Hyaluronic acid cross-linked conjugate loaded with capsaicin

Abstract

A composition comprising a hyaluronic acid cross-linked conjugate loaded with capsaicin by physical or chemical means, and a method for analgesia and treatment of arthritis using such compositions.

Description

Capsaicin-loaded hyaluronic acid crosslinks TECHNICAL FIELD

[0001] The present disclosure relates generally to a composition comprising hyaluronic acid crosslinks loaded with capsaicin by physical or chemical means, and the use of the composition in analgesia and treatment of arthritis. BACKGROUND

[0002] Pain can serve as a protective mechanism to keep healthy humans and animals from tissue damage and / or to prevent further damage to damaged tissue. However, in many cases, the persistence of pain goes beyond its usefulness, impairing the physical activity and mental performance of the subject.

[0003] In recent years, a great deal of resources has been invested in researching and developing drugs to alleviate pain in patients. Capsaicin is an activator of the capsaicin receptor (TRPV1), which acts on TRPV1, and is capable of selectively inactivating local pain fibers that transmit signals to the brain, thereby reducing pain. However, one of the challenging aspects of using capsaicin to treat pain, particularly when capsaicin is administered by injection, is that capsaicin also has strong irritancy, and its administration can cause initial neuronal excitation, which can cause an unbearable burning pain in a short period of time. In order to alleviate such strong burning pain, a cooling treatment of the affected part is usually required for a period of time when capsaicin is administered by injection, which increases the difficulty of drug administration and reduces the patient experience.

[0004] Therefore, there is a need to develop a low-irritant and long-acting capsaicin drug for treating pain, such as joint pain. SUMMARY

[0005] The present disclosure provides a composition comprising hyaluronic acid crosslinks loaded with capsaicin by physical or chemical means.

[0006] In one aspect, the present disclosure provides a composition comprising a hyaluronic acid matrix and capsaicin, wherein the hyaluronic acid matrix is formed by crosslinking hyaluronic acid polymers with a crosslinking agent.

[0007] In another aspect, the present disclosure provides a composition comprising a hyaluronic acid matrix and capsaicin, wherein the hyaluronic acid matrix is formed by crosslinking hyaluronic acid polymers with a crosslinking agent, and the capsaicin is conjugated to the hyaluronic acid polymers via a linker to form a conjugate moiety.

[0008] In another aspect, the present disclosure provides a composition comprising a hyaluronic acid matrix, wherein the hyaluronic acid matrix is obtained by crosslinking hyaluronic acid polymers with a crosslinking agent conjugated with capsaicin.

[0009] In another aspect, the present disclosure provides the composition of one or more of the foregoing for use in the treatment of bone or joint pain associated with inflammation or caused by injury, or arthritis associated with degenerative disease, rheumatoid arthritis and other arthritic conditions, in particular for use in the treatment of osteoarthritis.

[0010] In yet another aspect, the present disclosure provides the use of the composition of one or more of the foregoing in the manufacture of a medicament for the treatment of arthritis, in particular osteoarthritis.

[0011] In another aspect, the present disclosure provides a method of treating arthritis, in particular osteoarthritis, by using the composition of one or more of the foregoing. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 shows capsaicin release rate curves of five capsaicin-hyaluronic acid products.

[0013] Figure 2A shows a D-glucuronic acid carbazole method standard curve.

[0014] Figure 2B shows the enzymatic trend of crosslinked materials Exl a-3 and Exl a-13 of the present disclosure and a commercially available product Durolane.

[0015] Figure 2C shows the enzymatic trend of compositions Ex2b-6 and Ex2b-11 of the present disclosure and positive reference Exl a-3.

[0016] Figure 2D shows the enzymatic trend of compositions Exl-1, Ex4-1, Ex5-1 of the present disclosure and positive reference Exl a-3.

[0017] Figure 3 shows the rat paw withdrawal latency after administration of the solvent control group, the positive control capsaicin administration group and the administration of compositions Exl-1, Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1 of the present disclosure. DETAILED DESCRIPTION

[0018] Reference will now be made in detail to certain embodiments of the disclosure, examples of which are illustrated in the accompanying structures and formulas. While the disclosure will be described in conjunction with the enumerated embodiments, it will be understood that the disclosure is not limited to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents which can be included within the scope of the disclosure as defined by the claims. Persons skilled in the art will recognize many methods and materials similar or equivalent to those described herein as being suitable for use in practicing the disclosure. The disclosure is in no way limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials (including but not limited to definitions, use of terms by various authorities, described techniques, etc.) differ from the present application, the present disclosure controls. All references, patents, patent applications cited in this disclosure are hereby incorporated by reference in their entirety.

[0019] It should be understood that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable

[0020] Definitions

[0021] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional group terms are generally defined as described therein. Additionally, general principles of organic chemistry, and specific functional moieties and reactivity descriptions are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March’s Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; each of which is incorporated herein by reference in its entirety.

[0022] At various places in the present disclosure, connecting substituents are described. Where a structure explicitly requires a connecting group, the Markush variable recited in connection with that group should be understood as a connecting group. For example, if a structure requires a connecting group and the Markush group definition recitation lists “alkyl,” then it should be understood that the “alkyl” represents a connecting alkylene group.

[0023] As used herein, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety is replaced by a suitable substituent. It is understood that "substitution" or "substituted" includes the implicit proviso that such substitution is in accordance with permitted valence of the atom to which the moiety is substituted, and the substitution results in a stable or chemically feasible compound, e.g., a compound that does not spontaneously undergo rearrangement, cyclization, eliminations or other conversion in which the stability of the compound is adversely affected. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituent may, at each position, be either the same or different. The skilled artisan will appreciate that the substituents themselves can be further substituted, if appropriate. Unless specifically identified as "unsubstituted," a reference to a chemical moiety herein is understood to include substituted variants. For example, a reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0024] As used herein, the term "about," when used in reference to a given range or value (e.g., a temperature, time, amount, and concentration) refers to a range of values plus or minus ten percent, plus or minus five percent, or plus or minus one percent of the given range or value.

[0025] When a bond to a substituent appears cross-attached to two atoms of a ring, then the substituent can be bonded to any atom on the ring. When a listed substituent is not specified to be bonded to an atom of the rest of the formulaic compound to which it is drawn, then the substituent can be bonded via any atom in the formula. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0026] When any variable (e.g., R i ) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 to 2 R i groups, then at each occurrence the group can optionally be substituted with up to two R i groups, and the R i groups at each occurrence are independently selected from the definition of R i . Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0027] As used herein, the term "C i-j " indicates a range of carbon atoms, wherein i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and every integer point in between, and wherein j is greater than i. For example, C 1-6ranging from one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms, and six carbon atoms. In some embodiments, the term "C 1-12 ” indicates 1 to 12, especially 1 to 10, especially 1 to 8, especially 1 to 6, especially 1 to 5, especially 1 to 4, especially 1 to 3, or especially 1 to 2 carbon atoms.

[0028] As used herein, the term “alkyl,” whether used alone or as part of another term, refers to a saturated straight-chain or branched-chain hydrocarbon group. The term “C i-j alkyl” refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms. In some embodiments, the alkyl group contains 1 to 11 carbon atoms, 1 to 10 carbon atoms, 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1 -propyl (n-propyl), 2-propyl (i-propyl), 1 -butyl (n-butyl), 2-methyl- 1 -propyl (i-butyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (t-butyl), 1-pentyl (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, 1-heptyl, 1-octyl, and the like. “C 1- 12 Examples of “C 1-6 Examples of “C 1-6 Examples of “C

[0029] The alkyl groups can be optionally substituted with a substituent that independently replaces one or more hydrogen atoms on one or more carbons of the alkyl group. Examples of such substituents can include, but are not limited to, halogen, hydroxyl, cyano, nitro, azido, acyl, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, alkoxy, haloalkyl, haloalkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylaryl, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, formylamino, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfamido, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, or saturated or partially unsaturated heterocyclyl. Alkenyl, alkynyl, aryl, heteroaryl, saturated or partially unsaturated cyclic alkyl, and saturated or partially unsaturated heterocyclyl groups, as described below, can also be similarly substituted.

[0030] As used herein, the term "alkenyl" whether used alone or as part of another term, refers to a straight or branched hydrocarbon group having at least one carbon-carbon double bond, which can be optionally independently substituted with one or more substituents described herein, and includes groups having "cis" and "trans" orientations, or alternatively "E" and "Z" orientations. In some embodiments, an alkenyl group contains 2 to 12 carbon atoms. In some embodiments, an alkenyl group contains 2 to 11 carbon atoms. In some embodiments, an alkenyl group contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, an alkenyl group contains 2 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethylenyl (or vinyl), propenyl, butenyl, pentenyl, 1 -methyl-2but-1 -yl, 5-hexenyl, and the like.

[0031] The term "alkynyl," whether used alone or as part of another term, as used herein, refers to a straight or branched chain hydrocarbon group having at least one carbon-carbon triple bond, which can be optionally independently substituted with one or more substituents described herein. In some embodiments, the alkenyl group contains 2 to 12 carbon atoms. In some embodiments, the alkynyl group contains 2 to 11 carbon atoms. In some embodiments, the alkynyl group contains 2 to 11 carbon atoms, 2 to 10 carbon atoms, 2 to 9 carbon atoms, 2 to 8 carbon atoms, 2 to 7 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, and in some embodiments, the alkynyl group contains 2 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1 -propynyl, 2-propynyl, and the like.

[0032] The term "halo" or "halogen," as used herein, refers to an atom selected from fluorine, chlorine, bromine, and iodine.

[0033] The term "haloalkyl," as used herein, refers to an alkyl group substituted with one or more halogen atoms independently replacing one or more hydrogen atoms on the carbon(s) of the alkyl group.

[0034] The term "hydroxyl," as used herein, refers to an -OH group.

[0035] The term "hydroxyalkyl," as used herein, refers to an -alkyl-hydroxyl group.

[0036] The term "amino acid," as used herein, refers to naturally occurring and synthetic alpha, beta, gamma, or delta amino acids, including but not limited to, those found in proteins, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, and histidine. In certain embodiments, the amino acid is in the L-configuration. Alternatively, the amino acid can be a derivative of propionyl, valyl, leucyl, isoleucyl, prolyl, phenylalanyl, tryptophyl, methionyl, glycyl, seryl, threonyl, cysteinyl, tyrosyl, asparaginyl, glutaminyl, aspartyl, glutamyl, lysyl, arginyl, histidyl, beta-alanyl, beta-valyl, beta-leucyl, beta-isoleucyl, beta-prolyl, beta-phenylalanyl, beta-tryptophyl, beta-methionyl, beta-glycyl, beta-seryl, beta-threonyl, beta-cysteinyl, beta-tyrosyl, beta-asparaginyl, beta-glutaminyl, beta-aspartyl, beta-glutamyl, beta-lysyl, beta-arginyl, or beta-histidyl.

[0037] As used herein, the term "matrix metalloproteinase (MMP)-cleavable peptide" refers to an amino acid sequence that can be recognized and degraded by different matrix metalloproteinases (e.g., MMP-2, MMP-4, or MMP-9).

[0038] As used herein, the term "coupling agent" refers to a precursor compound comprising a capsaicin and a linker, which can be further conjugated to a hyaluronic acid polymer to form a conjugate moiety of Formula (I) of the present disclosure.

[0039] As used herein, the term "crosslinking agent" refers to an agent that acts as a bridge between molecular chains, thereby linking multiple molecular chains to each other to form a network structure. In particular, in the application of hyaluronic acid, the crosslinking agent links the polymer molecular chains of hyaluronic acid together to form a stable hyaluronic acid network.

[0040] Composition 1

[0041] In one aspect, the present disclosure provides a composition comprising a crosslinked hyaluronic acid conjugate physically loaded with capsaicin.

[0042] In some embodiments, the present disclosure provides a composition comprising a hyaluronic acid matrix and capsaicin, the hyaluronic acid matrix formed by crosslinking hyaluronic acid polymers with a crosslinking agent.

[0043] In some embodiments, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0044] In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.07-0.30. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.07-0.30, 0.07-0.25, 0.07-0.20, 0.10-0.30, 0.10-0.25, 0.10-0.20, 0.13-0.20, or 0.15-0.20. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.07-0.20. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.13-0.20. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.15-0.20. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20. In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.20.

[0045] In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 1000-3000 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 1000-2500 kDa, 1000-2000 kDa, or 1000-1500 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 3000 kDa, 2500 kDa, 2000 kDa, 1500 kDa, or 1000 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 2000 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 1000 kDa.

[0046] In some embodiments, the capsaicin is present in an amount of 1-10% wt based on the total weight of the composition. In some embodiments, the capsaicin is present in an amount of 1-10% wt, 1-9% wt, 1-8% wt, 1-7% wt, 1-6% wt, 1-5% wt, 2-10% wt, 3-10% wt, 4-10% wt, 5-10% wt, 2-9% wt, 3-9% wt, 4-9% wt, 5-9% wt, 2-8% wt, 3-8% wt, 4-8% wt, 5-8% wt, 2-7% wt, 3-7% wt, 4-7% wt, 5-7% wt, 2-6% wt, 3-6% wt, 4-6% wt, 5-6% wt, 2-5% wt, 3-5% wt, or 4-5% wt based on the total weight of the composition. In some embodiments, the capsaicin is present in an amount of 10% wt, 9% wt, 8% wt, 7% wt, 6% wt, 5% wt, 4% wt, 3% wt, 2% wt, or 1% wt based on the total weight of the composition. In some embodiments, the capsaicin is present in an amount of 5% wt based on the total weight of the composition.

[0047] In some embodiments, the molar ratio of the crosslinking agent to the hyaluronic acid polymer is 0.20, the weight average molecular weight Mw of the hyaluronic acid polymer is 1000 kDa, and the capsaicin is present in an amount of 5% wt based on the total weight of the composition.

[0048] In some embodiments, the hyaluronic acid matrix has the form of a nanoparticle, and the capsaicin is dispersed in the hyaluronic acid matrix.

[0049] Composition 2

[0050] In one aspect, the present disclosure provides a composition comprising a crosslinked hyaluronic acid chemically loaded with capsaicin.

[0051] In some embodiments, the present disclosure provides a composition comprising a hyaluronic acid matrix and a capsaicin, wherein the hyaluronic acid matrix is formed by crosslinking hyaluronic acid polymers with a crosslinking agent, and the capsaicin is conjugated to the hyaluronic acid polymers via a linker to form a conjugate moiety.

[0052] In some embodiments, the conjugate moiety has the following formula (I):

[0053] wherein,

[0054] X is a direct bond, *-C(O)-, *-C(O)N(R a )-, wherein the * end of X indicates the point of attachment to the O atom;

[0055] Y is a direct bond, -N(R a )-, -N(R a )-N(R a )-, or -O-;

[0056] L is a direct bond or **-L1-L2-L3-L4-, wherein the ** end of L indicates the point of attachment to X;

[0057] L1 is a direct bond, **-C(O)-alkyl-, **-C(O)-, **-C(O)O-alkyl-, an amino acid residue, or a matrix metalloproteinase (MMP) cleavable peptide, wherein the ** end of L1 indicates the point of attachment to X;

[0058] L2 is a direct bond, #-C(O)N(R a )-, #-C(O)-, #-C(O)-alkyl-, or #- (OCH2CH2) n -, wherein the # end of L2 indicates the point of attachment to L1;

[0059] L3 is a direct bond, alkyl, ##-C(O)-, ##-O-alkyl-C(O)-, ##-C(O)N(R a )-alkyl-, or ##- (OCH2CH2) n -, wherein the ## end of L3 indicates the point of attachment to L2;

[0060] L4 is a direct bond or ###-O-alkyl-C(O)-, wherein the ### end of L4 indicates the point of attachment to L3;

[0061] R a is hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, or hydroxyalkyl;

[0062] m is 100-20,000;

[0063] n is 1, 2, 3, 4, or 5.

[0064] In some embodiments, X is R a is hydrogen or alkyl (e.g., C 1-6 alkyl, C 1-5 alkyl, C 1-4 alkyl, C 1-3 alkyl, or C 1-2 alkyl). In some embodiments, X is R a is hydrogen.

[0065] In some embodiments, X is a direct bond.

[0066] In some embodiments, X is

[0067] In some embodiments, L1is **-C(O)-alkyl- (e.g., **-C(O)-C 1-6 alkyl-, **-C(O)-C 1-5 alkyl-, **-C(O)-C 1-4 alkyl-, **-C(O)-C 1-3 alkyl-, or **-C(O)-C 1-2 alkyl-), L2is a direct bond, #-C(O)N(R a )-, #-C(O)-, or #-(OCH2CH2) n -, L3is a direct bond, alkyl (e.g., C 1-6 alkyl, C 1-5 alkyl, C 1-4 alkyl, C 1-3 alkyl, or C 1-2 alkyl), or ##-O-alkyl-C(O)- (e.g., ##-O-C 1-6 alkyl-C(O)-, ##-O-C 1-5 alkyl-C(O)-, ##-O-C 1-4 alkyl-C(O)-, ##-O-C 1-3 alkyl-C(O)-, or ##-O-C 1-2 alkyl-C(O)-), and L4is a direct bond.

[0068] In some embodiments, L1is **-C(O)-C 1-6 alkyl-. In some embodiments, L1is **-C(O)-C6alkyl-, **-C(O)-C5alkyl-, **-C(O)-C4alkyl-, **-C(O)-C3alkyl-, **-C(O)-C2alkyl-, or **-C(O)-C1alkyl-.

[0069] In some embodiments, L2, L3, and L4 are each a direct bond. In some embodiments, L2 and L4 are direct bonds, and L3 is 1-6 alkyl-C(O)- (e.g., -O-C6alkyl-C(O)-, -O-C5alkyl-C(O)-, -O-C4alkyl-C(O)-, -O-C3alkyl-C(O)-, -O-C2alkyl-C(O)-, -O-C1alkyl-C(O)-). In some embodiments, L2 is -C(O)N(R a )-, R a is hydrogen, L3 is C 1-6 alkyl (e.g., C6alkyl, C5alkyl, C4alkyl, C3alkyl, C2alkyl, C1alkyl), and L4 is a direct bond. In some embodiments, L2 is -C(O)-, and L3 and L4 are direct bonds. In some embodiments, L2 is -(OCH2CH2) n -, L3 is -O-alkyl-C(O)-, and L4 is a direct bond. In some embodiments, L2 is -(OCH2CH2) n -, L3 is -O-C 1-6 alkyl-C(O)- (e.g., -O-C6alkyl-C(O)-, -O-C5alkyl-C(O)-, -O-C4alkyl-C(O)-, -O-C3alkyl-C(O)-, -O-C2alkyl-C(O)-, -O-C1alkyl-C(O)-), and L4 is a direct bond. In some embodiments, L2 is -(OCH2CH2) n -, L3 is -O-C1alkyl-C(O)-, and L4 is a direct bond.

[0070] In some embodiments, L1, L2, and L4 are each a direct bond, and L3 is alkyl. In some embodiments, L1, L2, and L4 are each a direct bond, and L3 is C 1-6 alkyl (e.g., C6alkyl, C5alkyl, C4alkyl, C3alkyl, C2alkyl, C1alkyl).

[0071] In some embodiments, L1 is **-C(O)O-alkyl-, L2 is -C(O)N(R a )- or -(OCH2CH2) n -, L3 is a direct bond, alkyl, or -O-alkyl-C(O)-, and L4 is a direct bond. In some embodiments, L1 is **-C(O)O-C 1-6In some embodiments, L2 is #-C(O)N(R a )-,R a is hydrogen, L3 is C 1-6 alkyl (e.g., C6 alkyl, C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, C1 alkyl), and L4 is a direct bond.

[0072] In some embodiments, L1 is an MMP cleavable peptide, L2 is #-C(O)-alkyl-, L3 is a straight bond, ##-C(O)-, ##-O-alkyl-C(O)-, ##-C(O)N(R a )-alkyl- or ##-(OCH2CH2) n -, L4 is a straight bond or ###-O-alkyl-C(O)-. In some embodiments, L2 is #-C(O)-C 1-6 In some embodiments, L3 is a straight bond, ##-C(O)-, ##-OC 1-6 Alkyl-C(O)- (e.g., ##-O-C6 alkyl-C(O)-, ##-O-C5 alkyl-C(O)-, ##-O-C4 alkyl-C(O)-, ##-O-C3 alkyl-C(O)-, ##-O-C2 alkyl-C(O)-, ##-O-C1 alkyl-C(O)-), ##-C(O)N(R a )-C 1-6 Alkyl-(e.g., ##-C(O)N(R a )-C6 alkyl-, ##-C(O)N(R a )-C5 alkyl-, ##-C(O)N(R a )-C4 alkyl-, ##-C(O)N(R a )-C3 alkyl-, ##-C(O)N(R a )-C2 alkyl-, ##-C(O)N(R a )-C1 alkyl-) or ##-(OCH2CH2) n -. In some embodiments, L4 is a direct bond. In some embodiments, L4 is ###-OC 1-6alkyl-C(O)-(e.g., ###-O-C6 alkyl-C(O)-, ###-O-C5 alkyl-C(O)-, ###-O-C4 alkyl-C(O)-, ###-O-C3 alkyl-C(O)-, ###-O-C2 alkyl-C(O)-, ###-O-C1 alkyl-C(O)-).

[0073] In some embodiments, L1is selected from GGFG**, PVGLIG**, PLGLAG**, FF**, GPQGIWGQ**, GPQGIAGQ**, VPMSMRGG**, QPQGLAK**, GPLGLSGK**, GPLGMHGK**, VPLSLYSG**, IPVSLRSG**, VPLSLTMG**, IPESLRAG**, GPLGLWAR**, SGESPAYYTA**, RPFSMIMG**, GGYAELRMGG**, GGPLGLYAGG**, CGPQGIWGQC**, GKKCGPQGIWGQCKKG**, CGGPLGLAGGC**, or GCRDGPQGIWGQDRCG**, where the **ends of L1represent the point of attachment to X. In some embodiments, L1is selected from GGFG** or PLGLAG**.

