RU2847196C1 - Drug delivery system for local delivery of therapeutic agents and use thereof - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: group of inventions relates to chemistry and pharmaceutics, specifically to a drug delivery system for local delivery of a therapeutic agent at a controlled rate (embodiments) and to a pharmaceutical composition for the inhibition of Janus kinase (JAK) or vascular endothelial growth factor (VEGF) comprising said delivery system together with a pharmaceutically acceptable excipient. Disclosed delivery system includes: biopolymer comprising a BG1 binding group and which is hyaluronic acid or chondroitin sulphate; a therapeutic agent comprising a BG2 binding group and selected from tofacitinib, oclacitinib, baricitinib, ruxolitinib, nintedanib, or sunitinib; linker having the structure where U is bonded to the biopolymer via BG1 and V is bonded to the therapeutic agent via BG2, wherein: BG1 is a carboxyl group; U is selected from -N(R¹)-, or -O-, wherein R¹ is hydrogen, C_1-6 alkyl or C_2-6 alkenyl, and is a nitrogen-containing heterocyclyl, optionally containing one or more additional N heteroatoms; A is a direct bond, C_1-6 alkyl, optionally substituted with one or more R^a groups, or -(CH₂CH₂O)_m-; B is a direct bond, C_1-6 alkyl, 3–8-membered cycloalkyl, , phenyl, pyridinyl or -O-phenyl, wherein each is optionally substituted with one or more R^b groups; C is direct connection, -C(=O)N(R²)-, -N(R²)C(=O)-, -[CH₂NHC(=O)]_n- or -[NHC(=O)CH₂]_n-, wherein R² is hydrogen; D is a direct bond, C_1-6 alkyl, optionally substituted with one or more groups R^c, or phenyl; BG2 is an amine group; V is direct communication, -N(R²)C(=O)-, -N(R²)C(S)-, OC(=O)-, -OC(=S)-, -OC(=O)OCH₂-, -C(=O)OCH₂-, -N(R²)C(=O)OCH₂-, -N(R²)P(=O)(OPh)- or -C(=O)-; R^a, R^b and R^c independently represent -C(=O)OR^e, where R^e is C_1-6 alkyl; m is integer from 0 to 4; n is integer from 1 to 4.

EFFECT: group of inventions provides retention of therapeutic agent locally in place of administration and release thereof at a controlled rate.

9 cl, 1 tbl, 103 ex

Description

FIELD OF INVENTION

The present invention relates to drug delivery systems, to methods for local delivery of therapeutic agents and to methods of using such drug delivery systems for the treatment of diseases.

PRIOR ART

Most therapeutic agents are delivered systemically through oral/gastrointestinal (GI) absorption or systemic injection. These delivery routes are convenient and suitable for treating systemic diseases. However, many diseases are localized disorders. Even though systemically administered therapeutic agents may be effective in treating these disorders, they may also affect other tissues or binding sites, potentially leading to side effects or adverse effects. To reduce the likelihood of systemic side effects, a locally administered drug delivery system is desirable. Delivering therapeutic agents to desired sites is not as straightforward as taking medications orally or by injection. Therefore, a sustained-release drug delivery system for local drug delivery offers a product that is highly acceptable to physicians and patients. Furthermore, the release profile of therapeutic agents, which ensures that an effective concentration is maintained at the delivery site after administration, can significantly impact the efficacy of therapeutic agents. Thus, drug delivery of a therapeutic agent to a specific target tissue or site in the body has been a long-standing challenge in the pharmaceutical industry.

To ensure controlled drug delivery, numerous drug delivery systems with tissue specificity or a desired release profile have been developed. The most common local drug delivery system relies on biodegradable polymers to control the release rate of therapeutic agents. These drug delivery systems release drugs through both biopolymer degradation and diffusion of the drug molecules. This complex release regulation poses a significant challenge in the production of finished dosage forms and quality control. Therefore, there is an ongoing need to develop drug delivery systems that can locally deliver therapeutic agents to specific tissues with controlled release and reduced side effects.

There are three critical characteristics for the successful performance of a local drug delivery system: the ability of the delivery system to be retained at the delivery site; the ability to release the therapeutic agent at a desired rate and profile; and the responsiveness of the localized disorder to treatment with the therapeutic agent. The present invention provides another approach to fully meet these critical characteristics for the performance of a local drug delivery system, namely: biopolymers, due to their large molecular size, must retain the drug delivery system at the delivery site; therapeutic agents must be selected from commercially available products or have activities demonstrated in late-stage clinical studies; and linkers covalently linking the biopolymers to the therapeutic agents must be chemically unstable and, upon degradation, facilitate the release of therapeutic agents at a rate desired for the specific delivery site and the specific disease.

BRIEF DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, a drug delivery system is provided for local delivery of a therapeutic agent at a controlled rate, wherein the drug delivery system comprises:

a biopolymer comprising at least a first linking group BG1 selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, and a combination thereof;

a therapeutic agent comprising at least a second BG2 linking group selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, an amine group, and a combination thereof; and

a linker that covalently connects the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration; wherein the linker comprises a structure of formula (I):

Where

U is connected to the biopolymer via BG1 in such a way that at least one bond is formed, selected from an ester or an amide, and U is selected from

group consisting of a direct bond, -N(R ¹ )-, -O-, -C(=O)- and Where is a nitrogen-containing heterocyclyl, optionally containing one or more additional heteroatoms selected from N, O or S;

A is selected from a direct bond, alkyl and -(CH ₂ CH ₂ O) _m -, wherein said alkyl is optionally substituted by one or more R ^a groups;

B is selected from the group consisting of a direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, -O-heteroaryl, wherein each of alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^b groups;

C is selected from a direct bond, -C(=O)-, -C(=O)N(R ² )-, -N(R ² )C(=O)-, -[CH ₂ NHC(=O)] _n -, -[NHC(=O)CH ₂ ] _n - and -NH(CH ₂ ) _p C(=O)-;

D is selected from a direct bond, alkyl and aryl, wherein said alkyl is optionally substituted by one or more R ^c groups;

V is linked to the therapeutic agent through BG2 such that at least one bond is formed selected from the group consisting of amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal and a combination 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)OCH ₂ -, -C(=O)OCH ₂ -, -N(R ² )C(=O)OCH ₂ -, -OP(=O)(OPh)- and -N(R ² )P(=O)(OPh)-;

R ¹ and R ² are independently selected from a hydrogen atom, alkyl, alkenyl and alkynyl;

R ^a , R ^b and R ^c are independently selected from halogen, hydroxyl, amino, cyano, nitro, alkyl, alkoxyl, -C(=O)OR ^e and =NH;

m is an integer from 0 to 4;

n is an integer from 1 to 4; and

p is an integer from 1 to 4.

In some embodiments, the linker in the drug delivery system of the present invention comprises a structure of formula (Ia) to (Im):

Where

U and V are as defined in paragraph 1;

M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, each of which is optionally substituted by one or more R ^b groups; and

q, r, s, t, and v are independently equal to an integer from 0 to 4.

In some embodiments, the biopolymer in the drug delivery system of the present invention is selected from the group consisting of hyaluronic acid, chitosan, chitin, chondroitin, or derivatives thereof.

In some embodiments, the therapeutic agent in the drug delivery system of the present invention 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 drugs that may have serious systemic toxic effects.

According to a first aspect of the present invention, there is provided a pharmaceutical composition comprising a drug delivery system as provided in the present invention and a pharmaceutically acceptable excipient.

According to another aspect of the present invention, there is provided a method of treating a disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided in this invention.

DETAILED DESCRIPTION OF THE INVENTION

Certain embodiments of the present invention will now be described in detail, examples of which are illustrated by the accompanying structures and claims. Although the present invention will be described in conjunction with the embodiments given, it is obvious that they are not intended to limit the present invention to these embodiments. On the contrary, it is intended that the present invention cover all alternatives, modifications and equivalents that may be included within the scope of the present invention, as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described in this application, which can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including, but not limited to, defined terms, use of terms, described methods or the like, then the description of the present invention controls. All references, patents, and patent applications mentioned in the description of the present invention are hereby incorporated by reference in their entirety.

It is obvious that some features of the present invention, which for brevity are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, other features of the present invention, which for brevity are described in the context of a single embodiment, may also be provided individually or in any suitable subcombination. It should be noted that, as used in the description and the appended claims, the singular forms "a," "an," and "the" include plural forms of the same, unless the context clearly dictates otherwise.

Definitions

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

Linking substituents are described throughout the present invention. Each linking substituent is specifically intended to include both forward and reverse linking forms of the 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 variables listed for that group are understood to represent linking groups. For example, if a structure requires a linking group, and the Markush group definition lists "alkyl" for that variable, then "alkyl" is understood to represent a linking alkylene group.

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

When any variable (e.g., R ⁱ ) occurs more than once in any component or formula for a compound, its definition in each instance is independent of its definition in any other instance. Thus, for example, if a group is shown to be substituted with 0-2 R ⁱ moieties, then the group may possibly be substituted with up to two R ⁱ moieties, and R ⁱ in each instance is selected independently of the definition for R ⁱ . Furthermore, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

As used herein, the term "C _ij " indicates a range of carbon atom numbers, wherein i and j are integers, and the range of carbon atom numbers includes the endpoints (i.e., i and j) and every integer between them, and wherein j is greater than i. As examples, 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 1-12 atoms, in particular 1-10, in particular 1-8, in particular 1-6, in particular 1-5, in particular 1-4, in particular 1-3, or in particular 1-2 carbon atoms.

As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated straight- or branched-chain hydrocarbon radical that may be optionally substituted independently with one or more substituents described below. The term " _Cij alkyl" refers to alkyl having from i to j carbon atoms. In some embodiments, alkyl groups contain from 1 to 10 carbon atoms. In some embodiments, alkyl groups contain from 1 to 9 carbon atoms. In some embodiments, alkyl groups contain from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or from 1 to 2 carbon atoms. Examples of "C _1-10 alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Examples of "C _1-6 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.

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

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

As used herein, the term "alkoxy" whether used as part of another term or independently refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom. The term "C _ij alkoxy" means that the alkyl moiety of the alkoxy group has from i to j carbon atoms. In some embodiments, alkoxy groups contain from 1 to 10 carbon atoms. In some embodiments, alkoxy groups contain from 1 to 9 carbon atoms. In some embodiments, alkoxy groups contain from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, or from 1 to 2 carbon atoms. Examples of "C _1-6 alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, neopentoxy, n-hexoxy, and the like.

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

As used herein, the term "amine" refers to ammonia derivatives in which one or more hydrogen atoms are replaced by a substituent and which may be represented by the group N(H) _n (R') _3-n , where n is 0, 1 or 2, and each R' is independently hydroxyl, 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.

As used herein, the term "amino" refers to -NH ₂ .

As used herein, the term "aryl," whether used as part of another term or independently, refers to monocyclic and polycyclic ring systems having a total of 5 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, anthracyl, and the like, which may bear one or more substituents. The term "aryl," as used herein, also includes a group in which an aromatic ring is fused to one or more additional rings. In the case of a polycyclic ring system, only one of the rings need be aromatic (e.g., 2,3-dihydroindole), although all rings may be aromatic (e.g., quinoline). The second ring may also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. Aryl groups may be substituted at one or more positions on the ring with the substituents described above.

As used herein, the term "aza-acetal" refers to -N-CH ₂ -O-.

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

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 atoms and which contains at least three ring-forming carbon atoms. In some embodiments, cycloalkyl may contain from 3 to 12 ring-forming carbon atoms, from 3 to 10 ring-forming carbon atoms, from 3 to 9 ring-forming carbon atoms, from 3 to 8 ring-forming carbon atoms, from 3 to 7 ring-forming carbon atoms, from 3 to 6 ring-forming carbon atoms, from 3 to 5 ring-forming carbon atoms, from 4 to 12 ring-forming carbon atoms, from 4 to 10 ring-forming carbon atoms, from 4 to 9 ring-forming carbon atoms, from 4 to 8 ring-forming carbon atoms, from 4 to 7 ring-forming carbon atoms, from 4 to 6 ring-forming carbon atoms, from 4 to 5 ring-forming carbon atoms. Cycloalkyl groups may be saturated or partially unsaturated. Cycloalkyl groups may be substituted. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system. In some embodiments, the cycloalkyl group may be monocyclic or polycyclic. Examples of a monocyclic cycloalkyl group 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 the polycyclic cycloalkyl group include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro[3.6]-decanyl, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, and the like.

As used herein, the term "cyano" refers to -CN. As used herein, the term "ester" refers to -C(=O)O-.

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

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

As used herein, the term "halogen" refers to an atom selected from fluorine (or a fluorine atom), chlorine (or a chlorine atom), bromine (or a bromine atom), and iodine (or an iodine atom).

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

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

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

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

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

The term "nitro" as used in this description refers to -NO ₂ .

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

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

As used herein, the term "phosphoramidate" refers to -(NR')P(=O)(OR') _a (NR'') _b -, where R' and R'' are independently absent, represent a hydrogen atom, alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl and other suitable organic groups, and a and b are independently 0, 1 or 2.

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

As used herein, the term "linkage" or "linker" refers to a bond or chemical grouping formed by a chemical reaction between the functional groups of at least two entities to be linked, thereby forming a single molecule or maintaining the entities in close proximity. The linker may be integrated into the resulting molecule or structure after linkage, with its interacted or unreacted functional groups. Such bonds may be covalent or non-covalent. The term "hydrolytically unstable or cleavable bonds" means that such bonds are cleavable in water or aqueous solutions, including, for example, in body fluids such as blood. The term "enzymatically unstable or cleavable bonds" means that such a bond can be cleaved by one or more enzymes. Such hydrolytically cleavable bonds include, but are not limited to, ester bonds formed between a carboxylic acid in one moiety and alcohol groups in the biologically active agent, which ester groups typically hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically cleavable bonds include, but are not limited to, carbonate bonds, imine bonds formed by the interaction of an amine and an aldehyde, phosphate ester bonds formed by the interaction of a phosphate group and an alcohol, hydrazone bonds formed by the interaction of a hydrazide and an aldehyde, acetal bonds formed by the interaction of an aldehyde and an alcohol, and amide bonds formed by the interaction of an amino group and a carboxyl group.

As used herein, the term "partially unsaturated" refers to a radical that contains 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.

As used herein, the term "pharmaceutically acceptable" indicates that the substance or composition is compatible chemically and/or toxicologically with the other ingredients contained in the composition and/or the subjects being treated therewith.

As used herein, the term "substituted," whether or not preceded by the term "optionally," means that one or more hydrogen atoms of the specified moiety are replaced by a suitable substituent. It will be understood that "substitution" or "substituted by" includes the implied condition that such substitution corresponds to the permissible valence of the atom being replaced and that the substitution results in a stable or chemically feasible compound, for example, one that does not undergo spontaneous transformation such as rearrangement, cyclization, elimination, and so on. Unless otherwise specified, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when in any given structure more than one position can be substituted by more than one substituent selected from the specified group, the substituents may be either the same or different at each position. Those skilled in the art will appreciate that the substituents themselves may be substituted, if appropriate. Unless substituents are specifically designated as "unsubstituted," references to chemical moieties in this specification are understood to include substituted versions. For example, a reference to an "aryl" group or moiety potentially includes both substituted and unsubstituted versions.

As used herein, the terms "therapeutic agent," "drug," "biologically active molecule," "biologically active agent," "active agent," and the like refer to any substance that can affect any physical or biochemical properties of a biological organism, including, but not limited to, viruses, bacteria, fungi, plants, animals, and humans. Specifically, therapeutic agents used in this invention include any substance intended for the diagnosis, cure, mitigation of adverse effects, treatment, or prevention of disease in humans or other animals, or for otherwise improving the physical or mental health of humans or animals.

Drug delivery of therapeutic agents to specific tissues or sites in the body presents many challenges, particularly where local delivery to a specific tissue of a therapeutic agent at a high dose that has poor water solubility is desired and where it is desirable to avoid high systemic concentrations of the therapeutic agent leading to toxic side effects.

Thus, according to one aspect of the present invention, a drug delivery system is provided that is 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 connects the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration.

Biopolymer

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

Various biopolymers are used as polymeric carriers for delivering therapeutic agents to target cells or tissues. Biopolymers suitable for a particular application are selected based on their ability to target specific tissues, organs, or cells and their in vivo stability, i.e., their in vivo residence time in the circulatory system or specific tissues, cells, or organs.

In some embodiments, the biopolymer is selected from biocompatible polymers comprising at least a first linking group BG1 that can react with a reactive functional group of a second entity (e.g., a linker as provided herein) to form a bond, thereby linking the biopolymer to this second entity (e.g., a linker). The term "biocompatible" as used herein refers to a substance that does not have any medically unacceptable toxic or harmful effects on biological function or that is tolerated by the body.

In some embodiments, the biopolymer is selected from biocompatible polymers comprising at least a first linking 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 linkers suitable for attaching therapeutic agents to the biopolymer. BG1 can be present anywhere within the biopolymer backbone, and thus, the bonds formed between the biopolymer and the linker can be present in any part of the biopolymer.

In some embodiments, the BG1s used to react with the reactive functional group of the linker may be the same or different. In some embodiments, the BG1s of the biopolymer are the same. In some embodiments, the BG1s of the biopolymer are different.

In some embodiments, the biopolymers are biocompatible polymers containing a carboxyl group as BG1 that can react with a reactive functional group of a suitable linker to form a bond connecting the carboxyl group-containing biopolymer to the linker.

In some embodiments, the reactive functional group of the linker is an amino group or an amine group that reacts with the carboxyl group of the biopolymer to form an amide bond.

In some embodiments, the reactive functional group of the linker is a hydroxy group that reacts with the carboxyl group of the biopolymer to form an ester bond.

In some embodiments, the reactive functional group of the linker is a halogen that reacts with the carboxyl group of the biopolymer to form an ester bond.

In some embodiments, the biopolymers are biocompatible polymers containing an amino group as BG1 that can react with a reactive functional group of a suitable linker to form a bond, thereby forming a biopolymer-linker conjugate.

In some embodiments, the reactive functional group of the linker is a carboxyl group that reacts with a hydroxy group of the biopolymer to form an ester bond.

In some embodiments, the bond formed as a result of the interaction 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 a hydrogen atom, alkyl, alkenyl and alkynyl, is a nitrogen-containing heterocyclyl, optionally containing one or more additional heteroatoms selected from N, O or S.

In some embodiments, R ¹ is a hydrogen atom.

In some incarnations selected from the group consisting of:

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

In some embodiments, the biopolymer may have a number average molecular weight ranging from 400 to 3,000,000 Da, such as from 1,000 to 3,000,000 Da, from 5,000 to 3,000,000 Da, from 10,000 to 3,000,000 Da, from 20,000 to 3,000,000 Da, from 30,000 to 3,000,000 Da, from 40,000 to 3,000,000 Da, from 50,000 to 3,000,000 Da, or from 50,000 to 2,000,000 Da.

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

In some embodiments, the biopolymer is HA. In some embodiments, HA can be isolated from any source.

In some embodiments, the HA may have a number average molecular weight ranging from 400 to 3,000,000 Da, such as from 1,000 to 3,000,000 Da, from 5,000 to 3,000,000 Da, from 10,000 to 3,000,000 Da, from 20,000 to 3,000,000 Da, from 30,000 to 3,000,000 Da, from 40,000 to 3,000,000 Da, from 50,000 to 3,000,000 Da, or from 50,000 to 2,000,000 Da.

Therapeutic agent

According to the present invention, an improved delivery system for local delivery of various therapeutic agents is provided.

In some embodiments, the therapeutic agent comprises at least a second linking group BG2 that can react with a reactive functional group of a second entity (e.g., a linker provided in this invention) and a possible coreactant to form a bond, thereby linking the therapeutic agent to the second entity (e.g., a linker).

In some embodiments, the therapeutic agent comprises at least a second linking group BG2 selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, an amide group, an amine group, and combinations thereof. BG2 serves as a binding site for conjugating linkers suitable for attaching therapeutic agents to the biopolymer.

In some embodiments, the therapeutic agent comprises an amine group as BG2 that can react with a reactive functional group of a suitable linker and a possible coreactant to form a bond connecting the amine-containing therapeutic agent to the linker.

In some embodiments, the amine group of the therapeutic agent reacts with a reactive functional group of the linker and a possible coreactant, resulting in the therapeutic agent being 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, and the like.

In some embodiments, the bond formed by the reaction involving BG2 of the therapeutic agent and the reactive functional group of the linker and a possible coreactant is selected from the group consisting of -N(R') ₂ -, -C(=O)N(R') ₂ , -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)OCH ₂ N(R') ₂ -, -N(R ² )C(=O)OCH ₂ N(R') ₂ -, -OP(=O)(OPh)N(R') ₂ - and -N(R ² )P(=O)(OPh)N(R') ₂ -, where R ² 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 heterocyclyl.

In some embodiments, R ² is a hydrogen atom.

In some embodiments, the therapeutic agent comprises a carboxyl group as BG2, which can react with a reactive functional group of a suitable linker and a possible coreactant to form a bond connecting the therapeutic agent containing the carboxyl group to the linker.

In some embodiments, a carboxyl group on the therapeutic agent reacts with a reactive functional group on the linker and a possible coreactant, resulting in the therapeutic agent being attached to the linker via an ester bond.

In some embodiments, the therapeutic agent comprises a hydroxyl group as BG2, which can react with a reactive functional group of a suitable linker and a possible coreactant to form a bond connecting the therapeutic agent containing the hydroxyl group to the linker.

In some embodiments, a hydroxyl group on the therapeutic agent reacts with a reactive functional group on the linker and a possible coreactant, resulting in the therapeutic agent being attached to the linker via an ester bond.

In some embodiments, the delivered therapeutic agent is selected from the group consisting of non-steroidal anti-inflammatory drugs (NSAIDs), Janus kinase (JAK) inhibitors, vascular endothelial growth factor (VEGF) inhibitors, anti-cancer drugs, and any drugs that may have serious systemic toxic effects.

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

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

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

In some embodiments, the delivered therapeutic agent is tofacitinib.

In some embodiments, the delivered therapeutic agent is upadacitinib.

In some embodiments, the delivered therapeutic agent is ruxolitinib.

In some embodiments, the delivered therapeutic agent is baricitinib.

In some embodiments, the delivered therapeutic agent is oclacitinib.

In some embodiments, the delivered therapeutic agent is nintedanib.

In some embodiments, the delivered therapeutic agent is sunitinib. Linker

Enhanced local delivery of therapeutic agents is achieved by linking the therapeutic agent to a biopolymer via a suitable linker. By selecting appropriate linkers, the release rate of the therapeutic agent from the biopolymer can be controlled, thereby ensuring improved delivery of the therapeutic agent to target cells or tissues.

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

A "cleavable bond" is a relatively labile bond that cleaves under physiological conditions. A typical detachable bond is a hydrolyzable bond, which cleaves upon interaction with water (i.e., hydrolyzes). The susceptibility of a bond to hydrolysis in water can depend not only on the general type of bond connecting the two atoms but also on the substituents attached to these atoms. Corresponding hydrolytically unstable or weak bonds include, but are not limited to, bonds in the following compounds: carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, and carbonates. Some functional groups contain atoms that can be chemically broken down by processes other than hydrolysis. Typical detachable bonds in this category include certain carbamate bonds and bonds formed by 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives. Some molecules containing these types of functional groups, when linked in this manner, can be chemically degraded (released) by base. In such cases, "cleavage" can occur at higher pH values or by biological molecules containing basic moieties (e.g., histidines). Another typical detachable bond is the enzymatically cleavable bond. "Enzymatically cleavable bond" means a bond that is cleavable by one or more enzymes.

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

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

Where

U is connected to the biopolymer via BG1 of the biopolymer such that at least one bond is formed, selected from an ester or amide bond, and U is selected from the group consisting of a direct bond, -N(R ¹ )-, -O-, -C(=O)- and Where is a nitrogen-containing heterocyclyl, optionally containing one or more additional heteroatoms selected from N, O or S;

A is selected from a direct bond, alkyl and -(CH ₂ CH ₂ O) _m -, wherein said alkyl is optionally substituted by one or more R ^a groups;

B is selected from the group consisting of a direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl, -O-heteroaryl, wherein each of alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted with one or more R ^b groups;

C is selected from a direct bond, -C(=O)-, -C(=O)N(R ² )-, -N(R ² )C(=O)-, -[CH ₂ NHC(=O)] _n -, -[NHC(=O)CH ₂ ] _n - and -NH(CH ₂ ) _p C(=O)-;

D is selected from a direct bond, alkyl and aryl, wherein said alkyl is optionally substituted by one or more R ^c groups;

V is linked to the therapeutic agent through BG2 in the therapeutic agent such that at least one bond is formed selected from the group consisting of amide, urea, thiourea, carbamate, thiocarbamate, phosphoramidate, aza-acetal and a combination 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)OCH ₂ -, -C(=O)OCH ₂ -, -N(R ² )C(=O)OCH ₂ -, -OP(=O)(OPh)- and -N(R ² )P(=O)(OPh)-;

R ¹ and R ² are independently selected from the group consisting of hydrogen, alkyl, alkenyl and alkynyl;

R ^a , R ^b and R ^c are independently selected from halogen, hydroxyl, amino, cyano, nitro, alkyl, alkoxyl, -C(=O)OR ^e and =NH;

R ^e is alkyl;

m is an integer from 0 to 4;

n is an integer from 1 to 4; and

p is an integer between 1 and 4.

In some embodiments, BG1 is a carboxyl group and U is -N(R ¹ )-, thereby forming an amide bond to attach the biopolymer to the linker.

In some embodiments, BG1 is a carboxyl group and U is resulting in the formation of an amide bond to attach the biopolymer to the linker.

In some embodiments, BG1 is a carboxyl group and U is a group selected from the group consisting of:

In some embodiments, BG1 is a hydroxyl group and U is -C(=O)-, thereby forming an ester bond to attach the biopolymer to the linker.

In some embodiments, BG1 is a carboxyl group and U is -O- or a direct bond, resulting in an ester bond for attaching the biopolymer to the linker.

In some embodiments, BG1 is an amino group and U is -C(=O)-, thereby forming an amide bond to attach the biopolymer to the linker.

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

(a) direct communication;

(b) a -N(R ² )C(=O)- group that is linked to the therapeutic agent through BG2 in such a way that a urea bond is formed for attaching the therapeutic agent to the linker;

(c) a -N(R ² )C(S)- group that is linked to the therapeutic agent through BG2 such that a thiourea bond is formed for attaching the therapeutic agent to the linker;

(d) an -OC(=O)- group which is linked to the therapeutic agent through BG2 in such a way that a carbamate bond is formed for attaching the therapeutic agent to the linker;

(d) an -OC(=S)- group that is linked to the therapeutic agent through BG2 such that a thiocarbamate bond is formed for attaching the therapeutic agent to the linker;

(e) a -OC(=O) _OCH2- group which is linked to the therapeutic agent through BG2 in such a way that an aza-acetal bond is formed for attaching the therapeutic agent to the linker;

(g) a -C(=O) _OCH2- group, which is linked to the therapeutic agent via BG2 in such a way that an aza-acetal bond is formed;

(z) a -N(R ² )C(=O)OCH ₂ - group which is linked to the therapeutic agent through BG2 in such a way that an aza-acetal bond is formed for attaching the therapeutic agent to the linker;

(i) an -OP(=O)(OPh)- group that is linked to the therapeutic agent via BG2 such that a phosphoramidate bond is formed for attaching the therapeutic agent to the linker; or

(k) a -N(R ² )P(=O)(OPh)- group that is linked to the therapeutic agent through BG2 in such a way that a phosphoramidate bond is formed for attaching the therapeutic agent to the linker;

(l) -C(=O)- group, which is linked to the therapeutic agent through BG2 in such a way that an amide bond is formed.

In some embodiments, BG2 is a carboxyl group and V is -O- or a direct bond, resulting in an ester bond for attaching the therapeutic agent to the linker.

In some embodiments, BG2 is a hydroxyl group and V is -C(=O)-, thereby forming an ester bond to attach the therapeutic agent to the linker.

In some embodiments, A represents a direct link.

In some embodiments, A is alkyl optionally substituted with one or more R ^a groups. In some embodiments, A is C _1-10 alkyl optionally substituted with one or more R ^a groups. In some embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups.

In some embodiments, R ^a is -C(=O)OR ^e , where R ^e is alkyl. In some embodiments, R ^a is -C(=O)OR ^e , where R ^e is C _1-8 alkyl, C _1-7 alkyl, C _1-6 alkyl, C _1-5 alkyl, C _1-4 alkyl, or C _1-3 alkyl. In some embodiments, R ^a is -C(=O)OCH ₃ .

In some embodiments _, A is -( _CH2CH2O ) _m- .

In some embodiments, m is an integer from 0 to 4. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

In some embodiments, B represents a direct bond.

In some embodiments, B is alkyl. In some embodiments, B is C _1-6 alkyl, C _1-5 alkyl, C _1-4 alkyl, C _1-3 alkyl, or C _1-2 alkyl. In some embodiments, B is ethyl.

In some embodiments, B is cycloalkyl, aryl, or heteroaryl.

In some embodiments, B is cycloalkyl.

In a certain embodiment, B is a saturated cycloalkyl. In a certain embodiment, B is a partially unsaturated cycloalkyl.

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.

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

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

In certain embodiments, B is phenyl.

In some embodiments, B is a heteroaryl. In certain embodiments, 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, B is pyridinyl.

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

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

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

In certain embodiments, A represents a direct link and B represents a direct link.

In some embodiments, A is a direct bond and 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. In certain embodiments, A is a direct bond and B is cyclohexyl.

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

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

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

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

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, and B is a direct bond, wherein R ^a is -C(=O)OR ^e , where R ^e is alkyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, and B is 5-12-membered aryl, 5-10-membered aryl, 5-8-membered aryl, or 5-6-membered aryl. In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, and B is phenyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more ^Ra groups and B is -O-aryl. In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more ^Ra groups and B is -O-phenyl.

In some embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and _B is selected from the group consisting of direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and _B is selected from the group consisting of a direct bond, alkyl, aryl, and heteroaryl.

In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and _B is a direct bond.

In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and _B is _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, or _C1-2 alkyl. In certain embodiments, A is -( _{CH2CH2O ) m-} , where m is an integer from 0 to 4, and B is ethyl.

In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and B is 5-12-membered aryl, 5-10-membered aryl, 5-8-membered aryl, or 5-6- _{membered aryl} . In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and B is phenyl.

In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and _B is 5-12-membered heteroaryl, 5-10-membered heteroaryl, 5-8-membered heteroaryl, or 5-6- _membered heteroaryl. In certain embodiments, A is -( _CH2CH2O ) _m- , where m is an integer from 0 to 4, and B is pyridinyl.

In some embodiment, C represents a direct connection.

In some embodiment, C is -C(=O)-.

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

In some embodiment, C is -N(R ² )C(=O)-.

In some embodiment, C is -[CH ₂ NHC(=O)] _n -.

In some embodiment, C is -[NHC(=O)CH ₂ ] _n -.

In some embodiment, C is -NH(CH ₂ )PC(=O)-.

In some embodiment, A is alkyl, B is selected from the group consisting of a direct bond, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-cycloalkyl, -O-heterocyclyl, -O-aryl and -O-heteroaryl, and C is selected from the group consisting of a direct bond, -C(=O)-, -N(R ² )C(=O)-, -[CH ₂ NHC(=O)] _n -, -[NHC(=O)CH ₂ ] _n - and -NH(CH ₂ )PC(=O)-.

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

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)-.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, 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 , wherein R ^e is alkyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, B is 5-12-membered aryl, 5-10-membered aryl, 5-8-membered aryl, or 5-6-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 is alkyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, B is phenyl, and C is a direct bond or -N(R ² )C(=O)-, wherein R ^a is -C(=O)OR ^e , wherein R ^e is alkyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, B is -O-aryl, and C is a direct bond, wherein R ^a is -C(=O)OR ^e , where R ^e is alkyl.

In certain embodiments, A is C _1-8 alkyl optionally substituted with one or more R ^a groups, B is -O-phenyl, and C is a direct bond, wherein R ^a is -C(=O)OR ^e , where R ^e is alkyl.

In some embodiments, A is -( _CH2CH2O ) _m- , _B is selected from the group consisting of a direct bond, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and C is a direct bond or -N( ^R2 )C(=O)-.

In some embodiments, A is -( _CH2CH2O ) _m- , where m is 1, 2, 3, or 4, _B is selected from the group consisting of a direct bond, alkyl, aryl, and heteroaryl, and C is a direct bond.

In some embodiments _, A is -( _CH2CH2O ) _m- , where m is 1, 2, 3, or 4, B is a direct bond, and C is a direct bond.

In some embodiments, A is -( _CH2CH2O ) _m- , where m is 1, 2, 3, or 4, B is _C1-6 alkyl, _C1-5 alkyl, _C1-4 alkyl, _C1-3 alkyl, or _C1-2 alkyl, and C is a direct _bond .

In some embodiments _, A is -( _CH2CH2O ) _m- , where m is 1, 2, 3, or 4, B is aryl, and C is a direct bond. In certain embodiments, A is - _{( CH2CH2O} ) _m- , where m is 1, 2, 3, or 4, B is phenyl, and C is a direct bond.

In some embodiments _, A is -( _CH2CH2O ) _m- , where m is 1, 2, 3, or 4, B is heteroaryl, and C is a direct bond. In certain embodiments, A is - _{( CH2CH2O} ) _m- , where m is 1, 2, 3, or 4, B is pyridinyl, and C is a direct bond.

In some embodiments, D represents a direct bond.

