0017] D is selected from a direct bond, an alkyl group and an aryl group, wherein the alkyl group is optionally substituted by one or more R c group substitution;

[0018] V is linked to the therapeutic agent through BG2 such that at least one bond selected from the group consisting of an amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal, and combinations thereof is formed, and V is selected from the group consisting of 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 2 )P(=O)(OPh)-;

[0019] R 1 and R 2 independently selected from hydrogen, alkyl, alkenyl, and alkynyl;

[0020] R a 、R b and R c independently selected from halogen, hydroxy, amino, cyano, nitro, alkyl, alkoxy, -C(=O)OR e and = NH;

[0021] m is an integer from 0 to 4;

[0022] n is an integer from 1 to 4; and

[0023] p is an integer from 1 to 4.

[0024] In some embodiments, the linker in the drug delivery system provided herein comprises a structure of Formula (Ia) to (Im):

[0025]

[0026]

[0027] in,

[0028] U and V are as defined in this application;

[0029] M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, each of which is optionally replaced by one or more R b group substitution; and

[0030] q, r, s, t, u and v are independently integers from 0 to 4.

[0031] 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.

[0032] 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, anti-cancer drugs, and any drug that may have severe systemic toxicity.

[0033] In another aspect, the present disclosure provides a pharmaceutical composition comprising the drug delivery system provided herein and a pharmaceutically acceptable excipient.

[0034] In another aspect, the present disclosure provides a method of treating a disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein. DETAILED DESCRIPTION

[0035] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are shown in the accompanying structures and formulas. Although the present disclosure will be described in conjunction with the enumerated embodiments, it should be understood that the embodiments are not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to encompass all alternatives, modifications and equivalents, which may be included within the scope of the present disclosure as defined herein. Those skilled in the art will recognize that many methods and materials similar to or equivalent to the methods and materials described herein may be used in practicing the present disclosure. The present disclosure is in no way limited to the methods and materials described. In the event that one or more (including but not limited to defined terms, term usage, described technology, etc.) of the incorporated references and similar materials are different from or contradictory to the present application, the present disclosure shall prevail. All references, patents, and patent applications cited in the present disclosure are hereby incorporated by reference in their entirety.

[0036] It should be understood that certain features of the present disclosure that are described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for the sake of brevity may also be provided individually or in any suitable subcombination. It must be noted that, as used in this application, the singular forms "a" and "an" include their plural forms unless the context clearly indicates otherwise.

[0037] definition

[0038] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described herein. In addition, general principles of organic chemistry as well as specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, 2nd ed., University Science Books, Sausalito, 2006; Smith and March, March's Advanced Organic Chemistry, 6th ed., John Wiley & Sons, Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3rd ed., VCH Publishers, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4th ed., Cambridge University Press, Cambridge, 2004; the entire contents of each of the foregoing are incorporated herein by reference.

[0039] Throughout this disclosure, linking substituents are described. It is particularly desirable that each linking substituent include both the forward and reverse forms of the linking substituent. For example, -NR(CR'R")- includes both -NR(CR'R")- and -(CR'R")NR-. Where a structure clearly requires a linking group, the Markush variable listed for that group is understood to be the linking group. For example, if a structure requires a linking group and the Markush group definition of the variable lists "alkyl", it is understood that "alkyl" means a linking alkylene group.

[0040] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, such substituent may be bonded to any atom in the ring. When a substituent is listed without specifying the atom via which such substituent is bonded to the rest of the compound of a given formula, such substituent may be bonded to any atom in that formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0041] In any variable (e.g., R i ) occurs more than once in any constituent or formula of a compound, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, if the group shown is represented by 0 to 2 R i Partially substituted, the group may optionally be replaced by up to two R i Partially substituted, and R i Each occurrence is independently selected from R i Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0042] As used herein, the term "C i-j " indicates a range of carbon atoms, where i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point therebetween, and where j is greater than i. For example, C 1-6 Indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term "C 1-12 ” indicates 1 to 12 carbon atoms, especially 1 to 10 carbon atoms, especially 1 to 8 carbon atoms, especially 1 to 6 carbon atoms, especially 1 to 5 carbon atoms, especially 1 to 4 carbon atoms, especially 1 to 3 carbon atoms or especially 1 to 2 carbon atoms.

[0043] As used herein, the term "alkyl", whether used as part of another term or by itself, refers to a saturated straight or branched chain hydrocarbon group that may be optionally substituted independently with one or more substituents as described below. i-j "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. "C 1-10 Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. 1-6Examples of "alkyl" are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0044] As used herein, the term "alkenyl", whether as part of another term or used independently, refers to a straight or branched hydrocarbon group with at least one carbon-carbon double bond that can be optionally independently substituted by one or more substituents described herein and includes groups with "cis" orientation and "trans" orientation or alternatively "E" orientation and "Z" orientation. In some embodiments, the alkenyl contains 2 to 12 carbon atoms. In some embodiments, the alkenyl contains 2 to 11 carbon atoms. In some embodiments, the alkenyl 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 contains 2 carbon atoms. The example of alkenyl includes but is not limited to vinyl (ethylenyl or vinyl), propenyl (allyl), butenyl, pentenyl, 1-methyl-2-butene-1-yl, 5-hexenyl etc.

[0045] As used herein, the term "alkynyl", whether used as part of another term or independently, refers to a straight or branched hydrocarbon group with at least one carbon-carbon triple bond that can be optionally independently substituted by 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, etc.

[0046] As used herein, the term "alkoxy", whether used as part of another term or by itself, refers to an alkyl group as previously defined attached to the parent molecular group through an oxygen atom. i-j"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. "C 1-6 Examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, neopentoxy, n-hexyloxy, and the like.

[0047] As used herein, the term "amide" refers to -C(=O)NR'-, where R' represents hydrogen, N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl and other suitable organic groups.

[0048] As used herein, the term "amine" refers to a derivative of ammonia in which one or more hydrogen atoms are replaced by a substituent and can be replaced by N(H) n (R') 3-n wherein n is 0, 1 or 2, and each R' is independently hydroxy, nitro, N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl and other suitable organic groups, or two R' together with the nitrogen atom to which they are attached form an optionally substituted heterocyclyl or heteroaryl group.

[0049] As used herein, the term "amino" refers to -NH2.

[0050] As used herein, the term "aryl", whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems with a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may carry one or more substituents. As used herein, the scope of the term "aryl" also includes groups in which an aromatic ring is fused to one or more additional rings. In the case of a polycyclic system, only one ring is required to be aromatic (e.g., 2,3-dihydroindole), but all rings can be aromatic (e.g., quinoline). The second ring can also be fused or bridged. Examples of polycyclic aryl groups include, but are not limited to, benzofuranyl, indanyl, phthalimido, naphthalimido, phenanthridinyl, or tetrahydronaphthyl, etc. Aryl can be substituted by substituents as described above at one or more ring positions.

[0051] As used herein, the term "aza-acetal" refers to -N-CH2-O-.

[0052] As used herein, the term "carboxylic group or carboxyl" refers to -COOH.

[0053] As used herein, the term "cycloalkyl", whether used as part of another term or independently, refers to a monovalent non-aromatic saturated or partially unsaturated monocyclic and polycyclic ring system in which all ring atoms are carbon and the system contains at least three ring-forming carbon atoms. In some embodiments, the cycloalkyl group 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, 4 to 5 ring-forming carbon atoms. Cycloalkyl groups can be saturated or partially unsaturated. Cycloalkyl groups can be substituted. In some embodiments, the cycloalkyl group can be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group can be a partially unsaturated cyclic alkyl group containing at least one double bond or triple bond in its ring system. In some embodiments, the cycloalkyl group can be monocyclic or polycyclic. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro[3.6]-decyl, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, etc.

[0054] As used herein, the term "cyano" refers to -CN.

[0055] As used herein, the term "ester" refers to -C(=O)O-.

[0056] As used herein, the term "carbamate" refers to -NR'(C=O)O-, where R' represents hydrogen, N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups.

[0057] As used herein, the term "thiocarbamate" refers to -NR'(C=S)O-, where R' represents hydrogen, N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, and other suitable organic groups.

[0058] As used herein, the term "halogen" refers to an atom selected from the group consisting of fluorine (fluoro), chlorine (chloro), bromine (bromo), and iodine (iodo).

[0059] As used herein, the term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen (including N-oxides).

[0060] As used herein, the term "heteroaryl", whether used as part of another term or independently, refers to an aryl group having one or more heteroatoms in addition to carbon atoms. Heteroaryl can be monocyclic. Examples of monocyclic heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. Heteroaryl also includes a polycyclic group in which a heteroaromatic ring is fused to one or more aryl, alicyclic or heterocyclic rings, wherein a linking group or point of attachment is located on the heteroaromatic ring. Examples of polycyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, benzothiophenyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0061] As used herein, the term "heterocyclic radical" refers to a saturated or partially unsaturated carbocyclic radical, wherein one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, wherein one or more ring atoms can optionally be independently substituted by one or more substituents. In some embodiments, the heterocyclic radical is a saturated heterocyclic radical. In some embodiments, the heterocyclic radical is a partially unsaturated heterocyclic radical with one or more double bonds in its ring system. In some embodiments, the heterocyclic radical can contain any oxidized form of carbon, nitrogen or sulfur and any quaternized form of basic nitrogen. "Heterocyclic radical" also includes a group in which a heterocyclic radical is fused to a saturated, partially unsaturated or completely unsaturated (i.e., aromatic) carbocyclic ring or heterocyclic ring. Where possible, the heterocyclic radical can be carbon-connected or nitrogen-connected. In some embodiments, the heterocycle is carbon-connected. In some embodiments, the heterocycle is nitrogen-connected. For example, the group derived from pyrrole can be pyrrole-1-yl (nitrogen-connected) or pyrrole-3-yl (carbon-connected). Furthermore, the group derived from imidazole may be imidazol-1-yl (nitrogen attached) or imidazol-3-yl (carbon attached).

[0062] 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 ring system having 1 to 3 heteroatoms, each independently selected from nitrogen, oxygen, or sulfur. Fused, spiro, and bridged ring systems are also included within the scope of this definition. Examples of monocyclic heterocyclyls include, but are not limited to, oxetanyl, 1,1-dioxothietane, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidinyl, piperazinyl, piperidinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazinonyl, pyrrolidinyl, triazinonyl, etc. Examples of fused heterocyclic groups include, but are not limited to, phenyl-fused rings or pyridyl-fused rings, such as quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthidinyl, imidazo[1,2-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, [1,2,3]triazolo[4,3-a]pyridinyl, etc. Examples of spiro heterocyclic groups include, but are not limited to, spiropyranyl, spirooxazinyl, etc. Examples of bridged heterocyclic groups include, but are not limited to, morphinyl, hexamethylenetetraminyl, 3-aza-bicyclo[3.1.0]hexane, 8-aza-bicyclo[3.2.1]octane, 1-aza-bicyclo[2.2.2]octane, 1,4-diazabicyclo[2.2.2]octane (DABCO), and the like.

[0063] As used herein, the term "hydroxy" refers to -OH.

[0064] As used herein, the term "nitro" refers to -NO2.

[0065] As used herein, the term "urea" refers to -NR'(C=O)NR"-, wherein R' and R" each independently represent hydrogen, an N-protecting group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, and other suitable organic groups.

[0066] As used herein, the term "thiourea" refers to -NR'H(C=S)NR"-, wherein R' and R" each independently represent hydrogen, an N-protecting group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, and other suitable organic groups.

[0067] As used herein, the term "phosphoramidate" refers to -(NR')P(=O)(OR') a (NR”) b -, wherein R' and R" are independently empty, hydrogen, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl and other suitable organic groups, and a and b are independently 0, 1 or 2.

[0068] As used herein, the term "binding group" or "BG" refers to a group at a specific position within a first entity (e.g., a biopolymer as provided herein, a therapeutic agent) that is capable of reacting with another group from a second entity (e.g., a linker as provided herein) to form a bond, thereby linking the two entities together to form one entity. For example, a carboxyl group included in one entity can react with an amino group included in another entity to form an amide bond linking the two entities together, wherein the carboxyl group and the amino group can be considered a binding group.

[0069] As used herein, the term "bond" or "linker" refers to a bond or chemical moiety formed by a chemical reaction between the functional groups of at least two entities to be connected, thereby forming a molecule or maintaining the entities in close enough proximity to associate. The linker can be integrated into the resulting connected molecule or structure with or without its reactive functional group. Such bonds can be covalent or non-covalent. A hydrolytically unstable or degradable bond means that the bond is capable of degrading in water or an aqueous solution (including, for example, body fluids such as blood). An enzymatically unstable or degradable bond means that the bond can be degraded by one or more enzymes. Such degradable bonds include, but are not limited to, ester bonds formed by a carboxylic acid in one entity and an alcohol group on a bioactive agent, wherein such ester groups are generally hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable bonds include, but are not limited to, carbonate bonds, imine bonds generated by the reaction of an amine with an aldehyde, phosphate bonds generated by the reaction of a phosphate group with an alcohol, hydrazone bonds generated by the reaction of a hydrazide with an aldehyde, acetal bonds generated by the reaction of an aldehyde with an alcohol, and amide bonds generated by the reaction of an amine group with a carboxyl group.

[0070] As used herein, the term "partially unsaturated" refers to a group that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings with multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0071] As used herein, the term "pharmaceutically acceptable" indicates that a substance or composition is chemically and / or toxicologically compatible with the other ingredients making up the formulation and / or the subject being treated.

[0072] As used herein, the term "substituted", whether or not there is the term "optionally" in front, means that one or more hydrogens of a specified portion are replaced by a suitable substituent. It should be understood that "substituted" or "substituted by..." includes implicit prerequisites, i.e., such substitutions are consistent with the allowed valence of the substituted atom, and that substitutions produce stable or chemically feasible compounds, for example, compounds that do not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. Unless otherwise indicated, "optionally substituted" groups can have suitable substituents at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a specified group, at each position, the substituents can be the same or different. It will be understood by those skilled in the art that, if appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", reference to a chemical moiety herein should be understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted variants and unsubstituted variants.

[0073] As used herein, the terms "therapeutic agent," "drug," "bioactive molecule," "bioactive agent," "active agent," and the like refer to any substance that may affect any physical or biochemical property of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, a therapeutic agent includes any substance intended for use in diagnosing, curing, mitigating, treating, or preventing disease in humans or other animals, or otherwise enhancing the physical or mental well-being of humans or animals.

[0074] Drug delivery of therapeutic agents to specific tissues or sites within the body presents various challenges, particularly when it is desired to deliver high doses of poorly water-soluble therapeutic agents locally to specific tissues and to avoid toxic side effects from high systemic concentrations of the therapeutic agent.

[0075] Therefore, in one aspect, the present disclosure provides a drug delivery system capable of locally delivering a therapeutic agent at a controlled rate. In some embodiments, the drug delivery system comprises a biopolymer, a therapeutic agent, and a linker that covalently links the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration.

[0076] biopolymers

[0077] Biopolymers are natural polymers produced by organisms, and contain monomeric units that are covalently bonded to form larger structures. According to the structure of the monomeric units used and the biopolymers formed, biopolymers are divided into three main categories: polynucleotides, polypeptides and polysaccharides. More specifically, polynucleotides (such as RNA and DNA) are long polymers consisting of 13 or more nucleotide monomers. Polypeptides or proteins are short polymers of amino acids, and some main examples include collagen, actin and fibrin. Polysaccharides are typically linearly bonded polymeric carbohydrate structures, and some examples include cellulose and alginate. Other examples of biopolymers include rubber, suberin, melanin and lignin.

[0078] A variety of biopolymers can be used as polymeric delivery vehicles to deliver therapeutic agents to target cells or tissues. The biopolymer suitable for a particular application is selected based on its ability to target specific tissues, organs or cells and its in vivo stability, that is, the in vivo residence time in the circulatory system or specific tissues, cells or organs.

[0079] In some embodiments, the biopolymer is selected from a biocompatible polymer comprising at least a first binding group BG1, wherein the first binding group is capable of reacting with a reactive functional group from a second entity (e.g., a linker as provided herein) to form a bond, thereby linking the biopolymer to the second entity (e.g., a linker). As used herein, the term "biocompatible" refers to a substance that has no medically unacceptable toxic or harmful effects on biological functions or is tolerable to the body.

[0080] In some embodiments, the biopolymer is selected from a biocompatible polymer comprising at least a first binding group, BG1, wherein BG1 is selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, and combinations thereof. BG1 serves as a binding site for conjugating a linker suitable for attaching a therapeutic agent to the biopolymer. BG1 can be present at any site within the backbone of the biopolymer, and thus the bond formed between the biopolymer and the linker can be present at any portion of the biopolymer.

[0081] In some embodiments, the BG1 used to react with the reactive functional group from the linker can be the same or different. In certain embodiments, the BG1 of the biopolymer is the same. In certain embodiments, the BG1 of the biopolymer is different.

[0082] In some embodiments, the biopolymer is a biocompatible polymer that includes a carboxyl group as BG1 that is capable of reacting with a reactive functional group of a suitable linker to form a bond connecting the carboxyl-containing biopolymer to the linker.

[0083] In certain embodiments, the reactive functional group of the linker is an amino group or an amine, which reacts with the carboxyl group of the biopolymer such that an amide bond is formed.

[0084] In certain embodiments, the reactive functional group of the linker is a hydroxyl group, which reacts with the carboxyl group of the biopolymer such that an ester bond is formed.

[0085] In certain embodiments, the reactive functional group of the linker is a halogen that reacts with the carboxyl group of the biopolymer such that an ester bond is formed.

[0086] In some embodiments, the biopolymer is a biocompatible polymer that includes an amino group as BG1 that is capable of reacting with a reactive functional group of a suitable linker to form a bond, thereby generating a biopolymer-linker conjugate.

[0087] In certain embodiments, the reactive functional group of the linker is a carboxyl group, which reacts with the hydroxyl group of the biopolymer such that an ester bond is formed.

[0088] In some embodiments, the bond formed by the reaction between BG1 of the biopolymer and the reactive functional group of the linker is selected from the group consisting of: -C(O)N(R 1 )-、 and -C(O)O-, where R 1 is selected from the group consisting of hydrogen, alkyl, alkenyl and alkynyl, is a nitrogen-containing heterocyclic group optionally including one or more additional heteroatoms selected from N, O or S.

[0089] In certain embodiments, R 1 It's hydrogen.

[0090] In certain embodiments, Selected from the group consisting of:

[0091]

[0092] In some embodiments, the biopolymer can be selected from the group consisting of hyaluronic acid (HA), dextran, cellulose, amylose, chitosan, chitin, chondroitin, gelatin, alginate, carrageenan, gellan gum, guar gum, pectin, scleroglucan, and xanthan gum.

[0093] In some embodiments, the number average molecular weight of the biopolymer can be in the range of 400 to 3,000,000 Da, e.g., 1,000 to 3,000,000 Da, 5,000 to 3,000,000 Da, 10,000 to 3,000,000 Da, 20,000 to 3,000,000 Da, 30,000 to 3,000,000 Da, 40,000 to 3,000,000 Da, 50,000 to 3,000,000 Da, or 50,000 to 2,000,000 Da.

[0094] In some embodiments, the biopolymer is selected from the group consisting of HA, chitosan, chitin, chondroitin, or derivatives thereof.

[0095] In certain embodiments, the biopolymer is HA.In certain embodiments, the HA can be derived from any source.

[0096] In certain embodiments, the number average molecular weight of HA may be in the range of 400 to 3,000,000 Da, e.g., 1,000 to 3,000,000 Da, 5,000 to 3,000,000 Da, 10,000 to 3,000,000 Da, 20,000 to 3,000,000 Da, 30,000 to 3,000,000 Da, 40,000 to 3,000,000 Da, 50,000 to 3,000,000 Da, or 50,000 to 2,000,000 Da.

[0097] therapeutic agents

[0098] The present disclosure provides improved delivery systems for the local delivery of various therapeutic agents.

[0099] In some embodiments, the therapeutic agent includes at least a second binding group BG2, which is capable of reacting with a reactive functional group from a second entity (e.g., a linker provided herein) and an optional co-reactant to form a bond, thereby linking the therapeutic agent to the second entity (e.g., a linker).

[0100] 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 serves as a binding site for conjugating a linker suitable for linking the therapeutic agent to the biopolymer.

[0101] In some embodiments, the therapeutic agent includes an amine group as BG2 that is capable of reacting with a reactive functional group of a suitable linker and an optional co-reactant to form a bond connecting the amine-containing therapeutic agent to the linker.

[0102] 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 attached to the linker via a direct bond, an amide bond, a urea bond, a thiourea bond, a carbamate bond, a thiocarbamate bond, an aza-acetal bond, a phosphoramidate bond, or the like.

[0103] In certain embodiments, the bond formed by the reaction involving BG2 of the therapeutic agent and the reactive functional group of the linker and the optional coreactant is selected from the group consisting of: -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(R2 )C(=O)OCH2N(R')2-, -OP(=O)(OPh)N(R')2- and -N(R 2 )P(=O)(OPh)N(R')2-, wherein 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' together with the nitrogen atom to which they are both attached form a heterocyclic group.

[0104] In certain embodiments, R 2 It's hydrogen.

[0105] In some embodiments, the therapeutic agent includes a carboxyl group as BG2 that is capable of reacting with a reactive functional group of a suitable linker and an optional co-reactant to form a bond connecting the carboxyl-containing therapeutic agent to the linker.

[0106] In certain embodiments, the carboxyl group of the therapeutic agent reacts with the reactive functional group of the linker and the optional co-reactant, such that the therapeutic agent is attached to the linker through an ester bond.

[0107] In some embodiments, the therapeutic agent includes a hydroxyl group as BG2 that is capable of reacting with a reactive functional group of a suitable linker and an optional co-reactant to form a bond connecting the hydroxyl-containing therapeutic agent to the linker.

[0108] In certain embodiments, the hydroxyl group in the therapeutic agent reacts with the reactive functional group of the linker and the optional co-reactant, such that the therapeutic agent is attached to the linker through an ester bond.

[0109] 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 drug that may have severe systemic toxicity.

[0110] In some embodiments, the therapeutic agent to be delivered is an NSAID selected from the group consisting of Piroxicam, Meloxicam, and Diclofenac.

[0111] In some embodiments, the therapeutic agent to be delivered is a JAK inhibitor selected from the group consisting of Tofacitinib, Ruxolitinib, Baricitinib, Pefitinib, Fedratinib, Oclacitinib, and Upadacitinib.

[0112] 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.

[0113] In some embodiments, the therapeutic agent to be delivered is tofacitinib.

[0114] In some embodiments, the therapeutic agent to be delivered is upadacitinib.

[0115] In some embodiments, the therapeutic agent to be delivered is ruxolitinib.

[0116] In some embodiments, the therapeutic agent to be delivered is baricitinib.

[0117] In some embodiments, the therapeutic agent to be delivered is oclacitinib.

[0118] In some embodiments, the therapeutic agent to be delivered is nintedanib.

[0119] In some embodiments, the therapeutic agent to be delivered is sunitinib.

[0120] Linker

[0121] Improved local delivery of therapeutic agents is achieved by linking the therapeutic agent to the biopolymer via a suitable linker. By selecting a suitable linker, the rate of release of the therapeutic agent from the biopolymer can be controlled, thereby providing improved delivery of the therapeutic agent to target cells or tissues.

[0122] In some embodiments, multiple linkers can be attached to the therapeutic agent via a cleavable bond that is cleaved under biological conditions, thereby releasing the therapeutic agent.

[0123] " Cleavable bond " is the relatively unstable bond that cracks under physiological conditions. Exemplary releasable bond is the hydrolyzable bond that cracks (that is, hydrolyzes) when reacting with water. The trend of bond hydrolysis in water may depend not only on the key of the general type connecting two atoms, but also on the substituent connected to these atoms. Suitable hydrolysis unstable or weak bonds include but are not limited to carboxylate, phosphate, anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester, peptide, oligonucleotide, thioester, urea, thiourea, carbamate, thiocarbamate, phosphoramidate and carbonate. Some functional groups have atoms that can be chemically degraded by the process except hydrolysis. This type of exemplary releasable bond includes some carbamate and Fmoc derivatives. Some molecules of the functional groups of the appropriate bonding containing these classes can experience chemical degradation (release) after alkali action. In such cases, " cracking " can occur under higher pH values ​​or occur by the effect of the biomolecule (for example, histidine) containing alkaline part. Another exemplary cleavable bond is an enzymatic cleavage bond. "Enzymatically cleavable bond" means a bond that is subject to cleavage by one or more enzymes.

[0124] In some embodiments, the linker is attached to the biopolymer via a bond formed between a reactive functional group of the linker and BG1 in the biopolymer, and is attached to the therapeutic agent via a bond formed between another reactive functional group of the linker and BG2 in the therapeutic agent.