[0074] In some embodiments, L1is an amino acid residue, and L2, L3, and L4are direct bonds. In some embodiments, L1is selected from glycine or phenylalanine, and L2, L3, and L4are direct bonds.

[0075] In some embodiments, L is selected from the group consisting of a direct bond, glycine, phenylalanine, **-C(O)(CH2) 1- 12 **-C(O)(CH2) 1-12 C(O)NH(CH2) 1-6 **-C(O)(CH2) 1-6 (OCH2CH2) n **-C(O)(CH2) 1-12 C(O)-, **-C(O)(CH2) 1-6 (OCH2CH2) n -O-(CH2) 1-6 C(O)-, **-GFGG-C(O)(CH2) 1-12 C(O)NH(CH2) 1-6 **-GALGLP-C(O)(CH2) 1-12 **-GALGLP-C(O)(OCH2CH2) n-**, -GALGLP-C(O)(CH2) 1- 12 (OCH2CH2) n -**, -GALGLP-C(O)(CH2) 1-12 C(O)-**, -GFGG-C(O)(CH2) 1-12 C(O)-**, -GALGLP-C(O)(CH2) 1-6 (OCH2CH2) n -O-(CH2) 1-6 C(O)-**, -FF-C(O)(CH2) 1-12 -**, -(CH2) 1-12 -**, -C(O)O(CH2) 1-6 C(O)-**, -C(O)O(CH2) 1-6 C(O)NH(CH2) 1-6 - and **-C(O)(OCH2CH2) n -O-(CH2) 1- 6C(O)-.

[0076] In some embodiments, n is 2, 3, or 4.

[0077] In some embodiments, -X-L-Y- is selected from the group consisting of:

[0078] a direct bond,

[0079] wherein the * end denotes the point of attachment to the O atom.

[0080] In some embodiments, -X-L-Y- is selected from the group consisting of:

[0081] In some embodiments, -X-L-Y- is selected from

[0082] In some embodiments, the conjugate moiety has a structure selected from the group consisting of:

[0083]

[0084]

[0085] or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the crosslinking agent is BDDE, 4,7,10-trioxa-l,13- tridecanediamine, or glutaraldehyde.

[0087] In some embodiments, the crosslinking agent is 4,7,10-trioxa-l,13-tridecanediamine.

[0088] In some embodiments, the molar ratio of the crosslinking agent to the conjugate moiety is 0.10-0.40. In some embodiments, the molar ratio of the crosslinking agent to the conjugate moiety is 0.10-0.40, 0.10-0.30, 0.10-0.20, 0.20-0.40, or 0.20-0.30. In some embodiments, the molar ratio of the crosslinking agent to the conjugate moiety is 0.10, 0.20, 0.30, or 0.40.

[0089] In some embodiments, the molar ratio of the crosslinking agent to the conjugate moiety is 0.20.

[0090] In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 50-2000 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 50-2000 kDa, 100-2000 kDa, 200-2000 kDa, 300-2000 kDa, 400-2000 kDa, 500-2000 kDa, 500-1500 kDa, or 500-1000 kDa.

[0091] In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 200-2000 kDa. In some embodiments, the hyaluronic acid polymer has a weight average molecular weight Mw of 200 kDa, 500 kDa, 1000 kDa, 1500 kDa, or 2000 kDa.

[0092] In some embodiments, the capsaicin is present in an amount of 10-20% wt based on the total weight of the composition. In some embodiments, the capsaicin is present in an amount of 10-20% wt, 10-18% wt, 12-18% wt, or 14-18% wt based on the total weight of the composition.

[0093] In some embodiments, -X-L-Y- is selected from the crosslinking agent is 4,7,10-trioxa-l,13-tridecanediamine, the molar ratio of the crosslinking agent to the conjugate moiety is 0.20, the hyaluronic acid polymer has a weight average molecular weight Mw of 1000 kDa, and the capsaicin is present in an amount of 10-20% wt based on the total weight of the composition.

[0094] Composition 3

[0095] In yet another aspect, the present disclosure provides a composition comprising a hyaluronic acid cross-linker chemically loaded with capsaicin.

[0096] In some embodiments, the present disclosure provides a composition comprising a hyaluronic acid matrix obtained by cross-linking hyaluronic acid polymers with a cross-linking agent conjugated with capsaicin.

[0097] In some embodiments, the cross-linking agent conjugated with capsaicin has the following formula (II):

[0098] wherein,

[0099] W is CH or N;

[0100] U and V are, independently of each other, -OH, -NH2, or -NHNH2;

[0101] L is -C(O)-, -C(O)-alkyl-C(O)-, -C(O)NH-alkyl-C(O)-, or -C(O)-alkyl-NHC(O)-.

[0102] In some embodiments, L is -C(O)- or -C(O)-alkyl-C(O)-. In some embodiments, L is -C(O)-C 1-6 alkyl-C(O)-. In some embodiments, L is -C(O)-C6alkyl-C(O)-, -C(O)-C5alkyl-C(O)-, -C(O)-C4alkyl-C(O)-, -C(O)-C3alkyl-C(O)-, -C(O)-C2alkyl-C(O)-, or -C(O)-C1alkyl-C(O)-.

[0103] In some embodiments, L is -C(O)- and W is CH.

[0104] In some embodiments, L is -C(O)-C 1-6 alkyl-C(O)- and W is N. In some embodiments, L is -C(O)-C2alkyl-C(O)-, -C(O)-C3alkyl-C(O)-, or -C(O)-C4alkyl-C(O)- and W is N.

[0105] In some embodiments, the cross-linking agent conjugated with capsaicin is selected from

[0106] In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.10-0.60. In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.10-0.60, 0.10-0.50, 0.10-0.40, 0.10-0.30, 0.20-0.30, 0.20-0.40, 0.20-0.50, or 0.20-0.60. In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.10, 0.20, 0.30, 0.40, 0.50, or 0.60.

[0107] In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.20-0.50. In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.20.

[0108] In some embodiments, the weight average molecular weight Mw of the hyaluronic acid polymer is 100-2000 kDa. In some embodiments, the weight average molecular weight Mw of the hyaluronic acid polymer is 100-2000 kDa, 200-2000 kDa, 300-2000 kDa, 400-2000 kDa, 500-2000 kDa, 500-1500 kDa, 500-1000 kDa, 200-1500 kDa, or 200-1000 kDa. In some embodiments, the weight average molecular weight Mw of the hyaluronic acid polymer is 100 kDa, 200 kDa, 500 kDa, 1000 kDa, 1500 kDa, or 2000 kDa.

[0109] In some embodiments, the weight average molecular weight Mw of the hyaluronic acid polymer is 200-1000 kDa.

[0110] In some embodiments, the content of capsaicin is 5-20% wt based on the total weight of the composition. In some embodiments, the content of capsaicin is 5-20% wt, 5-18% wt, 5-16% wt, 5-15% wt, 5-12% wt, or 5-10% wt based on the total weight of the composition.

[0111] In some embodiments, the content of capsaicin is 5-15% wt based on the total weight of the composition.

[0112] In some embodiments, the molar ratio of the capsaicin-conjugated crosslinking agent to the hyaluronic acid polymer is 0.20, the weight average molecular weight Mw of the hyaluronic acid polymer is 1000 kDa, and the content of capsaicin is 5-15% wt based on the total weight of the composition.

[0113] In some embodiments, the hyaluronan matrix has a structure selected from the group consisting of:

[0114] or a pharmaceutically acceptable salt thereof.

[0115] The compounds of the present disclosure also include prodrugs, active metabolic derivatives (active metabolites), active intermediates, and pharmaceutically acceptable salts thereof.

[0116] As used herein, the term "prodrug" refers to a compound, or a pharmaceutically acceptable salt thereof, that, when metabolized under physiological conditions or when converted by solvolysis, yields a desired active compound. Prodrugs include, but are not limited to, esters, amides, carbamates, carbonates, acylureas, solvates, or hydrates of the active compound. Typically, the prodrug is inactive or less active than the active compound, but can provide one or more of advantageous handling, use, and / or metabolic properties. For example, some prodrugs are esters of the active compound; during metabolism, the ester group is cleaved to yield the active drug. In addition, some prodrugs are enzymatically activated to yield the active compound, or are compounds that, upon further chemical reaction, yield the active compound. Prodrugs can develop from the prodrug form to the active form in a single step, or can have one or more intermediate forms that can or can not be active. The preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.

[0117] As used herein, the term "metabolite" (e.g., active metabolite) overlaps with prodrugs as described above. Thus, the metabolite is a pharmacologically active compound or a compound that is further metabolized to a pharmacologically active compound that is a derivative produced by metabolic processes within the body of an individual. For example, such metabolites can result from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like of the administered compound or salt or prodrug. Among these, the active metabolite is the derivative compound that is pharmacologically active. For a prodrug, the prodrug compound is generally inactive or less active than the metabolite. For an active metabolite, the parent compound can be the active compound or can be an inactive prodrug.

[0118] Prodrugs and active metabolites can be identified using routine techniques known in the art. See, e.g., Bertolini et al., 1997, J Med Chem 40:2011-2016; Shan et al., J Pharm Sci 86:756-757; Bagshawe, 1995, Drug Dev Res 34:220-230; Wermuth, supra.

[0119] As used herein, the term "active intermediate" refers to an intermediate compound in a synthesis process that exhibits the same or substantially the same biological activity as the final synthesized compound.

[0120] The compounds of the present disclosure can be formulated into or be in the form of a pharmaceutically acceptable salt. Unless otherwise specified, the compounds provided herein include pharmaceutically acceptable salts of such compounds.

[0121] As used herein, the term "pharmaceutically acceptable" indicates that the substance or composition is chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the subject being treated with it.

[0122] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that retain the biological effectiveness and non-antagonistic properties of the free acids and bases of the designated compounds, and which are not biologically or otherwise undesirable. Pharmaceutically acceptable salt forms contemplated include, but are not limited to, mono-, di-, tri-, tetra-, and the like, salts. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without adversely affecting its physiological function. Useful alterations in physical characteristics include reduction of melting point to facilitate transmucosal administration and increased solubility to facilitate use of higher concentrations of the drug.

[0123] Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts can be obtained from, for example, hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.

[0124] When acidic functional groups are present (e.g., carboxylic acid or phenol), pharmaceutically acceptable salts also include base addition salts, such as base addition salts containing benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, t-butyl amines, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkyl amines, and zinc. See, e.g., Remington's Pharmaceutical Sciences, 19th Ed., Mack Publishing Co., Easton, PA, Vol. 2, p. 1457, 1995; Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH, Weinheim, Germany, 2002. Such salts can be prepared using the appropriate corresponding bases.

[0125] Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of a compound can be dissolved in a suitable solvent, such as an aqueous or aqueous-alcohol solution containing the appropriate acid, and then isolated by evaporation of the solution. Thus, if a particular compound is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, such as by treatment with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.

[0126] Similarly, if the particular compound is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, by treatment with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali metal or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include: organic salts derived from amino acids such as L-glycine, L-lysine, and L-arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines such as hydroxylethylpiperazine, piperidine, morpholine, and piperazine; and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0127] Synthetic methods

[0128] The synthesis of the compounds provided herein, including salts thereof, is illustrated in the synthetic schemes in the Examples. The compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes, and thus, the schemes are merely illustrative and are not intended to limit other possible methods that can be used to prepare the compounds provided herein. Further, the steps in the schemes are for better illustration and can be changed as appropriate.

[0129] The reactions for preparing the compounds of the present disclosure can be carried out in suitable solvents which can be readily selected by one of ordinary skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperatures at which the reactions are carried out, e.g., at temperatures between the solvent's freezing temperature and boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. The choice of a suitable solvent will depend on the nature of the particular reaction step.

[0130] The preparation of the compounds of the present disclosure can involve 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 of ordinary skill in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rdEd., Wiley & Sons, Inc., New York (1999), which is hereby incorporated by reference in its entirety.

[0131] The reactions can be monitored by any method known in the art, for example, by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, or ultraviolet-visible spectroscopy, or by mass spectrometry, or by chromatographic means, such as high performance liquid chromatography or thin layer chromatography. 1 H or 13C), infrared spectroscopy (IR), spectrophotometry (e.g., UV-Visible), mass spectrometry (MS), or by chromatography, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). A person of skill in the art can purify compounds by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Compound Specific Method Optimization”, Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, J. Combi. Chem., 2004, 6(6), 874-883, incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0132] As used herein, the following abbreviations are defined as follows: “1x” or “x1” is once, “2x” or “x2” is twice, “3x” or “x3” is three times, “4x” or “x4” is four times, “5x” or “x5” is five times, “°C” is degrees Celsius, “eq” or “eq.” is equivalent, “g” is gram(s), “mg” is milligram(s), “L” is liter(s), “mL” or “ml” is milliliter(s), “pL” is microliter(s), “N” is normal, “M” is molar, “mmol” is millimole(s), “min” is minute(s), “h” or “hr” is hour(s), “r.t.” or “rt” is room temperature, “atm” is atmosphere, “psi” is pound(s) per square inch, “conc.” is concentration, “sat” or “sat’d” is saturated, “MS” or “Mass Spec” is mass spectrometry, “ESI” is electrospray ionization mass spectrometry, “LCMS” is liquid chromatography mass spectrometry, “HPLC” is high performance liquid chromatography, “RP” is reverse phase, “TLC” or “tlc” is thin layer chromatography, “SM” is starting material, “NMR” is nuclear magnetic resonance spectroscopy, “ 1H" is proton, "δ" is delta, "s" is singlet, "d" is doublet, "t" is triplet, "q" is quartet, "m" is multiplet, "br" is broad, and "Hz" is hertz. "α", "β", "R", "S", "E", and "Z" are stereochemical designations familiar to one of ordinary skill in the art.

[0133] Pharmaceutical compositions

[0134] The present disclosure provides pharmaceutical compositions comprising at least one composition of the present disclosure (i.e., Composition 1, Composition 2, or Composition 3). In some embodiments, the pharmaceutical composition comprises a composition of the present disclosure and one or more pharmaceutically acceptable carriers.

[0135] Pharmaceutically acceptable carriers are conventional pharmaceutical carriers that are well known in the art, and can be prepared in a manner well known in the pharmaceutical arts. In some embodiments, the compounds of the present disclosure can be mixed with a pharmaceutically acceptable carrier to produce a pharmaceutical composition.

[0136] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the compositions provided herein from one location, body fluid, tissue, organ (internal or external) or part of the body to another location, body fluid, tissue, organ or part of the body. A pharmaceutically acceptable carrier can be a vehicle, diluent, excipient or other material that can be used to facilitate the delivery of an active agent to an animal tissue, which is not biologically or otherwise undesirable. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, malt, gum, gelatin, Ringer's solution, alginic acid, isotonic saline, buffered solutions, and the like. Pharmaceutically acceptable carriers that can be used in the present disclosure include those generally known in the art, such as those disclosed in Remington Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

[0137] Some examples of materials that can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations, such as acetone.

[0138] The pharmaceutical compositions can contain, if desired, nontoxic auxiliary substances such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.

[0139] The form of the pharmaceutical composition depends on a variety of standards, including but not limited to, the route of administration, the extent of disease, or the dosage to be administered. The pharmaceutical composition can be formulated for oral, nasal, rectal, transdermal, intravenous, or intramuscular administration. For example, the dosage form for nasal administration can be suitably formulated as an aerosol, a solution, a drop, a gel, or a dry powder; the dosage form for intranasal administration can be formulated as a fluid formulation. Depending on the desired route of administration, the pharmaceutical composition can be formulated into tablets, capsules, pills, dragees, powders, granules, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (in solid form or in a liquid medium), sprays, ointments, pastes, creams, lotions, gels, patches, inhalers, or suppositories. In some embodiments, the pharmaceutical composition of the present disclosure is formulated as a dosage form for injectable administration. In some embodiments, the pharmaceutical composition of the present disclosure is formulated as a dosage form for intra-articular injectable administration.

[0140] The pharmaceutical compositions can also be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures known in the art of pharmaceutical formulation. In some embodiments, the pharmaceutical compositions are formulated in sustained release form. As used herein, the term "sustained release form" refers to the release of an active agent from a pharmaceutical composition such that it is bioabsorbed at the site of administration in a subject over an extended period of time (prolonged release) or at a location (controlled release). In some embodiments, the pharmaceutical compositions of the present disclosure can relieve joint pain for an extended period of time, e.g., about 1 hour to 24 hours, 2 hours to 12 hours, 3 hours to 8 hours, 4 hours to 6 hours, 1 to 2 days, 5 to 10 days, 30 to 60 days, 3 to 6 months, 6 to 12 months, or longer. In certain embodiments, the extended period of time is at least about 1 day, at least about 5 days, at least about 10 days, at least about 20 days, at least about 30 days, at least about 3 months, at least about 6 months, or at least about 1 year.

[0141] In some embodiments, the pharmaceutical composition comprises about 0.01 mg to about 5 mg of capsaicin (e.g., about 0.02 mg to about 5 mg, about 0.05 mg to about 5 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 4 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 2 mg, about 0.1 mg to about 1 mg, about 0.1 mg to about 0.5 mg, about 1 mg to about 5 mg, about 1 mg to about 4 mg, about 1 mg to about 3 mg, about 1 mg to about 2 mg). In some embodiments, a suitable dose per subject per day can be about 0.5 mg to about 5 mg, about 0.5 mg to about 4 mg, about 0.5 mg to about 3 mg, about 0.5 mg to about 2 mg, or about 0.5 mg to about 1 mg.

[0142] In certain embodiments, the pharmaceutical composition can be formulated in unit dosage form, each dosage containing from about 0.01 mg to about 10 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 4 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 2 mg, from about 0.1 mg to about 1 mg, from about 0.1 mg to about 0.5 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg of capsaicin. In some embodiments, each dosage contains about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1.0 mg, about 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2.0 mg, about 2.1 mg, about 2.2 mg, about 2.3 mg, about 2.4 mg, about 2.5 mg, about 2.6 mg, about 2.7 mg, about 2.8 mg, about 2.9 mg, about 3 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 6.0 mg, or about 7.0 mg of capsaicin. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier.

[0143] In some embodiments, the pharmaceutical composition can be formulated as an injectable formulation comprising capsaicin, wherein the concentration of capsaicin is about 0.1 mg / mL to about 20 mg / mL, about 0.1 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 4 mg / mL, about 0.1 mg / mL to about 3 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL. In some embodiments, the pharmaceutical composition can be formulated as an injectable formulation comprising capsaicin at a concentration of about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.325 mg / mL, about 0.35 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.4 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.475 mg / mL, about 0.5 mg / mL, about 0.55 mg / mL, about 0.575 mg / mL, about 0.6 mg / mL, about 0.625 mg / mL, about 0.65 mg / mL, about 0.675 mg / mL, about 0.7 mg / mL, about 0.75 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL.

[0144] In some embodiments, the pharmaceutical composition can be formulated as a topical preparation (e.g., in the form of a suspension, emulsion, solution, spray, ointment, paste, cream, lotion, gel, or patch) comprising capsaicin, wherein the concentration of capsaicin is about 0.1 mg / mL to about 20 mg / mL, about 0.1 mg / mL to about 15 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 4 mg / mL, about 0.1 mg / mL to about 3 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL. In some embodiments, the pharmaceutical composition can be formulated as a topical preparation comprising capsaicin at a concentration of about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.325 mg / mL, about 0.35 mg / mL, about 0.37 mg / mL, about 0.38 mg / mL, about 0.39 mg / mL, about 0.4 mg / mL, about 0.41 mg / mL, about 0.42 mg / mL, about 0.43 mg / mL, about 0.44 mg / mL, about 0.45 mg / mL, about 0.475 mg / mL, about 0.5 mg / mL, about 0.55 mg / mL, about 0.575 mg / mL, about 0.6 mg / mL, about 0.625 mg / mL, about 0.65 mg / mL, about 0.675 mg / mL, about 0.7 mg / mL, about 0.75 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL.

[0145] Methods of treatment

[0146] The present disclosure provides a method of treating arthritis comprising administering to a subject an effective amount of one or more compositions of the present disclosure.

[0147] The present disclosure provides a method of relieving joint pain comprising administering to a subject an effective amount of one or more compositions of the present disclosure.

[0148] In some embodiments, the arthritis includes, but is not limited to, osteoarthritis, rheumatoid arthritis, gout, septic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, reactive arthritis, septic arthritis, psoriatic arthritis, and the like. In some embodiments, the arthritis is osteoarthritis or rheumatoid arthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is rheumatoid arthritis.

[0149] In some embodiments, the joint pain is arthritic pain. In certain embodiments, the joint pain is osteoarthritic joint pain. In certain embodiments, the joint pain is rheumatoid arthritis joint pain. In certain embodiments, the joint is a painful knee joint. In certain embodiments, the joint is an osteoarthritic knee joint. In certain embodiments, the joint is a knee joint afflicted with rheumatoid arthritis.

[0150] In some embodiments, the joint is a knee joint, a hip joint, a shoulder joint, an elbow joint, an ankle joint, a wrist joint, a tarsal joint, or a metatarsal joint. In certain embodiments, the joint is a knee joint. In certain embodiments, the joint is a thumb joint. In certain embodiments, the joint is a hip joint. In certain embodiments, the joint has an intra-articular space surrounded by synovial membrane. In certain embodiments, the joint does not have an intra-articular space surrounded by synovial membrane.