In some embodiments, D is alkyl. In certain embodiments, D is C _1-6 alkyl, C _1-5 alkyl, C _1-4 alkyl, C _1-3 alkyl, or C _1-2 alkyl.

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

In some embodiments, a linker provided in this invention comprises a structure of formula (Ia) to (Im):

Where

U and V are defined as above;

M is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl, each of which is optionally substituted by one or more R ^b groups;

each of possibly substituted by the group -C(=O)OCH ₃ ; and

q, r, s, t, and v are independently equal to an integer from 0 to 4.

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.

In some embodiments, a linker provided by the present invention comprises a structure selected from the group consisting of:

where each of possibly substituted by the group -C(=O)OCH ₃ .

Drug delivery system

According to one aspect of the present invention, a therapeutic agent is attached to a biopolymer via a linker, thereby providing a drug delivery system for local delivery of the therapeutic agent to desired locations.

The biopolymer in the drug delivery system proposed in this invention may be coupled to one or more therapeutic agents conjugated via linkers. The biopolymer may be conjugated to one or more therapeutic agents via one or more linkers at a hydroxyl group, carboxyl group, and/or amino group in the biopolymer backbone.

The drug delivery system of the present invention is obtained by performing conjugation between a biopolymer and a therapeutic agent using a linker by forming bonds between the biopolymer and the linker and a bond between the therapeutic agent and the linker.

In some embodiments, the reactive functional group of the linker may first be reacted 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, which contains another reactive functional group at the end of the linker, may then be reacted with BG1 of the biopolymer to form a bond between the biopolymer and the linker, thereby providing the drug delivery system of the present invention.

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

In some embodiments, the biopolymer selected for the drug delivery system of the present invention is HA, and the therapeutic agent selected for the drug delivery system of the present invention is tofacitinib.

In some embodiments, the drug delivery system of the present invention is selected from the group consisting of:

In some embodiments, the therapeutic agent may be conjugated to the biopolymer via a linker with a degree of drug substitution (DSR) of the biopolymer, measured using nuclear magnetic resonance (NMR), of at least 1%, at least 2%, at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, wherein the degree of drug substitution (DSR) of the biopolymer refers to the ratio of the molar amount of groups on the biopolymer that are substituted with drugs to the total molar amount of groups on the biopolymer that can be substituted with drugs.

A therapeutic agent may be released from the drug delivery system of the present invention by cleaving the bond between the linker and the biopolymer or therapeutic agent. In some embodiments, release of the therapeutic agent occurs when the bond between the biopolymer and the linker is cleaved, releasing a therapeutic agent-linker conjugate, which can be considered a prodrug. Subsequent release of the therapeutic agent from the linker may involve enzymatic or non-enzymatic cleavage of the bond between the therapeutic agent and the linker. In some embodiments, release of the therapeutic agent occurs when the bond between the therapeutic agent and the linker is cleaved without or before cleavage of the bond between the biopolymer and the linker. Release of the therapeutic agent may also involve enzymatic or non-enzymatic processes.

The release of therapeutic agents can be influenced by many factors, such as the choice of a particular therapeutic agent, linker, and biopolymer, and the route of administration of the drug delivery system. The present invention encompasses biopolymers of varying molecular weights, with a BG1 binding group, and with linker-mediated binding; linkers with various reactive functional groups and subunits; and therapeutic agents with different BG2 binding groups, and with linker-mediated binding.

The present invention also provides for a variation in the method of local administration of the drug delivery system provided in the present invention. In some embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof. In certain embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof by injection. In certain embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof using an oral dosage form. In certain embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof by inhalation. In certain embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof by means of an implant. In certain embodiments, the drug delivery system provided in accordance with the present invention is locally administered to a subject in need thereof by topical application. Depending on the specific combination of therapeutic agent, linker, and biopolymer, release of therapeutic agents after administration to a subject may occur at various sites. For example, release of therapeutic agents may occur at the site of administration.

In some embodiments, administration to a subject of the drug delivery system of the present invention may provide release of the therapeutic agent over a period of time ranging from at least several days to at least several months.

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

Pharmaceutical compositions

According to another aspect, pharmaceutical compositions comprising the drug delivery system of the present invention are provided.

According to another aspect, pharmaceutical compositions are provided comprising a drug delivery system of the present invention and at least one pharmaceutically acceptable excipient.

As used herein, the term "pharmaceutical composition" refers to a composition comprising the drug delivery system of the present invention in a form suitable for administration to a subject.

As used herein, the term "pharmaceutically acceptable excipient" means an excipient useful for preparing a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes excipients acceptable for veterinary use as well as for the manufacture of human pharmaceuticals. The term "pharmaceutically acceptable excipient" as used herein includes both one and more than one such excipient. The term "pharmaceutically acceptable excipient" also encompasses "pharmaceutically acceptable carrier" and "pharmaceutically acceptable diluent."

The pharmaceutical compositions provided in this invention may be in any form that allows the composition to be administered to a subject, including, but not limited to, a human, and are prepared so as to be compatible with the intended route of administration.

A variety of routes are contemplated for the pharmaceutical compositions provided by this invention, and accordingly, the pharmaceutical composition provided by this invention may be supplied in bulk form or in unit dosage form depending on the intended route of administration. For example, for oral, buccal, and sublingual administration, powders, granules, tablets, pills, capsules, gelatin capsules, and caplets may be suitable as solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions may be suitable as liquid dosage forms. For injection administration, gels, solutions, emulsions, and suspensions may be suitable as liquid dosage forms, and a powder suitable for reconstitution with an appropriate solution may be suitable as solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols may be suitable dosage forms. For topical (including buccal and sublingual) or transdermal administration, acceptable dosage forms include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. For vaginal administration, acceptable dosage forms include pessaries, tampons, creams, gels, pastes, foams, and sprays. For implantation, acceptable dosage forms include solids, semi-solids, and gels.

In some embodiments, the pharmaceutical compositions of the present invention may be in the form of a composition for oral administration.

In some embodiments, the pharmaceutical compositions of the present invention may be in the form of a composition for administration by injection.

In some embodiments, the pharmaceutical compositions of the present invention may be in the form of a composition for inhalation administration.

In some embodiments, the pharmaceutical compositions of the present invention may be in the form of a composition for topical administration.

In certain embodiments, the pharmaceutical compositions provided in this invention may be formulated as skin patches, which are well known to those of ordinary skill in the art.

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

In some embodiments, the pharmaceutical compositions of the present invention may be formulated as a single dose. The amount of compounds provided herein in a single dose will vary depending on the subject being treated and the specific route of administration.

In some embodiments, the pharmaceutical compositions of the present invention may be prepared for administration to a subject at intervals of several days, several weeks, several months, or even longer.

According to a further aspect, pharmaceutical compositions are also provided comprising the drug delivery system of the present invention, in the form of a combination of two or more therapeutic agents.

Synthesis of a drug delivery system

The synthesis of the drug delivery system proposed in accordance with the present invention is illustrated in the synthesis schemes in the Examples section. The drug delivery system proposed in this invention can be prepared using any known methods of organic synthesis and can be synthesized according to any of numerous possible synthetic routes, and therefore such schemes are illustrative only and are not intended to limit other possible methods that can be used to prepare the compounds proposed in this invention. Furthermore, the steps in the schemes are provided for better illustration and can be modified if necessary. Embodiments of the compounds in the Examples section were synthesized for the purposes of research and possible submission to government regulatory agencies.

The reactions to produce the drug delivery system of the present invention can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents can exhibit substantial non-reactivity with the starting materials (reagents), intermediates, or products at the temperatures at which these reactions are carried out, for example, at temperatures that can range from the solvent's freezing point to the solvent's boiling point. This reaction can be carried out in a single solvent or in a mixture of two or more solvents. Depending on the specific reaction step, solvents suitable for a particular reaction step can be selected by one skilled in the art.

The preparation of the compounds of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art. Chemical reactions involving protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley & Sons, Inc., New York (1999), in P. Kocienski, Protecting Groups, Georg Thieme Verlag, 2003, and in 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.

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

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

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

Method of treating the disease

According to a further aspect, a method for treating a disorder in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of a drug delivery system or pharmaceutical composition provided in this invention.

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

In this context, the term "therapeutically effective amount" refers to the amount of a therapeutic agent selected for the drug delivery system of the present invention, or pharmaceutically acceptable salts thereof, that is effective to provide a "therapeutic effect" to a subject or to "treat" disorders, diseases, or conditions in a subject.

EXAMPLES

The following examples are included for illustrative purposes. However, it is to be understood that these examples do not limit the present invention and are intended merely to suggest a method for practicing the present invention. Those skilled in the art will recognize that the chemical reactions described can readily be adapted to prepare a variety of other compounds of the present invention, and alternative methods for preparing the compounds of the present invention are considered to be within the scope of the present invention. For example, the synthesis of compounds of the present invention that are not exemplified can be advantageously accomplished using modifications obvious to those skilled in the art, such as by appropriately protecting interfering groups, by using other suitable reagents and structural entities known in the art other than those described, and/or by making routine modifications to the reaction conditions. Alternatively, other reactions described herein or known in the art will be found applicable to the preparation of other compounds of the present invention.

Example 1

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

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

To a mixture of tofacitinib (1.5 g, 4.8 mmol, 1 eq.) and bis(4-nitrophenyl)carbonate (NPC; 1.61 g, 5.28 mmol, 1.1 eq.) in dichloromethane (DCM; 30 mL) was added triethylamine (1.2 g, 12 mmol, 2.5 eq.) under _N2 , the reaction mixture was heated to reflux for 3 h, then tert-butyl (4-aminobutyl)carbamate (0.9 g, 4.82 mmol, 1 eq.) was added and the resulting mixture was heated under reflux for 12 h. After removal of the solvent under reduced pressure, the residue was purified by chromatography on silica to give the title product (2.4 g, yield: 95%); MS (m/z): [M+H]+ calcd. for C ₂₆ H ₃₈ N ₈ O ₄ : 527.30; detected: 527.2.

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

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 (EA; 24 mL) was added 4 M HCl in ethyl acetate (9.6 mL; 38.4 mmol) dropwise at 0°C under N ₂ . The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the resulting solid was stirred in ethyl acetate (24 mL) for 0.5 h then filtered to give the desired product as the HCl salt (2.1 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₁ H ₃₀ N ₈ O ₂ : 427.25; found: 427.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.38 (d, J=6.7 Hz, 1H), 7.82 (d, J=3.9 Hz, 1H), 6.90 (s, 1H), 4.72-4.53 (m, 1H), 4.16-3.26 (m, 11H), 3.04 (d, J=6.4 Hz, 2H), 2.66-2.46 (m, 1H), 2.03-1.90 (m, 1H), 1.86-1.69 (m, 5H), 1.12 (d, J=7.1 Hz, 3H).

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

To a solution of sodium hyaluronate (molecular weight (MW) 50 kDa; 0.161 g; 0.432 mmol; 1 equiv.) in acetonitrile (22 mL) and _H2O (35 mL) were added 4-methylmorpholine (NMM; 0.066 g; 0.65 mmol; 1.5 equiv.) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT; 0.076 g; 0.432 mmol; 1 equiv.) at 0°C. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 1 h.

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) was added to the reaction mixture, and then the pH of the reaction mixture was adjusted to 6.5-7 using 4-methylmorpholine. The resulting reaction mixture was stirred for 3 days at room temperature.

To the above reaction mixture was added NaCl (257 mg; 10 equiv.) in _H2O (2 mL) and stirred for 0.5 h, then acetone (350 mL) was added dropwise to the above reaction mixture until a precipitate was formed. The mixture was filtered and the filter cake was washed with acetone (10 mL × 3). The wet filter cake was dissolved in acetonitrile (20 mL) and _H2O (40 mL) and then dialyzed using a 3.5 kDa MW cut-off membrane against 3 changes of deionized water and then lyophilized to give the title compound (0.15 g; yield: 43.2%; DSR (degree of drug substitution) = 17%). ¹ H-NMR (400 MHz, D ₂ O/d-DMSO (deuterated dimethyl sulfoxide) = 3:1) δ ^ppm 8.40-7.90 (m, 0.17H), 7.75-7.20 (m, 0.17H), 6.95-6.25 (m, 0.17H), 4.70-4.20 (m, 2.47Н), 4.00-3.23 (m, 11.91Н), 2.55-2.30 (m, 0.34Н), 1.99 (d, J=19.7 Hz, 3Н), 1.30 (t, J=6.8 Hz, 0.17Н), 1.20-1.10 (m, 0.51Н).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3, the corresponding product was obtained (0.16 g; yield: 46%; DSR=22%). NMR.

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3, the corresponding product was obtained (0.15 g; yield: 43.2%; DSR=17%). NMR.

Example 2

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

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

Following step 1 of Example 1 and using 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), the title product was obtained (1.8 g; yield: 67%); MS (m/z): [M+H]+ calcd for C ₂₉ H ₃₆ N ₈ O ₄ : 562.29; found: 562.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.33 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₄ H ₂₈ N ₈ O ₂ : 461.23; found: 461.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.30-8.08 (m, 1H), 7.7-7.3 (m, 5H), 6.80-6.50 (m, 1H), 4.55 (s, 2H), 4.19 (s, 2H), 4.09-3.88 (m, 3H), 3.6-3.19 (m, 5H), 2.44 (br, 1H), 2.0-1.5 (m, 2H), 1.25-1.0 (m, 3H).

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

Following step 3 of Example 1 and using a reaction mixture of sodium hyaluronate (MW 50 kDa; 0.163 g; 0.403 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.18 g; yield: 55%; DSR=38%); ¹ H-NMR (400 MHz, D ₂ O/d-DMSO = 3:1) δ ^ppm 9.0-7.0 (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).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.15 g; yield: 44%; DSR=21.4%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.18 g; yield: 53%; DSR=20.7%).

Example 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

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

Following step 1 of Example 1 and using tofacitinib (1.5 g, 4.8 mmol, 1 eq.) and tert-butyl (4-aminophenethyl)carbamate (1.14 g, 4.8 mmol, 1 eq.), the title product was obtained (2.2 g; yield: 79.7%); MS (m/z): [M+H]+ calcd for C ₃₀ H ₃₈ N ₈ O ₄ : 575.30; found: 575.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.33 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₀ N ₈ O ₂ : 475.25; found: 475.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.31-8.01 (m, 1H), 7.96-7.09 (m, 5H), 6.98-6.54 (m, 1H), 4.50 (dd, 2H), 4.10-3.42 (m, 5H), 3.25 (d, J=21.8 Hz, 5H), 3.02 (t, 2H), 2.43 (d, J=5.7 Hz, 1H), 1.83 (dd, J=72.1; 15.3 Hz, 2H), 1.13 (dd, J=18.5; 13.3 Hz, 3H).

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

Following step 3 of Example 1 and using 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.) the title compound was obtained (0.19 g; yield: 33.4%; DSR=19%). ¹ H-NMR (400 MHz, D ₂ O/DMSO = 3:1) δ ^ppm 8.5-8.25 (m, 0.09H), 7.90-6.72 (m, 1.23H), 4.75-4.25 (m, 1.98H), 4.02-3.17 (m, 12H), 3.10-2.75 (m, 0.76N), 2.54 (broad, 0.19N), 2.04 (s, 3H), 1.60-1.25 (m, 0.38N), 1.18-0.98 (m, 0.57N).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.147 g; yield: 44%; DSR=16%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.175 g; yield: 53%; DSR=22.9%).

Example 4

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

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

Following step 1 of Example 1 and using tofacitinib (3 g, 9.6 mmol, 1 eq.) and tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (2.38 g, 9.6 mmol, 1 eq.), the title product was obtained (3 g; yield: 54%); MS (m/z): [M+H]+ calcd for C ₂₈ H ₄₂ N ₈ O ₆ : 587.32; found: 587.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.34 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₄ N ₈ O ₄ : 487.32; found: 487.2. ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.40 (br, 1H), 7.83 (s, 1H), 6.91 (s, 1H), 4.7-4.5 (m, 1H), 4.06-3.92 (m, 3H), 3.83-3.36 (m, 16H), 3.17 (s, 2H), 2.56 (s, 1H), 2.00-1.72 (m, 2H), 1.14 (dd, J=16.1; 7.0 Hz, 3H).

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

Following step 3 of Example 1 and using 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.) the title compound was obtained (0.2 g; yield: 33%; DSR=36.3%). ¹ H-NMR (400 MHz, D ₂ O/d-DMSO = 3:1) δ ^ppm 8.52-7.93 (m, 0.37H), 7.82-7.20 (m, 0.33H), 6.99-6.26 (m, 0.39H), 4.75-3.8 (m, 5.09H), 3.8-2.75 (m, 14.93H), 2.5-2.25 (m, 0.36H), 2.05 (s, 3H), 1.82-1.30 (m, 0.73H), 1.2-0.8 (m, 1.09H).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.186 g; yield: 56.5%; DSR=32%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.198 g; yield: 60.2%; DSR=26.6%).

Example 5

Preparation of a conjugate of N-(2-(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

Step 1. Preparation of tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)carbamate

Following step 1 of Example 1 and using tofacitinib (1.5 g, 4.8 mmol, 1 eq.) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.41 g, 4.8 mmol, 1 eq.) the title product (1.8 g; yield: 60%) was obtained; MS (m/z): [M+H]+ calcd for C ₃₀ H ₄₆ N ₈ O ₇ : 631.35; found: 631.2.

Step 2. Preparation of N-(2-(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

Following step 2 of Example 1 and using tert-butyl (1-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)carbamate (1.5 g; 2.38 mmol; 1 eq.) gave the desired product as the HCl salt (1 g; yield: 80%); MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₈ N ₈ O ₅ : 531.30; found: 531.2. ¹ H NMR (400 MHz, DMSO) δ ^ppm 9.74 (s, 1H), 8.34 (d, J=7.0 Hz, 1H), 8.01 (s, 1H), 7.69 (s, 1H), 6.86 (s, 1H), 4.85 (d, J=3.1 Hz, 1H), 4.35-4.0 (m, 10H), 3.7-3.96 (m, 3H), 3.65-3.5 (m, 10H), 2.93 (d, J=4.3 Hz, 2H), 2.38 (s, 1H), 1.89-1.68 (m, 1H), 1.59 (s, 1H), 1.01 (d, J=4.8 Hz, 3H).

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

Following step 3 of Example 1 and using 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.) the title compound was obtained (0.2 g; yield: 62.7%; DSR=24%); ¹ H-NMR (400 MHz, D ₂ O-DMSO = 3:1) δ ^ppm 8.7-8.2 (m, 0.24H), 8.1-7.3 (m, 0.24H), 7.2-6.6 (m, 0.24H), 4.7-4.35 (m, 1H), 4.3-3.1 (m, 17Н), 2.7-2.5 (broad, 0.24Н), 2.14 (broad, 3Н), 1.93 (broad, 0.24Н), 1.46 (broad, 0.24Н), 1.24 (broad, 0.72Н).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.186 g; yield: 58.4%; DSR=26%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.198 g; yield: 62.2%; DSR=28%).

Example 6

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

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

Following step 1 of Example 1 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and tert-butyl hydrazinecarboxylate (0.845 g, 6.4 mmol, 1 eq.), the title product was obtained (2.23 g; yield: 75%); MS (m/z): [M+H]+ calcd for C ₂₂ H ₃₀ N ₈ O ₄ : 471.24; found: 471.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.5 g; yield: 85%); MS (m/z): [M+H]+ calcd for C ₁₇ H ₂₂ N ₈ O ₂ : 371.19; found: 371.1. ¹ H-NMR (400 MHz, CD ₃ OD) δ ^ppm 8.52 (s, 1H), 7.89 (d, J=3.1 Hz, 1H), 7.13 (d, J=3.8 Hz, 1H), 4.83 (s, 1H), 4.22-3.32 (m, 9H), 2.55 (br, 1H), 2.05-1.99 (m, 1H), 1.90-1.67 (m, 1H), 1.16 (d, J=7.0 Hz, 3H).

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

Following step 3 of Example 1 and using 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.) the title compound was obtained (0.15 g; yield: 43.2%; DSR=32%); ¹ H-NMR (400 MHz, D ₂ O-DMSO = 5:1): δ ^ppm 8.4-7.8 (m, 0.32H), 7.8-7.2 (m, 0.32H), 7.0-6.0 (m, 0.32H), 4.75-4.4 (m, 2.1H), 4.26-3.19 (m, 11.1H), 2.45 (br, 0.32H), 2.05 (br, 3H), 1.85-1.3 (m, 0.65H), 1.18 (br, 0.95H).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.18 g; yield: 48.5%; DSR=35%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.19 g; yield: 51.2%; DSR=30%).

Example 7

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

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

Following step 1 of Example 1 and using tofacitinib (3.2 g, 10.24 mmol, 1 eq.) and tert-butyl (2-aminoethyl)carbamate (1.64 g, 10.24 mmol, 1 eq.), the title product was obtained (3 g; yield: 59%); MS (m/z): [M+H]+ calcd for C ₂₄ H ₃₄ N ₈ O ₄ : 499.27; found: 499.1.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (870 mg; yield: 100%); MS (m/z): [M+H]+ calcd for C ₁₉ H ₂₆ N ₈ O ₂ : 399.22; found: 399.1. ¹ H-NMR (400 MHz, CD ₃ OD) δ ^ppm 8.49 (s, 1H), 7.95 (d, J=3.6 Hz, 1H), 7.08 (s, 1H), 4.08-3.73 (m, 7.5H), 3.71-3.41 (m, 4.5H), 3.27 (t, 2H), 2.66-2.44 (m, 1H), 2.00-1.67 (m, 2H), 1.16 (d, J=7.0 Hz, 3H).

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

Following step 3 of Example 1 and using 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.) the title compound was obtained (0.23 g; yield: 66%; DSR=18%). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm : 8.23 (m, 0.18H), 7.63 (m, 0.18H), 6.80 (m, 0.18H), 4.64-4.24 (m, 2.2H), 4.05-2.96 (m, 12.3H), 2.42 (m, 0.18Н), 1.98 (s, 3Н), 1.80-1.67 (m, 0.18Н), 1.26 (m, 0.18Н), 1.05 (m, 0.54Н). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm : 8.5-7.9 (m, 0.18H), 7.7-7.0 (m, 0.18H), 7.0-5.8 (m, 0.18H), 4.64-4.24 (m, 2.2H), 4.05-2.96 (m, 12.3H), 2.42 (br, 0.18H), 1.98 (s, 3H), 1.80-1.67 (m, 0.18H), 1.26 (br, 0.18H), 1.05 (m, 0.54H).

Example 8

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

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

Following step 1 of Example 1 and using tofacitinib (1 g, 3.2 mmol, 1 eq.) and tert-butyl (4-aminobutyl)(methyl)carbamate (0.65 g, 3.2 mmol, 1 eq.), the title product was obtained (1 g; yield: 58%); MS (m/z): [M+H]+ calcd for C ₂₇ H ₄₀ N ₈ O ₄ : 541.32; found: 541.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.88 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₂ H ₃₂ N ₈ O ₂ : 441.26; found: 441.1. ¹ H-NMR (400 MHz, CD ₃ OD) δ ^ppm 8.50 (d, J=4.7 Hz, 1H), 7.94 (d, J=3.7 Hz, 1H), 7.07 (s, 1H), 4.20-3.81 (m, 3H), 3.73-3.32 (m, 8H), 3.08 (t, 2H), 2.72 (s, 3H), 2.55 (br, 1H), 1.99 (br, 1H), 1.91-1.67 (m, 5H), 1.16 (d, J=6.9; 5.2 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.169 g; 0.42 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.236 g; yield: 34.4%; DSR=10%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.4-7.9 (t, 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).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.18 g; yield: 52.3%; DSR=6%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.17 g; yield: 49.4%; DSR=3%).

Example 9

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

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)-l-methylhydrazine-1-carboxylate

Following step 1 of Example 1 and using tofacitinib (3.124 g, 10 mmol, 1 eq.) and tert-butyl 1-methylhydrazine-1-carboxylate (1.45 g, 10 mmol, 1 eq.), the title product was obtained (2.2 g; yield: 46%); MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₂ N ₈ O ₄ : 485.25; found: 485.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (870 mg; yield: 100%); MS (m/z): [M+H]+ calcd for C ₁₈ H ₂₄ N ₈ O ₂ : 385.20; found: 385.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.44 (d, J=5.0 Hz, 1H), 7.79 (d, J=3.5 Hz, 1H), 6.98 (s, 1H), 4.75 (s, 1H), 4.15-3.79 (m, 4H), 3.72-3.32 (m, 5H), 3.03 (s, 3H), 2.55 (br, 1H), 1.97 (br, 1H), 1.83 (br, 1H), 1.13 (dd, J=15.1; 7.1 Hz, 3H).

Step 3. Preparation of the 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

Following step 2 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.192 g; 0.475 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.323 g; yield: 90%; DSR=15%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.4-8.2 (m, 0.15H), 7.8-7.5 (m, 0.15H), 7.05-6.75 (m, 0.15H), 4.75-4.2 (m, 2.9H), 4.0-3.2 (m, 11H), 2.47 (broad, 0.15N), 2.02 (broad, 3N), 1.30 (broad, 0.15N), 1.08 (m, 0.45N).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.303 g; yield: 83%; DSR=10%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.327 g; yield: 90.3%; DSR=15%).

Example 10

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

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

Following step 1 of Example 1 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and tert-butyl 1-methylhydrazine-1-carboxylate (1.1 g, 6.4 mmol, 1 eq.), the title product was obtained (0.8 g; yield: 25%); MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₄ N ₈ O ₄ : 511.27; found: 511.1.

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

Following step 2 of Example 1 and using 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), the desired product was obtained as the HCl salt (0.67 g; yield: 96%); MS (m/z): [M+H]+ calcd for C ₂₀ H ₂₆ N ₈ O ₂ : 411.22; found: 411.1. ¹ H-NMR (400 MHz, CD ₃ OD) δ ^ppm 8.50 (d, J=4.2 Hz, 1H), 7.86 (s, 1H), 7.10 (d, J=3.8 Hz, 1H), 6.08-5.98 (m, 1H), 5.61-5.52 (m, 2H), 4.34-3.73 (m, 6H), 3.73-3.32 (m, 6H), 2.53 (br, 1H), 2.05-1.9 (m, 1H), 1.87-1.64 (m, 1H), 1.17 (d, J=7.0 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.182 g; 0.45 mmol; 1 eq.) and N'-allyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methyl-piperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g; 0.45 mmol; 1 eq.) the title compound was obtained (0.172 g; yield: 48.6%; DSR=5%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.50-8.17 (m, 0.05H), 7.85-7.55 (m, 0.05H), 7.05-6.8 (m, 0.05H), 6.17-5.60 (m, 0.15H), 4.66-4.31 (m, 2H), 4.08-3.29 (m, 10.6H), 2.47 (broad, 0.05H), 2.01 (s, 3H), 1.30 (m, 0.1H), 1.19-0.98 (m, 0.15H).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.175 g; yield: 49%; DSR=3%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.186 g; yield: 52.3%; DSR=1%).

Example 11

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

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

Following step 1 of Example 1 and using 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.), the title product was obtained (1 g; yield: 62.5%); MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₅ N ₉ O ₄ : 526.28; found: 526.1.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.7 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₃ H ₂₇ N ₉ O ₂ : 462.23; found: 462.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.41-8.31 (m, 2H), 8.1-7.9 (m, 3H), 7.78-7.68 (m, 1H), 6.83 (d, J=2.4 Hz, 2H), 4.67-4.49 (m, 1H), 4.26 (s, 2H), 4.14-3.82 (m, 5H), 3.66-3.51 (m, 2H), 3.45-3.2 (m, 3H), 2.49 (br, 1H), 2.03-1.85 (m, 1H), 1.77 (m, 1H), 1.13 (dd, J=24.3; 6.3 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.161 g; 0.4 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.17 g; yield: 50%; DSR=20.5%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.6-7.3 (m, 1.12H), 7.2-6.8 (m, 0.11H), 4.7-4.3 (m, 2H), 4.02-3.17 (m, 10.46H), 2.40 (br., 0.21H), 2.00 (s, 3Н), 1.61 (broad, 0.21Н), 1.35-1.2 (m, 0.21Н), 1.15-0.85 (m, 0.65Н).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.177 g; yield: 52.6%; DSR=22.2%).

Example 12

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

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

Following step 1 of Example 1 and using 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.), the title product was obtained (0.65 g; yield: 65%); MS (m/z): [M+H]+ calcd for C ₃₂ H ₄₁ N ₉ O ₄ : 616.33; found: 616.1.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.538 mg; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₇ H ₃₃ N ₉ O ₂ : 516.28; found: 516.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.28 (s, 1H), 7.68 (s, 1H), 7.55-7.4 (m, 2H), 7.25-7.10 (m, 2H), 6.78 (s, 1H), 4.55 (s, 1H), 4.12-2.92 (m, 17H), 2.47 (broad, 1H), 2.0-1.85 (m, 1H), 1.85-1.65 (m, 1H), 1.11 (dd, J=16.6; 7.0 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.145 g; 0.36 mmol; 1 eq.) 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 eq.), the title compound (0.13 g; yield: 41%; DSR=19%) was obtained; ¹ H-NMR (400 MHz, D ₂ O) δ ppm ^-1 8.4-6.8 (m, 1.33H), 4.7-4.4 (m, 2H), 3.9-3.42 (m, 12Н), 3.34 (m, 1.42Н), 2.47 (broad, 0.19Н), 2.02 (s, 3Н), 1.29 (broad, 0.38Н), 1.06 (broad, 0.57Н).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.16 g; yield: 49.7%; DSR=20%).

Example 13

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

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

Following step 1 of Example 1 and using 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.), the title product was obtained (1.3 g; yield: 58.7%); MS (m/z): [M+H]+ calcd for C ₃₁ H ₄₀ N ₁₀ O ₄ : 617.32; found: 617.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.89 g; yield: 99%); MS (m/z): [M+H]+ calcd for C ₂₆ H ₃₂ N ₁₀ O ₂ : 517.28; found: 517.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.33 (d, J=11.6 Hz, 1H), 8.04 (d, J=13.2 Hz, 1H), 7.85 (d, J=19.7 Hz, 2H), 7.71 (d, J=8.7 Hz, 1H), 6.83 (s, 1H), 4.62-4.57 (m, 1H), 4.10-3.76 (m, 2H), 3.65-3.21 (m, 15H), 2.47 (m, 1H), 1.94 (m, 1H), 1.77 (m, 1H), 1.12 (dd, J=24.1; 7.0 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.145 g; 0.36 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.17 g; yield: 33%; DSR = 28%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.72-6.98 (m, 1.7H), 4.70-4.3 (m, 2H), 3.80-3.28 (m, 15H), 2.84-2.67 (m, 0.29H), 1.95 (s, 3H), 1.40-1.10 (m, 0.66H), 1.1-0.9 (m, 0.85H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.22 g; yield: 68%; DSR = 20%).

Example 14

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

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

Following step 1 of Example 1 and using tofacitinib (1.24 g, 3.96 mmol, 1 eq.) and tert-butyl (2-(4-aminophenoxy)ethyl)carbamate (1 g, 3.96 mmol, 1 eq.), the title product was obtained (1.3 g; yield: 55.6%); MS (m/z): [M+H] ⁺ calcd for C ₃₀ H ₃₈ N ₈ O ₅ : 591.30; found: 591.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.892 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₅ H ₃₀ N ₈ O ₃ : 491.24; found: 491.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.15 (s, 1H), 7.51 (s, 1H), 7.34 (dd, J=16.5; 8.8 Hz, 2H), 7.03 (t, 2H), 6.63 (s, 1H), 4.45 (s, 1H), 4.38-3.38 (m, 10H), 3.21 (d, J=15.1 Hz, 2H), 2.39 (s, 1H), 2.0-1.6 (m, 2H), 1.08 (dd, J=16.9; 6.8 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.153 g; 0.38 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.22 g; yield: 66%; DSR = 11%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.0-6.6 (m, 0.77H), 4.53 (br, 1.58H), 4.01-3.19 (m, 12H), 2.77-2.58 (m, 0.11H), 1.95 (s, 3H), 1.28 (broad, 0.22H), 1.15-0.95 (m, 0.33H).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.24 g; yield: 73%; DSR = 10%).

Example 15

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

Stage 1. Obtaining tert-butyl (4-(4-nitrophenoxy)butyl)carbamate

To a solution of tert-butyl (4-hydroxybutyl)carbamate (3.7 g; 19.5 mmol; 1.1 eq.) in tetrahydrofuran (THF; 40 mL) was added NaH (1.772 g; 44.3 mmol; 2.5 eq.) at 0 °C under N ₂ atmosphere. The reaction mixture was stirred at 0 °C for 30 min and then 1-fluoro-4-nitrobenzene (2.5 g; 17.72 mmol; 1 eq.) was added. The resulting mixture was heated under reflux for 12 h. After most of the 1-fluoro-4-nitrobenzene was consumed, the reaction mixture was quenched with a saturated solution of NH ₄ Cl in water (100 mL) and extracted with ethyl acetate (40 mL × 2). The combined organic phase was washed with a saturated solution of NaCl in water (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under high vacuum. The residue was purified by chromatography on silica to give the title product (4 g; yield: 73%); MS (m/z): [M+H] ⁺ calcd. for C ₁₅ H ₂₂ N ₂ O ₅ : 311.15; found: 311.1.