[0125] In some embodiments, the linker comprises a structure of formula (I):

[0126]

[0127] in

[0128] U is linked to the biopolymer via BG1 of the biopolymer such that at least one bond selected from ester or amide is formed, and U is selected from the group consisting of: a direct bond, N(R 1 )-, -O-, -C(=O)- and in is a nitrogen-containing heterocyclic group, which optionally includes one or more additional heteroatoms selected from N, O or S;

[0129] A is selected from a direct bond, an alkyl group and -(CH2CH2O) m -, wherein the alkyl group is optionally replaced by one or more R a group substitution;

[0130] B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, an -O-cycloalkyl group, an -O-heterocyclyl group, an -O-aryl group, an -O-heteroaryl group, wherein each of the alkyl group, the cycloalkyl group, the heterocyclyl group, the aryl group and the heteroaryl group is optionally replaced by one or more Rb group substitution;

[0131] 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)-;

[0132] D is selected from a direct bond, an alkyl group and an aryl group, wherein the alkyl group is optionally substituted by one or more R c group substitution;

[0133] V is linked to the therapeutic agent through BG2 in the therapeutic agent such that at least one bond is formed, the at least one bond being selected from the group consisting of amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal, and combinations thereof, and V is selected from the group consisting of 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 2 )P(=O)(OPh)-;

[0134] R 1 and R 2 independently selected from the group consisting of: hydrogen, alkyl, alkenyl, and alkynyl;

[0135] R a 、R b and R c independently selected from halogen, hydroxy, amino, cyano, nitro, alkyl, alkoxy, -C(=O)OR e and = NH;

[0136] R e is an alkyl group;

[0137] m is an integer from 0 to 4;

[0138] n is an integer from 1 to 4; and

[0139] p is an integer from 1 to 4.

[0140] In some embodiments, BG1 is carboxyl and U is -N(R 1)-, such that an amide bond is formed to link the biopolymer to the linker.

[0141] In some embodiments, BG1 is carboxyl and U is An amide bond is allowed to form to connect the biopolymer to the linker.

[0142] In certain embodiments, BG1 is carboxyl and U is selected from the group consisting of

[0143]

[0144] In some embodiments, BG1 is hydroxy and U is -C(=O)-, such that an ester bond is formed to connect the biopolymer to the linker.

[0145] In some embodiments, BG1 is a carboxyl group and U is -O- or a direct bond, such that an ester bond is formed to connect the biopolymer to the linker.

[0146] In some embodiments, BG1 is an amino group and U is -C(=O)-, such that an amide bond is formed to connect the biopolymer to the linker.

[0147] In some embodiments, BG2 is an amine group, and V is selected from one of the following:

[0148] (a) Direct key;

[0149] (b)-N(R 2 )C(=O)-, the -N(R 2 )C(═O)- is linked to the therapeutic agent through BG2, such that a urea bond is formed to link the therapeutic agent to the linker;

[0150] (c)-N(R 2 )C(S)-, said-N(R 2 )C(S)- is linked to the therapeutic agent through BG2, such that a thiourea bond is formed to link the therapeutic agent to the linker;

[0151] (d) -OC(=O)-, wherein the -OC(=O)- is linked to the therapeutic agent through BG2, such that a carbamate bond is formed to link the therapeutic agent to the linker;

[0152] (e) -OC(=S)-, wherein the -OC(=S)- is linked to the therapeutic agent through BG2, such that a thiocarbamate bond is formed to link the therapeutic agent to the linker;

[0153] (f) -OC(=O)OCH2-, wherein the -OC(=O)OCH2- is linked to the therapeutic agent through BG2, such that an aza-acetal bond is formed to link the therapeutic agent to the linker;

[0154] (g) -C(=O)OCH2-, wherein the -C(=O)OCH2- is linked to the therapeutic agent through BG2 such that an aza-acetal bond is formed;

[0155] (h)-N(R 2 )C(=O)OCH2-, the -N(R 2 )C(═O)OCH2- is linked to the therapeutic agent through BG2, such that an aza-acetal bond is formed to link the therapeutic agent to the linker;

[0156] (i) -OP(=O)(OPh)-, wherein the -OP(=O)(OPh)- is linked to the therapeutic agent through BG2, such that a phosphoramidate bond is formed to link the therapeutic agent to the linker; or

[0157] (j)-N(R 2 )P(=O)(OPh)-, the -N(R 2 )P(═O)(OPh)- is linked to the therapeutic agent through BG2, such that a phosphoramidate bond is formed to link the therapeutic agent to the linker;

[0158] (k) -C(=O)-, wherein the -C(=O)- is linked to the therapeutic agent through BG2 such that an amide bond is formed.

[0159] In certain embodiments, BG2 is a carboxyl group and V is -O- or a direct bond, such that an ester bond is formed to link the therapeutic agent to the linker.

[0160] In certain embodiments, BG2 is hydroxy and V is -C(=O)-, such that an ester bond is formed to link the therapeutic agent to the linker.

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

[0162] In some embodiments, A is optionally replaced by one or more R a In certain embodiments, A is optionally substituted with one or more R a C 1-10 In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl.

[0163] In some embodiments, R ais -C(=O)OR e , where R e In certain embodiments, R a is -C(=O)OR e , where R e It 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 In certain embodiments, R a It is -C(=O)OCH3.

[0164] In some embodiments, A is -(CH2CH2O) m -.

[0165] 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.

[0166] In some embodiments, B is a direct bond.

[0167] 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 In certain embodiments, B is ethyl.

[0168] In some embodiments, B is cycloalkyl, aryl, or heteroaryl.

[0169] In some embodiments, B is cycloalkyl.

[0170] In certain embodiments, B is a saturated cycloalkyl. In certain embodiments, B is a partially unsaturated cycloalkyl.

[0171] In certain embodiments, B is 3 to 8 membered cycloalkyl, 3 to 7 membered cycloalkyl, 3 to 6 membered cycloalkyl, 3 to 5 membered cycloalkyl, or 3 to 4 membered cycloalkyl.

[0172] In certain embodiments, B is a saturated 3- to 6-membered cycloalkyl. In certain embodiments, B is a cyclohexyl group.

[0173] In some embodiments, B is aryl. In certain embodiments, B is 5- to 12-membered aryl, 5- to 10-membered aryl, 5- to 8-membered aryl, or 5- to 6-membered aryl.

[0174] In certain embodiments, B is phenyl.

[0175] In some embodiments, B is heteroaryl. In certain embodiments, B is 5- to 12-membered heteroaryl, 5- to 10-membered heteroaryl, 5- to 8-membered heteroaryl, or 5- to 6-membered heteroaryl.

[0176] In certain embodiments, B is pyridinyl.

[0177] In some embodiments, B is -O-aryl. In certain embodiments, B is -O-phenyl.

[0178] In some embodiments, A is a direct bond, and B is selected from the group consisting of a direct bond, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0179] In certain embodiments, A is a direct bond, and B is selected from the group consisting of a direct bond, a cycloalkyl group, an aryl group, and a heteroaryl group.

[0180] In certain embodiments, A is a direct bond and B is a direct bond.

[0181] In certain embodiments, A is a direct bond and B is a 3- to 8-membered cycloalkyl, a 3- to 7-membered cycloalkyl, a 3- to 6-membered cycloalkyl, a 3- to 5-membered cycloalkyl, or a 3- to 4-membered cycloalkyl. In certain embodiments, A is a direct bond and B is a cyclohexyl.

[0182] In certain embodiments, A is a direct bond and B is a 5- to 12-membered aryl, a 5- to 10-membered aryl, a 5- to 8-membered aryl, or a 5- to 6-membered aryl. In certain embodiments, A is a direct bond and B is phenyl.

[0183] In certain embodiments, A is a direct bond and B is a 5- to 12-membered heteroaryl, a 5- to 10-membered heteroaryl, a 5- to 8-membered heteroaryl, or a 5- to 6-membered heteroaryl. In certain embodiments, A is a direct bond and B is pyridinyl.

[0184] In some embodiments, A is optionally substituted alkyl, and B is selected from the group consisting of a direct bond, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl.

[0185] In certain embodiments, A is optionally substituted alkyl and B is selected from the group consisting of a direct bond, aryl, and -O-aryl.

[0186] In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl, and B is a direct bond, where R a is -C(=O)ORe , where R e It is an alkyl group.

[0187] In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl, and B is a 5- to 12-membered aryl, a 5- to 10-membered aryl, a 5- to 8-membered aryl, or a 5- to 6-membered aryl. In certain embodiments, A is optionally substituted with one or more R a C 1-8 alkyl, and B is phenyl.

[0188] In certain embodiments, A is optionally replaced by one or more R a C 1-8 In certain embodiments, A is optionally substituted with one or more R a C 1-8 alkyl, and B is -O-phenyl.

[0189] In some embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group.

[0190] In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is selected from the group consisting of a direct bond, an alkyl group, an aryl group, and a heteroaryl group.

[0191] In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is a direct bond.

[0192] In certain embodiments, 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 In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is an ethyl group.

[0193] In certain embodiments, A is -(CH2CH2O) m-, wherein m is an integer from 0 to 4, and B is a 5- to 12-membered aryl, a 5- to 10-membered aryl, a 5- to 8-membered aryl, or a 5- to 6-membered aryl. In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is a phenyl group.

[0194] In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is a 5- to 12-membered heteroaryl, a 5- to 10-membered heteroaryl, a 5- to 8-membered heteroaryl, or a 5- to 6-membered heteroaryl. In certain embodiments, A is -(CH2CH2O) m -, wherein m is an integer from 0 to 4, and B is a pyridyl group.

[0195] In some embodiments, C is a direct bond.

[0196] In some embodiments, C is -C(=O)-.

[0197] In some embodiments, C is -C(=O)N(R 2 )-.

[0198] In some embodiments, C is -N(R 2 )C(=O)-.

[0199] In some embodiments, C is -[CH2NHC(=O)] n -.

[0200] In some embodiments, C is -[NHC(=O)CH2] n -.

[0201] In some embodiments, C is -NH(CH2) p C(=O)-.

[0202] In some embodiments, A is alkyl, B is selected from the group consisting of a direct bond, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, -O-cycloalkyl group, -O-heterocyclyl group, -O-aryl group, and -O-heteroaryl group, and C is selected from the group consisting of a direct bond, -C(=O)-, -N(R 2 )C(=O)-, -[CH2NHC(=O)] n -, -[NHC(=O)CH2] n - and -NH(CH2) p C(=O)-.

[0203] In some embodiments, A is alkyl, B is selected from the group consisting of a direct bond, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is a direct bond, -N(R 2 )C(=O)- or -[NHC(=O)CH2] n -.

[0204] In certain embodiments, A is alkyl, B is selected from the group consisting of a direct bond, aryl, and -O-aryl, and C is a direct bond or -N(R 2 )C(=O)-.

[0205] In certain embodiments, A is optionally replaced by one or more R a C 1-8 Alkyl, B is a direct bond, and C is a direct bond or -N(R 2 )C(=O), wherein R a is -C(=O)OR e , where R e It is an alkyl group.

[0206] In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl, B is a 5- to 12-membered aryl, a 5- to 10-membered aryl, a 5- to 8-membered aryl, or a 5- to 6-membered aryl, and C is a direct bond or -N(R 2 )C(=O)-, wherein R a is -C(=O)OR e , where R e It is an alkyl group.

[0207] In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl, B is phenyl, and C is a direct bond or -N(R 2 )C(=O)-, wherein R a is -C(=O)OR e , where R e It is an alkyl group.

[0208] In certain embodiments, A is optionally replaced by one or more R a C 1-8 alkyl, B is -O-aryl, and C is a direct bond, wherein R a is -C(=O)OR e , where R e It is an alkyl group.

[0209] In certain embodiments, A is optionally replaced by one or more Ra C 1-8 alkyl, B is -O-phenyl, and C is a direct bond, wherein R a is -C(=O)OR e , where R e It is an alkyl group.

[0210] In some embodiments, A is -(CH2CH2O) m -, B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group, and C is a direct bond or -N(R 2 )C(=O)-.

[0211] In some embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is selected from the group consisting of a direct bond, an alkyl group, an aryl group and a heteroaryl group, and C is a direct bond.

[0212] In some embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is a direct bond, and C is a direct bond.

[0213] 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 alkyl, and C is a direct bond.

[0214] In some embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is an aryl group, and C is a direct bond. In certain embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is phenyl, and C is a direct bond.

[0215] In some embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is heteroaryl, and C is a direct bond. In certain embodiments, A is -(CH2CH2O) m -, wherein m is 1, 2, 3 or 4, B is pyridyl, and C is a direct bond.

[0216] In some embodiments, D is a direct bond.

[0217] In some embodiments, D is alkyl. In certain embodiments, D is C1-6 Alkyl, C 1-5 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl or C 1-2 alkyl.

[0218] In some embodiments, D is aryl. In certain embodiments, D is 5- to 12-membered aryl, 5- to 10-membered aryl, 5- to 8-membered aryl, or 5- to 6-membered aryl. In certain embodiments, D is phenyl.

[0219] In some embodiments, the linkers provided herein include structures of Formula (Ia) to (Im):

[0220]

[0221]

[0222] in,

[0223] U and V are as defined above;

[0224] M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, each of which is optionally replaced by one or more R b group substitution;

[0225] Each of which is optionally substituted with -C(=O)OCH3; and

[0226] q, r, s, t, u and v are independently integers from 0 to 4.

[0227] 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.

[0228] In some embodiments, the linkers provided herein include a structure selected from the group consisting of:

[0229]

[0230]

[0231]

[0232] in Each of is optionally substituted with -C(=O)OCH3.

[0233] Drug delivery systems

[0234] In one aspect of the present disclosure, a therapeutic agent is attached to a biopolymer via a linker, thereby providing a drug delivery system for localized delivery of the therapeutic agent to a target site.

[0235] The biopolymers of the drug delivery systems provided herein can have one or more therapeutic agents conjugated via a linker. The biopolymers can be conjugated to the one or more therapeutic agents via one or more linkers at hydroxyl, carboxyl, and / or amino groups in the biopolymer backbone.

[0236] The drug delivery system of the present disclosure is obtained by conjugation between a biopolymer and a therapeutic agent via a linker by forming a bond between the biopolymer and the linker and between the therapeutic agent and the linker.

[0237] In some embodiments, the reactive functional group of the linker can first react with BG2 of the therapeutic agent to form a bond between the therapeutic agent and the linker, thereby providing a therapeutic agent-linker conjugate. The therapeutic agent-linker conjugate containing another reactive functional group at the end of the linker can then react with BG1 of the biopolymer to form a bond between the biopolymer and the linker, thereby providing the drug delivery system of the present disclosure.

[0238] In certain embodiments, BG1 of the biopolymer can be first reacted with a reactive functional group of the linker to form a biopolymer-linker conjugate, and then BG2 of the therapeutic agent can be reacted with another functional group of the linker in the biopolymer-linker conjugate, thereby providing the drug delivery system of the present disclosure.

[0239] In some embodiments, the biopolymer selected for the drug delivery systems provided herein is HA, and the therapeutic agent selected for the drug delivery systems provided herein is tofacitinib.

[0240] In certain embodiments, the drug delivery system provided herein is selected from the group consisting of:

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253] In some embodiments, the therapeutic agent can be conjugated to the biopolymer via a linker, and the therapeutic agent has a drug substitution ratio (DSR) with the biopolymer of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, as measured by NMR, wherein the drug substitution ratio (DSR) with the biopolymer refers to the ratio of the molar amount of groups on the biopolymer that are substituted by the drug to the total molar amount of groups on the biopolymer that can be substituted by the drug.

[0254] By cleaving the bond between the connexon and the biopolymer or therapeutic agent, the therapeutic agent can be released from the drug delivery system provided herein. In some embodiments, the release of the therapeutic agent occurs when the bond between the biopolymer and the connexon is cleaved to release the therapeutic agent-connexon conjugate, which can be considered a prodrug. Subsequently, releasing the therapeutic agent from the connexon can involve enzymatic or non-enzymatic cleavage of the bond between the therapeutic agent and the connexon. In some embodiments, the release of the therapeutic agent occurs when the bond between the therapeutic agent and the connexon is not cleaved or cleaved before the bond between the biopolymer and the connexon is cleaved. The release of the therapeutic agent can also involve enzymatic or non-enzymatic processes.

[0255] The release of therapeutic agents may be affected by a variety of factors, such as the choice of specific therapeutic agent, linker and biopolymer, and the administration of the drug delivery system. The present disclosure contemplates biopolymers with different molecular weights, binding groups BG1, and linkages to linkers; linkers with different reactive functional groups and subunits; and therapeutic agents with different binding groups BG2 and linkages to linkers.

[0256] The present disclosure also contemplates different local applications of the drug delivery system provided herein. In some embodiments, the drug delivery system provided herein is topically applied to a subject in need. In certain embodiments, the drug delivery system provided herein is topically applied to a subject in need by injection. In certain embodiments, the drug delivery system provided herein is topically applied to a subject in need by oral dosage forms. In certain embodiments, the drug delivery system provided herein is topically applied to a subject in need by suction. In certain embodiments, the drug delivery system provided herein is topically applied to a subject in need by implant. In certain embodiments, the drug delivery system provided herein is topically applied to a subject in need by topical application. According to specific therapeutic agent, linker and biopolymer combination, the release of therapeutic agent may occur in different locations when applied to a subject. Mode such as, the release of therapeutic agent may occur at the site of application.

[0257] In some embodiments, administration of the drug delivery systems provided herein to a subject can provide release of the therapeutic agent over a period of at least several days to at least several months.

[0258] The release of the therapeutic agent from the drug delivery system provided herein can be characterized by the percentage of the therapeutic agent released from the drug delivery system per day. In some embodiments, the release rate of the therapeutic agent can vary within the range of about 0.01% to about 20% / day, about 0.01% to about 15% / day, about 0.01% to about 10% / day, about 0.01% to about 9% / day, about 0.01% to about 8% / day, about 0.01% to about 7% / day, about 0.01% to about 6% / day, about 0.01% to about 5% / day, about 0.01% to about 4% / day, about 0.01% to about 3% / day, about 0.01% to about 5% / day, about 0.01% to about 6% / day, about 0.01% to about 7% / day, about 0.01% to about 8% / day, about 0.01% to about 9% / day, about 0.01% to about 10% / day, about 0.01% to about 11% / day, about 0.01% to about 12% / day, about 0.01% to about 13% / day, about 0.01% to about 14% / day, about 0.01% to about 15% / day, about 0.01% to about 16% / day, about 0.01% to about 17% / day, about 0.01% to about 18% / day, about 0.01% to about 19% / day, about 0.01% to about 20% / day, about 0.01% to about 25% / day, about 0.01% to about 26% / day, about 0.01% to about 27% / day, about 0.01% to about 0.01% to about 2% / day, about 0.01% to about 1% / day, about 0.01% to about 0.5% / day, about 0.01% to about 0.4% / day, about 0.01% to about 0.3% / day, about 0.01% to about 0.2% / day, about 0.01% to about 0.1% / day, about 0.01% to about 0.05% / day, about 0.01% to about 0.04% / day, about 0.01% to about 0.03% / day or about 0.01% to about 0.02% / day.

[0259] Pharmaceutical composition

[0260] In another aspect, a pharmaceutical composition comprising the drug delivery system of the present disclosure is provided.

[0261] In another aspect, provided is a pharmaceutical composition comprising the drug delivery system of the present disclosure and at least one pharmaceutically acceptable excipient.

[0262] As used herein, the term "pharmaceutical composition" refers to a formulation containing the drug delivery system of the present disclosure in a form suitable for administration to a subject.

[0263] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic, and biologically and otherwise desirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used herein, a "pharmaceutically acceptable excipient" includes one and more than one such excipient. The term "pharmaceutically acceptable excipient" also encompasses a "pharmaceutically acceptable carrier" and a "pharmaceutically acceptable diluent."

[0264] The pharmaceutical compositions provided herein can be in any form that permits administration of the composition to a subject, including but not limited to humans, and allows formulation of the composition compatible with the intended route of administration.

[0265] The pharmaceutical composition provided herein has been considered to have multiple approaches, and therefore the pharmaceutical composition provided herein can be supplied in bulk or unit dosage form depending on the expected route of administration. For example, for oral, buccal and sublingual administration, powders, granules, tablets, pills, capsules, soft capsules and caplets can be acceptable as solid dosage forms, and emulsions, syrups, elixirs, suspensions and solutions can be acceptable as liquid dosage forms. For injection, gels, solutions, emulsions and suspensions can be acceptable as liquid dosage forms, and powders suitable for reconstitution with suitable solutions can be acceptable as solid dosage forms. For inhalation, solutions, sprays, dry powders and aerosols can be acceptable dosage forms. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, emulsifiable pastes, lotions, gels, solutions and patches can be acceptable dosage forms. For vaginal administration, vaginal suppositories, tampons, emulsifiable pastes, gels, pastes, foams and sprays can be acceptable dosage forms. For implant administration, solids, semisolids, gels can be acceptable dosage forms.

[0266] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of orally administrable formulations.

[0267] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of injection formulations.

[0268] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of formulations for administration by inhalation.

[0269] In some embodiments, the pharmaceutical compositions of the present disclosure may be in the form of topical formulations.

[0270] In certain embodiments, the pharmaceutical compositions provided herein can be formulated in the form of a transdermal patch well known to those of ordinary skill in the art.

[0271] In addition to those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are therefore included in the present disclosure. Such excipients and carriers are described in, for example, Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991); Remington: The Science and Practice of Pharmacy, edited by University of the Sciences in Philadelphia, 21st edition, LWW (2005), which are incorporated herein by reference.

[0272] In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as a single dose. The amount of the compound provided herein in a single dose will vary depending on the subject being treated and the particular mode of administration.

[0273] In some embodiments, the pharmaceutical compositions of the present disclosure may be formulated for administration to a subject at intervals of days, weeks, months, or even longer.

[0274] In another aspect, pharmaceutical compositions comprising the drug delivery system of the present disclosure are also provided as two or more combination therapies.

[0275] Synthesis of drug delivery systems

[0276] The synthesis of the drug delivery system provided herein is described in the synthesis scheme in the embodiment. The drug delivery system provided herein can be prepared using any known organic synthesis technology and can be synthesized according to any of a variety of possible synthetic pathways, so these schemes are only illustrative and do not mean that restriction can be used for preparing other possible methods of the compound provided herein. In addition, the step in the scheme is to better illustrate and can be changed as appropriate. The embodiment of the compound in the embodiment is synthesized for the purpose of research and possibly being submitted to regulatory agencies.

[0277] The reaction for preparing the drug delivery system of the present disclosure can be carried out in a suitable solvent, and the technical staff of organic synthesis field can easily select suitable solvent.Suitable solvent can be substantially unreactive with starting material (reactant), intermediate or product under the temperature (for example, scope can be the temperature from the freezing temperature of solvent to the boiling temperature of solvent) of carrying out reaction.Given reaction can be carried out in a solvent or the mixture of more than a solvent.Depending on the specific reaction step, the suitable solvent of specific reaction step can be selected by those skilled in the art.

[0278] The preparation of compounds of the present disclosure may involve the protection and deprotection of various chemical groups. The demand for protection and deprotection and the selection of appropriate protecting groups can be easily determined by those skilled in the art. The chemistry of protecting groups can be found in, for example, the following references: TW Greene and PGM Wuts, " Protective Groups in Organic Synthesis ", 3rd edition, John Wiley & Sons Publishing Company, New York, (1999); P. Kocienski, " Protective Groups ", Georg Thieme Verlag, 2003; and Peter G.M. Wuts, " Greene's Protective Groups in Organic Synthesis ", 5th edition, Wiley, 2014, all of which are incorporated herein by reference in their entirety.

[0279] The reaction can be monitored by any suitable method known in the art. For example, the reaction can be monitored by methods such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV visible), mass spectrometry or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC) to monitor product formation. Those skilled in the art can purify the compound by various methods, including high performance liquid chromatography (HPLC) ("Preparative LC-MS Purification: Improved Compound Specific Method Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, "Journal of Combinatorial Chemistry (J. Combi. Chem.)" 2004, 6 (6), 874-883, which is incorporated herein by reference in its entirety) and normal phase silica gel chromatography.

[0280] Known starting materials of the present disclosure can be used or synthesized according to methods known in the art, or can be purchased from commercial suppliers. Unless otherwise stated, analytical grade solvents and commercially available reagents were used without further purification.

[0281] Unless otherwise noted, the reactions of the present disclosure were carried out under a positive pressure of nitrogen or argon or in anhydrous solvents using a drying tube, and the reaction flasks were typically fitted with rubber septa for the introduction of substrates and reagents by syringe. Glassware was oven-dried and / or heat-dried.