[0151] As used herein, the term "treatment" refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a genetic or other susceptibility). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0152] The compositions provided herein can be administered in pure form, in combination with other active ingredients, or as pharmaceutical compositions of the present disclosure. In some embodiments, the compositions provided herein can be administered simultaneously or sequentially with one or more other active agents known in the art to the subject in need thereof. The individual agents in such combinations are administered sequentially or simultaneously in separate or combined pharmaceutical compositions. Preferably, the individual agents will be administered concurrently in a combined pharmaceutical composition. The skilled artisan will appreciate that appropriate dosages of the known agents lie within the knowledge of those skilled in the art. In some embodiments, the other agents are, for example, anti-inflammatory agents (e.g., non-steroidal anti-inflammatory drugs, steroids, antibiotics, etc.), pain relieving agents (e.g., a local anesthetic). Examples of the anti-inflammatory agents include, but are not limited to, aspirin, indomethacin, naproxen, naprosene, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib, hydrocortisone, dexamethasone, erythromycin, roxithromycin, azithromycin, norfloxacin, ciprofloxacin, ofloxacin, levofloxacin, penicillin, ampicillin, amoxicillin, cefazolin, cephradine, etc. Examples of the local anesthetics include, but are not limited to, lidocaine, dibucaine, bupivacaine, ropivacaine, etidocaine, tetracaine, procaine, chloroprocaine, prilocaine, mepivacaine, xyllocaine, 2-chloroprocaine, and pharmaceutically acceptable salts thereof.

[0153] In some embodiments, the administration is once a day, twice a day, three times a day, or once every two days, once every three days, once every four days, once every five days, once every six days, once a week.

[0154] In some embodiments, the administration is by injection, although the compositions of the disclosure can be administered by any medically acceptable route of administration deemed safe and appropriate by a physician of ordinary skill. Exemplary routes of administration include injection into a joint, injection into a nerve or into tissue proximate to a nerve, and injection into the spinal canal. In certain embodiments, the administration comprises injection of a composition of the disclosure into or proximate to a painful site in a subject. In certain embodiments, the administration is intra-articular injection or intrathecal injection. In certain embodiments, the administration is intra-articular injection. In certain embodiments, the administration is intrathecal injection. In certain embodiments, the administration is epidural injection. In certain embodiments, the administration is injection into the spinal column, such as injection into an intervertebral disc.

[0155] In some embodiments, the administration comprises injection of a composition of the disclosure into the intra-articular space of a joint. In certain embodiments, the administration comprises injection of a composition of the disclosure into the intra-articular space of a knee joint to treat knee joint pain, e.g., osteoarthritic knee joint pain.

[0156] A therapeutically effective amount of a composition as provided herein will depend on various factors known in the art, such as body weight, age, prior medical history, current medications, the health status and cross-reactivity of the subject, the likelihood of allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. A person of skill in the art (e.g., a physician or veterinarian) can reduce or increase the dosage proportionally, as indicated by these and other conditions or requirements. In some embodiments, a suitable daily dose of one or more compositions provided herein is from about 0.001 mg to about 500 mg, from about 0.1 mg to about 200 mg, or from about 1 mg and 100 mg, and the administration is once a day, twice a day, three times a day, every day, or 3 to 5 days a week. In some embodiments, the daily dose of one or more compositions provided herein is 0.001 mg, 0.01 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg.

[0157] In another aspect, the present disclosure provides use of a composition of the present disclosure in the manufacture of a medicament for the treatment of arthritis. In some embodiments, the arthritis is osteoarthritis or rheumatoid arthritis.

[0158] The present disclosure also provides use of a composition of the present disclosure in the manufacture of a medicament for the relief of joint pain.

[0159] Examples

[0160] The general methods of the present disclosure are further illustrated below. The compounds of the present disclosure can be prepared by methods known in the art. The detailed preparation methods of preferred compounds of the present disclosure are described below. However, these are by no means limiting to the preparation methods of the compounds of the present disclosure.

[0161] The synthesis of the compounds provided herein, including pharmaceutically acceptable salts thereof, is illustrated in the synthetic schemes in the examples. The compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes, and thus, these schemes are merely illustrative and not intended to limit other possible methods that can be used to prepare the compounds provided herein. Furthermore, the steps in the schemes are modified as appropriate. Embodiments of the compounds in the examples are synthesized for research and possible submission to regulatory agencies.

[0162] The reactions for preparing the compounds of the present disclosure can be performed in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials, intermediates, or products, at the temperatures at which the reactions are carried out, e.g., at temperatures between the solvent's melting or freezing point and the boiling point. Given the teachings of the present disclosure, the suitable solvent for a particular reaction will be readily apparent. The given reaction can be performed in one solvent or a mixture of more than one solvent.

[0163] The preparation of the compounds of the present disclosure can involve 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. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rdEd., Wiley, New York (1999), which is incorporated herein by reference in its entirety.

[0164] The reactions can be monitored by any method known to one skilled in the art, e.g., spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C), infrared spectroscopy, or mass spectrometry, as well as by chromatographic means. 1 H or 13C), infrared spectroscopy, spectrophotometry (e.g., UV-Visible), mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS), or thin layer chromatography (TLC). A skilled artisan can purify compounds by a variety of methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Method Optimization for Compound Specificity,” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs, Journal of Comb. Chem. 2004, 6(6), 874-883, which is incorporated herein by reference in its entirety) and normal phase silica chromatography.

[0165] In the following examples, unless otherwise indicated, 1 H NMR spectra were typically recorded with a Bruker 400 MHz NMR and 500 MHz NMR nuclear magnetic resonance spectrometer, chemical shifts are expressed in δ (ppm). Mass spectra were recorded with an Agilent 1290 liquid chromatography + 6120B mass spectrometry LCMS. Silica gel column purification was performed using Biotage Selekt SEL-2SV or ISO-1SV. Preparative liquid chromatography purification was performed using Gilson 281 (column: waters Xbridge 19 mm x 250 mm, 5 μm or WELCH C18, 21.2 mm x 250 mm, 10 μm. Mobile phase: A: water (10 mM NH4HCO3 or 0.05% formic acid), B: acetonitrile (or containing 0.05% formic acid), flow rate: 20-30 mL / min, detection wavelength: 214 nm / 254 nm).

[0166] Abbreviations for chemical reagents used in the synthesis of the compounds provided herein are listed below:

[0167] Example A: Preparation of coupling reagent

[0168] Example A1: Preparation of coupling reagent A1

[0169] N-[(4-{[p-(5-bromopentylcarbonylamino)phenyl]methoxy}-3-methoxyphenyl)methyl]-(E)-8-methyl-6-nonenamide

[0170] Step A: N-p-(hydroxymethyl)phenyl 6-bromohexanamide

[0171] To a solution of 6-bromohexanoic acid (A1-1, 3.00 g, 15.4 mmol) in dichloromethane (20 mL) was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.01 g, 18.5 mmol) and N,N-diisopropylethylamine (4.96 g, 38.5 mmol) and stirred at room temperature for 5 min. p-Aminobenzyl alcohol (2.08 g, 16.9 mmol) was added to the reaction and stirred at room temperature for 3 h. The reaction was diluted with water (20 mL) and extracted with dichloromethane (20 mL) three times, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography PE / EtOAc = 80 / 20 to give a gray solid (A1-2, 1.3 g, yield 28.2%). MS-ESI [M+H] + : 300.0.

[0172] Step B: N-p-(chloromethyl)phenyl 6-bromohexanamide

[0173] To a solution of N-p-(hydroxymethyl)phenyl 6-bromohexanamide (A1-2, 1.00 g, 3.30 mmol) in dichloromethane (20 mL) was added a solution of dichlorosulfoxide (670 mg, 5.70 mmol) in dichloromethane (2 mL) and stirred at room temperature for 30 min. Concentrated under reduced pressure to give a light brown oil (A1-3, 660 mg, yield 62.1%). MS-ESI [M+H] + : 318.0.

[0174] Step C: N-[(4-{[p-(5-bromopentylcarbonylamino)phenyl]methoxy}-3- methoxyphenyl)methyl]-(E)-8-methyl-6-nonenamide

[0175] To a solution of N-p-(chloromethyl)phenyl 6-bromohexanamide (A1-3, 660 mg, 2.10 mmol) and capsaicin (630 mg, 2.10 mmol) in N,N-dimethylformamide (15 mL) was added potassium carbonate (0.63 g, 4.6 mmol) and stirred at room temperature overnight. The reaction was filtered and the filtrate was diluted with water (30 mL) and extracted with ethyl acetate (30 mL) three times, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography DCM / MeOH = 98 / 2 to give a gray solid (A1, 740 mg, yield 61%). 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.20 (t, J = 5.6 Hz, 1H), 7.62-7.54 (m, 2H), 7.37-7.26 (m, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.76-6.67 (m, 1H), 5.35-5.33 (m, 2H), 4.96 (s, 2H), 4.17 (d, J = 5.8 Hz, 2H), 3.73 (s, 3H), 3.54 (t, J = 6.8 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H), 2.23-2.19 (m, 1H), 2.11 (t, J = 7.2 Hz, 2H), 1.96-1.91 (m, 2H), 1.86-1.78 (m, 2H), 1.66-1.56 (m, 2H), 1.54-1.46 (m, 2H), 1.44-1.40 (m, 2H), 1.33-1.27 (m, 2H), 0.93 (d, J = 6.8 Hz, 6H). MS - ESI [M+H] + : 587.2.

[0176] Example A2: Preparation of Coupling Agent A2

[0177] N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl} 14-amino-3,6,9,12-tetraoxatetradecanamide

[0178] Step A: 13-[N-p-(hydroxymethyl)anilincarbonyl]-3,6,9,12-tetraoxatridecyl 2-methyl-2- propylaminocarbamate

[0179] To a solution of 14-[(tert-butyl)(oxycarbonylamino)]-3,6,9,12-tetraoxatetradecanoic acid (A2-1, 3.00 g, 8.60 mmol) and p-aminobenzyl alcohol (1.16 g, 9.40 mmol) in dichloromethane (60 mL) was added (2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.89 g, 10.2 mmol) and N,N- diisopropylethylamine (3.30 g, 25.6 mmol), and the resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give a crude product, which was purified by column chromatography (DCM / MeOH = 96 / 4) to give a gray solid (A2-2, 2.80 g, yield 71.9%). 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 7.65 - 7.47 (m, 2H), 7.31 - 7.18 (m, 2H), 6.75 (t, J = 5.8 Hz, 1H), 5.11 (t, J = 5.8 Hz, 1H), 4.44 (d, J = 5.6 Hz, 2H), 4.06 (s, 2H), 3.67 - 3.65 (m, 2H), 3.64 - 3.58 (m, 2H), 3.37 - 3.33 (m, 8H), 3.36 (t, J = 6.2 Hz, 2H), 3.05 (d, J = 6.0 Hz, 2H), 1.37 (s, 9H). MS-ESI [M+H] + : 457.3.

[0180] Step B: 13-[N-p-(chloromethyl)anilincarbonyl]-3,6,9,12-tetraoxatridecyl 2-methyl-2- propylaminocarbamate

[0181] To a solution of 13-[N-p-(hydroxymethyl)anilincarbonyl]-3,6,9,12-tetraoxatridecyl 2- methyl-2-propylaminocarbamate (A2-2, 400 mg, 0.877 mmol) in dichloromethane (15 mL) was added a solution of dichlorosulfoxide (240 mg, 2.00 mmol) in dichloromethane (2 mL), stirred at room temperature for 10 min, concentrated under reduced pressure to give the crude product as a light brown oil (A2-3, 410 mg). MS-ESI [M+Na] + : 497.3.

[0182] Step C: 13-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]anilincarbonyl}-3,6,9,12-tetraoxatridecyl 2-methyl-2- propylaminocarbamate

[0183] To a solution of capsaicin (216 mg, 0.700 mmol) and potassium carbonate (366 mg, 2.70 mmol) in acetonitrile (6 mL) was added a solution of 13-[N-p-(chloromethyl)anilincarbonyl]- 3,6,9,12-tetraoxatridecyl 2-methyl-2-propylaminocarbamate (A2-3, 410 mg, 0.863 mmol) in acetonitrile (2 mL), stirred at room temperature overnight. The reaction was filtered to remove the solid, the filtrate was concentrated to give the crude product, which was purified by column chromatography with DCM / MeOH = 90 / 10 to give a gray solid (A2-4, 610 mg, yield 95%). MS-ESI [M+Na] + : 766.4

[0184] Step D: N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 14-amino-3,6,9,12-tetraoxatetradecanoic amide

[0185] 13-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylamino carbonyl}-3,6,9,12-tetraoxatridecyl 2- methyl-2-propylcarbamic acid ester (A2-4, 460 mg, 0.621 mmol) and hexafluoroisopropanol (7 mL) were stirred in a 20 mL sealed tube at 110 °C overnight. Concentration under reduced pressure gave a crude product, which was slurried in a mixture of PE / EtOAc (2 / 1, 5 mL) and filtered to give a light gray solid (A2, 305 mg, yield 78.9%). 1 H NMR (400 MHz, Methanol-d4) δ 8.05-7.98 (m, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.40-7.30 (m, 2H), 7.20-7.17 (dd, J = 8.2, 2.0 Hz, 1H), 5.84-5.70 (m, 2H), 5.49 (s, 2H), 4.64 (s, 2H), 4.55 (s, 2H), 4.19-4.18 (m, 3H), 4.14-4.13 (m, 2H), 4.12-4.08 (m, 2H), 4.08-4.04 (m, 2H), 4.04-4.01 (m, 2H), 3.99-3.96 (m, 4H), 3.87 (t, J = 5.6 Hz, 2H), 3.12 (t, J = 5.6 Hz, 2H), 2.62-2.58 (t, J = 7.2 Hz, 3H), 2.36 (q, J = 6.8 Hz, 2H), 1.98-1.93 (m, 2H), 1.75-1.68 (m, 2H), 1.35 (d, J = 6.8 Hz, 6H). MS - ESI [M+H] + :644.4.

[0186] Example A3: Preparation of coupling agent A3

[0187] N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-hydrazinyl-6-carbonylhexanamide

[0188] Step A: methyl 5-[N-p-(hydroxymethyl)phenylaminocarbonyl]pentanoate

[0189] To a solution of methyl 5-carboxyvalerate (A3-1, 5.00 g, 31.3 mmol) and (p- aminophenyl)methanol (3.85 g, 31.3 mmol) in dichloromethane (50 mL) was added 1- ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (9.01 g, 47.0 mmol) and 1- hydroxybenzotriazole (6.35 g, 47.0 mmol), the resulting mixture was stirred at room temperature for 16 hours, after the reaction was completed, water (30 mL) was added, dichloromethane (30 mL) was extracted 3 times, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by column chromatography PE / EtOAc = 50 / 50 to give a light yellow solid (A3-2, 3.60 g, yield 43.5%). 1 H NMR (400 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.53-7.51 (m, 2H), 7.22-7.20 (m, 2H), 5.07 (t, J = 4.0 Hz, 1H), 4.41 (d, J = 4.0 Hz, 1H), 3.58 (s, 3H), 2.35-2.28 (m, 4H), 1.59-1.55 (m, 4H). MS-ESI [M+H] + : 266.1.

[0190] Step B: Methyl 5-[N-p-(hydroxymethyl)anilincarbonyl]valerate

[0191] To a solution of methyl 5-[N-p-(hydroxymethyl)anilincarbonyl]valerate (A3-2, 2.00 g, 7.54 mmol) in dichloromethane (50 mL) was added slowly dropwise dichlorosulfoxide (1.80 g, 15.1 mmol), the resulting mixture was stirred at room temperature for 2 hours, after the reaction was completed, it was concentrated under reduced pressure to give a light yellow crude product (A3-3, 2.20 g). MS-ESI [M+H] + : 284.1.

[0192] Step C: Methyl 5-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]anilincarbonyl}valerate

[0193] To a solution of methyl 5-[N-p-(chloromethyl)anilincarbonyl]valerate (A3-3, 2.10 g, 7.40 mmol) and capsaicin (2.30 g, 7.40 mmol) in acetonitrile (50 mL) was added potassium carbonate (3.06 g, 22.2 mmol), the resulting mixture was stirred at 75 °C for 16 hours, the reaction was cooled to room temperature, the solvent was removed under reduced pressure and poured into water (30 mL), a solid was precipitated, which was filtered to give a yellow solid (A3-4, 3.80 g, yield 92.3%). MS-ESI [M+H] + : 553.3.

[0194] Step D: 5-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]anilincarbonyl}valeric acid

[0195] To a solution of methyl 5-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}- 2-methoxyphenoxy)methyl]anilincarbonyl}valerate (A3-4, 3.80 g, 6.88 mmol) in tetrahydrofuran (90 mL) and water (36 mL) was added lithium hydroxide monohydrate (866 mg, 20.6 mmol), the resulting mixture was stirred at room temperature for 16 hours, the solvent was removed under reduced pressure, to the remaining aqueous solution was added dilute hydrochloric acid (1 M) to adjust to pH 5, a solid was precipitated, filtration gave a yellow solid (A3-5, 3.00 g, yield 81.1%). 1 H NMR (400 MHz, DMSO-d6) δ 10.01 (s, 1H), 8.23-8.20 (m, 1H), 7.62-7.52 (m, 2H), 7.37-7.24 (m, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.72-6.70 (m, 1H), 5.41-5.24 (m, 2H), 4.96 (s, 2H), 4.18 (s, 2H), 3.73 (s, 3H), 2.32-2.28 (m, 2H), 2.21-2.17 (m, 3H), 2.13-2.09 (m, 2H), 1.96-1.91 (m, 2H), 1.61-1.48 (m, 6H), 1.30-1.27 (m, 2H), 0.92 (d, J = 6.8 Hz, 6H). MS - ESI [M+H] + : 539.3.

[0196] Step E: N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-[(tert-butyl)-2-carboxyhydrazino]-6- carbonylhexanamide

[0197] To a solution of 5-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}valeric acid (A3-5, 1.00 g, 1.86 mmol) and tert- butylhydrazinecarboxylate (270 mg, 2.05 mmol) in N,N-dimethylformamide (50 mL) was added N,N-diisopropylethylamine (721 mg, 5.58 mmol) and 2-(7-azabenzotriazol- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (779 mg, 2.05 mmol), the resulting mixture was stirred at room temperature for 16 hours, the reaction was added to water (50 mL) and extracted with ethyl acetate (50 mL) three times, the organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by column chromatography PE / EtOAc = 40 / 60 to give a light yellow solid (A3-6, 1.10 g, yield 90.2%). MS-ESI [M+H] + : 653.4.

[0198] Step F: N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-hydrazino-6-oxohexanamide

[0199] To a solution of N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-[(tert-butyl)-2-carboxyhydrazino]-6-oxohexanamide (A3-6, 500 mg, 0.800 mmol) was added a solution of hexafluoroisopropanol (8 mL) and stirred at 120 °C for 2 hours in a microwave device, the reaction was cooled to room temperature and the solvent was removed under reduced pressure to give a crude product, which was purified by column chromatography DCM / MeOH = 90 / 10 to give a light yellow solid (A3, 380 mg, yield 89.7%). 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.94 (s, 1H), 8.21-8.19 (m, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.73-6.70 (m, 1H), 5.41-5.24 (m, 2H), 4.96 (s, 2H), 4.23-4.05 (m, 4H), 3.73 (s, 3H), 2.31-2.27 (m, 2H), 2.23-2.18 (m, 1H), 2.13-2.09 (m, 2H), 2.05-2.02 (m, 2H), 1.96-1.91 (m, 2H), 1.54-1.47 (m, 6H), 1.33-1.23 (m, 2H), 0.93 (d, J = 6.8 Hz, 6H). MS-ESI [M+H]+: 553.3.

[0200] Example A4: Preparation of coupling agent A4

[0201] N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]anilincarbonyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl}methyl)aminocarbonyl)methyl]-N-2- aminoethylpentanamide

[0202] Step A: N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]anilincarbonyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl}methyl)aminocarbonyl)methyl]5-(2,5- dicarbonyl-1-pyrrolidinyloxy)-5-oxopentanamide

[0203] To a solution of 4-{[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]anilincarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl]aminocarbonyl}butanoic acid (A5-10, 600 mg, 0.71 mmol) and N-hydroxysuccinimide (81.7 mg, 0.71 mmol) in N,N-dimethylformamide (6 mL) was added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (136 mg, 0.71 mmol) and the resulting mixture was stirred at room temperature for 16 hours. To the reaction was added methyl tert-butyl ether (60 mL) to precipitate a solid, which was filtered and the solid was slurried with methyl tert-butyl ether (20 mL) to give a brown solid (A4-1, 420 mg, yield 61.9%). MS-ESI [M+H] + : 940.4.

[0204] Step B: N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]anilincarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl]-N-2-aminoethylpentanamide

[0205] To a solution of N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]anilincarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl] 5-(2,5-dicarbonyl-l-pyrrolidinyloxy)-5-oxopentanamide (A4-1, 420 mg, 0.45 mmol) in N,N-dimethylformamide (5 mL) was added ethylenediamine (4.5 mL) and the resulting mixture was stirred in an ice water bath for 1 hour. The reaction was poured into water to precipitate a solid, which was filtered and the solid was lyophilized to give a brown solid (A4, 210 mg, yield 53.2%). 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.97 (s, 1H), 8.45 (t, J = 5.8 Hz, 1H), 8.26 - 8.11 (m, 6H), 7.64 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.20 - 7.18 (m, 6H), 6.94 (d, J = 8.2 Hz, 1H), 6.86 (s, 1H), 6.72 (dd, J = 8.2, 1.8 Hz, 1H), 6.17 (s, 1H), 6.05 (s, 1H), 5.43 - 5.25 (m, 2H), 4.97 (s, 2H), 4.53 - 4.48 (m, 1H), 4.17 (d, J = 5.8 Hz, 2H), 3.93 - 3.83 (m, 2H), 3.82 - 3.59 (m, 8H), 3.1 - 2.93 (m, 6H), 2.29 - 2.19 (m, 2H), 2.13 - 2.11 (m, 2H), 1.96 - 1.90 (m, 2H), 1.88 - 1.80 (m, 2H), 1.78 - 1.70 (m, 2H), 1.55 - 1.47 (m, 2H), 1.33 - 1.23 (m, 2H), 0.96 (d, J = 4 Hz, 6H). MS-ESI [M+H] + :885.4.