Step 2. Obtaining tert-butyl (4-(4-aminophenoxy)butyl)carbamate

To a solution of tert-butyl (4-(4-nitrophenoxy)butyl)carbamate (2 g, 6.444 mmol, 1 eq.) in methanol (30 mL) was added 10% Pd/C (0.2 g) and the reaction mixture was stirred under H ₂ using a balloon for 24 h at room temperature. After tert-butyl (4-(4-nitrophenoxy)butyl)carbamate was completely consumed, the reaction mixture was filtered through a pad of celite and the pad was washed with methanol (10 mL × 2). The combined filtrates were concentrated to give the title product (1.8 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd. for C ₁₅ H ₂₄ N ₂ O ₃ : 281.18; found: 281.1.

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

Following step 1 of Example 1 and using tofacitinib (tofa; 2.23 g; 7.133 mmol; 1 eq.) and tert-butyl (4-(4-aminophenoxy)butyl)carbamate (2 g; 7.133 mmol; 1 eq.), the title product was obtained (2.6 g; yield: 59%); MS (m/z): [M+H] ⁺ calcd for C ₃₂ H ₄₂ N ₈ O ₅ : 619.33; found: 619.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.1 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd. For C ₂₇ H ₃₄ N ₈ O ₃ : 519.28; found: 519.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.14-8.11 (d, J=10.8 Hz, 1H), 7.43-7.21 (m, 3H), 6.83 (d, J=8.6 Hz, 2H), 6.70-6.45 (m, 1H), 4.49 (br, 1H), 4.12-3.09 (m, 13H), 2.36 (m, 1H), 1.85-1.55 (m, 6H), 1.04 (d, J=7.4 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.153 g; 0.38 mmol; 1 eq.) 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 (0.2 g; 0.38 mmol; 1 eq.) the title compound was obtained (0.22 g; yield: 66%; DSR = 31%); ¹ n-nmr (400 MHz, D ₂ O) δ ^ppm 8.60-5.22 (m, 2.17H), 4.7-4.2 (m, 3.1H), 4.0-3.25 (m, 12H), 3.3-2.9 (m, 1.24H), 2.84-2.11 (m, 0.31H), 2.02 (s, 3H), 1.79 (broad, 1.24H), 1.30 (broad, 0.62H), 1.15-0.5 (m, 0.93H).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.123 g; yield: 36%; DSR = 4%).

Example 16

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

Stage 1. Obtaining tert-butyl (2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate

To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (3.2 g, 15.59 mmol, 1.1 eq.) in THF (40 mL) was added NaH (1.42 g, 35.425 mmol, 2.5 eq.) at 0 °C under n ₂ , the reaction mixture was stirred at 0 °C for 30 min, then 1-fluoro-4-nitrobenzene (2 g, 14.17 mmol, 1 eq.) was added, and the resulting mixture was refluxed for 12 h. After most of the 1-fluoro-4-nitrobenzene was consumed, the reaction mixture was quenched with a saturated solution of NH ₄ Cl in water (100 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phase was washed with a saturated solution of NaCl in water (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under high vacuum. The residue was purified by chromatography on silica to give the title product (2 g; yield: 43.3%); MS (m/z): [M+H] ⁺ calcd. for C ₁₅ H ₂₂ N ₂ O ₆ : 327.15; found: 327.1.

Step 2. Obtaining tert-butyl (2-(2-(4-aminophenoxy)ethoxy)ethyl)carbamate

To a solution of tert-butyl (2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate (2 g, 6.13 mmol, 1 eq.) in methanol (40 mL) was added 10% Pd/C (0.2 g) and stirred under H ₂ using a balloon for 24 h at room temperature. After tert-butyl (2-(2-(4-nitrophenoxy)ethoxy)ethyl)carbamate was completely consumed, the reaction mixture was filtered through a pad of celite 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): [M+H] ⁺ calcd. for C ₁₅ H ₂₄ N ₂ O ₄ : 297.17; found: 297.1.

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

Following step 1 of Example 1 and using 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.), the title product was obtained (1.8 g; yield: 57%); MS (m/z): [M+H] ⁺ calcd for C ₃₂ H ₄₂ N ₈ O ₆ : 635.32; found: 635.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.89 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₇ H ₃₄ N ₈ O ₄ : 535.27; found: 535.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.19 (d, J=9.4 Hz, 1H), 7.52 (d, J=15.7 Hz, 1H), 7.37 (d, J=8.8 Hz, 2H), 6.99 (d, J=8.8 Hz, 2H), 6.72-6.50 (m, 1H), 4.63-4.54 (m, 1H), 4.23 (s, 2H), 4.09-3.81 (m, 7H), 3.74-3.12 (m, 8H), 2.42 (s, 1H), 1.77 (m, 2H), 1.08 (d, J=6.8 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.141 g; 0.35 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.15 g; yield: 32%; DSR = 25%); ¹ h-nmr (400 MHz, D ₂ O) δ ^ppm 8.49-7.25 (m, 0.76H), 7.25-5.75 (m, l,0H), 4.78-4.02 (m, 3.23H), 4.02-3.22 (m, 12H), 3.2-2.75 (m, 1.23H), 2.5 (broad, 0.25H), 2.01 (s, 3H), 1.63-1.16 (m, 0.5H), 1.25-0.5 (m, 0.75H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.132 g; yield: 42%; DSR = 28%).

Example 17

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-oxopropanenitrile and HA

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

Following step 1 of Example 1 and using tofacitinib (1.68 g, 5.37 mmol, 1 eq.) and tert-butyl piperazine-1-carboxylate (1 g, 5.37 mmol, 1 eq.), the title product was obtained (0.9 g; yield: 32%); MS (m/z): [M+H] ⁺ calcd for C ₂₆ H ₃₆ N ₈ O ₄ : 525.29; found: 525.2.

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-oxopropanenitrile hydrochloride

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.7 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₁ H ₂₈ N ₈ O ₂ : 425.23; found: 425.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.41 (d, J=5.5 Hz, 1H), 7.59 (d, J=3.9 Hz, 1H), 7.04 (s, 1H), 4.74-4.57 (m, 1H), 4.17-3.82 (m, 7H), 3.80-3.06 (m, 10H), 2.68-2.51 (m, 1H), 2.06-1.90 (m, 1H), 1.89-1.71 (m, 1H), 1.16 (dd, J=14.8; 7.1 Hz, 3H).

Step 3. Preparation of the 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-oxopropanenitrile and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.175 g; 0.434 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-carboxamide hydrochloride (0.2 g; 0.434 mmol; 1 eq.) the title compound was obtained (0.2 g; yield: 57.3%; DSR = 24%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.21 (br, 0.24H), 7.33 (b, 0.24H), 6.91 (b, 0.24H), 4.7-4.3 (m, 2.32H), 4.18-3.12 (m, 14H), 2.46 (broad, 0.24H), 2.02 (s, 3H), 1.79 (broad, 0.24H), 1.30 (m, 0.24H), 1.18-0.93 (m, 0.72H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.162 g; yield: 46.4%; DSR = 28%).

Example 18

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-oxopropanenitrile and HA

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

Following step 1 of Example 1 and using tofacitinib (1.895 g, 6.066 mmol, 1 eq.) and tert-butyl (4-aminocyclohexyl)carbamate (1.3 g, 6.066 mmol, 1 eq.), the title product was obtained (1.785 g; yield: 53.2%); MS (m/z): [M+H] ⁺ calcd for C ₂₈ H ₄₀ N ₈ O ₄ : 553.32; found: 553.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1 g; yield: 77%); MS (m/z): [M+H] ⁺ calcd for C ₂₃ H ₃₂ N ₈ O ₂ : 453.26; found: 453.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.6-8.2 (m, 1H), 7.90-7.70 (m, 1H), 6.89 (broad, 1H), 4.69-4.59 (m, 1H), 4.14-3.13 (m, 11H), 2.62-2.49 (m, 1H), 2.31-2.11 (m, 1H), 2.05-1.58 (m, 9H), 1.15 (dd, J=6.6; 5.4 Hz, 3H).

Step 3. Preparation of the 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-oxopropanenitrile and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.178 g; 0.44 mmol; 1 eq.) 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-oxopropanenitrile hydrochloride (0.2 g; 0.44 mmol; 1 eq.), the title compound was obtained (0.2 g; yield: 55%; DSR = 25%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.4-7.8 (m, 0.25H), 7.7-7.2 (m, 0.25H), 6.90-6.0 (m, 0.25H), 4.45 (br., 2.5H), 3.77-3.27 (m, 12H), 3.17-3.06 (m, 0.51H), 2.42 (broad, 0.25H), 1.96 (s, 3H), 1.85-1.52 (m, 2H), 1.24 (broad, 0.5H), 1.1-0.9 (m, 0.75H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.109 g; yield: 30%; DSR = 9%).

Example 18

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-oxopropanenitrile and HA

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

Following step 1 of Example 1 and using tofacitinib (1.895 g, 6.066 mmol, 1 eq.) and tert-butyl (4-aminocyclohexyl)carbamate (1.3 g, 6.066 mmol, 1 eq.), the title product was obtained (1.785 g; yield: 53.2%); MS (m/z): [M+H] ⁺ calcd for C ₂₈ H ₄₀ N ₈ O ₄ : 553.32; found: 553.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1 g; yield: 77%); MS (m/z): [M+H] ⁺ calcd for C ₂₃ H ₃₂ N ₈ O ₂ : 453.26; found: 453.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.6-8.2 (m, 1H), 7.90-7.70 (m, 1H), 6.89 (broad, 1H), 4.69-4.59 (m, 1H), 4.14-3.13 (m, 11H), 2.62-2.49 (m, 1H), 2.31-2.11 (m, 1H), 2.05-1.58 (m, 9H), 1.15 (dd, J=6.6; 5.4 Hz, 3H).

Step 3. Preparation of the 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-oxopropanenitrile and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.178 g; 0.44 mmol; 1 eq.) 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-oxopropanenitrile hydrochloride (0.2 g; 0.44 mmol; 1 eq.), the title compound was obtained (0.2 g; yield: 55%; DSR = 25%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.4-7.8 (m, 0.25H), 7.7-7.2 (m, 0.25H), 6.90-6.0 (m, 0.25H), 4.45 (br., 2.5H), 3.77-3.27 (m, 12H), 3.17-3.06 (m, 0.51H), 2.42 (broad, 0.25H), 1.96 (s,3H), 1.85-1.52 (m, 2H), 1.24 (broad, 0.5H), 1.1-0.9 (m, 0.75H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.109 g; yield: 30%; DSR = 9%).

Example 19

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

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

Following step 1 of Example 1 and using tofacitinib (1.46 g, 4.67 mmol, 1 eq.) and tert-butyl (2-aminocyclohexyl)carbamate (1 g, 4.67 mmol, 1 eq.), the title product was obtained (2 g; yield: 78%); MS (m/z): [M+H] ⁺ calcd for C ₂₈ H ₄₀ N ₈ O ₄ : 553.32; found: 553.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (1.15 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₃ H ₃₂ N ₈ O ₂ : 453.26; found: 453.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.52-8.36 (m, 1H), 7.90-7.78 (m, 1H), 6.93 (s, 1H), 4.63 (m, 2H), 4.21-3.78 (m, 4H), 3.70-3.25 (m, 6H), 2.55 (broad, 1H), 2.14 (m, 1H), 2.04-1.31 (m, 9H), 1.20-1.04 (m, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.161 g; 0.4 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.2 g; yield: 59%; DSR = 18%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.62-7.85 (m, 0.18H), 7.82-7.41 (m, 0.18H), 6.86-6.70 (m, 0.18H), 4.7-4.2 (m, 2H), 4.23-2.54 (m, 12.16H), 2.42 (broad, 0.18H), 2.00 (s, 3H), 1.50-1.2 (m, 0.86H), 1.22-0.87 (m, 1.05H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.224 g; yield: 67%; DSR = 7%).

Example 20

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

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

Following step 1 of Example 1 and using tofacitinib (2.5 g, 8.114 mmol, 1 eq.) and tert-butyl 1-ethylhydrazine-1-carboxylate (1.3 g, 8.114 mmol, 1 eq.), the title product was obtained (1.745 g; yield: 43%); MS (m/z): [M+H] ⁺ calcd for C ₂₄ H ₃₄ N ₈ O ₄ : 499.27; found: 499.2.

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

Following step 2 of Example 1 and using 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 eq), the desired product was obtained as the HCl salt (1 g; yield: 82%); MS (m/z): [M+H] ⁺ calcd for C ₁₉ H ₂₆ N ₈ O ₂ : 399.22; found: 399.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.45 (d, J=4.8 Hz, 1H), 7.81 (d, J=3.6 Hz, 1H), 7.00 (s, 1H), 4.76-4.67 (m, 1H), 4.19-3.79 (m, 3H), 3.72-3.31 (m, 8H), 2.56 (broad, 1H), 2.06-1.70 (m, 2H), 1.35 (broad, 3H), 1.14 (dd, J=15.2; 7.1 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.186 g; 0.46 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.12 g; yield: 34%; DSR = 9%); ¹ n-nmr (400 MHz, D ₂ O) δ ^ppm 8.4-8.3 (m, 0.09H), 7.69 (d, J=23.0 Hz, 0.09H), 6.95 (d, J=10.7 Hz, 0.09H), 4.60-4.41 (m, 2.08H), 3.84-3.34 (m, 10H), 2.47 (m, 0.09H), 2.01 (s, 3H), 1.82-1.74 (m, 0.19H), 1.31 (broad, 0.27H), 1.07 (broad, 0.27H).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.198 g; yield: 55.3%; DSR = 5%).

Example 21

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

Step 1. Preparation of methyl 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-lysinate

Following step 1 of Example 1 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and methyl N6-(tert-butoxycarbonyl)-L-lysinate hydrochloride (1.9 g, 6.4 mmol, 1 eq.), the title product was obtained (2.2 g; yield: 57.4%); MS (m/z): [M+H] ⁺ calcd for C ₂₉ H ₄₂ N ₈ O ₆ : 599.32; found: 599.2.

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

Following step 2 of Example 1 and using methyl 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-lysinate (1.5 g; 2.5 mmol; 1 eq.), the desired product was obtained as the HCl salt (1.34 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₄ H ₃₄ N ₈ O ₄ : 499.27; found: 499.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.41 (d, J=4.4 Hz, 1H), 7.79 (dd, J=9.0; 4.1 Hz, 1H), 6.88 (dd, J=6.7; 2.3 Hz, 1H), 4.70-4.54 (m, 2H), 4.26-3.80 (m, 8H), 3.67-3.37 (m, 5H), 3.00 (t, J=7.6 Hz, 3H), 2.67-2.41 (m, 1H), 2.05-1.93 (m, 2H), 1.89-1.62 (m, 4H), 1.62-1.41 (m, 3H), 1.15 (dd, J=15.9; 7.1 Hz, 3N).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.151 g; 0.374 mmol; 1 eq.) and methyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysinate hydrochloride (0.2 g; 0.374 mmol; 1 eq.), the title compound was obtained (0.2 g; yield: 61%; DSR = 19%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.25 (m, 0.2H), 7.56 (m, 0.2H), 6.72 (m, 0.2H), 4.72-2.85 (m, 15.2H), 2.45 (m, 0.4H), 2.02 (s, 3H), 1.56 (m, 1.2H), 1.31 (m, 0.2H), 1.11 (m, 0.6H). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 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 (broad, 0.2H), 1.2-0.95 (m, 0.6H).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.2 g; yield: 61%; DSR = 23.2%).

Example 22

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-carbothioamide and HA

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-carbothioamido)butyl)carbamate

To a mixture of tofacitinib (2 g; 6.4 mmol; 1 eq.) and O-phenyl carbonochloridothioate (2.14 g; 7.04 mmol; 1.1 eq.) in dichloromethane (40 mL) was added N,N-dimethylpyridin-4-amine (DMAP; 2.15 g; 17.6 mmol; 2.5 eq.) under N ₂ . The reaction mixture was heated to reflux for 3 h, then tert-butyl (4-aminobutyl)carbamate (1.2 g, 6.4 mmol, 1 eq) was added and the mixture was heated at reflux for another 12 h. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure and the residue was purified by silica chromatography to give the title product (1.16 g, yield: 33.4%); MS (m/z): [M+H] ⁺ calcd for C ₂₆ H ₃₈ N ₈ O ₃ S; 543.28; found: 543.2.

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-carbothioamide hydrochloride

Following step 2 of Example 1 and using tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamido)butyl)carbamate (1.16 g, 2.14 mmol, 1 eq), the desired product was obtained as the HCl salt (1 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₁ H ₃₀ N ₈ OS; 443.23; found: 443.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.36 (d, J=36.1 Hz, 2H), 6.92 (s, 1H), 4.63 (d, J=1.2 Hz, 1H), 4.1-3.75 (m, 4H), 3.39 (s, 3H), 3.23 (s, 4H), 2.96 (t, J=7.5 Hz, 3H), 2.47 (s, 1H), 2.0-1.6 (m, 5H), 1.23-1.06 (m, 4H).

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-carbothioamide and HA

Following step 3 of Example 1 and using 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-carbothioamide hydrochloride (0.2 g; 0.42 mmol; 1 eq.), the title compound was obtained (0.18 g; yield: 53%; DSR = 20%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.8-7.4 (m, 0.48H), 7.0-6.0 (m, 0.12H), 4.7-4.25 (m, 2H), 4.18-3.03 (m, 12.8H), 2.47 (br., 0.2H), 2.03 (s, 3H), 1.83 (broad, 1H), 1.30 (broad, 0.2H), 1.15-1.0 (m, 0.6H).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.196 g; yield: 56.8%; DSR = 30%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.203 g; yield: 58.8%; DSR = 8%).

Example 23

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-carbothioamide and HA

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-carbothioamido)benzyl)carbamate

Following step 1 of Example 22 and using tofacitinib (3 g, 9.6 mmol, 1 eq.) and tert-butyl (4-aminobenzyl)carbamate (2.134 g, 9.6 mmol, 1 eq.), the title product was obtained (0.3 g; yield: 5.5%); MS (m/z): [M+H] ⁺ calcd for C ₂₉ H ₃₆ N ₈ O ₃ S; 577.26; found: 577.2.

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-carbothioamide hydrochloride

Following step 2 of Example 1 and using tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamido)benzyl)carbamate (0.375 g, 0.65 mmol, 1 eq), the desired product was obtained as the HCl salt (0.26 g; yield: 78.8%); MS (m/z): [M+H] ⁺ calcd for C ₂₄ H ₂₈ N ₈ OS; 477.21; found: 477.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.43-8.30 (m, 2H), 7.85 (d, J=8.1 Hz, 2H), 7.51 (d, J=8.1 Hz, 2H), 6.94 (s, 1H), 4.1-3.65 (m, 6H), 3.63-3.25 (m, 6H), 2.47 (s, 1H), 1.93-1.56 (m, 2H), 1.15-1.05 (m, 3H).

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-carbothioamide and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.157 g; 0.39 mmol; 1 eq.) and N-(4-(aminomethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamide hydrochloride (0.2 g; 0.39 mmol; 1 eq.) the title compound was obtained (0.18 g; yield: 55%; DSR = 12%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.5-6.75 (m, 0.91H), 4.52 (m, 3H), 4.24-2.85 (m, 10.45H), 2.55-2.42 (m, 0.12H), 2.03 (s, 3H), 1.30 (m, 0.24H), 1.13 (m, 0.36H).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.166 g; yield: 49.8%; DSR = 24.3%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.132 g; yield: 39.7%; DSR = 14.7%).

Example 24

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-carbothioamide and HA

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-carbothioamido)phenethyl)carbamate

Following step 1 of Example 22 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and tert-butyl (4-aminophenethyl)carbamate (1.512 g, 6.4 mmol, 1 eq.), the title product was obtained (0.78 g; yield: 20.6%); MS (m/z): [M+H] ⁺ calcd. For C ₃₀ H ₃₈ N ₈ O ₃ S; 591.28; found: 591.2.

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-carbothioamide hydrochloride

Following step 2 of Example 1 and using tert-butyl (4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioamido)phenethyl)carbamate (0.55 g; 0.931 mmol; 1 eq.), the desired product was obtained as the HCl salt (0.49 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₅ H ₃₀ N ₈ OS; 491.23; found: 491.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.43-8.30 (m, 2H), 7.73 (d, J=7.7 Hz, 2H), 7.34 (d, J=7.9 Hz, 2H), 6.95 (s, 1H), 4.69 (s, 1H), 3.97-3.71 (m, 3H), 3.61-3.37 (m, 4H), 3.23-3.04 (m, 4H), 2.95 (t, 2H), 2.48 (s, 1H), 1.92-1.62 (m, 2H), 1.12 (dd, J=13.9; 6.0 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.153 g; 0.38 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-carbothioamide hydrochloride (0.2 g; 0.38 mmol; 1 eq.) the title compound was obtained (0.18 g; yield: 55%; DSR = 14%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-6.85 (m, 0.98H), 4.46 (broad, 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).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.185 g; yield: 56%; DSR = 15%).

Example 25

Preparation of a conjugate of N-(2-(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-carbothioamide and HA

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-azatridecan-13-yl)carbamate

Following step 1 of Example 22 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (1.871 g, 6.4 mmol, 1 eq.), the title product was obtained (1.12 g; yield: 27%); MS (m/z): [M+H] ⁺ calcd for C ₃₀ H ₄₆ N ₈ O ₆ S: 647.33; found: 647.2.

Step 2. Preparation of N-(2-(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-carbothioamide hydrochloride

Following step 2 of Example 1 and using 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-azatridecan-13-yl)carbamate (1.1 g; 1.7 mmol; 1 eq.), the desired product was obtained as the HCl salt (0.84 g; yield: 85%); MS (m/z): [M+H] ⁺ calcd for C ₂₅ H ₃₈ N ₈ O ₄ S; 547.27; found: 547.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.5-8.0 (m, 2H), 6.61 (s, 1H), 4.75-4.40 (m, 2H), 4.05-3.5 (m, 18H), 3.40-3.3 (m, 6H), 2.45 (broad, 1H), 2.00-1.6 (m, 2H), 1.17-1.04 (m, 3H).

Step 3. Preparation of a conjugate of N-(2-(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-carbothioamide and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.137 g; 0.34 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-carbothioamide hydrochloride (0.2 g; 0.34 mmol, 1 eq.) gave the title compound (0.17 g; yield: 55%; DSR = 31%); ¹ H-NMR (400 MHz, D ₂ O) 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 (broad, 0.31H), 1.93 (s, 3H), 1.62-1.15 (m, 0.62H), 1.10-0.75 (m, 0.93H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.185 g; yield: 58.8%; DSR = 25%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.231 g; yield: 73.4%; DSR = 30%).

Example 26

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

Step 1. Preparation of methyl 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-lysinate

Following step 1 of Example 22 and using tofacitinib (2 g, 6.4 mmol, 1 eq.) and methyl N6-(tert-butoxycarbonyl)-L-lysinate hydrochloride (1.9 g, 6.4 mmol, 1 eq.), the title product was obtained (0.675 g; yield: 17.2%); MS (m/z): [M+H] ⁺ calcd for C ₂₉ H ₄₂ N ₈ O ₅ S; 615.30; found: 615.2.

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

Following step 2 of Example 1 and using methyl 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-lysinate (0.625 g; 1.017 mmol; 1 eq.), the desired product was obtained as the HCl salt (0.56 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₄ H ₃₄ N ₈ O ₃ S: 515.25; found: 515.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.45 (s, 1H), 8.33 (s, 1H), 6.92 (s, 1H), 5.09 (t, J=6.2 Hz, 1H), 4.63 (br, 1H), 3.95-3.67 (m, 5H), 3.60-3.28 (m, 4H), 3.23 (s, 3H), 2.88 (broad, 2H), 2.47 (s, 1H), 2.21-1.96 (m, 2H), 1.92-1.42 (m, 6H), 1.10 (d, J=6.8 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.145 g; 0.36 mmol; 1 eq.) and methyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonothioyl)-L-lysinate hydrochloride (0.2 g; 0.36 mmol; 1 eq.), the title compound was obtained (0.13 g; yield: 41%; DSR = 10%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.75-7.5 (m, 0.27H), 6.87 (broad, 0.03H), 5.25 (m, 0.14H), 4.65-4.21 (m, 1.86H), 4.0-2.8 (m, 11.6H), 2.44 (broad, 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).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.117 g; yield: 36.3%; DSR = 10%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.161 g; yield: 50%; DSR = 5.7%).

Example 27

Preparation of the 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 NA

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

Following step 1 of Example 1 and using tofacitinib (1.32 g, 4.214 mmol, 1 eq.) and tert-butyl (4-(hydroxymethyl)benzyl)carbamate (2 g, 4.214 mmol, 1 eq.), the title product was obtained (1.3 g; yield: 53.5%); MS (m/z): [M+H] ⁺ calcd for C ₃₀ H ₃₇ N ₇ O ₅ ; 576.29; found: 576.2.

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

Following step 2 of Example 1 and using 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 (1.3 g, 2.25 mmol, 1 eq), the desired product was obtained as the HCl salt (1 g; yield: 87%); MS (m/z): [M+H] ⁺ calcd for C ₂₅ H ₂₉ H ₇ O ₃ ; 476.23; found: 476.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.30 (dd, J=36.1; 9.5 Hz, 1H), 7.75-7.23 (m, 5H), 6.84 (d, J=15.6 Hz, 1H), 4.70 (broad, 3H), 4.35-3.71 (m, 6H), 3.71-3.23 (m, 5H), 2.50 (d, J=33.4 Hz, 1H), 2.03-1.62 (m, 2H), 1.17-0.99 (m, 3H).

Step 3. Preparation of the 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 NA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.157 g; 0.39 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.2 g; yield: 60%; DSR = 22%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.37-7.89 (m, 0.18H), 7.75-7.22 (m, 1.36H), 4.71-4.32 (m, 2.75H), 4.29-3.20 (m, 12.02H), 3.14 (broad, 0.34Н), 2.32 (broad, 0.22Н), 2.02 (s, 3Н), 1.31 (broad, 0.45Н), 1.06 (broad, 0.65Н).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.2 g; yield: 60%; DSR = 43.6%).

In the case of the reaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.175 g; yield: 52.6%; DSR = 35.3%).

Example 28

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

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

Following step 1 of Example 1 and using tofacitinib (3 g, 9.62 mmol, 1 eq), the title product was obtained (1.7 g; yield: 28%); MS (m/z): [M+H] ⁺ calcd for C ₃₂ H ₃₉ H ₇ O ₇ ; 634.29; found: 634.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.9 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₂₇ H ₃₁ N ₇ O ₅ ; 534.24; found: 534.2. ¹ h-nmr (400 MHz, CD ₃ OD) δ ^ppm 8.55-8.34 (m, 1H), 7.52-7.42 (m, 3H), 7.41-6.74 (m, 3H), 5.28 (s, 1H), 4.46-4.21 (m, 1H), 4.09-3.85 (m, 5H), 3.82 (d, J=4.2 Hz, 3H), 3.76-3.39 (m, 5H), 3.26-3.03 (m, 1H), 2.55 (broad, 1H), 2.0-1.65 (m, 2H), 1.16 (dd, J=13.2; 5.2 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.141 g; 0.35 mmol; 1 eq.) and 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 (0.2 g; 0.35 mmol; 1 eq.) the title compound was obtained (0.2 g; yield: 32%; DSR = 20%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 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).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.16 g; yield: 50%; DSR = 22%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.238 g; yield: 74.5%; DSR = 22%).

Example 29

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

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

Following step 1 of Example 1 and using tofacitinib (1.15 g, 3.67 mmol, 1 eq.) and tert-butyl (8-hydroxyoctyl)carbamate (0.9 g, 3.668 mmol, 1 eq.), the title product was obtained (0.7 g; yield: 33%); MS (m/z): [M+H] ⁺ calcd for C ₃₀ H ₄₅ N ₇ O ₅ ; 584.35; found: 584.3. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.46 (d, J=8.8 Hz, 1H), 7.46 (dd, J=10.6; 4.2 Hz, 1H), 6.63 (d, J=4.1 Hz, 1H), 5.13 (s, 1H), 4.65-4.33 (m, 2H), 4.16-3.99 (m, 1H), 3.89-3.74 (m, 1H), 3.69-3.43 (m, 4H), 3.37 (d, J=18.7 Hz, 2H), 3.10 (d, J=6.4 Hz, 2H), 2.62-2.41 (m, 1H), 2.02-1.70 (m, 4H), 1.45-1.3 (m, 12H), 1.08 (dd, J=14.0; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using 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 eq), the desired product was obtained as the HCl salt (0.6 g; yield: 96%); MS (m/z): [M+H] ⁺ calcd for C ₂₅ H ₃₇ N ₇ O ₃ ; 484.30; found: 484.2.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.153 g; 0.38 mmol; 1 eq.) 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 eq.) the title compound was obtained (0.19 g; yield: 54%; DSR = 14%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 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).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.191 g; yield: 58.23%; DSR = 5.3%).

Example 30

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

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

To a stirred mixture of tofacitinib (3000 mg, 9.6 mmol) and bis(4-nitrophenyl) carbonate (3.22 mg, 10.6 mmol) in dichloromethane (60 mL) was added triethylamine (2.7 mL, 19.2 mmol). The reaction mixture was heated to 45°C and stirred at this temperature for 5 h. Then, the reaction mixture was cooled to room temperature. 4-(tert-Butoxycarbonylamino)-1-butanol (2000 mg, 10.6 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with saturated NaHCO ₃ solution, water, and brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (ethyl acetate:hexane = 1:1) to give the title compound (2.64 g; yield: 52.1%). MS (m/z): [M+H] ⁺ calcd for C ₂₆ H ₃₇ N ₇ O ₅ ; 528.28; found: 528.2.

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

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

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 161.2 mg; 0.399 mmol) and 4-aminobutyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg; 0.399 mmol), the title compound was obtained (0.13 g; yield: 36.7%; DSR = 12%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.20-8.02 (m, 0.12H), 7.53-7.25 (m, 0.12H), 6.82-6.62 (m, 0.12H), 4.53-4.15 (m, 2.12H), 3.93-2.89 (m, 11.56N), 2.02-1.44 (m, 3.6N), 1.18-1.09 (m, 0.12N), 1.01-0.82(m, 0.36N).

In the case of the reaction with sodium hyaluronate (MM 500 kDa) carried out in accordance with step 3 of Example 1, the corresponding product was obtained (0.125 g; yield: 35.3%; DSR = 37%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.134 g; yield: 37.8%; DSR = 32%).

Example 31

Preparation of the conjugate of (4-aminophenyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and NA

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

Following step 1 of Example 1 and using tofacitinib (1562 mg, 5 mmol) and tert-butyl (4-hydroxyphenyl)carbamate (1256 mg, 6 mmol), the title compound was obtained (1.1 g; yield: 40%). MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₃ N ₇ O ₅ : 548.25; found: 548.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.48 (d,J = 8.0 Hz, 1H), 8.14-8.05 (m, 1H), 7.61 (dd, J = 8.0; 4.2 Hz, 1H), 7.42 (m, 1H), 7.23 (d, J = 8.9 Hz, 2H), 6.89-6.74 (m, 1H), 6.58 (s, 1H), 5.15 (d, J = 3.9 Hz, 1H), 4.11-4.01 (m, 1H), 3.88-3.78 (m, 1H), 3.67-3.45 (m, 4H), 3.43-3.31 (m, 3H), 2.59-2.44 (m, 1H), 1.94 (tdd, J = 23.2; 8.8; 4.5 Hz, 1H), 1.83-1.69 (m, 1H), 1.51 (m, 9H), 1.15-1.03 (m, 3H).

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

Following step 2 of Example 1 and using [4-(tert-butoxycarbonylamino)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.8 g, 1.46 mmol) the title compound was obtained as a white solid (0.52 g; yield: 73.6%); MS (m/z): [M+H]+ calcd for C ₂₃ H ₂₅ N ₇ O ₃ : 448.20; found: 448.3.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 116 mg; 0.288 mmol) and (4-aminophenyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (150 mg; 0.288 mmol) the title compound was obtained (0.126 g; yield: 48.3%; DSR: 10%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.26-8.17 (m, 0.1H), 7.72-7.51 (m, 0.2H), 7.38-7.11 (m, 0.3H), 6.90-6.78 (m, 0.1H), 4.53-4.25 (m, 2.1H), 4.07-3.09 (m, 10.9H), 2.49-2.34 (m, 0.1H), 2.12-1.78 (m, 3.1H), 1.61-1.50 (m, 0.1H), 1.26-0.92 (m, 0.3H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.123 g; yield: 47.1%; DSR: 24%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.138 g; yield: 52.9%; DSR: 8%).

Example 32

Preparation of a conjugate of azetidin-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

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

Following step 1 of Example 1 and using tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxyazetidine-1-carboxylate (831.4 mg, 4.8 mmol), the title compound was obtained (0.48 g; yield: 23.4%). MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₃ N ₇ O ₅ : 512.25; found: 512.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.47 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 11.6; 4.2 Hz, 1H), 6.65 (dd, J = 15.6; 4.0 Hz, 1H), 5.14 (d, J = 3.0 Hz, 1H), 4.38 (dd,J = 10.0; 7.0 Hz, 2H), 4.11-3.75 (m, 3H), 3.67-3.45 (m, 5H), 3.41-3.33 (m, 3H), 2.59-2.41 (m, 1H), 2.02-1.84 (m, 1Н), 1.76 (ddd, J = 23.8; 13.5; 8.5 Hz, 2H), 1.45 (d, J = 6.8 Hz, 9H), 1.11-1.02 (m, 3H).

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

Following step 2 of Example 1 and using (1-tert-butoxycarbonyl azetidin-3-yl)-4-[[(3R,4R-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.44 g, 0.86 mmol), the title compound was obtained as a white solid (0.366 g; yield: 95%). MS (m/z): [M+H]+ calcd for C ₂₀ H ₂₅ N ₇ O ₃ : 412.20; found: 412.3.

