Cleavable conjugates of TLR7 / 8 agonist compounds, methods for preparation and uses thereof

Abstract

To provide a method of treating disease.SOLUTION: The present disclosure relates to cleavable conjugates (for example, particle-based or antibody-based conjugates) of TLR7 / 8 agonists (for example, 1H-imidazo[4,5-c]quinoline derivatives) that contain a conjugation linker, a cleavable linker and a self-eliminating linker. The disclosure also relates to a method of preparing cleavable conjugate, the use for stimulating effective immune response, and the use for the treatment of cancer.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 586,110, filed November 14, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to Toll-like receptor 7 / 8 agonist compounds covalently conjugated to a drug, such as a tumor-specific targeting agent or a polymeric nanoparticle agent, via a cleavable linker moiety, which facilitates local release and / or local retention of a bioactive form of the TLR7 / 8 agonist and reduces undesirable systemic proinflammatory cytokine responses. The present disclosure also relates to methods for preparing the cleavable conjugates, their use for stimulating an effective immune response, and their use for treating cancer. [Background technology]

[0003] Background of the Invention Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize structurally conserved molecules derived from pathogens, called pathogen-associated molecular patterns. Thus, TLRs function as frontline sensors of pathogen-associated molecular patterns in the mammalian immune system, detecting the presence of invading pathogens (Takeuchi and Akira 2010, Cell 140:805-820). The human genome contains 10 known TLRs. Among these, TLR7 and TLR8 sense single-stranded RNA ligands and their (oligo)nucleotide degradation products. The distribution of TLR7 and TLR8 is restricted to the endolysosomal compartment, and these receptors are preferentially expressed in antigen-presenting cells (APCs), a key cell type that modulates immune system activation.

[0004] Engagement of TLRs on sentinel immune cells triggers the biosynthesis of select cytokines (e.g., interferon type I), induction of costimulatory molecules, and increased antigen-presenting capacity by APCs—key molecular mechanisms activating innate and adaptive immune responses. Engagement of TLR7 on plasmacytoid dendritic cells leads to the induction of IFN-α / β, which plays an essential role in regulating adaptive immunity. TLR8 is expressed on myeloid dendritic cells, monocytes, and monocyte-induced dendritic cells, and agonist engagement of TLR8 induces a pronounced proinflammatory cytokine profile characterized by increased production of tumor necrosis factor α (TNF-α), interleukin-12 (IL-12), and IL-18. Thus, virtually all major types of monocytic and dendritic cells can be activated by agonists of either TLR7 or TLR8 to become highly effective antigen-presenting cells. Because most antigen-presenting cell types express only one of these two receptors, agonists that can stimulate both receptors are potentially more effective adjuvants than agonists specific for only one of these TLRs (Wille-Reece, et al. Proc. Nat'l Acad. Sci. 2005, 102:15190-15194). Furthermore, TLR7 and TLR7 / 8 agonists may also be effective in stimulating antitumor immune responses in cancer, based on studies in animal models (Singh, et al. J Immunol 2014, 193:4722-4731). Therefore, TLR agonists have potential applications as cancer therapeutics and vaccine adjuvants. It has been extensively investigated as a stimulator of innate and adaptive immune responses, including for use in immunosuppressants (Sabado et al. 2015, Ca Immunol Res 3:278-287; Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819).

[0005] Several small molecule structural classes are known to interact at the guanosine / uridine ligand-binding site and possess varying levels of TLR7 and / or TLR8 agonist bioactivity, but many of these agonists are derivatives of the 1H-imidazo[4,5-c]quinoline-rich chemical template. One early example is 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod), a moderately effective TLR7 agonist approved in 1997 as a topical formulation for actinic keratosis, superficial basal cell carcinoma, and genital warts. Subsequent medicinal chemistry efforts have yielded several derivatives with markedly improved dual TLR7 / 8 agonist activity, most notably 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (R848, Resiquimod), as well as 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ, FIG. 1) and 1-(3-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (meta-IMDQ, FIG. 1; see, e.g., Beesu, M. et al. 2015, J Med Chem, vol. 1, pp. 111-115, incorporated herein by reference). 50:7833-7849; U.S. Patent Nos. 8,728,486 and 9,441,005). However, the rapid systemic diffusion of these small molecule compounds after local administration (e.g., subcutaneous [SC], intratumoral [IT], or intramuscular [IM]) of pharmacologically relevant doses results in the systemic induction of proinflammatory cytokine responses and an increased risk of adverse events (e.g., fever, malaise, lymphopenia, etc.) in humans. See, e.g., Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819; Smirnov, D. et al. 2011, Vaccine 29:5434-5442. Thus, there remains a need for immunotherapeutic agents that: 1) have TLR7 / 8 agonist activity that has potent biological activity against both receptors and potent immunostimulatory activity that activates a larger subset of APCs than exclusively TLR7 or TLR8 agonists alone; 2) preferentially target a stable prodrug form of the TLR7 / 8 agonist to the tumor microenvironment or, after SC or intravenous administration, locally maintain the stable prodrug form of the TLR7 / 8 agonist in the tumor microenvironment and subsequently release the active form within the tumor microenvironment; and 3) have physicochemical properties that limit the subsequent distribution of the released active form (i.e., unconjugated) of the TLR7 / 8 agonist from the tumor microenvironment. The present invention provides conjugates of TLR7 / 8 agonist compounds containing a cleavable linker and an agent for tissue-specific targeting or local retention after administration, methods for their preparation, their use for stimulating local immune responses and reducing unwanted systemic immune activation, and their use for treating cancer. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 8,728,486 [Patent Document 2] U.S. Patent No. 9,441,005 [Non-patent literature]

[0007] [Non-Patent Document 1] Takeuchi and Akira 2010, Cell 140:805-820 [Non-patent document 2] Wille-Reece, et al. Proc. Nat'l Acad. Sci. 2005, 102:15190-15194 [Non-patent document 3] Singh, et al. J Immunol 2014, 193:4722-4731 [Non-patent document 4] Sabado et al. 2015, Ca Immunol Res 3:278-287 [Non-Patent Document 5] Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819 [Non-patent document 6] Beesu, M. et al. 2015, J Med Chem 50:7833-7849 [Non-Patent Document 7] Smirnov, D. et al. 2011, Vaccine 29:5434-5442 Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention The present disclosure provides modified 1H-imidazo[4,5-c]quinoline derivatives, which are potent TLR7 / 8 agonists, covalently conjugated to drugs for tumor-specific targeting or local retention after administration via a combination of self-removal linkers, cleavable linkers, and conjugation linkers. These cleavable conjugates facilitate local release and / or local retention of the bioactive form of the TLR7 / 8 agonist, reducing undesirable systemic proinflammatory cytokine responses. The present disclosure also relates to methods for preparing the cleavable conjugates, their use for stimulating effective immune responses, and their use for treating cancer.

[0009] In one embodiment, a compound of formula (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is a TLR7 / 8 agonist moiety; L 1is a bond or a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10 is H or C1-C8 alkyl, x is an integer from 1 to 500; F is a conjugation moiety; The TLR7 / 8 agonist moiety is a 1H-imidazo[4,5-c]quinoline derivative. The compound of formula (I) is provided.

[0010] In one embodiment, D is of formula (D-1): [ka] (In the formula, R 1A is C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl, R 2 is NHR 2a and R 2a is H or C1-C8 alkyl, R 35 are each independently halogen or C1-C8 alkyl; R 4a and R 4b are independently H or C1-C8 alkyl; R 5 are each independently halogen or C1-C8 alkyl; p and q are independently 0, 1, 2, 3, or 4; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0011] In one embodiment, D is a group represented by formula (D-2): [ka] (In the formula, n is an integer from 4 to 21, X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0012] In another embodiment, D is of formula (D-2a) or (D-2b): [ka] (In the formula, n is an integer from 4 to 21, R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1cis a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0013] In another embodiment, D is a group represented by formula (D-3): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0014] In a further embodiment, D is of formula (D-3a) or (D-3b): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0015] In yet another embodiment, D is a group represented by formula (D-4): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, L is X or -CH2-X-; X is -NH- or -NH(C=O)-; A is independently a C6-C alkyl optionally substituted with 1 to 4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. 14 arylene or 5-14 membered heteroarylene optionally substituted by 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0016] In another embodiment, D is of formula (D-4a) or (D-4b): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, A is independently a C6-C alkyl optionally substituted with 1 to 4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. 14 arylene or 5-14 membered heteroarylene optionally substituted with 1 to 4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b Ma TAHA-(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). Compounds of formula (I) are provided, having the formula:

[0017] In one aspect, L 1 But, equation (L-1): [ka] (In the formula, Y 1 is S, O or NH, and Ar 1 is an optionally substituted arylene, and R 11 and R 12 are independently H or optionally substituted C1-C8 alkyl. Compounds of formula (I) are provided, having the formula:

[0018] In another embodiment, L 2 But, equation (L-2): [ka] (In the formula, Y 2 is NR 30 , O or S, and R 30 , R 31 , R 32 , R 33 and R 34 are independently H, C1-C8 alkyl, or C3-C8 cycloalkyl. 2 is a peptide linker that is cleavable by one or more endosomal or lysosomal peptidases or proteases, or one or more pericellular peptidases or proteases expressed by cells in the tumor microenvironment. 2 is a peptide linker cleavable by endosomal cathepsins or pericellular type II transmembrane serine proteases.

[0019] In another embodiment, L 3 But -L 3a -Y 3 -L 3b - and Y 3 , L 3aand L 3b are independently an optional spacer fragment. 3 teeth, [ka] In some embodiments, L 3a teeth, [ka] In some embodiments, L 3b is the expression: [ka] or an acyl spacer fragment of the formula: [ka] and n is 0 to 200. In some embodiments, L 3b is the expression: [ka] or an acyl spacer fragment of the formula: [ka] and n is 0 to 200. In some embodiments, L 3 is the following: [ka] is.

[0020] In another embodiment, -L 3 -L 2 -L 1 The -D part is: [ka] Compounds of formula (I) are provided, wherein:

[0021] In one aspect, provided herein are compounds of Formula (I), wherein F is a tumor targeting agent. In some embodiments, the tumor targeting agent is an antibody or binding ligand that preferentially binds to a tumor cell surface antigen, a unique structural element of the extracellular matrix of the tumor microenvironment, or a unique structural element of tumor vasculature. In some embodiments, F has physical properties or surface chemical modifications that are designed to result in preferential distribution to the tumor microenvironment.

[0022] In another aspect, provided herein are compounds of Formula (I), wherein F is an agent that facilitates local retention of the compound of Formula (I). In some embodiments, F is a liposome, a virus-like particle, a nanoparticle, a microparticle, a macromolecule or supramolecule, a dendrimer, or a polypeptide.

[0023] Further provided is a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable excipient. In some embodiments of the pharmaceutical composition, the excipient is a solvent, a filler, an emulsifier / surfactant, a buffer, a tonicity agent, and / or a preservative. In some embodiments of the pharmaceutical composition, The form may be a solution or a lyophilized solid.

[0024] In another aspect, there is provided a method of stimulating an immune response in a mammalian subject in need thereof, the method comprising the step of administering to the mammalian subject a pharmaceutical composition comprising a compound of formula (I) in an amount sufficient to stimulate an immune response in the mammalian subject.

[0025] Also provided is a method of inducing an antigen-specific antibody response and / or an antigen-specific T cell response in a mammalian subject in need thereof, comprising administering to the mammalian subject a pharmaceutical composition comprising a compound of formula (I) in an amount and at a dosage schedule sufficient to induce an antigen-specific antibody response and / or an antigen-specific T cell response in the mammalian subject.

[0026] Also provided are methods of treating cancer in a mammalian subject in need thereof, comprising administering an effective amount of the pharmaceutical composition by a parenteral route of administration, either as a single agent or in combination with other agents for treating cancer (e.g., chemotherapy, targeted therapy, and / or immunotherapy).Kits comprising the pharmaceutical composition of the invention and instructions for use in treating cancer are also provided herein. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows a representation of the chemical structures of 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ) and 1-(3-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (meta-IMDQ). The atom numbering is also shown on IMDQ for the imidazoquinoline core structure.

[0028] [Figure 2] FIG. 2 shows the mechanism of release of IMDQ from exemplary compounds of formula (I) containing a valine-citrulline dipeptide cleavable linker.

[0029] [Figure 3] FIG. 3 shows the mechanism of release of IMDQ from exemplary compounds of formula (I) containing a dimethyl disulfide cleavable linker.

[0030] [Figure 4] FIG. 4 shows the in vitro release of IMDQ (as a percentage of total IMDQ) over time from Compound No. 64-53, Compound No. 64-53a, Compound No. 64-54, and Compound No. 64-54a, or variants without a cleavable linker, after incubation with glutathione or 5 μM cathepsin B, respectively.

[0031] [Figure 4A]FIG. 4A shows the in vitro release of IMDQ (as a percentage of total IMDQ) over time from Compound Nos. 64-53a, 64-53b, and 64-54c after incubation with 30 nM cathepsin B.

[0032] [Figure 5] FIG. 5 shows the induction of interferon-related genes in the popliteal lymph nodes of BALB / c mice 24 hours after a single subcutaneous footpad injection of Compound No. 64-53, Compound No. 64-54, or an equimolar amount of unconjugated IMDQ.

[0033] [Figure 5A] Figure 5A shows the induction of interferon-related genes over time in subcutaneously implanted CT26 tumors in BALB / c mice following a single bolus intratumoral injection of Compound No. 64-54a, Compound No. 64-54b, or an IMDQ-equivalent mass of unconjugated IMDQ. Data points are expressed as fold-increase in gene expression relative to PBS-injected controls, N=5 mice.

[0034] [Figure 5B] Figure 5B shows the induction of pro-inflammatory genes over time in subcutaneously implanted CT26 tumors in BALB / c mice following a single bolus intratumoral injection of Compound No. 64-54a, Compound No. 64-54b, or an IMDQ-equivalent mass of unconjugated IMDQ. Data points are expressed as fold-increase in gene expression relative to PBS-injected controls, N=5 mice.

[0035] [Figure 6] FIG. 6 shows concentrations of cytokines ILs-12p40, IL-6, and TNFα in serum of BALB / c mice 2 hours after a single subcutaneous footpad injection of Compound No. 64-53, Compound No. 64-54, equimolar concentrations of unconjugated IMDQ, or PBS vehicle control.

[0036] [Figure 7]FIG. 7 shows the induction of maturation marker CD86 on various antigen-presenting cells in popliteal lymph nodes and spleens 24 hours after a single subcutaneous footpad injection of Compound No. 64-53, Compound No. 64-54, an equimolar amount of unconjugated IMDQ, or a PBS vehicle control.

