Compounds, compositions and methods of use comprising raltitrexed or 5-MTHF conjugated to a therapeutic agent

Abstract

Compounds comprising a radical of a therapeutic agent conjugated via a linker to a radical of raltitrexed, 5-methyltetrahydrofolate (5-MTHF), an analog of raltitrexed, or an analog of 5-MTHF; compositions comprising the same; and methods of immunomodulating Tregs.

Description

[Technical field]

[0001] Priority This patent application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 29,7938, filed January 12, 2022, and U.S. Provisional Patent Application No. 63 / 299,261, filed January 13, 2022, the contents of which are hereby incorporated by reference in their entireties into this disclosure.

[0002] Technical Field The present disclosure relates to compounds comprising a therapeutic agent conjugated (e.g., via a linker) to raltitrexed or 5-methyltetrahydrofolate (5-MTHF), compositions comprising same, and methods of use to immunomodulate regulatory T cells (Tregs), for example in patients exhibiting cancer or a fibrotic disease or disorder. [Background technology]

[0003] This section introduces aspects that may be helpful in facilitating a further understanding of the present disclosure and, as such, they should be read in this light and not be construed as admissions of what is or is not prior art.

[0004] Tumors are not simply masses of malignant cells, but rather may be a complex of many different components, some of which may surround cancer cells and directly affect their growth and malignant behavior, leading to invasion and metastasis. Currently, the majority of FDA-approved therapeutics focus on targeting and killing tumor cells. However, stromal cell types within the tumor microenvironment (TME) are genetically more stable. The TME contains multiple types of stromal cells, including immune cells, fibroblasts, and epithelial cells.

[0005] Successful targeting of stromal cells in the TME would allow cancer treatments with universal application; they would not be limited to a specific type of cancer. Therefore, identifying ways to reprogram stromal cells to be anti-tumorigenic is a major priority in cancer research.

[0006] Tumor cells in the TME are known to recruit regulatory T cells (Tregs) through several cytokines and chemokines. Tregs promote tumor growth and metastasis and inhibit antitumor immunity through complex and dynamic paracrine signaling by a network of cytokines as well as contact-dependent and contact-independent mechanisms. These mechanisms include direct cytotoxicity and inhibitory receptors, primarily the inhibition of CD8+ T cells. However, Tregs can be reprogrammed from inhibitors to promoters of antitumor immunity. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above, it is an object of the present disclosure to provide compounds, compositions containing same, and methods of using them to immunomodulate Tregs, for example in patients exhibiting cancer or fibrotic diseases or disorders. This and other objects and advantages, as well as features of the invention, will be apparent from the description provided herein. [Means for solving the problem]

[0008] summary Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof is provided, where T is a radical of raltitrexed, 5-methyltetrahydrofolic acid (5-MTHF), an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a radical of a therapeutic agent.

[0009] In certain embodiments, T has the structure of formula (II):

[0010] [ka] has.

[0011] In certain embodiments, T has a structure of formula (III):

[0012] [ka] Yes.

[0013] The therapeutic agent may be selected from the group consisting of Toll-like receptor 7 (TLR7) agonists, phosphoinositide 3-kinase (PI3k) inhibitors, steroids, nucleotide-binding oligomerization domain (NOD)-like receptor 2 (NLR2) agonists, stimulated interferon genes (STING) agonists, enhancer of zest homolog 2 (EZH2) inhibitors, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inhibitors, caspase I inhibitors, retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonists, absent in melanoma 2 (AIM2)-like receptor agonists, and receptor for advanced glycation end products (RAGE) agonists.

[0014] The therapeutic agent has the structure:

[0015] [ka] The compound may be an NLR2 agonist having the formula:

[0016] The therapeutic agent has the structure:

[0017] [ka] The compound may be a STING agonist having the following structure:

[0018] In certain embodiments, the therapeutic agent is an EZH2 inhibitor.

[0019] [ka] Or it may be tazemetostat.

[0020] In certain embodiments, the therapeutic agent has the structure:

[0021] [ka] It is an NLRP3 inhibitor having the following function:

[0022] In certain embodiments, the therapeutic agent has the structure:

[0023] [ka] It is a caspase I inhibitor having the following structure:

[0024] In certain embodiments, the therapeutic agent has the structure:

[0025] [ka] It is a PI3 kinase inhibitor having the following structure:

[0026] In certain embodiments, the therapeutic agent has the structure:

[0027] [ka] It is an RLR agonist having the following function:

[0028] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a TLR7 agonist of formula (IV):

[0029] [ka] or a radical of a pharma- ceutically acceptable salt thereof: R 1 , R 3 , R 4 , R 5 are each independently hydrogen (H), alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, aryl, halo, heteroaryl, -COR 2x ,

[0030] [ka] and; R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH 2、

[0031] [ka] and; Z is H, -OH, -NH2, -NHR 2x , -OR 2x , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0032] [ka] and; Each R 2x and R 2y is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q independently selected from the group consisting of: Each R 2q and R 2q’ are independently alkyl or H;

[0033] [ka] is a 3-10 membered N-containing non-aromatic, monocyclic or bicyclic heterocycle; R 21 is H or alkyl, n' is 0 to 30; In formula (IV), X 1 , X 2 , X 3 Each of the following is independently q or N, and each R q are independently H, halogen, or optionally substituted alkyl; n is 0 to 30; m is 0 to 4; When n is 0, Y is H, -OH, or -OR 2x isn't it).

[0034] E is a compound represented by formula (IVA):

[0035] [ka] or a radical of a pharma- ceutically acceptable salt thereof, R 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3: R 2x and R 2y are each independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl; Each R Z are independently hydrogen, halogen, or optionally substituted alkyl; R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; Z is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2, Each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, wherein the alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4.

[0036] In certain embodiments, the compound of formula (I) has formula (IVB) or formula (IVC):

[0037] [ka] or a pharma- ceutically acceptable salt thereof (wherein each R 1 is independently an optionally substituted C3-C8 alkyl; each R 2 are independently H, -OR z , -SO2N(R z )2, -NR 2x R 2y or N3; each R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z, -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; each R 4 and R 5 is independently alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; each Z 2 and Z 3 are independently represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; S 1 is a spacer; x is 0 to 3; n is 1 to 3, and m is 0 to 4).

[0038] In other embodiments, the compound of formula (I) has the formula (IVB):

[0039] [ka] or a pharma- ceutically acceptable salt thereof, 1is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3; R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; R 4 and R 5 each independently is alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; each Z 2 are represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x-alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; x is 0 to 3; n is 1 to 3; S 1 is a spacer ; m is 0 to 4.

[0040] In other embodiments, the compound of formula (I) has the formula (IVC):

[0041] [ka] or a pharma- ceutically acceptable salt thereof, 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3; R 2x and R 2y are each independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z, -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; R 4 and R 5 each independently is alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; each Z 3 are represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; x is 0 to 3; S 1 is a spacer; n is 1 to 3, and m is 0 to 4).

[0042] In certain embodiments where the compound of Formula (I) is represented by formula (IVB) or formula (IVC), R 1 is C1-C6 alkyl optionally substituted with 1-3 substituents, each of which is independently halogen or C1-C6 alkoxy; R 2 -NR 2x R 2y and R 2x and R2y are each independently H or C1-C6 alkyl; 3 are independently halogen, -CN, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, amino, hydroxy, carboxyl, or thiol; R 4 and R 5 are each independently C1 to C6 alkyl; 1 , X 2 , and X 3 is N;Z 2 and Z 3 is independently TL- or TLO-; n is 1; and m is 0 to 4. In certain embodiments where the compound of formula (I) is represented by formula (IVB) or formula (IVC), Z 2 and Z 3 Each of the above is TLO-.

[0043] In certain embodiments of formula (IV) or formula (IVA), Z is of the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2- groups: R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; S 1 is a spacer; and x is 0 to 3.

[0044] In certain embodiments of formula (IVA), R 1is C1-C6 alkyl optionally substituted with 1-3 substituents, each of which is independently halogen or C1-C6 alkoxy; R 2 -NR 2x R 2y and R 2x and R 2y are each independently H or C1-C6 alkyl; 3 are independently halogen, -CN, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, amino, hydroxy, carboxyl, or thiol; R 4 and R 5 are each independently C1 to C6 alkyl; 1 , X 2 , and X 3 is N; Z is TL- or TLO-; n is 1; and m is 0. Z may be TLO-.

[0045] R 1 R may be an optionally substituted C3-C6 alkyl. 1 R may be an optionally substituted acyclic C3-C6 alkyl. 2 -NR 2x R 2y R 2 may be -NH2.

[0046] The compound of formula (IVA) has the formula:

[0047] [ka] or one of its pharma- ceutically acceptable salts, 3 is optionally absent). Compounds of formula (IV) have the formula:

[0048] [ka] or one of the pharma- ceutically acceptable salts of any of the preceding formulas, 3 is not present as necessary).

[0049] R 1 may be a C1 to C6 alkyl. 2 R may be -NH2. 3 may not be present.

[0050] In certain embodiments, R 1 is C1-C6 alkyl; R 2 is -NH2; n is 1; R 3 does not exist.

[0051] In certain embodiments, the compound of formula (I) is a compound represented by formula (V).

[0052] [ka]

[0053] In certain embodiments, a radical (e.g., E) of the TLR7 agonist has the structure:

[0054] [ka] has.

[0055] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is

[0056] [ka] (It is.)

[0057] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a compound having the structure:

[0058] [ka] is a radical of X may be any of the following:

[0059] [ka]

[0060] E has the structure:

[0061] [ka] It may contain a radical of the formula:

[0062] In certain embodiments, the compound is of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E is the radical of a corticosteroid).

[0063] The corticosteroid may be betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone-17-valerate, budesonide, flumethasone, fluticasone propionate, flurocortisone, fludrocortisone, paramethasone, eplerenone, or an ester of any of the foregoing.

[0064] L may be a releasable linker. L may be a linker that is not releasable. L may comprise an optionally substituted heteroalkyl. In certain embodiments, the optionally substituted heteroalkyl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylate, and halo. L may comprise a substituted heteroalkyl having at least one disulfide bond in its backbone. L may comprise a peptide or peptidoglycan having at least one disulfide bond in its backbone. L may be a releasable linker that can be cleaved by an enzymatic reaction, reactive oxygen species (ROS), or reductive conditions. L may be a group of formula -NH-CH2-CR 6 R 7 -SS-CH2-CH2-O-CO-(wherein, R 6 and R 7 are each independently H, alkyl, or heteroalkyl. L may be a group of the formula:

[0065] [ka] or may contain these groups, where p is an integer from 0 to 30; d is an integer from 1 to 40; R 8 and R 9 are each independently H, alkyl, heterocyclyl, cycloalkyl, aryl, or heteroalkyl.

[0066] L may comprise one or more linker moieties, each of which is independently selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, carboxylate, carbonate, carbamate, amino acid, peptide, and peptidoglycan. L may be or comprise a peptide or peptidoglycan. L may be or comprise an amino acid. L may be or comprise a PEG group. L may be or comprise a polysaccharide. L has the structure:

[0067] [ka] (wherein w is 0 to 5 and p is 1 to 30). L is

[0068] [ka] where n″ is 0 to 30. L may be or include a linker moiety selected from the group consisting of:

[0069] Any of the above compounds may further comprise a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, wherein the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to a linker.

[0070] The compound may include an albumin binding group, such as:

[0071] [ka] an albumin binding group selected from the group consisting of for example:

[0072] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0073] In certain embodiments, the compound has the following structure:

[0074] [ka]

[0075] [ka] It comprises (e.g. consists of) one of:

[0076] In certain embodiments, the compound has the following structure:

[0077] [ka] It comprises (e.g. consists of) one of:

[0078] In certain embodiments, the compound has the following structure:

[0079] [ka]

[0080] [ka] It comprises (e.g. consists of) one of:

[0081] In certain embodiments, the compound has the following structure:

[0082] [ka] It comprises (e.g. consists of) one of:

[0083] Any of the above compounds may further comprise a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, where the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to a linker.

[0084] [ka] an albumin binding group selected from the group consisting of for example:

[0085] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0086] Pharmaceutical compositions are also provided. In certain embodiments, pharmaceutical compositions are provided herein that include a compound described herein (e.g., a compound of formula (I)) and one or more pharma- ceutically acceptable excipients. The pharmaceutical composition is a compound of formula F-L'-G The second compound or a pharma- ceutically acceptable salt thereof, wherein F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine.

[0087] Also provided is a combination of pharmaceutical compositions. The combination may comprise (i) the aforementioned pharmaceutical composition (e.g., a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof) and (ii) a pharmaceutical composition comprising a compound of formula F-L'-G or a pharma- ceutically acceptable salt thereof (wherein F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine). In certain embodiments, (i) and (ii) may be administered by the same route. In certain embodiments, (i) and (ii) may be administered by different routes. Whether administered by the same route or different routes, the combination may be administered simultaneously or sequentially in any order.

[0088] Further provided is a method of immunomodulating regulatory T cells (Treg) in a subject in need thereof. In certain embodiments, the method comprises administering to the subject an effective amount of a first compound (e.g., a compound of formula (I) or a pharma- ceutically acceptable salt thereof) or a pharmaceutical composition comprising the first compound. The method may further comprise administering a second compound of formula F-L'-G or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine. The administration of the second compound may be performed simultaneously with the first compound or the first pharmaceutical composition, or sequentially in any order, by the same or different routes.

[0089] In certain embodiments of the method, the subject has cancer, E (of the first compound) is a radical of TLR7 agonist, PI3K inhibitor, NLR2 agonist, STING agonist, EZH2 inhibitor, NLRP3 inhibitor, caspase I inhibitor, or RLR agonist, and administering an effective amount of the first compound or the first pharmaceutical composition changes the promotion of tumor growth and metastasis and / or the inhibition of anti-tumor immunity by Treg in the subject.The method may further comprise administering a third therapeutic agent, for example, an anti-cancer agent, to the subject.The anti-cancer agent may be, for example, a chemotherapeutic agent or a radiotherapeutic agent.

[0090] The method may further comprise administering to the subject a second compound of formula F-L'-G, or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine, and the administration of the second compound may be simultaneous with the administration of the first compound or sequentially, in any order, by the same or a different route.

[0091] In certain embodiments of the method, the subject has a fibrotic disease or disorder, and E of the first compound (e.g., a compound of formula (I) or a pharma- ceutically acceptable salt thereof) or the first pharmaceutical composition is a radical of a therapeutic agent selected from the group consisting of TLR7 agonists, PI3K inhibitors, steroids, NLR2 agonists, STING agonists, EZH2 inhibitors, NLRP3 inhibitors, caspase I inhibitors, and RLR agonists. The method may further comprise administering a second compound of formula F-L'-G or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine, and the administration of the second compound may be by the same or different route, simultaneously with the administration of the first compound, or sequentially in any order.

[0092] In yet another embodiment of the method, the subject has an inflammatory disease, and the method includes administering an effective amount of a first compound (e.g., a compound of formula (I) or a pharma- ceutically acceptable salt thereof) or a first pharmaceutical composition, where E is a radical of a steroid. The method may further include administering a second compound of formula F-L'-G or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine, and the administration of the second compound may be performed simultaneously with the administration of the first compound or sequentially in any order, by the same or different route.

[0093] Further provided herein is a method of treating cancer in a subject (e.g., a subject in need thereof), comprising administering to the subject a therapeutically effective amount of a first compound (e.g., a compound of Formula (I) or a pharma- ceutically acceptable salt thereof).

[0094] Further provided herein is a method of treating a fibrotic disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I)) or a pharma- ceutical acceptable salt thereof.

[0095] Further provided herein is a method of treating an inflammatory disease in a subject (e.g., a subject in need thereof), comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I)) or a pharma- ceutical acceptable salt thereof.

[0096] The disclosed embodiments, as well as other features, advantages, and aspects contained herein, and the methods for achieving the same, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when considered in conjunction with the accompanying drawings. [Brief description of the drawings]