[0282] Methods of treating diseases

[0283] In another aspect, provided is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided herein.

[0284] The condition to be treated depends on the therapeutic agent selected in the drug delivery system or the pharmaceutical composition provided herein. In some embodiments, the condition can be selected from the group consisting of: inflammation, cancer, cardiovascular disease, respiratory disease, diseases related to vascular endothelial growth factor (VEGF), osteoarthritis, neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), dermatitis, psoriasis, chronic obstructive pulmonary disease, asthma.

[0285] In this context, the term "therapeutically effective amount" refers to the amount of a selected therapeutic agent in the drug delivery system provided herein, or a pharmaceutically acceptable salt thereof, that is effective to provide "therapy" or "treatment" to a subject for a disorder, disease or condition in the subject.

[0286] The present disclosure also includes the following embodiments:

[0287] Embodiment 1. A drug delivery system for locally delivering a therapeutic agent at a controlled rate, the drug delivery system comprising:

[0288] A biopolymer comprising at least a first binding group BG1, wherein the first binding group BG1 is selected from the group consisting of: a hydroxyl group, a carboxyl group, an amino group, and a combination thereof;

[0289] a therapeutic agent, the 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; and

[0290] a linker that covalently attaches the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration;

[0291] Wherein the linker comprises a structure of formula (I):

[0292]

[0293] in

[0294] U is linked to the biopolymer via BG1 such that at least one bond selected from ester or amide is formed, and U is selected from the group consisting of: a direct bond, -N(R 1 )-, -O-, -C(=O)- and in is a nitrogen-containing heterocyclic group, which optionally includes one or more additional heteroatoms selected from N, O or S;

[0295] A is selected from a direct bond, an alkyl group and -(CH2CH2O) m -, wherein the alkyl group is optionally replaced by one or more R a group substitution;

[0296] B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, an -O-cycloalkyl group, an -O-heterocyclyl group, an -O-aryl group, an -O-heteroaryl group, wherein each of the alkyl group, the cycloalkyl group, the heterocyclyl group, the aryl group and the heteroaryl group is optionally replaced by one or more R b group substitution;

[0297] 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)-;

[0298] D is selected from a direct bond, an alkyl group and an aryl group, wherein the alkyl group is optionally substituted by one or more R c group substitution;

[0299] V is linked to the therapeutic agent through BG2 such that at least one bond selected from the group consisting of an amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal, and combinations thereof is formed, and V is selected from the group consisting of 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 2 )P(=O)(OPh)-;

[0300] R 1 and R 2 independently selected from the group consisting of: hydrogen, alkyl, alkenyl, and alkynyl;

[0301] R a 、R b and R c independently selected from halogen, hydroxy, amino, cyano, nitro, alkyl, alkoxy, -C(=O)OR e and = NH;

[0302] R e is an alkyl group;

[0303] m is an integer from 0 to 4;

[0304] n is an integer from 1 to 4; and

[0305] p is an integer from 1 to 4.

[0306] Embodiment 2. The drug delivery system according to embodiment 1, wherein BG1 is a carboxyl group and U is -N(R 1 )-, so that an amide bond is formed.

[0307] Embodiment 3. The drug delivery system according to embodiment 1, wherein BG1 is a carboxyl group and U is This allows an amide bond to be formed.

[0308] Embodiment 4. The drug delivery system according to embodiment 1, wherein BG1 is a carboxyl group and U is -O- or a direct bond, such that an ester bond is formed.

[0309] Embodiment 5. The drug delivery system according to embodiment 3, wherein U is selected from the group consisting of

[0310]

[0311] Embodiment 6. The drug delivery system according to embodiment 1, wherein BG1 is hydroxyl and U is -C(=O)-, such that an ester bond is formed.

[0312] Embodiment 7. The drug delivery system according to embodiment 1, wherein BG1 is amino and U is -C(=O)-, such that an amide bond is formed.

[0313] Embodiment 8. The drug delivery system according to embodiment 1, wherein BG2 is an amine group, and V is selected from one of the following:

[0314] (a) Direct key;

[0315] (b)-N(R 2 )C(=O)-, the -N(R 2 )C(=O)- is linked to the therapeutic agent through BG2, such that a urea bond is formed;

[0316] (c)-N(R 2 )C(S)-, said-N(R 2 )C(S)- is linked to the therapeutic agent through BG2 such that a thiourea bond is formed;

[0317] (d) -OC(=O)-, wherein the -OC(=O)- is linked to the therapeutic agent through BG2 such that a carbamate bond is formed;

[0318] (e) -OC(=S)-, wherein the -OC(=S)- is linked to the therapeutic agent via BG2 such that a thiocarbamate bond is formed;

[0319] (f) -OC(=O)OCH2-, wherein the -OC(=O)OCH2- is linked to the therapeutic agent via BG2 such that an aza-acetal bond is formed;

[0320] (g) -C(=O)OCH2-, wherein the -C(=O)OCH2- is linked to the therapeutic agent through BG2 such that an aza-acetal bond is formed;

[0321] (h)-N(R 2 )C(=O)OCH2-, the -N(R 2 )C(=O)OCH2- is linked to the therapeutic agent through BG2 such that an aza-acetal bond is formed;

[0322] (i) -OP(=O)(OPh)-, wherein the -OP(=O)(OPh)- is linked to the therapeutic agent via BG2 such that a phosphoramidate bond is formed;

[0323] (j)-N(R 2 )P(=O)(OPh)-, the -N(R 2 )P(=O)(OPh)- is linked to the therapeutic agent through BG2 such that a phosphoramidate bond is formed;

[0324] (k) -C(=O)-, wherein the -C(=O)- is linked to the therapeutic agent through BG2 such that an amide bond is formed.

[0325] Embodiment 9. The drug delivery system according to embodiment 1, wherein BG2 is a carboxyl group and V is -O- or a direct bond, such that an ester bond is formed.

[0326] Embodiment 10. The drug delivery system according to embodiment 1, wherein BG2 is hydroxyl and V is -C(=O)-, such that an ester bond is formed.

[0327] Embodiment 11. The drug delivery system according to embodiment 1, wherein A is a direct bond.

[0328] Embodiment 12. The drug delivery system according to embodiment 1, wherein A is an alkyl group.

[0329] Embodiment 13. The drug delivery system according to embodiment 1, wherein A is -(CH2CH2O) m -.

[0330] Embodiment 14. The drug delivery system according to embodiment 1, wherein B is a direct bond.

[0331] Embodiment 15. The drug delivery system according to embodiment 1, wherein B is an alkyl group.

[0332] Embodiment 16. The drug delivery system according to embodiment 1, wherein B is cycloalkyl, aryl or heteroaryl.

[0333] Embodiment 17. The drug delivery system according to embodiment 1, wherein B is -O-aryl.

[0334] Embodiment 18. The drug delivery system according to embodiment 1, wherein A is a direct bond, and B is selected from the group consisting of a direct bond, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group.

[0335] Embodiment 19. The drug delivery system according to embodiment 18, wherein A is a direct bond and B is selected from the group consisting of a direct bond, a cycloalkyl group, an aryl group, and a heteroaryl group.

[0336] Embodiment 20. The drug delivery system of embodiment 1, wherein A is alkyl and B is selected from the group consisting of a direct bond, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, an -O-cycloalkyl group, an -O-heterocyclyl group, an -O-aryl group, and an -O-heteroaryl group.

[0337] Embodiment 21. The drug delivery system according to embodiment 20, wherein A is an alkyl group, and B is selected from the group consisting of a direct bond, an aryl group, or an -O-aryl group.

[0338] Embodiment 22. The drug delivery system according to embodiment 1, wherein A is -(CH2CH2O) m -, B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group.

[0339] Embodiment 23. The drug delivery system according to embodiment 22, wherein A is -(CH2CH2O) m -, B is selected from the group consisting of a direct bond, an alkyl group, an aryl group, and a heteroaryl group.

[0340] Embodiment 24. The drug delivery system according to embodiment 1, wherein C is a direct bond.

[0341] Embodiment 25. The drug delivery system according to embodiment 1, wherein C is -C(=O)-.

[0342] Embodiment 26. The drug delivery system according to embodiment 1, wherein C is -N(R 2 )C(=O)- or -C(=O)N(R 2 )-.

[0343] Embodiment 27. The drug delivery system according to embodiment 1, wherein C is -[CH2NHC(=O)] n -.

[0344] Embodiment 28. The drug delivery system according to embodiment 1, wherein C is -[NHC(=O)CH2]n -.

[0345] Embodiment 29. The drug delivery system according to embodiment 1, wherein C is -NH(CH2) p C(=O)-.

[0346] Embodiment 30. The drug delivery system of embodiment 1, wherein A is alkyl, B is selected from the group consisting of a direct bond, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, -O-cycloalkyl group, -O-heterocyclyl group, -O-aryl group, and -O-heteroaryl group, and C is selected from the group consisting of a direct bond, -C(=O)-, -N(R 2 )C(=O)-, -[CH2NHC(=O)] n -, -[NHC(=O)CH2] n - and -NH(CH2) p C(=O)-.

[0347] Embodiment 31. The drug delivery system of embodiment 30, wherein A is alkyl, B is selected from the group consisting of a direct bond, a cycloalkyl, a heterocyclyl, an aryl, a heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, and -O-heteroaryl, and C is a direct bond, -N(R 2 )C(=O)- or -[NHC(=O)CH2] n -.

[0348] Embodiment 32. The drug delivery system according to embodiment 1, wherein A is -(CH2CH2O) m -, B is selected from the group consisting of a direct bond, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, and C is a direct bond or -N(R 2 )C(=O)-.

[0349] Embodiment 33. The drug delivery system according to embodiment 1, wherein D is a direct bond.

[0350] Embodiment 34. The drug delivery system according to embodiment 1, wherein D is an alkyl group.

[0351] Embodiment 35. The drug delivery system according to embodiment 1, wherein D is aryl.

[0352] Embodiment 36. The drug delivery system of embodiment 1, wherein the linker comprises a structure of Formula (Ia) to (Im):

[0353]

[0354]

[0355] in,

[0356] U and V are as defined in Embodiment 1;

[0357] M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, each of which is optionally replaced by one or more R b group substitution;

[0358] Each of which is optionally substituted with -C(=O)OCH3; and

[0359] q, r, s, t, u and v are independently integers from 0 to 5.

[0360] Embodiment 37. The drug delivery system according to embodiment 36, wherein M is selected from the group consisting of cyclohexyl, phenyl, pyridyl, thiazolyl, and adamantyl.

[0361] Embodiment 38. The drug delivery system of embodiment 36, wherein the linker comprises a structure selected from the group consisting of:

[0362]

[0363]

[0364]

[0365] in Each of is optionally substituted with -C(=O)OCH3.

[0366] Embodiment 39. The drug delivery system according to any one of embodiments 1 to 38, wherein the biopolymer is selected from the group consisting of hyaluronic acid, chitosan, chitin, chondroitin or derivatives thereof.

[0367] Embodiment 40. The drug delivery system of embodiment 39, wherein the biopolymer is hyaluronic acid.

[0368] Embodiment 41. The drug delivery system of embodiment 39, wherein the biopolymer is chondroitin.

[0369] Embodiment 42. The drug delivery system of any one of embodiments 1 to 41, wherein the therapeutic agent is selected from the group consisting of an anticancer drug, a nonsteroidal anti-inflammatory drug (NSAID), a Janus kinase (JAK) inhibitor, and a vascular endothelial growth factor (VEGF) inhibitor.

[0370] Embodiment 43. The drug delivery system of embodiment 42, wherein the therapeutic agent is an NSAID selected from the group consisting of piroxicam, meloxicam, and diclofenac.

[0371] Embodiment 44. The drug delivery system of embodiment 42, wherein the therapeutic agent is a JAK inhibitor selected from the group consisting of tofacitinib, ruxolitinib, baricitinib, peftinib, fuzotinib, oclacitinib, and upadacitinib.

[0372] Embodiment 45. The drug delivery system of embodiment 42, wherein the therapeutic agent is a VEGF inhibitor selected from the group consisting of axitinib, lapatinib, lenvatinib, pazopanib, nintedanib, sunitinib, and vandetanib.

[0373] Embodiment 46. The drug delivery system of embodiment 44, wherein the therapeutic agent is tofacitinib.

[0374] Embodiment 47. The drug delivery system of embodiment 44, wherein the therapeutic agent is upadacitinib.

[0375] Embodiment 48. The drug delivery system of embodiment 44, wherein the therapeutic agent is ruxolitinib.

[0376] Embodiment 49. The drug delivery system of embodiment 44, wherein the therapeutic agent is baricitinib.

[0377] Embodiment 50. The drug delivery system of embodiment 44, wherein the therapeutic agent is oclacitinib.

[0378] Embodiment 51. The drug delivery system of embodiment 44, wherein the therapeutic agent is nintedanib.

[0379] Embodiment 52. The drug delivery system of embodiment 44, wherein the therapeutic agent is sunitinib.

[0380] Embodiment 53. The drug delivery system of embodiment 1, which is selected from the group consisting of:

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393] Embodiment 54. The drug delivery system of any one of embodiments 1 to 53, wherein the drug delivery system is topically administered to a subject in need thereof.

[0394] Embodiment 55. The drug delivery system of embodiment 54, wherein the drug delivery system is locally administered to a subject in need thereof by injection.

[0395] Embodiment 56. The drug delivery system of embodiment 54, wherein the drug delivery system is topically administered to a subject in need thereof via an oral dosage form.

[0396] Embodiment 57. The drug delivery system of embodiment 54, wherein the drug delivery system is topically administered to a subject in need thereof by inhalation.

[0397] Embodiment 58. The drug delivery system of embodiment 54, wherein the drug delivery system is locally administered to a subject in need thereof via an implant.

[0398] Embodiment 59. The drug delivery system of embodiment 54, wherein the drug delivery system is topically administered to a subject in need thereof by topical application.

[0399] Embodiment 60. A pharmaceutical composition comprising the drug delivery system according to any one of embodiments 1 to 59 and a pharmaceutically acceptable excipient.

[0400] Embodiment 61. A method of treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the drug delivery system according to any one of embodiments 1 to 59 or the pharmaceutical composition according to embodiment 60.

[0401] Embodiment 62. The method of embodiment 61, wherein the condition is selected from the group consisting of: inflammation, cancer, cardiovascular disease, respiratory disease, diseases related to vascular endothelial growth factor (VEGF), osteoarthritis, neovascular (wet) age-related macular degeneration (AMD), macular edema after retinal vein occlusion (RVO), diabetic macular edema (DME), diabetic retinopathy (DR), myopic choroidal neovascularization (mCNV), dermatitis, psoriasis, chronic obstructive pulmonary disease and asthma.

[0402] Example

[0403] The following examples are included for illustrative purposes. However, it should be understood that these examples do not limit the present disclosure and are only intended to illustrate methods for practicing the present disclosure. Those skilled in the art will recognize that the described chemical reactions can be easily adapted to prepare a variety of other compounds of the present disclosure, and alternative methods for preparing compounds of the present disclosure are considered to be within the scope of the present disclosure. For example, non-exemplary compounds according to the present disclosure can be successfully synthesized by modifications apparent to those skilled in the art, such as by appropriately protecting interfering groups, by utilizing other suitable reagents and building blocks other than the described reagents and building blocks, and / or by making conventional modifications to reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to be suitable for preparing other compounds of the present disclosure.

[0404] Example 1

[0405] Preparation of a conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0406]

[0407] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)butyl)carbamate

[0408]

[0409] Triethylamine (1.2 g, 12 mmol, 2.5 eq) was added to a mixture of tofacitinib (1.5 g, 4.8 mmol, 1 eq) and bis(4-nitrophenyl) carbonate (1.61 g, 5.28 mmol, 1.1 eq) in dichloromethane (30 mL) under N2, and the reaction mixture was heated to reflux for 3 hours. Tert-butyl(4-aminobutyl)carbamate (0.9 g, 4.82 mmol, 1 eq) was then 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 give the title product (2.4 g, yield: 95%); MS (m / z): C 26 H 38 [M+H]+ calculated for N8O4, 527.30; found, 527.2.

[0410] Step 2: Preparation of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0411]

[0412] To a solution of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)butyl)carbamate (2.4 g, 4.56 mmol, 1 eq) in ethyl acetate (24 mL) was added dropwise under N at 0°C. The resulting reaction mixture was stirred at 0°C for 30 minutes and then 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 and then filtered to give the desired product as an HCl salt (2.1 g, yield: 100%); MS (m / z): C 21 H 30 [M+H]+ calculated for N8O2, 427.25; found, 427.2. 1 H-NMR(400MHz,D2O)δppm 8.38(d,J=6.7Hz,1H),7.82(d,J=3.9Hz 1H),6.90(s,1H),4.72-4.53(m,1H),4.16-3.26(m,11H),3.04(d,J=6.4Hz 2H),2.66-2.46(m,1H),2.03-1.90(m,1H),1.86-1.69(m,5H),1.12(d,J=7.1Hz,3H).

[0413] Step 3: Preparation of a conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0414]

[0415] 4-Methylmorpholine (0.066 g, 0.65 mmol, 1.5 eq) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.076 g, 0.432 mmol, 1 eq) were added to a solution of sodium hyaluronate (MW 50 KDa, 0.161 g, 0.432 mmol, 1 eq) in acetonitrile (22 mL) and H O (35 mL) at 0° C. The resulting reaction mixture was stirred at 0° C. for 30 minutes and then at room temperature for 1 hour.

[0416] To the reaction mixture was added N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.432 mmol, 1 equiv), and the pH of the reaction mixture was then adjusted to 6.5 to 7 with 4-methylmorpholine. The resulting reaction mixture was stirred at room temperature for 3 days.

[0417] H2O (2 mL) containing NaCl (257 mg, 10 equivalents) was added to the above reaction mixture and stirred for 0.5 hours. Acetone (350 mL) was then added dropwise to the above mixture to form a precipitate. The mixture was filtered and the filter cake was washed with propanol (10 mL*3). The wet cake was dissolved in acetonitrile (20 mL) and H2O (40 mL), and then dialyzed 3 times against deionized water using a 3.5 kDa MW cut-off membrane, and then lyophilized to obtain the title compound (0.15 g, yield: 43.2%, DSR (drug substitution rate) = 17%). 1 H-NMR (400MHz, D2O / d-DMSO=3:1) δppm8.40-7.90(m,0.17H),7.75-7.20(m,0.17H),6.95-6.25(m,0.17H),4.70-4.20(m,2.4 7H),4.00-3.23(m,11.91H),2.55-2.30(m.0.34H),1.99(d,J=19.7Hz,3H),1.30(t,J=6.8Hz,0.17H),1.20-1.10(m,0.51H).

[0418] In the case of step 3, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.16 g, yield: 46%, DSR = 22%).

[0419] In the case of step 3, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.15 g, yield: 43.2%, DSR=17%).

[0420] Example 2

[0421] Preparation of a conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0422]

[0423] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)benzyl)carbamate

[0424]

[0425] By following Step 1 in Example 1, tofacitinib (1.5 g, 4.8 mmol, 1 eq), bis(4-nitrophenyl) carbonate (1.61 g, 5.28 mmol, 1.1 eq) and tert-butyl (4-aminobutyl) carbamate (1.07 g, 4.8 mmol, 1 eq) produced the title product (1.8 g, yield: 67%); MS (m / z): C 29 H 36 [M+H]+ calculated for N8O4, 562.29; found, 562.2.

[0426] Step 2: Preparation of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0427]

[0428] By following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)benzyl)carbamate (1.5 g, 2.675 mmol, 1 equiv) gave the desired product as an HCl salt (1.33 g, yield: 100%); MS (m / z): C 24 H 28 [M+H]+ calculated for N8O2, 461.23; found, 461.2. 1 H-NMR(400MHz,D2O)δppm)δ8.30-8.08(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).

[0429] Step 3: Preparation of a conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0430]

[0431] By following Step 3 in Example 1, the reaction mixture of sodium hyaluronate (MW 50 KDa, 0.163 g, 0.403 mmol, 1 equiv) and N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.403 mmol, 1 equiv) afforded the title compound (0.18 g, 55% yield, DSR = 38%); 1 H-NMR (400MHz, D2O / d-DMSO=3:1) δppm9.0-7.0(m,2.7H),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).

[0432] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.15 g, yield: 44%, DSR = 21.4%).

[0433] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.18 g, yield: 53%, DSR = 20.7%)

[0434] Example 3

[0435] Preparation of a conjugate of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenethyl)carbamate and HA

[0436]

[0437] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenethyl)carbamate

[0438]

[0439] By following Step 1 in Example 1, tofacitinib (1.5 g, 4.8 mmol, 1 eq) and tert-butyl (4-aminophenethyl)carbamate (1.14 g, 4.8 mmol, 1 eq) gave the title product (2.2 g, yield: 79.7%); MS (m / z): C 30 H 38 [M+H]+ calculated for N8O4, 575.30; found, 575.2.

[0440] Step 2: Preparation of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0441]

[0442] By following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenethyl)carbamate (1.5 g, 2.61 mmol, 1 equiv) gave the desired product as an HCl salt (1.33 g, yield: 100%); MS (m / z): C 25 H 30 [M+H]+ calculated for N8O2, 475.25; found, 475.2. 1 H-NMR(400MHz,D2O)δppm 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=5.7Hz,1H),1.83(dd,J=72.1,15.3Hz,2H),1.13(dd,J=18.5,13.3Hz,3H).

[0443] Step 3: Preparation of a conjugate of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenethyl)carbamate and HA

[0444]

[0445] By following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.157 g, 0.39 mmol, 1 eq) and N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.39 mmol, 1 eq) provided the title compound (0.19 g, yield: 33.4%, DSR = 19%). 1 H-NMR(400MHz,D2O / DMSO=3:1)δppm 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).

[0446] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.147 g, yield: 44%, DSR = 16%),

[0447] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.175 g, yield: 53%, DSR = 22.9%).

[0448] Example 4

[0449] Preparation of a conjugate of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0450]

[0451] Step 1: Preparation of tert-butyl (2-(2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethoxy)ethoxy)ethyl)carbamate

[0452]

[0453] By following Step 1 in Example 1, tofacitinib (3 g, 9.6 mmol, 1 eq), tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (2.38 g, 9.6 mmol, 1 eq) gave the title product (3 g, yield: 54%); MS (m / z): C 28 H 42 [M+H]+ calculated for N8O6, 587.32; found, 587.2.

[0454] Step 2: Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0455]

[0456] By following Step 2 in Example 1, tert-butyl (2-(2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethoxy)ethoxy)ethyl)carbamate (1.5 g, 2.56 mmol, 1 equiv) gave the desired product (1.34 g, yield: 100%) as an HCl salt; MS (m / z): C 23 H 34 [M+H]+ calculated for N8O4, 487.32; found, 487.2. 1 H-NMR(400MHz,D2O)δppm 8.40(br,1H),7.83(s,1H),6.91(s,1H),4.7-4.5(m,1H),4.06-3.92(m,3H),3.83-3.3 6(m,16H),3.17(s,2H),2.56(s,1H),2.00-1.72(m,2H),1.14(dd,J=16.1,7.0Hz,3H).

[0457] Step 3: Preparation of a conjugate of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0458]

[0459] By following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.153 g, 0.38 mmol, 1 eq) and N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.38 mmol, 1 eq) provided the title compound (0.2 g, 33% yield, DSR = 36.3%). 1 H-NMR (400MHz, D2O / d-DMSO=3:1) δppm8.52-7.93(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).

[0460] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.186 g, yield: 56.5%, DSR = 32%)

[0461] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.198 g, yield: 60.2%, DSR = 26.6%).

[0462] Example 5

[0463] Preparation of a conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0464]

[0465] Step 1: Preparation of tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-oxo-5,8,11-trioxa-2-azatridec-13-yl)carbamate

[0466]

[0467] By following Step 1 in Example 1, tofacitinib (1.5 g, 4.8 mmol, 1 eq) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.41 g, 4.8 mmol, 1 eq) gave the title product (1.8 g, yield: 60%); MS (m / z): C 30 H 46 [M+H]+ calculated for N8O7, 631.35; found, 631.2.

[0468] Step 2: Preparation of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0469]

[0470] By following Step 2 in Example 1, tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-oxo-5,8,11-trioxa-2-azatridec-13-yl)carbamate (1.5 g, 2.38 mmol, 1 equiv) gave the desired product (1 g, yield: 80%) as an HCl salt; MS (m / z): C 25 H 38 [M+H]+ calculated for N8O5, 531.30; found, 531.2. 1 H-NMR (400MHz, DMSO) δppm9.74(s,1H),8.34(d,J=7.0Hz,1H),8.01(s,1H),7.69(s,1H),6.86(s,1H),4.85(d,J=3.1Hz,1H),4.35-4.0( m,10H),3.7-3.96(m,3H),3.65-3.5(m,10H),2.93(d,J=4.3Hz,2H),2.38(s,1H),1.89-1.68(m,1H),1.59(s,1H),1.01(d,J=4.8Hz,3H).