[0206] Example A5: Preparation of coupling agent A5

[0207] N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl]-5-hydrazinyl-5- oxopentanamide

[0208] Step A: Resorcinol potassium salt

[0209] To a solution of resorcinol (2.00 g, 6.55 mmol) in tetrahydrofuran (10 mL) was added aqueous potassium hydroxide (1 M, 6.55 mL, 6.55 mmol) and the resulting mixture was stirred at room temperature for 3 hours. Upon completion of the reaction, water (50 mL) was added to the reaction mixture and the tetrahydrofuran was removed under reduced pressure. The aqueous phase was lyophilized to give a white solid crude product (A5-1, 2.2 g). MS-ESI [M+H] + :306.4.

[0210] Step B: [(S)-2-({[({[(9H-fluoren-9-yl)methyl](oxo carbonyl amino)}methyl) carbonyl amino]methyl}carbonyl amino)-3-phenylpropanoyl amino] acetic acid

[0211] To a solution of tert-butyl [(S)-2-({[({[(9H-fluoren-9-yl)methyl](oxycarbonylamino)}methyl)carbonylamino]methyl}carbonylamino)-3- phenylpropanoylamino]acetate [(S)-2-({[({[[(9H-fluoren-9-yl)methyl](oxycarbonylamino)}methyl)carbonylamino]methyl}carbonylamino)-3- phenylpropanoyl]amino]acetic acid tert-butyl ester (A5-2, 2.00 g, 3.25 mmol) in dichloromethane (60 mL) was added trifluoroacetic acid (15 mL) and the resulting mixture was stirred at room temperature for 5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to give a crude product, which was filtered by slurry with methyl tert-butyl ether (50 mL) to give a white solid (A5-3, 1.50 g, yield 82.0%). MS-ESI [M+H] + : 559.2.

[0212] Step C: ({[N-(S)-1-({[N-p-(hydroxymethyl)phenylcarbamoyl]methyl}aminocarbonyl)-2- phenylethylcarbamoyl]methyl}aminocarbonyl)methyl (9H-fluoren-9-yl)methyl carbamate

[0213] To a solution of [(S)-2-({[({[(9H-fluoren-9-yl)methyl](oxycarbonylamino)}methyl)carbonylamino]methyl}carbonylamino)-3- phenylpropanoylamino]acetic acid (L5-3, 1.85 g, 3.31 mmol), p-aminobenzyl alcohol (407 mg, 3.31 mmol) and 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.39 g, 3.64 mmol) in N,N-dimethylformamide (15 mL) was added N,N- diisopropylethylamine (885 mg, 6.62 mmol) under ice-water bath, and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water (150 mL) and stirred to have solid precipitate, which was filtered and the solid was filtered by slurry with methyl tert-butyl ether (100 mL) to give a white solid (A5-4, 1.60 g, yield 72.7%). MS-ESI [M-H20+H] + : 646.3.

[0214] Step D: ({[N-(S)-1-({[N-p-(chloromethyl)phenylcarbamoyl]methyl}aminocarbonyl)-2- phenylethylcarbamoyl]methyl}aminocarbonyl)methyl (9H-fluoren-9-yl)methyl carbamate

[0215] To a solution of ({[N-(S)-1-({[N-p-(hydroxymethyl)phenylcarbamoyl]methyl} aminocarbonyl)-2-phenylethylcarbamoyl]methyl}aminoformyl)methyl (9H-fluoren-9- yl)methylcarbamate (A5-4, 1.50 g, 2.26 mmol) in tetrahydrofuran (15 mL) was added a solution of sulfurous dichloride in tetrahydrofuran (1 M, 4.52 mL, 4.52 mmol) and the resulting mixture was stirred at room temperature for 2 hours. Concentration under reduced pressure gave a yellow crude product (A5-5, 1.5 g). MS - ESI [M+H] + : 682.3.

[0216] Step E: ({[N-(S)-1-({[N-p-(iodomethyl)phenylcarbamoyl]methyl}aminocarbonyl)-2- phenylethylcarbamoyl]methyl}aminoformyl)methyl (9H-fluoren-9-yl)methylcarbamate

[0217] To a solution of ({[N-(S)-1-({[N-p-(chloromethyl)phenylcarbamoyl]methyl} aminocarbonyl)-2-phenylethylcarbamoyl]methyl}aminoformyl)methyl (9H-fluoren-9- yl)methylcarbamate (A5-5, 1.0 g, 5.50 mmol) in acetone (10 mL) was added sodium iodide (1.1 g, 7.5 mmol) and the resulting mixture was stirred at room temperature for 16 hours in the dark. The reaction was poured into water (100 mL) and a solid precipitated which was filtered and the solid was lyophilized to give a yellow crude product (A5-6, 1.0 g). MS - ESI [M+H] + : 774.2.

[0218] Step F: ({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylcarbamoyl]methyl}aminoformyl)methyl (9H-fluoren-9- yl)methylcarbamate

[0219] To a solution of ({[N-(S)-1-({[N-p-(iodomethyl)phenylcarbamoyl]methyl} aminocarbonyl)-2-phenylethylcarbamoyl]methyl}aminoformyl)methyl (9H-fluoren-9- yl)methylcarbamate (A5-6, 500 mg, 0.65 mmol) in N,N-dimethylformamide (5 mL) was added potassium capsaicin salt (222 mg, 0.65 mmol) and the resulting mixture was stirred at room temperature for 5 minutes. The reaction was poured into methyl tert-butyl ether (25 mL) and a solid precipitated which was filtered and the solid was slurried with methyl tert-butyl ether (15 mL) and filtered to give a yellow crude product (A5-7, 200 mg). MS - ESI [M+H] +:951.5.

[0220] Step G: N-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl}(S)-2-[(glycylamino- methyl)carbonylamino]-3-phenylpropanamide

[0221] To a solution of ({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}- 2-methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl]methyl}aminocarbonyl)methyl (9H-fluoren-9- yl)methylcarbamate (A5-7, 200 mg, 0.21 mmol) in dichloromethane (2 mL) was added diethylamine (2 mL), the resulting mixture was stirred at room temperature for 6 hours. Concentration under reduced pressure gave a crude product which was slurried with methyl tert-butyl ether (20 mL) and filtered to give a brown solid (A5-8, 140 mg, yield 91.5%). MS-ESI [M+H] + :729.4.

[0222] Step H: Methyl 4-{[({[N-(S)-1-[({N-p-[(4-{[[(E)-7-methyl-5-octenylcarbonylamino]methyl}- 2-methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl]methyl}aminocarbonyl)methyl]aminocarbonyl}butanoate

[0223] To a solution of N-{N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl}(S)-2-[(glycylamino- methyl)carbonylamino]-3-phenylpropanamide (A5-8, 150 mg, 0.21 mmol), 4- methoxycarbonylbutanoic acid (31.0 mg, 0.21 mmol), 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (48.0 mg, 0.25 mmol) and 1-hydroxybenzotriazole (34.0 mg, 0.25 mmol) in N,N-dimethylformamide (5 mL) was added N,N- diisopropylethylamine (54.0 mg, 0.42 mmol), the resulting mixture was stirred at room temperature for 2 hours. The reaction was poured into water (20 mL), a solid precipitated, which was filtered and the crude solid was purified by column chromatography with DCM / MeOH = 91 / 9 to give a brown solid (A5-9, 120 mg, yield 72.7%). MS-ESI [M+H] + :857.4.

[0224] Step I: 4-{[({[N-(S)-l-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylcarbamoyl}methyl)aminocarbonyl)methyl]aminocarbonyl}butanoic acid

[0225] To a solution of methyl 4-{[({[N-(S)-l-[({N-p-[(4-{[[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylcarbamoyl]methyl}aminocarbonyl)methyl]aminocarbonyl}butanoate (A5-9, 120 mg, 0.14 mmol), lithium hydroxide monohydrate (17.6 mg, 0.42 mmol) in tetrahydrofuran (3 mL) was added water (1 mL), the resulting mixture was stirred at room temperature for 2 hours. Concentrated under reduced pressure, the remaining aqueous solution was adjusted to pH 6 with dilute hydrochloric acid (0.1 M), a white solid precipitated, filtered, the solid was lyophilized to give off-white solid (A5-10, 110 mg, yield 94.0%). MS-ESI [M+H] + : 843.4.

[0226] Step J: N-[({[N-(S)-l-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylcarbamoyl]methyl}aminocarbonyl)methyl]-5-[(tert-butyl)-2- carboxyhydrazino]-5-oxopentanamide

[0227] To a solution of 4-{[({[N-(S)-l-[({N-p-[(4-{[[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]-2- phenylethylcarbamoyl]methyl}aminocarbonyl)methyl]aminocarbonyl}butanoic acid (A5-10, 300 mg, 0.36 mmol), tert-butyl hydrazinecarboxylate (47.0 mg, 0.36 mmol), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (142 mg, 0.37 mmol) in N,N-dimethylformamide (10 mL) was added N,N-diisopropylethylamine (92.0 mg, 0.71 mmol), the resulting mixture was stirred at room temperature for 2 hours. The reaction was poured into water (50 mL), a solid precipitated, filtered, the crude solid product was purified by column chromatography with DCM / MeOH = 91 / 9 to give yellow solid (A5-11, 170 mg, yield 49.9%). MS-ESI [M+H]+ 957.4.

[0228] Step K: N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl}methyl)aminocarbonyl)methyl]-5-hydrazinyl-5- oxovaleramide

[0229] A solution of N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2- phenylethylaminocarbonyl}methyl)aminocarbonyl)methyl]-5-[(tert-butyl)-2- carboxyhydrazinyl]-5-oxovaleramide (A5-11, 170 mg, 0.18 mmol) in hexafluoroisopropanol (5 mL) was microwaved at 100 °C for 2 h. The reaction was cooled to room temperature and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (DCM / MeOH = 85 / 15) to give a yellow solid (A5, 120 mg, yield 81.6%). 1 H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.93 (s, 1H), 8.41 (t, J = 5.8 Hz, 1H), 8.25 - 8.15 (m, 2H), 8.12 - 8.05 (m, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 4.4 Hz, 4H), 7.20 - 7.17 (m, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.75 - 6.69 (m, 1H), 5.40 - 5.27 (m, 2H), 4.97 (s, 2H), 4.56 - 4.48 (m, 1H), 4.17 (d, J = 5.8 Hz, 2H), 4.13 (s, 1H), 3.91 - 3.87 (m, 2H), 3.73 (s, 3H), 3.70 - 3.57 (m, 3H), 3.10 - 3.05 (m, 1H), 2.86 - 2.80 (m, 1H), 2.25 - 2.16 (m, 1H), 2.14 - 2.11 (m, 4H), 2.01 (t, J = 7.4 Hz, 2H), 1.99 - 1.91 (m, 2H), 1.75 - 1.69 (m, 2H), 1.53 - 1.47 (m, 2H), 1.34 - 1.24 (m, 2H), 0.93 (d, J = 6.6 Hz, 6H). MS - ESI [M+H] + 857.4.

[0230] Example A6: Preparation of Coupling Agent A6

[0231] N-[(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethyl] (S)-2-[({(S)-2-(S)-1- (14-amino-3,6,9,12-tetraoxatetradodecanoyl)-2-pyrrolidinylcarbonylamino]-4- methylpentanoyl}methyl)carbonylamino]-4-methylpentanamide

[0232] Step A: (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-tert-butoxycarbonyl-3- methylbutylaminocarbonyl]-1-pyrrolidinecarboxylate

[0233] To a solution of (S)-1-[(9H-fluoren-9-yl)methyl] -2-pyrrolidinecarboxylate (A6-1, 120 g, 0.356 mol) and tert-butyl (S)-2-amino-4-methylpentanoate hydrochloride (87.0 g, 0.392 mol) in N,N-dimethylformamide (1500 mL) was added N,N-diisopropylethylamine (140 g, 1.07 mol) and 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (148 g, 0.392 mmol), and the resulting mixture was stirred at 25 °C for 2 h. The reaction was poured into water (2000 mL), and a solid precipitated, which was filtered, and the solid was slurried with methyl tert-butyl ether and filtered to give a white solid (A6-2, 144 g, 79.1% yield). 1 H NMR (400 MHz, Methanol-d4) δ 7.72 (d, J = 8.0 Hz, 2H), 7.69 - 7.60 (m, 2H), 7.43 - 7.40 (m, 2H), 7.36 - 7.32 (m, 2H), 4.46 - 4.16 (m, 5H), 3.65 - 3.40 (m, 2H), 2.41 - 2.20 (m, 1H), 2.14 - 1.89 (m, 3H), 1.77 - 1.54 (m, 3H), 1.46 (d, J = 8.7 Hz, 9H), 0.98 - 0.92 (m, 3H), 0.83 - 0.75 (m, 3H). MS - ESI [M+H] + : 507.3.

[0234] Step B: (S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2-pyrrolidinylcarbonylamino}-4- methylpentanoic acid

[0235] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-tert-butoxycarbonyl-3- methylbutylcarbamoyl]-1-pyrrolidinecarboxylate (A6-2, 8.00 g, 14.2 mmol) in dichloromethane (40 mL) was added trifluoroacetic acid (10 mL). The resulting mixture was stirred at 25 °C for 8 h. Concentration under reduced pressure gave a crude product which was filtered by slurry with methyl tert-butyl ether (50 mL) to give a white solid (A6-3, 4.00 g, yield 62.5%). MS-ESI [M+H] + : 451.5.

[0236] Step C: (9H-fluoren-9-yl)methyl (S)-2-{N-(S)-1-[(tert-butoxycarbonylmethyl)carbamoyl]-3- methylbutylcarbamoyl}-1-pyrrolidinecarboxylate

[0237] To a solution of (S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2-pyrrolidinylcarbonylamino}-4- methylpentanoic acid (A6-3, 10 g, 22.20 mmol) in N,N-dimethylformamide (10 mL) and dichloromethane (20 mL) was added tert-butyl aminoacetate hydrochloride (4.09 g, 24.42 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.19 g, 22.20 mmol) and 2,4,6-trimethylpyridine (8.07 g, 66.59 mmol). The resulting mixture was stirred at 25 °C for 3 h. The reaction was diluted with 1 M dilute hydrochloric acid (20 mL), extracted with ethyl acetate (50 mL) three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography PE / EtOAc = 50 / 50 to give a white solid (A6-4, 6.2 g, yield 49.5%). 1H NMR (400 MHz, CDC13) δ 7.78 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.46 - 7.36 (m, 2H), 7.35 - 7.29 (m, 2H), 6.89 (s, 1H), 6.82 (d, J = 8.4 Hz, 1H), 4.56 - 4.31 (m, 4H), 4.31 - 4.17 (m, 1H), 4.01 (dd, J = 18.0, 5.6 Hz, 1H), 3.80 (dd, J = 18.0, 4.8 Hz, 1H), 3.62 - 3.42 (m, 2H), 2.28 - 2.18 (m, 1H), 2.14 - 2.06 (m, 1H), 2.02 - 1.88 (m, 2H), 1.87 - 1.75 (m, 1H), 1.64 - 1.50 (m, 2H), 1.47 - 1.31 (m, 9H), 0.89 (dd, J = 6.4, 3.6 Hz, 6H). MS - ESI [M+Na] + : 586.2.

[0238] Step D: [(S)-2-{(S)-l-[(9H-Fluoren-9-yl)methoxycarbonyl]-2-pyrrolidinylcarbonylamino}-4- methylpentanoylamino]acetic acid

[0239] To a solution of (9H-fluoren-9-yl)methyl (S)-2-{N-(S)-l-[(tert-butoxycarbonylmethyl)aminocarbonyl]-3-methylbutylaminocarbonyl}-l-pyrrolidinecarboxylate (A6-4, 5.7 g, 10.11 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL). The resulting mixture was stirred at 25 °C for 1 h. Concentration under reduced pressure gave the crude product (A6-5, 9 g) as a yellow oil. MS - ESI [M+H] + : 508.2.

[0240] Step E: (9H-Fluoren-9-yl)methyl (S)-2-[N-(S)-l-({[N-(S)-l-tert-butoxycarbonyl-3- methylbutylaminocarbonyl]methyl}aminocarbonyl)-3-methylbutylaminocarbonyl]-l- pyrrolidinecarboxylate

[0241] To a solution of [(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoylamino]acetic acid (A6-5, 3 g, 5.91 mmol) in N,N-dimethylformamide (3 mL) and dichloromethane (6 mL) was added tert-butyl 2-amino-4-methylpentanoate hydrochloride (1.45 g, 6.50 mmol), 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.25 g, 5.91 mmol) and 2,4,6-trimethylpyridine (1.43 g, 11.82 mmol). The resulting mixture was stirred at 25 °C for 1 h. The reaction was added water (20 mL), extracted with ethyl acetate (50 mL) for 3 times, the organic phase was combined, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by column chromatography (PE / EtOAc = 35 / 65) to give a white solid (A6-6, 3.26 g, yield 81.5%). 1 H NMR (400 MHz, CDC13) δ 7.78 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6, 1.3 Hz, 2H), 7.03 - 6.87 (m, 1H), 6.82 - 6.66 (m, 1H), 4.59 - 4.36 (m, 3H), 4.36 - 4.21 (m, 2H), 4.09 - 3.80 (m, 1H), 3.60 - 3.38 (m, 2H), 2.94 - 2.87 (m, 3H), 2.47 - 2.18 (m, 3H), 2.05 (s, 1H), 2.01 - 1.90 (m, 1H), 1.84 - 1.53 (m, 5H), 1.45 (s, 9H), 0.96 - 0.85 (m, 12H). MS-ESI [M+Na] + : 699.4.

[0242] Step F: (S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoylamino]methyl}carbonylamino)-4- methylpentanoic acid

[0243] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-tert- butyloxycarbonyl-3-methylbutylaminocarbonyl]methyl}aminocarbonyl)-3- methylbutylaminocarbonyl]-1-pyrrolidinecarboxylate (A6-6, 3 g, 4.43 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL). The resulting mixture was stirred at 25 °C for 1 h. Concentration under reduced pressure gave the crude product (A6-7, 5 g) as a yellow oil. MS-ESI [M+H]+ :621.3.

[0244] Step G: (9H-Fluoren-9-yl)methyl (S)-2-[N-(S)-1-[({N-(S)-1-[N-(S)-1-tert- butyloxycarbonyl ethyl aminocarbonyl]-3-methylbutyl aminocarbonyl}methyl) aminocarbonyl]-3-methylbutyl aminocarbonyl]-1-pyrrolidinecarboxylate

[0245] To a solution of (S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoyl amino]methyl}carbonylamino)-4- methylpentanoic acid (A6-7, 5 g, 4.72 mmol) in N,N-dimethylformamide (5 mL) and dichloromethane (10 mL) was added tert-butyl 2-aminopropanoate hydrochloride (943 mg, 5.19 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.79 g, 4.72 mmol) and 2,4,6-trimethylpyridine (1.72 g, 14.2 mmol). The resulting mixture was stirred at 25 °C for 1 h. The reaction was diluted with 1 M hydrochloric acid (20 mL) and extracted with ethyl acetate (20 mL) three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography PE / EtOAc = 20 / 80 to give a white solid (A6-8, 2 g, 56.7% yield). 1 H NMR (400 MHz, Methanol-d4) δ 7.80 (d, J = 7.6 Hz, 2H), 7.69 - 7.54 (m, 2H), 7.44 - 7.36 (m, 2H), 7.35 - 7.27 (m, 2H), 4.47 - 4.29 (m, 4H), 4.28 - 4.21 (m, 2H), 4.20 - 4.12 (m, 1H), 4.01 - 3.63 (m, 2H), 3.63 - 3.53 (m, 1H), 3.53 - 3.41 (m, 1H), 2.39 - 2.10 (m, 1H), 2.03 - 1.88 (m, 3H), 1.75 - 1.49 (m, 6H), 1.48 - 1.36 (m, 9H), 1.36 - 1.25 (m, 3H), 1.00 - 0.83 (m, 10H), 0.83 - 0.69 (m, 2H). MS-ESI [M+H] + :748.3.

[0246] Step H: (S)-2-[(S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoyl amino]methyl}carbonylamino)-4- methylpentanoyl amino]propanoic acid

[0247] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-[({N-(S)-1-[N-(S)-1- tert-butoxycarbonyl ethylcarbamoyl]-3-methylbutylcarbamoyl}methyl)aminocarbonyl]- 3-methylbutylcarbamoyl]-1-pyrrolidinecarboxylate (A6-8, 2 g, 2.67 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (10 mL). The resulting mixture was stirred at 25 °C for 1 h. Concentration under reduced pressure gave the crude product (A6-9, 4.1 g) as a yellow oil. MS-ESI [M+H] + : 692.3.

[0248] Step I: (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-{N-(S)-1-[(tert- butoxycarbonylmethyl)aminocarbonyl]ethylcarbamoyl}-3-methylbutylcarbamoyl]methyl} aminocarbonyl)-3-methylbutylcarbamoyl]-1-pyrrolidinecarboxylate

[0249] To a solution of (S)-2-[(S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoylamino]methyl}carbonylamino)-4- methylpentanoylamino]propanoic acid (A6-9, 4.1 g, 2.67 mmol) in N,N- dimethylformamide (5 mL) and dichloromethane (10 mL) was added tert-butyl aminoacetate hydrochloride (492 mg, 2.93 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (1.01 mg, 2.67 mmol) and 2,4,6- trimethylpyridine (970 mg, 8.00 mmol). The resulting mixture was stirred at 25 °C for 12 h. Solid was precipitated and filtered to give white solid (A6-10, 1.4 g, yield 65.1%). 1H NMR (400 MHz, DMSO-d6) δ 7.93 - 7.86 (m, 2H), 7.71 - 7.61 (m, 2H), 7.46 - 7.38 (m, 2H), 7.37 - 7.29 (m, 2H), 4.52 - 4.31 (m, 2H), 4.30 - 4.16 (m, 4H), 4.12 - 4.02 (m, 1H), 3.79 - 3.59 (m, 4H), 3.50 - 3.41 (m, 1H), 3.41 - 3.34 (m, 1H), 2.14 - 2.03 (m, 1H), 2.01 - 1.90 (m, 1H), 1.87 - 1.78 (m, 2H), 1.68 - 1.41 (m, 6H), 1.39 (s, 9H), 1.22 (d, J = 7.2 Hz, 3H), 0.91 - 0.75 (m, 9H), 0.72 - 0.60 (m, 3H). MS-ESI [M+H] + : 805.3.