[0037] [Figure 8] Figure 8 shows the effect of weekly intratumoral administration of Compound No. 64-53a, Compound No. 64-54a, Compound No. 64-10a, or PBS vehicle control on tumor growth in BALB / c mice bearing syngeneic CT26 tumors with a single subcutaneous tumor. Panel A shows the effect on tumor volume over 27 days after tumor implantation. Panel B shows a scatter plot of tumor growth versus treatment on day 27 (3 days after the last administration of compound at three weekly doses). Data points are the mean + / - standard error of the mean for groups of 8 mice.

[0038] [Figure 9] 9 shows the effect on tumor growth of weekly intratumoral administration of an IMDQ-equivalent mass of unconjugated IMDQ, Compound No. 64-53a, Compound No. 64-54a, Compound No. 64-70, or PBS vehicle control to BALB / c mice bearing syngeneic CT26 tumors with a single subcutaneous tumor. Time is days after tumor implantation, and data points are the mean + / - standard error of the mean for groups of 8 mice.

[0039] [Figure 10] 10 shows the effect on tumor growth of weekly intratumoral administration of 30, 125, or 500 ng of IMDQ equivalent mass of Compound No. 64-54a (Panel A) or Compound No. 64-54b (Panel B), and PBS vehicle control, to BALB / c mice bearing a single subcutaneous tumor and syngeneic CT26 tumors. Time is days after tumor implantation, and data points are the mean + / - standard error of the mean for groups of 10 mice.

[0040] [Figure 11] 11 shows the effect on tumor growth of weekly intratumoral administration of 500 ng of IMDQ, 500 ng of IMDQ equivalent mass of Compound No. 64-54a, or PBS vehicle control, with or without twice-weekly immune checkpoint inhibitor administration, into BALB / c mice bearing syngeneic CT26 tumors and two contralateral subcutaneous tumors. Time is days after tumor implantation, and data points are the mean + / - standard error of the mean for groups of 10 mice.

[0041] [Figure 12] FIG. 12 shows a synthetic scheme for preparing Compounds Nos. 64-57.

[0042] [Figure 13] FIG. 13 shows a synthetic scheme for preparing Compounds Nos. 64-75.

[0043] [Figure 14] FIG. 14 shows a synthetic scheme for preparing Compounds Nos. 64-76.

[0044] [Figure 15] FIG. 15 shows a synthetic scheme for preparing Compounds Nos. 64-77. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description of the Invention The present disclosure relates to a TLR7 / 8 agonist compound that exhibits potent biological activity against both TLR7 / 8 receptors and is covalently conjugated to a conjugation moiety that enables tumor-specific targeting or local retention after administration via a cleavable linker that facilitates local release of the bioactive form of the TLR7 / 8 agonist and reduces undesirable systemic proinflammatory cytokine responses. The TLR7 / 8 agonist compound comprises a modified 1H-imidazo[4,5-c]quinoline derivative modified with an alkyl or hydrocarbyl group, an aryl group, a heteroaryl group, or a combination thereof. The conjugation moiety is a tumor-specific targeting agent or an agent that facilitates local retention after administration. The conjugation moiety can be an antibody, ligand, liposome, virus-like particle, nanoparticle, microparticle, macromolecule or supramolecule, dendrimer, or polypeptide. The cleavable linker moiety enables release of the TLR7 / 8 agonist compound into the tumor microenvironment. The present disclosure also relates to the use of the cleavable conjugates to stimulate an immune response (e.g., an antigen-specific CD4+ / CD8+ T cell response), their use to treat cancer, and methods for preparing the cleavable conjugates. I. General Methods and Definitions

[0046] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic chemistry, analytical chemistry, molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully described in the literature; see, e.g., Fiesers' Reagents for Organic Synthesis, 25 th edition (Ho, ed., Wiley, 2016); Comprehensive Organic Functional Group Transformations, 2 nd edition (Katritsky and Taylor, eds., Elsevier, 2004); Comprehensive Organic Synthesis, version 1- 8 (Trost and Flemming, eds., Permagon Press, 1991); Beilsteins Handbuch der Organischen Chemie, 4 (Auflage, ed., Springer-Verlag, 1934); Animal Cell Culture, sixth edition (Freshney, Wiley-Blackwell, 2010); Current Protocols in Cell Biology (Bonifacino et al., ed., John Wiley and Sons, Inc., 1996, including supplements through 2014); Current Protocols in Immunology (Coligan et al., eds., John Wiley & Sons, Inc., 1991 including supplements through 2014); Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley and Sons, Inc., 1987, including supplements through 2014); Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russell, Cold Spring Harbor Laboratory Press, 2001);およびMolecular Cloning: A Laboratory Manual, fourth edition (Green and Sambrook, Cold Spring Harbor Laboratory Press, 2012)。

[0047] The terms "individual" and "subject" refer to mammals, including, but not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals (e.g., horses), rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0048] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or a T-cell antigen receptor. Antigens include peptides, polypeptides, proteins, glycoproteins, polysaccharides, It can include complex carbohydrates, sugars, gangliosides, lipids and phospholipids, moieties thereof, and combinations thereof.

[0049] When present in the composition of the present disclosure, antigen can be synthesized or isolated from nature.The antigen suitable for administration in the method of the present disclosure includes any molecule that can induce antigen-specific B cell or T cell response.Hapten is included in the scope of "antigen".A "hapten" is a low molecular weight compound that is not immunogenic by itself, but is generally made immunogenic when conjugated with a larger immunogenic molecule.

[0050] "Polypeptide antigens" can include purified native peptides, synthetic peptides, engineered peptides, recombinant peptides, crude peptide extracts, or peptides in their partially purified or unpurified active state (e.g., peptides that are portions of attenuated or inactivated viruses, microorganisms, or cells), or fragments of such peptides. Polypeptide antigens are preferably at least 6 amino acid residues in length, preferably 8 to 1800 amino acids in length, more preferably 9 to 1000 amino acids in length, or 10 to 100 amino acids in length. Similarly, in some embodiments, polypeptides are about 9 to about 2000, about 9 to about 1000, about 9 to about 100, or about 9 to about 60 amino acids in length. In some embodiments, polypeptides are at least (lower limit) 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 amino acids in length. In some embodiments, the polypeptide is at most (upper limit) 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 50, or 25 amino acids in length. In some embodiments, the polypeptide antigen is 9-35 amino acids in length.

[0051] As used herein, the term "immunogenic" refers to an agent (e.g., an endogenous or exogenously administered polypeptide antigen) that elicits an adaptive immune response when administered to a mammalian subject under suitable conditions. The immune response can be a B-cell (humoral) and / or T-cell (cellular) mediated response.

[0052] "Adjuvant" refers to a substance that, when mixed with an immunogenic agent such as an antigen, non-specifically enhances or stimulates the immune response to the immunogenic agent in a recipient upon exposure to the mixture.

[0053] The term "agonist" is used in the broadest sense and includes any molecule that activates signal transduction through a receptor. For example, a TLR7 agonist binds to toll-like receptor 7 protein and activates the TLR7 signaling pathway; a TLR8 agonist binds to toll-like receptor 8 protein and activates the TLR8 signaling pathway; and a dual TLR7 / 8 agonist binds to both toll-like receptor 7 and toll-like receptor 8 proteins and activates both the TLR7 and TLR8 signaling pathways.

[0054] "Stimulation" of a response or parameter includes eliciting and / or enhancing such response or parameter when compared to conditions that are otherwise the same except for the agent or molecule, or alternatively, when compared to another condition (e.g., increasing TLR signaling in the presence of a TLR agonist when compared to the absence of the TLR agonist). For example, "stimulation" of an immune response means an increase in that response.

[0055] An "effective amount" of a drug disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner in association with the stated purpose. An "effective amount" or "sufficient amount" of a drug is an amount adequate to produce a desired biological effect, such as a beneficial result, including a beneficial clinical result. The term "therapeutically effective amount" refers to an amount of an agent (e.g., a TLR modulator) effective to "treat" a disease or disorder in a subject (e.g., a mammal such as a human).

[0056] The term "treating" a disease or "treatment" refers to carrying out a protocol, which may include administering one or more drugs to an individual (human or otherwise) to alleviate signs or symptoms of the disease. Thus, "treating" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that have only a palliative effect on the individual. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, the alleviation or amelioration of one or more symptoms, whether detectable or undetectable, a reduction in the extent of the disease, a stabilized (i.e., no worsening) state of the disease, prevention of disease spread, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean a prolongation of survival compared to the expected survival of an individual not receiving treatment. " Alleviating " disease or disorder means that the severity and / or undesirable clinical symptoms of disease or disorder are reduced, and / or the progression of disease or disorder is slowed down compared with the expected outcome of untreated treatment.In particular, in the context of cancer, alleviation can occur when stable disease or disease remission is caused, which leads to an increase in overall survival rate.Furthermore, alleviation does not necessarily occur by administering a single dose, but often occurs when administering a series of doses.Therefore, the amount sufficient to respond or alleviate disorder can be administered in one or more doses.

[0057] "Alkyl," as used herein, refers to a monovalent linear (i.e., unbranched) or branched saturated hydrocarbon chain, or combinations thereof. Particular alkyl groups are those having a specified number of carbon atoms, e.g., 1 to 20 carbon atoms ("C1-C 20 alkyl"), having 1 to 10 carbon atoms ("C1-C 10" alkyl"), 1 to 8 carbon atoms ("C1-C8 alkyl"), 1 to 6 carbon atoms ("C1-C6 alkyl"), 2 to 6 carbon atoms ("C2-C6 alkyl"), or 1 to 4 carbon atoms ("C1-C4 alkyl"). Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0058] "Alkenyl," as used herein, refers to a monovalent linear (i.e., unbranched) or branched unsaturated hydrocarbon chain, or combinations thereof, having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C). Particular alkenyl groups are those having a specified number of carbon atoms, for example, 2 to 20 carbon atoms ("C2-C 20 alkenyl), having 2 to 10 carbon atoms ("C2-C 10 " alkenyl"), having 2 to 8 carbon atoms ("C2-C8 alkenyl"), having 2 to 6 carbon atoms ("C2-C6 alkenyl"), or having 2 to 4 carbon atoms ("C2-C4 alkenyl"). Alkenyl groups can be in the "cis" or "trans" configuration, or alternatively, the "E" or "Z" configuration. Examples of alkenyl groups include, but are not limited to, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs and isomers thereof.

[0059] "Alkynyl," as used herein, refers to a monovalent straight-chain (or any group) alkyl group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of formula C≡C). Specific alkynyl groups are those having a specified number of carbon atoms, for example, 2 to 20 carbon atoms ("C2-C6"). 20 alkynyl), having 2 to 10 carbon atoms ("C2-C 10 alkynyl groups having 2 to 8 carbon atoms ("C-C alkynyl"), 2 to 6 carbon atoms ("C-C alkynyl"), or 2 to 4 carbon atoms ("C-C alkynyl"). Examples of alkynyl groups include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs and isomers thereof.

[0060] "Alkylene," as used herein, refers to the same residue as alkyl, but with divalency. Particular alkylene groups are those having 1 to 6 carbon atoms ("C-C alkylene"), 1 to 5 carbon atoms ("C-C alkylene"), 1 to 4 carbon atoms ("C-C alkylene"), or 1 to 3 carbon atoms ("C-C alkylene"). Examples of alkylene groups include, but are not limited to, groups such as methylene (-CH- or =CH), ethylene (-CHCH- or =CHCH), propylene (-CHCHCH- or =CHCHCH), butylene (-CHCHCHCHCH- or =CHCHCHCH).

[0061] "Cycloalkyl," as used herein, refers to a monovalent non-aromatic, saturated or unsaturated cyclic hydrocarbon structure. Particular cycloalkyl groups have a specified number of annular (i.e., ring) carbon atoms, such as cycloalkyl groups having 3 to 12 ring carbon atoms ("C3-C6"). 12Preferred cycloalkyls are cyclic hydrocarbons having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), or 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). Cycloalkyls can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl, but exclude aryl groups. Cycloalkyls containing more than one ring can be fused, spiro, or bridged, or combinations thereof. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and the like.

[0062] "Cycloalkylene," as used herein, refers to a residue the same as cycloalkyl, but having a divalent atom. Particular cycloalkylene groups have 3 to 12 ring carbon atoms ("C3-C 12 cycloalkylene"), those having 3 to 8 ring carbon atoms ("C-C cycloalkylene"), or those having 3 to 6 ring carbon atoms ("C-C cycloalkylene"). Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, 1,2-cyclohexenylene, 1,3-cyclohexenylene, 1,4-cyclohexenylene, cycloheptyl, norbornyl, and the like.

[0063] "Hydrocarbyl," as used herein, refers to a group having a specified number of carbon atoms (i.e., C1-C 20"hydrocarbyl" refers to and includes monovalent groups formed by removing a hydrogen atom from a non-aromatic hydrocarbon, which may be fully saturated, monounsaturated, or polyunsaturated ("hydrocarbyl" means 1 to 20 carbon atoms). Hydrocarbyl groups can contain one or more linear, branched, or cyclic moieties, or combinations thereof. Alkyl, alkenyl, alkynyl, and cycloalkyl groups are particular subsets of hydrocarbyl groups. Hydrocarbyl groups are further substituted with one or more alkyl, alkenyl, or alkynyl groups, which are further substituted with one or more cycloalkyl groups, and / or one of further alkyl, alkenyl, and / or alkynyl groups. Examples of hydrocarbyl groups include, but are not limited to, the following: [ka] The hydrocarbyl groups may be substituted at one or more positions with one or more substituents such as halogen atoms, e.g., chlorine or fluorine. Examples of substituted hydrocarbyl groups include, but are not limited to, the following: [ka] It includes groups such as:

[0064] "Aryl," as used herein, refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), wherein one or more of the condensed rings may not be aromatic. Particular aryl groups are those having 6 to 14 annular (i.e., ring) carbon atoms ("C6-C6"). 14Aryl groups are groups having more than one ring, where at least one ring is non-aromatic, that can be attached to the parent structure at either an aromatic or non-aromatic ring position. In one variation, aryl groups having more than one ring, where at least one ring is non-aromatic, are attached to the parent structure at an aromatic ring position. Examples of aryl include, but are not limited to, groups such as phenyl, naphthyl, 1-naphthyl, 2-naphthyl, etc.

[0065] "Arylene," as used herein, is the same as aryl but has two valencies. A typical arylene group is one having 6 to 14 ring carbon atoms ("C6-C 14 Examples of arylene include, but are not limited to, groups such as phenylene, o-phenylene (i.e., 1,2-phenylene), m-phenylene (i.e., 1,3-phenylene), p-phenylene (i.e., 1,4-phenylene), naphthylene, 1,2-naphthylene, 1,3-naphthylene, 1,4-naphthylene, 2,7-naphthylene, and 2,6-naphthylene.