[0471] Step 3: Preparation of a conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0472]

[0473] By following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.141 g, 0.35 mmol, 1 eq) and N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.35 mmol, 1 eq) provided the title compound (0.2 g, 62.7% yield, DSR = 24%); 1H-NMR(400MHz,D2O-DMSO=3:1)δppm 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,17 H),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).

[0474] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.186 g, yield: 58.4%, DSR = 26%)

[0475] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.198 g, yield: 62.2%, DSR = 28%).

[0476] Example 6

[0477] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0478]

[0479] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate

[0480]

[0481] By following Step 1 in Example 1, tofacitinib (2 g, 6.4 mmol, 1 eq.) and tert-butyl hydrazinecarboxylate (0.845 g, 6.4 mmol, 1 eq.) gave the title product (2.23 g, yield: 75%); MS (m / z): C 22 H 30 [M+H]+ calculated for N8O4, 471.24; found, 471.2.

[0482] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

[0483]

[0484] By following Step 2 in Example 1, tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate (2 g, 4.25 mmol, 1 equiv) gave the desired product (1.5 g, yield: 85%) as an HCl salt; MS (m / z): C 17 H 22 [M+H]+ calculated for N8O2, 371.19; found, 371.1. 1 H-NMR(400MHz,CD3OD)δppm 8.52(s,1H),7.89(d,J=3.1Hz,1H),7.13(d,J=3.8Hz,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=7.0Hz,3H).

[0485] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0486]

[0487] By following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.202 g, 0.5 mmol, 1 eq) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g, 0.5 mmol, 1 eq) provided the title compound (0.15 g, yield: 43.2%, DSR = 32%); 1 H-NMR (400MHz, D2O-DMSO=5:1): δppm 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).

[0488] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.18 g, yield: 48.5%, DSR = 35%).

[0489] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.19 g, yield: 51.2%, DSR = 30%).

[0490] Example 7

[0491] Preparation of conjugates of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0492]

[0493] Step 1: Preparation of tert-butyl (2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)carbamate

[0494]

[0495] Following Step 1 in Example 1, tofacitinib (3.2 g, 10.24 mmol, 1 eq) and tert-butyl (2-aminoethyl)carbamate (1.64 g, 10.24 mmol, 1 eq) gave the title product (3 g, yield: 59%); MS (m / z): C 24 H 34 [M+H]+ calculated for N8O4, 499.27; found, 499.1.

[0496] Step 2: Preparation of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0497]

[0498] Following Step 2 in Example 1, tert-butyl (2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)ethyl)carbamate (1 g, 2 mmol, 1 equiv) gave the desired product as an HCl salt (870 mg, yield: 100%); MS (m / z): C19 H 26 [M+H]+ calculated for N8O2, 399.22; found, 399.1. 1 H-NMR(400MHz,CD3OD)δppm 8.49(s,1H),7.95(d,J=3.6Hz,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=7.0Hz,3H).

[0499] Step 3: Preparation of a conjugate of N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0500]

[0501] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.186 g, 0.46 mmol, 1 eq) and N-(2-aminoethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.46 mmol, 1 eq) provided the title compound (0.23 g, yield: 66%, DSR = 18%). 1 H-NMR(400MHz,D2O)δppm: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(400MHz,D2O)δppm: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).

[0502] Example 8

[0503] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0504]

[0505] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)butyl)(methyl)carbamate

[0506]

[0507] Following Step 1 in Example 1, tofacitinib (1 g, 3.2 mmol, 1 eq) and tert-butyl (4-aminobutyl)(methyl)carbamate (0.65 g, 3.2 mmol, 1 eq) gave the title product (1 g, yield: 58%); MS (m / z): C 27 H 40 [M+H]+ calculated for N8O4, 541.32; found, 541.2.

[0508] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0509]

[0510] Following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)butyl)(methyl)carbamate (1 g, 1.85 mmol, 1 equiv) gave the desired product as an HCl salt (0.88 g, yield: 100%); MS (m / z): C 22 H 32 [M+H]+ calculated for N8O2, 441.26; found, 441.1. 1H-NMR(400MHz,CD3OD)δppm 8.50(d,J=4.7Hz,1H),7.94(d,J=3.7Hz,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=6.9,5.2Hz,3H).

[0511] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0512]

[0513] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.169 g, 0.42 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(methylamino)butyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.42 mmol, 1 equiv) provided the title compound (0.236 g, yield: 34.4%, DSR = 10%); 1 H-NMR(400MHz,D2O)δppm 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).

[0514] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.18 g, yield: 52.3%, DSR = 6%).

[0515] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) provided the corresponding product KDa (0.17 g, yield: 49.4%, DSR = 3%).

[0516] Example 9

[0517] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0518]

[0519] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-methylhydrazine-1-carboxylate

[0520]

[0521] Following Step 1 in Example 1, tofacitinib (3.124 g, 10 mmol, 1 eq.) and tert-butyl 1-methylhydrazine-1-carboxylate (1.45 g, 10 mmol, 1 eq.) gave the title product (2.2 g, yield: 46%); MS (m / z): C 23 H 32 [M+H]+ calculated for N8O4, 485.25; found, 485.2.

[0522] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

[0523]

[0524] Following Step 2 in Example 1, tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-methylhydrazine-1-carboxylate (1 g, 2.06 mmol, 1 equiv) gave the desired product as an HCl salt (870 mg, yield: 100%); MS (m / z): C 18 H 24 [M+H]+ calculated for N8O2, 385.20; found, 385.1. 1H-NMR(400MHz,D2O)δppm 8.44(d,J=5.0Hz,1H),7.79(d,J=3.5Hz,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=15.1,7.1Hz,3H).

[0525] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0526]

[0527] Following Step 2 in Example 1, sodium hyaluronate (MW 50 KDa, 0.192 g, 0.475 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-methyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g, 0.475 mmol, 1 equiv) provided the title compound (0.323 g, yield: 90%, DSR = 15%); 1 H-NMR(400MHz,D2O)δppm 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).

[0528] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.303 g, yield: 83%, DSR = 10%).

[0529] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.327 g, yield: 90.3%, DSR = 15%).

[0530] Example 10

[0531] Preparation of a conjugate of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0532]

[0533] Step 1: Preparation of tert-butyl 1-allyl-2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate

[0534]

[0535] Following Step 1 in Example 1, tofacitinib (2 g, 6.4 mmol, 1 equiv) and tert-butyl 1-methylhydrazine-1-carboxylate (1.1 g, 6.4 mmol, 1 equiv) produced the title product (0.8 g, 25% yield); MS (m / z): [M+H]+ calculated for C25H34N8O4, 511.27; found, 511.1.

[0536] Step 2: Preparation of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

[0537]

[0538] Following Step 2 in Example 1, tert-butyl 1-allyl-2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazine-1-carboxylate (0.8 g, 1.57 mmol) gave the desired product as an HCl salt (0.67 g, yield: 96%); MS (m / z): C 20 H 26 [M+H]+ calculated for N8O2, 411.22; found, 411.1. 1H-NMR(400MHz,CD3OD)δppm 8.50(d,J=4.2Hz,1H),7.86(s,1H),7.10(d,J=3.8Hz,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=7.0Hz 3H).

[0539] Step 3: Preparation of a conjugate of N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0540]

[0541] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.182 g, 0.45 mmol, 1 equiv) and N′-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g, 0.45 mmol, 1 equiv) provided the title compound (0.172 g, yield: 48.6%, DSR = 5%); 1 H-NMR(400MHz,D2O)δppm 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).

[0542] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.175 g, yield: 49%, DSR = 3%)

[0543] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.186 g, yield: 52.3%, DSR = 1%).

[0544] Example 11

[0545] Preparation of a conjugate of N-(5-(aminomethyl)pyridin-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0546]

[0547] Step 1: Preparation of tert-butyl ((6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)pyridin-3-yl)methyl)carbamate

[0548]

[0549] Following Step 1 in Example 1, tofacitinib (0.9 g, 2.88 mmol, 1 eq) and tert-butyl ((6-aminopyridin-3-yl)methyl)carbamate (0.643 g, 2.88 mmol, 1 eq) gave the title product (1 g, yield: 62.5%); MS (m / z): C 28 H 35 [M+H]+ calculated for N9O4, 526.28; found, 526.1.

[0550] Step 2: Preparation of N-(5-(aminomethyl)pyridin-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0551]

[0552] Following Step 2 in Example 1, tert-butyl ((6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)pyridin-3-yl)(methyl)carbamate (0.8 g, 1.42 mmol, 1 equiv) gave the desired product as an HCl salt (0.7 g, yield: 100%); MS (m / z): C 23 H 27 [M+H]+ calculated for N9O2, 462.23; found, 462.1. 1H-NMR(400MHz,D2O)δppm 8.41-8.31(m,2H),8.1-7.9(m,3H),7.78-7.68(m,1H),6.83(d,J=2.4Hz,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=24.3,6.3Hz,3H).

[0553] Step 3: Preparation of a conjugate of N-(5-(aminomethyl)pyridin-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0554]

[0555] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.161 g, 0.4 mmol, 1 equiv) and N-(5-(aminomethyl)pyridin-2-yl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.4 mmol, 1 equiv) provided the title compound (0.17 g, yield: 50%, DSR = 20.5%); 1 H-NMR(400MHz,D2O)δppm 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).

[0556] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.177 g, yield: 52.6%, DSR = 22.2%).

[0557] Example 12

[0558] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(piperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0559]

[0560] Step 1: Preparation of tert-butyl 4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenyl)piperazine-1-carboxylate

[0561]

[0562] Following Step 1 in Example 1, tofacitinib (0.52 g, 1.66 mmol, 1 eq.) and tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (0.46 g, 1.66 mmol, 1 eq.) produced the title product (0.65 g, yield: 65%); MS (m / z): C 32 H 41 [M+H]+ calculated for N9O4, 616.33; found, 616.1.

[0563] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(piperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0564]

[0565] Following Step 2 in Example 1, tert-butyl 4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenethyl)piperazine-1-carboxylate (0.6 g, 0.975 mmol, 1 equiv) gave the desired product as an HCl salt (0.538 mg, yield: 100%); MS (m / z): C 27 H 33 [M+H]+ calculated for N9O2, 516.28; found, 516.1. 1H-NMR(400MHz,D2O)δppm 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=16.6,7.0Hz,3H).

[0566] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(piperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0567]

[0568] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.145 g, 0.36 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(piperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.36 mmol, 1 equiv) provided the title compound (0.13 g, yield: 41%, DSR = 19%); 1 H-NMR(400MHz,D2O)δppm 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).

[0569] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.16 g, yield: 49.7%, DSR = 20%).

[0570] Example 13

[0571] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(5-(piperazin-1-yl)pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0572]

[0573] Step 1: Preparation of tert-butyl 4-(6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)pyridin-3-yl)piperazine-1-carboxylate

[0574]

[0575] Following Step 1 in Example 1, tofacitinib (1.12 g, 3.59 mmol, 1 eq) and tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (1 g, 3.59 mmol, 1 eq) gave the title product (1.3 g, yield: 58.7%); MS (m / z): C 31 H 40 N 10 [M+H]+ calculated for O4, 617.32; found, 617.2.

[0576] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(5-(piperazin-1-yl)pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0577]

[0578] Following Step 2 in Example 1, tert-butyl 4-(6-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)pyridin-3-yl)piperazine-1-carboxylate (1 g, 1.621 mmol, 1 equiv) gave the desired product as an HCl salt (0.89 g, yield: 99%); MS (m / z): C 26 H 32 N 10 [M+H]+ calculated for O2, 517.28; found, 517.1. 1 H-NMR(400MHz,D2O)δppm 8.33(d,J=11.6Hz,1H),8.04(d,J=13.2Hz,1H),7.85(d,J=19.7Hz,2H),7.71(d,J=8.7Hz,1H),6.83(s,1H),4.62-4.5 7(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=24.1,7.0Hz,3H).

[0579] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(5-(piperazin-1-yl)pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0580]

[0581] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.145 g, 0.36 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(5-(piperazin-1-yl)pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.36 mmol, 1 equiv) provided the title compound (0.17 g, yield: 33%, DSR = 28%); 1 H-NMR(400MHz,D2O)δppm 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).

[0582] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.22 g, yield: 68%, DSR = 20%).

[0583] Example 14

[0584] Preparation of a conjugate of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0585]

[0586] Step 1: Preparation of tert-butyl (2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)ethyl)carbamate

[0587]

[0588] Following Step 1 in Example 1, tofacitinib (1.24 g, 3.96 mmol, 1 eq) and tert-butyl (2-(4-aminophenoxy)ethyl)carbamate (1 g, 3.96 mmol, 1 eq) gave the title product (1.3 g, yield: 55.6%); MS (m / z): C 30 H 38 [M+H]+ calculated for N8O5, 591.30; found, 591.2.

[0589] Step 2: Preparation of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0590]

[0591] Following Step 2 in Example 1, tert-butyl (2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)ethyl)carbamate (1 g, 1.69 mmol, 1 equiv) gave the desired product as an HCl salt (0.892 g, yield: 100%); MS (m / z): C 25 H 30 [M+H]+ calculated for N8O3, 491.24; found, 491.1. 1 H-NMR(400MHz,D2O)δppm 8.15(s,1H),7.51(s,1H),7.34(dd,J=16.5,8.8Hz,2H),7.03(t,2H),6.63(s,1H),4.45(s,1H),4.3 8-3.38(m,10H),3.21(d,J=15.1Hz,2H),2.39(s,1H),2.0-1.6(m,2H),1.08(dd,J=16.9,6.8Hz,3H).

[0592] Step 3: Preparation of a conjugate of N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0593]

[0594] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.153 g, 0.38 mmol, 1 equiv) and N-(4-(2-aminoethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.38 mmol, 1 equiv) provided the title compound (0.22 g, yield: 66%, DSR = 11%); 1 H-NMR(400MHz,D2O)δppm 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).

[0595] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.24 g, yield: 73%, DSR = 10%).

[0596] Example 15

[0597] Preparation of a conjugate of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0598]

[0599] Step 1: Preparation of tert-butyl (4-(4-nitrophenoxy)butyl)carbamate

[0600]

[0601] NaH (1.772 g, 44.3 mmol, 2.5 equivalents) was added to a solution of tert-butyl (4-hydroxybutyl) carbamate (3.7 g, 19.5 mmol, 1.1 equivalents) in THF (40 mL) under N 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 thereto. The resulting mixture was refluxed for 12 hours. After most of the 1-fluoro-4-nitrobenzene was consumed, the reaction was quenched with saturated NH4Cl aqueous solution (100 mL) and extracted with ethyl acetate (40 mL * 2). The combined organic phase was washed with saturated NaCl aqueous solution (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 [M+H]+ calculated for N2O5, 311.15; found, 311.1.

[0602] Step 2: Preparation of tert-butyl (4-(4-aminophenoxy)butyl)carbamate

[0603]

[0604] 10% Pd / C (0.2g) was added to a solution of tert-butyl (4- (4-nitrophenoxy) butyl) carbamate (2g, 6.444mmol, 1 equivalent) in methanol (30mL), and the reaction mixture was stirred under a H2 balloon at room temperature for 24 hours. After 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 (10mL * 2). The combined filtrate was concentrated to give the title product (1.8g, yield: 100%); MS (m / z): C 15 H 24 [M+H]+ calculated for N2O3, 281.18; found, 281.1.

[0605] Step 3: Preparation of tert-butyl (4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)butyl)carbamate

[0606]

[0607] Following Step 1 in Example 1, tofacitinib (2.23 g, 7.133 mmol, 1 eq) and tert-butyl (4-(4-aminophenoxy)butyl)carbamate (2 g, 7.133 mmol, 1 eq) gave the title product (2.6 g, yield: 59%); MS (m / z): C 32 H 42 [M+H]+ calculated for N8O5, 619.33; found, 619.2.

[0608] Step 4: Preparation of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0609]

[0610] Following Step 2 in Example 1, tert-butyl (4-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)butyl)carbamate (1.3 g, 2.1 mmol, 1 equiv) gave the desired product as an HCl salt (1.1 g, yield: 100%); MS (m / z): C 27 H 34 [M+H]+ calculated for N8O3, 519.28; found, 519.2. 1 H-NMR(400MHz,D2O)δppm 8.14-8.11(d,J=10.8Hz,1H),7.43-7.21(m,3H),6.83(d,J=8.6Hz,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=7.4Hz,3H).

[0611] Step 5: Preparation of a conjugate of N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0612]

[0613] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.153 g, 0.38 mmol, 1 equiv) and N-(4-(4-aminobutoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (00.2 g, 0.38 mmol, 1 equiv) provided the title compound (0.22 g, yield: 66%, DSR = 31%); 1 H-NMR(400MHz,D2O)δppm 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).

[0614] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.123 g, yield: 36%, DSR = 4%).

[0615] Example 16

[0616] Preparation of a conjugate of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0617]

[0618] Step 1: Preparation of tert-butyl (2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate

[0619]

[0620] NaH (1.42 g, 35.425 mmol, 2.5 eq) was added to a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (3.2 g, 15.59 mmol, 1.1 eq) in THF (40 mL) under N2 at 0°C, the reaction mixture was stirred at 0°C for 30 minutes, and then 1-fluoro-4-nitrobenzene (2 g, 14.17 mmol, 1 eq) was added thereto, and the resulting mixture was refluxed for 12 hours. After most of the 1-fluoro-4-nitrobenzene was consumed, the reaction was quenched with saturated aqueous NH4Cl solution (100 mL) and extracted with ethyl acetate (50 mL*2). The combined organic phases were washed with saturated aqueous NaCl solution (100 mL), dried over anhydrous Na2SO4 and concentrated under high vacuum. The residue was purified by silica gel chromatography to give the title product (2 g, yield: 43.3%); MS (m / z): C 15 H 22 [M+H]+ calculated for N2O6, 327.15; found, 327.1.

[0621] Step 2: Preparation of tert-butyl (2-(2-(4-aminophenoxy)ethoxy)ethyl)carbamate

[0622]

[0623] 10% Pd / C (0.2 g) was added to a solution of tert-butyl (2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate (2 g, 6.13 mmol, 1 eq) in methanol (40 mL), and the reaction mixture was stirred under a H2 balloon at room temperature for 24 hours. After 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*2). The combined filtrates were concentrated to give the title product (1.8 g, yield: 100%); MS (m / z): C 15 H 24 [M+H]+ calculated for N2O4, 297.17; found, 297.1.

[0624] Step 3: Preparation of tert-butyl (2-(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)ethoxy)ethyl)carbamate

[0625]

[0626] Following Step 1 in Example 1, tofacitinib (1.9 g, 6.07 mmol, 1 eq) and tert-butyl (2-(2-(4-aminophenoxy)ethoxy)ethyl)carbamate (1.8 g, 6.07 mmol, 1 eq) gave the title product (1.8 g, yield: 57%); MS (m / z): C 32 H 42 [M+H]+ calculated for N8O6, 635.32; found, 635.2.

[0627] Step 4: Preparation of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0628]

[0629] Following Step 2 in Example 1, tert-butyl (2-(2-(4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)phenoxy)ethoxy)ethyl)carbamate (1 g, 1.575 mmol, 1 equiv) gave the desired product as an HCl salt (0.89 g, yield: 100%); MS (m / z): C 27 H 34 [M+H]+ calculated for N8O4, 535.27; found, 535.1. 1 H-NMR(400MHz,D2O)δppm 8.19(d,J=9.4Hz,1H),7.52(d,J=15.7Hz,1H),7.37(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H),6.72-6.50(m,1H),4.6 3-4.54(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=6.8Hz,3H).

[0630] Step 5: Preparation of a conjugate of N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0631]

[0632] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.141 g, 0.35 mmol, 1 equiv) and N-(4-(2-(2-aminoethoxy)ethoxy)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.35 mmol, 1 equiv) provided the title compound (0.15 g, yield: 32%, DSR = 25%); 1 H-NMR(400MHz,D2O)δppm 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).

[0633] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.132 g, yield: 42%, DSR = 28%).

[0634] Example 17

[0635] Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile and HA

[0636]

[0637] Step 1: Preparation of tert-butyl 4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)piperazine-1-carboxylate

[0638]

[0639] Following Step 1 in Example 1, tofacitinib (1.68 g, 5.37 mmol, 1 eq.) and tert-butyl piperazine-1-carboxylate (1 g, 5.37 mmol, 1 eq.) gave the title product (0.9 g, yield: 32%); MS (m / z): C 26 H 36 [M+H]+ calculated for N8O4, 525.29; found, 525.2.

[0640] Step 2: Preparation of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile hydrochloride

[0641]

[0642] Following Step 2 in Example 1, tert-butyl 4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)piperazine-1-carboxylate (0.8 g, 1.525 mmol, 1 equiv) gave the desired product as an HCl salt (0.7 g, yield: 100%); MS (m / z): C 21 H 28 [M+H]+ calculated for N8O2, 425.23; found, 425.1. 1 H-NMR(400MHz,D2O)δppm 8.41(d,J=5.5Hz,1H),7.59(d,J=3.9Hz,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=14.8,7.1Hz,3H).

[0643] Step 3: Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile and HA

[0644]

[0645] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.175 g, 0.434 mmol, 1 equiv) and N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.434 mmol, 1 equiv) provided the title compound (0.2 g, yield: 57.3%, DSR = 24%); 11H-NMR(400MHz,D2O)δppm 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,14 H),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).

[0646] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.162 g, yield: 46.4%, DSR = 28%).

[0647] Example 18

[0648] Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile and HA

[0649]

[0650] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)cyclohexyl)carbamate

[0651]

[0652] Following Step 1 in Example 1, tofacitinib (1.895 g, 6.066 mmol, 1 eq) and tert-butyl (4-aminocyclohexyl)carbamate (1.3 g, 6.066 mmol, 1 eq) gave the title product (1.785 g, yield: 53.2%); MS (m / z): C 28 H 40 [M+H]+ calculated for N8O4, 553.32; found, 553.2.

[0653] Step 2: Preparation of N-(4-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0654]

[0655] Following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)cyclohexyl)carbamate (1.5 g, 2.71 mmol, 1 equiv) gave the desired product as an HCl salt (1 g, yield: 77%); MS (m / z): C 23 H 32 [M+H]+ calculated for N8O2, 453.26; found, 453.2. 1 H-NMR(400MHz,D2O)δppm 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=6.6,5.4Hz,3H).

[0656] Step 3: Preparation of a conjugate of 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile and HA

[0657]

[0658] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.178 g, 0.44 mmol, 1 equiv) and 3-((3R,4R)-4-methyl-3-(methyl(7-(piperazine-1-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile hydrochloride (0.2 g, 0.44 mmol, 1 equiv) provided the title compound (0.2 g, yield: 55%, DSR = 25%); 1 H-NMR(400MHz,D2O)δppm 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).

[0659] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.109 g, yield: 30%, DSR = 9%).

[0660] Example 19

[0661] Preparation of a conjugate of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0662]

[0663] Step 1: Preparation of tert-butyl (2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)cyclohexyl)carbamate

[0664]

[0665] Following Step 1 in Example 1, tofacitinib (1.46 g, 4.67 mmol, 1 eq) and tert-butyl (2-aminocyclohexyl)carbamate (1 g, 4.67 mmol, 1 eq) gave the title product (2 g, yield: 78%); MS (m / z): C 28 H 40 [M+H]+ calculated for N8O4, 553.32; found, 553.2.

[0666] Step 2: Preparation of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0667]

[0668] Following Step 2 in Example 1, tert-butyl (2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)cyclohexyl)carbamate (1.3 g, 2.352 mmol, 1 equiv) gave the desired product as an HCl salt (1.15 g, yield: 100%); MS (m / z): C 23 H 32 [M+H]+ calculated for N8O2, 453.26; found, 453.2. 1H-NMR(400MHz,D2O)δppm 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).

[0669] Step 3: Preparation of a conjugate of N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0670]

[0671] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.161 g, 0.4 mmol, 1 equiv) and N-(2-aminocyclohexyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.2 g, 0.4 mmol, 1 equiv) provided the title compound (0.2 g, yield: 59%, DSR = 18%); 1 H-NMR(400MHz,D2O)δppm 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).

[0672] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.224 g, yield: 67%, DSR = 7%).