[0097] [Figure 1-1] 1A-1D show data from an in silico analysis of the binding affinity of several folate analogs to the mouse and human folate receptor delta (FRδ), with FIG. 1A showing the structures of folic acid and raltitrexed. [Figure 1-2] FIG. 1B shows the predicted relative binding free energies (MMGBSA dGbind) and docking scores (XP GScore) for the binding of folate analogs to human FRδ (PDB: 5F4Q) and mouse FRδ (PDB: 5JYJ). [Figure 1-3]FIG. 1C shows the binding pose of raltitrexed to human FRδ; FIG. 1D shows the binding pose of raltitrexed to mouse FRδ. The ribbon diagram shows the ligand-protein interaction while the surface topography shows the electrostatic potential map and the binding orientation of the ligand in the FRδ binding cavity. In FIG. 1C and 1D, the areas labeled "blue" represent the positively charged regions of the binding site and the areas labeled "red" represent the negatively charged regions. [Figure 1-4] Figure ID shows the binding pose of raltitrexed to mouse FRδ. The ribbon diagram shows the ligand-protein interaction while the surface topography shows the electrostatic potential map and the binding orientation of the ligand in the FRδ binding cavity. In Figures 1C and 1D, the areas labeled "blue" represent the positively charged regions of the binding site and the areas labeled "red" represent the negatively charged regions. [Figure 2-1] Figures 2A and 2B show Raltitrexed-S0456 (Ral-S056) and Folate-S456 targeting to the tumor environment in vivo, with Figure 2A showing images of tumor-bearing mice taken 4 hours after injection with 10 nmol of Raltitrexed-S0456 (R-S0456) with or without 200-fold (200X) concentration of Raltitrexed-Glucosamine (Ral-Glucosamine) and 10 nmol of Folate-S0456 with or without 200-fold concentration of Folate-Glucosamine. The glucosamine conjugate serves as a competing ligand and is compared to a negative control. [Figure 2-2] FIG. 2B shows images of major organs taken from the treated mice shown in FIG. 2A. [Figure 2-3] FIG. 2B shows images of major organs taken from the treated mice shown in FIG. 2A. [Figure 3-1] 3A-3K show the targeting strategy and flow cytometry analysis of digested tumors and spleens from treated mice, with FIG. 3A showing the results of the flow cytometry scatter plots for viable cells. [Figure 3-2]FIG. 3B shows the results of a flow cytometry scatter plot for cells labeled with anti-CD45 antibody. [Figure 3-3] FIG. 3C shows the results of a flow cytometry scatter plot for cells labeled with anti-CD4 and anti-CD25 antibodies. [Diagram 3-4] FIG. 3D shows the results of a flow cytometry scatter plot for cells labeled with anti-CD127 and anti-FRδ antibodies. [Figure 3-5] FIG. 3E shows uptake of Ral-S0456 by CD45+CD4+CD25+CD127+FRδ+ Tregs isolated from murine tumors and spleens with or without 200-fold concentration of Ral-glucosamine competition compared to folate-S0456 and unstained negative controls. [Diagram 3-6] FIG. 3F shows a comparison of the uptake of Ral-S0456 and folate-S0456 by different leukocyte populations in the tumor. [Diagram 3-7] FIG. 3G shows results confirming the absence of binding of Ral-S0456 or folate-S0456 to CD45-cell conjugates. [Diagram 3-8] FIG. 3H shows graphical data supporting the absence of binding of Ral-S0456 or folate-S0456 to CD45+CD8+ cytotoxic T cells. [Diagram 3-9] FIG. 3I shows graphical results confirming the absence of binding of Ral-S0456 or folate-S0456 to CD45+CD4+CD25-FRδ- cells. [Figure 3-10] FIG. 3J shows graphical results confirming that although there is some binding of Ral-S0456, there is significantly more binding of folate-S0456 to CD45+CD11b+F4 / 80 macrophages. [Figure 3-11] FIG. 3K shows graphical data supporting the binding of Ral-S0456 to tumor Tregs in FRδ wild-type mice versus Ral-S0456 to Tregs in FRδ knockout mice. [Figure 4]FIG. 4 shows a scheme for the synthesis of raltitrexed-S0456 utilized in the binding studies shown in FIGS. 2A-3K. [Diagram 5] FIG. 5 shows an alternative scheme for the synthesis of an embodiment of the raltitrexed-S0456 conjugate described herein (ns=not significant; *=significant and P<0.05; **=significant and P<0.01). [Figure 6] FIG. 6 shows a scheme for the synthesis of a Toll-like receptor 7 (TLR7) agonist, referred to herein as TLR7-1A. [Figure 7] Figures 7A-7E show the immune modulating effect of TLR7-1A on murine CD45+CD4+CD25+ Tregs in vitro. Murine CD45+CD4+CD25- effector T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye and cell division was tracked by flow cytometry. Figure 7A shows representative graphical data of parental CFSE-labeled CD4+CD25- populations without co-cultured Tregs, treatment, or CD3 / CD28 bead activation; Figure 7B shows representative graphical data of split CFSE-labeled CD4+CD25- populations upon activation with co-cultured Tregs or without treatment; Figure 7C shows representative graphical data of the effect of Tregs on the division of CFSE-labeled CD4+CD25- effector T cells when co-cultured at a 1:4 ratio; Figure 7D shows representative graphical data of TLR7-1A after 10 nM TLR7-1A prior to co-culture with CFSE-labeled CD4+CD25- cells at a 1:4 ratio. Representative graphical data of the effect of Tregs pretreated with TLR7-1A for 3 hours are shown; FIG. 7E shows a bar graph representation of the data in FIGS. 7A-7D. [Figure 8]FIG. 8 shows graphical data depicting the immune modulating effect of TLR7-1A on murine CD45+CD4+CD25+ Tregs in vitro, where Tregs were pretreated with TLR7-1A for 3 hours and then co-cultured with murine CD45+CD4+CD25- effector T cells for 48 hours, after which supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) release. [Figure 9] FIG. 9 shows a scheme for the synthesis of a raltitrexed-TLR7-1A (Ral-TLR7-1A) releasable conjugate. [Figure 10-1] Figures 10A-10C show data regarding the effect of Ral-TLR7-1A on tumor growth, body weight, and immune cell composition of murine mammary 4T1 subcutaneous tumors in BALB / c mice, with Figure 10A showing a graph of 4T1 tumor volume change over the treatment course; Figure 10B showing a graph of mouse body weight change over the treatment course. [Figure 10-2] FIG. 10C shows bar graphs of the analysis of phenotypic markers of Tregs, CD8+ cytotoxic T cells, and macrophages, with the relevant phenotypic markers listed on each y-axis and treatment regimens indicated on each x-axis as 1 (untreated control), 2 (Ral-TLR7-1A), 3 (Ral-TLR7-1A+Ral-Gluc), or 4 (raltitrexed). [Figure 11] FIG. 11 shows a graph of data obtained from evaluation of the effect of Ral-TLR7-1A on phenotypic markers of splenic Tregs and CD8+ cytotoxic T cells isolated from the tumor-bearing mice of FIG. 10A, with the relevant phenotypic markers listed on each y-axis and treatment regimens indicated on each x-axis as 1 (untreated control), 2 (Ral-TLR7-1A), 3 (Ral-TLR7-1A+Ral-Gluc), or 4 (raltitrexed). [Figure 12] FIG. 12 shows a graph of data depicting the effect of Ral-TLR7-1A on tumor growth and body weight of murine colorectal CT26 subcutaneous tumors in BALB / c mice. [Figure 13-1]Figures 13A and 13B show graphical data regarding the effect of Ral-TLR7-1A on tumor growth, body weight, and immune cell composition of murine bladder MB49 subcutaneous tumors in FRβ knockout C57BL / 6 mice, with Figure 13A showing plots of MB49 tumor volume change and mouse body weight change over the course of treatment. [Figure 13-2] FIG. 13B shows a graph of the analysis of phenotypic markers of Treg and CD8+ cytotoxic T cells. [Figure 14] FIG. 14 shows a scheme for the synthesis of Ral-dexamethasone as described herein and used in Example 10. [Figure 15] Figures 15A and 15B show graphical data on the effect of Ral-TLR7-1A or Ral-dexamethasone on tumor growth and immune cell composition of murine mammary 4T1 subcutaneous tumors in BALB / c mice, with Figure 15A showing the change in 4T1 tumor volume over the course of treatment; Figure 15B showing analysis of phenotypic markers of Treg and CD8+ cytotoxic T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] While the present disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0099] Detailed Description For the purpose of promoting understanding of the principles of the present specification, reference will be made to the embodiments shown in the drawings and specific language will be used to describe them. Nevertheless, it will be understood that no limitation of scope is intended by the description of these embodiments. On the contrary, the present disclosure is intended to cover such alternatives, modifications, and equivalents as may be included within the spirit and scope of the present application as defined by the appended claims. As mentioned above, the technology is shown and described in one or more preferred embodiments, but the compounds, compositions, and their uses may include many different configurations, forms, materials, and accessories.

[0100] compound The present disclosure is based, at least in part, on the design of conjugates comprising immunomodulatory small molecules conjugated to suitable targeting ligands, such as raltitrexed or 5-methyltetrahydrofolate (5-MTHF). The compounds (e.g., immunomodulatory small molecules) can alter regulatory T cells (Tregs) to promote tumor growth and metastasis and / or inhibit anti-tumor immunity. Upon ligand binding, the compounds are internalized by target cells, thus reducing, if not eliminating, off-target effects and toxicity.

[0101] T may be any suitable targeting ligand, such as a folate mimetic (i.e., a compound other than folic acid that mimics folic acid and can be bound by folate receptor beta (FRβ) or folate receptor delta (FRδ)). T may be an antifolate. An antifolate can specifically bind to FRδ with a relative affinity compared to folic acid of about 0.05 or greater at about 20° C. / 25° C. / 30° C. / above physiological temperature.

[0102] A non-limiting example of a suitable antifolate is raltitrexed. Folic acid analogs and derivatives, such as 5-MTHF, may also be suitable targeting ligands. Other suitable targeting ligands may be screened, for example, for binding to FRδ on isolated Tregs and identified using IC 50 (See, for example, Example 2). Examples of targeting ligands that can be screened for binding to FRδ include, but are not limited to, folate analogs, such as folinic acid, pteropolyglutamic acid, folate receptor binding pteridines, such as tetrahydropterin, dihydrofolate, tetrahydrofolate, and their deaza and dideaza analogs. "Deaza" and "dideaza" analogs refer to art-recognized analogs in which one or two nitrogen atoms are substituted for carbon atoms in the naturally occurring folic acid structure. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. Dideaza analogs include, for example, 1,5 dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. Other examples of targeting ligands that can be screened for binding to FRδ include, but are not limited to, pemetrexed, proguanil, pyrimethamine, trimethaprim, pralatrexate, aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, and their deaza analogs, such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpentenoylglutamic acid (dichloromethotrexate).

[0103] In certain embodiments, T may have a higher binding affinity for FRδ than for FRβ (i.e., T may bind "preferentially" to FRδ over FRβ). In certain embodiments, T may bind preferentially to FRδ over FRβ in a ratio of about 1.5:1, about 2:1, or about 2.5:1 (see, e.g., the cell binding data shown in FIG. 3F, where the second to last column refers to uptake by Tregs (FRδ) and the last column refers to uptake by macrophages (FRβ), where the ratio of uptake by Tregs to uptake by macrophages is approximately the same as the ratio of FRδ binding to FRβ binding).

[0104] Binding assays can be used to determine the binding affinity and / or binding affinity ratio for the ligands herein for FRδ versus other folate isoforms (e.g., folate receptor alpha (FRα), FRβ, etc.). For example, but not by way of limitation, comparative binding affinities may be determined according to the methodology illustrated in Example 3. In certain embodiments, comparative binding affinities may be determined by transfecting either mouse or human folate receptor isoforms (e.g., FRα, FRβ, or FRδ) into a cell line (e.g., HEK293 cell line). If expression is confirmed, the binding affinity of the ligands can be tested for each receptor of interest by incubating cells with increasing concentrations of various analogs that are conjugated to a dye (e.g., S0456 NIR dye) that can be detected and quantified (e.g., by flow cytometry). The relative binding of each ligand to each receptor isoform can then be calculated based on the quantified binding data to determine which ligands have higher binding affinity (i.e., "preferential binding") for each receptor isoform compared to the other tested ligands.

[0105] The present disclosure relates in part to a compound of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, or an analog thereof; L is a linker; and E is a radical of a therapeutic agent. In certain embodiments, T is a radical of raltitrexed. In other embodiments, T is a radical of 5-MTHF.

[0106] The present disclosure further provides a compound of formula (I'): T'-LE (I') or a pharma- ceutically acceptable salt thereof, wherein T' is a targeting moiety (e.g., raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF, a compound of Formula I-VII, Formula II-VII, Formula III-VII, Formula IV-VII, Formula V-VII, Formula VI-VII, or Formula VII-VII described herein, or a radical of a compound of Table I, II, or III); L is a linker; and E is a radical of a therapeutic agent (e.g., a radical of a therapeutic agent described herein). In some embodiments, T binds to a receptor on a cell. In some embodiments, T binds to a pattern recognition receptor in a cell. In some embodiments, T binds to an immune cell receptor. In some embodiments, T selectively binds to a folate receptor. In some embodiments, T selectively binds to FRβ. In some embodiments, T selectively binds to FRδ or preferentially binds to FRδ over FRβ.

[0107] In some embodiments, T is a radical that can have the structure of Formula I-VII:

[0108] [ka] (In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2、 N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl; n is 0 to 1;

[0109] [ka] is a CC single bond or a C=C double bond).

[0110] In some embodiments, T is a radical of a compound of Formula I-VII, which may further have the structure of Formula II-VII or Formula III-VII:

[0111] [ka] (In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH 2、 N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl; n is 0 to 1;

[0112] [ka] is a C—C single bond or a C═C double bond; or

[0113] [ka] is: X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2, N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl; n is 0 to 1;

[0114] [ka] is a CC single bond or a C=C double bond).

[0115] In some embodiments, T is a radical of a compound of formula II-VII, which may further have the structure of formula IV-VII or formula V-VII:

[0116] [ka] (In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2, N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl;

[0117] [ka] is a C—C single bond or a C═C double bond); or

[0118] [ka] (In the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2, N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl;

[0119] [ka] is a CC single bond or a C=C double bond).

[0120] In some embodiments, T is a radical of a compound of formula III-VII, which may further have the structure of formula VI-VI or formula V-VII:

[0121] [ka] (In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2, N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl;

[0122] [ka] is a C—C single bond or a C═C double bond); or

[0123] [ka] (In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S; Y is C, CH, CH2, N, NH, O, or S; Z is glutamic acid, valine, or suberate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is H or alkyl;

[0124] [ka] is a CC single bond or a C=C double bond).

[0125] In some embodiments, T is a radical of a compound of Formula IV-VII, which may have a structure selected from Table I.

[0126] [Table 1-1]

[0127] [Table 1-2]

[0128] [Table 1-3]

[0129] In some embodiments, T is a radical of a compound of Formula V-VII, which may have a structure selected from Table II.

[0130] [Table 2-1]

[0131] [Table 2-2]

[0132] In some embodiments, T is a radical of a compound of Formula VI-VII, which may have a structure selected from Table III.

[0133] [Table 3]

[0134] In certain embodiments, T is any suitable targeting ligand to which FRδ can bind. In certain embodiments, T may be a radical of any of the following structures:

[0135] [ka]

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142]

change

[0143]

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[0144]

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[0145]

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[0146]

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[0147]

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[0148]

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[0149]

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[0150]

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[0151]

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[0152]

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[0153]

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[0154]

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[0155]

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[0156]

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[0157]

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[0158]

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[0159]

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[0160]

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[0161]

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[0162]

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[0163]

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[0164]

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[0165]

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[0166]

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[0167]

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[0168]

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[0169]

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[0170]

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[0171]

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[0172]

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[0173]

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[0174]

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[0175]

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[0176]

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[0177]

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[0178]

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[0179]

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[0180]

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[0181]

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[0182]

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[0183]

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[0184]

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[0185]

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[0186]

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[0187]

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[0188]

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[0189]

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[0190]

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[0191]

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[0192]

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[0193]

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[0194]

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[0195]

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[0196]

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[0197]

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[0198]

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[0199]

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[0200]

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[0201]

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[0202]

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[0203]

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[0204]

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[0205]

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[0206]

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[0207]

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[0208]

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[0209]

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[0210]

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[0211]

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[0212]

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[0213]

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[0214]

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[0215]

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[0216]

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[0217]

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[0218]

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[0219]

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[0220]

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[0221]

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[0222]

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[0223]

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[0224]

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[0225]

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[0226]

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[0227]

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[0228]

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[0229]

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[0230]

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[0231]

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[0232]

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[0233]

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[0234]

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[0235]

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[0236]

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[0237]

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[0238]

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[0239]

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[0240]

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[0241]

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[0242]

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[0243]

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[0244]

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[0245]

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[0246]

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[0247]

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[0248]

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[0249]

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[0250]

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[0251]

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[0252]

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[0253]

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[0254]

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[0255]

change

[0256]

change

[0257]

change

[0258] [ka]

[0259] [ka]

[0260] In certain embodiments, the compounds described herein have the structure of Formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, where T is a radical of raltitrexed, 5-MTHF, or an analog of either raltitrexed or 5-MTHF; L is a linker; and E is a radical of a therapeutic agent. In certain embodiments, L (i.e., the linker) is selected from the group consisting of a linker and a spacer (e.g., S 1 ) may also be included.

[0261] T is a structure of formula (II):

[0262] [ka]

[0043]

[0263] T is a structure of formula (III):

[0264] [ka]

[0043]

[0265] T may be an analogue of formula (II) or formula (III).

[0266] In certain embodiments, the compound has the structure of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, in which T is a radical of raltitrexed, 5-MTHF, or an analog of raltitrexed or 5-MTHF; L is a linker; and E is a TLR7 agonist of formula (IV):

[0267] [ka] or a radical of a pharma- ceutically acceptable salt thereof: R 1 , R 3 , R 4 , and R 5 are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, cycloalkyl, arylhalo, heteroaryl, -COR 2x ,

[0268] [ka] and; R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH 2、

[0269] [ka] and; Z is H, -OH, -NH2, -NHR 2x , -OR 2x , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0270] [ka] is: Each R 2x and R 2y is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q each R 2q and R 2q’ are independently alkyl or H;

[0271] [ka] is a 3-10 membered N-containing non-aromatic, monocyclic or bicyclic heterocycle; R 21 is H or alkyl, n' is 0 to 30; In formula (IV): X 1 , X 2 , X 3 Each of the following is independently q or N, and each R q are independently H, halogen, or optionally substituted alkyl; n is 0 to 30; m is 0 to 4; When n is 0, Y is H, -OH, or -OR 2x isn't it).

[0272] E of the compounds herein (e.g., therapeutic agent radical) can be any suitable therapeutic agent, e.g., an immunomodulatory small molecule. Non-limiting examples of E include immunostimulants that stimulate the immune system by inducing activation or increasing the activity of any of its components. The therapeutic agent may be selected from the group consisting of Toll-like receptor (TLR) agonists (e.g., TLR7 agonists), phosphoinositide 3-kinase (PI3K) inhibitors, steroids, nucleotide-binding oligomerization domain (NOD)-like receptor 2 (NLR2) agonists, stimulated interferon genes (STING) agonists, enhancer of zest homolog 2 (EZH2) inhibitors, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inhibitors, caspase I inhibitors, retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonists, absent-in-melanoma 2 (AIM2)-like receptor agonists, and receptor for advanced glycation end products (RAGE) agonists.

[0273] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., an NLR2 agonist) that binds to NLR2. For example, an NLR2 agonist may be

[0274] [ka] may be also possible.

[0275] E can be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., a STING agonist) that binds to STING. For example, a STING agonist can be

[0276] [ka] may be also possible.

[0277] Additional non-limiting examples of STING agonists include:

[0278] [ka]

[0279] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., an EZH2 inhibitor) that binds to EZH2. For example, and without limitation, an EZH2 inhibitor can be

[0280] [ka] may be also possible.

[0281] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., an NLRP3 inhibitor) that binds to NLRP3. For example, an NLRP3 inhibitor is

[0282] [ka] may be also possible.

[0283] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., a caspase I inhibitor) that binds to caspase I. For example, a caspase I inhibitor may be

[0284] [ka] may be also possible.

[0285] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., a PI3K agonist) that binds to PI3K. Non-limiting examples of PI3K agonists include, but are not limited to,

[0286] [ka] There is.

[0287] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., an RLR agonist) that binds to an RLR. Non-limiting examples of RLR agonists include:

[0288] [ka] It is.

[0289] E may be the radical of any suitable immunomodulatory (e.g., immunoinhibitory) small molecule (e.g., a TLR agonist) that binds to a TLR. In certain embodiments, E is the radical of a TLR7 agonist.

[0290] In certain embodiments of the compound, E is a radical of a TLR7 agonist of formula (IVA):

[0291] [ka] or a pharma- ceutically acceptable salt thereof, R 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3; Z is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2 and: R2x and R 2y are each independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl; Each R Z are independently H, halogen, or optionally substituted alkyl; R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; Each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, wherein the alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein alkyl, alkoxy, and cycloalkyl are optionally substituted; n is 1 to 6; m is 0 to 4.

[0292] In the compound having a radical of the structure of the TLR7 agonist of formula (IVA), Z is a radical of the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x-alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-, where x is 0 to 3; 1 is a spacer; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy.

[0293] In certain embodiments of a compound having the structure of a TLR7 agonist of formula (IVA), x is 1 or 2. In certain embodiments of a compound having the structure of a TLR7 agonist of formula (IVA), n is 1 to 3. In certain embodiments of a compound having the structure of a TLR7 agonist of formula (IVA), m is 0 to 4. In certain embodiments of a compound having the structure of a TLR7 agonist of formula (IVA), x is 0 to 3; n is 1 to 3; and m is 0 to 4.

[0294] R 1 may be C1-C6 alkyl optionally substituted with 1-3 substituents, each of which is independently halogen or C1-C6 alkoxy; R 2 -NR 2x R 2y R may be 2x and R 2y are each independently hydrogen or C1-C6 alkyl. 3 may be independently halogen, -CN, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, amino, hydroxy, carboxyl, or thiol; R 4 and R 5 may each independently be C1-C6 alkyl; 1 , X 2 , and X3 may be N; Z may be TL- or TLO-; n may be 1; m may be 0; Z may be TLO-.

[0295] R 1 R may be an optionally substituted C3-C6 alkyl. 1 R may be an optionally substituted acyclic C3-C6 alkyl. 2 -NR 2x R 2y (as defined herein). 2 may be -NH2.

[0296] The TLR7 agonist of the compound has the formula:

[0297] [ka] or a pharma- ceutically acceptable salt of any of the preceding formulas, 3 is optionally absent). The TLR7 agonist may have the formula:

[0298] [ka] or one of the pharma- ceutically acceptable salts of any of the preceding formulas, 3 is not present as needed). 1 may be a C1 to C6 alkyl. 2 may be -NH2. In certain embodiments, R 3 is absent. In certain embodiments, R 1 is C1-C6 alkyl; R 2 is -NH2; n is 1; R 3 does not exist.

[0299] The compound containing the TLR7 agonist radical of formula (IVA) may be a compound of formula (V).

[0300] [ka]

[0301] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, or an analog of raltitrexed or 5-MTHF; L is a linker; and E is a group having the structure:

[0302] [ka] (Having).

[0303] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a group having the structure:

[0304] [ka] is a radical of a compound having the formula X may be any of the following structures:

[0305] [ka] ).

[0306] Non-limiting examples of TLR agonists include TLR7 agonists, TLR8 agonists, and TLR7 / 8 agonists, such as:

[0307] [ka]

[0308] [ka]

[0309] [ka]

[0310] [ka] There is.

[0311] Polydeoxythymine (poly dt) is a molecule made from strings of deoxythymidines linked via 3'-5' phosphodiester bonds. Oligonucleotides may be used for E. Examples of TLR9 agonists include, but are not limited to, CpG-ODN (short synthetic ssDNA containing unmethylated CpG dinucleotide motifs in a specific sequence context), IMO-2055 (synthetic oligonucleotide containing unmethylated CpG dinucleotides), and 1018ISS (short synthetic unmethylated CpG oligodeoxynucleotide (CpGODN)). A non-limiting example of a TLR3 agonist is poly(I:C) (polyinosine homopolymer annealed to a strand of polycytidine homopolymer).

[0312] In some embodiments, E may be an imaging agent, such as an optical or radioactive imaging agent. Non-limiting examples of optical imaging agents include infrared, near infrared, and luminescent imaging agents. The optical imaging agent may be a rhodamine or indole-cyanine green-like dye S0456.