Step 3. Preparation of a conjugate of azetidin-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 83.2 mg; 0.207 mmol) and azetidin-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (100 mg; 0.207 mmol), the title compound was obtained (0.078 g; yield: 43.4%; DSR: 12%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-8.17 (s, 0.12H), 7.63-7.50 (m, 0.12H), 6.92-6.78 (m, 0.12H), 4.58-4.26 (m, 7.0 Hz, 2.12H), 4.07-3.05 (m, 11.56H), 2.45-2.35 (m, 0.12H), 2.13-1.55 (m, 3.24H), 1.23 (m, 0.12H), 1.11-0.89 (m, 0.36H).

Example 33

Preparation of a conjugate of pyrrolidin-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

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

Following step 1 of Example 1 and using tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 3-hydroxypyrrolidine-1-carboxylate (898.8 mg, 4.8 mmol), the title compound was obtained (0.94 g; yield: 44.7%). MS (m/z): [M+H]+ calcd for C ₂₆ H ₃₅ N ₇ O ₅ : 526.26; found: 526.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.47 (d, J = 6.3 Hz, 1H), 7.38 (dd, J = 11.2; 4.1 Hz, 1H), 6.63 (d, J = 4.1 Hz, 1H), 5.11 (t, J = 11.8 Hz, 1H), 4.08-3.77 (m, 2H), 3.73-3.47 (m, 8H), 3.36 (d, J = 17.7 Hz, 3H), 2.58-2.42 (m, 1H), 2.37-2.18 (m, 2H), 1.93 (dddd, J = 23.5; 18.7; 9.0; 4.5 Hz, 1H), 1.82-1.72 (m, 2H), 1.47 (s, 9H), 1.08 (dd, J = 14.4; 7.0 Hz, 3H).

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

Following step 2 of Example 1 and using (1-tert-butoxycarbonylpyrrolidin-3-yl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.6 g, 1.14 mmol), the title compound was obtained as a white solid (0.526 g; yield: 100%). MS (m/z): [M+H]+ calcd for C ₂₁ H ₂₇ N ₇ O ₃ : 426.21; found: 426.3.

Step 3. Preparation of a conjugate of pyrrolidin-3-yl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

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

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.185 g; yield: 52.2%; DSR: 33%).

Example 34

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

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

Following step 1 of Example 1 and using tofacitinib (1249.5 mg, 4 mmol) and 3-(Boc-amino)-1-propanol (Boc = tert-butyloxycarbonyl) (841.1 mg, 4.8 mmol), the title compound was obtained (1.03 g; yield: 50%). MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₅ N ₇ O ₅ : 514.26; found: 514.2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.53 (d, J = 10.4 Hz, 1H), 7.53 (dd, J = 10.8; 4.0 Hz, 1H), 7.15 (s, 1H), 6.67 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 30.4 Hz, 1H), 4.57 (dd, J = 14.1; 8.5 Hz, 2H), 4.06-3.78 (m, 2H), 3.67-3.48 (m, 4H), 3.47-3.41 (m, 2H), 3.41-3.33 (m, 3H), 2.56-2.36 (m, 1H), 2.09-2.01 (m, 1H), 1.93 (tdd, J = 22.0; 8.8; 4.5 Hz, 1H), 1.83-1.65 (m, 2H), 1.45 (s, 9H), 1.07 (dd, J = 16.9; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using 3-(tert-butoxycarbonylamino)propyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.03 g, 2 mmol) gave the title compound as a white solid (0.9 g, 100%). MS (m/z): [M+H]+ calcd for C ₂₀ H ₂₇ N ₇ O ₃ : 414.21; found: 414.3.

Step 3. Preparation of a conjugate of 3-aminopropyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-y]pyrimidine-7-carboxylate and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 165 mg; 0.411 mmol) and 3-aminopropyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg; 0.411 mmol), the title compound was obtained (0.162 g; yield: 45.2%; DSR: 15%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.34-8.20 (m, 0.15H), 7.63-7.50 (m, 0.15H), 6.91-6.78 (m, 0.15H), 4.49-4.31 (m, 2.15H), 4.06-3.07 (m, 11.95H), 2.44-2.35 (m, 0.15H), 2.16-1.56 (m, 3.3H), 1.21 (t, J = 7.0 Hz, 0.15H), 1.05-0.90 (m, 0.45H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.161 g; yield: 44.9%; DSR: 17%).

Example 35

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

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

Following step 1 of Example 1 and using tofacitinib (1249.5 mg, 4 mmol) and tert-butyl (4-hydroxycyclohexyl)carbamate (1033.4 mg, 4.8 mmol), the title compound was obtained (0.851 g; yield: 45%). MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₉ N ₇ O ₅ : 554.30; found: 554.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.46 (d,J = 5.8 Hz, 1H), 7.42 (dd, J = 9.5; 4.2 Hz, 1H), 6.61 (d, J = 4.1 Hz, 1H), 5.19-5.04 (m, 1H), 5.03-4.89 (m, 1H), 4.45 (s, 1H), 4.08-3.76 (m, 2H), 3.63-3.46 (m, 4H), 3.36 (d, J = 16.4 Hz, 3H), 2.49 (ddd, J = 24.3; 11.9; 6.4 Hz, 1H), 2.26-2.06 (m, 4H), 1.93 (dddd, J = 18.5; 13.7; 8.9; 4.4 Hz, 1H), 1.82-1.59 (m, 4H), 1.45 (s, 9H), 1.34 (td, J = 13.3; 2.7 Hz, 2H), 1.15-0.99 (m, 3H).

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

Following step 2 of Example 1 and using [4-(tert-butoxycarbonylamino)cyclohexyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.62 g, 1.12 mmol), the title compound was obtained as a white solid (0.55 g; yield: 100%). MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₁ N ₇ O ₃ ; 454.24; found: 454.3.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 153 mg; 0.38 mmol) and (4-aminocyclohexyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg; 0.38 mmol), the title compound was obtained (0.173 g; yield: 49.9%; DSR: 11%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.24-8.02 (m, 0.11H), 7.57-7.37 (m, 0.11H), 6.80-6.64 (m, 0.11H), 4.96-4.86 (m, 0.11H), 4.57-4.27 (m, 2H), 4.07-3.04 (m, 10.99H), 2.46-2.30 (m, 0.11H), 2.27-1.76 (m, 3.77H), 1.74-1.33 (m, 0.66H), 1.22 (d, J = 6.9 Hz, 0.11H), 1.09-0.85 (m, 0.33H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.162 g; yield: 46.7%; DSR: 27%).

Example 36

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

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

Following step 1 of Example 1 and using tofacitinib (1249.5 mg, 4 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (966 mg, 4.8 mmol), the title compound was obtained (0.537 g; yield: 24.9%). MS (m/z): [M+H]+ calcd for C ₂₇ H ₃₇ N ₇ O ₅ : 540.28; found: 540.2.

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

Following step 2 of Example 1 and using (1-tert-butoxycarbonyl-4-piperidyl)-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.47 g, 0.87 mmol) the title compound was obtained as a white solid 0.25 g; yield: 58%. MS (m/z): [M+H]+ calcd for C ₂₂ H ₂₉ N ₇ O ₃ : 440.23; found: 440.3.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 169.7 mg; 0.421 mmol) and 4-piperidyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg; 0.421 mmol), the title compound was obtained (0.161 g; yield: 44.4%; DSR: 11%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-8.04 (m, 0.11H), 7.61-7.38 (m, 0.11H), 6.84-6.68 (m, 0.11H), 4.61-4.28 (m, 2.11H), 4.12-3.05 (m, 11.43Н), 2.41-2.30 (m, 0.11Н), 2.19-1.58 (m, 3.66Н), 1.20 (t, J = 6.9 Hz, 0.11Н), 1.08-0.85 (m, 0.33Н).

Example 37

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

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

To a stirred mixture of tofacitinib (1250 mg, 4 mmol) and bis(4-nitrophenyl) carbonate (1460 mg, 4.8 mmol) in dichloromethane (40 mL) was added triethylamine (1.1 mL, 8 mmol). The reaction mixture was heated to 45°C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (985.2 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with saturated NaHCO ₃ solution, water, and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure to give the title compound (1.3 g; yield: 59.8%). MS (m/z): [M+H]+ calcd for C ₂₆ H ₃₇ N ₇ O ₆ : 544.28; found: 544.2.

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

Following step 2 of Example 1 and using 2-[2-(tert-butoxycarbonylamino)ethoxy]ethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (1.3 g, 2.39 mmol), the title compound was obtained as a white solid (1.1 g; yield: 95.9%). MS (m/z): [M+H]+ calcd for C ₂₁ H ₂₉ N ₇ C ₄ : 444.22; found: 444.2.

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

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (200 mg; 0.388 mmol), the title compound was obtained (0.175 g; yield: 50%; DSR: 15%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.23-8.09 (m, 0.15H), 7.58-7.39 (m, 0.15H), 6.86-6.65 (m, 0.15H), 4.58-4.25 (m, 2.15H), 4.02-2.94 (m, 12.55H), 2.43-2.28 (m, 0.15H), 2.16-1.56 (m, 3.3H), 1.10-0.89 (m, 0.45H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.182 g; yield: 52%; DSR: 17%).

Example 38

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

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1323 mg, 3 mmol), (S)-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 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:150-1:80) to give the title compound (1.726 g; yield: 83.3%). MS (m/z): [M+H]+ calcd. for C ₃₃ H ₄₉ N ₉ O ₇ : 684.37; found: 684.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 9.89 (dd, J = 13.5; 7.5 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 7.72 (dd, J = 11.9; 4.0 Hz, 1H), 6.75-6.50 (m, 2H), 5.14 (s, 1H), 4.75-4.48 (m, 2H), 4.12-3.76 (m, 2H), 3.73-3.32 (m, 9H), 3.11 (d, J = 5.4 Hz, 2H), 2.58-2.42 (m, 1H), 2.41-2.25 (m, 2H), 2.09-1.65 (m, 6H), 1.54-1.32 (m, 15H), 1.10 (dd, J = 12.7; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using methyl (2S)-6-(tert-butoxycarbonylamino)-2-[4-[[4-[[(3R4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate (1.5 g, 2.137 mmol) the title compound was obtained as a white solid (1.426 g; yield: 99.6%). MS (m/z): [M+H]+ calcd for C ₂₈ H ₄₁ N ₉ O ₅ : 584.32; found: 584.1.

Step 3. Preparation of a conjugate of methyl (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-1-dimethylacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 123 mg; 0.305 mmol) and methyl (2S)-6-amino-2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]hexanoate hydrochloride (200 mg; 0.36 mmol) the title compound was obtained (0.13 g; yield: 45%; DSR: 5%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.29-8.16 (m, 0.05H), 7.64-7.51 (m, 0.05H), 6.82-6.65 (m, 0.05H), 4.57-4.22 (m, 2.05H), 3.99-2.89 (m, 10.75H), 2.42-2.29 (m, 0.1H), 2.14-1.55 (m, 3.3H), 1.47-1.35 (m, 0.3H), 1.24-1.11 (m, 0.15H).

Example 39

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

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

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) was added triethylamine (33.9 g, 336.3 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 16 h. Then the reaction mixture was 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 h, the pH 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 brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:50) to give the title compound (10.3 g, 24%). MS (m/z): [M+H]+ calcd. for C ₂₁ H ₂₇ N ₇ O ₄ : 442.2; found: 442.3.

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1323 mg; 3 mmol), N-Boc-ethylenediamine (721 mg; 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI; 863 mg; 4.5 mmol) and 1-hydroxybenzotriazole (HOBt; 607.5 mg; 4.5 mmol) were dissolved in dichloromethane (40 ml) and triethylamine (1.25 ml; 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:50) to give the title compound (1.58 g; yield: 90%). MS (m/z): [M+H]+ calcd. for C ₂₈ H ₄₁ N ₉ O ₅ : 584.32; found: 584.2; ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.02-9.67 (m, 1H), 8.29 (d, J = 8.9 Hz, 1H), 7.72 (dd, J = 10.5; 4.0 Hz, 1H), 6.58 (dd, J = 14.2; 10.2 Hz, 2H), 5.32 (d, J = 17.1 Hz, 1H), 5.13 (s, 1H), 4.20-3.70 (m, 2H), 3.69-3.44 (m, 6H), 3.39-3.14 (m, 7H), 2.60-2.40 (m, 1H), 2.26 (dt, J = 15.3; 7.4 Hz, 2H), 2.11-1.83 (m, 3H), 1.83-1.70 (m, 1H), 1.41 (d,J = 28.2 Hz, 9H), 1.09 (dd, J = 12.6; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[2-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]ethyl]carbamate (1.18 g, 2 mmol) gave the title compound as a white solid (1.04 g; yield: 100%). MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₃ N ₉ O ₃ : 484.27; found: 484.3.

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

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.36 mmol) the title compound was obtained (0.174 g; yield: 51.4%; DSR: 30%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.24-8.13 (m, 0.2H), 7.61-7.47 (m, 0.2H), 6.79-6.61 (m, 0.2H), 4.58-4.16 (m, 2.2H), 4.10-2.77 (m, 13H), 2.42-2.18 (m, 0.6H), 2.04-1.55 (m, 4H), 1.06-0.85 (m, 0.6H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.186 g; yield: 54.9%; DSR: 39%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.189 g; yield: 55.8%; DSR: 30%).

Example 40

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

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (886 mg; 2 mmol), N-Boc-1,4-butanediamine (564 mg; 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI; 575.1 mg; 3 mmol) and 1-hydroxybenzotriazole (HOBt; 405 mg; 3 mmol) were dissolved in dichloromethane (30 ml) and triethylamine (0.84 ml; 6 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:50) to give the title compound (0.52 g; yield: 42.5%). MS (m/z): [M+H]+ calcd. for C ₃₀ H ₄₅ N ₉ O ₅ : 612.35; found: 612.1. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 9.62 (s, 1H), 8.29 (t, J = 14.4 Hz, 1H), 7.77 (s, 1H), 7.61 (d, J = 22.9 Hz, 1H), 6.77 (d, J = 28.6 Hz, 2H), 4.86 (s, 1H), 4.22-3.61 (m, 5H), 3.43-3.33 (m, 3H), 3.27 (s, 3H), 2.94 (d, J = 25.8 Hz, 2H), 2.86 (s, 2H), 2.37 (s, 1H), 2.13 (t, J = 7.0 Hz, 2H), 1.91-1.39 (m, 5H), 1.33 (s, 12H), 0.97 (t, J = 14.9 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]butyl]carbamate (0.5 g, 0.818 mmol) gave the title compound as a white solid (0.32 g; yield: 83%). MS (m/z): [M+H]+ calcd for C ₂₅ H ₃₇ N ₉ O ₃ : 512.30; found: 512.3.

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

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.365 mmol) the title compound was obtained (0.162 g; yield: 47.5%; DSR: 33%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.26-7.86 (m Hz, 0.33H), 7.64-7.18 (m, 0.33H), 6.81-6.32 (m, 0.33H), 4.52-4.28 (m, 2.33H), 4.06-2.81 (m, 14.95N), 2.41-2.21 (m, 0.99N), 1.99-1.63 (m, 4.32N), 1.47-1.26 (m, 1.32N), 1.11-0.91 (m, 0.99N).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.243 g; yield: 71.3%; DSR: 18%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.168 g; yield: 49.3%; DSR: 20%).

Example 41

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

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

Following step 1 of Example 1 and using tofacitinib (10 g, 32 mmol) and 4-aminobenzoic acid (8.7 g, 64 mmol), the title compound was obtained (9.5 g; yield: 62.5%). MS (m/z): [M+H]+ calcd for C ₂₄ H ₂₅ N ₇ O ₄ : 476.1; found: 476.3.

Step 2. Preparation of tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanocetyl)-4-methyl-3-gosheridyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg; 2 mmol), N-Boc-1,4-butanediamine (564 mg; 3 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 575.1 mg; 3 mmol) and 1-hydroxybenzotriazole (HOBt; 405 mg; 3 mmol) were dissolved in dichloromethane (24 ml) and triethylamine (0.84 ml; 6 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. 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 texane = 10:100-50:100) to give the title compound (0.89 g; yield: 69%). MS ₍ m/z): [M+H]+ calcd. for _C33H43N9O5 : _646.33 ; found: 646.1. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 12.21 (s, 1H), 8.42 (s, 2H), 7.89 (d, J = 7.6 Hz, 2H), 7.73 (d, J = 7.8 Hz, 3H), 6.84 (d, J = 55.3 Hz, 2H), 4.89 (s, 1H), 4.18-3.60 (m, 4H), 3.47-3.32 (m, 2H), 3.23 (s, 5H), 2.92 (s, 2H), 2.42-2.33 (m, 1H), 1.90-1.17 (m, 15H), 1.01 (d, J = 5.2 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[4-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.87 g, 1.35 mmol) gave the title compound as a white solid (1 g; yield: 83%). MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₅ N ₉ O ₃ : 546.28; found: 546.3.

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

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.344 mmol) gave the title compound (0.26 g; yield: 93.9%; DSR: 23%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.32-8.12 (m, 0.23H), 7.88-7.23 (m, 1.15H), 6.80-6.64 (m, 0.23H), 4.54-4.30 (m, 2.23H), 3.99-2.81 (m, 12.99H), 2.06-1.37 (m, 4.61H), 1.04-0.87(m, 0.69H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.145 g; yield: 52.4%; DSR: 15%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.104 g; yield: 37.5%; DSR: 33%).

Example 42

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

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

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 h. Then the reaction mixture was 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 (TBAB; 10.3 g) were added. The reaction mixture was stirred at 0-10°C for 16 h, the pH of the reaction mixture was adjusted to 3-4 with acetic acid, and the organic layer was washed with 0.5 N hydrochloric acid, water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:50-1:10) to give the title compound (89.8 g; yield: 67.8%). MS (m/z): [M+H]+ calcd. for C ₁₉ H ₂₃ N ₇ O ₄ : 414.1; found: 414.5.

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg; 1.5 mmol), N-Boc-1,4-butanediamine (423 mg; 2.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 431.3 mg; 2.25 mmol) and 1-hydroxybenzotriazole (HOBt; 303.7 mg; 2.25 mmol) were dissolved in dichloromethane (10 ml) and triethylamine (0.62 ml; 4.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:40) to give the title compound (0.65 g; yield: 74%). MS (m/z): [M+H]+ calcd. for C ₂₈ H ₄₁ N ₉ O ₅ : 584.32; found: 584.2. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 9.92 (t, J = 5.2 Hz, 1H), 8.32 (d, J = 5.8 Hz, 1H), 8.07 (t, J = 5.4 Hz, 1H), 7.63 (d, J = 4.0 Hz, 1H), 6.79 (d, J = 26.3 Hz, 2H), 4.86 (s, 1H), 4.16-3.89 (m, 4H), 3.86-3.54 (m, 2H), 3.43-3.33 (m, 2H), 3.28 (s, 3H), 3.03 (t, J = 19.0 Hz, 2H), 2.88 (d, J = 5.7 Hz, 2H), 2.38 (s, 1H), 1.72 (dt, J = 78.7; 27.2 Hz, 3H), 1.45-1.18 (m, 12H), 1.00 (d, J = 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]butyl]carbamate (0.3 g, 0.51 mmol) gave the title compound as a white solid (0.26 g; yield: 98%). MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₃ N ₉ O ₃ : 484.27; found: 484.3. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 9.90 (s, 1H), 8.34 (d, J = 6.7 Hz, 1H), 8.21 (s, 1H), 7.91 (s, 2H), 7.66 (s, 1H), 6.84 (s, 1H), 4.85 (s, 1H), 4.13-3.66 (m, 7H), 3.39 (s, 1H), 3.28 (s, 1H), 3.10 (d, J = 5.3 Hz, 2H), 2.70 (d, J = 37.4 Hz, 2H), 2.34 (d, J = 27.1 Hz, 2H), 1.77 (d, J = 41.2 Hz, 1H), 1.55-1.39 (m, 4H), 1.15 (t, J = 7.1 Hz, 2H), 1.00 (d, J = 6.5 Hz, 3H).

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

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (130 mg; 0.25 mmol) the title compound was obtained (0.102 g; yield: 45%; DSR: 40%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.26-7.85 (m, 0.4H), 7.63-7.18 (m, 0.4H), 6.83-6.31 (m, 0.4H), 4.58-4.28 (m, 2.4H), 4.15-2.90 (m, 16Н), 2.43-2.10 (m, 0.8Н), 2.06-1.69 (m, 4.4Н), 1.59-1.37 (m, 1.2Н), 1.19-0.87 (m, 0.8Н).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.092 g; yield: 40.6%; DSR: 26%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.143 g; yield: 63%; DSR: 21%).

Example 43

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

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (1239 mg; 3 mmol), N-Boc-ethylenediamine (721 mg; 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 863 mg; 4.5 mmol) and 1-hydroxybenzotriazole (HOBt; 607.5 mg; 4.5 mmol) were dissolved in dichloromethane (40 ml) and triethylamine (1.25 ml; 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:60) to give the title compound (1.1 g; yield: 66%). MS (m/z): [M+H]+ calcd. for C ₂₆ H ₃₇ N ₉ O ₅ : 556.29; found: 556.2. ¹ H-NMR (400 MHz, CDC13) δ ^ppm 10.37-10.14 (m, 1H), 8.31 (d, J = 6.3 Hz, 1H), 7.76-7.54 (m, 1H), 7.03 (t, J = 18.4 Hz, 1H), 6.59 (d, J = 3.9 Hz, 1H), 5.13 (s, 1H), 5.01 (s, 1H), 4.26-4.11 (m, 2H), 4.09-3.72 (m, 2H), 3.67-3.46 (m, 4H), 3.42-3.33 (m, 5H), 3.28 (dd, J = 10.8; 5.5 Hz, 2H), 2.50 (ddd, J = 18.5; 12.3; 6.0 Hz, 1H), 2.09-1.84 (m, 1H), 1.83-1.72 (m, 1H), 1.54-1.30 (m, 9H), 1.09 (dd,J = 11.8; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]ethyl]carbamate (0.89 g, 1.6 mmol) gave the title compound as a white solid (0.78 g; yield: 99%). MS (m/z): [M+H]+ calcd for C ₂₁ H ₂₉ N ₉ O ₃ : 456.23; found: 456.2.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 15.27 mg; 0.379 mmol) and N-[2-(2-aminoethylamino)-2-oxo-ethyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.379 mmol) gave the title compound 0.165 g; yield: 47.7%; DSR: 40%; ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.26-7.82 (m, 0.4H), 7.60-7.26 (m, 0.4H), 6.77-6.26 (m, 0.4H), 4.60-4.21 (m, 2.4H), 4.08-2.99 (m, 16Н), 2.42-2.18 (m, 0.4Н), 2.04-1.55 (m, 3.8Н), 1.06-0.85 (m, 1.2Н).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.173 g; yield: 50%; DSR: 28%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.18 g; yield: 52%; DSR: 40%).

Example 44

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

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-(1]pyrimidine-7-carbonyl]amino]acetic acid (1652 mg, 4 mmol), tert-butyl 4-aminophenethylcarbamate (1418 mg, 6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 1151 mg, 6 mmol) and 1-hydroxybenzotriazole (HOBt; 810 mg, 6 mmol) were dissolved in dichloromethane (40 mL) and triethylamine (1.665 mL, 12 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:150-1:100) to give the title compound (1.77 g; yield: 70%). MS (m/z): [M+H]+ calcd for C ₃₂ H ₄₁ N ₉ O ₅ : 632.32; found: 632.2. ¹ H NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.59-10.19 (m, 1H), 8.30 (dd, J = 17.1; 7.1 Hz, 2H), 7.67 (dd, J = 13.2; 4.0 Hz, 1H), 7.49 (t, J = 11.3 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 6.61 (t, J = 4.7 Hz, 1H), 5.13 (s, 1H), 4.54 (s, 1H), 4.38-4.23 (m, 2H), 4.13-3.72 (m, 2H), 3.68-3.44 (m, 4H), 3.42-3.24 (m, 5H), 2.75 (t, J = 6.8 Hz, 2H), 2.50 (ddd, J = 19.1; 11.9; 5.6 Hz, 1H), 2.08-1.83 (m, 1H), 1.83-1.64 (m, 1H), 1.43 (s, 9H), 1.09 (t, J = 8.2 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[2-[4-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]phenyl]ethyl]carbamate (1.48 g, 2.345 mmol) gave the title compound as a white solid (1.3 g; yield: 97.7%). MS (m/z): [M+H]+ calcd for C ₂₇ H ₃₃ N ₉ O ₃ : 532.27; found: 532.2.

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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.331 mmol) gave the title compound (0.136 g; yield: 41.5%; DSR: 20%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.33-8.06 (m, 0.2H), 7.68-7.50 (m, 0.2H), 7.48-7.13 (m, 0.8H), 6.85-6.67 (m, 0.2H), 4.59-4.23 (m, 2.2Н), 4.07-2.84 (m, 12.8Н), 2.46-2.22 (m, 0.4Н), 2.05-1.70 (m, 3.2Н), 1.21-1.25 (m, 0.2Н), 1.07-0.90 (m, 0.6Н).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.158 g; yield: 48.2%; DSR: 22%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.146 g; yield: 44.5%; DSR: 16%).

Example 45

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

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (1239 mg; 3 mmol), (S)-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 (2.1 ml; 15 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:150-1:80) to give the title compound (1.638 g; yield: 83.3%). MS (m/z): [M+H]+ calcd. for C ₃₁ H ₄₅ N ₉ O ₇ : 656.34; found: 656.1. ¹ H NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.36 (dd, J = 12.6; 7.1 Hz, 1H), 8.33 (d, J = 9.1 Hz, 1H), 7.72 (dd, J = 12.9; 4.0 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.61 (t, J = 4.2 Hz, 1H), 5.14 (s, 1H), 4.63 (dt, J = 33.6; 16.8 Hz, 2H), 4.19 (t, J = 14.2 Hz, 2H), 4.11-3.76 (m, 2H), 3.73 (s, 3H), 3.66-3.57 (m, 1H), 3.55-3.39 (m, 3H), 3.36 (s, 2H), 3.17-2.98 (m, 2H), 2.59-2.41 (m, 1H), 2.03-1.84 (m, 2H), 1.83-1.68 (m, 2H), 1.53-1.39 (m, 11H), 1.39-1.26 (m, 3H), 1.09 (dd,J = 12.3; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate (1.4 g, 2.137 mmol) gave the title compound as a white solid (1.26 g; yield: 99.6%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₂₆ H ₃₇ N ₉ O ₅ : 556.29; found: 556.2.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 128.2 mg; 0.318 mmol) and methyl (2S)-6-amino-2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]hexanoate hydrochloride (200 mg; 0.318 mmol) the title compound was obtained (0.131 g; yield: 40.6%; DSR: 30%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-7.99 (m, 0.3H), 7.64-7.33 (m, 0.3H), 6.82-6.51 (m, 0.3H), 4.58-4.30 (d, J = 28.8 Hz, 2.3H), 4.10-2.99 (m, 14.5N), 2.41-2.27 (m, 0.3N), 2.09-1.58 (m, 4.8N), 1.53-1.29 (m, 0.9N), 1.25-0.93 (m, 0.9N).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.134 g; yield: 41.5%; DSR: 20%).

Example 46

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

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (1764 mg; 4 mmol), tert-butyl 4-aminophenethylcarbamate (1418 mg; 6 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 1151 mg; 6 mmol) and 1-hydroxybenzotriazole (HOBt; 810 mg; 6 mmol) were dissolved in dichloromethane (40 ml) and triethylamine (1.66 ml; 12 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:150-1:100) to give the title compound (2.02 g; yield: 76.6%). MS (m/z): [M+H]+ calcd. for C ₃₄ H ₄₅ N ₉ O ₅ : 660.35; found: 660.3. ¹ H NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.00 (dd, J = 14.1; 8.0 Hz, 1H), 8.48 (d, J = 26.6 Hz, 1H), 8.28 (d, J = 9.5 Hz, 1H), 7.73 (dd, J = 10.8; 4.0 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 6.60 (t, J = 4.6 Hz, 1H), 5.14(s, 1H), 4.56 (s, 1H), 4.12-3.71 (m, 2H), 3.62 (t, J = 9.0 Hz, 3H), 3.48 (s, 3H), 3.35 (d, J = 8.6 Hz, 5H), 2.76 (t, J = 6.8 Hz, 2H), 2.60-2.35 (m, 3H), 2.14-2.04 (m, 2H), 2.02-1.84 (m, 1H), 1.84-1.68 (m, 1H), 1.43 (s, 9H), 1.10 (dd, J = 12.0; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl N-[2-[4-[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoylamino]phenyl]ethyl]carbamate (1.73 g, 2.625 mmol) gave the title compound as a white solid (1.56 g; yield: 99.8%). MS (m/z): [M+H]+ calcd for C ₂₉ H ₃₇ N ₉ O ₃ : 560.30; found: 560.1.

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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

Following step 3 of Example 1 and using 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-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.316 mmol) gave the title compound 0.154 g; yield: 47.8%; DSR: 33%; ¹ H-NMR (400 MHz, D20) δ ^ppm 8.10-7.85 (m, 0.33H), 7.54-7.32 (m, 0.33H), 7.24-6.81 (m, 1.32H), 6.68-6.42 (m, 0.33H), 4.59-4.21 (m, 2.33N), 4.04-2.92 (m, 14.29N), 2.73-2.63 (m, 0.66N), 2.50-2.23 (m, 0.99N), 2.11-1.56 (m, 3.99N), 1.30-1.16 (m, 0.33N), 1.13-0.87 (m, 0.99N).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 500 kDa), the corresponding product was obtained (0.136 g; yield: 42.2%; DSR: 18%).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.158 g; yield: 49.1%; DSR: 17%).

Example 47

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

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg; 1.5 mmol), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (624 mg; 2.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 43.3 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 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane=1:100-1:50) to give the title compound (0.55 g; yield: 54.5%). MS (m/z): [M+ _H ]+ calcd. for _{C34H44N10O5 : 673.34} ; found: 673.3. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.45 (dd, J=13.6; 7.8 Hz, 1H), 8.33 (d, J=9.1 Hz, 1H), 8.07 (d, J=10.2 Hz, 1H), 7.69 (dd, J=12.1; 4.0 Hz, 1H), 7.43 (d, J=8.9 Hz, 2H), 6.88 (d, J=8.9 Hz, 2H), 6.62 (t, J=4.3 Hz, 1H), 5.14 (s, 1H), 4.30 (d, J=5.8 Hz, 2H), 4.11-3.74 (m, 2H), 3.68-3.43 (m, 8H), 3.38 (d, J=20.1 Hz, 3H), 3.10 (dd, J=28.1; 23.2 Hz, 4H), 2.60-2.39 (m, 1H), 2.01-1.84 (m, 1H), 1.83-1.66 (m, 1H), 1.48 (s, 9H), 1.09 (t, J=8.5 Hz, 3H).

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

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

Step 3. Preparation of the conjugate of 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl-methyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylaniline)ethyl]pyrrolo[2,3-d]pyrimidane-7-carboxamide and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 99.54 mg; 0.247 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-(4-piperazin-1-ylaniline)ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg; 0.247 mmol) the title compound was obtained (0.121 g; yield: 49%; DSR: 25%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-8.09 (m, 0.25H), 7.62-6.96 (m, 0.25H), 6.79-6.64 (m, 0.25H), 4.57-4.24 (m, 2.15H), 4.09-2.77 (m, 14.25H), 2.41-2.31 (m, 0.25H), 2.16-1.45 (m, 4.25H), 1.24-1.12 (m, 0.25H), 1.07-0.78 (m, 0.75H).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.107 g; yield: 43.6%; DSR: 35%).

Example 48

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

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

2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl[-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (619.5 mg; 1.5 mmol), tert-butyl 4-(6-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). The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane=1: _100-1 :50) to give the title compound (0.4 g; yield: 39.6%). MS (m/z): [M+H]+ calcd. for _C33H43N11O5 : _674.34 ; found _: 674.3. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.54-10.40 (m, 1H), 8.55 (d, J=20.7 Hz, 1H), 8.34 (d, J=14.9 Hz, 1H), 8.12 (t, J=8.1 Hz, 1H), 7.93 (d, J=2.8 Hz, 1H), 7.70 (d, J=4.1 Hz, 1H), 7.33-7.28 (m, 1H), 6.62 (d, J=3.7 Hz, 1H), 5.15 (s, 1H), 4.33 (d, J=17.0 Hz, 2H), 4.14-3.77 (m, 2H), 3.73-3.45 (m, 8H), 3.39 (d,J=18.0 Hz, 3H), 3.18-2.99 (m, 4H), 2.52 (dd, J=16.9; 10.4 Hz, 1H), 2.01-1.85 (m, 1H), 1.83-1.69 (m, H), 1.48 (s, 9H), 1.10 (dd, J=12.7; 7.1 Hz, 3H).

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

Following step 2 of Example 1 and using tert-butyl 4-[6-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]-3-pyridyl]piperazine-1-carboxylate (0.36 g, 0.535 mmol) gave the title compound as a white solid (0.32 g; yield: 98%). MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₅ N ₁₁ O ₃ : 574.29; found: 574.3.

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 99.14 mg; 0.246 mmol) and 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]-N-[2-oxo-2-[(5-piperazin-1-yl-2-pyridyl)amino]ethyl]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (150 mg; 0.246 mmol) the title compound was obtained (0.126 g; yield: 52.5%; DSR: 22%); ¹ H-NMR (400 MHz, D ² O) δ ^ppm 8.26-8.07 (m, 0.22H), 8.01-7.82 (m, 0.22H), 7.71-7.40 (m, 0.44H), 6.81-6.57 (m, 0.22H), 4.64-4.28 (m, 2.22N), 4.20-2.73 (m, 13.74N), 2.42-2.32 (m, 0.22N), 2.20-1.54 (m, 3.44N), 1.09-0.78 (m, 0.66N).