[0673] Example 20

[0674] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0675]

[0676] Step 1: Preparation of tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-ethylhydrazine-1-carboxylate

[0677]

[0678] Following Step 1 in Example 1, tofacitinib (2.5 g, 8.114 mmol, 1 eq) and tert-butyl 1-ethylhydrazine-1-carboxylate (1.3 g, 8.114 mmol, 1 eq) gave the title product (1.745 g, yield: 43%); MS (m / z): C 24 H 34 [M+H]+ calculated for N8O4, 499.27; found, 499.2.

[0679] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

[0680]

[0681] Following Step 2 in Example 1, tert-butyl 2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-1-ethylhydrazine-1-carboxylate (1.4 g, 2.8 mmol, 1 equiv) gave the desired product as an HCl salt (1 g, yield: 82%); MS (m / z): C 19 H 26 [M+H]+ calculated for N8O2, 399.22; found, 399.1. 1 H-NMR(400MHz,D2O)δppm 8.45(d,J=4.8Hz,1H),7.81(d,J=3.6Hz,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=15.2,7.1Hz,3H)

[0682] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and HA

[0683]

[0684] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.186 g, 0.46 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-ethyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g, 0.46 mmol, 1 equiv) provided the title compound (0.12 g, yield: 34%, DSR = 9%); 1 H-NMR(400MHz,D2O)δppm 8.4-8.3(m,0.09H),7.69(d,J=23.0Hz,0.09H),6.95(d,J=10.7Hz,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).

[0685] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.198 g, yield: 55.3%, DSR = 5%).

[0686] Example 21

[0687] Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester and HA

[0688]

[0689] Step 1: Preparation of N6-(tert-Butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester

[0690]

[0691] Following Step 1 in Example 1, tofacitinib (2 g, 6.4 mmol, 1 eq.) and N6-(tert-butoxycarbonyl)-L-lysine methyl ester hydrochloride (1.9 g, 6.4 mmol, 1 eq.) gave the title product (2.2 g, yield: 57.4%); MS (m / z): C 29 H 42 [M+H]+ calculated for N8O6, 599.32; found, 599.2.

[0692] Step 2: Preparation of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester hydrochloride

[0693]

[0694] Following Step 2 in Example 1, N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester (1.5 g, 2.5 mmol, 1 equiv) gave the desired product as an HCl salt (1.34 g, yield: 100%); MS (m / z): C 24 H 34 [M+H]+ calculated for N8O4, 499.27; found, 499.2. 1 H-NMR(400MHz,D2O)δppm 8.41(d,J=4.4Hz,1H),7.79(dd,J=9.0,4.1Hz,1H),6.88(dd,J=6.7,2.3Hz,1H),4.70-4.54(m,2H),4.26-3.80(m,8H),3.67-3.37(m, 5H),3.00(t,J=7.6Hz,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=15.9,7.1Hz,3H).

[0695] Step 3: Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester and HA

[0696]

[0697] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.151 g, 0.374 mmol, 1 equiv) and (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysine methyl ester hydrochloride (0.2 g, 0.374 mmol, 1 equiv) provided the title compound (0.2 g, yield: 61%, DSR = 19%); 1 H-NMR(400MHz,D2O)δppm 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). 1 H-NMR(400MHz,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).

[0698] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.2 g, yield: 61%, DSR = 23.2%).

[0699] Example 22

[0700] Preparation of a conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0701]

[0702] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide)butyl)carbamate

[0703]

[0704] N, N-dimethylpyridine-4-amine (2.15g, 17.6mmol, 2.5 equivalents) was added to a mixture of tofacitinib (2g, 6.4mmol, 1 equivalent) and O-phenylchlorothioformate (2.14g, 7.04mmol, 1.1 equivalents) in dichloromethane (40mL) under N2. The reaction mixture was heated to reflux for 3 hours. Then tert-butyl (4-aminobutyl)carbamate (1.2g, 6.4mmol, 1 equivalent) was added, and the mixture was refluxed for another 12 hours. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure and the residue was purified by silica gel chromatography to give the title product (1.16g, yield: 33.4%); MS (m / z): C 26 H 38 [M+H]+ calculated for N8O3S, 543.28; found, 543.2.

[0705] Step 2: Preparation of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride

[0706]

[0707] Following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-methanethioamide)butyl)carbamate (1.16 g, 2.14 mmol, 1 equiv) gave the desired product as an HCl salt (1 g, yield: 100%); MS (m / z): C 21 H 30 [M+H]+ calculated for N8OS, 443.23; found, 443.2. 1 H-NMR(400MHz,D2O)δppm 8.36(d,J=36.1Hz,2H),6.92(s,1H),4.63(d,J=1.2Hz,1H),4.1-3.75(m,4H),3.39(s,3 H),3.23(s,4H),2.96(t,J=7.5Hz,3H),2.47(s,1H),2.0-1.6(m,5H),1.23-1.06(m,4H).

[0708] Step 3: Preparation of a conjugate of N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0709]

[0710] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.170 g, 0.42 mmol, 1 eq) and N-(4-aminobutyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride (0.2 g, 0.42 mmol, 1 eq) provided the title compound (0.18 g, yield: 53%, DSR = 20%); 1 H-NMR(400MHz,D2O)δppm 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).

[0711] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.196 g, yield: 56.8%, DSR = 30%)

[0712] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.203 g, yield: 58.8%, DSR = 8%).

[0713] Example 23

[0714] Preparation of a conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0715]

[0716] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide)benzyl)carbamate

[0717]

[0718] Following Step 1 in Example 22, tofacitinib (3 g, 9.6 mmol, 1 eq) and tert-butyl (4-aminobenzyl)carbamate (2.134 g, 9.6 mmol, 1 eq) gave the title product (0.3 g, yield: 5.5%); MS (m / z): C 29 H 36 [M+H]+ calculated for N8O3S, 577.26; found, 577.2.

[0719] Step 2: Preparation of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride

[0720]

[0721] Following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide)benzyl)carbamate (0.375 g, 0.65 mmol, 1 equiv) gave the desired product as an HCl salt (0.26 g, yield: 78.8%); MS (m / z): C 24 H 28 [M+H]+ calculated for N8OS, 477.21; found, 477.2. 1 H-NMR(400MHz,D2O)δppm 8.43-8.30(m,2H),7.85(d,J=8.1Hz,2H),7.51(d,J=8.1Hz,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).

[0722] Step 3: Preparation of a conjugate of N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0723]

[0724] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.157 g, 0.39 mmol, 1 equiv) and N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride (0.2 g, 0.39 mmol, 1 equiv) provided the title compound (0.18 g, yield: 55%, DSR = 12%); 1 H-NMR(400MHz,D2O)δppm 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).

[0725] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.166 g, yield: 49.8%, DSR = 24.3%).

[0726] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.132 g, yield: 39.7%, DSR = 14.7%).

[0727] Example 24

[0728] Preparation of a conjugate of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0729]

[0730] Step 1: Preparation of tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide)phenethyl)carbamate

[0731]

[0732] Following Step 1 in Example 22, tofacitinib (2 g, 6.4 mmol, 1 eq) and tert-butyl (4-aminophenethyl)carbamate (1.512 g, 6.4 mmol, 1 eq) gave the title product (0.78 g, yield: 20.6%); MS (m / z): C 30 H38 [M+H]+ calculated for N8O3S, 591.28; found, 591.2.

[0733] Step 2: Preparation of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride

[0734]

[0735] Following Step 2 in Example 1, tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide)phenethyl)carbamate (0.55 g, 0.931 mmol, 1 equiv) gave the desired product as an HCl salt (0.49 g, yield: 100%); MS (m / z): C 25 H 30 [M+H]+ calculated for N8OS, 491.23; found, 491.1. 1 H-NMR(400MHz,D2O)δppm 8.43-8.30(m,2H),7.73(d,J=7.7Hz,2H),7.34(d,J=7.9Hz,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=13.9,6.0Hz,3H).

[0736] Step 3: Preparation of a conjugate of N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0737]

[0738] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.153 g, 0.38 mmol, 1 equiv) and N-(4-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride (0.2 g, 0.38 mmol, 1 equiv) provided the title compound (0.18 g, yield: 55%, DSR = 14%); 1 H-NMR(400MHz,D2O)δppm 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).

[0739] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.185 g, yield: 56%, DSR = 15%).

[0740] Example 25

[0741] Preparation of a conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0742]

[0743] Step 1: Preparation of tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-thioxo-5,8,11-trioxa-2-azatridec-13-yl)carbamate

[0744]

[0745] Following Step 1 in Example 22, tofacitinib (2 g, 6.4 mmol, 1 eq) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.871 g, 6.4 mmol, 1 eq) gave the title product (1.12 g, 27% yield); MS (m / z): C 30 H 46[M+H]+ calculated for N8O6S, 647.33; found, 647.2.

[0746] Step 2: Preparation of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride

[0747]

[0748] Following Step 2 in Example 1, tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-thioxo-5,8,11-trioxa-2-azatridec-13-yl)carbamate (1.1 g, 1.7 mmol, 1 equiv) gave the desired product as an HCl salt (0.84 g, yield: 85%); MS (m / z): C 25 H 38 [M+H]+ calculated for N8O4S, 547.27; found, 547.2. 1 H-NMR(400MHz,D2O)δppm8.5-8.0(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).

[0749] Step 3: Preparation of a conjugate of N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide and HA

[0750]

[0751] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.137 g, 0.34 mmol, 1 equiv) and N-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-thioamide hydrochloride (0.2 g, 0.34, 1 equiv) afforded the title compound (0.17 g, 55% yield, DSR = 31%); 1 H-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). In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 kDa) provided the corresponding product (0.185 g, yield: 58.8%, DSR = 25%).

[0752] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.231 g, yield: 73.4%, DSR = 30%).

[0753] Example 26

[0754] Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysine methyl ester and HA

[0755]

[0756] Step 1: Preparation of N6-(tert-Butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonylthioate)-L-lysine methyl ester

[0757]

[0758] Following Step 1 in Example 22, tofacitinib (2 g, 6.4 mmol, 1 eq) and N6-(tert-butoxycarbonyl)-L-lysine methyl ester hydrochloride (1.9 g, 6.4 mmol, 1 eq) gave the title product (0.675 g, yield: 17.2%); MS (m / z): C 29 H 42 [M+H]+ calculated for N8O5S, 615.30; found, 615.2.

[0759] Step 2: Preparation of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysine methyl ester hydrochloride

[0760]

[0761] Following Step 2 in Example 1, N6-(tert-butoxycarbonyl)-N2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysine methyl ester (0.625 g, 1.017 mmol, 1 equiv) gave the desired product as an HCl salt (0.56 g, yield: 100%); MS (m / z): C 24 H 34 [M+H]+ calculated for N8O3S, 515.25; found, 515.2. 1 H-NMR(400MHz,D2O)δppm8.45(s,1H),8.33(s,1H),6.92(s,1H),5.09(t,J=6.2Hz,1H),4.63(br,1H),3.95-3.67(m,5 H),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=6.8Hz 3H).

[0762] Step 3: Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysine methyl ester and HA

[0763]

[0764] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.145 g, 0.36 mmol, 1 equiv) and (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysine methyl ester hydrochloride (0.2 g, 0.36 mmol, 1 equiv) provided the title compound (0.13 g, yield: 41%, DSR = 10%); 1 H-NMR(400MHz,D2O)δppm 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,1 1.6H),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).

[0765] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.117 g, yield: 36.3%, DSR = 10%).

[0766] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.161 g, yield: 50%, DSR = 5.7%).

[0767] Example 27

[0768] Preparation of a conjugate of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0769]

[0770] Step 1: Preparation of 4-(((tert-Butoxycarbonyl)amino)methyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate

[0771]

[0772] Following Step 1 in Example 1, tofacitinib (1.32 g, 4.214 mmol, 1 eq) and tert-butyl (4-(hydroxymethyl)benzyl)carbamate (2 g, 4.214 mmol, 1 eq) gave the title product (1.3 g, yield: 53.5%); MS (m / z): C 30 H 37 [M+H]+ calculated for N7O5, 576.29; found, 576.2.

[0773] Step 2: Preparation of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride

[0774]

[0775] Following Step 2 in Example 1, 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-(((tert-butoxycarbonyl)amino)methyl)benzyl ester (1.3 g, 2.25 mmol, 1 equiv) gave the desired product as an HCl salt (1 g, yield: 87%); MS (m / z): C 25 H 29 [M+H]+ calculated for N7O3, 476.23; found, 476.2. 1 H-NMR(400MHz,D2O)δppm 8.30(dd,J=36.1,9.5Hz,1H),7.75-7.23(m,5H),6.84(d,J=15.6Hz,1H),4.70(br,3H),4.35- 3.71(m,6H),3.71-3.23(m,5H),2.50(d,J=33.4Hz,1H),2.03-1.62(m,2H),1.17-0.99(m,3H).

[0776] Step 3: Preparation of a conjugate of 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0777]

[0778] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.157 g, 0.39 mmol, 1 equiv) and 4-(aminomethyl)benzyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (0.2 g, 0.39 mmol, 1 equiv) provided the title compound (0.2 g, yield: 60%, DSR = 22%); 1 H-NMR(400MHz,D2O)δppm 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,12.02 H),3.14(br,0.34H),2.32(br,0.22H),2.02(s,3H),1.31(br,0.45H),1.06(br,0.65H).

[0779] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.2 g, yield: 60%, DSR = 43.6%).

[0780] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.175 g, yield: 52.6%, DSR = 35.3%).

[0781] Example 28

[0782] Preparation of a conjugate of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0783]

[0784] Step 1: Preparation of 4-((S)-2-((tert-Butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate

[0785]

[0786] Following Step 1 in Example 1, tofacitinib (3 g, 9.62 mmol, 1 eq) gave the title product (1.7 g, yield: 28%); MS (m / z): C32 H 39 [M+H]+ calculated for N7O7, 634.29; found, 634.2.

[0787] Step 2: Preparation of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride

[0788]

[0789] Following Step 2 in Example 1, 4-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (1 g, 1.578 mmol, 1 equiv) gave the desired product as an HCl salt (0.9 g, yield: 100%); MS (m / z): C 27 H 31 [M+H]+ calculated for N7O5, 534.24; found, 534.2. 1 H-NMR (400MHz, CD3OD) δppm8.55-8.34(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=4.2Hz,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=13.2,5.2Hz,3H).

[0790] Step 3: Preparation of a conjugate of 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0791]

[0792] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.141 g, 0.35 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-((S)-2-amino-3-methoxy-3-oxopropyl)phenyl ester hydrochloride (0.2 g, 0.35 mmol, 1 equiv) provided the title compound (0.2 g, yield: 32%, DSR = 20%); 1 H-NMR(400MHz,D2O)δppm 7.87–6.43(m,1.4H),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).

[0793] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.16 g, yield: 50%, DSR = 22%).

[0794] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.238 g, yield: 74.5%, DSR = 22%).

[0795] Example 29

[0796] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 8-aminooctyl ester and HA

[0797]

[0798] Step 1: Preparation of 8-(tert-butoxycarbonyl)amino)octyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate

[0799]

[0800] Following Step 1 in Example 1, tofacitinib (1.15 g, 3.67 mmol, 1 eq) and tert-butyl (8-hydroxyoctyl)carbamate (0.9 g, 3.668 mmol, 1 eq) gave the title product (0.7 g, yield: 33%); MS (m / z): C 30 H 45[M+H]+ calculated for N7O5, 584.35; found, 584.3. 1 H-NMR (400MHz, CDCl3) δppm8.46 (d, J=8.8Hz, 1H), 7.46 (dd, J=10.6, 4.2Hz, 1H), 6 .63(d,J=4.1Hz,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=18.7Hz,2H),3.10(d,J=6.4Hz,2H),2.62 -2.41(m,1H),2.02-1.70(m,4H),1.45-1.3(m,12H),1.08(dd,J=14.0,7.1Hz,3H).

[0801] Step 2: Preparation of 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride

[0802]

[0803] Following Step 2 in Example 1, 8-((tert-butoxycarbonyl)amino)octyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.7 g, 1.2 mmol, 1 equiv) gave the desired product as an HCl salt (0.6 g, yield: 96%); MS (m / z): C 25 H 37 [M+H]+ calculated for N7O3, 484.30; found, 484.2.

[0804] Step 3: Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 8-aminooctyl ester and HA

[0805]

[0806] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.153 g, 0.38 mmol, 1 equiv) and 8-aminooctyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (0.2 g, 0.38 mmol, 1 equiv) provided the title compound (0.19 g, yield: 54%, DSR = 14%); 1 H-NMR(400MHz,D2O)δppm 8.39(m,0.14H),7.67(m,0.14H),6.95(m,0.14H),4.53(m,2H),3.73(m,12H),2 .51(m,0.24H),2.04(s,3H),1.87(m,0.82H),1.48(m,0.92H),1.11(m,0.57H).

[0807] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.191 g, yield: 58.23%, DSR = 5.3%).

[0808] Example 30

[0809] Preparation of the conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-aminobutyl ester and HA

[0810]

[0811] Step 1: Preparation of 4-[[(3S,4S)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-(tert-butoxycarbonylamino)butyl ester

[0812]

[0813] Triethylamine (2.7mL, 19.2mmol) is added to a stirred mixture of tofacitinib (3000mg, 9.6mmol) and bis(4-nitrophenyl) carbonate (3.22mg, 10.6mmol) in dichloromethane (60mL). The reaction mixture is heated to 45°C and stirred at this temperature for 5 hours. The reaction mixture is then cooled to room temperature. 4-(tert-butoxycarbonylamino)-1-butanol (2000mg, 10.6mmol) is added. The reaction mixture is stirred at room temperature for 16 hours. The solution is diluted with dichloromethane and washed with saturated NaHCO3 solution, water and saturated saline solution. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue is purified by column chromatography (ethyl acetate: hexane = 1: 1) to provide the title compound (2.64g, yield: 52.1%). MS (m / z): C 26 H 37 [M+H]+ calculated for N7O5, 528.28; found, 528.2.

[0814] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-aminobutyl ester hydrochloride

[0815]

[0816] Following Step 2 in Example 1, 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-(tert-butoxycarbonylamino)butyl ester (2.955 g, 5.6 mmol) provided the title compound as a white solid (2.59 g, yield: 100%). MS (m / z): C 21 H 29 [M+H]+ calculated for N7O3, 428.23; found, 428.2. 1 H NMR(400MHz,D2O)δppm 8.39(d,J=6.9Hz,1H),7.63(d,J=3.8Hz,1H),6.96(s,1H),5.04(s,1H),4.53(t,J=6.2Hz,2H),4.01–3.70(m,4H),3.67– 3.54(m,1H),3.41(d,J=30.2Hz,4H),3.02(dd,J=17.9,10.6Hz,2H),2.46(s,1H),1.97–1.59(m,6H),1.12–0.96(m,3H).

[0817] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-aminobutyl ester and HA

[0818]

[0819] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 161.2 mg, 0.399 mmol), 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-aminobutyl ester hydrochloride (200 mg, 0.399 mmol) provided the title compound (0.13 g, yield: 36.7%, DSR = 12%); 1 HNMR(400MHz,D2O)δppm 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).

[0820] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.125 g, yield: 35.3%, DSR = 37%).

[0821] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.134 g, yield: 37.8%, DSR = 32%).

[0822] Example 31

[0823] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminophenyl) ester and HA

[0824]

[0825] Step 1: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid [4-(tert-butoxycarbonylamino)phenyl ester]

[0826]

[0827] Following Step 1 in Example 1, tofacitinib (1562 mg, 5 mmol) and tert-butyl (4-hydroxyphenyl)carbamate (1256 mg, 6 mmol) provided the title compound (1.1 g, yield: 40%). MS (m / z): C 28 H 33 [M+H]+ calculated for N7O5, 548.25; found, 548.1. 1 H NMR (400MHz, CDCl3) δppm 8.48(d,J=8.0Hz,1H),8.14–8.05(m,1H),7.61(dd,J=8.0,4.2Hz,1H),7.42(m,1H),7. 23(d,J=8.9Hz,2H),6.89–6.74(m,1H),6.58(s,1H),5.15(d,J=3.9Hz,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=23.2,8.8,4.5Hz,1H),1.83–1.69(m,1H),1.51(m,9H),1.15–1.03(m,3H).

[0828] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminophenyl) ester hydrochloride

[0829]

[0830] Following step 2 in Example 1, 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid [4-(tert-butoxycarbonylamino)phenyl ester] (0.8 g, 1.46 mmol) provided the title compound as a white solid (0.52 g, yield: 73.6%); MS (m / z): C 23 H 25 [M+H]+ calculated for N7O3, 448.20; found, 448.3.

[0831] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminophenyl) ester and HA

[0832]

[0833] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 116 mg, 0.288 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminophenyl) ester hydrochloride (150 mg, 0.288 mmol) provided the title compound (0.126 g, yield: 48.3%, DSR: 10%); 1 H NMR(400MHz,D2O)δppm 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).

[0834] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.123 g, yield: 47.1%, DSR: 24%).

[0835] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.138 g, yield: 52.9%, DSR: 8%).

[0836] Example 32

[0837] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid azetidin-3-yl ester and HA

[0838]

[0839] Step 1: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonylamino-3-yl) ester

[0840]

[0841] Following Step 1 in Example 1, tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxyazetidine-1-carboxylate (831.4 mg, 4.8 mmol) provided the title compound (0.48 g, yield: 23.4%). MS (m / z): C 25 H 33 [M+H]+ calculated for N7O5, 512.25; found, 512.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.47(d,J=8.4Hz,1H),7.45(dd,J=11.6,4.2Hz,1H),6.65(dd,J=15.6,4.0Hz ,1H),5.14(d,J=3.0Hz,1H),4.38(dd,J=10.0,7.0Hz,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=23.8,13.5,8.5Hz,2H),1.45(d,J=6.8Hz,9H),1.11–1.02(m,3H).

[0842] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid azetidin-3-yl ester hydrochloride

[0843]

[0844] Following Step 2 in Example 1, 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonylazetidin-3-yl) ester (0.44 g, 0.86 mmol) provided the title compound as a white solid (0.366 g, yield: 95%). MS (m / z): C 20 H 25 [M+H]+ calculated for N7O3, 412.20; found, 412.3.

[0845] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid azetidin-3-yl ester and HA

[0846]

[0847] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 83.2 mg, 0.207 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid azetidin-3-yl ester hydrochloride (100 mg, 0.207 mmol) provided the title compound (0.078 g, yield: 43.4%, DSR: 12%); 1 H NMR(400MHz,D2O)δppm 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,7.0Hz,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).

[0848] Example 33

[0849] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid pyrrolidin-3-yl ester and HA

[0850]

[0851] Step 1: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonylpyrrolidin-3-yl) ester

[0852]

[0853] Following Step 1 in Example 1, tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxypyrrolidine-1-carboxylate (898.8 mg, 4.8 mmol) provided the title compound (0.94 g, yield: 44.7%). MS (m / z): C 26 H 35 [M+H]+ calculated for N7O5, 526.26; found, 526.1. 1H NMR (400 MHz, CDCl3) δ ppm 8.47(d,J=6.3Hz,1H),7.38(dd,J=11.2,4.1Hz,1H),6.63(d,J=4.1Hz,1H), 5.11(t,J=11.8Hz,1H),4.08–3.77(m,2H),3.73–3.47(m,8H),3.36(d,J=17. 7Hz,3H),2.58–2.42(m,1H),2.37–2.18(m,2H),1.93(dddd,J=23.5,18.7,9. 0,4.5Hz,1H),1.82–1.72(m,2H),1.47(s,9H),1.08(dd,J=14.4,7.0Hz,3H).

[0854] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid pyrrolidin-3-yl ester hydrochloride

[0855]

[0856] Following Step 2 in Example 1, 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonylpyrrolidin-3-yl) ester (0.6 g, 1.14 mmol) provided the title compound as a white solid (0.526 g, yield: 100%). MS (m / z): C 21 H 27 [M+H]+ calculated for N7O3, 426.21; found, 426.3.

[0857] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid pyrrolidin-3-yl ester and HA

[0858]

[0859] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 161.6 mg, 0.401 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid pyrrolidin-3-yl ester hydrochloride (200 mg, 0.401 mmol) provided the title compound (0.178 g, yield: 50%, DSR: 20%); 1HNMR(400MHz,D2O)δppm 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).

[0860] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.185 g, yield: 52.2%, DSR: 33%).