[0313] E has the structure:

[0314] [ka] ,for example,

[0315] [ka] It may contain a radical of the formula:

[0316] Another compound provided is of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, where T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a radical of a corticosteroid. The corticosteroid may be betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone-17-valerate, budesonide, flumethasone, fluticasone propionate, flurocortisone, fludrocortisone, paramethasone, eplerenone, or an ester of any of the foregoing.

[0317] E (e.g., a radical of a therapeutic agent) of the compounds herein is preferably a spacer (S 1 ) which may or may not further comprise

[0318] Linkers (e.g., L and L') L of the compounds herein may be a releasable linker. L of the compounds herein may be a non-releasable linker. A releasable linker is a linker that includes at least one bond that can be cleaved under physiological conditions, such as reductive, acidic, basic, oxidative, metabolic, biochemical, enzymatic (e.g., cathepsin B-cleavable), or other conditions (e.g., p-aminobenzyl-based polyvalent releasable bonds) (see, e.g., International Patent Application Publication No. WO 2017 / 0205661). A non-releasable linker is, for example, a linker that includes an amide, ester, ether, amine, or thioether (e.g., thiomaleimide).

[0319] L of the compounds herein may comprise an optionally substituted heteroalkyl. The optionally substituted heteroalkyl may be substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylate, and halo. L may comprise a substituted heteroalkyl having at least one disulfide bond in its backbone. L may comprise a peptide or peptidoglycan having at least one disulfide bond in its backbone. In certain embodiments, L is a releasable linker that can be cleaved by enzymatic reaction, reactive oxygen species (ROS), or reductive conditions.

[0320] L is a group of the formula -NH-CH2-CR 6 R 7 -SS-CH-CH-O-CO- (wherein R 6 and R 7 are each independently H, alkyl, or heteroalkyl.

[0321] L is a group represented by the formula:

[0322] [ka] or may contain these groups. (wherein p is an integer from 0 to 30; d is an integer from 1 to 40; R 8 and R 9 are each independently H, alkyl, heterocyclyl, cycloalkyl, aryl, or heteroalkyl.

[0323] L may comprise one or more linker moieties independently selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, carboxylate, carbonate, carbamate, amino acid, peptide, and peptidoglycan. L may be or comprise a peptide or peptidoglycan. L may be or comprise an amino acid. L may be or comprise a PEG group. L may be or comprise a polysaccharide. L has the structure:

[0324] [ka] (wherein w is 0 to 5 and p is 1 to 30. L is a group represented by the formula:

[0325] [ka] The linker moiety may be or may include a linker moiety selected from the group consisting of: (n" is 0 to 30). L may be a divalent linker. L may be a trivalent linker.

[0326] In some embodiments, L is a pyrido[2,3-d]pyrimidine analog having the following structure:

[0327] [ka]

[0328] The linker may include a pharmacokinetic extender, such as an albumin binder or a hapten. Examples of albumin binders include, but are not limited to,

[0329] [ka] There is.

[0330] Examples of haptens include, but are not limited to, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), rhamnose, galactose-α-1,3-galactose (α-Gal), or antibody binding agents. Examples of antibody binding agents include, but are not limited to, Fab, scFv, V H , V L , V H H, V-NAR, monobody, anticalin, affibody, or DARPin.

[0331] In the compounds herein, L is a spacer (S 1 ) may be optionally conjugated to and / or may optionally include S 1 may be any suitable spacer. Examples of spacers include, but are not limited to, an alkyl chain containing at least about 20 carbon atoms, e.g., at least 20 carbon atoms, a PEG containing at least about 20 units, e.g., at least 20 units, in the chain, a sugar, a peptidoglycan, a clickable linker (e.g., a triazole), a rigid linker (e.g., polyproline or polypiperidine), or a combination of two or more of the foregoing.

[0332] All of the compounds are S 1 and S 1can be a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, and the PEG group, peptide group, glycopeptide group, saccharide group, or albumin binding group radical is attached to L. The compound may also be an albumin binding group, e.g.

[0333] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0334] In certain embodiments, L comprises one or more releasable linkers that cleave under the conditions described herein by a chemical mechanism involving beta-elimination. Such releasable linkers include beta-thio, beta-hydroxy, and beta-amino substituted carboxylic acids and their derivatives, such as esters, amides, carbonates, carbamates, and ureas. Such linkers also include 2- and 4-thioaryl esters, carbamates, and carbonates.

[0335] Examples of releasable linkers include those of the formula:

[0336] [ka] (wherein X 4 is NR, n is an integer selected from 0, 1, 2, and 3, and R 32 is H or a substituent, including substituents that can stabilize a positive charge on the aryl ring inductively or by resonance, such as alkoxy. The releasable linker may be further substituted.

[0337] The auxiliary cleavage of the releasable moiety of L can be a mechanism involving a benzylium intermediate, a benzyne intermediate, lactone cyclization, an oxonium intermediate, beta-elimination, etc. In addition to fragmentation after cleavage of the releasable moiety of L, the initial cleavage of the releasable linker can be promoted by an anchimerically auxiliary mechanism. Thus, in the example of the releasable moiety of L given above, a hydroxyalkanoic acid that can cyclize can promote cleavage of the methylene bridge, for example by an oxonium ion, promoting bond cleavage or subsequent fragmentation of the releasable linker. Alternatively, an acid-catalyzed auxiliary cleavage of the methylene bridge by an oxonium ion can initiate a cascade of fragmentation of this exemplary bivalent linker or fragments thereof. Alternatively, acid-catalyzed hydrolysis of the carbamate can promote beta-elimination of a hydroxyalkanoic acid, which can cyclize and promote cleavage of the methylene bridge, for example by an oxonium ion. Other chemical mechanisms of bond cleavage under metabolic, physiological, or cellular conditions can initiate such a cascade of fragmentation.Other chemical mechanisms of bond cleavage under metabolic, physiological, or cellular conditions can initiate such a cascade of fragmentation.

[0338] Exemplary mechanisms for cleavage of the divalent linkers described herein include the following 1,4 and 1,6 fragmentation mechanisms for carbonates and carbamates:

[0339] [ka] (In the formula, Nuc - is an exogenous or endogenous nucleophile, glutathione, or a bioreductant, and R a and X a is attached via the other part of the bivalent linker). a and Xa The position of is, for example, such that the resulting product is X a -S-Nuc and HO-R a H2N-R a It may be replaced so that

[0340] Although the above fragmentation mechanism is illustrated as a concerted mechanism, any number of separate steps may occur to affect the final fragmentation of the bivalent linker to the final product shown. For example, bond cleavage may also occur by acid-catalyzed elimination of the carbamate moiety, which may be assisted anchimerically by the stabilization provided by either the beta sulfur or the aryl group of the disulfide shown in the above example. In these variations of this embodiment, the releasable linker is the carbamate moiety. Alternatively, fragmentation may be initiated by nucleophilic attack on the disulfide group, cleaving to form a thiolate. The thiolate can intermolecularly displace a carbonate or carbamate moiety to form the corresponding thiacyclopropane. In the case of a benzyl-containing bivalent linker, following cleavage of the illustrated disulfide bond, the resulting phenylthiolate is further fragmented to release the carbonate or carbamate moiety by forming a resonance-stabilized intermediate. In any of these cases, the releasable nature of the exemplary bivalent linkers described herein can be achieved by whatever mechanism is related to the chemical, metabolic, physiological, or biological conditions present.

[0341] Thus, as discussed above, the releasable linker may comprise a disulfide group. Further examples of releasable linkers within L include divalent radicals comprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl groups, each of which is optionally substituted. Additional examples of releasable linkers within L include methylene groups, 1-alkoxyalkylene groups, 1-alkoxycycloalkylene groups, 1-alkoxyalkylenecarbonyl groups, 1-alkoxycycloalkylenecarbonyl groups, carbonylarylcarbonyl groups, carbonyl(carboxyaryl)carbonyl groups, carbonyl(biscarboxyaryl)carbonyl groups, haloalkylenecarbonyl groups, alkylene(dialkylsilyl) groups, alkylene(alkylarylsilyl) groups, alkylene(diarylsilyl) groups, (dialkylarylsilyl) groups, and divalent radicals including (arylsilyl)aryl groups, (alkylarylsilyl)aryl groups, (diarylsilyl)aryl groups, oxycarbonyloxy groups, oxycarbonyloxyalkyl groups, sulfonyloxy groups, oxysulfonylalkyl groups, iminoalkylidenyl groups, carbonylalkylidenyliminyl groups, iminocycloalkylidenyl groups, carbonylcycloalkylidenyliminyl groups, alkylenethio groups, alkylenearylthio groups, or carbonylalkylthio groups, each releasable linker being optionally substituted.

[0342] Additional examples of releasable linkers included in L may include an oxygen atom and a methylene group, a 1-alkoxyalkylene group, a 1-alkoxycycloalkylene group, a 1-alkoxyalkylenecarbonyl group, or a 1-alkoxycycloalkylenecarbonyl group, each of which is optionally substituted. Alternatively, in some embodiments, the releasable linker includes an oxygen atom and a methylene group, the methylene group being substituted with an optionally substituted aryl, and the releasable linker is bonded to the oxygen to form an acetal or ketal. Furthermore, in some embodiments, the releasable linker includes an oxygen atom and a sulfonylalkyl group, and the releasable linker is bonded to the oxygen to form an alkyl sulfonate.

[0343] Additional examples of releasable linkers within L include an iminoalkylidenyl group, a carbonylalkylidenyliminyl group, an iminocycloalkylidenyl group, and a carbonylcycloalkylidenyliminyl group, each of which is optionally substituted, and which is attached to the nitrogen to form a hydrazone. In some embodiments, the hydrazone is acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form a variety of acylhydrazone releasable linkers.

[0344] Additional examples of releasable linkers within L include an oxygen atom and an alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl or (diarylsilyl)aryl group, each optionally substituted, where the releasable linker is bonded to the oxygen to form a silanol.

[0345] Additional examples of L containing releasable linkers include two independent nitrogens and a carbonylarylcarbonyl, a carbonyl(carboxyaryl)carbonyl, or a carbonyl(biscarboxyaryl)carbonyl. In some embodiments, the releasable linker is attached to a heteroatom nitrogen to form an amide.

[0346] Additional examples of L containing releasable linkers include an oxygen atom, a nitrogen, a carbonylarylcarbonyl, a carbonyl(carboxyaryl)carbonyl, or a carbonyl(biscarboxyaryl)carbonyl. In some embodiments, the releasable linker forms an amide.

[0347] In some embodiments, L comprises an optionally substituted 1-alkylenesuccinimid-3-yl group and a releasable moiety comprising a methylene group, a 1-alkoxyalkylene group, a 1-alkoxycycloalkylene group, a 1-alkoxyalkylenecarbonyl group, or a 1-alkoxycycloalkylenecarbonyl group, each of which can be optionally substituted to form a succinimid-1-yl alkyl acetal or ketal.

[0348] In some embodiments, L comprises carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, or 1-(carbonyltetrahydrofuranyl)succinimid-3-yl, each of which is optionally substituted. In some embodiments, L further comprises an additional nitrogen such that L comprises an alkylenecarbonyl group, a cycloalkylenecarbonyl group, a carbonylalkylcarbonyl group, or a 1-(carbonylalkyl)succinimid-3-yl group, each of which is optionally substituted, which is attached to the nitrogen to form an amide. In some embodiments, L further comprises a sulfur atom and an alkylene group or a cycloalkylene group, each of which is optionally substituted with carboxy, which is attached to the sulfur to form a thiol. In some embodiments, L comprises a sulfur atom and a 1-alkylenesuccinimid-3-yl, such that the 1-(carbonylalkyl)succinimid-3-yl group is attached to the sulfur to form a succinimide-3-ylthiol.

[0349] In some embodiments, L comprises a nitrogen and a releasable moiety comprising an alkyleneaziridin-1-yl, a carbonylalkylaziridin-1-yl, a sulfoxylalkylaziridin-1-yl, or a sulfonylalkylaziridin-1-yl, each of which is optionally substituted. In some embodiments, L comprises a carbonyl, a thionocarbonyl, an alkylenecarbonyl, a cycloalkylenecarbonyl, a carbonylalkylcarbonyl, or a 1-(carbonylalkyl)succinimide-3-yl, each of which is optionally substituted, which is attached to the releasable moiety to form an aziridine amide.

[0350] L may also include alkylene-amino-alkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimide-3-yl), and the like, as further depicted by the formula:

[0351] [ka] wherein x' and y' are each independently 1, 2, 3, 4, or 5.

[0352] L may have any suitable type of atom in the chain, including C (e.g., -CH2-, C(O)), N (e.g., NH, NR b , where R bis, for example, H, alkyl, alkylaryl, etc.), O (e.g., -O-), P (e.g., -OP(O)(OH)O-), and S (e.g., -S-). For example, the atoms used in forming L can be linked in any chemically appropriate manner, for example, a chain of carbon atoms forming an alkyl group, a chain of carbon and oxygen atoms forming a polyoxyalkyl group, a chain of carbon and nitrogen atoms forming a polyamine, rings, e.g., aryl and heterocyclyl groups (e.g., triazole, oxazole, etc.), and others. Furthermore, the bonds connecting the atoms of the chain in L can be either saturated or unsaturated, such as, for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, etc., can be divalent radicals included in L. Furthermore, the chain forming L can be substituted or unsubstituted.

[0353] Additional examples of L groups include 1-alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimid-3-yl, each of which may be substituted or unsubstituted. Any of the foregoing groups may be L or may be included as part of L. In some cases, any of the foregoing groups can be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may further include additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)-OR-NH-alkyl-), oxygen (e.g., -alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-). Examples of such L groups are alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, and succinimid-3-ylthiol, each group may be substituted or unsubstituted.

[0354] In some embodiments, L is formed via click chemistry / click chemistry-derived. Those skilled in the art will appreciate that the terms "click chemistry" and "click chemistry-derived" generally refer to certain small molecule reactions that are commonly used in conjugation to link selected substrates with specific molecules. Click chemistry is not a single specific reaction, but describes a method to generate products following examples from nature, and also to generate substances by combining small modular units. In many applications, click reactions link biomolecules with reporter molecules. Click chemistry is not limited to biological conditions: the concept of "click" reactions has been used in pharmacological and various biomimetic applications. However, they have been found to be particularly useful in the detection, localization and qualification of biomolecules.

[0355] Click reactions can occur in one pot, are typically not hindered by water, produce minimal by-products, are "spring loaded" i.e., are characterized by a high thermodynamic driving force, make the reaction fast and irreversible to give a single reaction product in high yield, and are associated with high reaction specificity (in some cases, both regiospecificity and stereospecificity). These properties make them well suited to the challenges of isolating and targeting molecules in complex biological environments. Thus, in such environments, the products need to be physiologically stable and any by-products non-toxic (as for in vivo systems).

[0356] Examples of click chemistry include those in which L can be derived from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (IEDDA), and Staudinger ligation (SL). a and R a can be linked together as shown in Schemes 1-5:

[0357] [ka] (In the formula, each R b are independently H, alkyl, arylalkyl, -alkyl-S-alkyl or arylalkyl or the side chain of any naturally occurring or unnatural amino acid, etc. a and R a The wavy line connected to X a and R a and the group to which they are attached. It should be understood that in Schemes 1-5, triazole, oxazole, and -NH-SO2-NH- groups would be considered to be part of L.

[0358] In some embodiments, L is a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers; L is either a non-releasable or releasable linker that is bivalently covalently attached to a folate ligand (or, in other embodiments, a folate analog or antifolate) and a steroid.

[0359] In some embodiments, L is

[0360] [ka] is (In the formula, x" is an integer from 0 to 10, and y″ is an integer from 3 to 100.

[0361] In some embodiments, x″ is an integer from 3 to 10.

[0362] In some embodiments, L is

[0363] [ka] is (In the formula, each R 33 and R 34 is independently H or C1-C6 alkyl; Z is an integer from 1 to 8.

[0364] In some embodiments, L is

[0365] [ka] It is.

[0366] In some embodiments, L is

[0367] [ka] is (In the formula, R 37 is H or C1-C6 alkyl; R 35a , R 35b , R 36a , and R 36b are each independently H or C1-C6 alkyl.

[0368] In some embodiments, L comprises an amino acid. In some embodiments, L comprises an amino acid selected from the group consisting of Lys, Asn, Thr, Ser, Ile, Met, Pro, His, Gln, Arg, Gly, Asp, Glu, Ala, Val, Phe, Leu, Tyr, Cys, and Trp. In some embodiments, L comprises at least two amino acids independently selected from the group consisting of Glu and Cys. In some embodiments, L comprises Glu-Glu, where the glutamic acids are covalently linked to each other via the carboxylic acid side chains.

[0369] In some embodiments, L comprises one or more hydrophilic spacer linkers that include multiple hydroxyl functional groups.

[0370] In some embodiments, L comprises at least one 2,3-diaminopropionic acid group, at least one glutamic acid group (e.g., the unnatural amino acid D-glutamic acid), and at least one cysteine ​​group. One example of such a linker is one with an unnatural amino acid, such as the formula:

[0371] [ka] is a linker having a repeat unit of where q is an integer from 1 to 10 (e.g., 1 to 3 and 2 to 5). In some embodiments, L has the general formula:

[0372] [ka] Includes wherein X can be O, NH, NR, or S, and q is an integer from 1 to 10. In some embodiments, L is a group represented by the formula:

[0373] [ka] Includes where the disulfide group is part of a self-immolative group which may be generally described as a group of the formula -CH2-SS-CH2-.

[0374] In some embodiments, the compounds described herein include a bond that allows the steroids described herein to be released by any suitable mechanism, including release mechanisms involving reduction, oxidation, or hydrolysis. An example of a reduction mechanism is the reduction of a disulfide group to two separate sulfhydryl groups. Thus, for example, a group of the formula -CH2-SS-CH2- will be reduced to two separate groups of the formula -CH2-SH, resulting in a linker of the formula:

[0375] [ka] If it was of The reduction product has the formula:

[0376] [ka] In this embodiment, the steroid is attached to the linker via an immolative moiety (eg, a disulfide group).

[0377] Examples of self-immolative disulfides also include sterically protected disulfide bonds.The steroid may be linked to the linker via other suitable self-immolative bonds, including a self-immolative cathepsin-cleavable amino acid sequence; a self-immolative furin-cleavable amino acid sequence; a self-immolative β-glucuronidase-cleavable moiety; a self-immolative phosphatase-cleavable moiety; or a self-immolative sulfatase-cleavable moiety.Multiple self-immolative bonds are also contemplated herein.

[0378] In some embodiments, the linker comprises a self-immolative moiety. In some embodiments, the linker comprises a self-immolative disulfide and / or a sterically protected disulfide bond. In some embodiments, the linker comprises a self-immolative cathepsin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative furin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative β-glucuronidase-cleavable moiety. In some embodiments, the linker comprises a self-immolative phosphatase-cleavable moiety. In some embodiments, the linker comprises a self-immolative sulfatase-cleavable moiety.

[0379] In some embodiments, the linker comprises a phosphate or pyrophosphate group. In some embodiments, the linker comprises a cathepsin B cleavable group. In some embodiments, the cathepsin B cleavable group is valine-citrulline. In some embodiments, the linker comprises a carbamate moiety. In some embodiments, the linker comprises a β-glucuronide.

[0380] In some embodiments, the compounds include a bond in which the steroid is attached to the linker via an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate, or an enzyme-cleavable amino acid sequence.

[0381] In some embodiments, the linker comprises an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate, or an enzyme-cleavable amino acid sequence.