[0066] "Heteroaryl," as used herein, refers to an unsaturated aromatic ring group having 1 to 14 ring carbon atoms and at least one ring heteroatom, including, but not limited to, nitrogen, oxygen, and sulfur. Heteroaryl groups can have a single ring (e.g., pyridyl or imidazolyl) or multiple condensed rings (e.g., indolizinyl or pyrazolo-pyridazinyl), wherein at least one of the condensed rings is aromatic. Particular heteroaryl groups are 5-14 membered rings having 1-12 ring carbon atoms and 1-6 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"), 5-10 membered rings having 1-8 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"), or 5-, 6-, or 7-membered rings having 1-5 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5-17 membered heteroaryl"). In one variation, heteroaryl includes 5-, 6-, or 7-membered monocyclic aromatic rings having 1-6 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In another variation, heteroaryl includes polycyclic aromatic rings having 1 to 12 ring carbon atoms and 1 to 6 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Heteroaryl groups having more than one ring, at least one of which is non-aromatic, can be attached to the parent structure at either an aromatic or non-aromatic ring position. In one variation, heteroaryl groups having more than one ring, at least one of which is non-aromatic, are attached to the parent structure at an aromatic ring position. Examples of heteroaryl include, but are not limited to, pyridyl, benzimidazolyl, benzotriazolyl, benzo[b]thienyl, quinolinyl, indolyl, benzothiazolyl, and the like.

[0067] "Heteroarylene," as used herein, refers to a divalent radical, similar to heteroaryl. Particular heteroarylene groups are 5- to 14-membered rings having 1-12 ring carbon atoms and 1-6 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5- to 14-membered heteroarylene"); 5- to 10-membered rings having 1-8 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5- to 10-membered heteroarylene"); or 5-, 6-, or 7-membered rings having 1-5 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur ("5- to 7-membered heteroarylene"). Examples of heteroarylene include, but are not limited to, pyridylene, benzimidazolylene, benzotriazolylene, benzo[b]thienylene, quinolinylene, indolylene, benzothiazolylene, and similar groups.

[0068] "Halo" or "halogen" refers to elements in the Group 17 series having atomic numbers 9-85. Preferred halo groups include fluoro, chloro, bromo, and iodo. When a residue is substituted with more than one halogen, it may be referred to using a prefix corresponding to the number of halogen moieties attached. For example, dihaloaryl, dihaloalkyl, and trihaloaryl refer to aryl and alkyl substituted with two ("di") or three ("tri") halo groups, which may, but need not, be the same halo. Thus, 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. An alkyl group in which each hydrogen is replaced with a halo group is referred to as "phenyl." A preferred perhaloalkyl group is trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" refers to an alkoxy group in which a halogen replaces each H in the hydrocarbon comprising the alkyl portion of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

[0069] "Amino" refers to the group -NH2.

[0070] "Substituted amino" refers to the group -NR'R" where R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one of R' and R" is not hydrogen.

[0071] "Optionally substituted" means, unless otherwise specified, that a group can be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) of the substituents listed for that group, which can be the same or different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has 1 to 2, 1 to 3, 1 to 4, or 1 to 5 substituents.

[0072] "Substituted alkyl," unless otherwise specified, refers to an alkyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from acyloxy, hydroxy, mercapto, acyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0073] "Substituted alkenyl," unless otherwise specified, refers to an alkenyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from acyloxy, hydroxy, mercapto, acyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0074] "Substituted alkynyl" means, unless otherwise specified, acyloxy, hydroxy, mercapto, acyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy ...

[0033] The term "alkynyl" refers to an alkynyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from oxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0075] "Substituted cycloalkyl," unless otherwise specified, refers to a cycloalkyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from acyloxy, hydroxy, thiol, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0076] "Substituted aryl" refers to an aryl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from acyloxy, hydroxy, thiol, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl, unless otherwise specified.

[0077] "Substituted heteroaryl" refers to a heteroaryl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from acyloxy, hydroxy, thiol, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, and trihalomethyl, unless otherwise specified.

[0078] "Substituted heterocyclyl" means, unless otherwise specified, acyloxy, hydroxy, thiol, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, " refers to a heterocyclyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyl ester, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, sulfonylamino, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0079] In addition to the groups disclosed for each individual term herein, one or more hydrogens on a saturated carbon atom in the specified group or radical (any two hydrogens on a single carbon can be replaced by ═O, ═NR 70 , =N-OR 70 , ═N2 or ═S), unless otherwise specified, is substituted with -R 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)R 70 , -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M+ )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heterocycloalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; R 70 are each independently hydrogen or R 60 and R 80 are each independently R70 or alternatively, two R 80 together with the nitrogen atom to which they are attached form a 3-, 4-, 5-, 6-, or 7-membered heterocycloalkyl, which can optionally contain 1 to 4 additional heteroatoms, the same or different, selected from the group consisting of O, N, and S, wherein N is —H, C1-C4 alkyl, —C(O)C 1~4 Alkyl, -CO2C 1~4 Alkyl or -SO2C 1~4 M may have alkyl substitution + are counterions each with a net positive charge of 1. + are each independently, e.g., K + , Na + , Li + Alkaline ions such as; + N(R 60 ) 4; or [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 or [Ba 2+ ] 0.5 (The "subscript 0.5" means that one of the counterions to such divalent alkaline earth ions can be the ionized form of a compound of the embodiments and the other can be a typical counterion such as chloride, or that a two-ionized compound disclosed herein can serve as a counterion to such divalent alkaline earth ions, or that a doubly-ionized compound of this embodiment can serve as a counterion to such divalent alkaline earth ions.)

[0080] Further to the disclosure herein, substituents for hydrogens on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl, and heteroaryl groups are defined as -R unless otherwise specified. 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M+ 、-NR 80 R 80 、トリハロメチル、-CF3、-CN、-OCN、-SCN、-NO、-NO2、 -N3、-S(O)R 70 、-SO2R 70 、-SO3 - M + 、-SO3R 70 、-OSO2R 70 、-OSO3 - M + 、-OSO3R 70 、-PO3 -2 (M + )2、-P(O)(OR 70 )O - M + 、-P(O)(OR 70 )2、-C(O)R 70 、-C(S)R 70 、-C(NR 70 )R 70 、-CO2 - M + 、-CO2R 70 、-C(S)OR 70 、-C(O)NR 80 R 80 、-C(NR 70 )NR 80 R 80 、-OC(O)R 70 、-OC(S)R 70 、-OCO2 - M + 、-OCO2R 70 、-OC(S)OR 70 、-NR 70 C(O)R 70 、-NR 70 C(S)R 70 、-NR 70 CO2 - M + 、-NR 70 CO2R 70 、-NR 70 C(S)OR 70 、-NR 70 C(O)NR 80 R 80 、-NR 70 C(NR 70 )R 70and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent may be -O - M + , -OR 70 , -SR 70 or -S - M + provided that it is not.

[0081] In addition to the substituents disclosed for each individual term herein, substituents for the hydrogen on a nitrogen atom in "substituted" heterocycloalkyl and cycloalkyl groups are defined as -R, unless otherwise specified. 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)R 70 , -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 and -NR 70 C(NR 70 )NR 80 R 80 and R 60 , R 70 , R 80 and M + is as defined above.

[0082] For all of the substituted groups defined above, it is understood that polymers arrived at by defining a substituent that itself has further substituents (e.g., a substituted aryl having a substituted aryl group as a substituent that is itself substituted with a substituted aryl group that is further substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0083] Unless otherwise indicated, naming of substituents not explicitly defined herein is accomplished by naming the terminal portion of that functionality and then the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0084] With respect to any of the groups disclosed herein that contain one or more substituents, it is understood that such groups do not contain any substitutions or substitution patterns that are sterically difficult and / or synthetically impractical. Furthermore, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0085] "Polyalkylene glycol" refers to linear or branched polyalkylene glycol polymers such as polyethylene glycol ("PEG" or polyethylene oxide), polypropylene glycol ("PPG" or polypropylene oxide), and polybutylene glycol. A polyalkylene glycol subunit is a single polyalkylene glycol. Poly(ethylene glycol) subunits are chain units. For example, examples of polyethylene glycol subunits are ethylene glycol, -[CH2-CH2-O]-; or propylene glycol, -[CH2-CH(CH3)-O]-, capped with hydrogen at the end chain points. Other examples of poly(alkylene glycols) include, but are not limited to, PEG; PEG derivatives such as methoxypoly(ethylene glycol) (mPEG); poly(ethylene oxide); PPG; poly(tetramethylene glycol) (also known as poly(tetrahydrofuran) or polyTHF); poly(ethylene oxide-co-propylene oxide); or copolymers and combinations thereof.

[0086] "Organic modifier," unless otherwise specified, means one of a group of solvents commonly used to dissolve organic chemical compounds. This group can include, but is not limited to, acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol (isopropanol), propyl acetate, and combinations thereof.

[0087] Further to the disclosure herein, the term "substituted," when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituents, as defined herein. In some embodiments, a substituted group has one, two, three, or four substituents, one, two, or three substituents, one or two substituents, or one substituent.

[0088] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0089] Unless a specific isotope of an element is indicated in a formula, the present invention also includes, for example, deuterated derivatives of the compounds (where H is 2H, which may be D. Isotopic substitutions may have isotopic substitutions at any or all positions in the structure, or may have atoms present at natural abundance at any or all positions in the structure.

[0090] "Solvate" refers to a complex formed by the combination of solvent molecules with solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0091] "Stereoisomer" and "stereoisomers" refer to compounds that have the same atom connectivity but differ in the arrangement of the atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0092] It will be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include all permutations of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compound.

[0093] For clarity, it is understood that certain features of the invention that are described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments with respect to chemical groups represented by variables are specifically embraced by the present invention, and each and every combination is disclosed herein exactly as if individually and explicitly disclosed, to the extent that such combinations result in stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). Furthermore, all subcombinations of chemical groups listed in embodiments describing such variables are also specifically embraced by the present invention, and each and every such subcombination of chemical groups is disclosed herein exactly as if individually and explicitly disclosed herein.

[0094] Aspects and embodiments described herein as "comprising" are understood to include "consisting of" and "consisting essentially of" embodiments.

[0095] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated or clear from the context.

[0096] The term "about," unless expressly indicated otherwise, is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself. For example, a description that refers to "about X" includes the description of "X." II. Tumor-targeting cleavable conjugates of TLR7 / 8 agonist compounds

[0097] The present disclosure provides potent TLR7 / 8 agonist modified 1H-imidazo[4,5-c]quinoline derivatives that can be covalently conjugated to drugs for tumor-specific targeting or local retention after administration using a combination of self-removal linkers, cleavable linkers, and conjugation linkers, facilitating the local release of the bioactive form of the TLR7 / 8 agonist. Within the tumor microenvironment, the covalently conjugated TLR7 / 8 agonist compounds of the present disclosure are chemically cleaved, facilitating the release of the TLR7 / 8 agonist in its most bioactive form (i.e., the unconjugated form). These locally released TLR7 / 8 agonists can therefore elicit effective immune responses. Furthermore, the systemic distribution of the covalently conjugated TLR7 / 8 agonist compounds of the present disclosure can be limited due to their chemical nature, thereby reducing undesirable systemic proinflammatory cytokine responses. The present disclosure also relates to methods for preparing therapeutic or vaccine adjuvants comprising covalent conjugates of TLR7 / 8 agonists, their use to reduce systemic proinflammatory cytokine responses and stimulate immune responses, and their use as therapeutic agents for treating cancer.

[0098] In one embodiment, a compound of formula (I): F-[WL 3 -L 2 -L 1 -D] X (I) (In the formula, D is a TLR7 / 8 agonist moiety; L 1 is a bond or a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10 is H or C1-C8 alkyl, x is an integer from 1 to 500; F is a conjugation moiety The compound of formula (I) is provided.

[0099] In another embodiment, a compound of formula (Ia): F-[WL 3 -L 2 -L 1 -D] X (Ia) (In the formula, D is a TLR7 / 8 agonist moiety; L 1 is a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10 is H or C1-C8 alkyl, x is an integer from 1 to 500; F is a conjugation moiety The compound of formula (I) is provided. A. TLR7 / 8 agonists

[0100] In some embodiments, the TLR7 / 8 agonist moiety of D in Formula (I) is a 1H-imidazo[4,5-c]quinoline derivative. In some embodiments, the TLR7 / 8 agonist moiety of D is a 2-butyl-1H-imidazo[4,5-c]quinolin-4-amine derivative.

[0101] In some embodiments, the TLR7 / 8 agonist moiety of D in formula (I) has the formula (D-1): [ka] (In the formula, R 1Ais C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl, R 2 is NHR 2a and R 2a is H or C1-C8 alkyl, R 35 are each independently halogen or C1-C8 alkyl; R 4a and R 4b are independently H or C1-C8 alkyl; R 5 are each independently halogen or C1-C8 alkyl; p and q are independently 0, 1, 2, 3, or 4; The wavy line represents the point of attachment of D in formula (I). It has.

[0102] In some embodiments, R 1A is optionally substituted C1-C8 alkyl, C1-C8 hydroxyalkyl, or optionally substituted C3-C8 cycloalkyl. 1A is C1-C8 alkyl optionally substituted with hydroxy. In a particular variation, R 1A is C1-C8 alkyl (e.g., n-butyl or isobutyl). In another particular variation, R 1A is C1-C8 hydroxyalkyl. In one variation, R 1A is a C3-C8 cycloalkyl.

[0103] In some embodiments, R 2 is NH2 or NHR 2a and R 2a is optionally substituted alkyl. In one variation, R 2 In another variation, R 2 is NHR 2a and R 2a is an optionally substituted C1-C8 alkyl. In a particular variation, R2 is NHR 2a and R 2a is a C1-C8 alkyl.

[0104] In some embodiments, q is 0 (i.e., R 35 In some embodiments, q is 1 and R 35 is attached to the imidazo[4,5-c]quinoline core at position 6, 7, 8, or 9. In some embodiments, q is 1 and R 35 is an amino or substituted amino attached to the 7- or 8-position of the imidazo[4,5-c]quinoline core. In some embodiments, q is 2 and two R 35 The groups are attached to the 7- and 8-positions of the imidazo[4,5-c]quinoline core and, together with the carbons to which they are attached, form a cycloalkyl, aryl, heteroaryl, or heterocyclyl. In some embodiments, q is 1 or 2, and R 35 is each independently halogen or C1-C8 alkyl.