[0861] Example 34

[0862] Preparation of the conjugate of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0863]

[0864] Step 1: Preparation of 3-(tert-butoxycarbonylamino)propyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate

[0865]

[0866] Following Step 1 in Example 1, tofacitinib (1249.5 mg, 4 mmol) and 3-(Boc-amino)-1-propanol (841.1 mg, 4.8 mmol) provided the title compound (1.03 g, yield: 50%). MS (m / z): C 25 H 35 [M+H]+ calculated for N7O5, 514.26; found, 514.2. 1H NMR (400MHz, CDCl3) δppm 8.53(d,J=10.4Hz,1H),7.53(dd,J=10.8,4.0Hz,1H),7.15(s,1H),6.67(d,J=4.1Hz,1H) ,5.12(d,J=30.4Hz,1H),4.57(dd,J=14.1,8.5Hz,2H),4.06–3.78(m,2H),3.67–3.48(m,4 H),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(td d,J=22.0,8.8,4.5Hz,1H),1.83–1.65(m,2H),1.45(s,9H),1.07(dd,J=16.9,7.1Hz,3H).

[0867] Step 2: Preparation of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride

[0868]

[0869] Following Step 2 in Example 1, 3-(tert-butoxycarbonylamino)propyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.03 g, 2 mmol) provided the title compound as a white solid (0.9 g, 100%). MS (m / z): C 20 H 27 [M+H]+ calculated for N7O3, 414.21; found, 414.3.

[0870] Step 3: Preparation of a conjugate of 3-aminopropyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0871]

[0872] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 165.6 mg, 0.411 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 3-aminopropyl ester hydrochloride (200 mg, 0.411 mmol) provided the title compound (0.162 g, yield: 45.2%, DSR: 15%); 1 HNMR(400MHz,D2O)δppm 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=7.0Hz,0.15H),1.05–0.90(m,0.45H).

[0873] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.161 g, yield: 44.9%, DSR: 17%).

[0874] Example 35

[0875] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminocyclohexyl) ester and HA

[0876]

[0877] Step 1: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid [4-(tert-butoxycarbonylamino)cyclohexyl ester]

[0878]

[0879] Following Step 1 in Example 1, tofacitinib (1249.5 mg, 4 mmol) and tert-butyl (4-hydroxycyclohexyl)carbamate (1033.4 mg, 4.8 mmol) provided the title compound (0.851 g, yield: 45%). MS (m / z): C 28 H 39 [M+H]+ calculated for N7O5, 554.30; found, 554.1. 1H NMR (400MHz, CDCl3) δppm 8.46(d,J=5.8Hz,1H),7.42(dd,J=9.5,4.2Hz,1H),6.61(d,J=4.1Hz,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=16.4Hz,3 H),2.49(dddd,J=24.3,11.9,6.4Hz,1H),2.26–2.06(m,4H),1.93(dddd,J=18.5,13.7,8.9,4 .4Hz,1H),1.82–1.59(m,4H),1.45(s,9H),1.34(td,J=13.3,2.7Hz,2H),1.15–0.99(m,3H).

[0880] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminocyclohexyl) ester hydrochloride

[0881]

[0882] Following Step 2 in Example 1, [4-(tert-butoxycarbonylamino)cyclohexyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (0.62 g, 1.12 mmol) provided the title compound as a white solid (0.55 g, yield: 100%). MS (m / z): C 23 H 31 [M+H]+ calculated for N7O3, 454.24; found, 454.3.

[0883] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminocyclohexyl) ester and HA

[0884]

[0885] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 153 mg, 0.38 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (4-aminocyclohexyl) ester hydrochloride (200 mg, 0.38 mmol) provided the title compound (0.173 g, yield: 49.9%, DSR: 11%); 1 HNMR(400MHz,D2O)δppm 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,1 0.99H), 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=6.9Hz, 0.11H), 1.09–0.85 (m, 0.33H).

[0886] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.162 g, yield: 46.7%, DSR: 27%).

[0887] Example 36

[0888] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-piperidinyl ester and HA

[0889]

[0890] Step 1: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonyl-4-piperidinyl) ester

[0891]

[0892] Following Step 1 in Example 1, tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (966 mg, 4.8 mmol) provided the title compound (0.537 g, yield: 24.9%). MS (m / z): C 27 H 37[M+H]+ calculated for N7O5, 540.28; found, 540.2.

[0893] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-piperidinyl ester hydrochloride

[0894]

[0895] Following Step 2 in Example 1, 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid (1-tert-butoxycarbonyl-4-piperidinyl) ester (0.47 g, 0.87 mmol) provided the title compound as a white solid (0.25 g, yield: 58%). MS (m / z): C 22 H 29 [M+H]+ calculated for N7O3, 440.23; found, 440.3.

[0896] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-piperidinyl ester and HA

[0897]

[0898] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 169.7 mg, 0.421 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylic acid 4-piperidinyl ester hydrochloride (200 mg, 0.421 mmol) provided the title compound 0.161 g, yield: 44.4%, DSR: 11%; 1 H NMR(400MHz,D2O)δppm 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),2.41–2.3 0(m,0.11H),2.19–1.58(m,3.66H),1.20(t,J=6.9Hz,0.11H),1.08–0.85(m,0.33H).

[0899] Example 37

[0900] Preparation of a conjugate of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0901]

[0902] Step 1: Preparation of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate

[0903]

[0904] Triethylamine (1.1mL, 8mmol) is added to a stirred mixture of tofacitinib (1250mg, 4mmol) and bis(4-nitrophenyl) carbonate (1460mg, 4.8mmol) in dichloromethane (40mL). The reaction mixture is heated to 45°C and stirred at this temperature for 6 hours. The reaction mixture is then cooled to room temperature. tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate (985.2mg, 4.8mmol) is added. The reaction mixture is stirred at room temperature for 16 hours. The solution is diluted with dichloromethane and washed with saturated NaHCO3 solution, water and saturated saline solution. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure to provide the title compound (1.3g, yield: 59.8%). MS (m / z): C 26 H 37 [M+H]+ calculated for N7O6, 544.28; found, 544.2.

[0905] Step 2: Preparation of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride

[0906]

[0907] Following Step 2 in Example 1, 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.3 g, 2.39 mmol) provided the title compound as a white solid (1.1 g, yield: 95.9%). MS (m / z): C 21 H 29[M+H]+ calculated for N7O4, 444.22; found, 444.2.

[0908] Step 3: Preparation of a conjugate of 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[0909]

[0910] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 156.4 mg, 0.388 mmol) and 2-(2-aminoethoxy)ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg, 0.388 mmol) provided the title compound (0.175 g, yield: 50%, DSR: 15%); 1 H NMR(400MHz,D2O)δppm 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).

[0911] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.182 g, yield: 52%, DSR: 17%).

[0912] Example 38

[0913] Preparation of conjugates of (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]hexanoic acid methyl ester

[0914]

[0915] Step 1: Preparation of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]hexanoate

[0916]

[0917] 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid (1323 mg, 3 mmol), (S)-methyl 6-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 a saturated saline 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 to 1:80) to provide the title compound (1.726 g, yield: 83.3%). MS (m / z): C 33 H 49 [M+H]+ calculated for N9O7, 684.37; found, 684.1. 1 H NMR (400MHz, CDCl3) δppm9.89 (dd, J=13.5, 7.5Hz, 1H), 8.29 (d, J=9.0Hz, 1H), 7.7 2(dd,J=11.9,4.0Hz,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=5.4Hz,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=12.7,7.1Hz,3H).

[0918] Step 2: Preparation of (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]hexanoic acid methyl ester hydrochloride

[0919]

[0920] Following Step 2 in Example 1, (2S)-methyl 6-(tert-butoxycarbonylamino)-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate (1.5 g, 2.137 mmol) provided the title compound as a white solid (1.426 g, yield: 99.6%). MS (m / z): C 28 H 41 [M+H]+ calculated for N9O5, 584.32; found, 584.1.

[0921] Step 3: Preparation of a conjugate of (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]hexanoic acid methyl ester

[0922]

[0923] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 123 mg, 0.305 mmol) and (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]hexanoic acid methyl ester hydrochloride (200 mg, 0.36 mmol) provided the title compound (0.13 g, yield: 45%, DSR: 5%); 1 H NMR(400MHz,D2O)δppm 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),3.99–2.8 9(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).

[0924] Example 39

[0925] Preparation of a conjugate of N-[4-(2-aminoethylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0926]

[0927] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid

[0928]

[0929] 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 of an aqueous solution 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 value of the reaction mixture was adjusted to 3-4 using 20% ​​aqueous citric acid solution, and the organic layer was washed with 10% aqueous citric acid solution, water and saturated saline 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 to 1:50) to provide the title compound (10.3 g, 24%). MS (m / z): C 21 H 27 [M+H]+ calculated for N7O4, 442.2; found, 442.3.

[0930] Step 2: Preparation of tert-butyl N-[2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]ethyl]carbamate

[0931]

[0932] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric 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 a saturated saline 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 to 1:50) to provide the title compound (1.58 g, yield: 90%). MS (m / z): C 28 H 41 [M+H]+ calculated for N9O5, 584.32; found, 584.2. 1 H NMR (400MHz, CDCl3) δppm 10.02–9.67(m,1H),8.29(d,J=8.9Hz,1H),7.72(dd,J=10.5,4.0Hz,1H),6.58(dd,J= 14.2,10.2Hz,2H),5.32(d,J=17.1Hz,1H),5.13(s,1H),4.20–3.70(m,2H),3.69–3.4 4(m,6H),3.39–3.14(m,7H),2.60–2.40(m,1H),2.26(dt,J=15.3,7.4Hz,2H),2.11–1 .83(m,3H),1.83–1.70(m,1H),1.41(d,J=28.2Hz,9H),1.09(dd,J=12.6,7.1Hz,3H).

[0933] Step 3: Preparation of N-[4-(2-aminoethylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0934]

[0935] Following step 2 in Example 1, tert-butyl N-[2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]ethyl]carbamate (1.18 g, 2 mmol) provided the title compound as a white solid (1.04 g, yield: 100%). MS (m / z): C 23 H 33 [M+H]+ calculated for N9O3, 484.27; found, 484.3.

[0936] Step 4: Preparation of a conjugate of N-[4-(2-aminoethylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0937]

[0938] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 145 mg, 0.36 mmol) and N-[4-(2-aminoethylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.36 mmol) provided the title compound (0.174 g, yield: 51.4%, DSR: 30%); 1 H NMR(400MHz,D2O)δppm 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).

[0939] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.186 g, yield: 54.9%, DSR: 39%).

[0940] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.189 g, yield: 55.8%, DSR: 30%).

[0941] Example 40

[0942] Preparation of a conjugate of N-[4-(4-aminobutylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0943]

[0944] Step 1: Preparation of tert-butyl N-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methylamino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]butyl]carbamate

[0945]

[0946] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 to 1:50) to provide the title compound (0.52 g, yield: 42.5%). MS (m / z): C 30 H 45 [M+H]+ calculated for N9O5, 612.35; found, 612.1. 1H NMR(400MHz,d-DMSO)δppm 9.62(s,1H),8.29(t,J=14.4Hz,1H),7.77(s,1H),7.61(d,J=22.9Hz,1H) ,6.77(d,J=28.6Hz,2H),4.86(s,1H),4.22–3.61(m,5H),3.43–3.33(m,3 H),3.27(s,3H),2.94(d,J=25.8Hz,2H),2.86(s,2H),2.37(s,1H),2.13( t,J=7.0Hz,2H),1.91–1.39(m,5H),1.33(s,12H),0.97(t,J=14.9Hz,3H).

[0947] Step 2: Preparation of N-[4-(4-aminobutylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0948]

[0949] Following step 2 in Example 1, tert-butyl N-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methylamino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]butyl]carbamate (0.5 g, 0.818 mmol) provided the title compound as a white solid (0.32 g, yield: 83%). MS (m / z): C 25 H 37 [M+H]+ calculated for N9O3, 512.30; found, 512.3.

[0950] Step 3: Preparation of a conjugate of N-[4-(4-aminobutylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0951]

[0952] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 147.2 mg, 0.365 mmol) and N-[4-(4-aminobutylamino)-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.365 mmol) provided the title compound (0.162 g, yield: 47.5%, DSR: 33%); 1 H NMR(400MHz,D2O)δppm 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).

[0953] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.243 g, yield: 71.3%, DSR: 18%).

[0954] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.168 g, yield: 49.3%, DSR: 20%).

[0955] Example 41

[0956] Preparation of a conjugate of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0957]

[0958] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid

[0959]

[0960] Following Step 1 in Example 1, tofacitinib (10 g, 32 mmol) and 4-aminobenzoic acid (8.7 g, 64 mmol) provided the title compound (9.5 g, yield: 62.5%). MS (m / z): C 24 H 25 [M+H]+ calculated for N7O4, 476.1; found, 476.3.

[0961] Step 2: Preparation of tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate

[0962]

[0963] 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 provide the title compound (0.89 g, yield: 69%). MS (m / z): C 33 H 43 [M+H]+ calculated for N9O5, 646.33; found, 646.1. 1 H NMR (400MHz, d-DMSO) δppm12.21(s,1H),8.42(s,2H),7.89(d,J=7.6Hz,2H),7.73(d,J=7.8Hz,3H),6.84(d,J=55.3Hz,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=5.2Hz,3H).

[0964] Step 3: Preparation of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0965]

[0966] Following step 2 in Example 1, tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.87 g, 1.35 mmol) provided the title compound as a white solid (1 g, yield: 83%). MS (m / z): C 28 H 35 [M+H]+ calculated for N9O3, 546.28; found, 546.3.

[0967] Step 4: Preparation of a conjugate of N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0968]

[0969] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 138.6 mg, 0.344 mmol) and N-[4-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.344 mmol) provided the title compound (0.26 g, yield: 93.9%, DSR: 23%); 1 H NMR(400MHz,D2O)δppm 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).

[0970] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.145 g, yield: 52.4%, DSR: 15%).

[0971] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.104 g, yield: 37.5%, DSR: 33%).

[0972] Example 42

[0973] Preparation of conjugates of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0974]

[0975] Step 1: Preparation of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid

[0976]

[0977] 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 of an aqueous solution 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 value of the reaction mixture was adjusted to 3-4 using acetic acid, and the organic layer was washed with 0.5 N hydrochloric acid solution, water and saturated saline 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 to 1:10) to provide the title compound (89.8 g, yield: 67.8%). MS (m / z): C 19 H 23 [M+H]+ calculated for N7O4, 414.1; found, 414.5.

[0978] Step 2: Preparation of tert-butyl N-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]butyl]carbamate

[0979]

[0980] 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 to 1:40) to provide the title compound (0.65 g, yield: 74%). MS (m / z): C 28 H 41 [M+H]+ calculated for N9O5, 584.32; found, 584.2. 1 H NMR(400MHz,d-DMSO)δppm 9.92(t,J=5.2Hz,1H),8.32(d,J=5.8Hz,1H),8.07(t,J=5.4Hz,1H),7.63(d,J=4.0 Hz,1H),6.79(d,J=26.3Hz,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=19.0Hz,2H),2.88(d,J=5.7Hz,2H),2.3 8(s,1H),1.72(dt,J=78.7,27.2Hz,3H),1.45–1.18(m,12H),1.00(d,J=7.1Hz,3H).

[0981] Step 3: Preparation of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0982]

[0983] Following step 2 in Example 1, tert-butyl N-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]butyl]carbamate (0.3 g, 0.51 mmol) provided the title compound as a white solid (0.26 g, yield: 98%). MS (m / z): C 23 H 33 [M+H]+ calculated for N9O3, 484.27; found, 484.3. 1 H NMR (400 MHz, d-DMSO) δ ppm 9.90(s,1H),8.34(d,J=6.7Hz,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=5.3Hz,2H),2.70(d,J=37.4Hz,2H),2.34(d,J=27.1Hz,2H),1.77(d,J=4 1.2Hz, 1H), 1.55–1.39 (m, 4H), 1.15 (t, J = 7.1Hz, 2H), 1.00 (d, J = 6.5Hz, 3H).

[0984] Step 4: Preparation of a conjugate of N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0985]

[0986] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 101 mg, 0.25 mmol) and N-[2-(4-aminobutylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (130 mg, 0.25 mmol) provided the title compound (0.102 g, yield: 45%, DSR: 40%); 1H NMR(400MHz,D2O)δppm 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),4.15–2.9 0(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).

[0987] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.092 g, yield: 40.6%, DSR: 26%).

[0988] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.143 g, yield: 63%, DSR: 21%).

[0989] Example 43

[0990] Preparation of conjugates of N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0991]

[0992] Step 1: Preparation of tert-butyl N-[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]ethyl]carbamate

[0993]

[0994] 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 to 1:60) to provide the title compound (1.1 g, yield: 66%). MS (m / z): C 26 H 37 [M+H]+ calculated for N9O5, 556.29; found, 556.2. 1 H NMR (400MHz, CDCl3) δppm 10.37–10.14(m,1H),8.31(d,J=6.3Hz,1H),7.76–7.54(m,1H),7.03(t,J=18.4Hz,1H),6.5 9(d,J=3.9Hz,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=10.8,5.5Hz,2H),2.50(ddd,J=18.5,12.3,6.0Hz, 1H),2.09–1.84(m,1H),1.83–1.72(m,1H),1.54–1.30(m,9H),1.09(dd,J=11.8,7.1Hz,3H).

[0995] Step 2: Preparation of N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[0996]

[0997] Following step 2 in Example 1, tert-butyl N-[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]ethyl]carbamate (0.89 g, 1.6 mmol) provided the title compound as a white solid (0.78 g, yield: 99%). MS (m / z): C 21 H 29 [M+H]+ calculated for N9O3, 456.23; found, 456.2.

[0998] Step 3: Preparation of a conjugate of N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[0999]

[1000] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 152.7 mg, 0.379 mmol) and N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.379 mmol) provided the title compound (0.165 g, yield: 47.7%, DSR: 40%); 1 H NMR(400MHz,D2O)δppm 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).

[1001] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.173 g, yield: 50%, DSR: 28%).

[1002] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.18 g, yield: 52%, DSR: 40%).

[1003] Example 44

[1004] Preparation of a conjugate of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1005]

[1006] Step 1: Preparation of tert-butyl N-[2-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]phenyl]amino]ethyl]carbamate

[1007]

[1008] By 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] acetic acid (1652mg, 4mmol), tert-butyl 4-aminophenethylcarbamate (1418mg, 6mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 1151mg, 6mmol) and 1-hydroxybenzotriazole (HOBt, 810mg, 6mmol) are dissolved in dichloromethane (40mL) and triethylamine (1.665mL, 12mmol) is added. The reaction mixture is stirred for 16 hours at room temperature. The solution is diluted with dichloromethane and washed with water and saturated saline solution. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:150 to 1:100) to provide the title compound (1.77 g, yield: 70%). MS (m / z): C 32 H 41 [M+H]+ calculated for N9O5, 632.32; found, 632.2. 1H NMR (400MHz, CDCl3) δppm10.59–10.19(m,1H),8.30(dd,J=17.1,7.1Hz,2H),7.67(dd,J=13.2,4.0Hz,1 H),7.49(t,J=11.3Hz,2H),7.13(d,J=8.2Hz,2H),6.61(t,J=4.7Hz,1H),5.13(s,1H),4.54(s,1H),4.3 8–4.23(m,2H),4.13–3.72(m,2H),3.68–3.44(m,4H),3.42–3.24(m,5H),2.75(t,J=6.8Hz,2H),2.50(d dd,J=19.1,11.9,5.6Hz,1H),2.08–1.83(m,1H),1.83–1.64(m,1H),1.43(s,9H),1.09(t,J=8.2Hz,3H).

[1009] Step 2: Preparation of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1010]

[1011] Following step 2 in Example 1, tert-butyl N-[2-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]ethyl]carbamate (1.48 g, 2.345 mmol) afforded the title compound as a white solid (1.3 g, yield: 97.7%). MS (m / z): C 27 H 33 [M+H]+ calculated for N9O3, 532.27; found, 532.2.

[1012] Step 3: Preparation of a conjugate of N-[2-[4-(2-aminoethyl)anilino]-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1013]

[1014] Following Step 3 in Example 1, sodium hyaluronate (MW 50 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-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.331 mmol) provided the title compound (0.136 g, yield: 41.5%, DSR: 20%); 1 H NMR(400MHz,D2O)δppm 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).

[1015] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.158 g, yield: 48.2%, DSR: 22%).

[1016] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.146 g, yield: 44.5%, DSR: 16%).

[1017] Example 45

[1018] Preparation of conjugate of HA with methyl (2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate

[1019]

[1020] Step 1: Preparation of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate

[1021]

[1022] By 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] acetic acid (1239mg, 3mmol), 6-amino-2-((tert-butoxycarbonyl)-amino) hexanoic acid (S)-methyl ester (1336mg, 4.5mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 863mg, 4.5mmol) and 1-hydroxybenzotriazole (HOBt, 608mg, 4.5mmol) are dissolved in dichloromethane (40mL) and triethylamine (2.1mL, 15mmol) is added. The reaction mixture is stirred for 16 hours at room temperature. The solution is diluted with dichloromethane and washed with water and saturated saline solution. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol / dichloromethane = 1:150 to 1:80) to provide the title compound (1.638 g, yield: 83.3%). MS (m / z): C 31 H 45 [M+H]+ calculated for N9O7, 656.34; found, 656.1. 1 H NMR (400MHz, CDCl3) δppm 10.36(dd,J=12.6,7.1Hz,1H),8.33(d,J=9.1Hz,1H),7.72(dd,J=12.9,4.0Hz,1H),6.72(d,J=7.8Hz,1H), 6.61(t,J=4.2Hz,1H),5.14(s,1H),4.63(dt,J=33.6,16.8Hz,2H),4.19(t,J=14.2Hz,2H),4.11–3.76(m,2 H),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=12.3,7.1Hz,3H).

[1023] Step 2: Preparation of (2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoic acid methyl ester hydrochloride

[1024]

[1025] Following Step 2 in Example 1, (2S)-methyl 6-(tert-butoxycarbonylamino)-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate (1.4 g, 2.137 mmol) provided the title compound as a white solid (1.26 g, 99.6% yield) which was used without further purification. MS (m / z): C 26 H 37 [M+H]+ calculated for N9O5, 556.29; found, 556.2.

[1026] Step 3: Preparation of a conjugate of methyl (2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate and HA

[1027]

[1028] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 128.2 mg, 0.318 mmol) and (2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoic acid methyl ester hydrochloride (200 mg, 0.318 mmol) provided the title compound (0.131 g, yield: 40.6%, DSR: 30%); 1 H NMR(400MHz,D2O)δppm 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=28.8Hz,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).

[1029] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.134 g, yield: 41.5%, DSR: 20%).

[1030] Example 46

[1031] Preparation of a conjugate of N-[4-[4-(2-aminoethyl)anilino]-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1032]

[1033] Step 1: Preparation of tert-butyl N-[2-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]phenyl]ethyl]carbamate

[1034]

[1035] 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric 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 a saturated saline 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 to 1:100) to provide the title compound (2.02 g, yield: 76.6%). MS (m / z): C 34 H 45 [M+H]+ calculated for N9O5, 660.35; found, 660.3. 1H NMR (400MHz, CDCl3) δppm10.00(dd,J=14.1,8.0Hz,1H),8.48(d,J=26.6Hz,1H),8.28(d,J=9.5Hz,1H),7.73(dd ,J=10.8,4.0Hz,1H),7.54(d,J=8.3Hz,2H),7.13(d,J=8.1Hz,2H),6.60(t,J=4.6Hz,1H),5.14(s,1H),4.56(s,1 H),4.12–3.71(m,2H),3.62(t,J=9.0Hz,3H),3.48(s,3H),3.35(d,J=8.6Hz,5H),2.76(t,J=6.8Hz,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=12.0,7.1Hz,3H).

[1036] Step 2: Preparation of N-[4-[4-(2-aminoethyl)anilino]-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1037]

[1038] Following step 2 in Example 1, tert-butyl N-[2-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyrylamino]phenyl]ethyl]carbamate (1.73 g, 2.625 mmol) provided the title compound as a white solid (1.56 g, yield: 99.8%). MS (m / z): C 29 H 37 [M+H]+ calculated for N9O3, 560.30; found, 560.1.

[1039] Step 3: Preparation of a conjugate of N-[4-[4-(2-aminoethyl)anilino]-4-oxo-butyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1040]

[1041] Following Step 3 in Example 1, sodium hyaluronate (MW 50 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-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg, 0.316 mmol) provided the title compound (0.154 g, yield: 47.8%, DSR: 33%); 1 H NMR(400MHz,D2O)δppm 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).

[1042] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 500 KDa) afforded the corresponding product (0.136 g, yield: 42.2%, DSR: 18%).

[1043] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.158 g, yield: 49.1%, DSR: 17%).