[0382] In some embodiments, L is one or more spacer linkers (e.g., S 1). The spacer linker may be a hydrophilic spacer linker that includes multiple hydroxyl functional groups. The spacer may include any stable atomic arrangement. Each spacer is independently selected from the group consisting of amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., polyethylene glycol), cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, the spacer includes any one or more of the following units: amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., PEG), cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, the spacer includes a solubility enhancer or a PK / PD modulator W. In some embodiments, the spacer includes a glycosylated amino acid. In some embodiments, the spacer comprises one or more monosaccharides, disaccharides, polysaccharides, glycans, or peptidoglycans. In some embodiments, the spacer comprises a releasable moiety (e.g., a disulfide bond, an ester, or other moiety that can be cleaved in vivo). In some embodiments, the spacer comprises one or more units, such as ethylene (e.g., polyethylene), ethylene glycol (e.g., PEG), ethanolamine, ethylenediamine, etc. (e.g., propylene glycol, propanolamine, propylenediamine). In some embodiments, the spacer comprises an oligoethylene, PEG, an alkyl chain, an oligopeptide, a polypeptide, a rigid functional group, a peptidoglycan, an oligoproline, an oligopiperidine, or any combination thereof. In some embodiments, the spacer comprises an oligoethylene glycol or PEG. The spacer may comprise an oligoethylene glycol.In some embodiments, the spacer comprises PEG. In some embodiments, the spacer comprises an oligopeptide or polypeptide. In some embodiments, the spacer comprises an oligopeptide. In some embodiments, the spacer comprises a polypeptide. In some embodiments, the spacer comprises a peptidoglycan. In some embodiments, the spacer does not comprise a glycan. In some embodiments, the spacer does not comprise a sugar. In some embodiments, the rigid functional group is an oligoproline or an oligopiperidine. In some embodiments, the rigid functional group is an oligoproline. In some embodiments, the rigid functional group is an oligopiperidine. In some embodiments, the rigid functional group is an oligophenyl. In some embodiments, the rigid functional group is an oligoalkyne. In some embodiments, the oligoproline or oligopiperidine has about 2 to about 20, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 15, about 2 to about 10, or about 2 to about 6 repeating units (e.g., proline or piperidine).

[0383] In some embodiments, L comprises a solubility enhancer or PK / PD modulator. In some embodiments, L comprises a PEG, a sugar, a peptide, or a peptidoglycan. In some embodiments, L comprises a PEG, a sugar, a peptide, or a peptidoglycan to achieve superior solubility and PK / PD properties. In some embodiments, L comprises one or more monosaccharides, disaccharides, peptides, peptidoglycans, and / or serum albumins. In some embodiments, L comprises one or more PEGs, peptides, peptidoglycans, or serum albumins. In some embodiments, W does not comprise a sugar. In some embodiments, W does not comprise a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, W does not comprise a glycan. In some embodiments, L comprises a glycosylated amino acid. In some embodiments, L comprises a glycosylated cysteine. In some embodiments, L comprises a free carboxylic acid. In some embodiments, L comprises a PEG.

[0384] In some embodiments, L comprises one or more monosaccharides, disaccharides, oligosaccharides, polysaccharides, peptides, peptidoglycans, serum albumins, solubility enhancers, PK / PD modulators, or combinations thereof. In some embodiments, L modulates a pharmacological, pharmacokinetic, pharmacodynamic, or physicochemical property. In some embodiments, L promotes internalization. In some embodiments, L improves water solubility. In some embodiments, L increases plasma protein binding. In some embodiments, W modulates (e.g., decreases) the excretion, elimination, metabolism, stability (e.g., enzyme stability, plasma stability), distribution, toxicity, or combinations thereof of the compound.

[0385] In some embodiments, monosaccharides, such as those found in W, exist in equilibrium between their linear and cyclic forms. In some embodiments, the monosaccharides are linear. In some embodiments, the monosaccharides are cyclic. In some embodiments, the monosaccharides exist as D-isomers. In some embodiments, the monosaccharides exist as L-isomers. As non-limiting examples, in some embodiments, L comprises one or more monosaccharides selected from the following: ribose, galactose, mannose, glucose fructose, N-acetylglucosamine, N-acetylmuramic acid, or derivatives thereof (e.g., cyclic or linear forms, methylated derivatives, acetylated derivatives, phosphorylated derivatives, aminated derivatives, oxidized or reduced derivatives, D or L isomers, isotopes, stereoisomers, positional isomers, tautomers, or combinations thereof).

[0386] In some embodiments, the disaccharide, oligosaccharide, or polysaccharide that can be disposed within W comprises O-linkages, N-linkages, C-linkages, or combinations thereof. In some embodiments, the disaccharide, oligosaccharide, or polysaccharide comprises glycosidic linkages in either alpha- or beta-orientation. In some embodiments, L comprises an oligosaccharide, polysaccharide, or glycan (e.g., glycoprotein, glycopeptide, glycolipid, glycogen, proteoglycan, peptidoglycan, etc.).

[0387] In some embodiments, L comprises an amino acid, a peptide, a polypeptide, or a protein. In some embodiments, the amino acid is a natural amino acid (e.g., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val)). Alternatively, in some embodiments, the amino acid is a non-natural or modified amino acid. L may comprise a sugar or sugar derivative covalently attached to the side chain of the amino acid (e.g., glutamic acid, aspartic acid).

[0388] In some embodiments, L is a glycosylated amino acid, such as:

[0389] [ka] Includes.

[0390] In some embodiments, the peptide or polypeptide comprises multiple amino acids, natural and / or unnatural. In some embodiments, the peptide (or peptidoglycan) has between about 2 and about 20 amino acids. In some embodiments, the amino acid, peptide, polypeptide, or protein has a pharmacological or physicochemical effect that enhances one or more properties of the compound (e.g., modulating solubility, solubility, size, permeability, protein binding, target binding, excretion, metabolism, toxicity, distribution, half-life, and / or duration of action). In some embodiments, L comprises a pharmacokinetic modulator. In some embodiments, the pharmacokinetic modulator is a peptide or protein that can modulate (e.g., enhance) protein binding. In some embodiments, the pharmacokinetic modulator enhances plasma protein binding. In some embodiments, the pharmacokinetic modulator reduces the rate of elimination, excretion, or metabolism. In some embodiments, the pharmacokinetic modulator increases the duration of action of the compound.

[0391] In some embodiments, the linker comprises an albumin ligand. In some embodiments, the albumin ligand is

[0392] [ka] Includes.

[0393] In some embodiments, L has the structure:

[0394] [ka] Includes.

[0395] In some embodiments, L comprises a template (eg, a multivalent template) linking multiple arms of the compound and comprises a template (eg, a repeat unit) of the following structure:

[0396] [ka]

[0397] In some embodiments, L comprises a template that binds multiple arms of a compound with a citrate-based template. In some embodiments, L comprises a template that binds multiple arms of a compound (e.g., a multivalent template) and has a (e.g., citrate-based) template of the following structure:

[0398] [ka]

[0399] In some embodiments, L comprises a template (eg, a multivalent template) that binds multiple arms of the compound and has a (eg, citrate-based) template of the following structure:

[0400] [ka]

[0401] In some embodiments, L comprises a template (eg, a multivalent template) that binds multiple arms of the compound and has a (eg, citrate-based) template of the following structure:

[0402] [ka]

[0403] In some embodiments, the linker comprises a dimethylcysteine ​​group. In some embodiments, the dimethylcysteine ​​group is linked to a succinimide to form:

[0404] [ka] Form.

[0405] L' of the compounds herein may comprise an optionally substituted heteroalkyl. The optionally substituted heteroalkyl may be substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylate, and halo. L' may comprise a substituted heteroalkyl having at least one disulfide bond in its backbone. L' may comprise a peptide or peptidoglycan having at least one disulfide bond in its backbone. L' may be a releasable linker that can be cleaved by enzymatic reaction, reactive oxygen species (ROS), or reductive conditions.

[0406] L' is a group of the formula -NH-CH2-CR 6 R 7 -SS-CH-CH-O-CO- (wherein R 6 and R 7 are each independently H, alkyl, or heteroalkyl.

[0407] L' is of the formula:

[0408] [ka] It may be or contain the group (wherein p is an integer from 0 to 30; d is an integer from 1 to 40; R 8 and R 9 are each independently H, alkyl, heterocyclyl, cycloalkyl, aryl, or heteroalkyl.

[0409] L' may comprise one or more linker moieties independently selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, carboxylate, carbonate, carbamate, amino acid, peptide, and peptidoglycan. L' may be or comprise a peptide or peptidoglycan. L' may be or comprise an amino acid. L' may be or comprise a PEG group. L' may be or comprise a polysaccharide. L' has the structure:

[0410] [ka] (wherein w is 0 to 5 and p is 1 to 30). L' is a group represented by the following formula or may contain these:

[0411] [ka] It may be or may include a linker moiety selected from the group consisting of: (n" is 0 to 30).

[0412] L' may be a divalent linker. L' may be a trivalent linker.

[0413] In some embodiments, L' is a pyrido[2,3-d]pyrimidine analog having the structure:

[0414] [ka]

[0415] The linker may include a pharmacokinetic extender, such as an albumin binder or a hapten. Examples of albumin binders include, but are not limited to,

[0416] [ka] There is.

[0417] Examples of haptens include, but are not limited to, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), rhamnose, galactose-α-1,3-galactose (α-Gal), or antibody binding agents. Examples of antibody binding agents include, but are not limited to, Fab, scFv, V H , V L , V H H, V-NAR, monobody, anticalin, affibody, or DARPin.

[0418] L' in the compounds herein is a spacer (S 1 ) may be optionally conjugated to and / or may optionally include S 1 may be any suitable spacer. Examples of spacers include, but are not limited to, an alkyl chain containing at least about 20 carbon atoms, e.g., at least 20 carbon atoms, a PEG containing at least about 20 units, e.g., at least 20 units, in the chain, a sugar, a peptidoglycan, a click-chemistry reactive linker (e.g., a triazole), a rigid linker (e.g., a polyproline or polypiperidine), or a combination of two or more of the foregoing.

[0419] All of the compounds are S 1 and S 1 can be a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, and the PEG group, peptide group, glycopeptide group, saccharide group, or albumin binding group radical is attached to L. The compound may also be an albumin binding group, e.g.

[0420] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0421] L' may contain one or more releasable linkers that cleave under the conditions described herein by a chemical mechanism involving beta-elimination. Such releasable linkers include beta-thio, beta-hydroxy, and beta-amino substituted carboxylic acids and their derivatives, such as esters, amides, carbonates, carbamates, and ureas. Such linkers also include 2- and 4-thioaryl esters, carbamates, and carbonates.

[0422] Examples of releasable linkers include those of the formula:

[0423] [ka] (wherein X 4 is NR, n is an integer selected from 0, 1, 2, and 3, and R 32 is hydrogen or a substituent, including substituents that can stabilize a positive charge on the aryl ring inductively or by resonance, such as alkoxy. The releasable linker may be further substituted.

[0424] The auxiliary cleavage of the releasable moiety of L' can be a mechanism involving a benzylium intermediate, a benzyne intermediate, lactone cyclization, an oxonium intermediate, beta-elimination, etc. In addition to fragmentation after cleavage of the releasable moiety of L, the initial cleavage of the releasable linker can be facilitated by an anchimeric auxiliary mechanism. Thus, in the example of the releasable moiety of L' given above, a hydroxyalkanoic acid that can cyclize can facilitate cleavage of the methylene bridge, for example by an oxonium ion, facilitating bond cleavage or subsequent fragmentation after bond cleavage of the releasable linker. Alternatively, an acid-catalyzed auxiliary cleavage of the methylene bridge by an oxonium ion can initiate a cascade of fragmentation of this exemplary bivalent linker or fragments thereof. Alternatively, acid-catalyzed hydrolysis of the carbamate can facilitate beta-elimination of a hydroxyalkanoic acid, which can cyclize and facilitate cleavage of the methylene bridge, for example by an oxonium ion. Other chemical mechanisms of bond cleavage under metabolic, physiological, or cellular conditions can initiate such a cascade of fragmentation.Other chemical mechanisms of bond cleavage under metabolic, physiological, or cellular conditions can initiate such a cascade of fragmentation.

[0425] Exemplary mechanisms for cleavage of the divalent linkers described herein include the following 1,4 and 1,6 fragmentation mechanisms for carbonates and carbamates:

[0426] [ka] (In the formula, Nuc - is an exogenous or endogenous nucleophile, glutathione, or a bioreductant, and R a and X a is attached via the other part of the bivalent linker). a and X aThe position of is, for example, such that the resulting product is X a -S-Nuc and HO-R a H2N-R a It may be replaced so that

[0427] Although the above fragmentation mechanism is illustrated as a concerted mechanism, any number of separate steps may occur to affect the final fragmentation of the bivalent linker to the final product shown. For example, bond cleavage may also occur by acid-catalyzed elimination of the carbamate moiety, which may be assisted anchimerically by the stabilization provided by either the beta sulfur or the aryl group of the disulfide shown in the above example. In these variations of this embodiment, the releasable linker is the carbamate moiety. Alternatively, fragmentation may be initiated by nucleophilic attack on the disulfide group, cleaving to form a thiolate. The thiolate can intermolecularly displace a carbonate or carbamate moiety to form the corresponding thiacyclopropane. In the case of a benzyl-containing bivalent linker, following cleavage of the illustrated disulfide bond, the resulting phenylthiolate is further fragmented to release the carbonate or carbamate moiety by forming a resonance-stabilized intermediate. In any of these cases, the releasable nature of the exemplary bivalent linkers described herein can be achieved by whatever mechanism may be related to the chemical, metabolic, physiological, or biological conditions present.

[0428] Thus, as discussed above, the releasable linker may comprise a disulfide group. Further examples of releasable linkers contained in L' include divalent radicals comprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl groups, each of which is optionally substituted. Additional examples of releasable linkers contained in L' include methylene groups, 1-alkoxyalkylene groups, 1-alkoxycycloalkylene groups, 1-alkoxyalkylenecarbonyl groups, 1-alkoxycycloalkylenecarbonyl groups, carbonylarylcarbonyl groups, carbonyl(carboxyaryl)carbonyl groups, carbonyl(biscarboxyaryl)carbonyl groups, haloalkylenecarbonyl groups, alkylene(dialkylsilyl) groups, alkylene(alkylarylsilyl) groups, alkylene(diarylsilyl) groups, (diaryl)alkylene(alkylaryl)carbonyl groups, (aryl)alkylene(di ... and divalent radicals including (alkylsilyl)aryl groups, (alkylarylsilyl)aryl groups, (diarylsilyl)aryl groups, oxycarbonyloxy groups, oxycarbonyloxyalkyl groups, sulfonyloxy groups, oxysulfonylalkyl groups, iminoalkylidenyl groups, carbonylalkylidenyliminyl groups, iminocycloalkylidenyl groups, carbonylcycloalkylidenyliminyl groups, alkylenethio groups, alkylenearylthio groups, or carbonylalkylthio groups, each releasable linker being optionally substituted.

[0429] Additional examples of releasable linkers included in L' may include an oxygen atom and a methylene group, a 1-alkoxyalkylene group, a 1-alkoxycycloalkylene group, a 1-alkoxyalkylenecarbonyl group, or a 1-alkoxycycloalkylenecarbonyl group, each of which is optionally substituted. Alternatively, in some embodiments, the releasable linker includes an oxygen atom and a methylene group, the methylene group being substituted with an optionally substituted aryl, and the releasable linker is bonded to the oxygen to form an acetal or ketal. Furthermore, in some embodiments, the releasable linker includes an oxygen atom and a sulfonylalkyl group, and the releasable linker is bonded to the oxygen to form an alkyl sulfonate.

[0430] Additional examples of releasable linkers contained in L' include an iminoalkylidenyl group, a carbonylalkylidenyliminyl group, an iminocycloalkylidenyl group, and a carbonylcycloalkylidenyliminyl group, each of which is optionally substituted, and which is attached to the nitrogen to form a hydrazone. In some embodiments, the hydrazone is acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form a variety of acylhydrazone releasable linkers.

[0431] Additional examples of releasable linkers contained in L' include an oxygen atom and an alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl or (diarylsilyl)aryl group, each optionally substituted, where the releasable linker is bonded to the oxygen to form a silanol.

[0432] Additional examples of releasable linkers contained in L' include two independent nitrogens and a carbonylarylcarbonyl, a carbonyl(carboxyaryl)carbonyl, or a carbonyl(biscarboxyaryl)carbonyl. In some embodiments, the releasable linker is attached to a heteroatom nitrogen to form an amide.

[0433] Additional examples of releasable linkers contained in L' include an oxygen atom, a nitrogen atom, a carbonylarylcarbonyl, a carbonyl(carboxyaryl)carbonyl, or a carbonyl(biscarboxyaryl)carbonyl. In some embodiments, the releasable linker forms an amide.

[0434] In some embodiments, L' comprises an optionally substituted 1-alkylenesuccinimide-3-yl group and a releasable moiety comprising a methylene group, a 1-alkoxyalkylene group, a 1-alkoxycycloalkylene group, a 1-alkoxyalkylenecarbonyl group, or a 1-alkoxycycloalkylenecarbonyl group, each of which can be optionally substituted to form a succinimid-1-yl alkyl acetal or ketal.

[0435] In some embodiments, L' comprises carbonyl, thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimide-3-yl, 1-(carbonylalkyl)succinimide-3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimide-3-yl, or 1-(carbonyltetrahydrofuranyl)succinimide-3-yl, each of which is optionally substituted. In some embodiments, L' further comprises an additional nitrogen such that L' comprises an alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, or 1-(carbonylalkyl)succinimid-3-yl group, each of which is optionally substituted, attached to the nitrogen to form an amide. In some embodiments, L' further comprises a sulfur atom and an alkylene or cycloalkylene group, each of which is optionally substituted with carboxy, attached to the sulfur to form a thiol. In some embodiments, L' comprises a sulfur atom and a 1-alkylenesuccinimid-3-yl, such that the 1-(carbonylalkyl)succinimid-3-yl group is attached to the sulfur to form a succinimide-3-ylthiol.

[0436] In some embodiments, L' comprises a nitrogen and a releasable moiety comprising an alkyleneaziridin-1-yl, carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, or sulfonylalkylaziridin-1-yl, each of which is optionally substituted. In some embodiments, L' comprises a carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, or 1-(carbonylalkyl)succinimide-3-yl, each of which is optionally substituted, which is attached to a releasable moiety to form an aziridine amide.

[0437] Examples of L' include alkylene-amino-alkylenecarbonyl, alkylene-thio-(carbonylalkylsuccinimid-3-yl), and the like, which are further illustrated by the formula:

[0438] [ka] wherein x' and y' are each independently 1, 2, 3, 4, or 5.

[0439] L may have any suitable type of atom in the chain, including C (e.g., -CH2-, C(O)), N (e.g., NH, NR b , where R bis, for example, H, alkyl, alkylaryl, etc.), O (e.g., -O-), P (e.g., -OP(O)(OH)O-), and S (e.g., -S-). For example, the atoms used in forming L' can be linked in any chemically appropriate manner, including, for example, a chain of carbon atoms forming an alkyl group, a chain of carbon and oxygen atoms forming a polyoxyalkyl group, a chain of carbon and nitrogen atoms forming a polyamine, and others including those forming aryl and heterocyclyl groups (e.g., triazole, oxazole, etc.). Furthermore, the bonds connecting the atoms of the chain in L' can be either saturated or unsaturated, such as, for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, etc. can be divalent radicals included in L. Furthermore, the chain forming L' can be substituted or unsubstituted.

[0440] Additional examples of L' groups include 1-alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1-(carbonyltetrahydrofuranyl)succinimid-3-yl, each of which may be substituted or unsubstituted. Any of the foregoing groups may be L' or may be included as part of L. In some cases, any of the foregoing groups can be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may further include additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)-OR-NH-alkyl-), oxygen (e.g., -alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-). Examples of such L' groups are alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, and succinimid-3-ylthiol, each group may be substituted or unsubstituted.

[0441] In some embodiments, L' is formed via click chemistry / click chemistry-derived. Those skilled in the art will appreciate that the terms "click chemistry" and "click chemistry-derived" generally refer to certain small molecule reactions that are commonly used in conjugation to link selected substrates with specific molecules. Click chemistry is not a single specific reaction, but describes a method to generate products according to examples in nature, and also to generate substances by combining small modular units. In many applications, click reactions link biomolecules with reporter molecules. Click chemistry is not limited to biological conditions: the concept of "click" reactions has been used in pharmacological and various biomimetic applications. However, they have been found to be particularly useful in the detection, localization and qualification of biomolecules.

[0442] Click reactions can occur in one pot, are typically not hindered by water, produce minimal by-products, are "spring loaded" i.e., are characterized by a high thermodynamic driving force, make the reaction fast and irreversible to give a single reaction product in high yield, and are associated with high reaction specificity (in some cases, both regiospecificity and stereospecificity). These properties make them well suited to the challenges of isolating and targeting molecules in complex biological environments. Thus, in such environments, the products need to be physiologically stable and any by-products non-toxic (as for in vivo systems).