[0250] Step J: [(S)-2-[(S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9-yl)methoxycarbonyl]-2- pyrrolidinylcarbonylamino}-4-methylpentanoylamino]methyl}carbonylamino)-4- methylpentanoylamino]propionylamino]acetic acid

[0251] (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-[(tert-butoxycarbonylmethyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl]methyl} aminocarbonyl)-3-methylbutylaminocarbonyl]-1-pyrrolidinecarboxylate (A6-10, 500 mg, 0.621 mmol) was added to trifluoroacetic acid (5 mL) and stirred at 25 °C for 10 minutes. The trifluoroacetic acid was removed by concentration under reduced pressure, and methyl tert-butyl ether (20 mL) was added. A solid precipitated, which was filtered to give the crude product as a white solid (A6-11, 500 mg). MS-ESI [M+H] + : 749.3.

[0252] Step K: (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-({[N-p- (hydroxymethyl)phenylaminocarbonyl]methyl}aminocarbonyl)ethylaminocarbonyl]-3- methylbutylaminocarbonyl]methyl}aminocarbonyl)-3-methylbutylaminocarbonyl]-1- pyrrolidinecarboxylate

[0253] To a solution of [(S)-2-[(S)-2-({[(S)-2-{(S)-1-[(9H-fluoren-9- yl)methoxycarbonyl]-2-pyrrolidinylcarbonylamino}-4-methylpentanoyl- amino]methyl}carbonylamino)-4-methylpentanoyl-amino]propionyl-amino]acetic acid (A6-11, 450 mg, 0.60 mmol) in N,N-dimethylformamide (9 mL) was added (p- aminophenyl)methanol (81 mg, 0.66 mmol), 2-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (229 mg, 0.60 mmol) and N,N- diisopropylethylamine (233 mg, 1.80 mmol). The resulting mixture was stirred at 25 °C for 1 h. Water (10 mL) was added to the reaction, and a solid precipitated, which was filtered to give the crude product as a white solid (A6-12, 500 mg). 1 H NMR (400 MHz, Methanol-d4) δ 7.80 (d, J = 7.6 Hz, 2H), 7.68 - 7.55 (m, 4H), 7.40 (t, J = 7.6 Hz, 2H), 7.35 - 7.25 (m, 4H), 4.55 (s, 2H), 4.47 - 4.19 (m, 7H), 4.08 - 3.67 (m, 4H), 3.61 - 3.37 (m, 2H), 2.38 - 2.17 (m, 1H), 2.13 - 1.83 (m, 3H), 1.73 - 1.50 (m, 6H), 1.41 (d, J = 7.2 Hz, 3H), 0.94 - 0.72 (m, 12H). MS - ESI [M-17] + : 836.3.

[0254] Step L: (9H-Fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-({[N-p- (chloromethyl)phenylcarbamoyl]methyl}carbamoyl)ethylcarbamoyl]-3- methylbutylcarbamoyl]methyl}carbamoyl)-3-methylbutylcarbamoyl]-1- pyrrolidinecarboxylate

[0255] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-({[N-p- (hydroxymethyl)phenylcarbamoyl]methyl}carbamoyl)ethylcarbamoyl]-3- methylbutylcarbamoyl}methyl)aminocarbonyl]-1-pyrrolidinecarboxylate (A6-12, 500 mg, 0.585 mmol) in tetrahydrofuran (5 mL) was added dichlorosulfoxide (1.76 mL). The resulting mixture was stirred at 25 °C for 1 h. Concentration under reduced pressure gave a crude product, which was purified by column chromatography DCM / THF = 65 / 35 to give a yellow solid (A6-13, 400 mg, yield 78.4%). MS-ESI [M+Na] + : 894.3.

[0256] Step M: (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-[({N-p-[(4-{[(E)-7- methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl- carbamoyl}methyl)aminocarbonyl]ethylcarbamoyl]-3-methylbutylcarbamoyl}methyl) aminocarbonyl]-1-pyrrolidinecarboxylate

[0257] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-({[N-p- (chloromethyl)phenylcarbamoyl]methyl}carbamoyl)ethylcarbamoyl]-3-methylbutyl- carbamoyl}methyl)aminocarbonyl]-1-pyrrolidinecarboxylate (A6-13, 100 mg, 0.115 mmol) in N,N-dimethylformamide (10 mL) was added a solution of potassium 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxide (39 mg, 0.115 mmol) in N,N-dimethylformamide (10 mL). The resulting mixture was stirred at 35 °C for 1 min. To the reaction was added acetic acid (0.5 mL). The reaction was poured into water (100 mL) and extracted with ethyl acetate (50 mL) three times. The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography DCM / THF = 65 / 35 to give a yellow solid (A6-14, 100 mg, yield 76.2%). MS-ESI [M+H] + : 1141.5.

[0258] Step N: N-[(S)-l-({[N-(S)-l-[N-(S)-l-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl]methyl}carbamoyl)-3-methylbutyl]-(S)-2-pyrrolidinecarboxamide

[0259] To a solution of (9H-fluoren-9-yl)methyl (S)-2-[N-(S)-l-({[N-(S)-l-[N-(S)-l- [({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl] phenylcarbamoyl}methyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl]methyl}carbamoyl)-3-methylbutylaminocarbonyl]-l-pyrrolidinecarboxylate] (A6-14, 330 mg, 0.173 mmol) in dichloromethane (8 mL) was added diethylamine (0.8 mL). The resulting mixture was stirred at 25 °C for 2 h. Concentration under reduced pressure gave the crude product as a yellow solid (A6-15, 230 mg). MS-ESI [M+H] + : 919.5.

[0260] Step O: 14-[(S)-2-[N-(S)-l-({[N-(S)-l-[N-(S)-l-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylcarbamoyl}methyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl]methyl}carbamoyl)-3-methylbutylaminocarbonyl]-l-pyrrolidinyl]-14-oxo-3,6,9,12-tetraoxatetradecane-2-methyl-2-propanaminecarboxylate

[0261] To a solution of N-[(S)-1-({[N-(S)-1-[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5- octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl}methyl)aminocarbonyl)-3-methylbutyl]-(S)-2-pyrrolidinecarboxamide (A6-15, 230 mg, 0.25 mmol) in dichloromethane (6 mL) was added tert-butoxycarbonyl tetrapolyethylene glycol acetic acid (88 mg, 0.25 mmol), 2-(7-azobenzo-triazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (95 mg, 0.25 mmol) and N,N- dimethylformamide solution of N,N-diisopropylethylamine (32 mg, 0.25 mmol) (0.5 mL). The resulting mixture was stirred at 25 °C for 1 h. Concentration under reduced pressure gave a crude product, which was purified by reverse column chromatography MeOH / H2O = 50 / 50 to give a white solid (A6-16, 60 mg, yield 19.2%). 1 H NMR (400 MHz, Methanol-d4) δ 7.71-7.64 (m, 2H), 7.42-7.36 (m, 2H), 6.91 (dd, J = 5.2, 3.2 Hz, 2H), 6.77 (dd, J = 8.4, 2.0 Hz, 1H), 5.39-5.32 (m, 2H), 5.04 (s, 2H), 4.42-4.17 (m, 8H), 4.06-3.85 (m, 4H), 3.83 (s, 3H), 3.71-3.50 (m, 14H), 3.50-3.45 (m, 2H), 3.21-3.19 (m, 2H), 2.28-2.16 (m, 4H), 2.07-1.89 (m, 5H), 1.76-1.56 (m, 9H), 1.45 (s, 1H), 1.45-1.42 (m, 9H), 1.31-1.28 (m, 3H), 1.19 (s, 4H), 0.98-0.87 (m, 18H). MS-ESI [M+H-Boc] + : 1152.6.

[0262] Step P: N-[(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethyl] (S)-2-[({(S)-2-[(S)-1-(14-amino-3,6,9,12-tetraoxatetradecanoyl)-2-pyrrolidinylcarbonyl]- 4-methylpentanoylamino}methyl)carbonylamino]-4-methylpentanamide

[0263] 14-[(S)-2-[N-(S)-1-({[N-(S)-1-[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethylaminocarbonyl]-3-methylbutylaminocarbonyl]methyl}aminocarbonyl)-3-methylbutylaminocarbonyl]-1-pyrrolidinyl]-14-oxo-3,6,9,12-tetraoxatetradecane-2-methyl-2-propanamine carbamate (A6-16, 200 mg, 0.160 mmol) in hexafluoroisopropanol (10 mL) was stirred at 120 °C under microwave for 5 h. After the reaction was cooled to room temperature, the crude product was concentrated under reduced pressure, and purified by reverse phase column chromatography with MeOH / H2O = 65 / 35 (FA, 0.1%) to give a white solid (A6, 100 mg, yield 54.2%). 1 H NMR (400 MHz, DMSO-d6) δ 9.88-9.76 (m, 1H), 8.39 (s, 1H), 8.35-7.88 (m, 5H), 7.64 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H), 6.71 (dd, J = 8.0, 2.0 Hz, 1H), 5.41-5.26 (m, 2H), 4.97 (s, 2H), 4.44-4.20 (m, 4H), 4.17 (d, J = 6.0 Hz, 2H), 4.15-4.10 (m, 1H), 3.86 (d, J = 6.0 Hz, 2H), 3.73 (s, 4H), 3.69 (d, J = 6.0 Hz, 2H), 2.87-2.78 (m, 2H), 2.26-2.15 (m, 1H), 2.11 (t, J = 7.2 Hz, 2H), 2.05-1.77 (m, 5H), 1.76-1.39 (m, 9H), 1.36-1.19 (m, 9H), 0.92 (d, J = 6.8 Hz, 6H), 0.89-0.85 (m, 6H), 0.84-0.80 (m, 6H). MS - ESI [M+H] + : 1152.7.

[0264] Example A7: Preparation of coupling agent A7

[0265] 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl bromoacetate

[0266] To a solution of capsaicin (800 mg, 2.60 mmol) in dichloromethane (15 mL) was added bromoacetyl bromide (1.06 g, 5.20 mmol) and N,N-diisopropylethylamine (1.35 g, 10.5 mmol) was added dropwise at ice water. The reaction was stirred at room temperature for 2 hours. Water (20 mL) was added to the reaction and extracted with dichloromethane (20 mL) three times. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product which was purified by column chromatography (DCM / EtOAc = 5 / 95) to give a white solid (A7, 240 mg, yield 21.5%). 1 H NMR (400 MHz, DMSO-d6) δ 8.32 (t, J = 6.0 Hz, 1H), 7.08-6.99 (m, 2H), 6.84-6.82 (m, 1H), 5.40-5.28 (m, 2H), 4.44 (s, 2H), 4.25 (d, J = 5.8 Hz, 2H), 3.75 (s, 3H), 2.25-2.18 (m, 1H), 2.14 (t, J = 7.2 Hz, 2H), 1.97-1.92 (m, 2H), 1.56-1.49 (m, 2H), 1.36-1.27 (m, 2H), 0.93 (d, J = 6.8 Hz, 6H). MS - ESI [M+H] + : 426.1.

[0267] Example A8: Preparation of coupling agent A8

[0268] 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl aminoacetate hydrochloride

[0269] Step A: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl [(tert- butyl)(oxycarbonylamino)]acetate

[0270] To a solution of capsaicin (1.00 g, 3.28 mmol) and [(tert-butyl)(oxycarbonylamino)] acetic acid (631 mg, 3.61 mmol) in dichloromethane (30 mL) was added N,N'- dicyclohexylcarbodiimide (812 mg, 3.94 mmol) and 4-dimethylaminopyridine (50.0 mg, 0.41 mmol) and the resulting mixture was stirred at room temperature for 16 hours. The reaction was filtered and the filtrate was concentrated to give the crude product which was purified by column chromatography (PE / EtOAc = 50 / 50) to give a white solid (A8-1, 1.00 g, yield 66.7%). 1H NMR (400 MHz, DMSO-d6) δ 8.33-8.30 (m, 1H), 7.39-7.36 (m, 1H), 7.00-6.98 (m, 2H), 6.83-6.80 (m, 1H), 5.40-5.29 (m, 2H), 4.24 (d, J = 4.0 Hz, 2H), 3.94 (d, J = 8.0 Hz, 2H), 3.73 (s, 3H), 2.23-2.20 (m, 1H), 2.18-2.12 (m, 2H), 1.97-1.92 (m, 2H), 1.54-1.48 (m, 2H), 1.38 (s, 9H), 1.34-1.23 (m, 2H), 0.93 (d, J = 4.0 Hz, 6H). MS-ESI [M+H] + : 463.6.

[0271] Step B: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl [(tert- butyl)(oxycarbonylamino)]acetate hydrochloride

[0272] To a solution of 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenyl [(tert-butyl)(oxycarbonylamino)]acetate (A8-1, 500 mg, 1.08 mmol) in dichloromethane (10 mL) was added slowly dropwise a hydrochloric acid dioxane solution (4 M, 2.70 mL, 10.8 mmol), and the resulting mixture was stirred at room temperature for 2 hours. Concentration under reduced pressure gave a crude product which was slurried with dichloromethane (10 mL) and filtered to give a white solid (A8, 390 mg, yield 90.7%). 1 H NMR (400 MHz, DMSO-d6) δ 8.33-8.30 (m, 1H), 7.39-7.36 (m, 1H), 7.00-6.98 (m, 2H), 6.83-6.80 (m, 1H), 5.40-5.29 (m, 2H), 4.24 (d, J = 4.0 Hz, 2H), 3.94 (d, J = 8.0 Hz, 2H), 3.73 (s, 3H), 2.23-2.20 (m, 1H), 2.18-2.12 (m, 2H), 1.97-1.92 (m, 2H), 1.54-1.48 (m, 2H), 1.38 (s, 9H), 1.34-1.23 (m, 2H), 0.93 (d, J = 4.0 Hz, 6H). MS-ESI [M+H] + : 363.1.

[0273] Example A9: Preparation of Coupling Agent A9

[0274] 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-amino-3- phenylpropanoate hydrochloride

[0275] Step A: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-[(tert-butyl)(oxo carbonylamino)]-3-phenylpropanoate

[0276] To a solution of capsaicin (1.05 g, 3.40 mmol), (S)-2-[(tert-butyl)(oxo carbonylamino)]-3-phenylpropanoic acid (1.00 g, 3.70 mmol) and 4-dimethylaminopyridine (51.0 mg, 0.41 mmol) in dichloromethane (10 mL) was added N,N'-dicyclohexylcarbodiimide (853 mg, 4.18 mmol) and the resulting mixture was stirred at room temperature for 3 hours. The reaction was filtered and the filtrate was concentrated under reduced pressure to give a crude product which was purified by column chromatography PE / EtOAc = 50 / 50 to give a white solid (A9-1, 1.80 g, yield 83.7%). MS-ESI [M+Na] 575.3. + :575.3.

[0277] Step B: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-amino-3-phenylpropanoate hydrochloride

[0278] To a solution of 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-[(tert-butyl)(oxo carbonylamino)]-3-phenylpropanoate (A9-1, 1.80 g, 3.30 mmol) in dichloromethane (15 mL) was added hydrochloric acid dioxane solution (4 M, 5 mL, 20 mmol) and the resulting mixture was stirred at room temperature for 5 hours. Concentrated under reduced pressure and the crude product was filtered by slurry with PE / EtOAc = 1 / 1 (30 mL) to give a white solid (A9, 1.50 g, yield 83.3%). 1 H NMR (400 MHz, DMSO-d6) δ 8.86 (s, 3H), 8.40 (t, J = 6.0 Hz, 1H), 7.45 - 7.35 (m, 4H), 7.32 - 7.28 (m, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.83 (dd, J = 8.2, 1.8 Hz, 1H), 5.44 - 5.25 (m, 2H), 4.61 - 4.51 (m, 1H), 4.25 (d, J = 5.8 Hz, 2H), 3.76 (s, 3H), 3.34 - 3.27 (m, 2H), 2.24 - 2.13 (m, 3H), 2.00 - 1.89 (m, 2H), 1.55 - 1.47 (m, 2H), 1.37 - 1.24 (m, 2H), 0.93 (d, J = 6.7 Hz, 6H). MS-ESI [M+H]+: 453.3.

[0279] Example A10: Preparation of Coupling Agent A10

[0280] p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl 3-(N-2- aminoethylcarbamoyl)-1 -propylaminoformate

[0281] Step A: p-{[(tert-butyl)bis(methyl)silyloxy]methyl}aniline

[0282] To a solution of p-aminobenzyl alcohol (A10-1, 2.00 g, 16.2 mmol) and imidazole (1.10 g, 16.2 mmol) in dichloromethane (100 mL) was added p-aminobenzyl alcohol (2.45 g, 16.2 mmol) and the resulting mixture was stirred at room temperature for 16 hours. To the reaction was added water (50 mL) and the mixture was extracted with dichloromethane (60 mL) three times. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product which was purified by column chromatography PE / EtOAc = 75 / 25 to give a light yellow liquid (A10-2, 3.20 g, yield 83.0%). MS-ESI [M+H] + : 238.1.

[0283] Step B: p-{[(tert-butyl)bis(methyl)silyloxy]methyl}phenyl p-nitrophenylcarbamate

[0284] To a solution of p-{[(tert-butyl)bis(methyl)silyloxy]methyl}aniline (A10-2, 1.22 g, 5.14 mmol) and pyridine (0.83 mL, 10.3 mmol) in dichloromethane (50 mL) was added p-nitrophenyl chloroformate (1.55 g, 7.71 mmol) slowly under ice-water bath condition and the resulting mixture was stirred at room temperature for 2 hours. The crude product was concentrated under reduced pressure and purified by column chromatography PE / EtOAc = 80 / 20 to give a white solid (A10-3, 1.50 g, yield 73.0%). MS-ESI [M+Na] + : 425.0.

[0285] Step C: 4-[(p-{[(tert-butyl)bis(methyl)silyloxy]methyl}phenyl)(aminocarbonyloxy)]butyric acid methyl ester

[0286] To a solution of p-{[(tert-butyl)bis(methyl)silyloxy]methyl}phenyl p-nitrophenylcarbamate (A10-3, 2.45 g, 6.10 mmol) and methyl 4-hydroxybutanoate (0.83 mL, 6.10 mmol) in dichloromethane (5 mL) was added 4-dimethylaminopyridine (0.72 g, 1.11 mmol) under ice-water bath, the resulting mixture was stirred at room temperature for 3 hours. Concentration under reduced pressure gave the crude product, which was purified by column chromatography PE / EtOAc = 80 / 20 to give white solid (A10-4, 1.90 g, yield 82.0%). MS-ESI [M+Na] + : 404.1.

[0287] Step D: 4-{[p-(hydroxymethyl)phenyl](aminocarbonyloxy)}butanoic acid methyl ester

[0288] To a solution of 4-[(p-{[(tert-butyl)bis(methyl)silyloxy]methyl}phenyl)(aminocarbonyloxy)]butanoic acid methyl ester (A10-4, 400 mg, 1.05 mmol) in tetrahydrofuran (4 mL) was added tetrabutylammonium fluoride in tetrahydrofuran (1 M, 4 mL, 4 mmol), the resulting mixture was stirred at room temperature for 1 hour. Concentration under reduced pressure gave the crude product, which was purified by column chromatography PE / EtOAc = 50 / 50 to give white solid (A10-5, 270 mg, yield 70.0%). MS-ESI [M+H] + : 268.0.

[0289] Step E: 4-{[p-(chloromethyl)phenyl](aminocarbonyloxy)}butanoic acid methyl ester

[0290] To a solution of 4-{[p-(hydroxymethyl)phenyl](aminocarbonyloxy)}butanoic acid methyl ester (A10-5, 1.5 g, 5.62 mmol) in tetrahydrofuran (4 mL) was added dichlorosulfoxide in tetrahydrofuran (1 M, 11.24 mL, 11.24 mmol), the resulting mixture was stirred at room temperature for 1 hour. Concentration under reduced pressure gave the crude product (A10-6, 1.50 g). MS-ESI [M+H] + : 286.1.

[0291] Step F: 4-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl}(aminocarbonyloxy))butanoic acid methyl ester

[0292] To a solution of methyl 4-{[p-(chloromethyl)phenyl](aminooxy)}butanoate (A10-6, 1.20 g, 4.20 mmol) and capsaicin (1.30 g, 4.20 mmol) in acetonitrile (12 mL) was added potassium carbonate (1.20 g, 8.40 mmol) and the resulting mixture was stirred at 70 °C for 2 h. The reaction was cooled to room temperature, water (50 mL) was added and the mixture was extracted with ethyl acetate (50 mL) three times. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product which was purified by column chromatography PE / EtOAc = 50 / 50 to give a white solid (A10-7, 1.70 g, 82.0% yield). MS-ESI [M+H] + : 555.3.

[0293] Step G: 4-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}(aminooxy))butanoic acid

[0294] To a solution of methyl 4-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}(aminooxy))butanoate (A10-7, 1.86 g, 3.35 mmol) and water (7 mL) in tetrahydrofuran (21 mL) was added lithium hydroxide monohydrate (422 mg, 10.1 mmol) and the resulting mixture was stirred at room temperature for 2 h. It was concentrated under reduced pressure and the remaining aqueous solution was adjusted to pH 5 with dilute hydrochloric acid (1 M). A solid precipitated and was filtered to give a white solid (A10-8, 1.70 g, 93.9% yield). MS-ESI [M+H] + : 541.3.