[1044] Example 47

[1045] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinylmethyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1046]

[1047] Step 1: Preparation of tert-butyl 4-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]piperazine-1-carboxylate

[1048]

[1049] 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 to 1:50) to provide the title compound (0.55 g, yield: 54.5%). MS (m / z): C 34 H 44 N 10 [M+H]+ calculated value for O5, 673.34; found value, 673.3. MS (m / z): C 34 H 44 N 10 [M+H]+ calculated for O5, 673.34; found, 673.3. 1 H NMR (400MHz, CDCl3) δppm10.45(dd,J=13.6,7.8Hz,1H),8.33(d,J=9.1Hz,1H),8.07(d,J=10.2Hz,1H),7. 69(dd,J=12.1,4.0Hz,1H),7.43(d,J=8.9Hz,2H),6.88(d,J=8.9Hz,2H),6.62(t,J=4.3Hz,1H),5.14(s,1H ),4.30(d,J=5.8Hz,2H),4.11–3.74(m,2H),3.68–3.43(m,8H),3.38(d,J=20.1Hz,3H),3.10(dd,J=28.1, 23.2Hz,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=8.5Hz,3H).

[1050] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1051]

[1052] Following Step 2 in Example 1, tert-butyl 4-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]piperazine-1-carboxylate (0.48 g, 0.714 mmol) provided the title compound as a white solid (0.43 g, yield: 99%). MS (m / z): C 29 H 36 N 10 O 31 Calcd. [M+H]+, 573.29; found, 573.2.

[1053] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinylmethyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1054]

[1055] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 99.54 mg, 0.247 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylanilino)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg, 0.247 mmol) provided the title compound (0.121 g, yield: 49%, DSR: 25%); 1 H NMR(400MHz,D2O)δppm 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).

[1056] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.107 g, yield: 43.6%, DSR: 35%).

[1057] Example 48

[1058] Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridinyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1059]

[1060] Step 1: Preparation of tert-butyl 4-[6-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]-3-pyridinyl]piperazine-1-carboxylate

[1061]

[1062] 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] acetic acid (619.5 mg, 1.5 mmol), tert-butyl 4-(6-aminopyridin-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 a saturated saline 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 to 1:50) to provide the title compound (0.4 g, yield: 39.6%). MS (m / z): C 33 H 43 N 11 [M+H]+ calculated for O5, 674.34; found, 674.3. 1H NMR (400MHz, CDCl3) δppm 10.54–10.40(m,1H),8.55(d,J=20.7Hz,1H),8.34(d,J=14.9Hz,1H),8.12(t,J=8.1Hz,1H),7.93(d,J =2.8Hz,1H),7.70(d,J=4.1Hz,1H),7.33–7.28(m,1H),6.62(d,J=3.7Hz,1H),5.15(s,1H),4.33(d,J=1 7.0Hz,2H),4.14–3.77(m,2H),3.73–3.45(m,8H),3.39(d,J=18.0Hz,3H),3.18–2.99(m,4H),2.52(dd, J=16.9,10.4Hz,1H),2.01–1.85(m,1H),1.83–1.69(m,1H),1.48(s,9H),1.10(dd,J=12.7,7.1Hz,3H).

[1063] Step 2: Preparation of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridinyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1064]

[1065] Following Step 2 in Example 1, tert-butyl 4-[6-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]-3-pyridinyl]piperazine-1-carboxylate (0.36 g, 0.535 mmol) provided the title compound as a white solid (0.32 g, yield: 98%). MS (m / z): C 28 H 35 N 11 [M+H]+ calculated for O3, 574.29; found, 574.3.

[1066] Step 3: Preparation of a conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridinyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1067]

[1068] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 99.14 mg, 0.246 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridinyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg, 0.246 mmol) provided the title compound (0.126 g, yield: 52.5%, DSR: 22%); 1 H NMR(400MHz,D2O)δppm 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).

[1069] In the case of step 3 in Example 1, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.116 g, yield: 48.3%, DSR: 26%).

[1070] Example 49

[1071] Preparation of a conjugate of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid and HA

[1072]

[1073] Step 1: Preparation of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (2,5-dioxopyrrolidin-1-yl) ester

[1074]

[1075] A solution of N, N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (20 mL) was slowly added to a stirred mixture of 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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) under 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 provide the title compound (1 g, yield: 98%), which was used without further purification. MS (m / z): C 23 H 26 [M+H]+ calculated for N8O6, 511.19; found, 511.1.

[1076] Step 2: Preparation of a conjugate of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid and HA

[1077]

[1078] Sodium hyaluronate is converted into TBA hyaluronic acid. Strong acid ion exchange resin Amberlite 732 is added in the sodium hyaluronate aqueous solution and at room temperature stirred mixture continues 8 hours.Solution is filtered, and subsequently with tetrabutylammonium hydroxide (TBA-OH) aqueous solution and filtrate.The gained aqueous solution is frozen immediately and lyophilized to hyaluronic TBA salt (HA-TBA).

[1079] Triethylamine (0.12 mL, 0.875 mmol) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL) at 0-15 ° C. Anhydrous DMSO (20 mL) containing 2-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] acetic acid (2,5-dioxopyrrolidin-1-yl) ester (255 mg, 0.5 mmol) was then added to the reaction mixture at 0-15 ° C. The reaction mixture was stirred at room temperature for 16 hours. 2.5 wt% sodium chloride solution (7 mL) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (250 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. Sodium hyaluronate (MW 10 kDa, MW 0.17 g, yield: 43.9%, DSR: 20%); 1 H NMR(400MHz,D2O)δppm 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).

[1080] In the case of this step, the reaction of TBA hyaluronic acid (MW 50 KDa) afforded the corresponding product (0.245 g, yield: 613.3%, DSR: 6%).

[1081] In the case of this step, the reaction of TBA hyaluronic acid (MW 500 KDa) afforded the corresponding product (0.274 g, yield: 70.8%, DSR: 23%).

[1082] In the case of this step, the reaction of TBA hyaluronic acid (MW 2000 KDa) afforded the corresponding product (0.237 g, yield: 61.2%, DSR: 24%).

[1083] Example 50

[1084] Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid and HA

[1085]

[1086] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid (2,5-dioxopyrrolidin-1-yl) ester

[1087]

[1088] A solution of N, N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (15 mL) was slowly added to a stirred mixture of 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid (882 mg, 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg, 2.4 mmol) in dichloromethane (25 mL) under 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 provide the title compound (1 g, yield: 93%), which was used without further purification. MS (m / z): C 25 H 30 [M+H]+ calculated for N8O6, 538.22; found, 538.1.

[1089] Step 2: Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butyric acid and HA

[1090]

[1091] Triethylamine (0.12 mL, 0.875 mmol) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL) at 0-15 ° C. Anhydrous DMSO (20 mL) containing 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] butyric acid (2,5-dioxopyrrolidin-1-yl) ester (269 mg, 0.5 mmol) was then added to the reaction mixture at 0-15 ° C. The reaction mixture was stirred at room temperature for 16 hours. 7 mL of 2.5 wt% sodium chloride solution was then added to the reaction mixture, the reaction mixture was stirred for 1 hour, and acetone (250 mL) was subsequently added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. Sodium hyaluronate MW 10 kDa, 0.166 g, yield: 41.3%, DSR: 10%; 1 H 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, 3.4.H), 1.11–0.86 (m, 0.3H). Sodium hyaluronate MW 50 kDa, 0.181 g, yield: 45.1%, DSR: 14%.

[1092] In the case of this step, the reaction of TBA hyaluronic acid (MW 500 KDa) afforded the corresponding product (0.263 g, yield: 65.5%, DSR: 5%).

[1093] In the case of this step, the reaction of TBA hyaluronic acid (MW 2000 KDa) afforded the corresponding product (0.17 g, yield: 42.3%, DSR: 7%).

[1094] Example 51

[1095] Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid and HA

[1096]

[1097] Step 1: Preparation of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (2,5-dioxopyrrolidin-1-yl) ester

[1098]

[1099] A solution of N, N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (20 mL) was slowly added to a stirred mixture of 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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) under 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 provide the title compound (1.1 g, yield: 96%), which was used without further purification. MS (m / z): C 28 H 28 [M+H]+ calculated for N8O6, 573.21; found, 573.1.

[1100] Step 2: Preparation of a conjugate of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid and HA

[1101]

[1102] Triethylamine (0.12 mL, 0.875 mmol) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL) at 0-15 ° C. Anhydrous DMSO (20 mL) containing 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (2,5-dioxopyrrolidin-1-yl) ester (286 mg, 0.5 mmol) was then added to the reaction mixture at 0-15 ° C. The reaction mixture was stirred at room temperature for 16 hours. 7 mL of 2.5 wt% sodium chloride solution was then added to the reaction mixture, the reaction mixture was stirred for 1 hour, and acetone (250 mL) was subsequently added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V = 3:1). Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. Sodium hyaluronate MW 10 kDa, 0.177: 42.3%, DSR: 10%; 1 H 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 50 kDa, 0.2 g, yield: 47.8%, DSR: 6%.

[1103] In the case of this step, the reaction of TBA hyaluronic acid (MW 500 KDa) afforded the corresponding product (0.187 g, yield: 44.7%, DSR: 5%).

[1104] In the case of this step, the reaction of TBA hyaluronic acid (MW 2000 KDa) afforded the corresponding product (0.1 g, yield: 23.9%, DSR: 2%).

[1105] Example 52

[1106] Preparation of a conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(4-aminophenyl) ester and HA

[1107]

[1108] Step 1: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(4-((tert-butoxycarbonyl)amino)phenyl)

[1109]

[1110] Thiophosgene (1.21 g, 10.5 mmol, 1.1 equivalents) was added to a mixture of tert-butyl (4-hydroxyphenyl)carbamate (2 g, 9.56 mmol, 1 equivalent) and N, N-dimethylpyridin-4-amine (2.92 g, 23.9 mmol, 2.5 equivalents) in dichloromethane (40 mL, 20 V) under N2, and the reaction mixture was stirred at room temperature for 0.5 hours. Tofacitinib (2.99 g, 9.56 mmol, 1 equivalent) was then added thereto, and the resulting mixture was stirred for another 12 hours at room temperature. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give the title product (1.827 g, yield: 34%); MS (m / z): C 28 H 33 [M+H]+ calculated for N7O4S, 564.23; found, 564.1.

[1111] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(4-aminophenyl) ester hydrochloride

[1112]

[1113] Following Step 2 in Example 1, O-(4-((tert-butoxycarbonyl)amino)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate (1.5 g, 2.661 mmol, 1 equiv) gave the desired product as an HCl salt (1.33 g, yield: 100%); MS (m / z): C 23 H 25 [M+H]+ calculated for N7O2S, 64.18; found, 464.1. 1H-NMR(400MHz,D2O)δppm 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=14.8,6.8Hz,3H).

[1114] Step 3: Preparation of a conjugate of HA with 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(4-aminophenyl) ester

[1115]

[1116] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.161 g, 0.4 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(4-aminophenyl) ester hydrochloride (0.2 g, 0.4 mmol, 1 equiv) provided the title compound (0.15 g, yield: 45%, DSR = 14.3%); 1 H-NMR(400MHz,D2O)δppm 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).

[1117] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.175 g, yield: 52%, DSR = 4.6%).

[1118] Example 53

[1119] Preparation of a conjugate of HA with 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(piperidin-4-yl) ester

[1120]

[1121] Step 1: Preparation of tert-butyl 4-((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)oxy)piperidine-1-carboxylate

[1122]

[1123] Thiophosgene (1.26 g, 10.934 mmol, 1.1 equivalents) was added to a mixture of tert-butyl 4-hydroxypiperidine-1-carboxylate (2 g, 9.94 mmol, 1 equivalent) and N, N-dimethylpyridin-4-amine (3.03 g, 9.94 mmol, 2.5 equivalents) in dichloromethane (40 mL, 20 V) under N2, and the reaction mixture was stirred at room temperature for 0.5 hours. Tofacitinib (3.1 g, 9.94 mmol, 1 equivalent) was then added thereto, and the resulting mixture was stirred for another 12 hours at room temperature. After most of the tofacitinib was 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 for N7O4S, 556.26; found, 556.2.

[1124] Step 2: Preparation of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(piperidin-4-yl) ester hydrochloride

[1125]

[1126] Following Step 2 in Example 1, tert-butyl 4-((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)oxy)piperidine-1-carboxylate (1 g, 1.8 mmol, 1 equiv) gave the desired product as an HCl salt (0.885 g, yield: 100%); MS (m / z): C 22 H 29 [M+H]+ calculated for N7O2S, 456.21; found, 456.1. 1H-NMR(400MHz,D2O)δppm 8.49(d,J=9.7Hz,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=17.1,7.1Hz,3H).

[1127] Step 3: Preparation of a conjugate of HA with 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(piperidin-4-yl) ester

[1128]

[1129] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.164 g, 0.406 mmol, 1 equiv) and 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioate O-(piperidin-4-yl)ester hydrochloride (0.2 g, 0.406 mmol, 1 equiv) provided the title compound (0.17 g, yield: 34%, DSR = 12.6%); 1 H-NMR(400MHz,D2O)δppmδ8.25-7.0(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).

[1130] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.192 g, yield: 56.6%, DSR = 4.6%).

[1131] Example 54

[1132] Preparation of a conjugate of HA with 4-aminobutyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)carbonate

[1133]

[1134] Step 1: Preparation of 3-((3R,4R)-4-methyl-3-(methyl(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile

[1135]

[1136] Ethyl DIP acetate (3.745 g, 28.81 mmol, 1 eq) was added to a mixture of tofacitinib (9 g, 28.81 mmol, 1 eq) in dichloromethane (180 mL, 20 V) under N2, and the reaction mixture was stirred at room temperature for 0.5 hours. (2-(chloromethoxy)ethyl)trimethylsilane (4.8 g, 28.81 mmol, 1 eq) was then added thereto, and the resulting mixture was stirred at room temperature overnight. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give the title product (9 g, yield: 71%); MS (m / z): C 22 H 34 [M+H]+ calculated for N6O2Si, 443.25; found, 443.2.

[1137] Step 2: Preparation of 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropionitrile

[1138]

[1139] TFA (6.44 g, 56.5 mmol, 5 eq) was added dropwise to a mixture of 3-((3R,4R)-4-methyl-3-(methyl(7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropionitrile (5 g, 11.3 mmol, 1 eq) in dichloromethane (100 mL, 20 V) at 0°C under N2. The resulting reaction mixture was stirred at 0°C for 30 minutes, allowed to warm to room temperature, and then stirred at this temperature for 24 hours. After most of the SM was consumed, NaHCO3 (saturated aqueous solution) was added to the above 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 NaCl (saturated aqueous solution), dried over Na 2 SO 4 and then concentrated to give the title product (3.5 g, yield: 90%); MS (m / z): C 17 H 22 [M+H]+ calculated for N6O2, 343.18; found, 343.1.

[1140] Step 3: Preparation of tert-butyl (4-((((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methoxy)carbonyl)oxy)butyl)carbamate

[1141]

[1142] Following Step 1 in Example 1, 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropionitrile (1 g, 2.92 mmol, 1 eq) and tert-butyl (4-hydroxybutyl)carbamate (1 g, 2.92 mmol, 1 eq) gave the title product (0.85 g, 52% yield); MS (m / z): C 27 H 39 [M+H]+ calculated for N7O6, 558.30; found, 558.2.

[1143] Step 4: Preparation of 4-aminobutyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)carbonate hydrochloride

[1144]

[1145] Following Step 2 in Example 1, tert-butyl (4-((((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methoxy)carbonyl)oxy)butyl)carbamate (0.85 g, 1.524 mmol, 1 equiv) gave the desired product as an HCl salt (0.753 mg, yield: 100%); MS (m / z): C 22 H 31 [M+H]+ calculated for N7O4, 458.24; found, 458.1. 1 H-NMR(400MHz,CD3OD)δppm 8.41(s,1H),7.64(d,J=3.7Hz,1H),7.01(d,J=3.4Hz,1H),6.28(s,2H),4.69(s,1H),4.21-3.40( m,11H),2.95(t,J=6.8Hz,1H),2.56(br,1H),2.05(br,1H),1.87-1.66(m,5H),1.23-1.14(m,3H).

[1146] Step 5: Preparation of a conjugate of HA with 4-aminobutyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)carbonate

[1147]

[1148] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.161 g, 0.4 mmol, 1 equiv) and 4-aminobutyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)carbonate hydrochloride (0.2 g, 0.4 mmol, 1 equiv) afforded the title compound (0.18 g, yield: 56%, DSR = 21%); 1H-NMR(400MHz,D2O)δppm 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=27.3Hz,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=6.9Hz,0.21H),1.05-0.75(m,0.7H).

[1149] In the case of this step, the reaction of TBA hyaluronic acid (MW 2000 KDa) afforded the corresponding product (0.172 g, yield: 47.6%, DSR = 14%).

[1150] Example 55

[1151] Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl(4-aminobutyl)carbamate and HA

[1152]

[1153] Step 1: Preparation of tert-butyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)butane-1,4-diyldicarbamate

[1154]

[1155] Following Step 1 in Example 1, 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropionitrile (0.6 g, 1.75 mmol, 1 eq) and bis(4-nitrophenyl)carbonate (0.587 g, 1.928 mmol, 1.1 eq) and tert-butyl (4-aminobutyl)carbamate (0.33 g, 1.75 mmol, 1 eq) gave the title product (0.878 g, 90% yield); m / z (ESI): 557.31 [M+H] + 。MS(m / z):C 27 H 40 [M+H]+ calculated for N8O5, 557.31; found, 557.2.

[1156] Step 2: Preparation of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl(4-aminobutyl)carbamate hydrochloride

[1157]

[1158] Following Step 2 in Example 1, tert-butyl ((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)butane-1,4-diyldicarbamate (0.8 g, 1.44 mmol, 1 equiv) gave the desired product as an HCl salt (0.7 g, yield: 100%); MS (m / z): C 22 H 32 [M+H]+ calculated for N8O3, 457.25; found, 457.2. 1 H-NMR(400MHz,D2O)δppm 8.33(t,J=6.7Hz,1H),7.52(dd,J=17.0,3.7Hz,1H),6.88(d,J=9.3Hz,1H),6.17(d,J=24.8Hz,2H),4.58(s,1H),4.10-3.81(m,3H),3.6 8-3.16(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=14.9,7.0Hz,3H).

[1159] Step 3: Preparation of a conjugate of (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl(4-aminobutyl)carbamate and HA

[1160]

[1161] Following Step 3 in Example 1, sodium hyaluronate (MW 50 KDa, 0.161 g, 0.4 mmol, 1 equiv) and 4-aminobutyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl(4-aminobutyl)carbamate hydrochloride (0.2 g, 0.4 mmol, 1 equiv) afforded the title compound (0.17 g, yield: 50%, DSR = 6%);1 H-NMR(400MHz,D2O)δppm 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),3.9 3-3.11(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).

[1162] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.150 g, yield: 44.9%, DSR = 8%).

[1163] Example 56

[1164] Preparation of conjugates of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1165]

[1166] Step 1: Preparation of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide

[1167]

[1168] Triethylamine (0.558mL, 8mmol) is added to a stirred mixture of tofacitinib (1.245g, 4mmol) in toluene (50mL). The reaction mixture is heated to 50 ° C. Then 2-chloroethyl isocyanate (1688mg, 16mmol) is added to the reaction mixture. The reaction mixture is stirred at room temperature for 4 hours. The reaction mixture is then filtered and then concentrated under reduced pressure. The crude residue is purified by column chromatography (methanol / dichloromethane=1: 100~1: 20) to provide the title compound (1.2g, 71.9%). MS (m / z): C 19 H 24 [M+H]+ calculated for ClN7O2, 418.16; found, 418.1.

[1169] Step 2: Preparation of a conjugate of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

[1170]

[1171] A solution of N-(2-chloroethyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide (209 mg, 0.5 mmol) in anhydrous DMSO (2 mL) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, 331 mg, 0.5 mmol, sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL). The reaction mixture was stirred at room temperature for 7 days and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 40 mL of deionized water. Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. Sodium hyaluronate MW 50 kDa, 0.177 g, yield: 42.3%, DSR: 10%; 1 H NMR(400MHz,d-DMSO)δppm 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).

[1172] Example 57

[1173] Preparation of the conjugate of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[1174]

[1175] Step 1: Preparation of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate

[1176]

[1177] 2-Chloroethyl chloroformate (0.1mL, 1mmol) is added to a stirred mixture of tofacitinib (156mg, 0.5mmol) and K2CO3 (138mg, 1mmol) in anhydrous DMF (2mL). The reaction mixture is stirred at room temperature for 30 minutes. The solution is diluted with dichloromethane and washed with water and a saturated saline solution. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue is purified by column chromatography (methanol / dichloromethane=1:100~1:50) to provide the title compound (0.08g, 40%). MS (m / z): C 19 H 23 [M+H]+ calculated for ClN6O3, 419.15; found, 419.3.

[1178] Step 2: Preparation of a conjugate of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

[1179]

[1180] A solution of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (209 mg, 0.5 mmol) in anhydrous DMSO (2 mL) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, MW 500 kDa; 331 mg, 0.5 mmol, sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL). The reaction mixture was stirred at room temperature for 7 days and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 40 mL of deionized water. Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. 0.28 g, yield: 73.5%, DSR: 25%; 1 HNMR(400MHz,D2O)δppm 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).

[1181] In the case of this step, the reaction of HA-TBA (sodium hyaluronate MW 2000 KDa) afforded the corresponding product (0.18 g, yield: 47.2%, DSR = 20%). 1 H-NMR

[1182] Example 58

[1183] Preparation of a conjugate of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidinyl]-3-oxo-propionitrile and HA

[1184]

[1185] Step 1: Preparation of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidinyl]-3-oxo-propionitrile

[1186]

[1187] 2-Chloromethoxyethyl chloride (142 mg, 1.1 mmol) was added to a stirred mixture of tofacitinib (156 mg, 0.5 mmol) and K2CO3 (138 mg, 1 mmol) in anhydrous DMF (2 mL). The reaction mixture was stirred at room temperature for 1 hour. The solution was diluted with dichloromethane and washed with water and a saturated saline 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 provide the title compound (0.11 g, 55%). MS (m / z): C 19 H 25 [M+H]+ calculated for ClN6O2, 405.17; found, 405.3.

[1188] Step 2: Preparation of a conjugate of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidinyl]-3-oxo-propionitrile and HA

[1189]

[1190] A solution of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidinyl]-3-oxo-propionitrile (202 mg, 0.5 mmol) in anhydrous DMSO (2 mL) was slowly added to a stirred mixture of the TBA salt of hyaluronic acid (HA-TBA, 331 mg, 0.5 mmol, sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL). The reaction mixture was stirred at room temperature for 4 days and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 40 mL of deionized water. Extensive dialysis of the solution against deionized water (MW cutoff 3.5 kDa) and lyophilization provided the title compound. 0.28 g, yield: 74.9%, DSR: 10%; 1 HNMR(400MHz,d-DMSO)δppm 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).

[1191] In the case of this step, the reaction of HA-TBA (sodium hyaluronate MW 2000 KDa) afforded the corresponding product (0.175 g, yield: 46.8%, DSR = 35%).

[1192] Example 59

[1193] Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]-hexanoic acid methyl ester

[1194]

[1195] Step 1: Preparation of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate

[1196]

[1197] 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol), (S)-methyl 6-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 a saturated saline 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 / DCM=1:150-1:80) to provide the title compound (1.1 g, yield: 76.7%). MS (m / z): C 36 H 47 [M+H]+ calculated for N9O7, 718.35; found, 718.3. 1 H NMR (400MHz, CDCl3) δppm12.29(d,J=30.5Hz,1H),8.39(d,J=8.5Hz,1H),7.87(d,J=8.6Hz,2H),7.78(dd,J=10.8,6.4Hz, 3H),6.85–6.70(m,1H),6.66(d,J=3.7Hz,1H),5.11(d,J=23.2Hz,1H),4.89–4.74(m,1H),4.62(s,1H),4.09(dd,J=13.0,3 .8Hz,1H),3.95–3.70(m,4H),3.66–3.45(m,4H),3.39(d,J=14.1Hz,3H),3.13(d,J=6.0Hz,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=19.4Hz,11H),1.27(d,J=12.0Hz,1H),1.10(t,J=7.0Hz,3H).

[1198] Step 2: Preparation of (2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoic acid methyl ester hydrochloride

[1199]

[1200] Ethyl acetate (commercially available) (4 mL) containing 4M HCl was slowly added to a stirred solution of (2S) -6- (tert-butoxycarbonylamino) -2- [[4- [[4- [[(3R, 4R) -1- (2-cyanoacetyl) -4-methyl -3-piperidinyl] -methyl-amino] pyrrolo [2,3-d] pyrimidine -7- carbonyl] amino] benzoyl] amino] hexanoic acid methyl ester (1 g, 1.39 mmol) in EtOAc (20 mL) under 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 EtOAc and concentrated under reduced pressure to provide the title compound (0.9 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 31 H 39 [M+H]+ calculated for N9O5, 618.30; found, 618.1.