[0443] Examples of click chemistry include those in which L' can be derived from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (IEDDA), and Staudinger ligation (SL). a and R a can be linked together as shown in Schemes 1-5:

[0444] [ka] (In the formula, each R b are independently H, alkyl, arylalkyl, -alkyl-S-alkyl or arylalkyl or the side chain of any naturally occurring or unnatural amino acid, etc. a and R a The wavy line connected to X a and R a and the group to which they are attached. It should be understood that in Schemes 1-5, triazole, oxazole, and -NH-SO2-NH- groups would be considered to be part of L.

[0445] In some embodiments, L' is a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide-based dual linkers; L' is either a non-releasable or releasable linker that is bivalently covalently attached to a folate ligand (or, in other embodiments, a folate analog or antifolate) and a steroid.

[0446] In some embodiments, L' is

[0447] [ka] is (In the formula, x" is an integer from 0 to 10, and y″ is an integer from 3 to 100.

[0448] In some embodiments, x″ is an integer from 3 to 10.

[0449] In some embodiments, L' is

[0450] [ka] is (In the formula, each R 33 and R 34 is independently H or C1-C6 alkyl; Z is an integer from 1 to 8.

[0451] In some embodiments, L' is

[0452] [ka] It is.

[0453] In some embodiments, L' is

[0454] [ka] is (In the formula, R 37 is H or C1-C6 alkyl; R 35a , R 35b , R 36a , and R 36b are each independently H or C1-C6 alkyl.

[0455] In some embodiments, L' comprises an amino acid. In some embodiments, L' comprises an amino acid selected from the group consisting of Lys, Asn, Thr, Ser, Ile, Met, Pro, His, Gln, Arg, Gly, Asp, Glu, Ala, Val, Phe, Leu, Tyr, Cys, and Trp. In some embodiments, L' comprises at least two amino acids independently selected from the group consisting of Glu and Cys. In some embodiments, L' comprises Glu-Glu, where the glutamic acids are covalently linked to each other via the carboxylic acid side chains.

[0456] In some embodiments, L' comprises one or more hydrophilic spacer linkers that contain multiple hydroxyl functional groups.

[0457] In some embodiments, L' comprises at least one 2,3-diaminopropionic acid group, at least one glutamic acid group (e.g., the unnatural amino acid D-glutamic acid), and at least one cysteine ​​group. One example of such a linker is one with an unnatural amino acid, such as the formula:

[0458] [ka] is a linker having a repeat unit of where q is an integer from 1 to 10 (e.g., 1 to 3 and 2 to 5). In some embodiments, L' has the general formula:

[0459] [ka] Includes wherein X can be O, NH, NR, or S, and q is an integer from 1 to 10. In some embodiments, L' is a group represented by the formula:

[0460] [ka] Includes where the disulfide group is part of a self-immolative group which may be generally described as a group of the formula -CH2-SS-CH2-.

[0461] In some embodiments, the compounds described herein include a bond that allows the steroid described herein to be released by any suitable mechanism, including release mechanisms involving reduction, oxidation, or hydrolysis. An example of a reduction mechanism is the reduction of a disulfide group to two separate sulfhydryl groups. Thus, for example, a group of the formula -CH2-SS-CH2- will be reduced to two separate groups of the formula -CH2-SH, resulting in a linker of the formula:

[0462] [ka] If it was of The reduction product has the formula:

[0463] [ka] It will be something like this. In this example, the steroid is attached to the linker via a self-immolative moiety (eg, a disulfide group).

[0464] Examples of self-immolative disulfides also include sterically protected disulfide bonds.The steroid may be linked to the linker via other suitable self-immolative bonds, including a self-immolative cathepsin-cleavable amino acid sequence; a self-immolative furin-cleavable amino acid sequence; a self-immolative β-glucuronidase-cleavable moiety; a self-immolative phosphatase-cleavable moiety; or a self-immolative sulfatase-cleavable moiety.Multiple self-immolative bonds are also contemplated herein.

[0465] In some embodiments, the linker comprises a self-immolative moiety. In some embodiments, the linker comprises a self-immolative disulfide and / or a sterically protected disulfide bond. In some embodiments, the linker comprises a self-immolative cathepsin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative furin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative β-glucuronidase-cleavable moiety. In some embodiments, the linker comprises a self-immolative phosphatase-cleavable moiety. In some embodiments, the linker comprises a self-immolative sulfatase-cleavable moiety.

[0466] In some embodiments, the linker comprises a phosphate or pyrophosphate group. In some embodiments, the linker comprises a cathepsin B cleavable group. In some embodiments, the cathepsin B cleavable group is valine-citrulline. In some embodiments, the linker comprises a carbamate moiety. In some embodiments, the linker comprises a β-glucuronide.

[0467] In some embodiments, the compounds described herein include a linkage in which the steroid is attached to the linker via an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate, or an enzyme-cleavable amino acid sequence.

[0468] In some embodiments, the linker comprises an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate, or an enzyme-cleavable amino acid sequence.

[0469] In some embodiments, L' comprises one or more spacer linkers. The spacer linker may be a hydrophilic spacer linker that comprises multiple hydroxyl functional groups. The spacer may comprise any stable atomic arrangement. Each spacer is independently selected from the group consisting of amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., polyethylene glycol), cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, the spacer comprises any one or more of the following units: amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., PEG), cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, the spacer comprises a solubility enhancer or a PK / PD modulator W. In some embodiments, the spacer comprises a glycosylated amino acid. In some embodiments, the spacer comprises one or more monosaccharides, disaccharides, polysaccharides, glycans, or peptidoglycans. In some embodiments, the spacer comprises a releasable moiety (e.g., a disulfide bond, an ester, or other moiety that can be cleaved in vivo). In some embodiments, the spacer comprises one or more units, such as ethylene (e.g., polyethylene), ethylene glycol (e.g., PEG), ethanolamine, ethylenediamine, etc. (e.g., propylene glycol, propanolamine, propylenediamine). In some embodiments, the spacer comprises an oligoethylene, PEG, an alkyl chain, an oligopeptide, a polypeptide, a rigid functional group, a peptidoglycan, an oligoproline, an oligopiperidine, or any combination thereof. In some embodiments, the spacer comprises an oligoethylene glycol or PEG.The spacer may comprise an oligoethylene glycol. In some embodiments, the spacer comprises PEG. In some embodiments, the spacer comprises an oligopeptide or a polypeptide. In some embodiments, the spacer comprises an oligopeptide. In some embodiments, the spacer comprises a polypeptide. In some embodiments, the spacer comprises a peptidoglycan. In some embodiments, the spacer does not comprise a glycan. In some embodiments, the spacer does not comprise a sugar. In some embodiments, the rigid functional group is an oligoproline or an oligopiperidine. In some embodiments, the rigid functional group is an oligoproline. In some embodiments, the rigid functional group is an oligopiperidine. In some embodiments, the rigid functional group is an oligophenyl. In some embodiments, the rigid functional group is an oligoalkyne. In some embodiments, the oligoproline or oligopiperidine has from about 2 to about 20, from about 2 to about 40, from about 2 to about 30, from about 2 to about 20, from about 2 to about 15, from about 2 to about 10, or from about 2 to about 6 repeating units (e.g., proline or piperidine).

[0470] In some embodiments, L' comprises a solubility enhancer or PK / PD modulator. In some embodiments, L' comprises a polyethylene glycol (PEG), a sugar, a peptide, or a peptidoglycan. In some embodiments, L' comprises a PEG, a sugar, a peptide, or a peptidoglycan to achieve superior solubility and PK / PD properties. In some embodiments, L' comprises one or more monosaccharides, disaccharides, peptides, peptidoglycans, and / or serum albumins. In some embodiments, L' comprises one or more PEGs, peptides, peptidoglycans, or serum albumins. In some embodiments, W does not comprise a sugar. In some embodiments, W does not comprise a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, W does not comprise a glycan. In some embodiments, L' comprises a glycosylated amino acid. In some embodiments, L' comprises a glycosylated cysteine. In some embodiments, L' comprises a free carboxylic acid. In some embodiments, L' comprises a PEG.

[0471] In some embodiments, L' comprises one or more monosaccharides, disaccharides, oligosaccharides, polysaccharides, peptides, peptidoglycans, serum albumins, solubility enhancers, PK / PD modulators, or combinations thereof. In some embodiments, L' modulates pharmacological, pharmacokinetic, pharmacodynamic, or physicochemical properties. In some embodiments, L' promotes internalization. In some embodiments, L' improves water solubility. In some embodiments, L' increases plasma protein binding. In some embodiments, W modulates (e.g., decreases) the excretion, elimination, metabolism, stability (e.g., enzyme stability, plasma stability), distribution, toxicity, or combinations thereof of the compound.

[0472] In some embodiments, monosaccharides, such as those found in W, exist in equilibrium between their linear and cyclic forms. In some embodiments, the monosaccharides are linear. In some embodiments, the monosaccharides are cyclic. In some embodiments, the monosaccharides exist as D-isomers. In some embodiments, the monosaccharides exist as L'-isomers. As non-limiting examples, in some embodiments, L' comprises one or more monosaccharides selected from the following: ribose, galactose, mannose, glucose fructose, N-acetylglucosamine, N-acetylmuramic acid or derivatives thereof (e.g., cyclic or linear forms, methylated derivatives, acetylated derivatives, phosphorylated derivatives, aminated derivatives, oxidized or reduced derivatives, D or L' isomers, isotopes, stereoisomers, positional isomers, tautomers, or combinations thereof).

[0473] In some embodiments, the disaccharide, oligosaccharide, or polysaccharide that can be disposed within W comprises O-linkages, N-linkages, C-linkages, or combinations thereof. In some embodiments, the disaccharide, oligosaccharide, or polysaccharide comprises glycosidic linkages in either alpha- or beta-orientation. In some embodiments, L' comprises an oligosaccharide, polysaccharide, or glycan (e.g., glycoprotein, glycopeptide, glycolipid, glycogen, proteoglycan, peptidoglycan, etc.).

[0474] In some embodiments, L' comprises an amino acid, a peptide, a polypeptide, or a protein. In some embodiments, the amino acid is a natural amino acid (e.g., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val)). Alternatively, in some embodiments, the amino acid is a non-natural or modified amino acid. L' may comprise a sugar or sugar derivative covalently attached to the side chain of the amino acid (e.g., glutamic acid, aspartic acid).

[0475] In some embodiments, L' is a glycosylated amino acid, such as:

[0476] [ka] Includes.

[0477] In some embodiments, the peptide or polypeptide comprises multiple amino acids, natural and / or unnatural. In some embodiments, the peptide (or peptidoglycan) has between about 2 and about 20 amino acids. In some embodiments, the amino acid, peptide, polypeptide, or protein has a pharmacological or physicochemical effect that enhances one or more properties of the compound (e.g., modulating solubility, solubility, size, permeability, protein binding, target binding, excretion, metabolism, toxicity, distribution, half-life, and / or duration of action). In some embodiments, L' comprises a pharmacokinetic modulator. In some embodiments, the pharmacokinetic modulator is a peptide or protein that can modulate (e.g., enhance) protein binding. In some embodiments, the pharmacokinetic modulator enhances plasma protein binding. In some embodiments, the pharmacokinetic modulator reduces the rate of elimination, excretion, or metabolism. In some embodiments, the pharmacokinetic modulator increases the duration of action of the compound.

[0478] In some embodiments, the linker comprises an albumin ligand. In some embodiments, the albumin ligand is

[0479] [ka] Includes.

[0480] In some embodiments, the linker comprises a dimethylcysteine ​​group. In some embodiments, the dimethylcysteine ​​group is linked to a succinimide to form:

[0481] [ka] Form.

[0482] The compounds herein may have or include the following structure:

[0483] [ka]

[0484] [ka]

[0485] The compounds herein may have or include the following structure:

[0486] [ka]

[0487] The compounds herein may have or include the following structure:

[0488] [ka]

[0489] [ka]

[0490] The compounds herein may have or include the following structure:

[0491] [ka]

[0492] Examples of compounds that include TLR7 / 8 agonists include, but are not limited to, those having the structure:

[0493] [ka] and a releasable targeting raltitrexed-TLR7-1A compound having the formula: The targeting raltitrexed-TLR7-1 compound that cannot be released has the structure:

[0494] [ka] having The non-releasable targeting raltitrexed-TLR7-1A compound has the structure:

[0495] [ka] has.

[0496] Examples of compounds that include EZH2 antagonists include, but are not limited to, compounds having the structure:

[0497] [ka] There is a releasable targeting raltitrexed-EZH2 antagonist having the following structure:

[0498] Any of the above compounds may further comprise a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, where the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to a linker.

[0499] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0500] Although the structures depicted above are represented as planar, one of skill in the art will appreciate that the ligands and conjugates depicted above include stereoisomers, i.e., ligands and conjugates that are identical in structure but differ in composition or spatial arrangement. Stereoisomerism is often due to chirality or "handedness," i.e., the existence of dextrorotatory (R) and levorotatory (L) forms of a drug that are not superimposable mirror images (i.e., "enantiomers").

[0501] Chiral conjugates (or conjugates that contain, for example, chiral ligands) may be administered as mixtures or as single enantiomers, especially when there are important differences in their activity and pharmacokinetics that need to be considered. The structural representations above are intended to encompass single enantiomers and mixtures thereof.

[0502] Those skilled in the art will further understand that the above ligands and conjugates may be "deuterated", meaning that one or more hydrogen atoms may be replaced with deuterium. Deuterium and hydrogen have almost the same physical properties, so deuterium replacement is the least possible structural change. Replacing hydrogen with deuterium may increase stability in the presence of other drugs, thus reducing undesirable drug-drug interactions, and can significantly reduce metabolic rate (due to kinetic isotope effect). By reducing metabolic rate, half-life may increase, toxic metabolite formation may decrease, and dosage and / or frequency may be reduced.

[0503] The above compounds may be synthesized using methods known in the art or exemplified herein, see, e.g., the Examples.

[0504] Pharmaceutical Compositions In view of the above, further provided herein is a pharmaceutical composition comprising any of the compounds herein.In certain embodiments, provided herein is a pharmaceutical composition comprising the compound described herein (e.g., the compound of formula (I)) and one or more pharma- ceutically acceptable excipients.

[0505] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof is provided, where T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a radical of a therapeutic agent.

[0506] In certain embodiments, T has the structure of formula (II):

[0507] [ka] has.

[0508] In certain embodiments, T has the structure of formula (III):

[0509] [ka] has.

[0510] The therapeutic agent may be selected from the group consisting of a TLR7 agonist, a PI3K inhibitor, a steroid, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an NLRP3 inhibitor, a caspase I inhibitor, an RLR agonist, an AIM2-like receptor agonist, and a RAGE agonist.

[0511] The therapeutic agent has the structure:

[0512] [ka] The compound may be an NLR2 agonist having the formula:

[0513] The therapeutic agent has the structure:

[0514] [ka] The compound may be a STING agonist having the following structure:

[0515] In certain embodiments, the therapeutic agent is an EZH2 inhibitor.

[0516] [ka] Or it may be tazemetostat.

[0517] In certain embodiments, the therapeutic agent has the structure:

[0518] [ka] It is an NLRP3 inhibitor having the following function:

[0519] In certain embodiments, the therapeutic agent has the structure:

[0520] [ka] It is a caspase I inhibitor having the following structure:

[0521] In certain embodiments, the therapeutic agent has the structure:

[0522] [ka] It is a PI3 kinase inhibitor having the following structure:

[0523] In certain embodiments, the therapeutic agent has the structure:

[0524] [ka] It is an RLR agonist having the following function:

[0525] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a TLR7 agonist of formula (IV):

[0526] [ka] or a radical of a pharma- ceutically acceptable salt thereof: R 1 , R 3 , R 4 , R 5 are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, cycloalkyl, arylhalo, heteroaryl, -COR 2x ,

[0527] [ka] and; R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH 2、

[0528] [ka] and; Z is H, -OH, -NH2, -NHR 2x , -OR 2x , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x, -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0529] [ka] and; Each R 2x and R 2y is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; Each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q independently selected from the group consisting of: Each R 2q and R 2q’ are independently alkyl or H;

[0530] [ka] is a 3-10 membered N-containing non-aromatic, monocyclic or bicyclic heterocycle; R 21 is H or alkyl, n' is 0 to 30; In formula (IV), X 1 , X 2 , X 3 Each of the following is independently q or N, and each R q are independently H, halogen, or optionally substituted alkyl; n is 0 to 30; m is 0 to 4; When n is 0, Y is H, -OH, or -OR 2x isn't it).

[0531] E is a radical of a compound of formula (IVA):

[0532] [ka] or a pharma- ceutically acceptable salt thereof, R 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3: R 2x and R 2y are each independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl; Each R Z are independently hydrogen, halogen, or optionally substituted alkyl; R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; Z is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2, Each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(Rz )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, wherein the alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4.

[0533] In certain embodiments, the compound of formula (I) has formula (IVB) or formula (IVC):

[0534] [ka] or a pharma- ceutically acceptable salt thereof (wherein each R 1 is independently an optionally substituted C3-C8 alkyl; each R 2 are independently H, -OR z , -SO2N(R z )2, -NR 2x R 2y or N3; each R 2x and R 2y are independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2ytaken together form an optionally substituted heterocycloalkyl; each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; 4 and R 5 is independently alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; and each Z 2 and Z 3 are independently represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; S 1 is a spacer; x is 0 to 3; n is 1 to 3, and m is 0 to 4).

[0535] In other embodiments, the compound of formula (I) has the formula (IVB):

[0536] [ka] or a pharma- ceutically acceptable salt thereof, 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3; R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; each R 3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; R 4 and R 5 each independently is alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; each Z 2 are represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1-, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; x is 0 to 3; n is 1 to 3; S 1 is a spacer ; m is 0 to 4.

[0537] In other embodiments, the compound of formula (I) has the formula (IVC):

[0538] [ka] or a pharma- ceutically acceptable salt thereof, 1 is an optionally substituted C3-C8 alkyl; R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3; R 2x and R 2y are each independently H, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl, and each R z are independently H, halogen, or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl; each R3 are independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z 2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; R 4 and R 5 each independently is alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4; each Z 3 are represented by the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2-; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; x is 0 to 3; S 1 is a spacer; n is 1 to 3, and m is 0 to 4).

[0539] In certain embodiments where the compound of Formula (I) is represented by formula (IVB) or formula (IVC), R 1is C1-C6 alkyl optionally substituted with 1-3 substituents, each of which is independently halogen or C1-C6 alkoxy; R 2 -NR 2x R 2y and R 2x and R 2y are each independently H or C1-C6 alkyl; 3 are independently halogen, -CN, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, amino, hydroxy, carboxyl, or thiol; R 4 and R 5 are each independently C1 to C6 alkyl; 1 , X 2 , and X 3 is N;Z 2 and Z 3 is independently TL- or TLO-; n is 1; and m is 0 to 4. In certain embodiments where the compound of formula (I) is represented by formula (IVB) or formula (IVC), Z 2 and Z 3 Each of the above is TLO-.

[0540] In certain embodiments of formula (IV) or formula (IVA), Z is of the formula TL-, TLO-, TLO-alkyl-, TLS 1 -, T-SO2-NH-, TL-NR a R b - TLS(O) x -alkyl-, TL-CO-, TL-aryl-, TL-NH-CO-NH-, TL-NH-O-, TL-NH-NH-, TL-NH-CS-NH, TLC(O)-alkyl-, or TL-SO2- groups: R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy; S1 is a spacer; and x is 0 to 3.

[0541] In certain embodiments of formula (IVA), R 1 is C1-C6 alkyl optionally substituted with 1-3 substituents, each of which is independently halogen or C1-C6 alkoxy; R 2 -NR 2x R 2y and R 2x and R 2y are each independently H or C1-C6 alkyl; 3 are independently halogen, -CN, C1-C6 alkyl, C1-C6 heteroalkyl, C3-C7 cycloalkyl, C1-C6 alkoxy, amino, hydroxy, carboxyl, or thiol; R 4 and R 5 are each independently C1 to C6 alkyl; 1 , X 2 , and X 3 is N; Z is TL- or TLO-; n is 1; and m is 0. Z may be TLO-.