In the case of the reaction carried out in accordance with step 3 of Example 1 with sodium hyaluronate (MM 2000 kDa), the corresponding product was obtained (0.116 g; yield: 48.3%; DSR: 26%).

Example 49

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

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

To a stirred mixture of 2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetic acid (826 mg; 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg; 2.4 mmol) in dichloromethane (20 mL) was slowly added a solution of N,N-dicyclohexylcarbodiimide (494.4 mg; 2.4 mmol) in dichloromethane (20 mL) using an ice bath. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered and then concentrated under reduced pressure to give the title compound (1 g; yield: 98%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₃ H ₂₆ N ₈ O ₆ : 511.19; detected: 511.1.

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

Conversion of sodium hyaluronate to tetrabutylammonium (TBA) hyaluronic acid. Amberlite 732, a strong ion-exchange resin in its acidic form, was added to an aqueous solution of sodium hyaluronate, and the mixture was stirred at room temperature for 8 hours. The solution was filtered, and the filtrate was then neutralized with an aqueous solution of tetrabutylammonium hydroxide (TBA-OH). The resulting aqueous solution was immediately frozen and lyophilized, yielding TBA hyaluronate (HA-TBA).

To a stirred mixture of hyaluronic acid TBA salt (HA-TBA; 331 mg; 0.5 mmol) in anhydrous DMSO (20 mL) was slowly added triethylamine (0.12 mL; 0.875 mmol) at 0-15°C. Then, (2,5-dioxopyrrolidin-1-yl)-2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetate (255 mg; 0.5 mmol) in anhydrous DMSO (20 mL) at 0-15°C was added to the reaction mixture. The reaction mixture was stirred for 16 h at room temperature. Then, a 2.5 wt % solution of sodium chloride (7 ml), the mixture was stirred for 1 h and then acetone (250 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). After extended dialysis of the solution (with a MW cut-off of 3.5 kDa) against deionized water and lyophilization, the title compound was obtained. MW of sodium hyaluronate 10 kDa; 0.17 g; yield: 43.9%; DSR: 20%; ¹ H-NMR (400 MHz, D ₂ O) δ ppm ^-1 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, 2.2N), 4.09-2.94 (m, 12.2N), 2.41-2.38 (m, 0.2N), 2.12-1.58 (m, 3.4N), 1.09-0.87 (m, 0.6N).

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 50 kDa), the corresponding product was obtained (0.245 g; yield: 613.3%; DSR: 6%).

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 500 kDa), the corresponding product was obtained (0.274 g; yield: 70.8%; DSR: 23%).

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 2000 kDa), the corresponding product was obtained (0.237 g; yield: 61.2%; DSR: 24%).

Example 50

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

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

To a stirred mixture of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]butanoic acid (882 mg; 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg; 2.4 mmol) in dichloromethane (25 mL) was slowly added a solution of N,N-dicyclohexylcarbodiimide (DCC; 494.4 mg; 2.4 mmol) in dichloromethane (15 mL) using an ice bath. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered and then concentrated under reduced pressure to give the title compound (1 g; yield: 93%), which was used without further purification. MS (m/z): [M+H]+ calc. for C ₂₅ H ₃₀ N ₈ O ₆ : 538.22; detected: 538.1.

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

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

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 500 kDa), the corresponding product was obtained (0.263 g; yield: 65.5%; DSR: 5%).

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 2000 kDa), the corresponding product was obtained (0.17 g; yield: 42.3%; DSR: 7%).

Example 51

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

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

To a stirred mixture of 4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol) and 1-hydroxypyrrolidine-2,5-dione (276 mg, 2.4 mmol) in dichloromethane (40 mL) was slowly added a solution of N,N-dicyclohexylcarbodiimide (494.4 mg, 2.4 mmol) in dichloromethane (20 mL) using an ice bath. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered and then concentrated under reduced pressure to give the title compound (1.1 g; yield: 96%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₈ H ₂₈ N ₈ O ₆ : 573.21; detected: 573.1.

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

To a stirred mixture of hyaluronic acid TBA salt (HA-TBA; 331 mg, 0.5 mmol) in anhydrous DMSO (20 mL) was slowly added triethylamine (0.12 mL, 0.875 mmol) at 0-15 °C. Then, (2,5-dioxopyrrolidin-1-yl)-4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoate (286 mg, 0.5 mmol) in anhydrous DMSO (20 mL) at 0-15 °C was added to the reaction mixture. The reaction mixture was stirred for 16 h at room temperature. Then, 7 mL of 2.5 wt. % sodium chloride solution, the mixture was stirred for 1 h and then acetone (250 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). After extended dialysis of the solution (with a MW cut-off of 3.5 kDa) against deionized water and lyophilization, the title compound was obtained. MW of sodium hyaluronate 10 kDa; 0.177 g; 42.3%; DSR: 10%; ¹ H-NMR (400 MHz, D ₂ O) 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). MW of sodium hyaluronate 50 kDa; 0.2 g; yield: 47.8%; DSR: 6%.

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 500 kDa), the corresponding product was obtained (0.187 g; yield: 44.7%; DSR: 5%).

In the case of the reaction carried out in accordance with this step with the TBA salt of hyaluronic acid (MM 2000 kDa), the corresponding product was obtained (0.1 g; yield: 23.9%; DSR: 2%).

Example 52

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

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

To a mixture of tert-butyl (4-hydroxyphenyl)carbamate (2 g, 9.56 mmol, 1 eq.) and N,N-dimethylpyridin-4-amine (2.92 g, 23.9 mmol, 2.5 eq.) in dichloromethane (40 mL, 20V) was added thiophosgene (1.21 g, 10.5 mmol, 1.1 eq.) under N ₂ , and the reaction mixture was stirred at room temperature for 0.5 h. Tofacitinib (2.99 g, 9.56 mmol, 1 eq.) was then added, and the resulting mixture was stirred at room temperature for another 12 h. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure, and the residue was purified by silica chromatography to give the title compound title product (1.827 g; yield: 34%); MS (m/z): [M+H]+ calcd. for C ₂₈ H ₃₃ N ₇ O ₄ S: 564.23; detected: 564.1.

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

Following step 2 of Example 1 and using O-(4-((tert-butoxycarbonyl)amino)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate (1.5 g, 2.661 mmol, 1 eq), the desired product was obtained as the HCl salt (1.33 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₃ H ₂₅ N ₇ O ₂ S: 464.18; found: 464.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.45-8.30 (m, 1H), 7.66-7.44 (m, 2H), 7.44-7.22 (m, 2H), 7.09-6.74 (m, 2H), 4.73 (s, 1H), 4.06-3.94 (m, 3H), 3.71-3.17 (m, 6H), 2.56 (s, 1H), 2.03-1.70 (m, 2H), 1.13 (dd, J=14.8; 6.8 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.161 g; 0.4 mmol; 1 eq.) and O-(4-aminophenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride (0.2 g; 0.4 mmol; 1 eq.) the title compound was obtained (0.15 g; yield: 45%; DSR=14.3%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 7.7-7.55 (m, 0.5H), 7.35-7.2 (m, 0.5H), 4.6-4.45 (m, 1.43H), 3.85-3.4 (m, 12H), 2.46-2.43 (m, 0.14N), 2.00 (s, 3.0N), 1.9-1.45 (m, 2.86N), 1.3-1.25 (m, 0.43N).

In the case of interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this method, the corresponding product was obtained (0.175 g; yield: 52%; DSR=4.6%).

Example 53

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

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

To a mixture of tert-butyl 4-hydroxypiperidine-1-carboxylate (2 g, 9.94 mmol, 1 eq.) and N,N-dimethylpyridin-4-amine (3.03 g, 9.94 mmol, 2.5 eq.) in dichloromethane (40 mL, 20V) was added thiophosgene (1.26 g, 10.934 mmol, 1.1 eq.) under _N2 , and the reaction mixture was stirred at room temperature for 0.5 h. Tofacitinib (3.1 g, 9.94 mmol, 1 eq.) was then added to the reaction mixture, and the resulting mixture was stirred at room temperature for another 12 h. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure, and the residue was purified by chromatography on dioxide silicon to give the title product (1.2 g; yield: 22%); MS (m/z): [M+H]+ calcd for C ₂₇ H ₃₇ N ₇ O ₄ S: 556.26; found: 556.2.

Step 2. Preparation of O-(piperidin-4-yl)-4-(((3R,4R)-1-(2-1-methylacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.885 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₂ H ₂₉ N ₇ O ₂ S: 456.21; found: 456.1. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.49 (d, J = 9.7 Hz, 1H), 8.00 (s, 1H), 7.03 (s, 1H), 5.93 (br, 1H), 5.03 (s, 1H), 4.15-4.0 (m, 1H), 3.79-3.31 (m, 11H), 3.29-3.05 (m, 1H), 2.52 (br, 1H), 2.45-2.28 (m, 4H), 2.0-1.7 (m, 3H), 1.11 (dd, J = 17.1; 7.1 Hz, 3H).

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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.164 g; 0.406 mmol; 1 eq.) and O-(piperidin-4-yl)4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)-(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbothioate hydrochloride (0.2 g; 0.406 mmol; 1 eq.) the title compound was obtained (0.17 g; yield: 34%; DSR=12.6%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.25-7.0 (m, 0.22H), 7.0-6.5 (m, 0.08H), 5.95-5.49(m, 0.08H), 4.45-4.25 (m, 1.26H), 3.8-2.9(m, 12.63Н), 2.33 (broad, 0.13Н), 1.88 (s, 3Н), 1.81-1.72 (m, 0.51Н), 1.23-1.07 (m, 0.25Н), 0.99-0.82 (m, 0.38Н).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this stage, the corresponding product was obtained (0.192 g; yield: 56.6%; DSR=182.6%).

Example 54

Preparation of a conjugate 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 and HA

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

To a mixture of tofacitinib (9 g, 28.81 mmol, 1 eq.) in dichloromethane (180 mL, 20V) was added diisopropyl (DIP)ethyl acetate (3.745 g, 28.81 mmol, 1 eq.) under _N2 , and the reaction mixture was stirred at room temperature for 0.5 h. Then, (2-(chloromethoxy)ethyl)trimethylsilane (4.8 g, 28.81 mmol, 1 eq.) was added to the reaction mixture, and the resulting mixture was stirred overnight at room temperature. After most of the tofacitinib was consumed, the solvent was removed under reduced pressure, the residue was purified by silica chromatography to give the title product (9 g; yield: 71%); MS (m/z): [M+H]+ calcd. for C ₂₂ H ₃₄ N ₆ O ₂ Si: 443.25; detected: 443.2.

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-oxopropanenitrile

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-oxopropanenitrile (5 g; 11.3 mmol; 1 eq.) in dichloromethane (100 mL; 20V) was added trifluoroacetic acid (TFA; 6.44 g; 56.5 mmol; 5 eq.) dropwise under _N2 at 0°C. The resulting reaction mixture was stirred at 0 °C for 30 min, allowed to warm to room temperature, and then stirred at this temperature for 24 h. After most of the starting material (SM) was consumed, NaHCO ₃ solution (sat.) 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 solution (sat.), dried over Na ₂ SO ₄ and then concentrated to give the title product (3.5 g; yield: 90%); MS (m/z): [M+H]+ calcd. for C ₁₇ H ₂₂ N ₆ O ₂ : 343.18; found: 343.1.

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

Following step 1 of Example 1 and using 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (1 g, 2.92 mmol, 1 eq) and tert-butyl (4-hydroxybutyl)carbamate (1 g, 2.92 mmol, 1 eq), the title product (0.85 g; yield: 52%) was obtained; MS (m/z): [M+H]+ calcd for C ₂₇ H ₃₉ N ₇ O ₆ : 558.30; found: 558.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.753 mg; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₂ H ₃₁ N ₇ O ₄ : 458.24; found: 458.1. ¹ H-NMR (400 MHz, CD ₃ OD) δ ^ppm 8.41 (s, 1H), 7.64 (d, J=3.7 Hz, 1H), 7.01 (d, J=3.4 Hz, 1H), 6.28 (s, 2H), 4.69 (s, 1H), 4.21-3.40 (m, 11H), 2.95 (t, J=6.8 Hz, 1H), 2.56 (broad, 1H), 2.05 (br, 1H), 1.87-1.66 (m, 5H), 1.23-1.14 (m, 3H).

Step 5. Preparation of a conjugate 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 and HA

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.161 g; 0.4 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.18 g; yield: 56%; DSR=21%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.2-7.9 (m, 0.21H), 7.3-7.0 (m, 0.21H), 6.8-6.3 (m, 0.21H), 6.1-5.1(m, 0.42H), 4.45 (d, J=27.3 Hz, 2.84Н), 3.8-3.3 (m, 12Н), 2.40 (broad, 0.21H), 1.97 (s, 3Н), 1.65-1.45 (m, 0.94Н), 1.25 (t, J=6.9 Hz, 0.21Н), 1.05-0.75 (m, 0.7Н).

In the case of the interaction with the TBA salt of hyaluronic acid (MM 2000 kDa) carried out in accordance with this stage, the corresponding product was obtained (0.172 g; yield: 185.6%; DSR=14%).

Example 55

Preparation of the conjugate (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

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

Following step 1 of Example 1 and using 3-((3R,4R)-3-((7-(hydroxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile (0.6 g, 1.75 mmol, 1 eq), 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), the title product (0.878 g; yield: 90%); m/z (electrospray ionization (ESI)): 557.31 [M+H] ⁺ . MS (m/z): [M+H]+ calc. for C ₂₇ H ₄₀ N ₈ O ₅ : 557.31; detected: 557.2.

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

Following step 2 of Example 1 and using 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 eq.), the desired product was obtained as the HCl salt (0.7 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₂ H ₃₂ N ₈ O ₃ : 457.25; found: 457.2. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.33 (t, J=6.7 Hz, 1H), 7.52 (dd, J=17.0; 3.7 Hz, 1H), 6.88 (d, J=9.3 Hz, 1H), 6.17 (d, J=24.8 Hz, 2H), 4.58 (s, 1H), 4.10-3.81 (m, 3H), 3.68-3.16 (m, 5H), 3.16-2.88 (m, 5H), 2.56 (broad, 1H), 2.0-1.97 (m, 1H), 1.83-1.72 (m, 1H), 1.67-1.49 (m, 4H), 1.13 (dd, J=14.9; 7.0 Hz, 3H).

Step 3. Preparation of the 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

Following step 3 of Example 1 and using sodium hyaluronate (MW 50 kDa; 0.161 g; 0.4 mmol; 1 eq.) 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 eq.), the title compound was obtained (0.17 g; yield: 50%; DSR=6%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.15-7.95 (m, 0.06H), 7.27 (broad, 0.06H), 6.68 (broad, 0.06H), 6.1-5.9 (m, 0.12H), 4.40 (d, 2H), 3.93-3.11 (m, 10.8N), 2.37 (broad, 0.06N), 2.01-1.79 (s, 3H), 1.6-1.5 (m, 0.36N), 1.0-0.9 (m, 0.18N).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.150 g; yield: 187.9%; DSR=8%).

Example 56

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

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

To a stirred mixture of tofacitinib (1.245 g, 4 mmol) in toluene (50 mL) was added triethylamine (0.558 mL, 8 mmol). The reaction mixture was heated to 50 °C. Then 2-chloroethyl isocyanate (1688 mg, 16 mmol) was added to the reaction mixture. The reaction mixture was stirred at this temperature for 4 h. Then, the reaction mixture was filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:20) to give the title compound (1.2 g, 71.9%). MS (m/z): [M+H]+ calcd. for C ₁₉ H ₂₄ ClN ₇ O ₂ : 418.16; found: 418.1.

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

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

Example 57

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

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

To a stirred mixture _of tofacitinib (156 mg, 0.5 mmol) and _K2CO3 (138 mg, 1 mmol) in anhydrous dimethylformamide (DMF, 2 mL) was added 2-chloroethyl chloroformate (0.1 mL, 1 mmol). The reaction mixture was stirred for 30 min at room temperature. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/ _{dichloromethane} = 1: _100-1 :50) to give the title compound (0.08 g, 40%). MS (m/z): [M+H]+ calcd. for _C19H23ClN6O3 : _419.15 ; found: 419.3.

Step 2. Preparation of a conjugate of 2-chloroethyl-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

To a stirred mixture of hyaluronic acid TBA salt (HA-TBA; MW 500 kDa; 331 mg; 0.5 mmol; sodium hyaluronate MW 50 kDa) in anhydrous DMSO (20 mL) was slowly added a solution of 2-chloroethyl 4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxylate (209 mg; 0.5 mmol) in anhydrous DMSO (2 mL). The reaction mixture was stirred for 7 days at room temperature, and then acetone (200 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 40 mL of deionized water. After prolonged dialysis of the solution (with a MW cut-off of 3.5 kDa) against deionized water and lyophilization, the title compound was obtained (0.28 g; yield: 73.5%; DSR: 25%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.21-7.99 (m, 0.25H), 7.57-7.10 (m, 0.25H), 6.83-6.57 (m, 0.25H), 4.54-4.31 (m, 2.25H), 4.09-2.73 (m, 12.75H), 2.39-2.23 (m, 0.25H), 2.02-1.50 (m, 3.75H), 1.32-1.21 (m, 0.25H), 1.00-0.79 (m, 0.75H).

In the case of the reaction with HA-TBA (MW sodium hyaluronate 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.18 g; yield: 190.2%; DSR=20%). ¹ H-NMR.

Example 58

Preparation of a conjugate of 3-[(3R,4R)-3-[[7-(2-chloroethoxymethyl)pyrrolo[2,3-d]pyrimidin-4 yl]|-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile and HA

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

To a stirred mixture _of tofacitinib (156 mg, 0.5 mmol) and _K2CO3 (138 mg, 1 mmol) in anhydrous DMF (2 mL) was added 2-chloromethoxyethyl chloride (142 mg, 1.1 mmol). The reaction mixture was stirred for 1 h at room temperature. The solution was diluted with dichloromethane and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/dichloromethane = 1:100-1:50) to _{give the title compound (0.11 g, 55%). MS (m/z): [M+H]+ calcd for C19H25ClN6O2 : 405.17} ; found: 405.3.

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

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

In the case of the interaction with HA-TBA (MW sodium hyaluronate 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.175 g; yield: 46.8%; DSR=35%).

Example 59

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

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (950 mg, 2 mmol), (S)-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 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/DCM=1:150-1:80) to _give the title compound (1.1 g; yield: 76.7%). MS (m/z): _[ M ₊ H]+ calcd for _C36H47N9O7 : 718.35; found: 718.3. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 12.29 (d, J=30.5 Hz, 1H), 8.39 (d, J=8.5 Hz, 1H), 7.87 (d, J=8.6 Hz, 2H), 7.78 (dd, J=10.8; 6.4 Hz, 3H), 6.85-6.70 (m, 1H), 6.66 (d, J=3.7 Hz, 1H), 5.11 (d, J=23.2 Hz, 1H), 4.89-4.74 (m, 1H), 4.62 (s, 1H), 4.09 (dd, J=13.0; 3.8 Hz, 1H), 3.95-3.70 (m, 4H), 3.66-3.45 (m, 4H), 3.39 (d, J=14.1 Hz, 3H), 3.13 (d, J=6.0 Hz, 2H), 2.60-2.43 (m, 1H), 2.06-1.91 (m, 2H), 1.91-1.75 (m, 2H), 1.59-1.49 (m, 2H), 1.40 (d, J=19.4 Hz, 11H), 1.27 (d, J=12.0 Hz, 1H), 1.10 (t, J=7.0 Hz, 3H).

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

To a stirred solution of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate (1 g, 1.39 mmol) in EtOAc (20 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.9 g _{; yield} : 99%), which was used without further purification. MS (m/z): [M+H]+ calcd for _C31H39N9O5 : _618.30 ; found: 618.1.

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

Sodium hyaluronate (117 mg; 0.29 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 23.4 mL of deionized water in a 100 mL round-bottomed flask, then 16 mL of acetonitrile was added dropwise with stirring. 4-Methylmorpholine (NMM; 59 mg; 0.58 mmol) was added to the solution, which temporarily increased the viscosity. The solution was then cooled to 0°C, 2-chloro-4,6-dimethoxy-1,3,5-triazine (51 mg; 0.29 mmol) was added, and the mixture was stirred at room temperature for 1 h. The solution was mixed with methyl (2S)-6-amino-2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]hexanoate hydrochloride (200 mg; 0.29 mmol) and stirred for 72 h at room temperature. Then NaCl (170 mg; 2.9 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (290 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). After extended dialysing of the solution (with a MW cut-off of 3.5 kDa) against deionised water and lyophilisation, the title compound was obtained (0.119 g; yield: 41.9%; DSR: 20%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.35-8.23 (m, 0.2H), 7.89-7.40 (m, 1H), 6.83-6.71 (m, 0.2H), 4.61-4.16 (m, 2.2H), 4.13-2.47 (m, 13.4H), 2.09-1.29 (m, 4.6H), 1.23-1.16 (m, 0.2H), 1.06-0.81 (m, 0.6H).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.119 g; yield: 195.9%; DSR=21%).

Example 60

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

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

To a stirred mixture of tofacitinib (1248 mg; 4 mmol) and bis(4-nitrophenyl) carbonate (1459 mg; 4.8 mmol) in DCM (40 mL) was added triethylamine (1.12 mL; 8 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 h. Then, the reaction mixture was cooled to room temperature. 3-Aminobenzoic acid (658 mg; 4.8 mmol) was added. The reaction mixture was stirred at 45 °C for 16 h. The reaction mixture was filtered. The filtrate was washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=20:1) to give the title compound (1.8 g; yield: 94.7%). MS (m/z): [M+H]+ calcd for C ₂₄ H ₂₅ N ₇ O ₄ : 476.19; detected: 476.1.

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

3-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (1188 mg; 2.5 mmol), N-Boc-1,4-butanediamine (705 mg; 3.75 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 719 mg; 3.75 mmol) and 1-hydroxybenzotriazole (HOBt; 507 mg; 3.75 mmol) were dissolved in DCM (40 mL) and triethylamine (1.04 mL; 7.5 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/DCM=1:150-1:80) to _{give the} title compound (1 g; yield: 62%). MS (m/z): _[ M+H]+ calcd for _C33H43N9O5 : 646.33; found: 646.3.

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

To a stirred solution of tert-butyl N-[4-[[3-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]butyl]carbamate (0.6 g; 0.93 mmol) in EtOAc (12 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.54 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calc. for C ₂₈ H ₃₅ N ₉ O ₃ : 546.28; detected: 546.3.

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

Sodium hyaluronate (186 mg; 0.462 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 37.2 mL of deionized water in a 100 mL round-bottomed flask, and 24.2 mL of acetonitrile was then added dropwise with stirring. N-[3-(4-aminobutylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.324 mmol) and 4-methylmorpholine (NMM; 32 mg; 0.324 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 128 mg; 0.462 mmol) was added to the solution and the mixture was stirred for 72 hours at room temperature. Then NaCl (270 mg; 4.62 mmol) was added to the reaction mixture, the reaction mixture was stirred for 1 hour and then acetone (250 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). After extended dialysing of the solution (with a MW cut-off of 3.5 kDa) against deionised water and lyophilisation, the title compound was obtained (0.209 g; yield: 49.9%; DSR: 22%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.06-7.86 (m, 0.22H), 7.56-7.41 (m, 1.1H), 6.56-6.42 (m, 0.22H), 4.63-4.18 (m, 2.22H), 4.05-2.37 (m, 12.86H), 2.13-1.70 (m, 3.66H), 1.69-1.07 (m, 1.54H).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.214 g; yield: 51.1%; DSR=18%).

Example 61

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

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

4-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoic acid (1425 mg, 3 mmol), N-Boc-ethylenediamine (721 mg, 4.5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 863 mg, 4.5 mmol) and 1-hydroxybenzotriazole (HOBt; 607.5 mg, 4.5 mmol) were dissolved in DCM (40 mL) and triethylamine (1.25 mL, 9 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/DCM=1:150-1:80) to give the title compound (1.23 g; yield: 66.4%). MS (m/z): _[ M ₊ H]+ calcd for _C31H39N9O5 : _618.30 ; found: 618.2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 12.26 (d, J=23.3 Hz, 1H), 8.38 (d, J=9.3 Hz, 1H), 7.81 (dd, J=39.5; 8.6 Hz, 5H), 7.24 (s, 1H), 6.65 (d, J=4.0 Hz, 1H), 5.11 (d, J=33.0 Hz, 2H), 4.13-3.74 (m, 2H), 3.68-3.11 (m, 11H), 2.62-2.45 (m, 1H), 2.08-1.72 (m, 2H), 1.44 (s, 9H), 1.10 (t, J=8.6 Hz, 3H).

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

To a stirred solution of tert-butyl N-[2-[[4-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]benzoyl]amino]ethyl]carbamate (0.6 g, 0.97 mmol) in EtOAc (6 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.5 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calc. for C ₂₆ H ₃₁ N ₉ O ₃ : 518.25; detected: 518.2.

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

Sodium hyaluronate (208 mg; 0.516 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 41.6 mL of deionized water in a 100 mL round-bottomed flask, and 27 mL of acetonitrile were then added dropwise with stirring. N-[4-(2-aminoethylcarbamoyl)phenyl]-4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (200 mg; 0.361 mmol) and 4-methylmorpholine (NMM; 36.5 mg; 0.361 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 142 mg; 0.516 mmol) was added to the solution and the mixture was stirred for 72 hours at room temperature. Then NaCl (303 mg; 5.16 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour and then acetone (250 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). After extended dialysing of the solution (with a MW cut-off of 3.5 kDa) against deionised water and lyophilisation, the title compound was obtained (0.25 g; yield: 55.1%; DSR: 22%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.36-8.18 (m, 0.22 H), 7.92-6.84 (m, 1.1 H), 6.68-6.55 (m, 0.22 H), 4.61-4.22 (m, 2.22 H), 4.13-2.65 (m, 12.86 H), 2.24-1.52 (m, 3.66 H), 1.30-1.09 (m, 0.66 H). In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.24 g; yield: 52.9%; DSR=26%).

Example 62

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

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

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) was added triethylamine (1.25 mL, 9 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 h. Then the reaction mixture was 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 h. 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 give the title compound ( _0.66 g; yield: 35.3%). MS (m/z): _[ M+H]+ calcd for _C28H45N7O7S : _624.31 ; found: 624.2. ¹ H NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.38 (d, J=7.6 Hz, 1H), 8.36 (s, 1H), 7.67 (d, J=4.1 Hz, 1H), 6.59 (d, J=4.0 Hz, 1H), 4.78-4.69 (m, 2H), 4.58 (s, 1H), 4.22-4.05 (m, 1H), 3.81 (s, 3H), 3.24 (s, 3H), 3.14 (d, J=5.4 Hz, 2H), 3.01 (dd, J=15.2; 5.9 Hz, 2H), 2.86 (d, J=5.3 Hz, 3H), 2.21 (d, J=11.6 Hz, 2H), 2.07-1.87 (m, 5H), 1.71 (dd, J=23.8; 11.5 Hz, 2H), 1.59-1.48 (m, 4H), 1.47-1.32 (m, 11H).

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

To a stirred solution of methyl (2S)-6-(tert-butoxycarbonylamino)-2-[[trans-4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (0.61 g, 0.97 mmol) in EtOAc (12 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to 0 °C and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.54 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₃ H ₃₇ N ₇ O ₅ S: 524.25; detected: 524.2.

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

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

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.105 g; yield: 47.1%; DSR=20%).

Example 63

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide

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

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 at room temperature for 4 h. Then tert-butyl hydrazinecarboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into _{a saturated Na2CO3 solution} . The mixture was filtered. The filter cake was washed with a saturated _NaHCO3 solution and water. Then the filter cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure to give the title compound (1.67 g; yield: 84.3%). MS (m/z): [M+H]+ calcd for C ₂₁ H ₃₃ N ₇ O ₅ S: 496.22; found: 496.1. ¹ H-NMR (400 MHz, CDCCl ₃ ) δ ^ppm 11.38 (s, 1H), 8.30 (s, 1H), 7.63 (d, J=4.1 Hz, 1H), 6.56 (dd, J=25.8; 3.5 Hz, 2H), 4.75 (s, 1H), 4.36 (q, J=5.1 Hz, 1H), 3.22 (d, J=11.1 Hz, 3H), 2.97 (d, J=6.3 Hz, 2H), 2.83 (d, J=5.3 Hz, 3H), 2.20 (t, J=14.4 Hz, 2H), 2.04-1.94 (m, 1H), 1.87 (d, J=10.8 Hz, 2H), 1.74-1.62 (m, 2H), 1.51 (s, 9H), 1.43-1.32 (m, 2H).

Step 2. Preparation of 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride

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) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.52 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₁₆ H ₂₅ N ₇ O ₃ S: 396.17; detected: 396.1.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide

Sodium hyaluronate (246 mg; 0.61 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 49.2 mL of deionized water in a 100 mL round-bottomed flask, and 32 mL of acetonitrile was then added dropwise with stirring. 4-Methylmorpholine (NMM; 43 mg; 0.427 mmol) was added to the solution, which resulted in a temporary increase in viscosity. 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-trans-N-methyl-methanesulfonamide hydrochloride (200 mg; 0.427 mmol) and 4-methylmorpholine (NMM; 43 mg; 0.427 mmol) were added to the solution at room temperature, which resulted in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 169 mg; 0.61 mmol) was added to the solution, and the mixture was stirred for 72 hours at room temperature. Then, NaCl (357 mg; 6.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then acetone (400 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of a mixture of deionized water and acetonitrile (v/v=3:1). Then, NaCl (357 mg; 6.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then acetone (200 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone, and dried in vacuo to give the title compound as a white solid (0.29 g; yield: 62.8%; DSR: 23%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.26-8.09 (m, 0.23H), 7.63-7.52 (m, 0.23H), 6.85-6.67 (m, 0.23H), 4.70-4.28 (m, 2.23H), 4.20-2.53 (m, 11.84N), 2.23-1.40 (m, 4.61N), 1.34-1.06 (m, 0.46N).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.305 g; yield: 66.1%; DSR=31%).

Example 64

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate

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

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 (1114 mg, 3 mmol) and bis(4-nitrophenyl)carbonate (1094 mg, 3.6 mmol) in DCM (40 mL) was added triethylamine (1.25 mL, 9 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 5 h. Then the reaction mixture was 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 h. The solution was diluted with DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography ₍ MSM:methanol=100:1) to give the title compound (1.3 g; yield: 65.9%). MS (m/z): _[ M+H]+ calcd _{for C29H39N9O7S} : 658.26; found: 658.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 9.93 (d, J=7.6 Hz, 1H), 8.95 (s, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 8.04 (d, J=4.0 Hz, 1H), 6.85 (d, J=4.1 Hz, 1H), 5.45-5.34 (m, 1H), 4.76-4.71 (m, 1H), 4.66 (d, J=9.4 Hz, 2H), 4.63-4.56 (m, 1H), 4.52-4.42 (m, 1H), 4.28 (d, J=9.6 Hz, 2H), 3.84 (s, 3H), 3.44 (s, 2H), 3.20-3.07 (m, 2H), 2.10-1.94 (m, 2H), 1.88-1.76 (m, 1H), 1.61-1.35 (m, 12H), 1.27 (s, 3H).

Step 2. Preparation of methyl (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate hydrochloride

To a stirred solution 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 (1.23 g, 1.87 mmol) in EtOAc (25 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (4.92 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (1.1 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₄ H ₃₁ N ₉ O ₅ S: 558.21; detected: 558.1.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate

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

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.154 g; yield: 38.6%; DSR=30%).

Example 65

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

Step 1. Preparation of tert-butyl N-[[4-[1-[3-(1-methyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate

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 h. Then tert-butyl hydrazinecarboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into a saturated _Na2CO3 solution. The mixture was filtered. The filter cake _was washed with a saturated _NaHCO3 solution and water. Then the filter cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (MSM:methanol=100:1) _to give the title compound ( _0.758 g; yield: 35.8%). MS (m/z): _[ M+H]+ calcd for _C22H27N9O5S : 530.18; found: 530.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.93 (d, J = 1.8 Hz, 1H), 8.87 (s, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 7.95 (dd, J = 28.4; 4.1 Hz, 1H), 6.82 (t, J = 17.0 Hz, 1H), 6.70 (s, 1H), 4.64 (d, J = 9.3 Hz, 2H), 4.23 (t, J = 23.2 Hz, 2H), 3.42 (s, 2H), 3.09 (q, J = 7.4 Hz, 2H), 1.52 (s, 9H), 1.42 (t, J = 7.4 Hz, 3H).

Step 2. Preparation of 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide trifluoroacetate

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) was slowly added trifluoroacetic acid (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was concentrated under reduced pressure to give the title compound as a white solid (0.61 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₁₇ H ₁₉ N ₉ O ₃ S: 430.13; detected: 430.0.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

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

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.154 g; yield: 37%; DSR=40%).