[1201] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]-hexanoic acid methyl ester

[1202]

[1203] 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, followed by dropwise addition of 16 mL of acetonitrile while stirring. 4-Methylmorpholine (NMM, 59 mg, 0.58 mmol) was added to the solution to temporarily increase 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 stirred at room temperature for 1 hour. The solution was mixed with (2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoic acid methyl ester hydrochloride (200 mg, 0.29 mmol) and stirred at room temperature for 72 hours. NaCl (170 mg, 2.9 mmol) was then added to the reaction mixture, which was stirred for 1 hour and then acetone (290 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Extensive dialysis of the solution against deionized water (Mw cutoff value was 3.5 kDa) and lyophilization provided the title compound. 0.119 g, yield: 41.9%, DSR: 20%; 1 H NMR(400MHz,D2O)δppm 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).

[1204] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.119 g, yield: 41.9%, DSR = 21%).

[1205] Example 60

[1206] Preparation of a conjugate between HA (sodium hyaluronate) and N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide

[1207]

[1208] Step 1: Preparation of 3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid

[1209]

[1210] Triethylamine (1.12mL, 8mmol) is added to a stirred mixture of tofacitinib (1248mg, 4mmol) and bis(4-nitrophenyl) carbonate (1459mg, 4.8mmol) in DCM (40mL). The reaction mixture is heated to 45 ° C and stirred at this temperature for 5 hours. The reaction mixture is then cooled to room temperature. 3-aminobenzoic acid (658mg, 4.8mmol) is added. The reaction mixture is stirred at 45 ° C for 16 hours. The reaction mixture is filtered. The filtrate is washed with water. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue is purified by column chromatography (DCM: methanol = 20: 1) to provide the title compound (1.8g, yield: 94.7%). MS (m / z): C 24 H 25 [M+H]+ calculated for N7O4, 476.19; found, 476.1.

[1211] Step 2: Preparation of tert-butyl N-[4-[[3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate

[1212]

[1213] 3-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 / DCM=1:150-1:80) to provide the title compound (1 g, yield: 62%). MS (m / z): C 33 H 43 [M+H]+ calculated for N9O5, 646.33; found, 646.3.

[1214] Step 3: Preparation of N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1215]

[1216] Ethyl acetate (commercially available) (2.4 mL) containing 4M HCl was slowly added to a stirred solution of tert-butyl N-[4-[[3-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.6 g, 0.93 mmol) in EtOAc (12 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to provide the title compound (0.54 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 28 H 35 [M+H]+ calculated for N9O3, 546.28; found, 546.3.

[1217] 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-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide

[1218]

[1219] 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, followed by the addition of 24.2 mL of acetonitrile dropwise with stirring. N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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, causing a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 128 mg, 0.462 mmol) was added to the solution and stirred at room temperature for 72 hours. NaCl (270 mg, 4.62 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (250 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Extensive dialysis (Mw cutoff value 3.5 kDa) and lyophilization of the solution against deionized water provided the title compound. 0.209 g, yield: 49.9%, DSR: 22%; 1 H NMR(400MHz,D2O)δppm 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).

[1220] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.214 g, yield: 51.1%, DSR = 18%).

[1221] Example 61

[1222] Preparation of a conjugate between HA (sodium hyaluronate) and N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide

[1223]

[1224] Step 1: Preparation of tert-butyl N-[2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]ethyl]carbamate

[1225]

[1226] 4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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 a saturated saline 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 / DCM=1:150-1:80) to provide the title compound (1.23 g, yield: 66.4%). MS (m / z): C 31 H 39 [M+H]+ calculated for N9O5, 618.30; found, 618.2. 1 H NMR (400MHz, CDCl3) δppm 12.26(d,J=23.3Hz,1H),8.38(d,J=9.3Hz,1H),7.81(dd,J=39.5,8.6Hz,5H),7.24(s,1H),6.65(d,J=4.0Hz,1H),5.11(d,J=3 3.0Hz,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=8.6Hz,3H).

[1227] Step 2: Preparation of N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride

[1228]

[1229] Ethyl acetate (commercially available) (2.4 mL) containing 4M HCl was slowly added to a stirred solution of tert-butyl N-[2-[[4-[[4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]ethyl]carbamate (0.6 g, 0.97 mmol) in EtOAc (6 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to provide the title compound (0.5 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 26 H 31 [M+H]+ calculated for N9O3, 518.25; found, 518.2.

[1230] 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-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide

[1231]

[1232] 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, followed by the addition of 27 mL of acetonitrile dropwise with stirring. N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-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, causing a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 142.8 mg, 0.516 mmol) was added to the solution and stirred at room temperature for 72 hours. NaCl (303 mg, 5.16 mmol) was then added to the reaction mixture, which was stirred for 1 hour and then acetone (250 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). Extensive dialysis of the solution against deionized water (Mw cutoff value was 3.5 kDa) and lyophilization provided the title compound. 0.25 g, yield: 55.1%, DSR: 22%; 1 H 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). In this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.24 g, yield: 52.9%, DSR = 26%).

[1233] Example 62

[1234] Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1235]

[1236] 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

[1237]

[1238] Triethylamine (1.25 mL, 9 mmol) was added to a stirred mixture of trans-N-methyl-1-[4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-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)-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 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 provide the title compound (0.66 g, yield: 35.3%). MS(m / z):C 28 H 45 [M+H]+ calculated for N7O7S, 624.31; found, 624.2. 1 H NMR (400MHz, CDCl3) δppm 10.38(d,J=7.6Hz,1H),8.36(s,1H),7.67(d,J=4.1Hz,1H),6.59(d,J=4.0Hz,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=15.2,5.9Hz,2H),2.86(d,J=5.3Hz,3H),2.21(d,J=11.6Hz,2H),2. 07–1.87(m,5H),1.71(dd,J=23.8,11.5Hz,2H),1.59–1.48(m,4H),1.47–1.32(m,11H).

[1239] 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

[1240]

[1241] Ethyl acetate (commercially available) (2.4 mL) containing 4M HCl was slowly added to a stirred solution of (2S)-6-(tert-butoxycarbonylamino)-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester (0.61 g, 0.97 mmol) in EtOAc (12 mL) under 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 EtOAc and concentrated under reduced pressure to provide the title compound (0.54 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 23 H 37 [M+H]+ calculated for N7O5, 524.25; found, 524.2.

[1242] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1243]

[1244] Sodium hyaluronate (101.6 mg, 0.252 mmol carboxylic acid, MW 50 kDa) is dissolved in 21 mL of deionized water in a 100 mL round-bottom flask, and 13.2 mL of acetonitrile is subsequently added dropwise while stirring. 4-Methylmorpholine (NMM, 51 mg, 0.504 mmol) is added to the solution, thereby temporarily increasing viscosity. The solution is then cooled to 0 ° C., and 2-chloro-4,6-dimethoxy-1,3,5-triazine (44.3 mg, 0.252 mmol) is added and stirred at room temperature for 1 hour. The solution is mixed with (2S)-6-amino-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester 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, the reaction mixture was stirred for 1 hour and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter 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, the reaction mixture was stirred for 1 hour and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.118 g, yield: 52.9%, DSR: 16%; 1 H NMR(400MHz,D2O)δppm 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).

[1245] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.105 g, yield: 47.1%, DSR = 20%).

[1246] Example 63

[1247] Preparation of conjugate between HA (sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide

[1248]

[1249] Step 1: Preparation of tert-butyl N-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate

[1250]

[1251] A mixture of trans-N-methyl-1-[4-[methyl(7H-pyrrolo[2,3-d]pyrimidin-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 for 4 hours at room temperature. Then tert-butyl hydrazinecarboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filter cake was washed with a NaHCO3 solution and water. The filter cake was then dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure to provide the title compound (1.67 g, yield: 84.3%). MS (m / z): C 21 H 33 [M+H]+ calculated for N7O5S, 496.22; found, 496.1. 1 H NMR (400MHz, CDCl3) δppm11.38(s,1H),8.30(s,1H),7.63(d,J=4.1Hz,1H),6.56( dd,J=25.8,3.5Hz,2H),4.75(s,1H),4.36(q,J=5.1Hz,1H),3.22(d,J=11.1Hz,3H) ,2.97(d,J=6.3Hz,2H),2.83(d,J=5.3Hz,3H),2.20(t,J=14.4Hz,2H),2.04–1.94( m,1H),1.87(d,J=10.8Hz,2H),1.74–1.62(m,2H),1.51(s,9H),1.43–1.32(m,2H).

[1252] Step 2: Preparation of 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride

[1253]

[1254] Ethyl acetate (commercially available) (2.4 mL) containing 4M HCl was slowly added 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 EtOAc (12 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to provide the title compound (0.52 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 16 H 25 [M+H]+ calculated for N7O3, 396.17; found, 396.1.

[1255] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide

[1256]

[1257] Sodium hyaluronate (246mg, 0.61mmol formic acid, MW 50kDa) is dissolved in the deionized water of 49.2mL in a 100mL round-bottom flask, and 32mL of acetonitrile is subsequently added dropwise when stirring.4-methylmorpholine (NMM, 43mg, 0.427mmol) is added to the solution, thereby temporarily increasing viscosity.At room temperature 1-[4-[[7-(hydrazine carbonyl) pyrrolo[2,3-d] pyrimidin-4-yl]-methyl-amino] cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride (200mg, 0.427mmol) and 4-methylmorpholine (NMM, 43mg, 0.427mmol) are added to the solution, thereby temporarily increasing viscosity.Reactant mixture is stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 169 mg, 0.61 mmol) was added to the solution and stirred at room temperature for 72 hours. NaCl (357 mg, 6.1 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (400 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). NaCl (357 mg, 6.1 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.29 g, yield: 62.8%, DSR: 23%; 1 H NMR(400MHz,D2O)δppm8.26–8.09(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).

[1258] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.305 g, yield: 66.1%, DSR = 31%).

[1259] Example 64

[1260] Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1261]

[1262] Step 1: Preparation of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate

[1263]

[1264] Triethylamine (1.25mL, 9mmol) is added to a stirred mixture of 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]azetidin-3-yl]acetonitrile (1114mg, 3mmol) and bis(4-nitrophenyl) carbonate (1094mg, 3.6mmol) in DCM (40mL). The reaction mixture is heated to 45°C and stirred at this temperature for 5 hours. The reaction mixture is then cooled to room temperature. (S)-methyl 6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1069mg, 3.6mmol) is added. The reaction mixture is stirred at 45°C for 16 hours. The solution is diluted with DCM and washed with water. The organic layer is dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=100:1) to provide the title compound (1.3 g, yield: 65.9%). MS (m / z): C 29 H 39 [M+H]+ calculated for N9O7S, 658.26; found, 658.1. 1 H NMR (400MHz, CDCl3) δppm9.93 (d, J = 7.6Hz, 1H), 8.95 (s, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 8.04 (d,J=4.0Hz,1H),6.85(d,J=4.1Hz,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=9.6Hz,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).

[1265] Step 2: Preparation of (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester hydrochloride

[1266]

[1267] Ethyl acetate (commercially available) (4.92 mL) containing 4M HCl was slowly added to a stirred solution of (2S) -6- (tert-butoxycarbonylamino) -2- [ [4- [1- [3- (cyanomethyl) -1- ethylsulfonyl -azetidin-3-yl] pyrazol-4-yl] pyrrolo [2,3-d] pyrimidine -7- carbonyl] amino] hexanoic acid methyl ester (1.23 g, 1.87 mmol) in EtOAc (25 mL) under 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 EtOAc and concentrated under reduced pressure to provide the title compound (1.1 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 24 H 31 [M+H]+ calculated for N9O5S, 558.21; found, 558.1.

[1268] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1269]

[1270] 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, followed by the addition of 23 mL of acetonitrile dropwise while stirring. (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester 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 viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 120 mg, 0.434 mmol) was added to the solution and stirred at room temperature for 72 hours. NaCl (254 mg, 4.34 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (300 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). NaCl (254 mg, 4.34 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.2 g, yield: 50.1%, DSR: 20%; 1 H NMR(400MHz,D2O)δppm 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).

[1271] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.154 g, yield: 38.6%, DSR = 30%).

[1272] Example 65

[1273] Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

[1274]

[1275] Step 1: Preparation of tert-butyl N-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate

[1276]

[1277] A mixture of 2-[1-ethylsulfonyl-3-[4(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]azetidin-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. Tert-butyl hydrazinecarboxylate (634.4 mg, 4.8 mmol) was then 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 filter cake was washed with a NaHCO3 solution and water. The filter cake was then 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 provide the title compound (0.758 g, yield: 35.8%). MS (m / z): C 22 H 27 [M+H]+ calculated for N9O5S, 530.18; found, 530.1. 1 HNMR(400MHz,CDCl3)δppm 10.93(d,J=1.8Hz,1H),8.87(s,1H),8.48(s,1H),8.32(s,1H),7.95(dd,J=28.4,4.1Hz,1H),6.82(t,J=17.0Hz,1H),6.70(s ,1H),4.64(d,J=9.3Hz,2H),4.23(t,J=23.2Hz,2H),3.42(s,2H),3.09(q,J=7.4Hz,2H),1.52(s,9H),1.42(t,J=7.4Hz,3H).

[1278] Step 2: Preparation of 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide trifluoroacetate

[1279]

[1280] Trifluoroacetic acid (2.4 mL) was slowly added to a stirred mixture of tert-butyl N-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate (0.6 g, 1.13 mmol) in DCM (9.6 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure to provide the title compound (0.61 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 17 H 19 [M+H]+ calculated for N9O3, 430.13; found, 430.0.

[1281] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

[1282]

[1283] Sodium hyaluronate (212mg, 0.526mmol carboxylic acid, MW 50kDa) is dissolved in the deionized water of 42.4mL in 100mL round-bottom flask, then 28mL acetonitrile is added dropwise when stirring.At room temperature by 4- [1- [3- (cyanomethyl) -1- ethylsulfonyl -azetidin -3- bases] pyrazole -4- bases] pyrrolo [2,3-d] pyrimidine -7- carbohydrazide hydrochloride (200mg, 0.368mmol) and 4- methylmorpholine (NMM, 37mg, 0.368mmol) are added to solution, so that viscosity is temporarily increased.Reactant mixture is stirred at this temperature for 2 hours.4- (4,6- dimethoxy -1,3,5- triazine -2- bases) -4- methylmorpholinium chloride (DMTMM, 146mg, 0.526mmol) is added to solution and stirred at room temperature for 72 hours. Then NaCl (308 mg, 5.26 mmol) was added to the reaction mixture, the reaction mixture was stirred for 1 hour and then acetone (300 mL) was added dropwise while stirring. The mixture was filtered. The filter 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, the reaction mixture was stirred for 1 hour and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.2 g, yield: 48.1%, DSR: 40%; 1H NMR(400MHz,D2O)δppm 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=34.6Hz,3.6H),3.46(dd,J=86.0,63.7Hz,11.6H),1.93(s,3H),1.26(d,J=43.7Hz,1.2H).

[1284] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.154 g, yield: 37%, DSR = 40%).

[1285] Example 66

[1286] Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1287]

[1288] Step 1: Preparation of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate

[1289]

[1290] A mixture of (3R)-3-cyclopentyl-3-[4(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propionitrile (1486 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1141 mg, 3.754 mmol) in DMSO (12 mL) was stirred for 7 hours at room temperature. (S)-methyl 6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (1448 mg, 4.88 mmol) was added. The reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was poured into a saturated Na2CO3 solution. The mixture was filtered. The filter cake was washed with a NaHCO3 solution and water. The filter cake was then 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 provide the title compound (0.828 g, yield: 34.9%). MS(m / z):C 30 H 40[M+H]+ calculated for N8O5, 593.31; found, 593.2. 1 H NMR (400MHz, CDCl3) δppm 9.95(d,J=7.5Hz,1H),8.91(s,1H),8.32(d,J=11.9Hz,2H),7.99(d,J=4.0Hz,1H),6.82 (d,J=4.0Hz,1H),4.76(dd,J=12.8,7.5Hz,1H),4.60(s,1H),4.28(td,J=9.9,3.8Hz,1H ),3.81(s,3H),3.22–3.05(m,3H),2.97(dd,J=17.0,3.8Hz,1H),2.61(dt,J=16.5,8.4H z,1H),2.13–1.88(m,3H),1.65–1.50(m,7H),1.44–1.35(m,9H),1.27(d,J=12.8Hz,4H).

[1291] Step 2: Preparation of (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester hydrochloride

[1292]

[1293] Ethyl acetate (commercially available) (2.4 mL) containing 4M HCl was slowly added to a stirred solution of (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester (0.6 g, 1.01 mmol) in EtOAc (12 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to provide the title compound (0.53 g, yield: 99%) as a white solid, which was used without further purification. MS (m / z): C 25 H 32 [M+H]+ calculated for N8O3, 493.25; found, 493.2.

[1294] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester

[1295]

[1296] 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, followed by the addition of 26.5 mL of acetonitrile dropwise while stirring. (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoic acid methyl ester hydrochloride (200 mg, 0.354 mmol) and 4-methylmorpholine (NMM, 36 mg, 0.354 mmol) were added to the solution at room temperature, causing a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM, 140 mg, 0.506 mmol) was added to the solution and stirred at room temperature for 72 hours. NaCl (207 mg, 3.54 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (300 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of deionized water and acetonitrile (V / V=3:1). NaCl (207 mg, 3.54 mmol) was then added to the reaction mixture, which was stirred for 1 hour and subsequently acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.195 g, yield: 45.1%, DSR: 27%; 1 H NMR(400MHz,D2O)δppm 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).

[1297] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.197 g, yield: 45.5%, DSR = 16%).

[1298] Example 67

[1299] Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

[1300]

[1301] Step 1: Preparation of tert-butyl N-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate

[1302]

[1303] A mixture of 2-[1-ethylsulfonyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]azetidin-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 for 7 hours at room temperature. Then tert-butyl hydrazinecarboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred for 16 hours at room temperature. The reaction mixture was poured into saturated Na2CO3 solution. The mixture was filtered. The filter cake was washed with NaHCO3 solution and water. The filter cake was then 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 provide the title compound (1.02 g, yield: 54.9%). MS (m / z): C 23 H 28 [M+H]+ calculated for N8O3, 465.22; found, 465.2. 1 H NMR (400 MHz, CDCl3) δ ppm 10.97(d,J=1.9Hz,1H),8.86(s,1H),8.31(d,J=15.2Hz,2H),7.97(d,J=4.0Hz, 1H),6.87–6.84(m,1H),6.62(s,1H),4.27(td,J=10.0,3.9Hz,1H),3.14(dd,J= 17.0,8.6Hz,1H),2.97(dd,J=17.0,3.9Hz,1H),2.61(dt,J=17.0,8.6Hz,1H),2 .04–1.92(m,1H),1.79–1.62(m,5H),1.54(d,J=13.3Hz,9H),1.35–1.19(m,3H).

[1304] Step 2: Preparation of 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

[1305]

[1306] Ethyl acetate (commercially available) (3 mL) containing 4M HCl was slowly added to a stirred solution of tert-butyl N-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl] pyrazol-4-yl] pyrrolo[2,3-d] pyrimidine-7-carbonyl] amino] carbamate (0.75 g, 1.61 mmol) in EtOAc (15 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was diluted with EtOAc and concentrated under reduced pressure to provide the title compound (0.557 g, yield: 95%) as a white solid, which was used without further purification. MS (m / z): C 18 H 20 [M+H]+ calculated for N8O, 365.17; found, 365.1.

[1307] Step 3: Preparation of a conjugate between HA (sodium hyaluronate) and 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

[1308]

[1309] Sodium hyaluronate (263.5mg, 0.654mmol carboxylic acid, MW 50KDa) is dissolved in the deionized water of 53mL in 100mL round-bottom flask, then 34mL acetonitrile is added dropwise when stirring.At room temperature by 4- [1- [(1R) -2- cyano -1- cyclopentyl -ethyl] pyrazole -4- bases] pyrrolo [2,3-d] pyrimidine -7- carbohydrazide hydrochloride (200mg, 0.458mmol) and 4- methylmorpholine (NMM, 46mg, 0.458mmol) are added in solution, so that viscosity is temporarily increased.Reactant mixture is stirred at this temperature for 2 hours.4- (4,6- dimethoxy -1,3,5- triazine -2- bases) -4- methylmorpholinium chloride (DMTMM, 181mg, 0.654mmol) is added in solution and at room temperature stirred 72 hours. Then NaCl (382 mg, 6.54 mmol) was added to the reaction mixture, the reaction mixture was stirred for 1 hour and then acetone (300 mL) was added dropwise while stirring. The mixture was filtered. The filter 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, the reaction mixture was stirred for 1 hour and then acetone (200 mL) was added dropwise while stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to obtain the title compound as a white solid. 0.316 g, yield: 66.5%, DSR: 33%; 1 H NMR(400MHz,D2O)δppm 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).

[1310] In the case of this step, the reaction of sodium hyaluronate (MW 2000 KDa) afforded the corresponding product (0.308 g, yield: 64.8%, DSR = 21%).

[1311] Example 68

[1312] Preparation of a conjugate between HA (sodium hyaluronate) and 2-aminoacetic acid [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl ester

[1313]

[1314] Step 1: Preparation of [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 2-(tert-butoxycarbonylamino)acetate

[1315]

[1316] A mixture of 3-[(3R, 4R)-3-[[7-(hydroxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidinyl]-3-oxo-propionitrile (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 for 10 minutes at 0°C to 10°C. Diisopropyl azodicarboxylate (1420 mg, 7.02 mmol) was then added dropwise. The reaction mixture was stirred at this temperature for 16 hours. The reaction mixture was poured into a saturated NaHCO solution under an ice bath. The mixture was diluted with EtOAc and washed with water and a saturated saline solution. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (EtOAc: hexane = 1:2) to provide the title compound (400 mg, yield: 45.7%). MS (m / z): C 24 H 33 [M+H]+ calculated for N7O5, 500.25; found, 500.1. 1 H NMR (400 MHz, CDCl3) δ ppm 8.31(t,J=16.1Hz,1H),7.15(dd,J=13.4,3.7Hz,1H),6.56(t,J=3.9Hz,1H),6. 23(d,J=2.8Hz,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,1 H),1.82–1.69(m,1H),1.51–1.32(m,9H),1.27–1.24(m,2H),1.13–1.09(m,1H).

[1317] Step 2: Preparation of 2-aminoacetic acid [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl ester trifluoroacetate

[1318]

[1319] Trifluoroacetic acid (2 mL) was slowly added to a stirred mixture of 2-(tert-butoxycarbonylamino)acetic acid [4-[[(3R, 4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidinyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl ester (0.35 g, 0.7 mmol) in DCM (10 mL) under an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 hours. The solution was concentrated under reduced pressure to provide the title compo...

Claims

1. A drug delivery system for locally delivering a therapeutic agent at a controlled rate, the drug delivery system comprising: A biopolymer, the biopolymer comprising at least a first binding group BG1, wherein the biopolymer is hyaluronic acid; A therapeutic agent, wherein the therapeutic agent is nintedanib or sunitinib, and comprises at least a second binding group BG2, wherein the second binding group BG2 is an amine group; as well as a linker that covalently links the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration; Wherein, when the therapeutic agent is nintedanib, the linker comprises a structure selected from the group consisting of: Where q is an integer from 0 to 5, In Optionally substituted with -C(=O)OCH3; Wherein, when the therapeutic agent is sunitinib, the linker comprises a structure selected from the group consisting of: Where q is an integer from 0 to 5, In Optionally substituted with -C(=O)OCH3; Wherein, BG1 is a carboxyl group, which forms an amide bond with the linker; R 1 and R 2 are independently selected from hydrogen or C 1-6 alkyl.

2. The drug delivery system according to claim 1, wherein the therapeutic agent is nintedanib.

3. The drug delivery system according to claim 1, wherein the therapeutic agent is sunitinib.

4. The drug delivery system according to claim 1, selected from the group consisting of:

5. The drug delivery system according to any one of claims 1 to 4, wherein the drug delivery system is topically administered to a subject in need thereof.

6. The drug delivery system of claim 5, wherein the drug delivery system is topically administered to a subject in need thereof by injection.

7. The drug delivery system of claim 5, wherein the drug delivery system is topically administered to a subject in need thereof via an oral dosage form.

8. The drug delivery system of claim 5, wherein the drug delivery system is topically administered to a subject in need thereof by inhalation.

9. The drug delivery system of claim 5, wherein the drug delivery system is locally administered to a subject in need thereof via an implant.

10. The drug delivery system of claim 5, wherein the drug delivery system is topically administered to a subject in need thereof by topical application.

11. A pharmaceutical composition comprising the drug delivery system according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

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