[0542] R 1 R may be an optionally substituted C3-C6 alkyl. 1 R may be an optionally substituted acyclic C3-C6 alkyl. 2 -NR 2x R 2y R 2 may be -NH2.

[0543] The compound of formula (IVA) is one of the formulae:

[0544] [ka] or a pharma- ceutically acceptable salt thereof, 3 is optionally absent). Compounds of formula (IV) have the formula:

[0545] [ka] or a pharma- ceutically acceptable salt of any of the preceding formulas, 3 is not present as necessary).

[0546] R 1 may be a C1 to C6 alkyl. 2 R may be -NH2. 3 may not be present.

[0547] In certain embodiments, R 1 is C1-C6 alkyl; R 2 is -NH2; n is 1; R 3 does not exist.

[0548] In certain embodiments, the compound of formula (I) is a compound represented by formula (V).

[0549] [ka]

[0550] In certain embodiments, a radical (e.g., E) of the TLR7 agonist has the structure:

[0551] [ka] has.

[0552] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is

[0553] [ka] (It is.)

[0554] Compounds of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, wherein T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; and E is a compound having the structure:

[0555] [ka] and X may be any of the following:

[0556] [ka] ).

[0557] E has the structure:

[0558] [ka] It may contain a radical of the formula:

[0559] In certain embodiments, the compound is of formula (I): TLE (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E is the radical of a corticosteroid).

[0560] The corticosteroid may be betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone-17-valerate, budesonide, flumethasone, fluticasone propionate, flurocortisone, fludrocortisone, paramethasone, eplerenone, or an ester of any of the foregoing.

[0561] L may be a releasable linker. L may be a linker that is not releasable. L may comprise an optionally substituted heteroalkyl. In certain embodiments, the optionally substituted heteroalkyl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylate, and halo. L may comprise a substituted heteroalkyl having at least one disulfide bond in its backbone. L may comprise a peptide or peptidoglycan having at least one disulfide bond in its backbone. L may be a releasable linker that can be cleaved by an enzymatic reaction, reactive oxygen species (ROS), or reductive conditions. L may be a group of formula -NH-CH2-CR 6 R 7 -SS-CH2-CH2-O-CO-(wherein, R 6 and R 7 are each independently H, alkyl, or heteroalkyl. L may be a group of the formula:

[0562] [ka] or may contain these groups, where p is an integer from 0 to 30; d is an integer from 1 to 40; R 8 and R 9 are each independently H, alkyl, heterocyclyl, cycloalkyl, aryl, or heteroalkyl.

[0563] L may comprise one or more linker moieties, each of which is independently selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, carboxylate, carbonate, carbamate, amino acid, peptide, and peptidoglycan. L may be or comprise a peptide or peptidoglycan. L may be or comprise an amino acid. L may be or comprise a PEG group. L may be or comprise a polysaccharide. L has the structure:

[0564] [ka] (wherein w is 0 to 5 and p is 1 to 30). L is

[0565] [ka] where n″ is 0 to 30. L may be or include a linker moiety selected from the group consisting of:

[0566] Any of the above compounds may further comprise a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, wherein the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to a linker.

[0567] The compound may include an albumin binding group, such as:

[0568] [ka] Albumin binding groups selected from the group consisting of, for example:

[0569] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0570] In certain embodiments, the compound has the following structure:

[0571] [ka]

[0572] [ka] It comprises (e.g. consists of) one of:

[0573] In certain embodiments, the compound has the following structure:

[0574] [ka] It comprises (e.g. consists of) one of:

[0575] In certain embodiments, the compound has the following structure:

[0576] [ka]

[0577] [ka] It comprises (e.g. consists of) one of:

[0578] In certain embodiments, the compound has the following structure:

[0579] [ka] It comprises (e.g. consists of) one of:

[0580] Any of the above compounds may further comprise a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, where the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to a linker.

[0581] [ka] Albumin binding groups selected from the group consisting of, for example:

[0582] [ka] The antibody may further comprise an albumin binding group selected from the group consisting of:

[0583] The pharmaceutical composition may further comprise a compound of formula F-L'-G, or a pharma- ceutically acceptable salt thereof, where F is a radical of a folate or an analog thereof; L' is a linker; and G is a radical of a sugar (e.g., glucosamine).

[0584] The pharmaceutical composition may further comprise a compound of formula F-L'-G, or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine.

[0585] Also provided is a combination of pharmaceutical compositions. In certain embodiments, the combination comprises: (i) a first pharmaceutical composition comprising any of the compounds herein (e.g., compounds of formula (I)); and (ii) a second pharmaceutical composition comprising a compound of formula F-L'-G, or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine. The first and second pharmaceutical compositions may be administered by the same or different routes, e.g., simultaneously or sequentially in any order, and / or with the same or different dosing regimens. In certain embodiments, the first and second pharmaceutical compositions are administered sequentially, e.g., sequentially in any order. In certain embodiments, the first and second pharmaceutical compositions are administered contemporaneously, simultaneously, sequentially, or separately.

[0586] The pharmaceutical compositions may include one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles (e.g., conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles), and combinations thereof.

[0587] Pharmaceutical compositions can be, for example, formulated for a given route of administration and formulated according to methods known in the art, for example, as described in Remington, The Science and Practice of Pharmacy, 22 nd edition (2012). For example, and without limitation, the composition may be an injectable composition, e.g., a composition that can be injected subcutaneously.

[0588] Pharmaceutical compositions may be administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration. In certain embodiments, pharmaceutical compositions are formulated to be administered subcutaneously. In certain embodiments, pharmaceutical compositions are formulated to be administered orally. In certain embodiments, pharmaceutical compositions are formulated to be administered intramuscularly, intravenously, intraarterially, intraperitoneally, or other art-recognized parenteral routes of administration.

[0589] In certain embodiments, pharmaceutical compositions are administered systemically in combination with pharmaceutically acceptable vehicles.The percentage of components of compositions and preparations may vary and may be between about 1% and about 99% by weight of active ingredient (e.g., compound), as well as binders, excipients, disintegrants, lubricants, and / or sweeteners (as known in the art).The amount of active compound in such therapeutically useful compositions is such that it can obtain an effective dosage level.

[0590] Exemplary means of parenteral administration include needle (including microneedle) injectors, needleless injectors and injection techniques, and other means of parenteral administration recognized in the art.Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates and buffers (preferably with a pH ranging from about 3 to about 9), but for some applications, they may be more suitably formulated as sterile non-aqueous solutions or as dry forms that will be used with suitable vehicles such as sterile pyrogen-free water.Preparation of parenteral formulations under sterile conditions, for example by lyophilization, can be easily achieved using standard pharmaceutical techniques well known to those skilled in the art.

[0591] Pharmaceutical dosage forms suitable for administration may be sterile aqueous solutions or dispersions or sterile powders containing the active ingredient suitable for extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes, nanocrystals, or polymer nanoparticles. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles may be solvents or liquid dispersion media, including, for example, but not limited to, water, electrolytes, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the desired fluidity may be maintained by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants.

[0592] Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount of suitable solvent with one or more of the other components listed above as necessary, followed by filter sterilization.For the preparation of sterile injectable solutions, in the case of sterile powder, vacuum drying and freeze-drying technology can be used, which can obtain powder of active ingredient and any additional desired ingredient that are present in the solution previously sterile-filtered.

[0593] The compounds and pharmaceutical compositions may be administered as unit dosage forms and / or compositions. As used herein, the term "administering" and variations thereof include any means of introducing the compounds and compositions described herein into a subject, including, but not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation (e.g., intranasal (in)), buccal, intraocular, sublingual, vaginal, rectal, etc.

[0594] For the methods described herein, the compounds and compositions may be administered in a single dose or via a combination of multiple doses, which may be administered contemporaneously, simultaneously, sequentially, or separately by any suitable means.When the doses are administered in separate dosage forms, the number of doses administered per day for each compound or composition may be the same or different.The doses of the compounds and / or compositions may be administered via the same or different administration routes.The compounds or compositions may be administered in separate or single forms at the same or different times during the course of treatment, according to simultaneous or alternating regimens.

[0595] The compounds / compositions may be administered more than once, for example, daily (1-3 or more times per day; qd (once a day), bid (twice a day), tid (three times a day)), weekly (including 1-3 or more times on a given day), biweekly (including 1-3 or more times on a given day), monthly (including 1-3 or more times on a given day), or bimonthly (including 1-3 or more times on a given day). In any case, it is understood that the effective amounts described herein correspond to the total dose when administered as determined by the dosing protocol, or daily, weekly, monthly, or quarterly.

[0596] The effective amount of the compound or pharmaceutical composition containing it may be determined according to methods known in the art (e.g., animal models, human data, and human data for compounds used in a similar manner). The dosage / effective amount may be determined by taking into account several factors, including the mode of administration, the potency of the compound, the specific disease or disorder involved, the response of the individual subject, the severity and / or details of the subject's current condition, the use of concomitant medications, the age, weight, and health of the subject, and other appropriate circumstances. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of an antigen or composition) information for a particular patient may affect the dosage used.

[0597] A wide range of acceptable dosages is considered depending on the route of administration.For example, the effective amount of the compound and / or pharmaceutical composition may be in the range of about 0.1 μg / kg / day, for example, 0.5 μg / kg / day, 0.7 μg / kg / day, or 0.01 mg / kg / day to about 1,000 mg / kg / day.Intravenous dosage may be several orders of magnitude smaller.

[0598] Methods for immunomodulating Tregs Further provided is a method for immunomodulating Treg in a subject (e.g., in need thereof).In certain embodiments, the method comprises administering to the subject an effective amount of a first compound or a pharmaceutical composition comprising a first compound.By administering an effective amount of a first compound or a pharmaceutical composition comprising a first compound, Treg can be activated, inhibited (e.g., in the case of cancer), proliferated or killed.

[0599] In certain embodiments, the subject has cancer; E of the first compound is a radical of a TLR7 agonist, a PI3K inhibitor, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an NLRP3 inhibitor, a caspase I inhibitor, or an RLR agonist; and administration of the first compound or a pharmaceutical composition comprising the first compound alters Treg promotion of tumor growth and metastasis and / or inhibition of anti-tumor immunity.

[0600] Cancers include lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, epithelial cancer, leiomyosarcoma, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, and small cell lung cancer. The cancer may be a cancer of the adrenal gland, a soft tissue sarcoma, a cancer of the urethra, a cancer of the penis, a prostate cancer, a chronic leukemia, an acute leukemia, a lymphocytic lymphoma, a pleural mesothelioma, a cancer of the bladder, a cancer of the stomach, a Burkitt's lymphoma, a cancer of the ureter, a cancer of the kidney, a renal cell carcinoma, a cancer of the renal pelvis, a neoplasm of the central nervous system (CNS), a primary CNS lymphoma, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, a cholangiocarcinoma, a Hurth's cell thyroid cancer, or a gastroesophageal junction adenocarcinoma. The cancer may be a cancer of the lung, a triple negative breast cancer, a colon cancer, a cancer of the stomach, a cancer of the bladder, a prostate cancer, or a pancreatic cancer.

[0601] In certain embodiments, the method (eg, administering an effective amount of a first compound or a pharmaceutical composition comprising the first compound) does not induce undesirable inflammation in the subject.

[0602] In certain embodiments, the method includes administering to the subject an effective amount of a first compound or a pharmaceutical composition comprising the first compound, wherein E of the first compound is a radical of a TLR agonist (e.g., a TLR7 agonist), a PI3K inhibitor, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an NLRP3 inhibitor, a caspase I inhibitor, or an RLR agonist; the administration of the first compound or a pharmaceutical composition comprising the first compound alters the promotion of tumor growth and metastasis and / or the inhibition of anti-tumor immunity by Treg. The method may further include administering a second compound of formula F-L'-G, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the second compound, wherein F is a radical of folate or an analog thereof; L' is a linker; and G is a radical of glucosamine. Administration of the second compound or a pharmaceutical composition comprising the second compound may occur simultaneously with the first compound or a pharmaceutical composition comprising the first compound, or sequentially in any order, by the same or different routes.

[0603] The method may further include administering an additional therapeutic agent (e.g., a third therapeutic agent), such as an anti-cancer agent. The anti-cancer agent may be, for example, a chemotherapeutic agent or a radiotherapeutic agent. The administration of the additional therapeutic agent may be by the same or different route as the first compound or a pharmaceutical composition comprising the first compound and / or the second compound or a pharmaceutical composition comprising the second compound, simultaneously or sequentially in any order.

[0604] In another embodiment of the method, the subject has a fibrotic disease or disorder, and the method includes administering an effective amount of a compound, wherein E is a radical of a TLR7 agonist, a PI3K inhibitor, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an NLRP3 inhibitor, a caspase I inhibitor, or an RLR agonist, or a pharmaceutical composition comprising the same. The administration of an effective amount of the first compound or a pharmaceutical composition comprising the first compound may alter the promotion of tumor growth and metastasis and / or the inhibition of anti-cancer immunity by Treg in the subject. The fibrotic disease or disorder may be arthrofibrosis, autoimmune pancreatitis, bladder fibrosis, chronic kidney disease, chronic wound, Crohn's disease, desmoid tumor, Dupuytren's contracture, endometrial fibrosis, fibromatosis, graft-versus-host disease, cardiac fibrosis, keloid, liver fibrosis, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, Peyronie's disease, pulmonary fibrosis, retroperitoneal fibrosis, scleroderma or systemic sclerosis, or skin fibrosis. The fibrotic disease or disorder may be pulmonary fibrosis, liver fibrosis, scleroderma, myelofibrosis, Crohn's disease, or chronic kidney disease. The pulmonary fibrosis may be idiopathic pulmonary fibrosis (IPF). The liver fibrosis may be NASH or liver cirrhosis.

[0605] In certain embodiments of the method, the subject has an inflammatory disease and the method includes administering an effective amount of a first compound or a pharmaceutical composition comprising the first compound, where E is a radical of a steroid. The inflammatory disease may be Crohn's disease, lupus, inflammatory bowel disease (IBS), Addison's disease, Graves' disease, Sjogren's syndrome, celiac disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, reactive arthritis, psoriatic arthritis, pernicious anemia, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, organ transplant rejection, multiple sclerosis, graft-versus-host disease (GVHD), fatty liver disease, asthma, osteoporosis, sarcoidosis, ischemia-reperfusion injury, osteolysis of prosthetic joints, glomerulonephritis, scleroderma, psoriasis, autoimmune myocarditis, spinal cord injury, central nervous system inflammation, viral infection, influenza, coronavirus infection, cytokine storm syndrome, bone injury, inflammatory brain disease, or atherosclerosis.

[0606] The method may further comprise administering a second compound of formula F-L'-G or a pharma- ceutically acceptable salt thereof, where F is a radical of folate or an analogue thereof; L' is a linker; and G is a radical of glucosamine, and the administration of the second compound or pharmaceutical composition comprising the second compound may be by the same or different route, simultaneously with the first compound or pharmaceutical composition comprising the first compound, or sequentially in any order.

[0607] While the invention has been shown and described in detail in the drawings and foregoing description, the same are to be considered as illustrative and not restrictive in nature, it being understood that only certain specific embodiments have been shown and described, and that all changes and modifications within the spirit of the invention are desired to be protected.

[0608] The scope of the compounds, compositions and methods of the present invention is intended to be defined by the following claims.However, it should be understood that the present disclosure may be carried out in a manner other than as specifically described and illustrated without departing from its spirit or scope.It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be used in the practice of the following claims without departing from the spirit and scope defined in the following claims.

[0609] Treatment Further provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I) or a pharma- ceutically acceptable salt thereof).

[0610] Cancers include lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, epithelial cancer, leiomyosarcoma, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, and small cell lung cancer. The cancer may be a cancer of the adrenal gland, a soft tissue sarcoma, a cancer of the urethra, a cancer of the penis, a prostate cancer, a chronic leukemia, an acute leukemia, a lymphocytic lymphoma, a pleural mesothelioma, a cancer of the bladder, a cancer of the stomach, a Burkitt's lymphoma, a cancer of the ureter, a cancer of the kidney, a renal cell carcinoma, a cancer of the renal pelvis, a neoplasm of the central nervous system (CNS), a primary CNS lymphoma, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, a cholangiocarcinoma, a Hurth's cell thyroid cancer, or a gastroesophageal junction adenocarcinoma. The cancer may be a cancer of the lung, a triple negative breast cancer, a colon cancer, a cancer of the stomach, a cancer of the bladder, a prostate cancer, or a pancreatic cancer.

[0611] Further provided herein is a method of treating a fibrotic disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I) or a pharma- ceutically acceptable salt thereof).

[0612] The fibrotic disease or disorder may be arthrofibrosis, autoimmune pancreatitis, bladder fibrosis, chronic kidney disease, chronic wound, Crohn's disease, desmoid tumor, Dupuytren's contracture, endometrial fibrosis, fibromatosis, graft-versus-host disease, cardiac fibrosis, keloid, liver fibrosis, mediastinal fibrosis, myelofibrosis, nephrogenic systemic fibrosis, Peyronie's disease, pulmonary fibrosis, retroperitoneal fibrosis, scleroderma or systemic sclerosis, or skin fibrosis. The fibrotic disease or disorder may be pulmonary fibrosis, liver fibrosis, scleroderma, myelofibrosis, Crohn's disease, or chronic kidney disease. The pulmonary fibrosis may be idiopathic pulmonary fibrosis (IPF). The liver fibrosis may be NASH or liver cirrhosis.

[0613] Further provided herein is a method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of Formula (I) or a pharma- ceutically acceptable salt thereof).

[0614] Additional Usage Also provided is a method of binding a compound of formula (I') or a pharma- ceutically acceptable salt thereof to a receptor in a cell (e.g., in a subject, e.g., in a subject in need thereof). The method may include binding a compound of formula (I') or a pharma- ceutically acceptable salt thereof to a receptor in a cell (e.g., in a subject, e.g., in a subject in need thereof), where formula (I') has the structure: T'-LE (I') and T' is a targeting moiety (e.g., raltitrexed, 5-MTHF, an analog of raltitrexed, or a radical of an analog of 5-MTHF, a compound of Formula I-VII, II-VII, III-VII, IV-VII, V-VII, VI-VII, or VII-VII described herein, or a compound of Table I, II, or III; L is a linker; and E is a radical of a therapeutic agent (e.g., a radical of a therapeutic agent described herein); contacting a cell with the compound. In some embodiments, T may bind to a receptor on the cell. In some embodiments, T may bind to a pattern recognition receptor in the cell. In some embodiments, T may bind to an immune cell receptor. In some embodiments, T selectively binds to a folate receptor. In some embodiments, T selectively binds to FRβ. In some embodiments, T selectively binds to FRδ. In some embodiments, T binds to FRδ with a higher affinity than T binds to FRβ. In some embodiments, the cell is a macrophage. In some embodiments, the cell is a tumor associated macrophage. In some embodiments, the cell is a tumor associated macrophage. In some embodiments, the cell is a M1-macrophage. In some embodiments, the cell is a M2-macrophage.

[0615] A method of binding a compound of formula (I') or a pharma- ceutically acceptable salt thereof to a receptor in a subject (e.g., in a subject in need thereof), wherein formula (I') has the formula: T'-LE (I') Also provided are methods that include contacting a cell with the compound, T' being a targeting moiety (e.g., raltitrexed, 5-MTHF, an analog of raltitrexed, or a radical of an analog of 5-MTHF, a compound of Formula I-VII, II-VII, III-VII, IV-VII, V-VII, VI-VII, or VII-VII described herein, or a compound of Table I, II, or III); L is a linker; and E is a radical of a therapeutic agent (e.g., a radical of a therapeutic agent described herein); in some embodiments, T binds to a receptor on the cell. In some embodiments, T binds to a pattern recognition receptor in the cell. In some embodiments, T binds to an immune cell receptor. In some embodiments, T selectively binds to a folate receptor. In some embodiments, T selectively binds to FRβ. In some embodiments, T selectively binds to FRδ. In some embodiments, T binds to FRδ with higher affinity than T binds to FRβ. In some embodiments, the subject has cancer (e.g., a cancer described herein). In some embodiments, the subject has a fibrotic disease or disorder (e.g., a fibrotic disease or disorder described herein). In some embodiments, the subject has an inflammatory disease (e.g., an inflammatory disease described herein).