[0295] Step H: p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl-4-(2,5-dioxo-1-pyrrolidinooxy)-4-oxo-1- butanaminocarbodithioate

[0296] To a solution of 4-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}(aminooxy))butanoic acid (A10-8, 100 mg, 0.18 mmol) and N-hydroxysuccinimide (21 mg, 0.18 mmol) in tetrahydrofuran (5 mL) was added N,N'-dicyclohexylcarbodiimide (74.0 mg, 0.36 mmol) and the resulting mixture was stirred at room temperature for 24 h. It was concentrated under reduced pressure to give a crude product which was purified by column chromatography PE / EtOAc = 20 / 80 to give a white solid (A10-9, 100 mg, 87.0% yield). MS-ESI [M+H]+ 638.3.

[0297] Step I: p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl 3-(N-2-aminoethylcarbamoyl)-1- propylaminoformate

[0298] To a solution of ethylenediamine (6 mL) in tetrahydrofuran (12 mL) was added p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl-4-(2,5-dioxo-l-pyrrolidinyloxy)-4- oxo-l-butanaminocarboxylate (A10-9, 677 mg, 1.1 mmol) slowly and the resulting mixture was stirred in an ice water bath for 1 h. A solid precipitated and was filtered, slurried with a mixture of methyl tert-butyl ether and water (5 / 1, 12 mL), and filtered to give the crude yellow product (A10, 390 mg). 1 HNMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.20 (t, J = 5.8 Hz, 1H), 7.83 (t, J = 5.6 Hz, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.2 Hz, 1H), 6.85 (s, 1H), 6.71 (dd, J = 8.2, 2.0 Hz, 1H), 5.43-5.23 (m, 2H), 4.95 (s, 2H), 4.17 (d, J = 5.8 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 3.00-3.02 (m, 2H), 2.55 (t, J = 6.4 Hz, 2H), 2.28-2.06 (m, 5H), 1.96-1.91 (m, 2H), 1.88-1.81 (m, 2H), 1.77-1.67 (m, 1H), 1.64-1.59 (m, 1H), 1.54-1.47 (m, 2H), 1.33-1.27 (m, 2H), 0.93 (d, J = 6.7 Hz, 6H). MS - ESI [M+H] + 583.3.

[0299] Example A11: Preparation of Coupling Agent A11

[0300] p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl 14-hydrazinyl-14-oxo-3,6,9,12- tetraoxa-1-tetradecanaminocarboxylate

[0301] Step A: Methyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate

[0302] To a solution of 14-hydroxy-3,6,9,12-tetraoxatetradecanoic acid (Al l-1, 1.90 g, 7.54 mmol) in methanol (20 mL) was added concentrated sulfuric acid (784 mg, 8.00 mmol) dropwise, and the resulting mixture was heated to reflux for 6 h. After cooling to room temperature, saturated aqueous sodium carbonate solution was added to the reaction to adjust pH 7.0, methanol was removed by concentration under reduced pressure, and the remaining aqueous solution was extracted with dichloromethane (20 mL) 7 times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a colorless oil (Al l-2, 1.70 g, 84.7% yield). MS-ESI [M+H] + :267.1.

[0303] Step B: Methyl 14-({p-[(l,l,2,2-tetramethyl-silanyloxy)methyl]phenyl}(aminooxy))- 3,6,9,12-tetraoxatetradecanoate

[0304] To a solution of methyl 14-hydroxy-3,6,9,12-tetraoxatetradecanoate (Al l-2, 1.70 g, 6.39 mmol) and p-{[(tert-butyl)di(methyl)silyloxy]methyl}phenyl p-nitrophenylcarbamate (AlO-3, 2.60 g, 6.39 mmol) in dichloromethane (20 mL) was added 4-dimethylaminopyridine (2.20 g, 9.84 mmol), and the resulting mixture was stirred at room temperature for 2 h. The crude product was concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 96 / 4) to give a gray solid (Al l-3, 1.30 g, 38.5% yield). MS-ESI [M+H2O] + :547.3.

[0305] Step C: Methyl 14-{[p-(hydroxymethyl)phenyl](aminooxy)}-3,6,9,12- tetraoxatetradecanoate

[0306] To a solution of methyl 14-({p-[(l,l,2,2-tetramethyl-silanyloxy)methyl]phenyl}(aminooxy))- 3,6,9,12-tetraoxatetradecanoate (Al l-3, 3.00 g, 5.67 mmol) in tetrahydrofuran (20 mL) was added tetrabutylammonium fluoride (1 M in THF, 6.80 mL, 6.80 mmol), and the resulting mixture was stirred at room temperature for 6 h. The crude product was concentrated under reduced pressure, and purified by column chromatography (DCM / MeOH = 95 / 5) to give a light brown oil (Al l-4, 2.20 g, 93.5% yield).

[0307] Step D: Methyl 14-{[p-(chloromethyl)phenyl](aminooxy)}-3,6,9,12- tetraoxatetradecanoate

[0308] To a solution of methyl 14-{[p-(hydroxymethyl)phenyl](aminooxy)}-3,6,9,12- tetraoxatetradecanoate (A11-4, 2.20 g, 5.30 mmol) in dichloromethane (20 mL) was added dichlorosulfoxide (1.2 mL, 15.9 mmol) and the resulting mixture was stirred at room temperature for 1 h. Concentration under reduced pressure afforded the crude product as a light brown oil (A11-5, 2.29 g).

[0309] Step E: Methyl 14-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}(aminooxy))-3,6,9,12-tetraoxatetradecanoate

[0310] To a solution of capsaicin (1.62 g, 5.30 mmol) in acetonitrile (20 mL) was added potassium carbonate (1.46 g, 10.6 mmol) and methyl 14-{[p-(chloromethyl)phenyl](aminooxy)}- 3,6,9,12-tetraoxatetradecanoate (A11-5, 2.29 g, 5.30 mmol) and the resulting mixture was stirred at 70 °C for 8 h. The reaction was cooled to room temperature and the solids were removed by filtration. The filtrate was concentrated to afford the crude product which was purified by column chromatography (EtOAc = 100) to afford a grey solid (A11-6, 2.10 g, 56.4% yield). MS-ESI [M+H] + : 703.4.

[0311] Step F: p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl 14-hydrazino-14-oxo-3,6,9,12-tetraoxa-1-tetradecanaminocarboxylate

[0312] To a solution of 14-({p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl}(aminooxy))-3,6,9,12-tetraoxatetradecanoate (A11-6, 1.00 g, 1.42 mmol) in methanol (15 mL) was added 50% hydrazine hydrate (3 mL) and the resulting mixture was stirred at room temperature for 0.5 h. Water (10 mL) was added to the reaction and the resulting suspension was concentrated under reduced pressure. The supernatant was separated by centrifugation to afford a cream solid which was lyophilized to afford a white solid (A11, 1.00 g, 100% yield). 1H NMR (400 MHz, Methanol-d4) δ 7.43 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 6.93 - 6.88 (m, 2H), 6.78 - 6.75 (m, 1H), 5.37 - 5.32 (m, 2H), 5.00 (s, 2H), 4.26 (d, J = 7.0 Hz, 4H), 4.01 (s, 2H), 3.82 (s, 3H), 3.76 - 3.70 (m, 2H), 3.68 - 3.61 (m, 12H), 2.22 (d, J = 7.2 Hz, 2H), 2.00 - 1.95 (m, 2H), 1.65 - 1.58 (m, 2H), 1.41 - 1.27 (m, 3H), 0.94 (d, J = 6.8 Hz, 6H). MS - ESI [M+H] + : 703.4.

[0313] Example B: Preparation of crosslinking agents

[0314] Examples B1, B2, B3:

[0315] Crosslinking agent B1 is 1,4-butanediol diglycidyl ether, CAS# 2425-79-8

[0316] Crosslinking agent B2 is 4,7,10,-trioxa-1,13-tridecanediamine, CAS# 4246-51-9

[0317] Crosslinking agent B3 is glutaraldehyde, CAS# 111-30-8

[0318] All three crosslinking agents are purchased from chemical reagent suppliers.

[0319] Example B4: Preparation of crosslinking agent B4

[0320] 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2,6- diaminohexanoate dihydrochloride

[0321] Step A: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)- 2,6-di[(tert-butyl)(oxycarbonylamino)]hexanoate

[0322] To a solution of (S)-2,6-bis[(tert-butyl)(oxycarbonylamino)]hexanoic acid (B4-1, 1.24 g, 3.36 mmol), capsaicin (1.00 g, 3.30 mmol) and 4-dimethylaminopyridine (43.0 mg, 0.34 mmol) in dichloromethane (10 mL) was added N,N'-dicyclohexylcarbodiimide (1.20 g, 3.96 mmol), the resulting mixture was stirred at room temperature for 16 h. Filtration, the filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography PE / EtOAc = 50 / 50 to give white solid (B4-2, 1.43 g, yield 68.1%). MS-ESI [M+Na] + : 656.4.

[0323] Step B: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2,6- diaminohexanoate dihydrochloride

[0324] To a solution of 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2,6-bis[(tert-butyl)(oxycarbonylamino)]hexanoate (B4-2, 1.43 g, 2.26 mmol) in dichloromethane (30 mL) was added hydrochloric acid dioxane solution (4 M, 10 mL, 40 mmol), the resulting mixture was stirred at room temperature for 1 h. Concentration under reduced pressure, the resulting crude product was slurried with PE / EtOAc (1 / 1, 50 mL) and filtered to give white solid (B4, 720 mg, yield 63.2%). 1 H NMR (400 MHz, DMSO-d6) δ 8.87 (s, 3H), 8.45-8.41 (m, 1H), 8.15 (s, 3H), 7.11 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 1.8 Hz, 1H), 6.86 (dd, J = 8.2, 1.8 Hz, 1H), 5.40-5.28 (m, 2H), 4.26 (d, J = 6.2 Hz, 2H), 3.73 (s, 3H), 3.35 (s, 1H), 2.79 (s, 2H), 2.25-2.13 (m, 3H), 2.03-1.92 (m, 4H), 1.74-1.59 (m, 3H), 1.59-1.49 (m, 3H), 1.34-1.27 (m, 2H), 0.93 (d, J = 6.8 Hz, 6H). MS-ESI [M+H] + : 434.3.

[0325] Example B5: Preparation of crosslinker B5

[0326] 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2- aminoethyl)carbamoyl]propionate dihydrochloride

[0327] Step A: 3-(bis{2-[(tert-butyloxy carbonylamino)]ethyl}carbamoyl) methyl 3- methoxypropionate

[0328] To a solution of 3-methoxycarbonylpropionic acid (B5-1, 0.3 g, 2.27 mmol), 2-{2-[(tert-butyloxy carbonylamino)]ethyl}amino}ethyl 2-methylpropane-2- carboxylate (0.724 g, 2.38 mmol) and triethylamine (3.43 g, 4.77 mmol) in N,N- dimethylformamide (8 mL) was added 1-butylphosphonic anhydride (0.689 g, 6.81 mmol, 50% in ethyl acetate) and the resulting mixture was stirred at room temperature for 3 hours. The reaction was added to water (30 mL) and extracted with ethyl acetate (60 mL) three times, the organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a colorless oil (B5-2, 770 mg, 81.2% yield). MS-ESI [M+H] + : 418.3.

[0329] Step B: 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)propionic acid

[0330] To a solution of 3-(bis{2-[(tert-butyloxy carbonylamino)]ethyl}carbamoyl) methyl 3- methoxypropionate (B5-2, 770 mg, 1.84 mmol) in a mixture of methanol and water (15 mL, 2 / 1) was added sodium hydroxide (590 mg, 14.8 mmol) and the resulting mixture was stirred at 50 °C for 12 hours. Concentrated under reduced pressure, the pH was adjusted to 5 with 1M dilute hydrochloric acid and extracted with ethyl acetate (80 mL) four times, dried over anhydrous sodium sulfate, filtered and concentrated to give a colorless oil (B5-3, 465 mg, 62.6% yield). MS-ESI [M+Na] + : 426.2.

[0331] Step C: 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-(bis{2- [(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)propionate

[0332] To a solution of 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)propanoic acid (B5-3, 407 mg, 1.01 mmol), capsaicin (220 mg, 0.72 mmol) and 4-dimethylaminopyridine (220 mg, 0.72 mmol) in dichloromethane (20 mL) was added N,N'-dicyclohexylcarbodiimide (222.8 mg, 1.08 mmol) under ice-water bath, the resulting mixture was stirred at room temperature for 12 h. The reaction was added water (15 mL), extracted with dichloromethane (50 mL) for 3 times, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by column chromatography (DCM / MeOH = 95 / 5) to give a white solid (B5-4, 480 mg, yield 68.8%). 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 6.0 Hz, 1H), 6.98-6.94 (m, 2H), 6.80 (dd, J = 8.0, 2.0 Hz, 1H), 5.44-5.23 (m, 2H), 4.24 (d, J = 6.0 Hz, 2H), 3.72 (s, 3H), 3.32-3.22 (m, 4H), 3.09-2.99 (m, 4H), 2.77-2.71 (m, 2H), 2.69-2.61 (m, 2H), 2.25-2.18 (m, 1H), 2.13 (d, J = 7.2 Hz, 2H), 1.97-1.91 (m, 2H), 1.55-1.49 (m, 2H), 1.38-1.35 (m, 18H), 1.33-1.27 (m, 2H), 0.93 (d, J = 6.8 Hz, 6H). MS-ESI [M+H-Boc] + :591.4.

[0333] Step D: 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2- aminoethyl)carbamoyl]propanoate dihydrochloride

[0334] To a solution of 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)propanoate (B5-4, 380 mg, 0.55 mmol) in dichloromethane (12 mL) was added hydrochloric acid dioxane solution (4 M, 3 mL, 12 mmol) under ice-water bath, the resulting mixture was stirred at room temperature for 2 h. Concentrated under reduced pressure to give a white solid (B5, 280 mg, yield 90.3%). MS-ESI [M+H] + :491.3.

[0335] Example B6: Preparation of crosslinker B6

[0336] 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2- aminoethyl)carbamoyl]-2,2-dimethylpropionate dihydrochloride

[0337] Step A: methyl 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2- dimethylpropanoate

[0338] To a solution of 2,2-dimethylsuccinic acid-1 -monomethyl ester (2.00 g, 6.60 mmol), 1 -butylphosphonic anhydride (9.50 g, 13.2 mmol, 50% in ethyl acetate) and triethylamine (2.00 g, 19.8 mmol) in N,N-dimethylformamide (20 mL) was added 2-{2-[(tert-butyl)(oxycarbonylamino)]ethyl}ethyl 2-methyl-2-propylcarbamate (B6-1, 1.06 g, 6.60 mmol) and the resulting mixture was stirred at room temperature overnight. The reaction was quenched with water (20 mL) and extracted with ethyl acetate (30 mL) three times. The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a white solid (B6-2, 1.50 g, 51.0% yield). MS-ESI [M+H] + : 446.3.

[0339] Step B: 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2-dimethyl acid

[0340] To a solution of methyl 3-(bis{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2- dimethylpropanoate (B6-2, 1.50 g, 3.40 mmol) in methanol (10 mL) was added a solution of sodium hydroxide (1.07 g, 27.0 mmol) in water (5 mL) and the resulting mixture was stirred at 50 °C for 3 hours. The methanol was removed by concentration under reduced pressure, water (5 mL) was added and the pH was adjusted to 6 with 1 M dilute hydrochloric acid. The mixture was extracted with ethyl acetate (20 mL) three times, dried over anhydrous sodium sulfate, filtered and concentrated to give a white solid (B6-3, 910 mg, 62.5% yield). MS-ESI [M+H] + : 432.3.

[0341] Step C: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl 3-(bis{2- [(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2-dimethylpropanoate dihydrochloride

[0342] To a solution of 3-(di{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2- dimethylpropanoic acid (B6-3, 800 mg, 2.60 mmol) and capsaicin (1.28 g, 2.90 mmol) in dichloromethane (30 mL) was added N,N'-dicyclohexylcarbodiimide (640 mg, 3.20 mmol) and 4-dimethylaminopyridine (40.0 mg, 0.30 mmol), the resulting mixture was stirred at room temperature overnight. The reaction was diluted with water (20 mL) and extracted with dichloromethane (30 mL) for 3 times, dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product, which was purified by column chromatography (DCM / MeOH = 92 / 8) to give a gray solid (B6-4, 960 mg, yield 46%). 1 HNMR (400 MHz, DMSO-d6) δ 8.29 (t, J = 6.0 Hz, 1H), 6.99-6.92 (m, 2H), 6.89 (d, J = 8.0 Hz, 1H), 6.82-6.76 (m, 2H), 5.42-5.27 (m, 2H), 4.23 (d, J = 5.8 Hz, 2H), 3.70 (s, 3H), 3.26 (d, J = 6.8 Hz, 2H), 3.04 (d, J = 8.6 Hz, 4H), 2.74 (s, 2H), 2.24-2.18 (m, 1H), 2.13 (t, J = 7.2 Hz, 2H), 1.96-1.91 (m, 2H), 1.72 (d, J = 12.2 Hz, 1H), 1.65-1.58 (m, 1H), 1.56-1.48 (m, 2H), 1.38-1.36 (m, 18H), 1.30-1.23 (m, 8H), 0.93 (d, J = 6.8 Hz, 6H). MS-ESI [M+H-Boc] + : 619.4.

[0343] Step D: 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-[di(2- aminoethyl)carbamoyl]-2,2-dimethylpropanoate dihydrochloride

[0344] To a solution of 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-(di{2-[(tert-butyl)(oxycarbonylamino)]ethyl}carbamoyl)-2,2-dimethylpropanoate (B6-4, 150 mg, 0.21 mmol) in dichloromethane (9 mL) was added hydrochloric acid in dioxane solution (4 M, 3 mL, 12 mmol) dropwise, the resulting mixture was stirred at room temperature for 2 hours. Concentration under reduced pressure gave a white solid crude product (B6, 130 mg). MS-ESI [M+H] + : 519.4.

[0345] Example C: Preparation of capsaicin-hyaluronan conjugate moiety

[0346] Example C1: Preparation of conjugate moiety C1

[0347] Capsaicin-hyaluronan conjugate moiety

[0348] The preparation of conjugate moiety C1 is described below using conjugate moiety C1-3 as an example.

[0349] Step A: Hyaluronan tetrabutylammonium salt

[0350] To a solution of sodium hyaluronate (HA, 5.00 g, 12.4 mmol) in pure water (500 mL) was added ion exchange resin Dowex 50wx4 200-400(H) (5.00 g) and the resulting mixture was stirred at room temperature for 4 hours. The mixture was filtered and to the filtrate was added dropwise an aqueous solution of tetrabutylhydroxylamine (1 M) to adjust the pH to 7.05. The solution was lyophilized to give a white solid (HA-TBA, 4.8 g). 1 HNMR (400 MHz, D20) δ 4.45-4.35 (m, 2H), 3.87-3.19 (m, 8H), 3.15-3.06 (m, 8H), 1.93 (s, 3H), 1.59-1.52 (m, 8H), 1.31-1.22 (m, 8H), 0.85 (t, J = 7.2 Hz, 12H).

[0351] Step B: Capsaicin-hyaluronan conjugate moiety

[0352] To a solution of hyaluronan tetrabutylammonium salt (HA-TBA, 100 mg, 0.161 mmol, 500 kDa) in dimethyl sulfoxide (7 mL) was added a solution of N-[(4-{[p-(5-bromopentylcarbonylamino)phenyl]methoxy}-3-methoxyphenyl)methyl]-(E)-8-methyl-6- nonenamide (A1, 42.0 mg, 0.072 mmol) in dimethyl sulfoxide (3.5 mL) and the resulting mixture was stirred at 40 °C overnight. To the reaction mixture was added 10% aqueous sodium chloride solution (5 mL) and stirred at room temperature for 20 minutes. Anhydrous ethanol (80 mL) was added dropwise and a white solid precipitated. The supernatant was separated by centrifugation to give a white solid which was washed twice with 85% ethanol (20 mL) and tetrahydrofuran (20 mL), respectively, and then dispersed in pure water (30 mL) and lyophilized to give a white solid (C1, 75.1 mg, capsaicin loading 13.0% wt).

[0353] The following conjugates were prepared according to the synthetic route of C1 using hyaluronic acid of different molecular weights.

[0354] Example C2: Preparation of Conjugate Part C2

[0355] Hyaluronic acid-N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl}14-amino-3,6,9,12-tetraoxatetradecanoamide

[0356] The preparation of conjugate part C2 is described below using conjugate part C2-3 as an example.

[0357] Sodium hyaluronate (30.0 mg, 0.075 mmol, 500 kDa) was added to a mixed solution of dioxane (3 mL) and water (3 mL) and stirred for half an hour to completely dissolve. A solution of N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl}14-amino-3,6,9,12-tetraoxatetradecanoamide (A2, 19.0 mg, 0.030 mmol) in dioxane (1 / 1, 0.4 mL) was added, followed by a solution of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (22.0 mg, 0.075 mmol) in dioxane (1 / 1, 0.4 mL). The resulting mixture was stirred at room temperature overnight. After the reaction, 5% aqueous sodium bicarbonate solution (0.45 mL) was added to the reaction solution and stirred at room temperature for 4 hours. Then, 50% aqueous acetic acid solution (13.5 μL) was added to adjust the reaction pH to 7. Solid sodium chloride (150 mg) was added to the reaction solution and stirred to dissolve it. Then, anhydrous ethanol (24 mL) was added dropwise to precipitate a white solid. The supernatant was separated by centrifugation to obtain a white solid, which was washed twice with 85% ethanol (10 mL) and tetrahydrofuran (10 mL), respectively, and then dispersed in pure water (20 mL). The product was lyophilized to obtain a white solid (C2, 27.6 mg, capsaicin loading rate 16.9% wt).

[0358] The following conjugates were prepared according to the synthetic route of C2 using hyaluronic acid of different molecular weights.

[0359] Example C3: Preparation of Conjugate Part C3

[0360] Hyaluronan-N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-hydrazino-6-oxohexanamide

[0361] The preparation of conjugate moiety C3 is described below using conjugate moiety C3-3 as an example.