[0105] In some embodiments, R 4a and R 4b are each H. In some embodiments, R 4a and R 4b taken together with the carbon to which they are attached form an optionally substituted C3-C8 cycloalkyl (e.g., cyclopropyl). In some embodiments, p is 0 (i.e., R 5 In some embodiments, p is 1 or 2 and R 5 is each independently halogen or C1-C8 alkyl. R as detailed herein with respect to formula (D-1) 1A , R 2 , R 35 , R 4a , R 4b , R 5Any and all variations of p and q are within the R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , R 37 , R 38 , R 39 , R 40 , R 41 , R 42 , 1A , R 2 , R 35 , R 4a , R 4b , R 5 It is intended and understood that D can be combined with any and all other variations of p and q. For example, in some embodiments, D can be selected from R 1A is C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl, and R 2 But NH2 or NHR 2a and R 2a is C1-C8 alkyl, and R 35 are each independently halogen or C1-C8 alkyl, and R 4a and R 4b are independently H or C1-C8 alkyl, and R 5 is each independently halogen or C1-C8 alkyl; and p and q are independently 0, 1, 2, 3, or 4. In some embodiments, the TLR7 / 8 agonist moiety of formula (D-1) is a p-aminomethylbenzyl-1H-imidazo[4,5-c]quinoline moiety of formula (D-1a) or an m-aminomethylbenzyl-1H-imidazo[4,5-c]quinoline moiety of formula (D-1b): [ka] (wherein the wavy line represents the point of attachment of D in formula (I)). In some embodiments, D has the formula (D-1a) or (D-1b), and R 1A is butyl, and R 2 is NH and q is 0. In one variation, R 4a and R 4b are H, respectively.

[0106] In some embodiments, D is a group represented by formula (D-1a-1) or (D-1b-1): [ka] (wherein the wavy line represents the attachment point of D in formula (I)) It has.

[0107] Other TLR7 / 8 agonists known in the art are also encompassed by the present disclosure. In some embodiments, the TLR7 / 8 agonist is an amino acid sequence similar to that described in Beesu et al. 2015, J Med Chem 58:7833-7849, which is incorporated herein by reference in its entirety. Norrin TLR8 agonist compounds. For example, the compounds containing reactive amino groups listed in Table 1 in Beesu et al. J. Med. Chem. 2015, 58:7833-7849 can be converted into the conjugate of formula (D-1) or any variant thereof as described herein.

[0108] In some embodiments, D is of formula (D-1c) or (D-1d): [ka] where the wavy line represents the point of attachment of D in formula (I).

[0109] In some embodiments, the TLR7 / 8 agonist is selected from the group consisting of Shukla et al. J. Med. Chem. 2010, 53:4450-4465; and and WO2015 / 023958. For example, Shukla et al. J. Med. Chem. 2010, 53:4450-446 The compounds listed in Table 1 in 5, or their derivatives, are aryl groups derived from benzyl groups. The compounds of Formula 11 or Formula 11A described in WO2015 / 023958, or derivatives thereof, may be covalently linked (or conjugated) to a conjugation moiety (i.e., moiety F in Formula (I)) via an amino group or any other applicable portion of the molecule. Similarly, the compounds of Formula 11 or Formula 11A described in WO2015 / 023958, or derivatives thereof, may be covalently linked (or conjugated) to a conjugation moiety (i.e., moiety F in Formula (I)) via an amino group derived from a benzyl group. [ka] Formula 11 and Formula 11A (wherein R 9 , R 10 and R 11 as described in WO2015 / 023958).

[0110] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2): [ka] (In the formula, n is an integer from 4 to 21, X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4bare independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0111] In some embodiments, X is -NH-. In some embodiments, X is -NH(C=O)-.

[0112] In some embodiments, X is -NH-. In some embodiments, n is an integer from 4 to 15. In some embodiments, n is 4, 5, 6, or 7. In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 16, 17, 18, 19, 20, or 21.

[0113] In some embodiments, X is -NH(C=O)-. In some embodiments, n is 11, 12, 13, or 14. In some embodiments, n is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 15, 16, 17, 18, 19, 20, or 21.

[0114] In some embodiments, R 1 is C3-C6 alkyl. In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1 is n-pentyl.

[0115] In some embodiments, R 1 is -(CH2) p OR 1a where p is 1 or 2, and R 1a is C1-C3 alkyl. In some embodiments, R 1 is -CH2OCH2CH3.

[0116] In some embodiments, R 1 is -(CH2) p NHR 1band R 1b is C1-C3 alkyl. In some embodiments, R 1 is -CH2NHCH2CH3.

[0117] In some embodiments, R 1 is -(CH2) p R 1c where p is 1 or 2, and R 1c is cyclopropyl or cyclobutyl. In some embodiments, R 1 is -CH2-cyclopropyl or -CH2CH2-cyclopropyl.

[0118] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with one, two, three, or four substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0119] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0120] Any and all variations of X and n described with respect to formula (D-2), when present, are to be combined with any and all combinations of R described with respect to formula (D-2) as if each and every combination were specifically and individually described. 1 , q, p, R 3 , R 4a and R 4b It is intended and understood that R may be combined with any and all variations of R. For example, in some embodiments, 1is C3-C6 alkyl (e.g., n-butyl), q is 0, X is -NH-, and n is 4, 5, 6, or 7. In some embodiments, R 1 is C3-C6 alkyl (for example, n-butyl), q is 0, X is -NH(C=O)-, and n is 11, 12, 13, or 14.

[0121] In some embodiments, the TLR7 / 8 agonist moiety of D has formula (D-2a) or (D-2b): [ka] (In the formula, n is an integer from 4 to 21, R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0122] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2a): In other embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2b):

[0123] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2a) and n is an integer from 4 to 15. In some variations, n is 4, 5, 6, or 7. In some variations, n is 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2a) and n is 16, 17, 18, 19, 20, or 21.

[0124] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2b) and n is 11, 12, 13, or 14. In some variations, n is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-2b) and n is 15, 16, 17, 18, 19, 20, or 21.

[0125] In some embodiments, R 1 is C3-C6 alkyl. In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1 is n-pentyl.

[0126] In some embodiments, R 1 is -(CH2) p OR 1a where p is 1 or 2, and R 1a is C1-C3 alkyl. In some embodiments, R 1 is -CH2OCH2CH3.

[0127] In some embodiments, R 1 is -(CH2) p NHR 1b and R 1bis C1-C3 alkyl. In some embodiments, R 1 is -CH2NHCH2CH3.

[0128] In some embodiments, R 1 is -(CH2) p R 1c where p is 1 or 2, and R 1c is cyclopropyl or cyclobutyl. In some embodiments, R 1 is -CH2-cyclopropyl or -CH2CH2-cyclopropyl.

[0129] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with one, two, three, or four substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0130] In some embodiments, R 20 is NHR 20a and R 20a are H, OH, and NH2 or methyl. In some embodiments, R 20 is NH. In some embodiments, R 20 is NHOH, NHNH2 or NHCH3.

[0131] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0132] Any and all variations of n described with respect to formula (D-2a), when present, are equivalent to any and all combinations of R described with respect to formula (D-2a), as if each and every combination were specifically and individually described. 1 , R 20 , q, p, R 3 , R 4a and R 4b Similarly, it is intended and understood that any and all variations of n described with respect to formula (D-2b) can be combined with any and all variations of R described with respect to formula (D-2b), when present, just as if each and every combination were specifically and individually described. 1 , R 20 , q, p, R 3 , R 4a and R 4b For example, in some embodiments of formula (D-2a), R 1 is C3-C6 alkyl (e.g., n-butyl), and R 20 is NH, q is 0, and n is 4, 5, 6, or 7. In some embodiments of Formula (D-2b), R 1 is C3-C6 alkyl (e.g., n-butyl), and R 20 is NH2, q is 0, and n is 11, 12, 13, or 14.

[0133] Representative compounds of formula (D-2), (D-2a) and (D-2b) are listed in Table 1, where the wavy line represents the point of attachment of D in formula (I). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] 1 The listed chemical names are the corresponding non- The chemical name of the conjugate compound (i.e., a primary or secondary amine).

[0134] The TLR7 / 8 agonist moieties of formulas (D-2), (D-2a) and (D-2b) are generated from the corresponding unconjugated compounds, which can be synthesized according to Scheme D-2 and / or using methods known in the art. Scheme D-2 [ka] (In the formula, R 1 , q and R 3 is as defined for formulas (D-2), (D-2a) and (D-2b), and R 20 is NH2 or as defined for formulas (D-2a) and (D-2b), and R and R″ are straight-chain alkyl groups).

[0135] In some embodiments, R 1 When q is C3-C6 alkyl (e.g., n-butyl), 0 is NH2, and q is 0, the compound is synthesized according to Scheme D-2-a. Individual compounds useful for preparing the starting compound (IMDQ) in Scheme D-2-a are: For a more detailed description of the reaction steps, see, for example, US Pat. Nos. 8,728,486 and 9,441,005. Scheme D-2-a [ka] (wherein R and R″ are straight chain alkyl groups).

[0136] Those skilled in the art will recognize that other synthetic routes can be used to synthesize the TLR7 / 8 agonist moieties of formula (D-2), (D-2a) or (D-2b) described herein, including different solvents, catalysts, reducing agents, temperatures, reaction times and atmospheric conditions.

[0137] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0138] In some embodiments, R 0 is C4~C 21 In some embodiments, R 0 is C4~C 14 In some embodiments, R 0 is C5~C 10 In some embodiments, R 0 is C 10 ~C 14 In some embodiments, R 0 is a C5-C7 hydrocarbyl. 0 is C 15 ~C 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 21 It is a hydrocarbyl. In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 14 In some embodiments, R 0 is a C4-C alkyl group substituted with 1-2 halogen atoms. 10 In some embodiments, R 0 is C substituted with 1-2 halogen atoms 10 ~C 14 In some embodiments, R 0 is a C4-C7 hydrocarbyl substituted with 1-2 halogen atoms. 0 is a C4-C7 hydrocarbyl substituted with one halogen atom. 0 is C substituted with 1 to 4 halogen atoms 15 ~C 21 It is a hydrocarbyl.

[0139] In some embodiments, X is -NH(C=O)-. In other embodiments, X is -NH-.

[0140] In some embodiments, R 0 Branched C4 to C 14 Alkyl or -(CH2) m R A m is 0, 1, 2 or 3; R A is independently a C3-C8 cycloalkyl optionally substituted with 1 to 4 groups selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene.

[0141] In some embodiments, R 0 Branched C4 to C 14 In some embodiments, R 0 Branched C5~C 10 In some embodiments, R 0 is a branched C 10 ~C 14 In some embodiments, R 0 is a branched C5-C7 alkyl. In some embodiments, R 0 is a branched C 15 ~C 21 It is alkyl.

[0142] In some embodiments, R 0 is -(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0143] In some embodiments, R A is a C3-C8 cycloalkyl.

[0144] In some embodiments, R A is C-C cycloalkyl optionally substituted with 1-3 groups independently selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2. In another variation, m is 0 and R Ais cyclobutyl, cyclopentyl or cyclohexyl.

[0145] In some embodiments, R A is cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, and m is 1 or 2.

[0146] In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0147] In some embodiments, R A is a C3-C6 cycloalkyl optionally substituted with 1-4 halogen atoms. A is a C3-C6 cycloalkyl optionally substituted with 1-3 chlorine or fluorine atoms. A is a C-C cycloalkyl optionally substituted with 1-2 chlorine or fluorine atoms. A is cyclobutyl optionally substituted with 1 to 2 fluorine atoms. In one variation, m is 1.

[0148] In some embodiments, R 0 is -(CH2) z (C(CH3)2)R A In one variation, z is 1 or 2 and R A is cyclopropyl, cyclobutyl or cyclo In one variation, z is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0149] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0150] In some embodiments, R 0 is the following: [ka] [ka] is selected from the group consisting of:

[0151] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0152] In some embodiments, X is —NH— and R 0 is -(CH2) m R A where m is 2 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0153] In some embodiments, X is —NH— and R 0 is -(CH2) z (C(CH3)2)R A where z is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0154] In some embodiments, X is —NH— and R 0 is -(CH2) m R A where m is 0 and R A is cyclobutyl, cyclopentyl or cyclohexyl.

[0155] In some embodiments, X is —NH(C═O)— and R 0 is -(CH2) m RA where m is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0156] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 is -(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 is -(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 is -(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0157] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is It is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is independently substituted with one, two, three, or four substituents selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0158] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0159] X and R described with respect to formula (D-3) 0 Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-3), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c , p, q, m, R A , R 3 , R 4a and R 4b It is intended and understood that the above-mentioned expressions can be combined with any and all variations of the above.

[0160] In some embodiments, the TLR7 / 8 agonist moiety of D has formula (D-3a) or (D-3b): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0161] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3a): In other embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3b):

[0162] In some embodiments, R 0 is C4~C 21 In some embodiments, R 0 is C4~C 14 In some embodiments, R 0 is C5~C 10 In some embodiments, R 0 is C 10 ~C 14 In some embodiments, R 0 is a C5-C7 hydrocarbyl. 0 is C 15 ~C 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 21 In some embodiments, R 0 is a C4-C1 substituted with 1-4 halogen atoms 4 hydrocarbyl. In some embodiments, R 0 is a C4-C alkyl group substituted with 1-2 halogen atoms. 10 In some embodiments, R 0 is C substituted with 1-2 halogen atoms 10 ~C 14 In some embodiments, R 0 is a C4-C7 hydrocarbyl substituted with 1-2 halogen atoms. 0is a C4-C7 hydrocarbyl substituted with one halogen atom. 0 is C substituted with 1 to 4 halogen atoms 15 ~C 21 It is a hydrocarbyl.

[0163] In some embodiments, R 0 Branched C4 to C 14 Alkyl or -(CH2) m R A m is 0, 1, 2 or 3; R A is independently a C3-C8 cycloalkyl optionally substituted with 1 to 4 groups selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene.

[0164] In some embodiments, R 0 Branched C4 to C 14 In some embodiments, R 0 Branched C5~C 10 In some embodiments, R 0 is a branched C 10 ~C 14 In some embodiments, R 0 is a branched C5-C7 alkyl. In some embodiments, R 0 is a branched C 15 ~C 21 It is alkyl.

[0165] In some embodiments, R 0 is -(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In another variation, m is 0 and R A is cyclobutyl, cyclopentyl or cyclohexyl.

[0166] In some embodiments, R A is a C3-C8 cycloalkyl.

[0167] In some embodiments, R A is C-C cycloalkyl optionally substituted with 1-3 groups independently selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2.

[0168] In some embodiments, R A is cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, and m is 1 or 2.