Example 66

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate

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

A mixture of (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile (1486 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1141 mg, 3.754 mmol) in DMSO (12 mL) was stirred at room temperature for 7 h. Then (S)-methyl 6-amino-2-((tert-butoxycarbonyl)amino)hexanoate (1448 mg, 4.88 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into _a saturated _Na2CO3 solution. The mixture was filtered. The filter cake was washed with a saturated _NaHCO3 solution and water. Then the filter cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=150:1) to _give the title compound (0.828 g; yield: 34.9%). MS (m/z): [M ₊ H]+ calcd. for _C30H40N8O5 : _593.31 ; found: 593.2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 9.95 (d, J=7.5 Hz, 1H), 8.91 (s, 1H), 8.32 (d, J=11.9 Hz, 2H), 7.99 (d, J=4.0 Hz, 1H), 6.82 (d, J=4.0 Hz, 1H), 4.76 (dd, J=12.8; 7.5 Hz, 1H), 4.60 (s, 1H), 4.28 (td, J=9.9; 3.8 Hz, 1H), 3.81 (s, 3H), 3.22-3.05 (m, 3H), 2.97 (dd, J=17.0; 3.8 Hz, 1H), 2.61 (dt, J=16.5; 8.4 Hz, 1H), 2.13-1.88 (m, 3H), 1.65-1.50 (m, 7H), 1.44-1.35 (m, 9H), 1.27 (d, J=12.8 Hz, 4H).

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

To a stirred solution 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 (0.6 g, 1.01 mmol) in EtOAc (12 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.53 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calc. for C ₂₅ H ₃₂ N ₈ O ₃ : 493.25; detected: 493.2.

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

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

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.197 g; yield: 45.5%; DSR=16%).

Example 67

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

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

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 at room temperature for 7 h. Then tert-butyl hydrazine carboxylate (634.4 mg, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into _a saturated _Na2CO3 solution. The mixture was filtered. The filter cake was washed with a saturated _NaHCO3 solution and water. Then the filter cake was dissolved in DCM and washed with water. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (ESM:methanol=100:1) to _give the title compound ( _1.02 g; yield: 54.9%). MS (m/z): [M+H]+ calcd. for _C23H28N8O3 : _465.22 ; found: 465.2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.97 (d, J=1.9 Hz, 1H), 8.86 (s, 1H), 8.31 (d, J=15.2 Hz, 2H), 7.97 (d, J=4.0 Hz, 1H), 6.87-6.84 (m, 1H), 6.62 (s, 1H), 4.27 (td, J=10.0; 3.9 Hz, 1H), 3.14 (dd, J=17.0; 8.6 Hz, 1H), 2.97 (dd, J=17.0; 3.9 Hz, 1H), 2.61 (dt, J=17.0; 8.6 Hz, 1H), 2.04-1.92 (m, 1H), 1.79-1.62 (m, 5H), 1.54 (d, J=13.3 Hz, 9H), 1.35-1.19 (m, 3H).

Step 2. Preparation of 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride

To a stirred solution of tert-butyl N-[[4-[1-[(1H)-2-cyano-1-cyclopentylethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]carbamate (0.75 g, 1.61 mmol) in EtOAc (15 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (3 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.557 g; yield: 95%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₁₈ H ₂₀ N ₈ O: 365.17; detected: 365.1.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and 4-[1-[(1R)-2-cyano-l-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide

Sodium hyaluronate (263.5 mg, 0.654 mmol as the carboxylic acid, MW 50 kDa) was dissolved in 53 mL of deionized water in a 100 mL round-bottomed flask, and 34 mL of acetonitrile was then added dropwise with stirring. 4-[1-[(1R)-2-cyano-1-cyclopentylethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (200 mg, 0.458 mmol) and 4-methylmorpholine (NMM, 46 mg, 0.458 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 h. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 181 mg; 0.654 mmol) was added to the solution and stirred for 72 h at room temperature. Then NaCl (382 mg; 6.54 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (300 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (382 mg; 6.54 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (200 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid (0.316 g; yield: 66.5%; DSR: 33%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.88-8.65 (m, 0.33H), 8.45-7.28 (m, 0.99H), 7.07-6.82 (m, 0.33H), 4.68-4.27 (m, 2.33H), 4.21-2.62 (m, 10.66N), 2.47-2.23 (m, 0.33N), 2.15-1.32 (m, 4.32N), 1.31-1.02 (m, 0.99N).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.308 g; yield: 64.8%; DSR=21%).

Example 68

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl-2-aminoacetate

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

A mixture of 3-[(36R,4R)-3-[[7-(hydroxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile (600 mg; 1.754 mmol), 2-(tert-butoxycarbonylamino)acetic acid (1.23 mg; 7.02 mmol) and triphenylphosphine (PPh ₃ ; 1840 mg; 7.02 mmol) in THF (120 mL) was stirred at 0°C-10°C for 10 min. Diisopropyl azodicarboxylate (DIAD; 1420 mg; 7.02 mmol) was then added dropwise. The reaction mixture was stirred at this temperature for 16 h. The reaction mixture was poured into saturated _NaHCO3 solution using an ice bath. The mixture was diluted with EtOAc and washed with saturated brine. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (EtOAc:hexane=1:2) to give the title compound (400 mg; yield: 45.7%). MS (m _/ z): [M+H]+ calcd. for _{C24H33N7O5 : 500.25} ; found: 500.1; ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.31 (t, J=16.1 Hz, 1H), 7.15 (dd, J=13.4; 3.7 Hz, 1H), 6.56 (t, J=3.9 Hz, 1H), 6.23 (d, J=2.8 Hz, 2H), 5.14 (s, 1H), 4.96 (s, 1H), 4.10-3.98 (m, 1H), 3.97-3.76 (m, 3H), 3.68-3.45 (m, 4H), 3.42-3.30 (m, 3H), 2.57-2.43 (m, 1H), 2.01-1.85 (m, 1H), 1.82-1.69 (m, 1H), 1.51-1.32 (m, 9H), 1.27-1.24 (m, 2H), 1.13-1.09 (m, 1H).

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

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

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl-aminoacetate

Sodium hyaluronate (178 mg; 0.443 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 53 mL of deionized water in a 100 mL round flask. To the solution were added [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 2-aminoacetate trifluoroacetate (150 mg; 0.443 mmol) and 4-methylmorpholine (NMM; 44.8 mg; 0.443 mmol) at room temperature, which resulted in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 h. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 122 mg; 0.443 mmol) was added to the solution and stirred for 72 h at room temperature. Then NaCl (259 mg; 4.43 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (300 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (259 mg; 4.43 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (200 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried in vacuo to give the title compound as a white solid (0.15 g; yield: 44.5%; DSR: 21%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.24-8.11 (m, 0.21H), 7.40-7.27 (m, 0.21H), 6.78-6.67 (m, 0.21H), 6.31-6.07 (m, 0.42H), 4.59-4.17 (m, 2.21H), 4.11-2.95 (m, 12.31H), 2.49-2.35 (m, 0.21H), 2.00-1.61 (m, 3.42H), 1.27-1.16 (m, 0.2 1H), 1.07-0.90 (m, 0.42H).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.108 g; yield: 45.7%; DSR=19%).

Example 69

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl-4-aminobutanoate

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

A mixture of 3-[(3R,4R)-3-[[7-(hydroxymethyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]-4-methyl-1-piperidyl]-3-oxo-propanenitrile (300 mg; 0.877 mmol), 4-(tert-butoxycarbonylamino)butanoic acid (713 mg; 3.51 mmol) and triphenylphosphine (920 mg; 3.51 mmol) in THF (60 mL) was stirred at 0°C-10°C for 10 min. Then diisopropyl azodicarboxylate (709.5 mg; 3.51 mmol) was added dropwise. The reaction mixture was stirred at this temperature for 16 h. The reaction mixture was poured into saturated NaHCO ₃ solution using an ice bath. The mixture was diluted with EtOAc and washed with brine. 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 give _the title compound (240 mg; yield: 51.9%). MS (m/z): [M ₊ H]+ calcd for _C26H37N7O5 , _528.28 ; found: 528.1. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 8.34 (d, J=7.5 Hz, 1H), 7.16 (dd, J=13.8; 3.7 Hz, 1H), 6.55 (t, J=4.1 Hz, 1H), 6.17 (d, J=3.4 Hz, 2H), 5.14 (s, 1H), 4.61 (s, 1H), 4.09-3.99 (m, 1H), 3.89-3.56 (m, 3H), 3.54-3.31 (m, 5H), 3.12 (d, J=6.1 Hz, 2H), 2.50 (d, J=26.3 Hz, 1H), 2.37 (t, J=7.3 Hz, 2H), 2.01-1.86 (m, 1H), 1.78 (dd, J=14.2; 7.1 Hz, 2H), 1.50-1.30 (m, 9H), 1.31-1.21 (m, 3H), 1.13-1.09 (m, 1H).

Step 2. Preparation of trifluoroacetate [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 4-aminobutanoate

To a stirred mixture of [5-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 4-(tert-butoxycarbonylamino)butanoate (0.12 g, 0.221 mmol) in DCM (5 mL) was added trifluoroacetic acid (0.5 mL) slowly using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was concentrated under reduced pressure to give the title compound as a white solid (0.9 g; yield: 95%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₁ H ₂₉ N ₇ O ₃ : 428.23; found: 428.1.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 4-aminobutanoate

Sodium hyaluronate (127 mg; 0.31 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 53 mL of deionized water in a 100 mL round-bottomed flask. Trifluoroacetate [4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidin-7-yl]methyl 4-aminobutanoate (119 mg; 0.22 mmol) and 4-methylmorpholine (NMM; 22 mg; 0.22 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 h. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 85.7 mg; 0.31 mmol) was added to the solution and stirred for 72 h at room temperature. Then NaCl (181 mg; 3.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (300 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 mL of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (181 mg; 3.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (200 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried in vacuo to give the title compound as a white solid (0.119 g, yield: 48.7%; DSR: 24%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.23-7.99 (m, 0.24H), 7.41-7.09 (m, 0.24H), 6.83-6.63 (m, 0.24H), 6.18-5.89 (m, 0.48H), 4.59-4.16 (m, 2.24H), 4.24-2.66 (m, 12.64H), 2.50-2.18 (m, 0.72H), 2.04-1.48 (m, 3.72H), 1.27-1.18 (m, 0.24H), 1.12-0.76 (m, 0.72H).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.109 g; yield: 44.6%; DSR=22%).

Example 70

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (3Z)-1-[[(1S)-5-amino-1-methoxycarbonyl-pentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylniperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate

Step 1. Preparation of methyl (3Z)-1-[[(1S)-5-(tert-butoxycarbonylamino)-1-methoxycarbonyl-pentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indolin-6-carboxylate

A mixture of methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indolin-6-carboxylate (1079 mg, 2 mmol) and bis(4-nitrophenyl)carbonate (668.8 mg, 2.2 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then (S)-methyl 6-amino-2-((tert-butoxycarbonyl)amino)hexanoate (593.6 mg, 2 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was poured into water and lyophilized. The crude residue was purified by column chromatography (MSM:methanol=5:1) to give the title compound (1 g; yield: 60.6%). MS (m/z): [M+H]+ calcd for C ₄₄ H ₅₀ N ₇ O ₉ : 826.40; found: 826.4. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 12.03 (s, 1H), 9.60 (d, J=7.5 Hz, 1H), 8.93 (d, J=1.3 Hz, 1H), 7.63 2.50-2.18 (m, 0.72H), (dd, J=12.7; 7.4 Hz, 1H), 3.83 (d, J=16.3 Hz, 6H), 3.27-3.09 (m, 5H), 2.80 (s, 2H), 2.64-2.44 (m, 6H), 2.37 (s, 3H), 2.05-1.99 (m, 2H), 1.91 (dd, J=13.9; 8.9 Hz, 4H), 1.54 (dd, J=18.8; 7.6 Hz, 3H), 1.42 (s, 9H).

Step 2. Preparation of methyl (3Z)-1-[[(1S)-5-amino-1-methoxycarbonyl-pentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate hydrochloride

To a stirred solution of methyl (3Z)-1-[[(1S)-5-(tert-butoxycarbonyl amino)-1-methoxycarbonylpentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indolin-6-carboxylate (0.6 g, 0.686 mmol) in EtOAc (12 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (2.4 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.51 g; yield: 97%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₃₉ H ₄₇ N ₇ O ₇ : 726.35; found: 726.2.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (3Z)-1-[[(1S)-5-amino-1-methoxycarbonyl-pentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate

Sodium hyaluronate (121 mg; 0.3 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 24.2 mL of deionized water in a 100 mL round-bottomed flask, and 15.7 mL of acetonitrile was then added dropwise with stirring. Methyl (3Z)-1-[[(1S)-5-amino-1-methoxycarbonylpentyl]carbamoyl]-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate (160 mg; 0.21 mmol) and 4-methylmorpholine (NMM; 21 mg; 0.21 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 83 mg; 0.3 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (123 mg; 2.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour and then acetone (200 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (123 mg; 2.1 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour and then acetone (200 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried in vacuo to give the title compound as a white solid (0.142 g; yield: 43.5%; DSR: 34%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.44-8.37 (m, 0.34 H), 7.50-6.59 (m, 3.4 H), 6.13-6.04 (m, 0.34 H), 4.58-4.12 (m, 2.34 H), 4.10-2.51 (m, 17.48 H), 2.12-1.82 (m, 2.04 H), 1.74-1.00 (m, 4.02 H).

In the case of the interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.13 g; yield: 39.9%).

Example 71

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate

Step 1. Preparation of methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate

A mixture of methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indoline-6-carboxylate (1079 mg, 2 mmol) and bis(4-nitrophenyl)carbonate (668.8 mg, 2.2 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then tert-butyl hydrazinecarboxylate (264.3 mg, 2 mmol) was added. The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (MSM:methanol=8:1) to give the title compound (0.5 g; yield: 35.8%). MS ₍ m/z): _[ M ₊ H]+ calcd for _C37H43N7O7 : 698.32; found: 698.1. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 11.79 (s, 1H), 10.29 (s, 1H), 9.26 (s, 1H), 8.69 (s, 1H), 7.76-7.33 (m, 6H), 7.19 (d, J=8.3 Hz, 2H), 7.06 (d, J=7.7 Hz, 2H), 5.76 (s, 1H), 3.80 (s, 3H), 3.08 (s, 3H), 2.74-2.53 (m, 2H), 2.19 (d, J=28.7 Hz, 10H), 1.46 (s, 9H).

Step 2. Preparation of methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate hydrochloride

To a stirred solution of methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indolin-6-carboxylate (0.4 g, 0.57 mmol) in EtOAc (4 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (1.6 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.36 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₃₂ H ₃₅ N ₇ O ₅ : 598.26; detected: 598.1.

Step 3. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate

Sodium hyaluronate (136 mg; 0.337 mmol based on the carboxylic acid, MW 50 kDa) was dissolved in 30 mL of deionized water in a 100 mL round-bottomed flask, then 19.5 mL of acetonitrile was added dropwise with stirring. Methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]-phenyl-methylene]-2-oxo-indoline-6-carboxylate hydrochloride (150 mg; 0.236 mmol) and 4-methylmorpholine (NMM; 23.9 mg; 0.236 mmol) were added to the solution at room temperature, which resulted in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 93 mg; 0.337 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour and then acetone (200 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour and then acetone (200 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid (0.153 g; yield: 47.3%; DSR: 21%); ¹ H NMR (400 MHz, D ₂ O) δ ^ppm 8.63-8.47 (m, 0.2 1H), 7.75-6.81 (m, 2.1H), 5.91-5.78 (m, 0.2 1H), 4.66-4.34 (m, 2H), 4.21-2.51 (m, 11.68H), 2.44-1.63 (m, 5.31H).

In the case of interaction with sodium hyaluronate (MM 2000 kDa) carried out in accordance with this stage, the corresponding product was obtained.

Example 72

Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (2R)-6-amino-2-[[2-[[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetyl]amino]hexanoate

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

A mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1248 mg; 4 mmol) and bis(4-nitrophenyl)carbonate (1.337 mg; 4.4 mmol) in DMSO (30 ml) was stirred at room temperature for 7 hours. Then, glycyl-glycyl-glycine (758.8 mg; 4 mmol) was added. The reaction mixture was stirred at 50°C for 16 hours. The reaction mixture was poured into water and lyophilized. The crude residue was purified by column chromatography (RMS:methanol=4:1) to give the title compound (0.7 g; yield: 33.2%). MS (m/z): [M+H]+ calcd. for C ₂₃ H ₂₉ N ₉ O ₆ : 528.22; detected: 528.1. ¹ H NMR (400 MHz, d-DMSO) δ ^ppm 12.78-12.38 (m, 1H), 9.98 (t, J=5.3 Hz, 1H), 8.46 (d, J=5.7 Hz, 1H), 8.35 (d, J=5.7 Hz, 1H), 8.21 (s, 1H), 7.67 (d, J=4.0 Hz, 1H), 6.85 (s, 1H), 4.87 (s, 1H), 4.21-4.02 (m, 4H), 4.01-3.60 (m, 8H), 3.39 (d, J=16.8 Hz, 3H), 2.39 (d, J=6.4 Hz, 1H), 1.78 (d, J=47.7 Hz, 1H), 1.60 (d, J=8.7 Hz, 1H), 1.02 (d, J=7.1 Hz, 3H).

Stage 2. Receipt methyl-(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidium l]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetyl]amino]hexanoate

2-[[2-[[2-[[4-[[(3R,4R)-1-(2-Cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetic acid (768 mg; 1.46 mmol), (S)-methyl 6-amino-2-((tert-butoxycarbonyl)-amino)hexanoate (454 mg; 1.53 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 335 mg; 1.75 mmol) and 1-hydroxybenzotriazole (HOBt; 236 mg; 1.75 mmol) were dissolved in DCM (20 mL), DMF (2 ml) and triethylamine (368 mg, 3.64 mmol). The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (methanol/DCM=1:150-1:80) to give the title compound (0.66 g; yield: 76.7%). MS (m/z): [M+H]+ calcd for C ₃₅ H ₅₁ N ₁₁ O ₉ : 770.38; found: 770.2. ¹ H-NMR (400 MHz, CDCl ₃ ) δ ^ppm 10.41 (s, 1H), 8.29 (d, J=8.0 Hz, 1H), 7.63 (dd, J=20.0; 3.8 Hz, 1H), 7.41 (s, 1H), 7.05-6.86 (m, 1H), 6.60 (dd, J=12.6; 4.0 Hz, 1H), 5.12 (s, 1H), 4.89 (s, 1H), 4.51 (s, 1H), 4.16 (d, J=36.3 Hz, 2H), 4.13-3.80 (m, 5H), 3.79-3.43 (m, 9H), 3.42-3.28 (m, 3H), 3.01 (d, J=26.8 Hz, 2H), 2.51 (d, J=6.2 Hz, 1H), 2.05-1.92 (m, 1H), 1.86-1.76 (m, 1H), 1.43 (s, 9H), 1.38-1.16 (m, 6H), 1.14-0.99 (m, 3H).

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

To a stirred solution of methyl (2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetyl]amino]hexanoate (0.51 g, 0.663 mmol) in EtOAc (20 mL) was slowly added 4 M HCl in ethyl acetate (commercially available) (8.5 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.44 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₃₀ H ₄₃ N ₁₁ O ₇ : 670.33; found: 670.1.

Step 4. Preparation of a conjugate between HA (in the form of sodium hyaluronate) and methyl (2R)-6-amino-2-[[2-[[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]-methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetyl]amino]hexanoate

Sodium hyaluronate (2456 mg; 0.607 mmol in terms of carboxylic acid, MW 50 kDa) was dissolved in 60 ml of deionized water in a 100 ml round flask, then 39 ml of acetonitrile was added dropwise with stirring. Methyl (2R)-6-amino-2-[[2-[[2-[[2-[[4-[[(3R,4R)-1-(2-cyanoacetyl)-4-methyl-3-piperidyl]methyl-amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]acetyl]amino]acetyl]amino]acetyl]amino]hexanoate hydrochloride (300 mg; 0.425 mmol) and 4-methylmorpholine (NMM; 43 mg; 0.425 mmol) were added to the solution at room temperature, resulting in a temporary increase in viscosity. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 168 mg; 0.607 mmol) was added to the solution and stirred for 72 h at room temperature. Then NaCl (355 mg; 6.07 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (400 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was dissolved in 80 ml of a mixture of deionized water and acetonitrile (v/v=3:1). Then NaCl (355 mg; 6.7 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (400 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid (0.25 g; yield: 41%; DSR: 15%); ¹ H NMR (400 MHz, D ₂ O) δ _ppm 8.26-8.13 (m, 0.15 H), 7.60-7.47 (m, 0.15 H), 6.80-6.66 (m, 0.15 H), 4.66-4.28 (m, 2.15 H), 4.24-2.69 (m, 13.15 H), 2.41-2.24 (m, 0.15 H), 2.04-1.42 (m, 3.3 H), 1.37-0.89 (m, 1.35 H).

In the case of the reaction with sodium hyaluronate (MW 2000 kDa) carried out in accordance with this step, the corresponding product was obtained (0.26 g; yield: 42.6%; DSR=14%). ¹ H-NMR (400 MHz, D ₂ O) δ ppm ^-1 8.26-8.13 (m, 0.14H), 7.59-7.48 (m, 0.14H), 6.79-6.66 (m, 0.14H), 4.66-4.28 (m, 2.14H), 4.26-2.66 (m, 12.94H), 2.40-2.24 (m, 0.14H), 2.03-1.42 (m, 3.28H), 1.35-0.89 (m, 1.26H).

Example 73

Preparation of a conjugate of 2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

Step 1. Preparation of 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (812 mg, 2.6 mmol) and bis(4-nitrophenyl)carbonate (870 mg, 2.86 mmol) in DCM (60 mL) was added triethylamine (1.19 mL, 8.58 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (832 mg, 3.9 mmol) was added. The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (EtOc:hexane=5:1-1:1) to give the title compound (0.58 g; yield: 40%). MS ₍ m/z): [M+H]+ calcd. _{for C28H37N7O5} : _552.65 ; found: 552.2.

Step 2. Preparation of 2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate as a trifluoroacetic acid salt

To a stirred solution of 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (166 mg, 0.3 mmol) in DCM (16.5 mL) was slowly added _CF3COOH (3.3 mL) using an ice bath. The reaction mixture was warmed to room temperature and then stirred at room temperature for 3 h. The solution was diluted with DCM and concentrated under reduced pressure to give the title compound as a brown oil (0.2 g; yield: 100%), which was used in the next step without further purification. MS (m/z): [M+H]+ calcd. for C ₂₃ H ₂₉ N ₇ O ₃ : 452.53; detected: 452.3.

Step 3. Preparation of a conjugate of 2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate and HA

To a solution of hyaluronic acid (161 mg; 0.4 mmol) in 32 ml of deionized water and 20 ml of acetonitrile, 4-methylmorpholine (NMM; 28 mg; 0.28 mmol) was added, and the solution was then cooled to 0°C. 2-chloro-4,6-dimethoxy-1,3,5-triazine (166 mg; 0.6 mmol) was added, and the mixture was stirred at room temperature for 1 hour. Then, a solution of 2-azaspiro[3.3]heptan-6-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (157 mg; 0.28 mmol) in water (5 mL) was added and stirred for 72 h at room temperature. Then, NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then acetone (300 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (MW of sodium hyaluronate 2000 kDa; 0.14 g; yield: 61.5%, DSR: 3%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.27-8.18 (m, 0.03H), 7.63-7.51 (m, 0.03H), 6.90-6.77 (m, 0.03H), 5.29-5.16 (m, 0.03H), 4.57-4.28 (m, 2.03N), 4.11-2.87 (m, 10.39N), 2.64-2.47 (m, 0.12N), 2.30-1.46 (m, 3.06N), 1.07-0.97 (m, 0.09N).

Example 74

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

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

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1249.5 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg; 4.8 mmol) in DCM (120 mL) was added triethylamine (1012 mg; 1.39 mL; 10 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl (3-hydroxycyclobutyl)carbamate (1123 mg; 6 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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: _10-1 :5) to give the title compound (1.5 g; yield: 71.4%). MS (m/z): [M ₊ H]+ calcd. for _C26H35N7O5 : _526.61 ; found: 526.2. ¹ H NMR (400 MHz, chloroform-d) δ ^ppm 8.47 (d, J=9.3 Hz, 1H), 7.44 (dd, J=11.3; 4.2 Hz, 1H), 6.64 (d, J=4.2 Hz, 1H), 5.19 (d, J=33.2 Hz, 2H), 5.05 (t, J=6.8 Hz, 1H), 4.04 (dd, J=13.2; 4.6 Hz, 1H), 3.82 (dd, J=13.7; 7.4 Hz, 1H), 3.69-3.57 (m, 1H), 3.56-3.44 (m, 2H), 3.37 (d, J=18.8 Hz, 3H), 3.09-2.95 (m, 2H), 2.50 (dt, J=14.1; 6.3 Hz, 1H), 2.18 (d, J=10.0 Hz, 2H), 2.01-1.86 (m, 1H), 1.83-1.69 (m, 1H), 1.68-1.53 (m, 2H), 1.45 (s, 9H), 1.08 (dd, J=15.2; 7.1 Hz, 3H).

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

To a solution of 3-((tert-butoxycarbonyl)amino)cyclobutyl 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.525 g, 1 mmol) in ethyl acetate (20 mL) was added 4 M HCl in ethyl acetate (4 mL, 16 mmol) dropwise at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.46 g; yield: 99.6%). MS (m/z): [M+H]+ calc. for C ₂₁ H ₂₇ N ₇ O ₃ : 426.49; detected: 426.2.

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

To a solution of hyaluronic acid (201 mg; 0.5 mmol) in 40 ml of deionized water and 20 ml of acetonitrile, 4-methylmorpholine (NMM; 50 mg; 0.5 mmol) was added, and then the solution was cooled to 0°C. 2-chloro-4,6-dimethoxy-1,3,5-triazine (277.5 mg; 1 mmol) was added, and then stirred at room temperature for 1 hour. 3-Aminocyclobutyl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (231 mg; 0.5 mmol) was added, and then stirred for 72 hours at room temperature. After this, NaCl (439 mg; 7.5 mmol) was added to the reaction mixture and stirred for 1 h, and then acetone (300 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (MW sodium hyaluronate 50 kDa; 0.31 g; yield: 78.8%, DSR: 33%); ¹ H-NMR (400 MHz, deuterium oxide) δ ^ppm 8.25-8.13 (m, 0.33H), 7.60-7.47 (m, 0.33H), 6.88-6.74 (m, 0.33H), 5.12-4.95 (m, 0.66H), 4.56-4.31 (m, 2Н), 4.15-2.88 (m, 13.3Н), 2.76-2.61 (m, 0.33Н), 2.43-2.19 (m, 0.66Н), 2.05-1.41 (m, 4.32Н), 1.09-0.80 (m, 0.99Н).

(MW sodium hyaluronate 500 kDa; 0.22 g; yield: 55.9%, DSR: 41%), ¹ H-NMR (400 MHz, deuterium oxide) δ ppm ^-1 8.19-8.04 (m, 0.41H), 7.54-7.38 (m, 0.41H), 6.85-6.61 (m, 0.41H), 5.10-4.95 (m, 0.82H), 4.56-4.20 (m, 2H), 4.06-2.70 (m, 14.1H), 2.69-2.49 (m, 0.41H), 2.40-2.15 (m, 0.82H), 2.10-1.44 (m, 4.64H), 1.08-0.68 (m, 1.23H).

(Sodium hyaluronate MM 2000 kDa; 0.2 g; yield: 50.8%, DSR: 49%); ¹ H-NMR (400 MHz, deuterium oxide) δ ^ppm 8.20-7.89 (m, 0.49H), 7.60-6.98 (m, 0.49H), 6.61-6.54 (m, 0.49H), 5.22-4.87 (m, 0.98H), 4.64-4.26 (m, 2Н), 4.16-2.80 (m, 14.9Н), 2.78-2.63 (m, 0.49Н), 2.48-2.24 (m, 0.98Н), 2.07-1.47 (m, 4.96Н), 1.11-0.76 (m, 1.47Н).

Example 75

Preparation of a conjugate of azepan-4-yl-4-(((3K,4K)-1-(2-cyanoacetyl)-4-methyl1 speridin-3-ylXmethyl)amino)-7H-pyrrolo[2.3^]pyrimidine-7-carboxylate and HA

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

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (937 mg, 3 mmol) and bis(4-nitrophenyl)carbonate (1094 mg, 3.6 mmol) in DCM (30 mL) was added triethylamine (2.78 mL, 20 mmol). The reaction mixture was heated to 40 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl 4-hydroxyazepane-1-carboxylate (775 mg, 3.6 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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=1:1-4:1) to give _the title compound (0.93 g; yield: 56%). MS (m/z): [M+H]+ calcd for _C28H39N7O5 , _{554.66 ;} found: 554.2. ₁ H-NMR (400 MHz, chloroform-d) δ ^ppm 8.45 (d, J=8.3 Hz, 1H), 7.44 (dd, J=12.1; 4.1 Hz, 1H), 6.62 (d, J=4.0 Hz, 1H), 5.28 (s, 1H), 5.14 (s, 1H), 4.09-3.75 (m, 2H), 3.69-3.28 (m, 11H), 2.59-2.37 (m, 1H), 2.14-1.84 (m, 6H), 1.86-1.71 (m, 2H), 1.48 (s, 9H), 1.08 (dd, J=15.2; 7.1 Hz, 3H).

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

To a solution of 1-(tert-butoxycarbonyl)azepan-4-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (0.63 g, 1.14 mmol, 1 eq.) in ethyl acetate (12 mL) was added 4 M HCl in ethyl acetate (4 mL, 16 mmol) dropwise at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 16 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.55 g; yield: 98.5%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₂₃ H ₃₁ N ₇ O ₃ : 454.55; found: 454.2.

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

To a solution of hyaluronic acid (201 mg; 0.5 mmol carboxylic acid) in 40 ml deionized water and 20 ml acetonitrile was added 4-methylmorpholine (NMM; 50 mg; 0.5 mmol) and then the solution was cooled to 0°C. 2-chloro-4,6-dimethoxy-1,3,5-triazine (207.5 ml; 0.75 mmol) was added and stirred at room temperature for 1 hour. The solution was mixed with azepan-4-yl-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate hydrochloride (245 mg; 0.5 mmol) and stirred for 72 h at room temperature. Then, NaCl (439 mg; 7.5 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then acetone (300 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.18 g; yield: 44.2%, DSR: 12%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.31-8.13 (m, 0.12H), 7.59-7.42 (m, 0.12H), 6.89-6.76 (m, 0.12H), 5.28-5.16 (m, 0.24H), 4.60-4.30 (m, 2H), 4.14-3.04 (m, 11.56H), 2.47-2.34 (d, J=31.1 Hz, 0.12H), 2.19-1.49 (m, 3.96H), 1.10-0.91 (m, 0.36H).

(MM sodium hyaluronate 2000 kDa; 0.16 g; yield: 39.3%, DSR: 18%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.29-8.17 (m, 0.18H), 7.57-7.41 (m, 0.18H), 6.86-6.74 (m, 0.18H), 5.29-5.16 (m, 0.36H), 4.56-4.25 (m, 2Н), 4.13-2.77 (m, 12.34Н), 2.46-2.31 (d, J=31.1 Hz, 0.18Н), 2.19-1.36 (m, 4.44Н), 1.08-0.82 (m, 0.54Н).

Example 76

Preparation of the conjugate 3-((3R,4R)-3-((7-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile and HA

Step 1. Preparation of tert-butyl (1S,4S)-5-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate

A mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1250 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DMSO (25 mL) was stirred at room temperature for 7 h. Then tert-butyl (1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate (992 mg, 5 mmol) was added. The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (MSM:methanol=50:1) to give the title compound (2 g; yield: 93.1%). MS (m/z ₎ : _[ M+H]+ calcd for _C27H36N8O4 : _537.64 ; found: 537.2. ¹ H-NMR (400 MHz, chloroform-d) δ 8.30 (d, J=5.9 Hz, 1H), 7.30 (d, J=9.4 Hz, 1H), 6.74-6.52 (m, 1H), 5.10 (s, 1H), 4.68 (d, J=124.9 Hz, 2H), 4.17-4.02 (m, 1H), 3.81 (d, J=53.0 Hz, 3H), 3.66-3.24 (m, 9H), 2.59-2.45 (m, 1H), 1.96 (qd, J=11.8; 9.6; 7.0 Hz, 2H), 1.77 (s, 2H), 1.46 (s, 9H), 1.11 (dd, J=10.0; 7.1 Hz, 3H).

Step 2. Preparation of 3-((3R,4R)-3-((7-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile hydrochloride

To a solution of tert-butyl (1 S,4S)-5-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate (0.52 g; 0.97 mmol) in ethyl acetate (10 mL) was added dropwise 4 M HCl solution in ethyl acetate (2 mL; 8 mmol) at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 16 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.45 g; yield: 98.2%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₂₂ H ₂₈ N ₈ O ₂ : 437.52; found: 437.3.

Step 3. Preparation of the conjugate of 3-((3R,4R)-3-((7-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile and HA

Sodium hyaluronate (161 mg; 0.4 mmol) was dissolved in 32 ml of deionized water in a 100 ml round-bottomed flask, then 21 ml of acetonitrile was added dropwise with stirring. 3-((3R,4R-3-((7-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile hydrochloride (132 mg; 0.28 mmol) and 4-methylmorpholine (NMM; 28 mg; 0.28 mmol) were added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then acetone (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid. (MW sodium hyaluronate 2000 kDa; 0.19 g; yield: 59.5%, DSR: 29%); ¹ H NMR (400 MHz, D ₂ O) δ 8.23-8.08 (m, 0.29H), 7.36-7.21 (m, 0.29H), 6.85-6.73 (m, 0.29H), 4.65-4.30 (m, 2.29H), 4.10-2.72 (m, 14.35N), 2.47-2.33 (m, 0.29N), 2.03-1.44 (m, 2.58N), 1.14-0.86 (m, 0.87N).