[0362] Hyaluronan-N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-hydrazino-6-oxohexanamide

[0363] The following conjugate moieties were prepared according to the synthetic route described above for C3 using hyaluronan of different molecular weights.

[0364] Example C4: Preparation of conjugate moiety C4

[0365] Hyaluronan-N-{p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl} 6-hydrazino-6-oxohexanamide

[0366] The preparation of conjugate moiety C4 is described below using conjugate moiety C4-3 as an example.

[0367] Sodium hyaluronate (50.0 mg, 0.13 mmol, 500 kDa) was added to a mixture of N,N-dimethylformamide (5 mL) and pure water (5 mL) and stirred for half an hour to completely dissolve, N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl]-N-2- aminoethylpentanamide (A4, 46.0 mg, 0.05 mmol) in aqueous solution of N,N-dimethylformamide (1 / 1, 4 mL) was added, followed by the addition of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (34.6 mg, 0.13 mmol) in aqueous solution (1.5 mL), and the resulting mixture was stirred at room temperature for 16 hours. After the reaction was completed, 5% aqueous sodium bicarbonate solution (0.75 mL) was added and stirred for 4 hours, 50% aqueous acetic acid solution (20 μL) was added to adjust the reaction pH to 7, sodium chloride solid (250 mg) was added to the reaction solution and stirred to dissolve, followed by the dropwise addition of anhydrous ethanol (40 mL), and a white solid was precipitated, the supernatant was separated by centrifugation, and the white solid was washed twice with 90% ethanol (10 mL) and anhydrous ethanol (10 mL), and then dispersed with pure water (20 mL) and lyophilized to obtain a white solid (C4, 60.0 mg, capsaicin loading rate 11.8% wt).

[0368] The following conjugate fractions were prepared according to the above synthesis route of C4 using hyaluronic acid of different molecular weights.

[0369] Example C5: Preparation of conjugate fraction C5

[0370] Hyaluronan-N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl]methyl}aminocarbonyl)methyl]-5-hydrazinyl-5-oxopentanamide

[0371] The preparation of conjugate fraction C5 is described below using conjugate fraction C5-3 as an example.

[0372] Sodium hyaluronate (50.0 mg, 0.13 mmol, 50 kDa) was added to a mixture of N,N-dimethylformamide (5 mL) and pure water (5 mL) and stirred for half an hour to completely dissolve, N-[({[N-(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]-2-phenylethylaminocarbonyl}methyl]aminocarbonyl)methyl]-5-hydrazino-5-oxopentanamide (A5, 43.0 mg, 0.05 mmol) in aqueous solution of N,N-dimethylformamide (1 / 1, 4 mL) was added, followed by adding 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (34.6 mg, 0.13 mmol) in aqueous solution (1.5 mL), the resulting mixture was stirred at room temperature for 16 hours. After the reaction was completed, 5% aqueous solution of sodium bicarbonate (0.75 mL) was added and stirred for 4 hours, 50% aqueous solution of acetic acid (20 μL) was added to adjust the pH of the reaction to 7, sodium chloride solid (250 mg) was added to the reaction solution and stirred to dissolve, followed by adding anhydrous ethanol (40 mL) dropwise, white solid was precipitated, the supernatant was separated by centrifugation, the white solid was washed twice with 90% ethanol (15 mL) and anhydrous ethanol (15 mL), then pure water (20 mL) was added to disperse, and freeze-drying was performed to obtain a white solid (C5, 76.0 mg, capsaicin loading rate 16.7% wt).

[0373] The following conjugate fractions were prepared according to the above synthesis route of C5 using hyaluronic acid of different molecular weights.

[0374] Example C6: Preparation of conjugate fraction C6

[0375] Hyaluronic acid-N-[(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethyl] (S)-2-[({(S)-2-(S)-1-(14-amino-3,6,9,12-tetraoxatetradecanoyl)-2-pyrrolidinylcarbonylamino]-4-methylpentanoyl}methyl)carbonylamino]-4-methylpentanamide

[0376] The preparation of conjugate fraction C6 is described below taking conjugate fraction C6-3 as an example.

[0377] Sodium hyaluronate (17.4 mg, 0.0434 mmol, 1000 kDa) was added to a mixture of N,N-dimethylformamide (10 mL) and water (10 mL) and stirred for half an hour to completely dissolve, N-[(S)-1-[({N-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenylaminocarbonyl}methyl)aminocarbonyl]ethyl] (S)-2-[({(S)-2-(S)-1-(14-amino-3,6,9,12-tetraoxatetradecanoyl)-2-pyrrolidinylcarbonylamino]-4-methylpentanoyl-amino}methyl)carbonylamino]-4-methylpentanoic amide (A6, 20.0 mg, 0.0174 mmol) in aqueous N,N-dimethylformamide (1 / 1, 4 mL) was added, followed by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (12.8 mg, 0.0434 mmol) in aqueous N,N-dimethylformamide (1 / 1, 1 mL), and the resulting mixture was stirred at room temperature for 24 hours. After the reaction was completed, sodium chloride solid (100 mg) was added to the reaction solution and stirred to dissolve, followed by dropwise addition of anhydrous ethanol (50 mL), and a white solid was precipitated, which was separated by centrifugation, and the supernatant was washed twice with 90% ethanol (10 mL) and anhydrous ethanol (10 mL), and then dispersed with pure water (10 mL), and lyophilized to obtain a white solid (14.1 mg, capsaicin loading rate 10.1% wt).

[0378] The following conjugate fractions were prepared according to the above synthesis route of C6 using hyaluronic acid of different molecular weights.

[0379] Example C7: Preparation of conjugate fraction C7

[0380] Hyaluronate-4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl bromoacetate

[0381] The preparation of conjugate fraction C7 is described below using conjugate fraction C7-3 as an example.

[0382] To a solution of hyaluronic acid tetrabutylammonium salt (HA-TBA, 100 mg, 0.161 mmol, 500 kDa) in dimethyl sulfoxide (7 mL) was added a solution of 4-{[(E)-7-methyl-5- octenylcarbonylamino]methyl}-2-methoxyphenyl bromoacetate (A7, 31.0 mg, 0.073 mmol) in dimethyl sulfoxide (3 mL) and the resulting mixture was stirred at 40 °C overnight. To the reaction mixture was added 10% aqueous sodium chloride solution (5 mL) and stirred at room temperature for 30 minutes. To the reaction mixture was added absolute ethanol (80 mL) dropwise and a white solid precipitated. The supernatant was removed by centrifugation and the white solid was washed with 85% ethanol (20 mL) and tetrahydrofuran (20 mL) twice, respectively. The white solid was then dispersed in purified water (20 mL) and lyophilized to give a white solid (57.4 mg, capsaicin loading 21.4% wt).

[0383] The following conjugate fractions were prepared according to the above synthesis route of C7 using different molecular weight of hyaluronic acid.

[0384] Example C8: Preparation of conjugate fraction C8

[0385] Hyaluronic acid-4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenylaminoacetate

[0386] The following describes the preparation of conjugate fraction C8 using conjugate fraction C8-3 as an example.

[0387] Sodium hyaluronate (70.0 mg, 0.175 mmol, 500 kDa) was added to a mixture of dioxane (7 mL) and purified water (7 mL) and stirred for half an hour to completely dissolve. A solution of 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenylaminoacetate hydrochloride (A8, 28.0 mg, 0.07 mmol) in dioxane and water (1 / 1, 1.5 mL) was added to the solution, followed by the addition of a solution of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (52.0 mg, 0.175 mmol) in water (1.5 mL). The resulting mixture was stirred at room temperature for 16 hours. After the reaction was completed, sodium chloride solid (350 mg) was added to the reaction mixture and stirred to dissolve. Absolute ethanol (50 mL) was then added dropwise and a white solid precipitated. The supernatant was removed by centrifugation and the white solid was washed with 90% ethanol (10 mL) and absolute ethanol (10 mL) twice, respectively. The white solid was then dispersed in purified water (20 mL) and lyophilized to give a white solid (C8, 78.0 mg, capsaicin loading 24.6% wt).

[0388] The following conjugate fractions were prepared according to the above synthesis route of C8 using different molecular weight of hyaluronic acid.

[0389] Example C9: Preparation of conjugate moiety C9

[0390] Hyaluronan-4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-amino-3-phenylpropionic acid

[0391] The preparation of conjugate moiety C9 is described below using conjugate moiety C9-3 as an example.

[0392] Sodium hyaluronate (100.0 mg, 0.25 mmol, 500 kDa) was added to a mixed solution of dioxane (10 mL) and pure water (10 mL) and stirred for half an hour to completely dissolve, 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl (S)-2-amino-3-phenylpropionic acid (A9, 45.3 mg, 0.10 mmol) in dioxane aqueous solution (1 / 1, 4 mL) was added, followed by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (69.2 mg, 0.25 mmol) in aqueous solution (1.5 mL), and the resulting mixture was stirred at room temperature for 16 hours. After the reaction was completed, sodium chloride solid (500 mg) was added to the reaction solution and stirred to dissolve, followed by dropwise addition of anhydrous ethanol (80 mL), and a white solid was precipitated, which was separated by centrifugation to obtain a white solid, which was washed twice with 90% ethanol (20 mL) and anhydrous ethanol (20 mL), and then dispersed with pure water (20 mL) and lyophilized to obtain a white solid (C9, 100.0 mg, capsaicin loading rate 22.6% wt).

[0393] The following conjugate moieties were prepared according to the above synthesis route of C9 using hyaluronic acid of different molecular weights.

[0394] Example C10: Preparation of conjugate moiety C10

[0395] Hyaluronan-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl 3-(N-2-aminoethylcarbamoyl)-1-propylamino carboxylate

[0396] The preparation of conjugate moiety C10 is described below using conjugate moiety C10-3 as an example.

[0397] Sodium hyaluronate (100 mg, 0.25 mmol, 500 kDa) was added to a mixture of dioxane (10 mL) and pure water (10 mL) and stirred for half an hour to completely dissolve, then added a solution of p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl 3-(N-2- aminoethylcarbamoyl)-1 -propylaminoformate (A10, 58.2 mg, 0.10 mmol) in dioxane and water (1 / 1, 8 mL), followed by adding a solution of 4-(4,6-dimethoxytriazin-2-yl)-4- methylmorpholine hydrochloride (69.8 mg, 0.25 mmol) in water (1.5 mL), the resulting mixture was stirred at room temperature for 16 hours. After the reaction was completed, added a 5% aqueous solution of sodium bicarbonate (1.5 mL) and stirred for 4 hours, then added a 50% aqueous solution of acetic acid (40 μί) to adjust the reaction pH to 7, added sodium chloride solid (500 mg) to the reaction solution and stirred to dissolve, then added anhydrous ethanol (80 mL) dropwise, white solid precipitated, the supernatant was separated by centrifugation, the white solid was washed twice with 90% ethanol (15 mL) and anhydrous ethanol (15 mL), then added pure water (20 mL) to disperse, and freeze-dried to obtain a white solid (C10, 114 mg, capsaicin loading rate 21.6% wt).

[0398] The following conjugate moieties were prepared according to the above synthesis route of C10 using different molecular weight of hyaluronic acid.

[0399] Example C11 : Preparation of conjugate moiety C11

[0400] Hyaluronic acid-p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2- methoxyphenoxy)methyl]phenyl 14-hydrazinyl-14-oxo-3,6,9,12-tetraoxo-1- tetradecylaminocarbamate

[0401] The preparation of conjugate moiety C11 is described below using conjugate moiety C11-3 as an example.

[0402] Sodium hyaluronate (60.0 mg, 0.150 mmol, 50 kDa) was added to a mixture of dioxane (6 mL) and water (6 mL) and stirred for half an hour to completely dissolve, then p-[(4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenoxy)methyl]phenyl 14-hydrazino-14-oxo-3,6,9,12-tetraoxo-l-tetradecylaminocarbamate (A11, 42.0 mg, 0.059 mmol) in dioxane water solution (1 / 1, 2 mL) was added, followed by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride (44.0 mg, 0.149 mmol) in dioxane water solution (1 / 1, 1 mL), and the resulting mixture was stirred at room temperature overnight. After the reaction was completed, 5% sodium bicarbonate aqueous solution (0.9 mL) was added and stirred for 4 hours, then 50% acetic acid aqueous solution (27 μL) was added to adjust the reaction pH to 7, sodium chloride solid (300 mg) was added to the reaction solution and stirred to dissolve, followed by dropwise addition of anhydrous ethanol (60 mL), and a white solid was precipitated, which was separated by centrifugation, and the supernatant was washed with 85% ethanol (20 mL) and tetrahydrofuran (20 mL) twice, then dispersed with pure water (20 mL), and lyophilized to obtain a white solid (C11, 75.0 mg, capsaicin loading rate 12.6% wt).

[0403] The following conjugate parts were prepared according to the synthesis route of C11 above using different molecular weights of hyaluronic acid.

[0404] Preparation of capsaicin-hyaluronic acid composition

[0405] Example 1: Hyaluronic acid-3-[4-(2,3-dihydroxypropoxy)butyloxy]-l,2-propanediol nanoparticle-loaded capsaicin

[0406] Step A: Hyaluronic acid-3-[4-(2,3-dihydroxypropoxy)butyloxy]-l,2-propanediol

[0407] In a ziplock bag, sodium hyaluronate (5 g, 1000 kDa) was added to pure water (50 mL) and mixed well to dissolve. To the mixture, 1,4-butanediol diglycidyl ether (450 μL, 0.9%) was added and mixed well. The resulting mixture was left to stand at 25°C for 12 hours. After the reaction was completed, the gel was removed and placed in a dialysis bag and dialyzed against PBS (pH 7.2-7.4, 2000 mL) three times for 8 hours each time. Then, it was dialyzed against pure water (2000 mL) three times for 8 hours each time. After dialysis was completed, the gel was filtered to obtain Ex la gel (550 g).

[0408] The following crosslinked materials were prepared according to the procedure of Step A of Example 1 above, using different molecular weights of hyaluronic acid and crosslinker B1.

[0409] Step B: Hyaluronic acid-3-[4-(2,3-dihydroxypropoxy)butyloxy]-1,2-propanediol nanoparticle loaded capsaicin

[0410] Transfer 0.2 volumes of the capsaicin stock solution (5 mg / mL capsaicin in PEG 300) to a sample bottle, add 0.8 volumes of an aqueous solution of sulfobutyl betacyclodextrin sodium (20% w / v), and mix well to obtain a 1 mg / mL solution of capsaicin. Weigh Ex 1a-3 gel (density taken as 1.0 g / mL) into the sample bottle, and start stirring at a speed that allows the gel in the sample bottle to rotate with the stir bar in a vortex. Slowly add an equal volume of the capsaicin solution (1 mg / mL) described above, and continue stirring overnight. Finally, measure the pH of the gel system, and adjust the pH to the range of 6.0-7.5 using hydrochloric acid or sodium hydroxide solution, as necessary, to obtain composition Ex 1-1. Composition Ex 1-2 was prepared according to the procedure of Step B above, using 2 volumes of Ex 1a-3 gel and 1 volume of capsaicin solution (1 mg / mL).

[0411] Example 2: Hyaluronic acid-4,7,10-trio-1,13-tridecanediamine-4-{[(E)-7-methyl-5- octenylcarbonylamino]methyl}-2-methoxyphenylaminoacetic acid ester

[0412] The preparation of composition Ex 2 is described below using composition Ex 2b-6 as an example.

[0413] C8 (1 g, 2.5 mmol, 200 kDa) was added to a mixture of 2-(N-morpholino)ethanesulfonic acid buffer (20 mL, pH 5.5, 0.1 M) and acetonitrile (20 mL) and stirred until completely dissolved. To the solution was added a mixture of 4,7,10-trioxa-1,13-tridecanediamine (B2, 110 mg, 0.5 mmol) in 2-(N-morpholino)ethanesulfonic acid buffer (pH 5.5, 0.1 M, 1 mL) and acetonitrile (1 mL) and a mixture of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (415 mg, 1.5 mmol) in 2-(N-morpholino)ethanesulfonic acid buffer (pH 5.5, 0.1 M, 4 mL) and acetonitrile (4 mL), respectively. The resulting mixture was stirred at 25 °C for 12 hours. After the reaction was completed, pure water (200 mL) was added to soak, and the pure water (200 mL) was replaced every 8 hours. After three times of replacement, the pure water was removed by filtration, and 0.1% sodium chloride aqueous solution (200 mL) was added to soak, and the 0.1% sodium chloride aqueous solution was replaced every 8 hours. After three times of replacement, the 0.1% sodium chloride aqueous solution was removed by filtration, and pure water (200 mL) was added again, and the pure water was replaced every 8 hours. After three times of replacement, the pure water was removed by filtration. Composition Ex2b-6 (48 g, capsaicin loading rate 16.0% wt) was obtained.

[0414] The following compositions were prepared according to the synthesis route of Example 2 above, using different molecular weights of the conjugate moiety C8 and equivalents of the crosslinker B2.

[0415] Example 3: 4-{[(E)-7-methyl-5-octenylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2- aminoethyl)carbamoyl]-2,2-dimethylpropanoate dihydrochloride

[0416] The preparation of composition Ex3 is described below using composition Ex3-4 as an example.

[0417] Sodium hyaluronate (100 mg, 0.25 mmol, 1000 kDa) was added to pure water (1.5 mL) and stirred at room temperature for 1 hour until complete dissolution. Then, a solution of 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 2,6-diaminohexanoate hydrochloride (B4, 63.3 mg, 0.125 mmol) in dioxane / water (3 / 2, 7.5 mL) was added dropwise and stirred at room temperature for 0.5 hours. Then, a solution of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (138.4 mg, 0.5 mmol) in water (1 mL) was added and the resulting mixture was stirred at room temperature for 40 hours. After completion of the reaction, the mixture was soaked in ethanol (40 mL) for 5 times, changing every hour, and the gel was filtered. The gel was then soaked in pure water (100 mL) for 4 times, changing every hour, and finally filtered to obtain composition Ex3-4 (1.07 g, capsaicin loading 11.2% wt).

[0418] The following compositions were prepared according to the synthetic route of Example 3 above, using different molecular weights of hyaluronic acid and different equivalents of crosslinker B4.

[0419] Example 4: Hyaluronic acid-4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2- methoxyphenyl 3-[bis(2-aminoethyl)carbamoyl]propionate

[0420] The following describes the preparation of composition Ex4-1 as an example of composition Ex4.

[0421] Sodium hyaluronate (500 mg, 1.25 mmol, 1000 kDa) was added to a mixture of acetonitrile (5 mL) and pure water (10 mL) and stirred at room temperature for 1.5 hours until complete dissolution. Then, a solution of 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2-aminoethyl)carbamoyl]propionate hydrochloride (B5, 156 mg, 0.25 mmol) in acetonitrile / water (1 / 2, 30 mL) was added and stirred at room temperature for 0.5 hours. Then, a solution of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (276 mg, 1 mmol) in water (5 mL) was added and the resulting mixture was stirred at room temperature for 40 hours. After completion of the reaction, the mixture was soaked in ethanol (400 mL) for 5 times, changing every hour, and the gel was filtered. The gel was then soaked in pure water (800 mL) for 5 times, changing every hour, and finally filtered to obtain composition Ex4-1 (38 g, capsaicin loading 11.3% wt).

[0422] The following cross-linking materials were prepared according to the synthetic route of Example 4 above, using different molecular weights of hyaluronic acid and equivalent amounts of cross-linking agent B5.

[0423] Example 5: Hyaluronic acid-4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2- methoxyphenyl 3-[bis(2-aminoethyl)carbamoyl]-2,2-dimethylpropanoate

[0424] The following describes the preparation of composition Ex5-1 as an example of composition Ex5.

[0425] Sodium hyaluronate (500.0 mg, 1.25 mmol, 1000 kDa) was added to a mixed solution of acetonitrile (5 mL) and pure water (10 mL), and stirred at room temperature for 1.5 hours until completely dissolved. An aqueous solution (1 / 2, 30 mL) of 4-{[(E)-7-methyloct-5-enylcarbonylamino]methyl}-2-methoxyphenyl 3-[bis(2-aminoethyl)carbamoyl]-2,2-dimethylpropanoate hydrochloride (B6, 164 mg, 0.25 mmol) was added, and stirred at room temperature for 0.5 hours. An aqueous solution (5 mL) of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (276 mg, 1 mmol) was added, and the resulting mixture was stirred at room temperature for 40 hours. After completion of the reaction, ethanol (400 mL) was added and soaked for 5 hours, and changed every hour. The gelatinous material was filtered, soaked in pure water (800 mL) for 4 hours, and changed every hour. The filtered composition Ex5-1 (36 g, capsaicin loading rate 8.0% wt) was obtained.

[0426] The following compositions were prepared according to the synthetic route of Example 5 above, using different molecular weights of hyaluronic acid and equivalent amounts of cross-linking agent B6.

[0427] Example 6: Determination of capsaicin loading rate

[0428] Spectrophotometry: 2 mg of the sample was weighed, added to MeCN / H2O (1 / 1, 2 mL), and allowed to stand for 1 hour to dissolve. 200 μL was added to a 96-well quartz microplate, and the absorbance was measured at λ = 405 nm using a microplate reader (Molecular Devices SpectraMax®). max The absorbance was read, and the average value of triplicate wells was obtained. The capsaicin loading rate was determined by comparison with a standard curve of the coupling agent.

[0429] Basic dissociation method: Take 2 mg sample, add methanol 2 mL and 1 M NaOH aqueous solution 2 mL, mix well, stir at 50 °C, react for 2 h. Take 150 μL of the reaction solution, add a mixed solution of methanol and 1 M HC1 aqueous solution (1 / 1, 150 μL), mix well, and inject 20 μL for HPLC analysis. Repeat twice, take the average value, and compare with the calibration curve to obtain the capsaicin loading rate.

[0430] The capsaicin loading rates measured using the spectrophotometry and the basic dissociation method under the above experimental conditions are comparable. The following table lists the capsaicin loading rates of exemplary compositions measured using the basic dissociation method.