[0169] In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0170] In some embodiments, R A is a C3-C6 cycloalkyl optionally substituted with 1-4 halogen atoms. A is a C3-C6 cycloalkyl optionally substituted with 1-3 chlorine or fluorine atoms. A is a C-C cycloalkyl optionally substituted with 1-2 chlorine or fluorine atoms. A is cyclobutyl optionally substituted with 1 to 2 fluorine atoms. In one variation, m is 1.

[0171] In some embodiments, R 0 is -(CH2) z (C(CH3)2)R A In one variation, z is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In one variation, z is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0172] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0173] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0174] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0175] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3a), and R 0 is -(CH2) m R A where m is 2 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0176] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3a), and R 0 is -(CH2) z (C(CH3)2)R A where z is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0177] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3a), and R 0 is -(CH2) m R A where m is 0 and R A is cyclobutyl, cyclopentyl or cyclohexyl.

[0178] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-3b), and R 0 is -(CH2) m R A where m is 1 and R A is cyclopropyl, cyclobutyl or cyclopentyl.

[0179] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 is -(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 is -(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 is -(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0180] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with one, two, three, or four substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0181] In some embodiments, R 20 is NHR20a and R 20a is H, OH, NH, or methyl. In some embodiments, R 20 is NH. In some embodiments, R 20 is NHOH, NHNH2 or NHCH3.

[0182] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0183] R described with respect to formula (D-3a) 0 Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-3a), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c , R 20 , p, q, m, z, R A , R 3 , R 4a and R 4b Similarly, the R described with respect to formula (D-3b) can be combined with any and all variations of the R 0 Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-3b), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c , R 20 , p, q, m, z, R A , R 3 , R 4a and R 4b It is intended and understood that the above-mentioned expressions can be combined with any and all variations of the above.

[0184] Representative compounds of formula (D-3), (D-3a) and (D-3b) are listed in Table 2, where the wavy line represents the point of attachment of D in formula (I). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] 1 The listed chemical names are the corresponding non- Chemical names for conjugate compounds (i.e., primary and secondary amines).

[0185] The TLR7 / 8 agonist moieties of formulas (D-3), (D-3a) and (D-3b) are generated from the corresponding unconjugated compounds, which can be synthesized according to Scheme D-3 and / or using methods known in the art. Scheme D-3 [ka] (In the formula, R 1 , q and R 3 is as defined for formulas (D-3), (D-3a) and (D-3b), and R 20 is NH or as defined for formulas (D-3a) and (D-3b), and R and R 0 is an optionally substituted hydrocarbyl group).

[0186] R 1 is C3-C6 alkyl (e.g., n-butyl), and R 20In some embodiments, where q is NH and q is 0, the compound is synthesized according to Scheme D-3-a. For a more detailed description of the individual reaction steps useful for preparing the starting compound (IMDQ) in Scheme D-3-a, see, for example, U.S. Pat. Nos. 8,728,486 and 9,441,005. Scheme D-3-a [ka] (Wherein R and R 0 is an optionally substituted hydrocarbyl group).

[0187] Some embodiments of formula (D-3) wherein X is —NH—; or R 1 is C3-C6 alkyl (e.g., n-butyl), and R 0 -(CH2) m R A where m is 0 and R A In some embodiments of Formula (D-3a), where is cycloalkyl, the compound is synthesized according to Scheme D-3-b. Scheme D-3-b [ka] (In the formula, R 1 , q and R 3 is as defined for formulae (D-3) and (D-3a), and R is a cycloalkyl group).

[0188] Those skilled in the art will recognize that other synthetic routes can be used to synthesize the TLR7 / 8 agonist moieties of formulas (D-3), (D-3a) and (D-3b) described herein, including different solvents, catalysts, reducing agents, temperatures, reaction times and atmospheric conditions.

[0189] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-4): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, L is X or -CH2-X-; X is -NH- or -NH(C=O)-; A is independently a C6-C alkyl optionally substituted with 1 to 4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. 14 arylene or 5-14 membered heteroarylene optionally substituted by 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0190] In some embodiments, A is independently selected from halogen and 1 to 8 halogen atoms. C6-C alkyl optionally substituted with 1 to 4 groups selected from the group consisting of C1-C8 alkyl optionally substituted with 10 It is an arylene.

[0191] In some embodiments, A is phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of F, Cl, CF3, and methyl.

[0192] In some embodiments, A is 2,6-dimethyl-1,4-phenylene; 2,3-dimethyl-1,4-phenylene; 2,6-difluoro-1,4-phenylene; 2,3-difluoro-1,4-phenylene; 2,6-dichloro-1,4-phenylene; 2,6-dichloro-1,4-phenylene; 2,3,5,6-tetramethyl-1,4-phenylene; or 2,3,5,6-tetrafluoro-1,4-phenylene.

[0193] In some embodiments, A is 1,3-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms.

[0194] In some embodiments, A is a 5-10 membered heteroarylene optionally substituted with 1-4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1-8 halogen atoms.

[0195] In some embodiments, A is naphthylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-naphthylene, 1,3-naphthylene, or 2,7-naphthylene. In some embodiments, A is 2,6-naphthylene.

[0196] In some embodiments, A is 4,7-benzo[b]thiophene. In some embodiments, A is 2,5-1H-benzo[d]imidazole.

[0197] In some embodiments, L is X. In other embodiments, L is -CH2-X-.

[0198] In some embodiments, X is -NH-. In some embodiments, X is -NH(C=O)-.

[0199] In some embodiments, R 0 is C4~C 14 In some embodiments, R 0 is C5~C 10 In some embodiments, R 0 is C 10 ~C 14 In some embodiments, R 0 is a C5-C7 hydrocarbyl. 0 is C 15 ~C 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 14 In some embodiments, R 0 is a C4-C alkyl group substituted with 1-2 halogen atoms. 10In some embodiments, R 0 is C substituted with 1-2 halogen atoms 10 ~C 14 Hydrocal In some embodiments, R 0 is a C4-C7 hydrocarbyl substituted with 1-2 halogen atoms. 0 is a C4-C7 hydrocarbyl substituted with one halogen atom. 0 is C substituted with 1 to 4 halogen atoms 15 ~C 21 It is a hydrocarbyl.

[0200] In some embodiments, R 0 is -(CH2) m R A m is 0, 1, 2 or 3; R A is C3-C8 cycloalkyl optionally substituted with 1 to 4 groups independently selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene. In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. A is independently a C3-C6 cycloalkyl optionally substituted with 1-3 groups selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2. In some embodiments, R A is cyclopropyl optionally substituted with 1 to 3 groups selected from the group consisting of methyl and methylene, and m is 1 or 2. In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0201] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0202] In some embodiments, R 0 (cyclopropyl)methyl, 2-(cyclopropyl)ethoxy 2-(cyclobutyl)ethyl, 2-(cyclopentyl)ethyl, or 2-(cyclohexyl)ethyl.

[0203] In some embodiments, R 0 Branched C4 to C 14 In some embodiments, R 0 Branched C5~C 10 In some embodiments, R 0 is a branched C 10 ~C 14 In some embodiments, R 0 is a branched C5-C7 alkyl. In some embodiments, R 0 is a branched C 15 ~C 21 It is alkyl.

[0204] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 is -(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 is -(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 is -(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0205] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0206] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with one, two, three, or four substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0207] A, L and R as described with respect to formula (D-4) 0 Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-4), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c ,p,X,R A , m, q, R 3 , R 4a and R 4b It is intended and understood that R can be combined with any and all variations of R. 1 is n-butyl, q is 0, and R 4a and R 4b are each H, A is 1,4-naphthylene, L is -CH2-X-, X is -NH-, and R 0 is -(CH2) m R A m is 1 or 2, and R A is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0208] In some embodiments, the TLR7 / 8 agonist moiety of D has formula (D-4a) or (D-4b): [ka] (In the formula, R 0 is a C4-C optionally substituted with 1-4 halogen atoms 21 is a hydrocarbyl, A is independently a C6-C alkyl optionally substituted with 1 to 4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. 14 arylene or 5-14 membered heteroarylene optionally substituted by 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b or -(CH2) p R 1c and R 1a and R 1b are independently C1-C3 alkyl, and R 1c is a C3-C4 cycloalkyl, and p is 1 or 2; R 20 is NHR 20a and R 20a is H, OH, NH2 or methyl, R 3 are each independently halogen, C1-C8 alkyl, -(C1-C7 alkylene)-NH2, or -CH2-phenylene-CH2NH2; q is 0, 1, 2, 3 or 4; R 4a and R 4b are independently H or C1-C8 alkyl; The wavy line represents the point of attachment of D in formula (I). It has.

[0209] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-4a): In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-4b):

[0210] In some embodiments, A is independently a C6-C alkyl optionally substituted with 1-4 groups selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1-8 halogen atoms. 10 It is an arylene.

[0211] In some embodiments, A is phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of F, Cl, CF3, and methyl.

[0212] In some embodiments, A is 2,6-dimethyl-1,4-phenylene; 2,3-dimethyl-1,4-phenylene; 2,6-difluoro-1,4-phenylene; 2,3-difluoro-1,4-phenylene; 2,6-dichloro-1,4-phenylene; 2,6-dichloro-1,4-phenylene; 2,3,5,6-tetramethyl-1,4-phenylene; or 2,3,5,6-tetrafluoro-1,4-phenylene.

[0213] In some embodiments, A is 1,3-phenylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms.

[0214] In some embodiments, A is a 5-10 membered heteroarylene optionally substituted with 1-4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1-8 halogen atoms.

[0215] In some embodiments, A is naphthylene optionally substituted with 1 to 4 groups independently selected from the group consisting of halogen and C1-C8 alkyl optionally substituted with 1 to 8 halogen atoms. , 1,4-naphthylene, 1,3-naphthylene, or 2,7-naphthylene. In some embodiments, A is 2,6-naphthylene.

[0216] In some embodiments, A is 4,7-benzo[b]thiophene. In some embodiments, A is 2,5-1H-benzo[d]imidazole.

[0217] In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-4a): In some embodiments, the TLR7 / 8 agonist moiety of D has the formula (D-4b):

[0218] In some embodiments, R 0 is C4~C 14 In some embodiments, R 0 is C5~C 10 In some embodiments, R 0 is C 10 ~C 14 In some embodiments, R 0 is a C5-C7 hydrocarbyl. 0is C 15 ~C 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 21 In some embodiments, R 0 is a C4-C alkyl group substituted with 1 to 4 halogen atoms. 14 In some embodiments, R 0 is a C4-C alkyl group substituted with 1-2 halogen atoms. 10 In some embodiments, R 0 is C substituted with 1-2 halogen atoms 10 ~C 14 In some embodiments, R 0 is a C4-C7 hydrocarbyl substituted with 1-2 halogen atoms. 0 is a C4-C7 hydrocarbyl substituted with one halogen atom. 0 is C substituted with 1 to 4 halogen atoms 15 ~C 21 It is a hydrocarbyl.

[0219] In some embodiments, R 0 is -(CH2) m R A m is 0, 1, 2 or 3; R A is C3-C8 cycloalkyl optionally substituted with 1 to 4 groups independently selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene. In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. A is C-C cycloalkyl optionally substituted with 1-3 groups independently selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2. In some embodiments, R Ais cyclopropyl, optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, and m is 1 or 2. In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0220] In some embodiments, R 0 is the following: [ka] is selected from the group consisting of:

[0221] In some embodiments, R 0 is (cyclopropyl)methyl, 2-(cyclopropyl)ethyl, 2-(cyclobutyl)ethyl, 2-(cyclopentyl)ethyl or 2-(cyclohexyl)ethyl.

[0222] In some embodiments, R 0 Branched C4 to C 14 In some embodiments, R 0 Branched C5~C 10 In some embodiments, R 0 is a branched C 10 ~C 14 In some embodiments, R 0 is a branched C5-C7 alkyl. In some embodiments, R 0 is a branched C 15 ~C 21 It is alkyl.

[0223] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). 1 is propyl, butyl, pentyl, or hexyl. 1 is n-butyl. In some embodiments, R 1is n-pentyl. In some embodiments, R 1 is -(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 is -(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 is -(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0224] In some embodiments, R 20 is NHR 20a and R 20a is H, OH, NH, or methyl. In some embodiments, R 20 is NH. In some embodiments, R 20 is NHOH, NHNH2 or NHCH3.

[0225] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b are H, respectively.

[0226] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is independently substituted with one, two, three, or four substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0227] A and R as described with respect to formula (D-4a) 0Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-4a), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c , R 20 ,p,R A , m, q, R 3 , R 4a and R 4b Similarly, the A and R groups described with respect to formula (D-4b) can be combined with any and all variations of the formula. 0 Any and all variations of R, when present, are intended to be included in the R groups described with respect to formula (D-4b), just as if each and every combination were specifically and individually described. 1 , R 1a , R 1b , R 1c , R 20 ,p,R A , m, q, R 3 , R 4a and R 4b For example, in some embodiments of formula (D-4a), R 1 is n-butyl, and R 20 is NH2, q is 0, and R 4a and R 4b are each H, A is 1,4-naphthylene, and R 0 is -(CH2) m R A m is 1 or 2, and R A is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0228] Representative compounds of formula (D-4), (D-4a) and (D-4b) are listed in Table 3, where the wavy line represents the point of attachment of D in formula (I). [Table 3-1] [Table 3-2] [Table 3-3] 1 The listed chemical names are the corresponding non- Chemical names for conjugate compounds (i.e., primary and secondary amines).

[0229] The TLR7 / 8 agonist moieties of formulas (D-4), (D-4a) and (D-4b) are generated from the corresponding unconjugated compounds, which can be synthesized according to Scheme D-4 and / or using methods known in the art. Scheme D-4 [ka] (In the formula, R 1 , q, R 3 and A are as defined for formulas (D-4), (D-4a) and (D-4b), and R 20 is NH or as defined for formulas (D-4a) and (D-4b), and R and R 0 is an optionally substituted hydrocarbyl group).

[0230] Those skilled in the art will recognize that other synthetic routes can be used to synthesize the TLR7 / 8 agonist moieties of formulas (D-4), (D-4a) and (D-4b) described herein, including different solvents, catalysts, reducing agents, temperatures, reaction times and atmospheric conditions. B. Self-Removal Linker

[0231] Those skilled in the art will recognize that, with respect to ADCs, cleavable conjugates of TLR7 / 8 agonist compounds, such as those of Formula (I) described herein, have lower biological activity when stably conjugated, and greater biological activity of the TLR7 / 8 agonist when cleavage occurs in the tumor microenvironment to release the original (i.e., unconjugated) chemical form of the TLR7 / 8 agonist (Beck et al. 2017, Nature Reviews 16:315-337; Dubowchik et al. 2002, Bioconj Chem 13:855-869). In the present disclosure, the self-eliminating linker moiety (L in formula (I)) 1 ) is a compound that combines a TLR7 / 8 agonist compound (D in formula (I)) with a cleavable linker moiety (L in formula (I)). 2 The self-eliminating linker L in formula (I) is used to covalently link the 1 The presence of the TLR7 / 8 agonist moiety D maintains the TLR7 / 8 agonist moiety D in a relatively inactive state. 2 After hydrolysis of the self-eliminating linker L 1 undergoes a spontaneous chemical rearrangement that dissociates the moiety from the TLR7 / 8 agonist conjugate, releasing the unconjugated chemical form of the TLR7 / 8 agonist.