Example 77

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

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)(methyl)carbamate

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (3442 mg, 11 mmol) and bis(4-nitrophenyl)carbonate (3352 mg, 11 mmol) in DCM (103 mL) was added triethylamine (2330 mg, 3.2 mL, 23 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl (4-aminophenethyl)(methyl)carbamate (2300 mg, 9.19 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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=1:2-4:1) to give the title compound (2.9 g; yield: 53.7%). MS (m/z): [M ₊ H]+ calcd for _{C31H40N8O4 : 589.71} ; found: 589.2.

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

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)phenethyl)(methyl)carbamate (0.588 g, 1 mmol, 1 eq.) in ethyl acetate (20 mL) was added 4 M HCl in ethyl acetate (8 mL, 32 mmol) dropwise at 0°C under _N2 . The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 16 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.48 g; yield: 98.3%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₂₆ H ₃₂ N ₈ O ₂ : 489.60; found: 489.3.

Step 3. Preparation of the conjugate of 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(2-(methylamino)ethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide and HA

4-methylmorpholine (NMM; 21 mg; 0.21 mmol) was added to a solution of hyaluronic acid (121 mg; 0.3 mmol) in 24 ml of deionized water and 14 ml of acetonitrile, and then cooled to 0°C. 2-chloro-4,6-dimethoxy-1,3,5-triazine (83 mg; 0.3 mmol) was added and stirred at room temperature for 1 hour. 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(2-(methylamino)ethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (110 mg; 0.21 mmol) was added and stirred for 72 h at room temperature. NaCl (175.5 mg; 3 mmol) was added to the reaction mixture and stirred for 1 h. Acetone (300 ml) was added dropwise with stirring, and then the mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.22 g; yield: 86.3%, DSR: 30%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.04-7.93 (m, 0.3H), 7.65-7.09 (m, 1.20H), 6.87-6.63 (m, 0.60H), 4.54-4.28 (m, 2.30H), 4.04-2.64 (m, 14.8H), 2.32-2.14 (m, 0.30H), 2.04-1.41(m, 3.60H), 1.07-0.82 (m, 0.90H).

(MW sodium hyaluronate 2000 kDa; 0.13 g; yield: 51%, DSR: 15%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.02-7.87 (m, 0.15H), 7.34-6.88 (m, 0.60H), 6.79-6.36 (m, 0.30H), 4.55-4.14 (m, 2.15H), 4.07-2.51 (m, 12.4H), 2.36-2.22 (m, 0.15H), 1.87 (s, 3H), 1.47-1.37 (m, 0.30H), 1.05-0.80 (m, 0.45H).

Example 78

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

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

To a mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1190 mg, 3.81 mmol) and bis(4-nitrophenyl)carbonate (1390 mg, 4.57 mmol) in DCM (24 mL) was added triethylamine (1.3 mL, 9.5 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. The reaction mixture was cooled to room temperature and tert-butyl (3-aminophenethyl)carbamate (900 mg, 3.81 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The mixture was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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: _2-4 :5) to give the title compound (0.687 g; yield: 31.4%). MS (m/z): [M ₊ H]+ calcd for _C30H38N8O4 : _575.69 ; found: 575.2.

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

To a stirred solution of tert-butyl N-(3-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.66 g, 1.15 mmol) in EtOAc (13.2 mL) was slowly added 4 M HCl in ethyl acetate (2.64 mL) using an ice bath. The reaction mixture was allowed to warm to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.55 g; yield: 93.5%), which was used without further purification. MS (m/z): [M+H]+ calc. for C ₂₅ H ₃₀ N ₈ O ₂ : 475.57; detected: 475.2.

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

To a solution of sodium hyaluronate (161 mg; 0.4 mmol; MW 50 kDa) in 32 ml of deionized water and 21 ml of acetonitrile at room temperature were added N-(3-(2-aminoethyl)phenyl)-4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (143 mg; 0.28 mmol) and 4-methylmorpholine (NMM; 28 mg; 0.28 mmol). The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added to the solution and stirred for 72 h at room temperature. Then, NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.234 g; yield: 70%, DSR: 50%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.99-7.85 (m, 0.50H), 7.22-6.88 (m, 2H), 6.66-6.42 (m, 1H), 4.53-4.25 (m, 2.5H), 4.09-2.44 (m, 16.5H), 2.31-2.22 (m, 0.5H), 2.02-1.70 (m, 3H), 1.67-1.36 (m, 1H), 0.99-0.83 (m, 1.5H).

(MW sodium hyaluronate 2000 kDa; 0.229 g; yield: 68.5%; DSR: 40%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.98-7.83 (m, 0.40H), 7.22-6.89 (m, 1.6H), 6.67-6.38 (m, 0.8H), 4.51-4.23 (m, 2.40H), 4.08-2.42 (m, 15.2H), 2.31-2.15 (m, 0.40H), 2.01-1.70 (m, 3H), 1.65-1.35 (m, 0.80H), 0.96-0.80 (m, 1.20H).

Example 79

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

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)pyridin-2-yl)oxy)ethyl)carbamate

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1190 mg, 3.81 mmol) and bis(4-nitrophenyl)carbonate (1390 mg, 4.57 mmol) in DCM (24 mL) was added triethylamine (1.3 mL, 9.5 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl (2-((4-aminopyridin-2-yl)oxy)ethyl)carbamate (964 mg, 3.81 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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: _2-4 :5) to give the title compound (0.45 g; yield: 19.9%). MS ₍ m/z): [M+H]+ calcd for _C29H37N9O5 : _592.67 ; found: 592.3.

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

To the solution of tert-butyl-(2-((4-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-

To (methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamido)pyridin-2-yl)oxy)ethyl)carbamate (450 mg, 0.76 mmol) in ethyl acetate (9 mL) was added 4 M HCl in ethyl acetate (1.8 mL, 7.2 mmol) dropwise at 0 °C under N ₂ . The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.4 g, yield: 99.7%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₄ H ₂₉ N ₉ O ₃ : 492.56; detected: 492.3.

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

To a solution of sodium hyaluronate (0.153 g; 0.38 mmol; 1 equiv.) in acetonitrile (22 ml) and _H2O (30 ml) were added 4-methylmorpholine (0.058 g; 0.38 mmol; 1.5 equiv.) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.067 g; 0.38 mmol; 1 equiv.) at 0°C. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 1 h. N-(2-(2-aminoethoxy)pyridin-4-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; 0.38 mmol) was added to the reaction mixture, and then the pH of the reaction mixture was adjusted to 6.5-7 using 4-methylmorpholine. The resulting reaction mixture was stirred at room temperature for 3 days. To the above reaction mixture was added NaCl (222 mg; 10 eq.) in _H2O (2 mL) and stirred for 0.5 h. Then, acetone (350 mL) was added dropwise to the above reaction mixture until a precipitate formed. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid. (MW sodium hyaluronate 50 kDa; 0.167 g; yield: 51.6%; DSR: 18%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.52-8.19 (m, 0.18H), 7.87-7.34 (m, 0.54H), 7.08-6.68 (m, 0.36H), 4.63-4.31 (m, 2.18H), 4.13-2.93 (m, 12.34H), 2.50-2.29 (m, 0.18H), 2.22-1.52 (m, 3.36H), 1.14-0.86 (m, 0.54H).

(MM sodium hyaluronate 2000 kDa; 0.137 g; yield: 42.3%; DSR: 30%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.42-8.28 (m, 0.30H), 7.86-7.45 (m, 0.90H), 7.12-6.77 (m, 0.60H), 4.59-4.29 (m, 2.30H), 4.13-2.82 (m, 13.90H), 2.47-2.29 (m, 0.30H), 2.03-1.47 (m, 3.60H), 1.06-0.85 (m, 0.90H).

Example 80

Preparation of a conjugate of methyl N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate and HA

Step 1. Preparation of methyl N ² -(tert-butoxycarbonyl)-N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1561 mg, 5 mmol) and bis(4-nitrophenyl)carbonate (1824 mg, 6 mmol) in DCM (46 mL) was added triethylamine (1265 mg, 1.7 mL, 12.5 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 16 h. Then the reaction mixture was cooled to room temperature. Then methyl (tert-butoxycarbonyl) lysinate (1300 mg, 5 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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=1:5-3:1) to give the title compound ( _0.6 g; yield: 20%). MS (m/z): [M+ _H ]+ calcd. for _C29H42N8O6 : _599.71 ; found: 599.2.

Step 2. Preparation of methyl N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate hydrochloride

To a stirred solution of N ² -(tert-butoxycarbonyl)-N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate (0.6 g, 1 mmol) in EtOAc (30 mL) was slowly added 4 M HCl in ethyl acetate (2.7 mL) using an ice bath. The reaction mixture was allowed to warm to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.528 g; yield: 98.8%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₄ H ₃₄ N ₈ O ₄ : 499.59; detected: 499.2.

Step 3. Preparation of a conjugate of methyl N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate and HA

Methyl N ⁶ -(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)lysinate hydrochloride (150 mg; 0.28 mmol) and 4-methylmorpholine (NMM; 28 mg; 0.28 mmol) were added to a solution of sodium hyaluronate (161 mg; 0.4 mmol) in 32 ml and 21 ml of acetonitrile at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added to the solution and stirred for 72 h at room temperature. Then, NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.21 g; yield: 61.1%, DSR: 29%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.00-7.43 (m, 0.29H), 7.29-6.64 (m, 0.29H), 6.44-5.78 (m, 0.29H), 4.63-4.22 (m, 2.29H), 4.12-2.67 (m, 14.35N), 2.44-1.29 (m, 4.32N), 1.12-0.81 (m, 0.87N).

(MW of sodium hyaluronate 2000 kDa; 0.19 g; yield: 55.2%; DSR: 52%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.00-7.53 (m, 0.52H), 7.31-6.67 (m, 0.52H), 6.43-5.87 (m, 0.52H), 4.62-4.18 (m, 2.52H), 4.10-2.52 (m, 17.80N), 2.41-1.29 (m, 7.16N), 1.11-0.78 (m, 1.56N).

Example 81

Preparation of the conjugate 3-((3R,4R)-3-((7-(6-amino-2-azaspiro[3.3]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile and HA

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)-2-azaspiro[3.3]heptan-6-yl)carbamate

To a stirred mixture of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidyl]-3-oxopropanenitrile (1249.5 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DCM (120 mL) was added triethylamine (1012 mg, 1.39 mL, 10 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 6 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl (2-azaspiro[3.3]heptan-6-yl)carbamate (938 mg, 4.4 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. 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=1:20-1:7) to give the title compound (1.5 g; yield: 68.1%). MS (m/z): [M ₊ H]+ calcd _{for C28H38N8O4} : _551.66 ; found: 551.2.

Step 2. Preparation of 3-((3R,4R)-3-((7-(6-amino-2-azaspiro[3.3]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-metalpiperidin-1-yl)-3-oxopropanenitrile as a trifluoroacetic acid salt

To a stirred solution of tert-butyl (2-(4-(((3R,,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-2-azaspiro[3.3]heptan-6-yl)carbamate (275 mg, 0.5 mmol) in DCM (10 mL) was slowly added _CF3COOH (2 mL) using an ice bath. The reaction mixture was allowed to cool to room temperature and then stirred at room temperature for 4 h. The solution was diluted with DCM and concentrated under reduced pressure to give the title compound as a brown oil (0.26 g; yield: 78%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₂₃ H ₃₀ N ₈ O ₂ : 451.55; detected: 451.3.

Step 3. Preparation of the conjugate of 3-((3R,4R)-3-((7-(6-amino-2-azaspiro[3.3]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile and HA

4-Methylmorpholine (NMM; 36 mg; 0.35 mmol) was added to a solution of hyaluronic acid (201 mg; 0.5 mmol) in 40 ml of deionized water and acetonitrile (ACN; 17 ml). The solution was then cooled to 0°C, 2-chloro-4,6-dimethoxy-1,3,5-triazine (201 mg; 0.5 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The solution was mixed with 3-((3R,4R)-3-((7-(6-amino-2-azaspiro[3.3]heptane-2-carbonyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)(methyl)amino)-4-methylpiperidin-1-yl)-3-oxopropanenitrile as trifluoroacetic acid salt (197 mg; 0.35 mmol) and stirred for 72 h at room temperature. Then NaCl (292.5 mg; 5 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (300 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid.

(Sodium hyaluronate, MW 50 kDa; 0.21 g; yield: 51.7%, DSR: 15%); ¹ H NMR (400 MHz, deuterium oxide) δ 8.24-8.15 (m, 0.15H), 7.35-7.22 (m, 0.15H), 6.90-6.71 (m, 0.15H), 4.61-4.37 (m, 2.15H), 4.33-3.11 (m, 12H), 2.77-2.51 (m, 0.30H), 2.48-2.30 (m, 0.15H), 2.11-1.60 (m, 3.6H), 1.09-0.87 (m, 0.45H).

(MM sodium hyaluronate 2000 kDa; 0.2 g; yield: 49.3%; DSR: 11%); ¹ H-NMR (400 MHz, deuterium oxide) δ 8.26-7.99 (m, 0.11H), 7.36-7.17 (m, 0.11H), 6.88-5.73 (m, 0.11H), 4.62-4.35 (m, 2.11H), 4.34-3.04 (m, 11.54H), 2.73-2.52 (m, 0.22H), 2.46-2.29 (m, 0.11H), 2.12-1.54 (m, 3.44H), 1.11-0.81 (m, 0.33H).

Example 82

Preparation of the conjugate of methyl-(Z)-3-(((4-N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-1-(piperazine-1-carbonyl)indoline-6-carboxylate and NA

Step 1. Preparation of methyl (Z)-1-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate

A mixture of methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indoline-6-carboxylate (1079 mg, 2 mmol) and bis(4-nitrophenyl)carbonate (668.8 mg, 2.2 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then tert-butyl hydrazinecarboxylate (264.3 mg, 2 mmol) was added. The reaction mixture was stirred at 50 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. 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=8:1) to give the title compound (0.5 g; yield: 35.8%). MS (m/z): [M+H]+ calcd for C ₄₁ H ₄₉ N ₇ O ₇ : 752.89; found: 752.2. ¹ H-NMR (400 MHz, chloroform-d) δ ^ppm 12.08 (s, 1H), 7.58 (dt, J=14.8; 7.2 Hz, 3H), 7.45 (dd, J=13.8; 6.9 Hz, 3H), 6.98 (d, J=8.2 Hz, 2H), 6.80 (d, J=8.2 Hz, 2H), 5.97 (d, J=8.3 Hz, 1H), 3.85 (s, 12H), 3.18 (s, 3H), 2.85 (d, J=36.9 Hz, 4H), 2.51 (d, J=21.2 Hz, 8H), 1.49 (s, 9H).

Step 2. Preparation of methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-1-(piperazine-1-carbonyl)indoline-6-carboxylate hydrochloride

To a stirred solution of methyl (Z)-1-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxylate (0.4 g, 0.57 mmol) in EtOAc (4 mL) was slowly added 4 M HCl in ethyl acetate (1.6 mL) using an ice bath. The reaction mixture was allowed to warm to room temperature and then stirred at room temperature for 16 h. The solution was diluted with EtOAc and concentrated under reduced pressure to give the title compound as a white solid (0.36 g; yield: 99%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₃₆ H ₄₁ N ₇ O ₅ : 652.77; detected: 652.2.

Step 3. Preparation of a conjugate of methyl (Z)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxo-1-(piperazine-1-carbonyl)indoline-6-carboxylate and HA

To a solution of sodium hyaluronate (136 mg; 0.337 mmol) in 30 ml of deionized water and 19.5 ml of acetonitrile were added methyl (3Z)-1-(hydrazinecarbonyl)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indoline-6-carboxylate hydrochloride (150 mg; 0.236 mmol) and 4-methylmorpholine (NMM; 23.9 mg; 0.236 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 93 mg; 0.337 mmol) was added and stirred for 72 h at room temperature. Then NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (200 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to yield the title compound as a white solid. Sodium hyaluronate MW 50 kDa; 0.153 g; yield: 44.8%, DSR: 12%;

¹ H-NMR (400 MHz, deuterium oxide) δ 7.70-7.18 (m, 0.84H), 7.10-6.79 (m, 0.36H), 5.99-5.76 (m, 0.12H), 4.59-4.30 (m, 2H), 4.06-2.62 (m, 12.28Н), 2.34-2.12 (m, 0.96Н), 2.07-1.67 (m, 3Н).

Example 83

Preparation of a conjugate of methyl (Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate and HA

Step 1. Preparation of methyl (Z)-1-((1S,4S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate

A mixture of methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indoline-6-carboxylate (1349 mg, 2.5 mmol) and bis(4-nitrophenyl)carbonate (912 mg, 3 mmol) in DMSO (27 mL) was stirred at room temperature for 7 h. Then tert-butyl (1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate (620 mg, 3.125 mmol) was added. The reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=20:1) to give the title compound (1.15 g, yield: 60.3%). MS (m/z): [M+H]+ calcd for C ₄₂ H ₄₉ N ₇ O ₇ : 764.90; found: 764.2. ¹ H-NMR (400 MHz, chloroform-d) δ 12.08 (s, 1H), 7.91 (d, J = 15.4 Hz, 1H), 7.65-7.51 (m, 3H), 7.42 (dt, J = 24.9; 7.1 Hz, 3H), 6.99 (dd, J = 8.2; 4.9 Hz, 2H), 6.79 (t, J=7.8 Hz, 2H), 5.96 (dd, J=8.3; 5.2 Hz, 1H), 4.93 (s, 1H), 4.61 (s, 1H), 3.81 (d, J=38.8 Hz, 5H), 3.51 (s, 2H), 3.18 (s, 3H), 2.78 (s, 2H), 2.41 (s, 7H), 2.12-1.88 (m, 5H), 1.49 (m, 9H).

Step 2. Preparation of methyl (2)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate hydrochloride

To a solution of methyl (Z)-1-((1S,4S)-5-(tert-butoxycarbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate (600 mg, 0.786 mmol) in ethyl acetate (12 mL) was added dropwise at 0°C under N ₂ atmosphere 4 M HCl solution in ethyl acetate (2.4 mL, 9.6 mmol). The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (540 mg; yield: 98%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₃₇ H ₄₁ N ₇ O ₅ : 664.32; found: 664.3.

Step 3. Preparation of a conjugate of methyl (Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate and HA

Methyl (Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate hydrochloride (56 mg; 0.08 mmol) and 4-methylmorpholine (NMM; 8 mg; 0.08 mmol) were added to a solution of sodium hyaluronate (161 mg; 0.4 mmol) in 32 ml of deionized water and 21 ml of acetonitrile at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added to the solution and stirred for 72 h at room temperature. Then, NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was collected, washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.169 g; yield: 41.2%; DSR: 14%); ¹ H-NMR (400 MHz, deuterium oxide) δ 7.69-7.22 (m, 0.84H), 7.21-6.78 (m, 0.56H), 5.93-5.82 (m, 0.14H), 4.96-4.79 (m, 0.14H), 4.50-4.16 (m, 2.14H), 4.00-2.49 (m, 12.66H), 2.30-2.16 (m, 0.7H), 2.00-1.52 (m, 3H).

Example 84

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 chondroitin sulfate

Chondroitin sulfate (231.5 mg, 0.5 mmol) was dissolved in 40 ml of deionized water and 26 ml of acetonitrile. 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-c1]pyrimidine-7-carbohydrazide (129.5 mg, 0.35 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 138 mg, 0.5 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (293 mg; 5 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.23 g; yield: 55.5%; DSR: 19%). ¹ H-NMR (400 MHz, D ₂ O): δ ^ppm 8.32-8.24 (m, 0.19H), 7.67-7.58 (m, 0.19H), 6.89-6.79 (m, 0.19H), 4.64-4.33 (m, 2.19H), 4.26-3.19 (m, 11.71H), 2.05-1.44 (m, 3.57H), 1.16-0.83 (m, 0.57H).

Example 85

Preparation of a conjugate of 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and chondroitin sulfate

Chondroitin sulfate (266 mg; 0.58 mmol) was dissolved in 50 ml of deionized water and 25 ml of acetonitrile. 4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide (230 mg; 0.58 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 189 mg; 0.68 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (339 mg; 5.8 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.135 g; yield: 28.9%; DSR: 7%). ¹ H-NMR (400 MHz, D ₂ O): δ ^ppm 8.94-8.57 (m, 0.14H), 8.44-8.34 (m, 0.07H), 8.03-7.88 (m, 0.07H), 7.39-7.29 (m, 0.07H), 4.67-4.34 (m, 2.07H), 4.33-3.04 (m, 10.14H), 2.23-1.78 (m, 3.14H), 1.77-1.26 (m, 0.56H).

Example 86

Preparation of a conjugate of 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide and chondroitin sulfate

Chondroitin sulfate (200 mg; 0.43 mmol) was dissolved in 40 ml of deionized water and 20 ml of acetonitrile. 4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbohydrazide (200 mg; 0.43 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 141 mg; 0.51 mmol) was added to the solution, and the mixture was stirred for 72 hours at room temperature. Then NaCl (252 mg; 4.3 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.177 g; yield: 47.3%; DSR: 3%). ¹ H-NMR (400 MHz, D ₂ O): δ ^ppm 9.06-8.86 (m, 0.06H), 8.50-8.42 (m, 0.03H), 8.14-8.06 (m, 0.03H), 7.51-7.31 (m, 0.03H), 4.62-4.28 (m, 2.12H), 4.24-3.05 (m, 10.12H), 2.55-1.57 (m, 3.09H).

Example 87

Preparation of a conjugate of 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-N-methyl-methanesulfonamide and chondroitin sulfate

Chondroitin sulfate (250 mg; 0.54 mmol) was dissolved in 50 ml of deionized water and 25 ml of acetonitrile with stirring. 1-[4-[[7-(hydrazinecarbonyl)pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-amino]cyclohexyl]-N-methyl-methanesulfonamide (230 mg; 0.54 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 175 mg; 0.54 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (316 mg; 5.4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.314 g; yield: 69.5%; DSR: 16%). ¹ H-NMR (400 MHz, D ₂ O): δ ^ppm 8.44-8.35 (m, 0.16H), 7.91-7.80 (m, 0.16H), 7.04-6.92 (m, 0.16H), 4.64-4.36 (m, 2H), 4.35-3.05 (m, 10.48H), 3.03-2.92 (m, 0.32H), 2.77-2.60 (m, 0.48H), 2.56-2.39 (m, 0.16H), 2.20-1.58 (m, 3.8H), 1.49-1.30 (m, 0.64H).

Example 88

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

Chondroitin sulfate (231.5 mg, 0.5 mmol) was dissolved in 46 ml of deionized water and 23 ml of acetonitrile with stirring. Methyl (4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)-L-lysinate hydrochloride (187 mg, 0.35 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 145 mg, 0.53 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (293 mg; 5 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.23 g; yield: 48.1%; DSR: 35%). ¹ H-NMR (400 MHz, D ₂ O) δ 8.20-7.68 (m, 0.35H), 7.33-6.95 (m, 0.35H), 6.47-6.02 (m, 0.35H), 4.80-4.18 (m, 2.35H), 4.11-2.78 (m, 15.25N), 2.33-1.16 (m, 5.8N), 1.11-0.78 (m, 1.05N).

Example 89

Preparation of a conjugate of methyl (2S)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentyl-ethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate and chondroitin sulfate

Chondroitin sulfate (184 mg, 0.4 mmol) was dissolved in 42 ml of deionized water and 21 ml of acetonitrile with stirring. Methyl (28)-6-amino-2-[[4-[1-[(1R)-2-cyano-1-cyclopentylethyl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (210 mg, 0.4 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 130 mg, 0.47 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (234 mg; 4 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.153 g; yield: 39%, DSR: 5%); ¹ H NMR (400 MHz, D ₂ O): δ ^ppm 8.96-8.8 (m, 0.1H), 8.44-8.34 (m, 0.05H), 8.05-7.92 (m, 0.05H), 7.40-7.27 (m, 0.05H), 4.62-4.36 (m, 2.1H), 4.29-3.03 (m, 10.25H), 3.02-2.9 (m, 0.1H), 2.47-2.25 (m, 0.05H), 2.20-1.81 (m, 3.15H), 1.72-1.24 (m, 0.55H).

Example 90

Preparation of a conjugate of methyl (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate and chondroitin sulfate

Chondroitin sulfate (117 mg; 0.25 mmol) was dissolved in 35 ml of deionized water and 20 ml of acetonitrile with stirring. Methyl (2S)-6-amino-2-[[4-[1-[3-(cyanomethyl)-1-ethylsulfonyl-azetidin-3-yl]pyrazol-4-yl]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (150 mg; 0.25 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 83 mg; 0.3 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (146 mg; 2.5 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.123 g; yield: 50%, DSR: 4%). ¹ H-NMR (400 MHz, D ₂ O): δ ^ppm 9.04-8.96 (m, 0.08H), 8.49-8.41 (m, 0.04H), 8.12-8.03 (m, 0.04H), 7.49-7.33 (m, 0.04H), 4.62-4.29 (m, 2.16N), 4.25-3.05 (m, 10.32N), 3.03-2.84 (m, 0.08N), 2.55-1.50 (m, 3.08N), 1.47-1.19 (m, 0.28N).

Example 91

Preparation of a conjugate of methyl (2S)-6-amino-2-[[4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate and chondroitin sulfate

Chondroitin sulfate (331 mg, 0.72 mmol) was dissolved in 35 mL of deionized water and 20 mL of acetonitrile with stirring. Methyl (28)-6-amino-2-[[4-[methyl-[4-(methylsulfamoylmethyl)cyclohexyl]amino]pyrrolo[2,3-d]pyrimidine-7-carbonyl]amino]hexanoate (400 mg, 0.72 mmol) was added to the solution at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 235 mg, 0.85 mmol) was added to the solution and stirred for 72 hours at room temperature. Then NaCl (421 mg; 7.2 mmol) was added to the reaction mixture, the mixture was stirred for 1 h, and then anhydrous alcohol (40 mL) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with anhydrous alcohol and dried under vacuum to give the title compound as a white solid (0.255 g; yield: 35%; DSR: 5%). ¹ H-NMR (400 MHz, D ₂ O): δ ppm 8.44-8.34 (m, 0.05H), 7.91-7.79 (m, 0.05H), 7.02-6.92 (m, 0.05H), 4.64-4.39 (m, 2.05H), 4.31-3.06 (m, 10.5H), 3.03-2.90 (m, 0.1H), 2.52-2.35 (m, 0.15H), 2.34-1.54 (m, 3.4H), 1.48-1.24 (m, 0.35H).

Example 92

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

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)piperidine-1-carboxylate

To a mixture of tofacitinib (1.8 g, 5.76 mmol, 1 eq.) and bis(4-nitrophenyl)carbonate (1.93 g, 6.34 mmol, 1.1 eq.) in DCM (30 mL) was added triethylamine (1.46 g, 14.4 mmol, 2.5 eq.) under N ₂ and the reaction mixture was heated to reflux for 3 h. Tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (1.591 g, 5.76 mmol, 1 equiv) was then added and the resulting mixture was heated under reflux for 12 h. The solvent was removed under reduced pressure and the residue was purified by chromatography on silica to give the title product (1.25 g, yield: 35.3%); MS (m/z): [M+H]+ calcd for C ₃₃ H ₄₂ N ₈ O ₄ : 615.33; found: 615.3.

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

To a solution 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)piperidine-1-carboxylate (1 g; 3.18 mmol) in ethyl acetate (10 mL) was added dropwise at 0°C under N ₂ atmosphere 4 M HCl solution in ethyl acetate (4 mL; 16 mmol). The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 2 days. After removing the solvent under reduced pressure, the resulting solid was stirred in ethyl acetate (24 mL) for 0.5 h and then filtered to give the desired product as a solid (0.9 g; yield: 100%); MS (m/z): [M+H]+ calcd for C ₂₈ H ₃₄ N ₈ O ₂ : 515.28; found: 515.3.

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

Sodium hyaluronate (0.174 g; 0.432 mmol) was dissolved in MeCN (22 mL) and _H2O (35 mL). Then 4-methylmorpholine (0.066 g; 0.65 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.076 g; 0.432 mmol) were added to the above solution at 0°C. The resulting reaction mixture was stirred at 0°C for 30 min, then warmed to room temperature and stirred for 1 h. Then, 4-(((3r,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N-(4-(piperidin-4-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxamide hydrochloride (0.238 g, 0.432 mmol) was added to the solution and the resulting solution was stirred for 3 days at room temperature. NaCl (253 mg, 4.32 mmol) in _H2O (1 mL) was added to the above reaction mixture and stirred for 0.5 h. Then, acetone (350 mL) was added dropwise to the above mixture until a precipitate was formed. The mixture was filtered and the filter cake was washed with acetone. The solid was dissolved in MeCN (20 mL) and _H2O (40 mL) to form a homogeneous solution, dialyzed using a 3.5 kDa MW cutoff membrane against deionized water, and then lyophilized to afford the title compound (0.15 g; yield: 43.2%, DSR: 7.6%). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 7.49-7.29 (m, 0.53H), 4.70-4.20 (m, 2H), 4.00-3.23 (m, 9.14H), 2.50-2.30 (m, 0.19H), 1.99 (d, 3H), 1.60-1.40 (m, 0.51H), 1.3-1.15 (m, 0.2H), 1.15-0.85 (m, 0.2H).

Using the reaction conditions of step 3 and employing sodium hyaluronate (0.174 g; MW 2000 kDa), the corresponding product (0.16 g; yield: 46%, DSR=2.3%) was obtained. ¹ H-NMR (400 MHz, D ₂ O) δ ppm ^-1 7.75-7.15 (m, 0.16H), 4.50-4.25 (m, 2H), 4.15-2.90 (m, 10.34H), 2.45-2.35 (m, 0.09H), 1.99 (s, 3H), 1.25-1.15 (m, 0.05H), 1.05-0.95 (m, 0.1H).

Example 93

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

Step 1. Preparation of tert-butyl (2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazinyl)-2-oxoethyl)carbamate

To a solution of (tert-butoxycarbonyl)glycine (0.414 g; 2.364 mmol) in MeCN (10 mL) were added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 0.898 g; 2.364 mmol), N,N-diisopropylethylamine (DIPEA; 0.512 g; 3.94 mmol) at room temperature (rt). The resulting reaction mixture was stirred at rt for 30 min, then 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.8 g, 1.97 mmol, 1 eq) was added to the above solution. The resulting solution was stirred at room temperature for 10 h, then the solvent was removed under reduced pressure, the residue was purified by chromatography on silica to give the title product (0.642 g; yield: 62%); MS (m/z): [M+H] ⁺ calcd. for C ₂₄ H ₃₃ N ₉ O ₅ : 528.26; found: 528.3.

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

To a solution of tert-butyl (2-(2-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidine-7-carbonyl)hydrazinyl)-2-oxoethyl)carbamate (0.642 g; 1.217 mmol) in ethyl acetate (10 mL) was added 4 M HCl in ethyl acetate (4 mL; 16 mmol) dropwise at 0°C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the resulting solid was stirred in ethyl acetate (10 mL) for 0.5 h then filtered to give the desired product as a solid (0.56 g; yield: 100%); MS (m/z): [M+H] ⁺ calcd for C ₁₉ H ₂₅ N ₉ O ₃ : 428.21; found: 428.2.

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

To a mixture of sodium hyaluronate (0.174 g; 0.431 mmol) in MeCN (22 ml) and _H2O (35 ml) were added 4-methylmorpholine (0.066 g; 0.65 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.076 g; 0.431 mmol) at 0°C. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 1 h. Then, 4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-N'-glycyl-7H-pyrrolo[2,3-d]pyrimidine-7-carbohydrazide hydrochloride (0.2 g; 0.431 mmol) was added to the solution and stirred for 3 days at room temperature. NaCl (253 mg) in _H2O (2 ml) was added and stirred for 0.5 h. Acetone (350 ml) was added dropwise to the above mixture until a precipitate formed. The mixture was filtered, the filter cake was washed with acetone and then dissolved in MeCN (20 mL) and _H2O (40 mL) to form a homogeneous solution. After dialyzing the solution using a 3.5 kDa MW cutoff membrane against deionized water and then lyophilizing, the title compound (0.183 g; yield: 49%, DSR: 11%) was obtained. ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.90-7.40 (m, 0.11H), 7.75-7.35 (m, 0.11H), 6.95-6.50 (m, 0.11H), 4.75-4.20 (m, 2H), 4.00-3.23 (m, 11.36H), 2.60-2.40 (m, 0.11H), 1.99 (s, 3H), 1.80-1.65 (m, 0.22H), 1.15-0.85 (m, 0.33H).

Using the reaction conditions of step 3 and employing sodium hyaluronate (0.174 g; MW 2000 kDa), the corresponding product was obtained (0.19 g; yield: 51%, DSR=11%). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.30-7.90 (m, 0.11H), 7.65-7.20 (m, 0.11H), 6.9-6.30 (m, 0.11H), 4.60-4.20 (m, 2H), 4.00-3.00 (m, 11.36H), 2.50-2.30 (m, 0.11H), 1.99 (s, 3H), 1.70-1.60 (m, 0.22H), 1.10-0.85 (m, 0.33H).