[0431] Example 7: Capsaicin release experiment in rabbit synovial fluid

[0432] Experimental method: LC-MS / MS method was used to evaluate the release kinetics of capsaicin in exemplary compositions Exl-1, Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1.

[0433] Experimental instrument: QTRAP 6500+ system from AB Sciex.

[0434] Experimental procedure:

[0435] 1) For compositions Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1: an amount of sample was removed and added to a certain volume of rabbit synovial fluid, mixed well and incubated at 37 °C for 20 h. 10 μL of sample was removed from the incubated sample at 0, 1, 4 and 20 h, respectively, and added to 490 μL of blank matrix (diluted 50x). 15 μL of the diluted sample was transferred to 235 μL of quenching agent containing internal standard (acetonitrile / methanol = 1 / 1). All samples were vortexed for 5 min, then centrifuged at 3000 g, 4 °C for 5 min. 20 μL of supernatant was transferred to a new 96-well plate, and 180 μL of diluent (methanol / water = 1 / 1) was added and mixed well. The same post-treatment steps were used to prepare capsaicin standard curve points, and the LC-MS / MS instrument was used to quantitatively analyze the concentration of capsaicin released in the sample.

[0436] 2) For Composition Ex1-1 : An aliquot of sample was taken and added to a volume of blank matrix, mixed and 200 μΐ^was taken and added to the red well of a RED Equilibrium Dialysis device, 350 μΐ^of blank matrix was added to the corresponding white well, and then incubated at 37 °C for 22 hours. At 0, 2, 5, and 22 hours, 10 μΐ^of sample was taken from the white well and added to 990 μΐ^of blank matrix (100x dilution). The diluted sample was transferred to 15 μΐ^and added to 235 μΐ^of quenching solution containing internal standard (acetonitrile / methanol = 1 / 1). All samples were vortexed for 5 minutes and then centrifuged at 3000 g, 4 °C for 5 minutes. 20 μΐ^of supernatant was transferred to a new 96 well plate and 180 μΐ^of diluent (methanol / water = 1 / 1) was added and mixed. The same post-treatment procedure was used to prepare the capsaicin standard curve points, and the LC-MS / MS instrument was used to quantify the concentration of capsaicin released in the sample.

[0437] Data analysis: The capsaicin release rate was calculated by the following formula.

[0438] As shown in Figure 1, the composition of the present disclosure has a good capsaicin release rate. Compared with the complete release (100% release percentage) of pure capsaicin at the initial stage of administration (0-4h after administration), the release of the composition of the present disclosure at the initial stage of administration is controlled, and the irritability of capsaicin is greatly reduced.

[0439] Example 8: Enzymatic degradation stability test

[0440] A. Preparation of D-glucuronide carbazole standard curve

[0441] (1) Preparation of pH 7.2 PBS solution: Dissolve sodium dihydrogen phosphate (6.00 g) in deionized water (2500 mL); dissolve sodium hydrogen phosphate (7.20 g) in deionized water (2500 mL). Slowly add the sodium dihydrogen phosphate solution to the sodium hydrogen phosphate solution, and measure the pH to 7.2 with an acidity meter.

[0442] (2) Preparation of D-glucuronic acid solution: Add D-glucuronic acid (100 mg) to a 100 mL volumetric flask, and dilute to the mark with pH 7.2 PBS to prepare a 1 mg / mL sugar solution. Take 7.5 mL of the solution in a 100 mL volumetric flask, and dilute to the mark with pH 7.2 PBS to prepare a 75 ug / mL sugar solution.

[0443] (3) Preparation of carbazole ethanol solution: Add carbazole (125 mg) to a 250 mL brown wide-mouth bottle, add anhydrous ethanol (100 mL), and ultrasonically dissolve and clarify, and store at 4 °C.

[0444] (4) Sodium tetraborate solution preparation: Sodium tetraborate (4.76 g) was added into 500 mL of concentrated sulfuric acid with constant shaking until clear, and then poured into a 1000 mL jar and stored at room temperature.

[0445] (5) Different volumes of 75 μg / mL sugar solution were mixed with different volumes of pH 7.2 PBS solution to prepare different concentrations of D-glucuronic acid solution with a total volume of 1 mL, and then added into a test tube with a stopper.

[0446] (6) Under ice-salt bath cooling, sodium tetraborate solution (5 mL) was slowly added into each concentration of D-glucuronic acid solution, and then shaken well. After the addition was completed, the solution was transferred into an ice-water bath for 3 minutes, and then transferred into a 100°C oil bath for heating for 15 minutes (during which the sugar solution was shaken once in the oil bath for 7 minutes). The solution was then transferred into an ice-water bath to reduce the temperature to 0°C, and then carbazole ethanol solution (0.2 mL) was added, and then shaken well. The solution was then transferred into a 100°C oil bath for heating for 20 minutes (during which the sugar solution was shaken once in the oil bath for 10 minutes). The solution was then transferred into an ice-water bath to reduce the temperature to 0°C, and then allowed to return to room temperature. 200 μL of each concentration of sugar reaction solution was added into a 96-well plate, and each concentration of sugar reaction solution was taken three times for parallel experiments. The absorbance was measured by an enzyme marker (λ = 530 nm), and the average of three absorbance values of each sample was plotted against the concentration of each glucuronic acid to obtain a standard curve shown in FIG. 2A.

[0447] B. Enzymatic experiment

[0448] B-1. Enzymatic experiment of cross-linked product

[0449] The cross-linked product Ex1a-3 (490 mg) was added into a 40 mL sample bottle, and then pH 7.2 PBS solution (11.5 mL) was added. The cross-linked product Ex1a-13 (510 mg) was added into a 40 mL sample bottle, and then pH 7.2 PBS solution (11.5 mL) was added. 0.6 mL of commercially available product DUROLANE (20 mg / mL) was taken and added into a 40 mL sample bottle, and then pH 7.2 PBS solution (11.4 mL) was added.

[0450] To each sample solution, 10 mL of 0.25 mg / mL hyaluronidase solution was added, and then incubated on a shaking table at 42°C. 1 mL of enzyme hydrolysis supernatant was taken from each sample at 0, 0.25, 0.5, 2, 4 and 20 hours, and then inactivated at 100°C for 10 minutes. After cooling to room temperature, the enzyme hydrolysis solution was filtered through a 0.22 μm filter head, and then the filter head was washed with pH 7.2 PBS solution for 3 times. The filtrate was diluted to 5 mL to obtain a test solution, and then 1 mL of the test solution was taken and used to measure the absorbance value according to the operation of the D-glucuronic acid carbazole method standard curve. The sugar concentration was calculated by substituting the absorbance value into the D-glucuronic acid carbazole method standard curve equation.

[0451] Enzymatic rate = C t / C20h x 100

[0452] C t D-glucuronide concentration at different enzymolysis time points

[0453] C 20h D-glucuronide concentration after 20h enzymolysis

[0454] The enzymolysis rates of each sample are listed in the following table, and the enzymolysis trend of each sample is shown in FIG. 2B.

[0455] As shown in FIG. 2B, the enzymolysis stability of the cross-linking products Ex1a-3 and Ex1a-13 of the present disclosure is better than that of the commercially available product DUROLANE.

[0456] B-2. Enzymolysis experiment of the composition

[0457] The composition Ex2b-6 (0.72 g) was added to a 40 mL sample bottle, and pH 7.2 PBS (11.28 mL) was added; the composition Ex2b-11 (1.27 g) was added to a 40 mL sample bottle, and pH 7.2 PBS (10.73 mL) was added; the composition Ex1-1 (1.32 g) was added to a 40 mL sample bottle, and pH 7.2 PBS (10.68 mL) was added; the composition Ex4-1 (1.58 g) was added to a 40 mL sample bottle, and pH 7.2 PBS (10.42 mL) was added; the composition Ex5-1 (1.09 g) was added to a 40 mL sample bottle, and pH 7.2 PBS (10.91 mL) was added; and the cross-linking product Ex1a-3 (1.11 g) as a positive reference was added to a 40 mL sample bottle, and pH 7.2 PBS (10.89 mL) was added.

[0458] To each of the above sample solutions, 10 mL of 0.25 mg / mL HAase solution was added, and incubation was performed on a 42°C shaking table. 1 mL of the enzymolysis supernatant was taken from each sample at 0, 0.25, 0.5, 2, 4 and 20 hours, respectively, and was inactivated at 100°C for 10 min, cooled to room temperature, filtered with a 0.22 μm filter head, and the filter head was rinsed with pH 7.2 PBS solution for 3 times. The filtrate was diluted to 5 mL to obtain a test solution, and 1 mL of the test solution was taken and used to measure the absorbance value according to the operation of the D-glucuronide carbazole method standard curve. The sugar concentration was converted by substituting the absorbance value into the D-glucuronide carbazole method standard curve equation.

[0459] Figure 2C shows the enzymolysis trend of compositions Ex2b-6 and Ex2b-11 and positive control Ex1a-3, wherein the enzymolysis level is low due to good anti-enzymolysis performance of compositions Ex2b-6 and Ex2b-11, and therefore the trend graph is plotted using the uronic acid concentration as the vertical coordinate. Figure 2D shows the enzymolysis trend of compositions Ex1-1, Ex4-1, Ex5-1 and positive control Ex1a-3, wherein the trend graph is plotted using the enzymolysis rate as the vertical coordinate, and the enzymolysis rate of each sample is listed in the table below.

[0460] As shown in Figures 2B-2D, the enzymolysis stability of compositions Ex1-1, Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1 of the present disclosure is better than that of commercially available DROLANE.

[0461] Example 9: Acute pain evaluation test of intra-articular injection in rats

[0462] The test was conducted to explore whether compositions Ex1-1, Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1 can shorten the paw withdrawal response time of rats after intra-articular injection.

[0463] Experimental materials:

[0464] SD rats, weighing 312-380 g, male, purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd. Disposable sterile insulin syringe (Yushu Medical, item number: 2302294). Capsaicin (Chengdu Ruifen Siden Biological Technology Co., Ltd., batch number: RDD-L03502305025).

[0465] Experimental method:

[0466] Twenty-eight SD rats were randomly divided into 7 experimental groups, 4 rats in each group. The 1st to 7th groups were respectively given solvent control (5 mg / mL hyaluronic acid), positive control capsaicin, Ex1-1, Ex2b-6, Ex2b-11, Ex4-1 and Ex5-1, and the dosage (converted to capsaicin) was 0.25 mg / mL. The left hind ankle joint cavity of each rat was injected with the drug once on the day of grouping, and the volume of the drug was 50 μL. The duration of rat paw withdrawal after drug administration was used as an indicator to evaluate the acute pain response, and the data was recorded. The experimental design is shown in the table below.

[0467] The experimental results are shown in the following table and FIG. 3. Injection of the solvent control did not cause the rats to exhibit a paw withdrawal response; injection of the positive control, capsaicin, caused the rats to exhibit an average paw withdrawal duration of 37.0 seconds; injection of the compositions of the present disclosure, Ex1-1, Ex2b-6, Ex2b-11, Ex4-1, and Ex5-1, caused the rats to exhibit an average paw withdrawal duration of 14.0, 17.0, 2.8, 6.5, and 3.5 seconds, respectively. Compared to the group administered the positive control, capsaicin, the groups administered the compositions of the present disclosure significantly reduced the acute pain response in the rats and had a statistical difference (p<0.05). In addition, 3, 3, and 2 of the 4 animals in the groups administered Ex2b-11, Ex5-1, and Ex4-1, respectively, of the compositions of the present disclosure did not exhibit a paw withdrawal response, which had a significant difference (p<0.01) compared to the group administered capsaicin. Thus, compared to direct injection of capsaicin, intra-articular injection of the compositions of the present disclosure, Ex1-1, Ex2b-6, Ex2b-11, Ex4-1, and Ex5-1, into rats was able to reduce the acute pain caused by capsaicin. Note: Independent sample T test method was used to compare whether there was a statistical difference between the group administered each composition and the group administered capsaicin. All data were analyzed using SPSS 22.0. p<0.05 was considered to have a statistical difference, and p<0.01 was considered to have a significant difference.

[0468] While the present disclosure has been particularly and specifically shown and described with reference to particular embodiments, some of which are preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the disclosure as disclosed herein.

Claims

1. A composition comprising a hyaluronic acid matrix and capsaicin, wherein the hyaluronic acid matrix is ​​formed by cross-linking hyaluronic acid polymers with a cross-linking agent.

2. The composition according to claim 1, wherein: The cross-linking agent is BDDE; and / or The molar ratio of the cross-linking agent to the hyaluronic acid polymer is 0.07-0.30; and / or The weight average molecular weight Mw of the hyaluronic acid polymer is 1000-2000 kDa; and / or Based on the total weight of the composition, the content of capsaicin is 1-10%wt.

3. The composition according to claim 1 or 2, wherein the molar ratio of the cross-linking agent to the hyaluronic acid polymer is 0.20, the weight average molecular weight Mw of the hyaluronic acid polymer is 1000 kDa, and the content of capsaicin is 5% wt based on the total weight of the composition.

4. The composition according to any one of claims 1 to 3, wherein the hyaluronic acid matrix has the form of nanoparticles and the capsaicin is dispersed in the hyaluronic acid matrix.

5. A composition comprising a hyaluronic acid matrix and capsaicin, wherein the hyaluronic acid matrix is ​​formed by cross-linking a hyaluronic acid polymer through a cross-linking agent, and the capsaicin is conjugated to the hyaluronic acid polymer through a linker to form a conjugate portion.

6. The composition of claim 5, wherein the conjugate moiety has the following formula (I): in, X is a direct bond, *-C(O)-, *-C(O)N(R a )-、 The * end of X represents the connection point with the O atom; Y is a straight bond, -N(R a )-、-N(R a )-N(R a )-or-O-; L is a straight bond or **-L1-L2-L3-L4-, where the ** end of L represents the connection point with X; L1 is a direct bond, **-C(O)-alkyl-, **-C(O)-, **-C(O)O-alkyl-, an amino acid residue, or a matrix metalloproteinase (MMP) cleavable peptide, wherein the ** end of L1 represents the point of attachment to X; L2 is a straight bond, #-C(O)N(R a )-, #-C(O)-, #-C(O)-alkyl-, or #-(OCH2CH2) n -, where the # end of L2 represents the connection point with L1; L3 is a straight bond, alkyl, ##-C(O)-, ##-O-alkyl-C(O)-, ##-C(O)N(R a )-alkyl-, or ##-(OCH2CH2) n -, where the ## end of L3 represents the connection point with L2; L4 is a straight bond or ###-O-alkyl-C(O)-, wherein the ### end of L4 represents the point of connection with L3; R a is hydrogen, alkyl, alkenyl, alkynyl, haloalkyl or hydroxyalkyl; m is 100-20000; n is 1, 2, 3, 4 or 5.

7. The composition according to claim 6, wherein X is R a For hydrogen.

8. The composition according to claim 6, wherein X is a direct bond.

9. The composition according to claim 6, wherein X is 10. The composition according to any one of claims 6 to 9, wherein L1 is **-C(O)-alkyl-, L2 is a direct bond, #-C(O)N(R a )-, #-C(O)- or #-(OCH2CH2) n -, L3 is a direct bond, alkyl or ##-O-alkyl-C(O)-, and L4 is a direct bond.

11. The composition according to any one of claims 6 to 9, wherein L1, L2 and L4 are direct bonds, and L3 is an alkyl group.

12. The composition according to any one of claims 6 to 9, wherein L1 is **-C(O)O-alkyl-, L2 is #-C(O)N(R a )- or #-(OCH2CH2) n -, L3 is a direct bond, alkyl or ##-O-alkyl-C(O)-, and L4 is a direct bond.

13. The composition according to any one of claims 6 to 9, wherein L1 is an MMP cleavable peptide, L2 is #-C(O)-alkyl-, and L3 is a straight bond, ##-C(O)-, ##-O-alkyl-C(O)-, ##-C(O)N(R a )-alkyl- or ##-(OCH2CH2) n -, L4 is a straight bond or ###-O-alkyl-C(O)-.

14. The composition of claim 13, wherein L1 is selected from GGFG**, PVGLIG**, PLGLAG**, FF**, GPQGIWGQ**, GPQGIAGQ**, VPMSMRGG**, QPQGLAK**, GPLGLSGK**, GPLGMHGK**, VPLSLYSG**, IPVSLRSG**, VPLSLTMG**, IPESLRAG**, GPLGLWAR**, SGESPAYYTA**, RPFSMIMG**, GGYAELRMGG**, GGPLGLYAGG**, CGPQGIWGQC**, GKKCGPQGIWGQCKKG**, CGGPLGLAGGC**, or GCRDGPQGIWGQDRCG**, wherein the ** end of L1 represents the point of attachment to X.

15. The composition according to any one of claims 6 to 9, wherein: L1 is an amino acid residue, optionally, L1 is selected from glycine or phenylalanine; and L2, L3 and L4 are direct bonds.

16. The composition of claim 6, wherein L is selected from the group consisting of a direct bond, glycine, phenylalanine, **-C(O)(CH2) 1-12 -, **-C(O)(CH2) 1-12 C(O)NH(CH2) 1-6 -, **-C(O)(CH2) 1- 6(OCH2CH2) n -, **-C(O)(CH2) 1-12 C(O)-, **-C(O)(CH2) 1-6 (OCH2CH2) n -O-(CH2) 1-6 C(O)-, **-GFGG-C(O)(CH2) 1-12 C(O)NH(CH2) 1-6 -,**-GALGLP-C(O)(CH2) 1-12 -,**-GALGLP-C(O)(OCH2CH2) n -,**-GALGLP-C(O)(CH2) 1-12 (OCH2CH2) n -,**-GALGLP-C(O)(CH2) 1- 12 C(O)-, **-GFGG-C(O)(CH2) 1-12 C(O)-, **-GALGLP-C(O)(CH2) 1-6 (OCH2CH2) n -O-(CH2) 1- 6C(O)-、**-FF-C(O)(CH2) 1-12 -, **-(CH2) 1-12 -, **-C(O)O(CH2) 1-6 C(O)-, **-C(O)O(CH2) 1- 6C(O)NH(CH2) 1-6 - and **-C(O)(OCH2CH2) n -O-(CH2) 1-6 C(O)-.

17. The composition of any one of claims 6 to 16, wherein n is 2, 3 or 4.

18. The composition of any one of claims 6-17, wherein -XLY- is selected from the group consisting of: a direct bond, The * end indicates the connection point to the O atom; Optionally, -XLY- is selected from the group consisting of: Optionally, -XLY- is selected from 19. The composition of any one of claims 5-18, wherein the conjugate moiety has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

20. The composition according to any one of claims 5 to 19, wherein: The cross-linking agent is BDDE, 4,7,10-trioxa-1,13-tridecanediamine or glutaraldehyde; optionally, the cross-linking agent is 4,7,10-trioxa-1,13-tridecanediamine; and / or The molar ratio of the cross-linking agent to the conjugate portion is 0.10-0.40; optionally, the molar ratio of the cross-linking agent to the conjugate portion is 0.20; and / or The weight average molecular weight Mw of the hyaluronic acid polymer is 50-2000 kDa, optionally, the weight average molecular weight Mw of the hyaluronic acid polymer is 200-2000 kDa; and / or Based on the total weight of the composition, the content of capsaicin is 10-20%wt.

21. The composition according to any one of claims 5 to 20, wherein -XLY- is selected from The cross-linking agent is 4,7,10-trioxa-1,13-tridecanediamine, the molar ratio of the cross-linking agent to the conjugate portion is 0.20, the weight average molecular weight Mw of the hyaluronic acid polymer is 1000 kDa, and the content of capsaicin is 10-20% wt based on the total weight of the composition.

22. A composition comprising a hyaluronic acid matrix obtained by cross-linking hyaluronic acid polymers with a cross-linking agent conjugated with capsaicin.

23. The composition of claim 22, wherein the capsaicin-conjugated cross-linker has the following formula (II): in, W is CH or N; U and V are independently -OH, -NH2 or -NHNH2; L is -C(O)-, -C(O)-alkyl-C(O)-, -C(O)NH-alkyl-C(O)-, or -C(O)-alkyl-NHC(O)-.

24. The composition of claim 23, wherein L is -C(O)- or -C(O)-alkyl-C(O)-.

25. The composition according to claim 23 or 24, wherein: L is -C(O)-, and W is CH; or L is -C(O)(CH2) 1-6 C(O)-, and W is N.

26. The composition according to any one of claims 22 to 26, wherein the cross-linking agent conjugated with capsaicin is selected from 27. The composition according to any one of claims 22 to 26, wherein: The molar ratio of the cross-linking agent to the hyaluronic acid polymer is 0.10-0.60; optionally, the molar ratio of the cross-linking agent to the hyaluronic acid polymer is 0.20-0.50; and / or wherein the weight average molecular weight Mw of the hyaluronic acid polymer is 100-2000 kDa, optionally, the weight average molecular weight Mw of the hyaluronic acid polymer is 200-1000 kDa; and / or Based on the total weight of the composition, the content of capsaicin is 5-20% wt. Optionally, the content of capsaicin is 5-15% wt.

28. The composition according to any one of claims 22 to 27, wherein the molar ratio of the cross-linking agent to the hyaluronic acid polymer is 0.20, the weight average molecular weight Mw of the hyaluronic acid polymer is 1000 kDa, and the content of capsaicin is 5-15% wt based on the total weight of the composition.

29. The composition of any one of claims 22-28, wherein the hyaluronic acid matrix has a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

30. A pharmaceutical composition comprising the composition according to any one of claims 1 to 29 and one or more pharmaceutically acceptable carriers.

31. Use of the composition according to any one of claims 1 to 29 or the pharmaceutical composition according to claim 30 in the preparation of a medicament for treating arthritis.

32. A method of treating arthritis in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1-29 or the pharmaceutical composition of claim 30.

33. The method of claim 32, wherein the composition is administered topically to the subject, optionally by injection.

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