[0232] The amino group in the TLR7 / 8 agonist moiety (D) in formula (I) can serve as a reactive chemical group that can turn the chemical conjugate into a self-eliminating linker. In some embodiments, the primary amine on the N1-benzyl group of 1-(aminomethylbenzyl)-1H-imidazo[4,5-c]quinolin-4-amine (see, for example, compounds of formula (D-1a-1) or (D-1b-1)) can be linked to L 1 can be used to conjugate to D in Formula (I). In some embodiments, the primary amine on the quinoline ring of the modified 1H-imidazo[4,5-c]quinolin-4-amine derivatives (see, for example, compound numbers 64-01 to 64-50 and 64-58 to 64-69 in Tables 1 to 3) can be conjugated to L1 can be used to conjugate to D in formula (I). In some embodiments, the secondary amine (i.e., substituent X in formulas (D-2), (D-3), and (D-4)) of the modified 1H-imidazo[4,5-c]quinolin-4-amine derivatives (see, for example, compound numbers 64-01a to 64-50a and 64-58a to 64-69a in Tables 1 to 3) can be selected from the group consisting of L 1 can be used to conjugate to D in formula (I). However, derivatization of these amino groups can result in a loss of TLR7 and / or TLR8 agonist activity. Therefore, it is desirable to have a linker that releases the TLR7 / 8 agonist moiety in its original form (i.e., unconjugated form; i.e., as a compound with a free primary amine group) once the conjugate is cleaved. The self-eliminating linkers of the present disclosure provide a system in which cleavage of a specific bond triggers a series of 1,6- and / or 1,4-elimination reactions that result in the self-destruction of the linker and the regeneration of the original (i.e., unconjugated) TLR7 / 8 agonist moiety.

[0233] In some embodiments, the self-eliminating linker L in Formula (I) 1 is a moiety that can be eliminated from compounds of formula (I) upon cleavage of specific bonds by one or more 1,6- and / or 1,4-elimination reactions.

[0234] In some embodiments, the self-eliminating linker L in the compound of Formula (I) 1 is expressed as equation (L-1): [ka] (Y 1 is S, O or NH, and Ar 1 is an optionally substituted arylene, and R 11 and R 12 are independently H or optionally substituted alkyl It has.

[0235] In some embodiments, Y 1 is S or NH. In some embodiments, Y 1 is S. In some embodiments, Y 1 is NH.

[0236] In some embodiments, R 11 and R 12 are independently H or optionally substituted C1-C8 alkyl. In some embodiments, R 11 and R 12 are H, respectively.

[0237] In some embodiments, Ar 1 is optionally substituted phenylene. In some embodiments, Ar 1 is optionally substituted 1,4-phenylene. In some embodiments, Ar 1 is optionally substituted 1,2-phenylene. In some embodiments, Ar 1 is optionally substituted naphthylene. In some embodiments, Ar 1 is optionally substituted 1,4-naphthylene, optionally substituted 1,2-naphthylene, or optionally substituted 2,6-naphthylene. 1 is 1,4-phenylene. In some embodiments, Ar 1 is 1,2-phenylene.

[0238] In some embodiments, Ar 1 is 1,4-phenylene, and R 11 and R 12 are H and L, respectively. 1 is expressed by the formula (L-1a): [ka] It has.

[0239] In some embodiments, Ar 1 is 1,2-phenylene, and R11 and R 12 are H and L, respectively. 1 is expressed by the formula (L-1b): [ka] It has.

[0240] In some embodiments, Y 1 is NH and L 1 is the following: [ka] is.

[0241] In some embodiments, Y 1 is S and L 1 is the following: [ka] is.

[0242] Other self-eliminating linkers useful for drug conjugates are known in the art, such as those described in Blencowe et al. 2011, Polymer Chem 2:773-790; and U.S. Patent No. 6,180,095, the disclosures of which are incorporated herein by reference.Those skilled in the art will recognize that other self-eliminating linkers may be functionally equivalent, and therefore the description of self-eliminating linkers provided herein is not intended to limit the scope of the present invention.

[0243] The self-releasable linker moieties of the present invention impart two important properties to the cleavable conjugates of TLR7 / 8 agonist compounds. First, when the self-releasable linker moieties are attached to the TLR7 / 8 agonist, they maintain the TLR7 / 8 agonist in a relatively inactive state. Second, the self-releasable linker moieties are attached to the cleavable linker L in Formula (I). 2Upon hydrolysis of L, L self-eliminates to yield its original (i.e., unconjugated) TLR7 / 8 agonist moiety. 1 -D is the formula (L1-D-1), (L1-D-2), (L1-D-3) or (L1-D-4): [ka] [ka] It has.

[0244] In some embodiments, L in formula (I) 1 -D is the formula (L1-D-2a), (L1-D-2b), (L1-D-3a), (L1-D-3b), (L1-D-4a) or (L1-D-4b): [ka] It has.

[0245] In some embodiments, L in formula (I) 1 -D is: [ka] is.

[0246] In some embodiments, L in formula (I) 1 -D is: [ka] [ka] is.

[0247] In some embodiments, L in the compound of Formula (I) 1 is a bond and the compound of formula (I) is F-[WL 3 -L 2 -D] x In some embodiments, L in formula (I) is 1 -D is: [ka] (L 1 is a bond). C. Cleavable Linker

[0248] The cleavable linker moiety L in formula (I) 2 allows for the subsequent release of the original (i.e., unconjugated) form of the TLR7 / 8 agonist moiety D upon localization or retention of the compound of Formula (I) in the tumor microenvironment. In some embodiments, L 1 is a self-eliminating linker, and is a conjugation moiety F and a conjugation linker moiety WL 3 (i.e., FWL in formula (I) 3 ) is a cleavable linker moiety L 2 via a TLR7 / 8 agonist moiety D and a self-eliminating linker L 1 (i.e., -L in formula (I) 1 In some embodiments, L 1 is a bond, and the conjugation moiety F and the conjugation linker moiety WL 3 (i.e., FWL in formula (I) 3 ) is a cleavable linker moiety L 2 and the compound of formula (I) is covalently conjugated to a TLR7 / 8 agonist moiety D via F-[WL 3 -L 2 -D] x is.

[0249] Those skilled in the art have utilized cleavable linker systems in the development of ADCs to antibody target highly potent chemotherapeutic drugs (e.g., Beck et al. 2017, Nature Reviews 16:315-337 (see, e.g., 2013, Methods Mol Biol 1045:71-100), recognizing that these linkers address the narrow therapeutic window observed with the use of increasingly potent cytotoxic agents as cancer therapeutics. Cleavable linkers used in the construction of ADCs generally fall into three classes: 1) enzyme-labile peptide-based linkers, 2) disulfide linkers cleavable by gluthione reduction, and 3) linkers susceptible to hydrolysis under acidic conditions.

[0250] In some embodiments, the cleavable linker moiety L in Formula (I) 2 is a peptide-based cleavable linker. In some embodiments, the cleavable linker L of the present disclosure 2 is an amino acid motif that can be cleaved by proteolytic enzymes such as cathepsin B. In such embodiments, L includes a hydroxyl group, which generates an unstable intermediate that self-eliminates in a manner that yields the original (i.e., unconjugated) TLR7 / 8 agonist moiety D (see, e.g., FIG. 2). 2 is a peptide, such as a peptide that is cleavable by a proteolytic enzyme (e.g., cathepsin B). 2 is a peptide linker of 2 to 10, 2 to 8, 2 to 6, or 2 to 4 amino acid residues. 2 is a dipeptide. In some embodiments, L 2 is a peptide of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

[0251] Amino acid residues of the present disclosure include proteinogenic amino acids such as Ala, Asp, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr; and those not found in proteins, such as homoserine, homoarginine, citrulline ("Cit"), phenylglycine, taurine, iodotyrosine, seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthalanine, ornithine ("Orn").

[0252] Peptide-based cleavable linkers rely on the differential expression of certain key enzymes in the tumor microenvironment and / or the specific uptake of targeted therapeutic agents into the endolysosomal compartment of tumor cells (e.g., Mason, SD and Joyce, JA 2011, Trends Cell Biol 21:228-237; Weidle, UH et al. 2014, Cancer Genomics Proteomics 11:67-79; Doronina, SO et al. 2003, Nature Biotechol 21:778-784). In some embodiments, L 2is a peptide linker that is cleavable by one or more endosomal or lysosomal peptidases or proteases, or one or more pericellular peptidases or proteases expressed by cells in the tumor microenvironment (see, e.g., Tanabe, LM and List, K. 2017, FEBS J 284:1421-1436; Ulisse, S. et al. 2009, Curr Cancer Drug Targets 9:32-71; Uhland, K. 2006, Cell Mol Life Sci 63:2968-2978; LeBeau, AM et al. 2013, Proc Nat Acad Sci USA 110:93-98; Liu, C. et al. 2003, Cancer Res 63:2957-2964).

[0253] In some embodiments, L 2 is a peptide linker cleavable by an endosomal cathepsin, including cathepsin B, C, D, H, L, Z, and / or S. In some embodiments, L 2 is a peptide linker cleavable by pericellular proteases, including urokinase-type plasminogen activator (uPA), membrane-type serine protease 1 (matriptase), matriptase-2, and / or legumain.

[0254] In some embodiments, L 2 is a peptide linker that is cleaved by cathepsin B. In some embodiments, L 2 is a peptide linker cleaved by cathepsin B described by the amino acid sequence AA1-AA2-AA3-AA4, where AA1 is absent or is alanine, β-alanine, isoleucine, leucine, valine, or glycine, AA2 is absent or is alanine, β-alanine, isoleucine, leucine, or valine, AA3 is alanine, β-alanine, isoleucine, leucine, or valine, and AA4 is arginine, serine, alanine, β-alanine leucine, ornithine, or citrulline.

[0255] In some embodiments, L 2 is the following: [ka] [ka] is.

[0256] In some embodiments, L 2 is a peptide linker that is specifically cleavable by members of the type II transmembrane serine protease family of enzymes, including 1) matriptase, 2) hepsin / transmembrane protease / serine, 3) human airway trypsin-like / differentially expressed in squamous cell carcinoma, and 4) choline. 2 is a peptide linker that is specifically cleavable by urokinase-type plasminogen activator (uPA), matriptase (including membrane serine protease 1 / ST14 and matriptase-2 / TMPRSS6 and / or legumain / LGMN).

[0257] In some embodiments, L 2 is the following: [ka] is.

[0258] In some embodiments, the cleavable linker moiety L in Formula (I) 2 represents the self-eliminating linker L in formula (I). 1 and forms a disulfide linker (-SS-), which can undergo reductive thiolysis by high levels of reducing agents in the tumor microenvironment to give rise to an unstable intermediate that self-eliminates in such a way as to yield the original (i.e., unconjugated) TLR7 / 8 agonist moiety D (see, e.g., Figure 3). Disulfide linker cleavage depends on the relatively high levels of reduced glutathione found in the tumor microenvironment and tumor cell cytosol compared to the lower levels of free cysteine ​​in plasma (see, e.g., Brulisauer, L. et al. 2014, J Controlled Release 195:147-154; Flygare et al. 2013, Chem Biol Drug Des 81:113-121; Yang et al. 2006, PNAS 103:13872-13877). It will be appreciated by those skilled in the art that the balance between stability and oncolytic cleavage (relative to reductive thiolysis) can be modulated by the absence or presence of adjacent alkyl groups (see, e.g., Hamann, PR et al. 2002, Bioconjugate Chem 13:40-46; Lambert, JM 2013, Br J Clin Pharmacol 76:248-262).

[0259] In some embodiments, the cleavable linker L in Formula (I) 2 is expressed as equation (L-2): [ka] (In the formula, Y 2 is NR 30 , O or S, and R 30 , R 31 , R 32 , R 33 and R 34 are independently H, optionally substituted alkyl or optionally substituted cycloalkyl, or R 30 , R 31 , R 32 , R 33 and R 34 two of which, together with the atom(s) to which they are attached, form an optionally substituted cycloalkyl or an optionally substituted heterocyclyl The sulfur atom in formula (L-2) has the formula (I). 1 It forms a disulfide bond with the sulfur atom of the Y moiety of (L-2). 2 is the conjugation linker L 3 Combine with.

[0260] In some embodiments, Y 2 is NR 30 , O or S, and R 30 , R 31 , R 32 , R 33 and R 34 is independently H, C1-C8 alkyl, or C3-C8 cycloalkyl. 2 is NH and R 31 , R 32 , R 33 and R 34 is independently H or C1-C8 alkyl. 2 is NH and R 33 and R 34 are H and R, respectively. 31 and R 32 is independently H or C1-C8 alkyl (e.g., methyl). 2 is NR 30 and R 30 is C1-C8 alkyl (e.g., methyl). 31 and R 32 are each H. In some embodiments, R 31 is H and R 32 is C1-C8 alkyl (e.g., methyl). 31 and R 32 is independently C1-C8 alkyl (e.g., methyl). 2 is NH and R 33 and R 34 are H and R, respectively. 31 and R 32taken together with the carbon atom to which they are attached form a C3-C8 cycloalkyl or a 3-8 membered heterocyclyl. 31 and R 32 taken together form a C2-C6 alkylene or a C2-C6 alkylene in which one or more of the carbon atoms is replaced by a heteroatom selected from N, O and S (e.g., -CH2CH2OCH2CH2-).

[0261] In some embodiments, L 2 is represented by the formula (L-2a), (L-2b), (L-2c) or (L-2d): [ka] [ka] It has.

[0262] Other disulfide linkers useful in drug conjugates are known in the art, such as those described in U.S. Pat. Nos. 7,276,248 and 7,592,307, the disclosures of which are incorporated herein by reference.

[0263] Cleavable linker L 2 represents the self-eliminating linker L in formula (I). 1 is covalently conjugated to (i.e., -L in formula (I) 2 -L 1 In some embodiments, L 2 is the self-eliminating linker L 1 is covalently conjugated to L 1 is expressed as equation (L-1): [ka] (In the formula, Y 1 is S, O or NH, and Ar 1 is an optionally substituted arylene, and R 11 and R12 are independently H or optionally substituted C1-C8 alkyl. It has.