[0616] A specific definition As used herein, the following terms and phrases shall have the meanings indicated below: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0617] The term "about" may allow for a degree of variation in value or range of a stated value or limit of a stated range, e.g., within 10%, within 5%, or within 1%. The term "substantially" may allow for a degree of variation in value or range of a stated value or limit of a stated range, e.g., within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

[0618] The terms "a", "an", or "the" are used to include one or more than one unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. Furthermore, it is to be understood that expressions or terminology used herein, unless otherwise defined, are for descriptive purposes only and are not limiting. Any use of section headings is intended for purposes of interpretation of the document and should not be considered as limiting. Furthermore, information related to a section heading may occur within or outside that particular section. Furthermore, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety as if each was individually incorporated by reference. In the event of a conflict in usage between this document and documents incorporated by reference, the usage in the incorporated references should be considered supplementary to the usage in this document; in the event of an unresolved conflict, the usage in this document shall take precedence.

[0619] The term "pharmacologically acceptable carrier" is recognized in the art and refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in the delivery or transport of any subject composition or its components. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not harmful to the patient. Some examples of materials that can serve as pharma-ceutically 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, cocoa butter, and suppository wax; (10) glycerin, such as glycerin, glycerin, glycerol ... (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) buffers, 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 preparations.

[0620] The terms "patient" and "subject" are used interchangeably and include human patients, experimental animals, such as rodents (e.g., mice, rats, or hamsters), rabbits, monkeys, chimpanzees, livestock, such as dogs, cats, or rabbits, agricultural animals, such as cows, horses, pigs, sheep, or goats, or domesticated wild animals, such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales. The patient to be treated is preferably a mammal, in particular a human. EXAMPLES

[0621] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claimed invention in any way.

[0622] [Example 1] High affinity binding of raltitrexed to folate receptor delta (FR.DELTA.) in silico Virtual binding studies of raltitrexed to FRδ (previously known as the JUNO protein (PDB:5F4E)) were performed. These studies showed a relatively high binding affinity for FRδ in silico. Folate was shown to have approximately 1,000-fold lower binding affinity for FRδ compared to its binding affinity for folate receptor alpha (FRα) and folate receptor beta (FRβ). Figure 1A shows the structures of raltitrexed and folate. Figure 1B shows the predicted relative binding free energies (MMGBSA dGbind) and docking scores (XP GScore) for the binding of folate analogs to human (PDB:5F4Q) and mouse (PDB:5JYJ) FRδ. Figure 1C shows the binding pose of raltitrexed to human FRδ. Figure 1D shows the binding pose of raltitrexed to mouse FRδ. The ribbon diagram shows the ligand-protein interaction while the surface topography shows the electrostatic potential map and the binding orientation of the ligand in the FRδ binding cavity. Blue represents the positively charged regions of the binding site and red represents the negatively charged regions.

[0623] [Example 2] Raltitrexed selectively targets Tregs in vivo via FRδ Female BALB / c mice and C57BL / 6FRδ knockout mice were subcutaneously implanted with 250,000 cells of the 4T1 breast cancer cell line and the MB49 bladder cancer cell line (a folate receptor negative cell line), respectively. Tumor volumes were approximately 400 mm. 3When the IL-10 expression level reached 10 nmol, mice were tail vein injected with 10 nmol of Raltitrexed-S0456 (Ral-S056) (left mouse), 10 nmol of Raltitrexed-S0456 with 200-fold competition with Raltitrexed-glucosamine (middle mouse in the top image of Fig. 2A and second mouse from the left in the bottom image of Fig. 2A), 10 nmol of Folate-S0456 with 200-fold competition with Folate-glucosamine (second mouse from the right in the bottom image of Fig. 2A), or vehicle as a control (right mouse), and 10 nmol of Folate-S0456 as another control (middle mouse in the bottom image of Fig. 2A). Images were taken 4 hours after injection.

[0624] Figure 2A shows an image of a tumor-bearing mouse taken 4 hours after injection. The tumor and other major organs were then excised and imaged separately (Figure 2B, which shows images of most of the major organs from treated mice). The glucosamine conjugate serves as a competing ligand and is compared to a negative control.

[0625] After digestion of the tumor and spleen, comprehensive flow cytometric analysis of most immune cells was performed.

[0626] FIG. 3A shows the results of flow cytometry scatter plots for viable cells, and FIG. 3B shows the results of flow cytometry scatter plots for cells labeled with anti-CD45 antibodies. FIG. 3C shows the results of flow cytometry scatter plots for cells labeled with anti-CD4 and anti-CD25 antibodies. FIG. 3D shows the results of flow cytometry scatter plots for cells labeled with anti-CD127 and anti-FRδ antibodies. FIG. 3E shows the uptake of Ral-S0356 by CD45+CD4+CD25+CD127+FRδ+ Tregs isolated from murine tumors and spleens with or without 200-fold concentration of Ral-glucosamine competition, compared to folate-S0456 and unstained controls. FIG. 3F shows a comparison of the uptake of Ral-S0456 and folate-S0456 by different leukocyte populations in tumors. Figure 3G shows that there is no binding of Ral-S0456 or folate-S0456 to CD45-cell conjugates. Figure 3H shows that there is no binding of Ral-S0456 or folate-S0456 to CD45+CD8+ cytotoxic T cells. Figure 3I shows that there is no binding of Ral-S0456 or folate-S0456 to CD45+CD4+CD25-FRδ- cells, while Ral-S0456, but not folate-S0456, shows binding to CD45+CD4+CD25-FRδ+ memory T cells. Figure 3J shows that although there is some binding of Ral-S0456, there is significantly more binding of folate-S0456 to CD45+CD11b+F4 / 80 macrophages. FIG. 3K shows flow cytometry data demonstrating that Ral-S0456 accumulates in tumor Tregs from wild-type but not FRδ knockout mice, demonstrating that Ral-S0456 uptake is FRδ-receptor mediated.

[0627] In summary, we demonstrated that raltitrexed-S0456 was selectively taken up by activated tumor Tregs and that there was no uptake in resting splenic Tregs. Analysis also demonstrated that there was no uptake of folate-S0456 by Tregs. Competition of raltitrexed-S0456 with 200-fold higher concentrations of raltitrexed-glucosamine did not show any signal, supporting the idea that binding was receptor-mediated. Finally, FRδ knockout mice showed reduced uptake of raltitrexed-S0456 compared to wild-type mice.

[0628] Thus, raltitrexed, but not folate, targets activated Tregs in the TME but not resting Tregs in the spleen, and the data support that this uptake is mediated by FRδ receptors.

[0629] [Example 3] Synthesis of Raltitrexed-S0456 Figures 4 and 5 show the synthetic scheme for producing raltitrexed-S0456 utilized in the binding studies presented above.

[0630] Raltitrexed (I) (0.109 mmol, 1 eq) was dissolved in dimethyl sulfoxide (DMSO) (150 μL). N,N'-diisopropylethylamine (DIPEA) (0.33 mmol, 3 eq) and hexafluorophosphate azabenzotriazole tetramethyluronium (HATU) (0.23 mmol, 2 eq) were added, resulting in a brown material. The solution was stirred for 15 min, after which N-hydroxy-succinamide (NHS) (0.109 mmol, 1 eq) dissolved in DMSO (250 μL) was added to the reaction mixture, and the reaction mixture was stirred for 6 h. The product was precipitated with excess ether (15 mL x 3) and centrifuged, resulting in the removal of all impurities and the formation of a sticky brownish oily material, which was used in the next step without further purification.

[0631] Next, NH2-PEG-NBoc (75 μL) was dissolved in DMSO (150 μL) along with triethylamine (0.359 mmol) and the reaction mixture was stirred for 15 minutes, after which the crude from the previous step (dissolved in 150 μL DMSO) was added. The reaction mixture was stirred overnight. The reaction was purified by high performance liquid chromatography (HPLC) with a yield of 44% (30 mg of compound (II)).

[0632] Compound (II) (0.039 mmol) was dissolved in 500 μL of 50:50 2,2,2-trifluoroacetic acid (TFA):dichloromethane (DCM) and the reaction mixture was stirred for 2 hours. After that, the solvent was evaporated and further washed with DCM (1 mL×3). The deprotected product was then dissolved in DMSO (100 μL) along with triethylamine (TEA) (0.25 mmol) and the reaction mixture was stirred for 15 minutes, after which S0456-Cl near infrared (NIR) dye (0.078 mmol, 2 eq) dissolved in DMSO (100 μL) was added and the reaction mixture was stirred for 3 hours. The final product (III) (M.Wt. 1,512 g / mol) was purified by HPLC with a yield % of 25% (15 mg) (Final product (III)).

[0633] [Example 4] Synthesis of TLR7-1A agonists FIG. 6 shows the synthesis scheme of the TLR7-1A agonists used in the therapeutic evaluation and targeting studies herein.

[0634] 3-Amino-2,2-dimethyl-1-propanol (2.4 mmol) was added to a solution of 4-chloro-3-nitroquinoline (1.2 mmol) and triethylamine (Et3N) (3 mmol) in a 4:1 mixture of toluene and 2-propanol. The mixture was heated to 70° C. for 0.5 h until precipitation of a solid began. The reaction mixture was then cooled, filtered, and washed with toluene / 2-propanol (7:3), ether, and cold water. The residue was dried at 80° C. to give 2,2-dimethyl-3-((3-nitroquinolin-4-yl)amino)propan-1-ol. LCMS: [M+H]+m / z=275.31.

[0635] 2,2-Dimethyl-3-((3-nitroquinolin-4-yl)amino)propan-1-ol (1.72 mmol) was dissolved in methanol and hydrogenated with a hydrogen balloon over Pd / C for 4 h. The solution was then filtered using Celite, followed by evaporation of the solvent under reduced pressure to give 3-((3-aminoquinolin-4-yl)amino)-2,2-dimethylpropan-1-ol. LCMS: [M+H]+m / z=245.33.

[0636] To a solution of 3-((3-aminoquinolin-4-yl)amino)-2,2-dimethylpropan-1-ol (0.43 mmol) in anhydrous tetrahydrofuran (THF) was added triethylamine (0.65 mmol) and valeryl chloride (0.52 mmol). The reaction mixture was then stirred for 6-8 h, followed by removal of the solvent under vacuum. The residue was dissolved in ethyl acetate (EtOAc), washed with water and brine, and then dried over Na2SO4 to give the intermediate amide compound. This was dissolved in MeOH, followed by addition of calcium oxide and heating at 110 °C in a microwave for 1 h. The solvent was then removed and the residue was purified using column chromatography (9% MeOH / dichloromethane) to give 3-(2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2,2-dimethylpropan-1-ol (58 mg). LCMS: [M+H]+m / z=311.43.

[0637] To a solution of 3-(2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2,2-dimethylpropan-1-ol (0.186 mmol) in a solvent mixture of MeOH:dichloromethane:chloroform (0.1:1:1) was added 3-chloroperoxybenzoic acid (0.49 mmol) and the solution was refluxed at 45-50 °C for 40 min. The solvent was then removed and the residue was purified using column chromatography (20% MeOH / dichloromethane) to give the oxide derivative (55 mg). This was then dissolved in anhydrous dichloromethane followed by the addition of benzoyl isocyanate (0.26 mmol) and heated at 45 °C for 15 min. The solvent was then removed under vacuum and the residue was dissolved in anhydrous MeOH followed by the addition of excess sodium methoxide. The reaction mixture was then heated at 80 °C for 1 h. The solvent was removed under vacuum and the residue was purified using column chromatography (11% MeOH / dichloromethane) to give 3-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2,2-dimethylpropan-1-ol. LCMS: [M+H]+m / z=313.44

[0638] [Example 5] TLR7-1A agonists inhibit the immunosuppressive properties of murine Tregs ex vivo Murine CD45+CD4+CD25+ Treg and CD45+CD4+CD25- effector T cells were isolated from healthy mice using the StemCell Mouse Treg Isolation Kit (StemCell Technologies, Cambridge, MA). The cells were used in two different assays.

[0639] First, Tregs pretreated with 10 nM TLR7-1A agonist for 3 h or left untreated were co-cultured with CFSE-labeled effector T cells at a 1:4 ratio with or without CD3 / CD28 activation beads. Incubation was continued for 4 days before flow cytometry analysis.

[0640] In the second assay, Tregs pretreated with 10 nM TLR7-1A agonist for 3 h or left untreated were co-cultured with effector T cells at a ratio of 1:4 with or without CD3 / CD28 activating beads. Incubation was continued for 48 h, after which supernatants were analyzed for interleukin-10 (IL-10) and transforming growth factor beta (TGF-β) release using enzyme-linked immunosorbent assay (ELISA).

[0641] Tregs inhibited CD4+ T cell proliferation. However, TLR7-1A agonists reversed the suppressive activity and restored CD4+ cell proliferation. Furthermore, TLR7-1A-mediated reversal of Treg suppressive activity was evident by a reduction in the release of IL-10 and TGF-beta immunosuppressant cytokines.

[0642] Representative data are shown in Figures 7A-7E and 8 below. Figure 7A shows the parental CFSE-labeled CD4+CD25- population without co-cultured Tregs, treatment, or CD3 / CD28 bead activation. Figure 7B shows the divided CFSE-labeled CD4+CD25- population upon activation with co-cultured Tregs or without treatment. Figure 7C shows the effect of Tregs on the division of CFSE-labeled CD4+CD25- effector T cells when co-cultured at a 1:4 ratio. Figure 7D shows the effect of Tregs pre-treated with 10 nM TLR7-1A before co-culture with CFSE-labeled CD4+CD25- cells at a 1:4 ratio. Figure 7E shows a bar graph representation of the data, and Figure 8 shows the immune modulating effect of TLR7-1A on murine CD45+CD4+CD25+ Tregs in vitro, where Tregs were pretreated with TLR7-1A for 3 hours and then co-cultured with murine CD45+CD4+CD25- effector T cells for 48 hours, after which supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) for IL-10 and TGF-beta release.

[0643] Therefore, TLR7-1A agonists may be good candidates for targeting Tregs in vivo.

[0644] [Example 6] Synthesis of Raltitrexed-TLR7-1A Agonist Releasable Conjugate (Compound 11) FIG. 9 shows the synthesis scheme of the Ral-TLR7-1A agonist used in the in vivo targeting studies herein.

[0645] To synthesize compound 10, DIPEA (100 μl) and 1H-benzo[d][1,2,3]triazol-1-yl(2-(pyridin-2-yldisulfanayl)ethyl)carbonate 9 (0.3 mmol) were added to a solution of TLR7-1A (0.1 mmol) (see Example 4) in DMSO and stirred for 12 h. The crude reaction mixture was then purified by HPLC using 5-95% water in acetonitrile mobile phase and the combined purified fractions were lyophilized to give the product 10 as a colorless crystalline solid (yield approx. 33%).

[0646] To synthesize compound 11, compound 10 (0.3 mmol) was added to a solution of raltitrexed-cysteine ​​compound 6 (0.1 mmol) in a 1:1 mixture of aqueous ammonium acetate (pH-5) and dimethylformamide (DMF) and stirred for 12 h. After complete consumption of the starting material, the crude reaction mixture was purified by ultra-high performance liquid chromatography (UHPLC) using 5-95% water acetonitrile in the mobile phase. The combined purified fractions were lyophilized to obtain the raltitrexed-TLR7-1A conjugate (compound 11) as a colorless solid (yield about 63%). The product is confirmed by liquid chromatography-mass spectrometry (LCMS) (M.Wt. 976.15 g / mol).

[0647] [Example 7] Inhibition of tumor growth in 4T1 breast cancer model in BALB / c mice in vivo using a Ral-TLR7-1A agonist releasable conjugate (compound 11) 50,000 cells of the 4T1 breast cancer cell line (folate receptor negative cell line) were subcutaneously transplanted into 8-10 week old female BALB / c mice. The tumor size was approximately 50 mm. 3 When tumors reached 10 nmol / day, treatment was initiated with raltitrexed-TLR7-1A agonist at a daily dose of 10 nmol, raltitrexed at a daily dose of 10 nmol, raltitrexed-TLR7-1A agonist at a daily dose of 10 nmol and 200-fold concentrated raltitrexed-glucosamine competing ligand, or phosphate-buffered saline (PBS) alone, 5 days / week. Tumors were measured every other day, with some of the untreated mouse tumors reaching approximately 1500 mm. 3 Mice were sacrificed when the tumor reached a mass of 0.01 mg / kg / day. At the end of the study, tumors were digested, single cell suspensions were obtained, and cells were analyzed by flow cytometry to obtain a comprehensive immune cell analysis.

[0648] Tumor growth was significantly reduced in the ral-TLR7-1A agonist (Figure 10A, which shows 4T1 breast tumor volume / mm compared to untreated controls versus days after treatment initiation). 3 Body weight generally serves as a reflection of toxicity, but this did not change significantly over the course of treatment (Fig. 10B). Finally, Tregs, CD8 + Analysis of cytotoxic T cells and macrophages showed a shift in their profile towards a more pro-inflammatory phenotype, whereas the same cells isolated from the spleen showed no change (as shown in FIG. 10C, where relevant phenotypic markers are listed on the respective y-axis and treatment regimens are indicated on the respective x-axis). FIG. 11 shows splenic Treg and CD8 isolated from tumor-bearing mice in FIG. 10A. + 1 shows an assessment of the effect of ral-TLR7-1A on phenotypic markers of cytotoxic T cells.

[0649] Thus, raltitrexed-TLR7-1A agonist releasable conjugate therapy showed promising results in tumor growth inhibition by targeting Tregs to reprogram the tumor immune environment, but not in Tregs present in healthy tissues.

[0650] [Example 8] Inhibition of tumor growth in CT26 colorectal cancer model in BALB / c mice in vivo using a ral-TLR7-1A agonist releasable conjugate (compound 11) 50,000 cells of the CT26 colorectal cancer cell line (folate receptor negative cell line) were subcutaneously implanted into 8-10 week old female BALB / c mice. The tumor size was approximately 50 mm. 3 When tumors reached 10 nmol / day, treatment was initiated with raltitrexed-TLR7-1A agonist at a daily dose of 10 nmol or PBS alone, 5 days / week. Tumors were measured every other day, with some of the untreated mouse tumors reaching approximately 1500 mm 3 Mice were sacrificed when the .

[0651] Tumor growth was significantly reduced in the ral-TLR71A agonist-treated group (Figure 12). Body weight did not change significantly over the course of treatment.

[0652] Thus, ral-TLR7-1A agonist releasable conjugate therapy was effective in inhibiting tumor growth in another tumor model, supporting that this treatment approach may be universally applicable to multiple tumor models.

[0653] [Example 9] Inhibition of tumor growth in the MB49 bladder cancer model in FRβ knockout C57BL / 6 mice in vivo using a Ral-TLR7-1A agonist releasable conjugate (compound 11) 50,000 cells of the MB49 bladder cancer cell line (a folate receptor-negative cell line) were subcutaneously implanted into 8-10 week-old female FRβ knockout C57BL / 6 mice. The tumor size was approximately 50 mm. 3 When tumors reached 10 nmol / day, treatment was initiated with raltitrexed-TLR7-1A agonist at a daily dose of 10 nmol or PBS alone, 5 days / week. Tumors were measured every other day, with some of the untreated mouse tumors reaching approximately 1500 mm 3Mice were sacrificed when they reached the Treg and CD8 + Phenotypic markers of cytotoxic T cells were assessed.

[0654] As shown in Figure 13A, tumor growth was significantly reduced in the group treated with ral-TLR7-1A agonist. Body weight did not change significantly over the course of treatment. Treg and CD8 + Analysis of cytotoxic T cells showed a shift in their profile towards a more pro-inflammatory phenotype (Figure 13B).

[0655] Raltitrexed-TLR7-1A agonist releasable conjugate therapy inhibited tumor growth despite not targeting any macrophages that normally express FRβ, and the results were solely attributable to targeting reprogramming Tregs.

[0656] [Example 10] Synthesis of Raltitrexed-Dexamethasone Releasable Conjugate (Compound 19) FIG. 14 shows the synthesis scheme of ral-dexamethasone (compound 19) used in the in vivo targeting studies described herein.

[0657] To synthesize compound 13, dexamethasone (500 mg) was added to a 50 mL round bottom flask, followed by anhydrous THF (2.5 mL). The reaction was then cooled to -40°C using a dry ice / acetonitrile cooling bath. 0.53 mL (3 eq) of diphosphoryl chloride was added and stirred at -40°C for 1 h. The reaction was quenched with water and titrated to pH 8 with saturated aqueous sodium bicarbonate solution. The solution was acidified to pH 2 using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was dried over anhydrous Na2SO4, then removed under vacuum and used in the next step without any further purification (yield = 0.51 g).