Example 94

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

Step 1. 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)(phenoxy)phosphoryl)amino)butyl)carbamate

To a mixture of tofacitinib (1 g, 3.2 mmol), tert-butyl (4-aminobutyl)carbamate (603 mg, 3.2 mmol), and phenyl phosphodichloridate (675 mg, 3.2 mmol) in MeCN (20 mL) was added N,N-diisopropylethylamine (1.45 g, 16 mmol) under N ₂ , and the reaction mixture was heated to reflux for 16 h. After removing the solvent under reduced pressure, the residue was purified by chromatography on silica to give the title product (400 mg; yield: 19.6%); MS (m/z): [M+H] ⁺ calcd. for C ₃₁ H ₄₃ N ₈ O ₅ P: 639.31; found: 639.3.

Step 2. Preparation of phenyl N-(4-aminobutyl)-P-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phosphonamidate hydrochloride

To a solution of tert-butyl (4-(((4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)(phenoxy)phosphoryl)amino)butyl)carbamate (200 mg; 0.313 mmol) in EA (10 ml) was added dropwise 4.5 M HCl solution in EA (0.9 ml; 4.5 mmol) at 0°C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 2 days. After removing the solvent under reduced pressure, the resulting solid was stirred in EA (20 mL) for 0.5 h and then filtered to give the desired product (150 mg; yield: 83.4%); MS (m/z): [M+H] ⁺ calcd for C ₂₆ H ₃₅ N ₈ O ₃ P: 539.26; found: 539.3.

Step 3. Preparation of a conjugate of phenyl N-(4-aminobutyl)-P-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phosphonamidate and HA

To a mixture of sodium hyaluronate (84 mg; 0.21 mmol) in MeCN (15.6 ml) and _H2O (24 ml) were added 4-methylmorpholine (31.8 mg; 0.31 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine (36.6 mg; 0.21 mmol) at 0°C. The resulting reaction mixture was stirred at 0°C for 30 min and stirred at room temperature for 1 h. Then, phenyl N-(4-aminobutyl)-P-(4-(((3R,4R)-1-(2-cyanoacetyl)-4-methylpiperidin-3-yl)(methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phosphonamidate hydrochloride (120 mg; 0.21 mmol) was added to the solution and the pH of the reaction mixture was adjusted to a value in the range from 6.5 to 7 using NMM. The resulting solution was stirred for 3 days at room temperature. NaCl (122 mg) in H ₂ O (1 mL) was added and stirred for 0.5 h. Then, acetone (168 mL) was added dropwise and the resulting mixture was filtered. The filter cake was washed with acetone and dissolved in MeCN (8.4 mL) and H ₂ O (16.8 mL) to form a homogeneous solution. Dialysis of the solution against three changes of deionized water followed by lyophilization afforded the title compound (82 mg; yield: 43.2%, DSR=31%). ¹ H-NMR (400 MHz, D ₂ O) δ 8.35-8.25 (m, 0.28H), 7.55-7.35 (m, 0.86H), 7.30-7.15 (m, 0.94H), 6.90-6.80 (m, 0.42H), 4.60-4.20 (m, 2Н), 4.10-3.20 (m, 15.37Н), 2.65-2.55 (m, 0.53Н), 1.99 (s, 3Н), 1.75-1.40 (m, 2Н), 1.20-1.15 (m, 0.95Н).

Example 95

Preparation of the conjugate of methyl-(Z)-1-glycyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindoline-6-carboxylate and NA

Step 1. Preparation of methyl (Z)-1-((tert-butoxycarbonyl)glycyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate

Methyl (3Z)-3-[[4-[methyl-[2-(4-methylpiperazin-1-yl)acetyl]amino]anilino]phenyl-methylene]-2-oxo-indolin-6-carboxylate (1079 mg, 2 mmol), (tert-butoxycarbonyl)glycine (701 mg, 4 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 864.5 mg, 4.5 mmol), N,N-dimethylpyridin-4-amine (DMAP; 1098 mg, 9 mmol) were dissolved in DCM (80 mL). The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM/MeOH=50:1-20:1) to give the title compound (700 mg; yield: 50.2%). MS (m/z): [M+H] ⁺ calcd for C ₃₈ H ₄₄ N ₆ O ₇ : 697.81; found: 697.2. ¹ H-NMR (400 MHz, chloroform-d) δ 12.11 (s, 1H), 8.91 (d, J=1.7 Hz, 1H), 7.64-7.50 (m, 4H), 7.42-7.37 (m, 2H), 7.06-7.00 (m, 2H), 6.82 (d, J=8.4 Hz, 2H), 5.92 (d, J=8.3 Hz, 1H), 5.45 (s, 1H), 4.83 (d, J=5.4 Hz, 2H), 3.87 (s, 3H), 3.19 (s, 3H), 2.96 (q, J=7.3 Hz, 2H), 2.79 (s, 2H), 2.56-2.34 (m, 6H), 2.28 (s, 3H), 1.50 (s, 9H).

Step 2. Preparation of methyl (Z)-1-glycyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate hydrochloride

To a solution of methyl (Z)-1-((tert-butoxycarbonyl)glycyl)-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate (139 mg, 0.2 mmol) in ethyl acetate (10 mL) was added 4 M HCl in ethyl acetate (2 mL, 8 mmol) dropwise at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (83 mg, yield: 70%), which was used without further purification. MS (m/z): [M+H]+ calcd. for C ₃₃ H ₃₆ N ₆ O ₅ : 597.69; detected: 597.2.

Step 3. Obtaining a conjugate of methyl (Z)-1-glycyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate and HA

To a solution of sodium hyaluronate (201 mg, 0.5 mmol) in deionized water (40 mL) and acetonitrile (26 mL) were added methyl (Z)-1-glycyl-3-(((4-(N-methyl-2-(4-methylpiperazin-1-yl)acetamido)phenyl)amino)(phenyl)methylene)-2-oxoindolin-6-carboxylate (149 mg, 0.25 mmol) and 4-methylmorpholine (NMM; 25 mg, 0.25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 104 mg; 0.75 mmol) was added to the solution and stirred for 72 h at room temperature. NaCl (293 mg) was added to the reaction mixture, stirred for 1 h, and then acetone (200 ml) was added dropwise. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (250 mg; yield: 52.2%, DSR=17%); ¹ H-NMR (400 MHz, D ₂ O) δ 8.58-8.40 (m, 0.17H), 7.65-7.15 (m, 1.02H), 7.07-6.75 (m, 0.68H), 4.52-4.21 (m, 2H), 4.01-2.50 (m, 12.89H), 2.33-2.13 (m, 0.51H), 2.00-1.59 (m, 3H).

Example 96

Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-(hydrazinecarbonyl)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of methyl (Z)-N ⁶ -(tert-butyloxycarbonyl)-N2-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-L-lysinate

A mixture of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (1594 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then methyl N6-(tert-butoxycarbonyl)-L-lysinate (1250 mg, 4.8 mmol) was added. The reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol=20:1) to give the title compound (0.77 g; yield: 28.1%). MS (m/z): [M+H]+ calcd for C ₃₅ H ₄₉ FN ₆ O ₇ : 685.81; found: 685.2. ¹ H-NMR (400 MHz, chloroform-d) δ ^ppm 12.75 (s, 1H), 9.31 (d, J=7.6 Hz, 1H), 8.17 (d, J=4.7 Hz, 1H), 7.42 (s, 1H), 7.17 (dd, J=8.4; 2.5 Hz, 1H), 6.91 (td, J=9.0; 2.6 Hz, 1H), 6.63 (s, 1H), 4.75-4.64 (m, 1H), 4.60 (s, 1H), 3.80 (d, J=6.4 Hz, 3H), 3.52 (dd, J=10.9; 5.2 Hz, 2H), 3.14 (d, J=5.5 Hz, 2H), 2.76-2.46 (m, 12H), 2.08-1.79 (m, 2H), 1.60-1.37 (m, 13H), 1.06 (t, J=7.1 Hz, 6H).

Step 2. Preparation of methyl (Z)-(3-((4-((2-(diethiamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-L-lysinate hydrochloride

To a solution of methyl (Z)-N ⁶ -(tert-butoxycarbonyl)-N ² -(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-L-lysinate (600 mg; 0.877 mmol) in ethyl acetate (18 mL) was added dropwise 4 M HCl in ethyl acetate (3.6 mL; 14.4 mmol) at 0°C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a solid (498 mg; yield: 91.5%). MS (m/z): [M+H]+ calcd for C ₃₀ H ₄₁ FN ₆ O ₅ : 585.69; found: 585.2.

Step 3. Preparation of a conjugate of methyl (Z)-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-L-lysinate and HA

To a solution of sodium hyaluronate (130 mg; 0.21 mmol) in deionized water (24 ml) and acetonitrile (16 ml) were added methyl (Z)-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-L-lysinate hydrochloride (130 mg; 0.21 mmol) and 4-methylmorpholine (NMM; 21 mg; 0.21 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 83 mg; 0.3 mmol) was added and stirred for 72 h at room temperature. Then, NaCl (176 mg) was added to the reaction mixture and stirred for 1 h, and then acetone (40 ml) was added dropwise. The mixture was filtered and the filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.19 g; yield: 95.7%, DSR: 6%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.82-7.62 (m, 0.06H), 7.38-7.13 (m, 0.06H), 6.97-6.72 (m, 0.12H), 4.55-4.27 (m, 2.06H), 4.06-2.75 (m, 10.42H), 2.53-1.48 (m, 4.08H), 1.39-1.26 (m, 0.06H).

Example 97

Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-(hydrazinecarbonyl)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of tert-butyl (Z)-2-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)hydrazine-1-carboxylate

A mixture of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (1594 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then tert-butyl hydrazine carboxylate (635 mg, 4.8 mmol) was added and the reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. 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=40:1) to give the title compound (1.06 g; Yield: 47.6%). MS (m/z): [M ₊ H]+ calcd for _C28H37FN6O5 : _557.64 ; found _: 557.2. ¹ H-NMR (400 MHz, chloroform-d) δ ^ppm 12.62 (s, 1H), 10.16 (s, 1H), 8.07 (s, 1H), 7.25 (d, J=10.1 Hz, 2H), 7.07 (d, J=8.2 Hz, 1H), 6.87 (t, J=8.5 Hz, 1H), 6.57 (s, 1H), 3.51 (q, J=5.3 Hz, 2H), 2.68 (t, J=5.9 Hz, 2H), 2.60 (dd, J=15.2; 8.0 Hz, 7H), 2.43 (s, 3H), 1.53 (s, 9H), 1.05 (t, J=7.1 Hz, 6H).

Step 2. Preparation of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-(hydrazinecarbonyl)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride

To a solution of tert-butyl (Z)-2-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)hydrazine-1-carboxylate (1000 mg, 1.8 mmol) in ethyl acetate (40 mL) was added dropwise 4 M HCl solution in ethyl acetate (8 mL, 32 mmol) at 0°C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (410 mg; yield: 82.8%), which was used without further purification. MS (m/z): [M+H]+ calcd for C ₂₃ H ₂₉ FN ₆ O ₃ : 457.52; found: 457.2.

Step 3. Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-l-(hydrazinecarbonyl)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

To a solution of sodium hyaluronate (161 mg; 0.4 mmol) in deionized water (32 ml) and acetonitrile (21 ml) were added (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-(hydrazinecarbonyl)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride (138 mg; 0.28 mmol) and 4-methylmorpholine (NMM; 28 mg; 0.28 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h, then 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added and stirred for 72 h at room temperature. Then NaCl (234 mg) was added to the reaction mixture, stirred for 1 h, and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered and the filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.185 g; yield: 56.6%; DSR: 10%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.76-7.19 (m, 0.20H), 7.08-6.66 (m, 0.20H), 4.56-4.24 (m, 2H), 4.07-2.88 (m, 10.20H), 2.54-1.55 (m, 4.20N), 1.41-1.22(m, 0.60N).

Example 98

Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxo-1-(niperazine-1-carbonyl)indolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of tert-butyl (Z)-4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)piperazine-1-carboxylate

A mixture of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (1594 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then tert-butyl piperazine-1-carboxylate (894 mg, 4.8 mmol) was added and the reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (ESM:methanol=40:1) to give the title compound (1.5 g; yield: 61.4%). MS (m/z): [M+H] ⁺ calcd for C ₃₂ H ₄₃ FN ₆ O ₅ : 611.73; found: 611.2. ¹ H NMR (400 MHz, chloroform-d) δ ^ppm 13.05 (s, 1H), 7.39 (s, 1H), 7.19 (dd, J=8.6; 2.5 Hz, 1H), 7.11 (dd, J=8.7; 4.3 Hz, 1H), 6.90 (td, J=8.9; 2.5 Hz, 1H), 6.66 (s, 1H), 3.89-3.30 (m, 10H), 2.71 (t, J=5.9 Hz, 2H), 2.68-2.57 (m, 7H), 2.51 (s, 3H), 1.49 (s, 9H), 1.07 (t, J=7.1 Hz, 6H).

Step 2. Preparation of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxo-1-(piperazine-1-carbonyl)indolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride

To a solution of tert-butyl (Z)-4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)piperazine-1-carboxylate (1000 mg, 1.64 mmol) in ethyl acetate (50 mL) was added 4 M HCl in ethyl acetate (8 mL, 32 mmol) dropwise at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (890 mg, yield: 99%). MS (m/z): [M+H] ⁺ calc. for C ₂₇ H ₃₅ FN ₆ O ₃ : 511.61; detected: 511.2.

Step 3. Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxo-1-(piperazine-1-carbonyl)indolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

To a solution of sodium hyaluronate (161 mg; 0.4 mmol) in 32 ml of deionized water and 22 ml of acetonitrile were added (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxo-1-(piperazine-1-carbonyl)indolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride (109 mg; 0.2 mmol) and 4-methylmorpholine (NMM; 20 mg; 0.2 mmol) at room temperature with stirring. The reaction mixture was stirred at room temperature for 2 h, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added and stirred for 72 h at room temperature. Then, NaCl (234 mg) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered and the filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.197 g; yield: 56.5%, DSR: 10%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.64-7.46 (m, 0.10H), 7.41-7.21 (m, 0.10H), 7.04-6.71 (m, 0.20H), 4.53-4.27 (m, 2H), 4.06-2.97 (m, 11Н), 2.51-1.64 (m, 4.20Н), 1.39-1.24 (m, 0.60Н).

Example 99

Preparation of the conjugate of 5-(((Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of tert-butyl (1S,4S)-5-((Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate

A mixture of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (1594 mg, 4 mmol) and bis(4-nitrophenyl)carbonate (1460 mg, 4.8 mmol) in DMSO (30 mL) was stirred at room temperature for 7 h. Then tert-butyl (1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate (992 mg, 5 mmol) was added. The reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The crude residue was purified by column chromatography (MSM:methanol = 8:1) to give the title compound (1.3 g; yield: 52%). MS (m/z): [M+H] ⁺ calcd for C ₃₃ H ₄₃ FN ₆ O ₅ : 623.74; found: 623.2. ¹ H NMR (400 MHz, chloroform-d) δ ^ppm 13.07 (s, 1H), 7.39 (d, J=9.9 Hz, 1H), 7.18 (h, J=6.6; 5.5 Hz, 2H), 6.91 (td, J=8.6; 2.7 Hz, 1H), 6.57 (s, 1H), 4.98-4.43 (m, 2H), 3.70 (d, J=14.5 Hz, 2H), 3.58-3.34 (m, 4H), 2.68 (t, J=5.9 Hz, 2H), 2.64-2.56 (m, 7H), 2.51 (d, J=3.1 Hz, 3H), 2.03 (dd, J=32.4; 7.3 Hz, 2H), 1.47 (dd, J=20.0, 8.9 Hz, 9H), 1.05 (t, J=7.1 Hz, 6H).

Step 2. Preparation of 5-(((Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride

To a solution of tert-butyl (1S,4S)-5-((Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carbonyl)-2,5-diazabicyclo[2,2.1]heptane-2-carboxylate (730 mg, 1.17 mmol, 1 eq.) in ethyl acetate (15 mL) was added 4 M HCl in ethyl acetate (2.9 mL, 11.6 mmol) dropwise at 0°C under _N2 . The resulting reaction mixture was stirred at 0°C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.65 g; yield: 99%), which was used without further purification. MS (m/z): [M+H] ⁺ calcd for C ₂₈ H ₃₅ FN ₆ O ₃ : 523.63; found: 522.3.

Step 3. Preparation of the conjugate of 5-(((Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

To a solution of sodium hyaluronate (161 mg; 0.4 mmol) in 32 ml of deionized water and 22 ml of acetonitrile were added 5-(((Z)-1-((1S,4S)-2,5-diazabicyclo[2,2.1]heptane-2-carbonyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride (45 mg; 0.08 mmol) and 4-methylmorpholine (NMM; 8.1 mg; 0.08 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 111 mg; 0.4 mmol) was added to the solution and stirred for 72 h at room temperature. Then, NaCl (234 mg) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.17 g; yield: 48.1%, DSR: 12%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.61-6.69 (m, 0.48H), 4.59-4.29 (m, 2.24H), 4.20-2.93 (m, 10.72H), 2.54-1.58 (m, 4.68H), 1.41-1.23 (m, 0.72H).

Example 100

Preparation of the conjugate (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-glycyl-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of tert-butyl (Z)-(2-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-2-oxoethyl)carbamate

(Z)-N-(2-(Diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (996 mg, 2.5 mmol), (tert-butoxycarbonyl)glycine (876 mg, 5 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 1081 mg, 5.625 mmol), N,N-dimethylpyridin-4-amine (DMAP; 1372 mg, 11.25 mmol) were dissolved in DCM (48 mL) and DMF (12 mL). The reaction mixture was stirred at room temperature for 18 h. The solution was diluted with DCM and washed with water and brine. 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 = 50:1-20:1) to give the title compound (440 mg; yield: 31.7%). MS (m/z): [M+H] ⁺ calcd for C ₂₉ H ₃₈ FN ₅ O ₅ : 556.65; found: 556.2. ¹ H NMR (400 MHz, chloroform-d) δ ^ppm 12.93 (s, 1H), 7.81 (dd, J=8.9; 4.5 Hz, 2H), 7.38 (s, 1H), 7.18 (dd, J=8.4; 2.6 Hz, 1H), 6.97-6.91 (m, 1H), 4.72 (s, 1H), 3.81 (s, 2H), 3.28-3.00 (m, 8H), 2.65 (s, 3H), 2.58 (s, 3H), 1.40 (d, J=28.3 Hz, 15H).

Step 2. Preparation of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-glycyl-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride

To a solution of tert-butyl (Z)-(2-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-2-oxoethyl)carbamate (222 mg, 0.4 mmol) in ethyl acetate (20 mL) was added 4 M HCl in ethyl acetate (3 mL, 12 mmol) dropwise at 0 °C under N ₂ atmosphere. The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.14 g, yield: 71.2%). MS (m/z): [M+H] ⁺ calc. for C ₂₄ H ₃₀ FN ₅ O ₃ : 456.53; detected: 456.2.

Step 3. Obtaining a conjugate of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-glycyl-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

To a solution of sodium hyaluronate (201 mg, 0.5 mmol) in 40 ml of deionized water and 26 ml of acetonitrile were added (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-1-glycyl-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide hydrochloride (56 mg, 0.1 mmol) and 4-methylmorpholine (NMM; 10 mg, 0.1 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 hours, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 28 mg, 0.1 mmol) was added and stirred for 72 hours at room temperature. Then NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 mL) was added dropwise with stirring. The mixture was filtered and the filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (MW sodium hyaluronate 50 kDa; 0.2 g; yield: 49%, DSR: 9%); ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.03-7.80 (m, 0.09H), 7.51-7.21 (m, 0.18H), 6.99-6.73 (m, 0.09H), 4.43-4.25 (m, 2.18H), 4.11-2.81 (m, 10.90H), 2.51-1.60 (m, 3.54H), 1.40-1.22 (m, 0.54H).

Example 101

Preparation of a conjugate of 4-aminobutyl-(Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carboxylate and HA

Step 1. Preparation of 4-((mpem-butoxycarbonyl)amino)butyl-(Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindoline-1-carboxylate

To a stirred mixture of (Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (598 mg, 1.5 mmol) and bis(4-nitrophenyl)carbonate (548 mg, 1.8 mmol) in DCM (30 mL) was added triethylamine (379.5 mg, 3.75 mmol). The reaction mixture was heated to 45 °C and stirred at this temperature for 20 h. Then the reaction mixture was cooled to room temperature. Then tert-butyl (4-hydroxybutyl)carbamate (426 mg, 2.25 mmol) was added. The reaction mixture was stirred at room temperature for 22 h. The solution was diluted with DCM and washed with saturated _NaHCO3 solution, water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM:methanol = 20: _1-10 :1) to give the title compound (0.6 g; yield: 65.2%). MS (m/z): [M ₊ H] ⁺ calcd. for _C32H44FN5O6 : _614.73 ; found: 614.3. ¹ H-NMR (400 MHz, chloroform-d) δ 12.93 (s, 1H), 7.81 (dd, J=8.9; 4.5 Hz, 1H), 7.38 (s, 1H), 7.28 (s, 1H), 7.18 (dd, J=8.4; 2.6 Hz, 1H), 6.93 (td, J=9.0; 2.6 Hz, 1H), 4.72 (s, 1H), 4.49 (t, J=6.6 Hz, 2H), 3.81 (s, 2H), 3.27-3.03 (m, 8H), 2.65 (s, 3H), 2.58 (s, 3H), 1.90 (t, J=7.5 Hz, 2H), 1.71 (q, J=7.3 Hz, 2H), 1.44 (s, 9H), 1.38 (d, J=9.3 Hz, 6H).

Step 2. Preparation of 4-aminobutyl-(Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carboxylate hydrochloride

To a solution of 4-((tert-butoxycarbonyl)amino)butyl (Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carboxylate (184 mg, 0.3 mmol) in ethyl acetate (15 mL) was added dropwise a 4 M solution of HCl in ethyl acetate (3 mL, 12 mmol) at 0 °C under N ₂ . The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.12 g; yield: 72.7%), which was used without further purification. MS (m/z): [M+H] ⁺ calcd for C ₂₇ H ₃₆ FN ₅ O ₄ : 514.61; found: 514.2.

Step 3. Preparation of a conjugate of 4-aminobutyl-(Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carboxylate and HA

To a solution of sodium hyaluronate (201 mg; 0.5 mmol) in 40 ml of deionized water and 26 ml of acetonitrile were added 4-aminobutyl-(Z)-3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-carboxylate hydrochloride (55 mg; 0.1 mmol) and 4-methylmorpholine (NMM; 10 mg; 0.1 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 42 mg; 0.15 mmol) was added to the solution and stirred for 72 h at room temperature. Then, NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 h and then acetone (40 ml) was added dropwise with stirring. The mixture was filtered. The filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (Sodium hyaluronate MW 50 kDa; 0.2 g; yield: 45.7%, DSR: 28%); ¹ H-NMR (400 MHz, D ₂ O) δ 7.48-6.42 (m, 1.12H), 4.57-4.18 (m, 2.56H), 4.04-2.60 (m, 12.80H), 2.41-1.58 (m, 4.8H), 1.43-1.17 (m, 0.84H).

Example 102

Preparation of the conjugate (Z)-5-((l-(4-aminobutanoyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethiamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

Step 1. Preparation of tert-butyl (Z)-(4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-4-oxobutyl)carbamate

(Z)-N-(2-(Diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (797 mg, 2 mmol), 4-((tert-butoxycarbonyl)amino)butanoic acid (813 mg, 4 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI; 864.5 mg, 4.5 mmol), N,N-dimethylpyridin-4-amine (DMAP; 1098 mg, 9 mmol) and DIPEA (1034 mg, 8 mmol) were dissolved in DCM (48 mL) and DMF (12 mL). The reaction mixture was stirred at room temperature for 16 h. The solution was diluted with DCM and washed with water and brine. The organic layer was dried over sodium sulfate, filtered and then concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM/MeOH = 50:1-10:1) to give the title compound (560 mg; yield: 48%). MS (m/z): [M+H] ⁺ calcd. for C ₃₁ H ₄₂ FN ₅ O ₅ : 584.71; found: 584.2. ¹ H-NMR (400 MHz, chloroform-d) δ ^ppm 12.85 (s, 1H), 8.21 (dd, J=9.0; 4.7 Hz, 1H), 7.98 (s, 1H), 7.37 (s, 1H), 7.16 (dd, J=8.5; 2.7 Hz, 1H), 6.92 (td, J=9.0; 2.6 Hz, 1H), 4.73 (s, 1H), 3.86 (s, 2H), 3.33-3.19 (m, 5H), 3.14 (d, J=7.6 Hz, 3H), 2.69 (s, 3H), 2.58 (s, 3H), 1.98 (q, J=7.1 Hz, 2H), 1.47-1.36 (m, 11H), 1.35-1.18 (m, 6H).

Step 2. Preparation of (Z)-5-((1-(4-aminobutanoyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-lH-pyrrole-3-carboxamide hydrochloride

To a solution of tert-butyl (Z)-(4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-4-oxobutyl)carbamate (204 mg; 0.35 mmol) in ethyl acetate (15 mL) was added dropwise a 4 M solution of HCl in ethyl acetate (3 mL; 12 mmol) at 0 °C under N ₂ . The resulting reaction mixture was stirred at 0 °C for 30 min and then stirred at room temperature for 20 h. The solution was diluted with ethyl acetate (20 mL) and concentrated under reduced pressure to give the title compound as a white solid (0.16 g; yield: 94.6%), which was used without further purification. MS (m/z): [M+H] ⁺ calcd for C ₂₆ H ₃₄ FN ₅ O ₃ : 484.59; found: 484.2.

Step 3. Preparation of the conjugate (Z)-5-((1-(4-aminobutanoyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide and HA

To a solution of sodium hyaluronate (136 mg; 0.337 mmol) in 30 ml of deionized water and 19.5 ml of acetonitrile were added (Z)-5-((1-(4-aminobutanoyl)-5-fluoro-2-oxoindolin-3-ylidene)methyl)-N-(2-(diethylamino)ethyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (150 mg; 0.236 mmol) and 4-methylmorpholine (NMM; 23.9 mg; 0.236 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 hours. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM; 93 mg; 0.337 mmol) was added to the solution and stirred for 72 hours at room temperature. Then, NaCl (197 mg; 3.37 mmol) was added to the reaction mixture, the mixture was stirred for 1 hour, and then acetone (200 ml) was added dropwise. The mixture was filtered, and the filter cake was washed with acetone and dried under vacuum to give the title compound as a white solid. (153 mg; yield: 53%, DSR=10%). ¹ H-NMR (400 MHz, D ₂ O) δ ^ppm 8.03-7.89 (m, 0.1H), 7.47-7.25 (m, 0.2H), 7.02-6.87 (m, 0.1H), 4.58-4.17 (m, 2H), 4.12-2.92 (m, 11H), 2.56-2.45 (m, 0.6H), 2.12-1.70 (m, 3.2H), 1.58-1.46 (m, 0.2H), 1.42-1.25 (m, 0.6H).

Example 103

Release of drug from drug delivery system

Method

Drug release and stability experiments were performed using test compounds. A solution of each test compound at a concentration of approximately 2.5 ± 1.0 mg/mL (based on the conjugate) was prepared using 10 mM phosphate-buffered saline (PBS) buffer (pH 7.4) in a 0.5 mL Millipore Amicon® Ultra 30 kDa ultrafiltration centrifuge tube. The mixture was allowed to swell for 1 h at room temperature and then placed on a shaker at 100 rpm, 37°C for the duration of the experiment. At the same time point each day, samples were centrifuged at 10,000 rpm for 1 h. An aliquot was transferred to a vial for HPLC analysis. 0.4 ml of 10 mM PBS buffer was added to the centrifuge tube and the experiment was continued.

To conduct the HPLC analysis, the peak areas of all relevant peaks in the chromatograms were read at each time point, and the concentration of free drug was calculated. The average free drug release rate was calculated based on the total amount of free drug and the number of days of the experiment. The calculation equation is given below.

The sample degradation rate was calculated based on the concentration and degree of substitution (by NMR) of the conjugated drug relative to the initial starting point of the experiment (at t=0). The equation for the calculation is given below.

Results

The drug release results for typical drug delivery systems of the present invention are shown in Table 1.

The above description is intended to be illustrative of the principles of the present invention. Furthermore, since numerous modifications and variations will be readily apparent to those skilled in the art, it is not desirable to limit the invention to the precise arrangement and method illustrated as described above. Accordingly, all suitable modifications and equivalents are intended to be within the scope of the invention as defined by the following claims.

Claims (114)

1. A drug delivery system for local delivery of a therapeutic agent at a controlled rate, comprising: a biopolymer comprising at least a first linking group BG1, wherein said biopolymer is hyaluronic acid or chondroitin sulfate; a therapeutic agent comprising at least a second BG2 binding group, wherein said therapeutic agent is tofacitinib, oclacitinib, baricitinib, ruxolitinib, nintedanib or sunitinib; and a linker that covalently connects the biopolymer to the therapeutic agent and is capable of retaining the therapeutic agent at the site of administration; where the linker comprises a structure of formula (I) (I), where U is connected to the biopolymer via BG1; BG1 is a carboxyl group and U is -N(R ¹ )- or , resulting in the formation of an amide bond, where R ¹ is hydrogen, C _1-6 alkyl, or C _2-6 alkenyl, is a nitrogen-containing heterocyclyl, optionally containing one or more additional N heteroatoms; or BG1 is a carboxyl group and U is -O-, resulting in an ester bond; A is selected from a direct bond, C _1-6 alkyl and -(CH ₂ CH ₂ O) _m -, wherein said C _1-6 alkyl is optionally substituted with one or more R ^a groups; B is selected from the group consisting of a direct bond, C _1-6 alkyl, 3-8-membered cycloalkyl, , phenyl, pyridinyl and -O-phenyl, where each of C _1-6 alkyl, 3-8-membered cycloalkyl, , phenyl, pyridinyl and -O-phenyl may be substituted by one or more R ^b groups; C is selected from a direct bond, -C(=O)N(R ² )-, -N(R ² )C(=O)-, -[CH ₂ NHC(=O)] _n - or -[NHC(=O)CH ₂ ] _n -, where R ² is hydrogen; D is selected from a direct bond, C _1-6 alkyl and phenyl, wherein said C _1-6 alkyl is optionally substituted with one or more R ^c groups; BG2 is an amine group; V is connected to the therapeutic agent via BG2 and is selected from one of the following (a)-(k): (a) direct communication; (b) the -N(R ² )C(=O)- group, which is linked to the therapeutic agent via BG2, resulting in the formation of a urea bond; (c) the -N(R ² )C(S)- group, which is linked to the therapeutic agent via BG2, resulting in the formation of a thiourea bond; (d) an -OC(=O)- group, which is linked to the therapeutic agent via BG2, resulting in the formation of a carbamate bond; (d) the -OC(=S)- group, which is linked to the therapeutic agent via BG2, resulting in the formation of a thiocarbamate bond; (e) the -OC(=O)OCH ₂ - group, which is linked to the therapeutic agent via BG2, resulting in the formation of an aza-acetal linkage; (g) a -C(=O)OCH ₂ - group, which is linked to the therapeutic agent via BG2, resulting in the formation of an aza-acetal linkage; (3) the -N(R ² )C(=O)OCH ₂ - group, which is linked to the therapeutic agent via BG2, resulting in the formation of an aza-acetal linkage; (i) the -N(R ² )P(=O)(OPh)- group, which is linked to the therapeutic agent via BG2, resulting in the formation of a phosphoramidate bond; (k) a -C(=O)- group, which is linked to the therapeutic agent through BG2, resulting in the formation of an amide bond; R ^a , R ^b and R ^c independently represent -C(=O)OR ^e ; R ^e is C _1-6 alkyl; m is an integer from 0 to 4; n is an integer between 1 and 4. 2. The drug delivery system according to paragraph 1, where A is a direct bond, B is selected from the group consisting of a direct bond, 3-8 membered cycloalkyl, phenyl and pyridinyl, or A is C _1-6 alkyl, B is selected from the group consisting of a direct bond, phenyl or -O-phenyl; or A is -( _CH2CH2O ) _m- , _B is selected from the group consisting of direct bond, _C1-6 alkyl, phenyl and pyridinyl. 3. The drug delivery system according to paragraph 1, where A is C _1-6 alkyl, B is selected from the group consisting of a direct bond, 3-8 membered cycloalkyl, , phenyl, pyridinyl and -O-phenyl, and C is a direct bond, -N(R ² )C(=O)- or -[NHC(=O)CH ₂ ] _n -; or A is -(CH ₂ CH ₂ O) _m -, B is selected from the group consisting of a direct bond, C _1-6 alkyl, 3-8 membered cycloalkyl, , phenyl and pyridinyl, and C represents a direct bond or -N(R ² )C(=O)-. 4. The drug delivery system of claim 1, wherein the linker comprises a structure of the formula: (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ii), (Ij), (Ik), (Im), (In) or (Io), where U and V are as defined in paragraph 1; M is selected from the group consisting of cyclohexyl, phenyl and pyridinyl, each of which is optionally substituted by one or more R ^b groups; each of , , And possibly substituted by a -C(=O)OCH ₃ group; and q, r, s, t and u are independently equal to an integer from 0 to 5. 5. The drug delivery system of claim 4, wherein the linker comprises a structure selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , And , where each of , And possibly substituted by the group -C(=O)OCH ₃ . 6. The drug delivery system of claim 1, wherein the therapeutic agent is tofacitinib. 7. A drug delivery system selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , And . 8. A drug delivery system according to any one of claims 1-7 for local administration to a subject in need thereof, preferably by injection, using an oral dosage form, by inhalation, by implant or by topical application. 9. A pharmaceutical composition for inhibiting Janus kinase (JAK) or vascular endothelial growth factor (VEGF), comprising a drug delivery system according to any one of claims 1-8 and a pharmaceutically acceptable excipient.