[0264] In some embodiments, L 2 is a peptide linker, and Y 1 is NH, -L 2 -L 1 - part of the formula (L-2-L-1): [ka] (In the formula, Ar 1 , R 11 and R 12 is as defined for formula (L-1) or any variation described herein, and AA 1 and A.A. 2 are independently amino acid residues) In some embodiments, AA 1 comprises a residue of an amino acid selected from the group consisting of lysine, lysine protected by acetyl or formyl, arginine, arginine protected by a tosyl group or a nitro group, histidine, ornithine, ornithine protected by acetyl or formyl, and citrulline (e.g., lysine or citrulline), and AA 2 comprises a residue of an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline (e.g., phenylalanine, leucine, or valine). 1 is a citrulline residue (Cit), and AA 2 is a valine residue (Val).

[0265] In some embodiments, L 2 is a self-eliminating linker L of formula (I) 1 In some embodiments, Y forms a disulfide bond with 1 is S and -L 2 -L 1 - is the formula (L-2a-L-1a): [ka] (In the formula, Ar 1 , R 11 and R 12 is as defined for formula (L-1) or any variation described herein; Y 2 , R 31 , R 32 , R 33 and R 34 is as defined for formula (L-2) or any variation described herein) It has.

[0266] The description of cleavable linkers provided herein is not intended to limit the scope of the invention, as one of skill in the art will recognize that other cleavable linkers may be functionally equivalent.

[0267] In some embodiments, L 1 is a bond and a cleavable linker L 2 is directly linked to the TLR7 / 8 agonist moiety D. D. Conjugation Moieties and Conjugation Linkers

[0268] Those skilled in the art will recognize several conjugation moieties / linkers (i.e., FWL in Formula (I)) that can be used to either facilitate preferential accumulation of the compound of Formula (I) within the tumor microenvironment or to facilitate retention of the compound of Formula (I) at the site of local administration. 3-). In some embodiments, the compound of formula (I) is a therapeutic agent. In some embodiments, the compound of formula (I) is a vaccine adjuvant. In some embodiments, the conjugation moiety / linker of the compound of formula (I) is comprised of a nano / microparticle-based conjugation moiety that is administered directly to the tumor by intratumoral injection or to tissue adjacent to the tumor lesion by subcutaneous or intramuscular injection for local peritumoral retention. In some embodiments, the conjugation moiety / linker of the compound of formula (I) is comprised of a tumor-targeting antibody conjugation moiety (which is administered by intravenous, intraperitoneal, or subcutaneous injection for preferential accumulation of the compound of formula (I) within the tumor microenvironment). The nano / microparticle-based or antibody-based conjugation moiety (FWL in formula (I)) 3 -) can facilitate preferential accumulation and / or retention of the compound of formula (I) within the tumor microenvironment or at the site of local administration, thereby allowing the cleavable linker moiety L in formula (I) to 2 Cleavage and self-elimination linker L 1 Upon self-elimination of D, the original (ie, unconjugated) form of the TLR7 / 8 agonist moiety D in formula (I) can be released in the target tissue. Particle-Based Conjugation Moieties

[0269] In some embodiments, the conjugation moiety F in Formula (I) is a micro / nanoparticle-based conjugation moiety that can facilitate 1) preferential accumulation of the compound of Formula (I) in the tumor microenvironment, or 2) retention of the compound of Formula (I) at the site of local administration. In some embodiments, the conjugation moiety F in Formula (I) has physical properties (e.g., charge, size, shape, hydrophobicity, etc.) or surface chemical modifications (e.g., tumor cell-specific binding ligands) that can facilitate 1) preferential accumulation of the compound of Formula (I) in the tumor microenvironment, or 2) retention of the compound of Formula (I) at the site of local administration. Subsequently, the preferential accumulation or retention of the compound of Formula (I) in the target tissue facilitated by the conjugation moiety F allows the cleavable linker L 2 Upon subsequent cleavage of the self-eliminating linker L 1 Upon self-detachment of Tissue release is allowed to occur.

[0270] In some embodiments, the particle-based conjugation moiety is a nanoparticle polymer (e.g., a branched copolymer of sucrose and epichlorohydrin, more particularly, Ficoll®). In some embodiments, the conjugation moiety is a high molecular weight polysaccharide (e.g., dextran). In some embodiments, the conjugation moiety is a synthetic polymer (e.g., poly(hydroxypropylmethacrylamide)). In some embodiments, the conjugation moiety is a dendrimer (e.g., a tris(2-aminoethyl)amine-derived dendrimer). In some embodiments, the conjugation moiety is a polypeptide (e.g., a cancer antigen). In some embodiments, the conjugation moiety is a protein. In some embodiments, the conjugation moiety is a virus-like particle. In some embodiments, the conjugation moiety is monovalent, i.e., each conjugation moiety is attached to one TLR7 / 8 agonist moiety (e.g., a compound of Formula (I) where x is 1). In some embodiments, the conjugation moiety is multivalent, i.e., each conjugation moiety is capable of binding to multiple TLR7 / 8 agonist moieties of the compound of formula (I), e.g., x is greater than 1 (e.g., up to 500). Macromolecules, supramolecules, nanoparticles, microparticles

[0271] Macromolecules and supramolecules that preferentially target TLR7 / 8 agonist compounds to the tumor microenvironment or enhance local retention at a desired anatomical site, particularly macromolecules and supramolecules having a molecular weight of about 5,000 daltons to about 2,000,000 daltons, such as nanoparticle polymers (e.g., branched copolymers of sucrose and epichlorohydrin, more specifically, Ficoll®), high molecular weight polysaccharides (e.g., dextran), and synthetic polymers (e.g., poly(hydroxypropylmethacrylamide)), are useful as conjugation moieties for the cleavable conjugates of the present disclosure.

[0272] In some embodiments, the macromolecular or supramolecular conjugation moiety is a nanoparticle polymer having a diameter of ≥10 nm. In some embodiments, the nanoparticle polymer has a diameter of 10-150 nm. In some embodiments, the macromolecules or supramolecules are branched copolymers of sucrose and epichlorohydrin, or epichlorohydrin-crosslinked sucrose, such as the branched copolymer of sucrose and epichlorohydrin sold under the trademark FICOLL® by GE Healthcare. In some embodiments, the macromolecular or supramolecular conjugation moiety is a high molecular weight polysaccharide. In some embodiments, the macromolecular or supramolecular conjugation moiety is a synthetic polymer (e.g., poly(hydroxypropylmethacrylamide)).

[0273] Polysaccharides that are derivatized to allow them to be linked to a TLR7 / 8 agonist moiety can be used as conjugation moieties for the cleavable conjugates of the present disclosure. Suitable polysaccharides can be naturally occurring or synthetic polysaccharides. Exemplary polysaccharides include, for example, dextran, mannin, chitosan, agarose, and starch. In some embodiments, the polysaccharide is cross-linked.

[0274] In some embodiments, the present disclosure provides a cleavable conjugate of Formula (I), wherein the conjugation moiety F is a macromolecule or supramolecule. In some embodiments, the macromolecule or supramolecule is a branched copolymer of sucrose and epichlorohydrin having a molecular weight of about 50,000 to about 1,000,000 daltons, which is linked to L via an ether linkage. 3 The ether linkages are derived from the hydroxyl groups of the sucrose in the copolymer. In some embodiments, the macromolecular or supramolecular conjugate of F In some embodiments, the conjugation moiety F is a branched copolymer of sucrose and epichlorohydrin having a molecular weight greater than (lower limit) about 50,000, 60,000, 70,000, 80,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Daltons. In some embodiments, the macromolecular or supramolecular conjugation moiety F is a branched copolymer of sucrose and epichlorohydrin having a molecular weight less than (upper limit) about 1,000,000, 900,000, 800,000, 700,000, 600,000, 500,000, 400,000, 300,000, or 200,000 Daltons. That is, the molecular weight of the macromolecular or supramolecular conjugation moiety F can be anywhere in the size range of about 50,000 to about 1,000,000 daltons, where the lower limit is less than the upper limit. In some embodiments, the macromolecular or supramolecular conjugation moiety F has a molecular weight of about 300,000 to 1,000,000 daltons (e.g., GE Healthcare's FICOLL® PM400). In some embodiments, the macromolecular or supramolecular conjugation moiety F has a molecular weight of about 20,000 to 100,000 daltons (e.g., GE Healthcare's FICOLL® PM70).

[0275] In some embodiments, the conjugation moiety F is attached to L via an ether linkage derived from a hydroxyl group in the polysaccharide. 3In some embodiments, conjugation moiety F is a polysaccharide (e.g., dextran) having a molecular weight of about 5,000 to about 2,000,000 daltons, conjugated to a polysaccharide (e.g., dextran) having a molecular weight greater than (lower limit) about 5,000, 10,000, 25,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 daltons. In some embodiments, conjugation moiety F is a polysaccharide having a molecular weight less than (upper limit) about 2,000,000, 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 daltons. That is, the molecular weight of the polysaccharide can be anywhere in the range of about 5,000 to about 2,000,000 daltons, with the lower limit being smaller than the upper limit.

[0276] For conjugates in which the conjugation moiety F is a polysaccharide (e.g., dextran) or a branched copolymer of sucrose and epichlorohydrin (e.g., Ficoll®), the number of TLR7 / 8 agonists D in the compound of Formula (I) can range from 3 to about 500. That is, x is an integer from 3 to 500. In some embodiments, x is an integer greater than (lower limit) 3, 6, 9, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 180, 200, 250, or 300. In some embodiments, x is an integer less than (upper limit) 500, 400, 300, 275, 250, 225, 200, 190, 180, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30. That is, x can be an integer in the range of about 3 to 500, in which case the lower limit is less than the upper limit. For example, in some embodiments, x is 20 to 500, 20 to 200, 30 to 180, 30 to 150, 50 to 100, 60 to 180, 90 to 150, 100 to 140, or 110 to 130. In some embodiments, x is about 120±30 or about 70±20.

[0277] The loading level of TLR7 / 8 agonist moiety D in a compound of Formula (I) can also be expressed as the number of TLR7 / 8 agonist moieties D relative to the molecular weight of the compound of Formula (I), e.g., the number of TLR7 / 8 agonist moieties D per 100,000 daltons (i.e., 100 kDa) of a compound of Formula (I) ("relative loading ratio"). In some embodiments, a compound of Formula (I) contains between about 1 and about 200 TLR7 / 8 agonist moieties D per 100 kDa of molecular weight of conjugation moiety F, i.e., a relative loading ratio of between about 1 and about 200 D per 100 kDa of F. In some embodiments, The relative loading ratio is about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 D per 100 kDa of F. In some embodiments, the relative loading ratio is greater than (lower limit) 3, 6, 9, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 180 D per 100 kDa of F. In some embodiments, the relative loading ratio is a D less than (upper limit) 200, 190, 180, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 per 100 kDa of F. The relative loading ratio can range from about 1 to 200 D per 100 kDa of F, where the lower limit is less than the upper limit. For example, in some embodiments, the relative loading ratio is between about 5 and about 200 D per 100 kDa F, between about 10 and about 180 D per 100 kDa F, between about 10 and about 50 D per 100 kDa F, between about 10 and about 20 D per 100 kDa F, between about 20 and about 200 D per 100 kDa F, between about 20 and about 100 D per 100 kDa F, between about 20 and about 50 D per 100 kDa F, between about 30 and about 150 D per 100 kDa F, between about 40 and about 100 D per 100 kDa F, between about 50 and about 100 D per 100 kDa F, between about 50 and about 100 D per 100 kDa F, between about 60 and about 100 D per 100 kDa F, between about 70 and about 100 D per 100 kDa F, between about 80 and about 100 D per 100 kDa F, between about 90 and about 100 D per 100 kDa F, between about 100 and about 200 D per 100 kDa F, between about 100 and about 200 D per 100 kDa F, between about 20 and about 100 D per 100 kDa F, between about 20 and about 50 D per 100 kDa F, between about 30 and about 150 D per 100 kDa F, between about 40 and about 100 D per 100 kDa F, between about 100 and about 200 D per 100 kDa F, between about 100 and about 200 D per 10 D per F between about 50 and about 150, D per 100 kDa F between about 50 and about 100, D per 100 kDa F between about 80 and about 200, D per 100 kDa F between about 80 and about 160, D per 100 kDa F between about 80 and about 120, D per 100 kDa F between about 100 and about 200, D per 100 kDa F between about 100 and about 150, D per 100 kDa F between about 120 and about 200, D per 100 kDa F between about 120 and about 150, or D per 100 kDa F between about 150 and about 200. dendrimer

[0278] Dendrimers can also be used to preferentially target TLR7 / 8 agonist compounds to the tumor microenvironment or to enhance local retention at a desired anatomical site. In some embodiments, the present disclosure provides compounds of Formula (I), wherein the conjugation moiety F is a dendrimer and the TLR7 / 8 agonist moiety D is covalently linked to a terminal functional group of the dendrimer via a cleavable linker.

[0279] In some embodiments, the dendrimer is a tris(2-aminoethyl)amine-derived dendrimer, which is linked via the terminal amino group to a conjugation linker, L 3 In some embodiments, the compound of formula (II): {N(CH2CH2N)3}[-L 3 -L 2 -L 1 -D]6(II) (In the formula, L 3 , L 2 , L 1 and D is as defined in relation to formula (I) or any variation thereof detailed herein. The compound of formula (I) is provided.

[0280] Other dendrimers, such as the 2,2-bis(2,2-diol) dendrimers described in Carlmark et al. 2013 Chem. Soc. Rev., 42:5858-5879, the disclosure of which is incorporated herein by reference. Dendrimers based on thyrolpropionic acid (bis-MPA) can also be used as the conjugation moiety F for the cleavable conjugates of the present disclosure.

[0281] In some embodiments, the conjugation moiety F of formula (I) is attached to the TLR7 / 8 agonist D via an ether linkage derived from a hydroxyl group in a bis-MPA dendrimer of formula (F-1): [ka] (In the formula, L 3 , L 2 , L 1 and D is as defined for formula (I) or any variation thereof detailed herein, and x is 16 to 64.

[0282] In some embodiments, the conjugation moiety F of formula (I) is attached to the TLR7 / 8 agonist D via an ether linkage derived from a hydroxyl group, formula (F-2): [ka] (wherein n is 100 to 1000, and L 3 , L 2 , L 1 and D is as defined for formula (I) or any variation thereof detailed herein, and x is 4 to 32. In some embodiments, the poly(ethylene glycol)-based bis-MPA dendrimer has a molecular weight of about 5,000 to about 50,000 daltons (e.g., PEG-core dendrimers available from Sigma-Aldrich®).