[0658] To synthesize compound 15, (9H-fluoren-9-yl)methyl(2-hydroxyethyl)carbamate (500 mg) was added to a 50 mL round bottom flask, followed by 3.4 mL of anhydrous THF. The reaction was then cooled to -40°C using a dry ice / acetonitrile cooling bath. 0.6 mL of diphosphoryl chloride was added and stirred at -40°C for 1 hour. The reaction was quenched with water and titrated to pH 8 using saturated aqueous sodium bicarbonate solution. The solution was acidified to pH 2 using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was dried over anhydrous Na2SO4, then dried under vacuum, and used in the next step without any further purification (yield=0.64 g).

[0659] Triethylamine (0.16 mL) and CDI (0.45 g) were then added to a stirred solution of 395 mg of Fmoc-phosphate in 3 mL of DMF. The resulting solution was stirred at room temperature for 30 min. To this mixture was added dexamethasone 21-phosphate (500 mg) and ZnCl2 (1.18 g) and the mixture was stirred at room temperature overnight. The reaction was diluted with 1N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated and subjected to reverse phase column chromatography using a CombiFlash (yield=255 mg).

[0660] To synthesize compound 16, diethylamine (1 mL) was added to a stirred solution of Fmoc-pyro-Dex (255 mg) in DCM (4 mL) and the resulting mixture was stirred at room temperature for 2 h. The DCM / DEA mixture was removed under vacuum (yield=185 mg).

[0661] To synthesize compound 17, a solution of 95 mg of Fmoc-β-Ala-acid was activated with 85 mg of HATU, 85 mg of HOBtCl, 11 mL of DMF, and 0.3 mL of N-methylmorpholine (NMM). This mixture was then added to the Fmoc-deprotected pyro-Dex residue (185 mg) and stirred for 3 h to complete the reaction as monitored by LCMS. The reaction mixture was purified by reversed-phase column chromatography using a CombiFlash (yield=120 mg).

[0662] To synthesize compound 18, diethylamine (1 mL) was added to a stirred solution of FmocNH-β-Ala-pyro-Dex (120 mg) in DCM (4 mL). The resulting mixture was stirred at room temperature for 2 h. The DCM / DEA mixture was removed under vacuum (yield=80 mg).

[0663] To synthesize compound 19, raltitrexed (100 mg), N-hydroxyl-succinimide (31.3 mg), and dicyclohexylcarbodiimide (46.7 mg) were dissolved in dry DMSO (10 mL). The reaction mixture was stirred for 15 h at room temperature in the dark. NHS-raltitrexed was precipitated by adding ethyl acetate, filtered, and washed once with ethyl acetate and three times with anhydrous diethyl ether. NHS-raltitrexed was dried in a desiccator and used immediately. Fmoc-deprotected compound (15 mg) was dissolved in DMSO (1 mL), then NHS-raltitrexed and DIPEA were added. The reaction was monitored by LCMS and, once complete, purified by RP-HPLC and verified by LCMS (yield=3.5 mg).

[0664] [Example 11] Tumor growth inhibition and enhancement in 4T1 breast cancer model in BALB / c mice in vivo using ral-TLR71A agonist and ral-dexamethasone releasable conjugates (compounds 11 and 19, respectively) 50,000 cells of the 4T1 breast cancer cell line were subcutaneously implanted into 8-10 week-old female BALB / c mice. The tumor size was approximately 50 mm.3 When tumors reached 10 nmol / day, treatment was initiated with either ral-TLR7-1A agonist (compound 11) or ral-dexamethasone (compound 19) at a daily dose of 10 nmol or PBS alone, 5 days / week. Tumors were measured every other day, with some of the tumors in untreated mice reaching approximately 1500 mm 3 Mice were sacrificed when they reached the Treg and CD8 + Phenotypic markers of cytotoxic T cells were assessed.

[0665] Tumor growth was significantly reduced in the ral-TLR7-1A agonist-treated group and enhanced in the ral-dexamethasone-treated group (Figure 15A). + Analysis of cytotoxic T cells showed that the immunosuppressive Treg phenotype was significantly enriched and the cytotoxic properties of CD8+ T cells were reduced (Figure 15B).

[0666] In summary, raltitrexed-dexamethasone releasable conjugate therapy enhanced tumor growth, which was attributed to the enhanced immunosuppressant capacity of Tregs.

Claims

1. Compounds of formula (I): T-L-E (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-methyltetrahydrofolic acid (5-MTHF), an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E is a radical of a therapeutic agent).

2. T is a structure of formula (II): 【Chemistry 1】 2. The compound of claim 1 having the formula:

3. T is a structure of formula (III): 【Chemistry 2】 2. The compound of claim 1 having the formula:

4. 4. The compound of any one of claims 1 to 3, wherein the therapeutic agent is selected from the group consisting of Toll-like receptor 7 (TLR7) agonists, phosphoinositide 3-kinase (PI3k) inhibitors, steroids, nucleotide-binding oligomerization domain (NOD)-like receptor 2 (NLR2) agonists, stimulated interferon genes (STING) agonists, zest homolog 2 (EZH2) enhancer inhibitors, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inhibitors, caspase I inhibitors, retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonists, absent-in-melanoma 2 (AIM2)-like receptor agonists, and receptor for advanced glycation end products (RAGE) agonists.

5. 40. The therapeutic agent having the structure: 【Chemistry 3】 2. The compound of claim 1 which is an NLR2 agonist having the formula:

6. 40. The therapeutic agent having the structure: 【Chemistry 4】 2. The compound of claim 1, which is a STING agonist having the formula:

7. The compound of claim 1 , wherein the therapeutic agent is an EZH2 inhibitor.

8. The EZH2 inhibitor comprises: 【Chemistry 5】 or tazemetostat.

9. 40. The therapeutic agent having the structure: 【Chemistry 6】 2. The compound of claim 1, which is an NLRP3 inhibitor having the formula:

10. 40. The therapeutic agent having the structure: 【Chemistry 7】 2. The compound of claim 1 which is a caspase I inhibitor having the formula:

11. 40. The therapeutic agent having the structure: 【Chemistry 8】 2. The compound of claim 1 which is a PI3 kinase inhibitor having the formula:

12. 40. The therapeutic agent having the structure: 【Chemistry 9】 2. The compound of claim 1 which is an RLR agonist having the formula:

13. Compounds of formula (I): T-L-E (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E is a TLR7 agonist represented by formula (IV): 【Chemistry 10】 or a radical of a pharma- ceutically acceptable salt thereof: R １ , R ３ , R ４ , R ５ are each independently H, an alkyl group, an alkoxy group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, a halo group, a heteroaryl group, -COR ２ｘ , 【Chemistry 11】 and R ２ は、H、-OH、-NH ２ -NHR ２ｘ 、N ３ -NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ -NH-CS-NH ２、 【Chemistry 12】 and Z is H, -OH, -NH ２ , -NHR ２ｘ , -O-R ２ｘ , -SO-R ２ｘ , -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ N.H. ２ , alkenyl, alkynyl, alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ N.H. ２ , -NH-CS-NH ２ , 【Chemistry 13】 is: Each R ２ｘ and R ２ｙ is H, -OH, -CH ２ -OH, -NH ２ , -CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ , alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl; Each R ２ｚ is -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ and -COR ２ｑ each R ２ｑ and R ２ｑ’ is independently alkyl or H; 【Chemistry 14】 is a 3-10 membered N-containing non-aromatic, monocyclic or bicyclic heterocycle; R ２１ is H or alkyl, n' is 0 to 30; In formula (IV): X １ , X ２ , X ３ Each of the above may be independently selected from the group consisting of CR ｑ or N, and each R ｑ is independently H, halogen, or optionally substituted alkyl; n is 0 to 30; m is 0 to 4; n is 0 and Y is H, —OH, or —O—R ２ｘ isn't it).

14. E is a compound represented by formula (IVA): 【Chemistry 15】 or a pharma- ceutically acceptable salt thereof. R １ is optionally substituted C ３ ~C ８ is alkyl; R ２ is H, -OR ｚ , -SO ２ N (R ｚ ) ２ , -NR ２ｘ R ２ｙ , or N ３ and Z is H, -OR ｚ , -NR ２ｘ R ２ｙ , -SR ｚ , -SOR ｚ , -SO ３ R ｚ , -N ３ , -COR ｚ , -COOR ｚ , -CON(R ｚ ) ２ , -COSR ｚ , -SO ２ N (R ｚ ) ２ , or -CON(R ｚ ) ２ , where: R ２ｘ and R ２ｙ are each independently hydrogen, -N(R ｚ ) ２ , -CON(R ｚ ) ２ , -C(R ｚ ) ２ -N(R ｚ ) ２ , -CS-N(R ｚ ) ２ or optionally substituted alkyl; Each R ｚ are independently H, halogen, or optionally substituted alkyl; R ２ｘ and R ２ｙ taken together form an optionally substituted heterocycloalkyl; Each R ３ are independently halogen, -N ３ , -CN, -NO ２ , -COR ｚ , -COOR ｚ , -CON(R ｚ ) ２ , -COSR ｚ , -SO ２ N (R ｚ ) ２ , or -CON(R ｚ ) ２ , alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; R ４ and R ５ are each independently alkyl, alkoxy, halogen, or cycloalkyl, each of which is optionally substituted; n is 1 to 6; m is 0 to 4).

15. The compound of formula (I) is represented by formula (IVB) or formula (IVC): 【Chemistry 16】 or a pharma- ceutically acceptable salt thereof, Each R １ independently represents an optionally substituted C ３ ~C ８ is alkyl; Each R ２ are independently H, -OR ｚ , -SO ２ N (R ｚ ) ２ , -NR ２ｘ R ２ｙ , or N ３ and Each R ２ｘ and R ２ｙ are independently hydrogen, -N(R ｚ ) ２ , -CON(R ｚ ) ２ , -C(R ｚ ) ２ -N(R ｚ ) ２ , -CS-N(R ｚ ) ２ or optionally substituted alkyl; Each R ｚ are independently H, halogen, or optionally substituted alkyl; R ２ｘ and R ２ｙ taken together form an optionally substituted heterocycloalkyl; Each R ３ are independently halogen, -N ３ , -CN, -NO ２ , -COR ｚ , -COOR ｚ , -CON(R ｚ ) ２ , -COSR ｚ , -SO ２ N (R ｚ ) ２ , or -CON(R ｚ ) ２ , alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, each of which is optionally substituted; Each R ４ and R ５ is independently alkyl, alkoxy, halogen, or cycloalkyl, each of alkyl, alkoxy, and cycloalkyl being optionally substituted; n is 1 to 6; m is 0 to 4; Each Z ２ and Z ３ are independently represented by the formulas TL-, TLO-, TLO-alkyl-, TL-S １ -, T-SO ２ -NH-, TL-NR ａ R ｂ -, TLS(O) ｘ -alkyl-, T-L-CO-, T-L-aryl-, T-L-NH-CO-NH-, T-L-NH-O-, T-L-NH-NH-, T-L-NH-CS-NH, T-L-C(O)-alkyl-, or T-L-SO ２ - is a group; R ａ and R ｂ are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R ｃ and R ｃ is alkyl, aryl, oxy or alkoxy; S １ is a spacer; x is 0 to 3; n is 1 to 3 and m is 0 to 4).

16. R １ is optionally substituted with 1 to 3 substituents １ ~C ６ alkyl, each substituent independently being halogen or C １ ~C ６ is alkoxy; R ２ -NR ２ｘ R ２ｙ and R ２ｘ and R ２ｙ are each independently H or C １ ~C ６ is alkyl; Each R ３ are independently halogen, -CN, C １ ~C ６ Alkyl, C １ ~C ６ Heteroalkyl, C ３ ~C ７ Cycloalkyl, C １ ~C ６ alkoxy, amino, hydroxy, carboxyl, or thiol; R ４ and R ５ However, each independently, C １ ~C ６ is alkyl; each X １ , X ２ , and X ３ is N; Z ２ and Z ３ each of is independently T-L- or T-L-O-; n is 1; The compound according to claim 15, wherein m is 0 to 4.

17. Z ２ and Z ３ The compound of claim 15, wherein each of is T-L-O-.

18. R １ is optionally substituted C ３ ~C ６ 15. The compound of claim 13 or 14, which is alkyl.

19. R １ is an optionally substituted acyclic C ３ ~C ６ 15. The compound of claim 13 or 14, which is alkyl.

20. R ２ -NR ２ｘ R ２ｙ 15. The compound according to claim 13 or 14,

21. R ２ Ga-NH ２ 15. The compound according to claim 13 or 14,

22. The compound of formula (IVA) has the formula: 【Chemistry 17】 or a pharma- ceutically acceptable salt thereof, ３ is not present as needed).

23. The compound of formula (IV) has the formula: 【Chemistry 18】 or a pharma- ceutically acceptable salt thereof, ３ is not present as needed).

24. R １ C １ ~C ６ 24. The compound of claim 23, which is alkyl.

25. R ２ Ga-NH ２ 25. The compound of claim 23 or 24,

26. R ３ 25. The compound of claim 23 or 24, wherein is absent.

27. R ２ Ga-NH ２ and R ３ 25. The compound of claim 23 or 24, wherein is absent.

28. R １ C １ ~C ６ alkyl, R ２ Ga-NH ２ n is 1, and R ３ 24. The compound of claim 23, wherein is absent.

29. 29. The compound according to claim 28, wherein the compound of formula (I) is a compound represented by formula (V). 【Chemistry 19】

30. Compounds of formula (I): T-L-E (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E has the structure: 【Chemistry 20】 (Having the following characteristics).

31. Compounds of formula (I): T-L-E (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E has the structure: 【Chemistry 21】 is a radical of X is any of the following structures: 【Chemical 22】 ）。

32. E has the structure: 【Chemistry 23】 32. The compound of claim 31 comprising the radical:

33. Compounds of formula (I): T-L-E (I) or a pharma- ceutically acceptable salt thereof, T is a radical of raltitrexed, 5-MTHF, an analog of raltitrexed, or an analog of 5-MTHF; L is a linker; E is a radical of a corticosteroid).

34. 34. The compound of claim 33, wherein the corticosteroid is betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone-17-valerate, budesonide, flumethasone, fluticasone propionate, flurocortisone, fludrocortisone, paramethasone, eplerenone, or an ester of any of the foregoing.

35. 35. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, wherein L is a releasable linker.

36. 35. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, wherein L is a non-releasable linker.

37. The compound of claim 1 , wherein L comprises an optionally substituted heteroalkyl.

38. 38. The compound of claim 37, wherein the optionally substituted heteroalkyl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylate, and halo.

39. 40. The compound of any one of claims 1-3, 5-14, 22-24, and 28-34, 37, and 38, wherein L comprises a substituted heteroalkyl having at least one disulfide bond in its backbone.

40. 40. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, and 38, wherein L comprises a peptide or peptidoglycan having at least one disulfide bond in its backbone.

41. 40. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, and 38, wherein L is a releasable linker that can be cleaved by an enzymatic reaction, a reactive oxygen species (ROS), or reductive conditions.

42. L is a group of the formula -NH-CH ２ -CR ６ R ７ -S-S-CH ２ -CH ２ The compound according to any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, and 38, which comprises -O-CO-, wherein R ６ and R ７ are each independently H, alkyl, or heteroalkyl.

43. L is of the formula: 【Chemistry 24】 40. A compound according to any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, and 38, which is or contains a group (In the formula, p is an integer from 0 to 30; d is an integer from 1 to 40; R ８ and R ９ are each independently H, alkyl, heterocyclyl, cycloalkyl, aryl, or heteroalkyl.

44. 2. The compound of claim 1, wherein L comprises one or more linker moieties, each of said one or more linker moieties independently selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, carboxylate, carbonate, carbamate, amino acid, peptide, and peptidoglycan.

45. 45. The compound of claim 44, wherein L is or comprises a peptide or peptidoglycan.

46. 50. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44, and 45, wherein L is or comprises an amino acid.

47. 50. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44, and 45, wherein L is or comprises a PEG group.

48. 50. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44, and 45, wherein L is or comprises a polysaccharide.

49. L has the structure: 【Chemistry 25】 The compound according to any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44 and 45, which is or contains a group represented by the formula:

50. L, 【Chemistry 26】 50. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44, and 45, which is or comprises a linker moiety selected from the group consisting of:

51. 50. The compound of any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44, and 45, wherein L is a divalent linker or a trivalent linker.

52. 2. The compound of claim 1, further comprising a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, wherein the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to the linker.

53. 【Catalogue 27】 53. The compound of claim 52, further comprising an albumin binding group selected from the group consisting of:

54. The structure: 【Chemistry 28-1】 【Chemistry 28-2】 A compound comprising one of the following:

55. The structure: 【Chemical 29】 A compound comprising one of the following:

56. The structure: 【Chemistry 30-1】 【Chemistry 30-2】 A compound comprising one of the following:

57. The following structure: 【Chemistry 31】 A compound comprising one of the following:

58. 58. The compound of any one of claims 54 to 57, further comprising a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, or an albumin binding group, wherein the radical of the PEG group, the peptide group, the glycopeptide group, the saccharide group, or the albumin binding group is attached to the linker.

59. 【Chemical 32】 58. The compound of any one of claims 54 to 57, further comprising an albumin binding group selected from the group consisting of:

60. 13. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44 and 45, and a pharma- ceutically acceptable carrier or excipient.

61. formula: F-L'-G or a pharma- ceutically acceptable salt thereof, F is a radical of folate or an analog thereof; L' is a linker; G is the radical of glucosamine).

62. (i) the pharmaceutical composition of claim 60; (ii) Formula: F-L'-G or a pharma- ceutical composition comprising a compound of the formula: F is a radical of folate or an analog thereof; L' is a linker; G is a radical of glucosamine; A combination pharmaceutical composition, wherein (i) and (ii) can be administered to a subject by the same or different routes.

63. A method for immunomodulating regulatory T cells (Tregs) in a subject, comprising administering to the subject an effective amount of a first compound described in any one of claims 1 to 57 or a first pharmaceutical composition described in claim 60.

64. formula: F-L'-G or a pharma- ceutically acceptable salt thereof, F is a radical of folate or an analog thereof; L' is a linker; G is the radical of glucosamine); 64. The method of claim 63, wherein administration of the second compound may occur simultaneously with administration of the first compound or the first pharmaceutical composition, or sequentially in any order, by the same or different route.

65. the subject has cancer; E of said first compound or said first pharmaceutical composition is a radical of a TLR7 agonist, a PI3K inhibitor, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an NLRP3 inhibitor, a caspase I inhibitor, or an RLR agonist; 64. The method of claim 63, wherein administration of an effective amount of the first compound or first pharmaceutical composition modulates Treg-mediated promotion of tumor growth and metastasis and / or inhibition of anti-tumor immunity in the subject.

66. 66. The method of claim 65, further comprising administering a third therapeutic agent to the subject.

67. 67. The method of claim 66, wherein the third therapeutic agent is an anti-cancer agent, such as a chemotherapeutic agent or a radiotherapeutic agent.

68. 64. The method of claim 63, wherein the subject has a fibrotic disease or disorder and the E of the first compound or the first pharmaceutical composition is a radical of a therapeutic agent selected from the group consisting of a TLR7 agonist, a PI3K inhibitor, a steroid, an NLR2 agonist, a STING agonist, an EZH2 inhibitor, an aNLRP3 inhibitor, a caspase I inhibitor, and an RLR agonist.

69. 64. The method of claim 63, wherein the subject has an inflammatory disease and the E of the first compound or the first pharmaceutical composition is a radical of a steroid.

70. formula: F-L'-G or a pharma- ceutically acceptable salt thereof, F is a radical of folate or an analog thereof; L' is a linker; G is the radical of glucosamine).

71. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound described in any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44 and 45, or a pharma- ceutically acceptable salt thereof.

72. A method of treating a fibrotic disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44 and 45, or a pharma- ceutically acceptable salt thereof.

73. A method of treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound described in any one of claims 1 to 3, 5 to 14, 22 to 24, and 28 to 34, 37, 38, 44 and 45, or a pharma- ceutically acceptable salt thereof.

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