[0283] In some embodiments, the conjugation moiety F is a polyamidoamine (PAMAM)-based dendrimer, for example, as shown in Scheme F-3: [ka] (In the formula, L 3 , L 2 , L 1and D is as defined with respect to formula (I) or any variation thereof detailed herein, and x is 16 (or larger, up to 4000, if the core structure is further extended), the dendrimer may be attached to the TLR7 / 8 agonist moiety D, for example, via an ether linkage derived from the surface hydroxyl groups in the PAMAM molecule. Compounds of formula (I) in which the conjugation moiety F is a dendrimer can be made using methods known in the art and described herein. For example, the surface hydroxyl groups on the dendrimers described herein can be converted to propargylamine-carboxymethyl (PACM) groups for reaction with azide-linked IMDQ compounds using click chemistry similar to the methods described herein for linking IMDQ to the surface hydroxyl groups of Ficoll® (e.g., Kolb, HC et al. 2001, Angew Chem Int Ed Engl 40:2004-2021; Kolb, HC and Sharpless, KB 2003, Drug Discov Today 8:1128-1137). Polypeptides

[0284] In some embodiments, the present disclosure provides compounds of Formula (I), wherein the conjugation moiety F is a polypeptide comprising, for example, at least 9 amino acid residues. In some embodiments, the F polypeptide conjugation moiety is a polypeptide antigen. In some embodiments, the F polypeptide is a cancer antigen. In some embodiments, the F polypeptide is a viral antigen, bacterial antigen, or allergen antigen. In some embodiments, the F polypeptide comprises polyalanine. In some embodiments, the F polypeptide comprises polyglutamic acid. In some embodiments, the F polypeptide is not an antigen.

[0285] A TLR7 / 8 agonist D conjugated to an antigen can serve to enhance the immunological response of the antigen (e.g., a cancer antigen). In the case of antigen conjugates, typically, 1 to 30 TLR7 / 8 agonist moieties D can be linked to each polypeptide molecule F (as detailed herein, where applicable, -WL). 3 -L 2 -L 1 -via either a linker) (i.e., x is an integer between 1 and 30). In some embodiments, x is 1 to 20, 1 to 10, 1 to 5, 2 to 30, 2 to 20, 2 to 10, 2 to 5, 3 to 30, 3 to 20, 3 to 15, 3 to 10, 5 to 30, 5 to 20, 5 to 15, or 5 to 10. The tumor antigen comprises the amino acid sequence of at least one full-length protein or a fragment thereof. Suitable tumor antigens are described in the art (e.g., Cheever et al. , 2009, Clinical Cancer Research, 15:5323-5337; and Caballero and Chen, 2009, Cancer Science, 100:2014-2021). For example, suitable tumor antigens include, but are not limited to, WT1, MUC1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, p53, NY-ESO-1 (CTAG1B), PSMA, GD2, CEA, MelanA / Mart1, Ras, gp100, proteinase 3, bcr-able, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint, EphA2, PAP, MP-IAP, AFP, EpCAM, ERG, NA17-A, PAX3, ALK, androgen receptor, cyclin B1, MYCN, PhoC, TRP-2, mesothelin, PSCA, MAGE A1, CYP1B1, PLAC1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7-H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PAP, PDGFR-beta, MAD-CT-2, CEA, TRP-1(gp75), BAGE1, BAGE2, BAGE3, BAGE4, ​​BAGE5, CAMEL, MAGE-A2, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, and Fos-related antigen 1. The amino acid sequences of representative tumor antigens are listed in the UniProtKB database under the accession numbers listed in Table 4, which are incorporated herein by reference. [Table 4-1] [Table 4-2] [Table 4-3]

[0286] In some embodiments, the tumor antigen comprises an amino acid sequence or fragment thereof from one or more of the group consisting of gp100, hTERT, MAGE A1, MAGE A3, MAGE A10, MelanA / Mart1, NY-ESO-1 (CTAG1B), PSA, Ras, survivin, TRP1 (gp75), TRP2, and tyrosinase. In some embodiments, the tumor antigen comprises a mammalian antigen (e.g., a triple peptide) or a viral antigen (e.g., HPV1 E6 and / or HPV E7) expressed by a tumor. In some embodiments, the mammalian antigen is a neoantigen or is encoded by a gene that contains a mutation relative to a gene present in normal cells derived from the mammalian subject. Neoantigens are thought to be particularly useful in enabling T cells to distinguish between cancer and non-cancerous cells (e.g., Schumacher and Schreiber, 2015, Science 348:69-74; Desrichard et al., 2016, Clinical Cancer Res, 22:8-7-812; Wang and Wang, 2017, Cell Research 27:11-37).

[0287] The polypeptide of F may be linked to the TLR7 / 8 agonist-containing portion of the compound of formula (I) via a cysteine, N-terminal amine, lysine, tyrosine, methionine, arginine, glutamic acid, or aspartic acid residue (-L 3 -L 2 -L 1 In some embodiments, W is S and F is a polypeptide moiety (L via a cysteine ​​thiol). 3 In some embodiments, W is NH and F is a polypeptide moiety (e.g., linked to L via an amino acid at the N-terminal amine or lysine residue). 3 (bonded to

[0288] In some embodiments, the conjugation moiety F of formula (I) is a virus-like particle (VLP).

[0289] Compounds of formula (I) in which the conjugation moiety F is a polypeptide or VLP can be synthesized using methods known in the art and described herein. The polypeptide or VLP can be attached to the TLR7 / 8 agonist-containing portion of the compound of formula (I) by using hydroxyl, thiol, amino, or carboxy groups present in the amino acid residues of the polypeptide or VLP (-L 3 -L 2 -L 1 -D). For example, hydroxyl groups can be converted to ether linkages using the methods described herein for conjugating Ficoll® via hydroxyl groups. Thiol groups can be converted to thioether linkages or succinimide spacers by reaction with maleimide compounds.

[0290] It is intended and understood that any and all variations of F detailed herein for compounds of formula (I) can be combined with any and all variations of D detailed herein for compounds of formula (I), just as if each and every combination were set forth individually. For example, in some embodiments, compounds of formula (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is a TLR7 / 8 agonist moiety; L 1 is a bond or a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10 is H or C1-C8 alkyl, x is an integer from 3 to 300; F is a branched copolymer of sucrose and epichlorohydrin having a molecular weight of about 50,000 to about 700,000 daltons (e.g., FICOLL® PM400 from GE Healthcare); D is a 1H-imidazo[4,5-c]quinoline derivative The compound of formula (I) is provided.

[0291] In some embodiments, W is O (i.e., F is connected to L via an ether linkage). 3 connected to the

[0292] In some embodiments, the compound of Formula (I) may comprise a cleavable linker, L 2 , a self-eliminating linker L 1 , conjugation linker L 3 , and through the connection W (-WL 3 -L 2 -L 1 -), a polymeric prodrug comprising a TLR7 / 8 agonist moiety D linked to a conjugation moiety F, 1 is a self-eliminating linker, and L 2 is a cleavable linker, and L 3 is a conjugation linker and W is O, S or NR 10 and R 10 is H or C1-C8 alkyl. 1 In some embodiments, where R is a bond, the compound of Formula (I) may further comprise a cleavable linker, L 2 , conjugation linker L 3 , and through the connection W (-WL 3 -L 2 -), a polymeric prodrug comprising a TLR7 / 8 agonist moiety D linked to a conjugation moiety F, 2 is a cleavable linker, and L 3 is a conjugation linker and W is O, S or NR 10 and R 10 is H or C1-C8 alkyl. Antibody-Based Conjugation Moieties

[0293] In some embodiments, the conjugation moiety F in Formula (I) is a tumor-targeting antibody conjugation moiety that causes preferential accumulation of the compound of Formula (I) in the tumor microenvironment due to the ability of the antibody to preferentially bind to tumor cell surface antigens, unique structural elements of the extracellular matrix of the tumor microenvironment, or unique structural elements of the tumor vasculature. This allows the cleavable linker L 2 Upon subsequent hydrolysis of, and L 1 is a self-eliminating linker, the self-eliminating linker L 1 Upon self-detachment of L, local release of the TLR7 / 8 agonist moiety is possible. 1 is the bond and local release of the TLR7 / 8 agonist moiety is achieved by the cleavable linker L 2 This occurs during the subsequent hydrolysis of

[0294] Antibody drug conjugates (ADCs) consist of three main structural units: a recombinant antibody that specifically binds to a protein target in the tumor microenvironment, a highly potent cytotoxic agent, and a stable synthetic linker that connects the recombinant antibody to the cytotoxic agent via a covalent conjugate (see, e.g., Beck, A. et al. 2017, Nature Rev Drug Discovery 16:315-337; Sau, S. et al. 2017, Drug Discov Today 22:1547-1556). Conjugation to tumor-targeting antibodies allows for the delivery of The preferential accumulation of ADCs in tumor microenvironment improves the therapeutic window of these highly potent cytotoxic agents, and the original (i.e., unconjugated) cytotoxic agent is then released by preferential cleavage of the cleavable linker in this tumor microenvironment. Currently, over 60 ADC therapeutics have either been approved by the U.S. Food and Drug Administration (FDA) or are currently in clinical trials. Key properties that contribute to the antitumor efficacy of ADCs are: , tumor-targeting antibodies, and research in the field has shown that a wide range of antibody targets are currently under consideration (e.g., Beck, A. et al. 2017, Nature Rev Drug Discovery 16:315-337; Sau, S. et al. 2017, Drug Discov Today 22:1547-1556; and and Wagh, A. et al. 2018, MABS 10:222-243).

[0295] Another important property of ADCs is the range and uniformity of the loading of the cleavable linker / cytotoxic agent onto the antibody, commonly referred to as the antibody:drug ratio. Studies have shown that ADCs with controlled and uniform stoichiometry (generally, antibody:drug ratios between 2 and 6-8 for highly hydrophobic cytotoxic agents) exhibit superior plasma half-lives, which translate into enhanced in vivo efficacy (Sun, X. et al. 2017, Bioconjug Chem 28:1371-1381; Lyon, RP et al. 2015, Nat Biotechnol 33:733-735). Control of the extent and uniformity of the loading of the cleavable linker / cytotoxic agent onto the antibody is achieved by the specific conjugation chemistry used, including the choice of synthetic linker and reactive moiety on the antibody. The first ADC constructs involved cleavable linker-cytotoxic agent conjugates to natural lysine and cysteine ​​amino acids within the primary sequence of antibodies (see, e.g., Lu, J. et al. 2016, Int J Mol Sci 14:561; Jain N. et al. 2015, Pharma Res 32:3526-3540). However, cleavable linker-cytotoxic agent conjugates have been used in many ADCs. More recent developments in drug conjugate chemistry, along with more stable linker chemistries in plasma, allow for more precise control of antibody:drug ratios by creating a more highly defined number of reactive sites on the antibody (e.g., Agarwal, P. et al. 2013, Bioconjug Chem 24:846-851; Kato, A. et al. 2017, Bioconjug Chem 28:2099-2108; Sadowsky, J. et al. 2017, Bioconjug Chem 28:2086-2098; Grunewald, J. et al. 2017, Bioconjug Chem 28:1906-1915; Tang, F. et al. 2017, Nature Protocols 12:1702-1721).

[0296] In some embodiments, the conjugation moiety F in formula (I) is an antibody or antibody derivative. In some embodiments, F is an antibody having a murine sequence, a "humanized" murine sequence, or a fully human sequence. In some embodiments, F is an antibody of the IgG1, 2, or 4 class, or a derivative or fragment thereof, including, but not limited to, bivalent monospecific antibodies, bivalent bispecific antibodies, etc., or derivatives without an Fc region, including single-chain variable fragments (scFvs), and derivatives such as tandem bivalent scFvs, diabodies, tandem trivalent scFvs, triabodies, bispecific tandem bivalent scFvs, etc. (See, e.g., Weidle, UH et al. 2014, Seminars in Oncology 41:653-660). In some embodiments, the IgG scaffold is engineered to modulate the effector function of the molecule (see, e.g., Warncke, M. et al. 2012, J Immunol 188:4405-4411; Jacobsen, FW et al. 2017, J Biol Chem 292:1865-1875, etc.). .

[0297] The description of ADCs provided herein is not intended to limit the scope of the invention, as one of skill in the art will recognize that other ADCs may be functionally equivalent.

[0298] In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor microenvironment-specific antigen. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor cell surface marker. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor microenvironment-specific antigen. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor cell surface marker. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor microenvironment-specific antigen. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor cell surface marker. In some embodiments, the antibody, or derivative or fragment thereof, specifically binds to a tumor microenvironment-specific antigen. CD174 (Lewis Y ag), CD227 (MUC1), CD303, CD352, ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), TIM3, LY6E, LIV1, nectin 4, SLITRK6, HGFR (cMet), SLAM7, BCMA, AXL, NaPi2B, GCC, STEAP1, mesothelin, ETBR, EphA2, 5T4, FOLR1, Binds to CEACAM5 (CD66e), LAMP1, cadherin, FGFR2, FGFR3, EpCAM, CA6, MUC16, integrin αV, cripto1 (TDGF1), DLL3, TROP2, mesothelin, PTK7, NOTCH3, C4.4A, FLT3, LIV-1, SLC44A4, CA-IX, CanAg, guanylyl cyclase C, B7H3, and ROR-1. In some embodiments, the antibody is a bispecific antibody, or a derivative or fragment thereof, that binds to EpCAMxCD3, HER2xCD3, HER2xCD63, CD19xCD3, CEAxCD3, PSMAxCD3, CD123xCD3, CD30xCD16A, CD20xCD3, CD22xCD19, CEACAM5xCD303, CEACAM5xCD103, CEACAM5xCD11b, HER2xCD303, HER2xCD103, HER2xCD11b, etc.

[0299] In some embodiments, the tumor-targeting antibody F in Formula (I) is linked to a conjugation linker L well known to those skilled in the art, including N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-butanoate (sulfo-SPDB), maleimidomethylcyclohexane-I-carboxylate (MCC), 4-(4-acetylphenoxy)butanoic acid (AcBut), succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), derivatives thereof, and the like. 3 using a cleavable linker / self-eliminating linker / TLR7 / 8 agonist moiety (-L 2 -L 1 In some embodiments, L 1 is a bond, and the tumor-targeting antibody F in formula (I) can be linked to a conjugation linker L well known to those skilled in the art, including N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-butanoate (sulfo-SPDB), maleimidomethylcyclohexane-I-carboxylate (MCC), 4-(4-acetylphenoxy)butanoic acid (AcBut), succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), derivatives thereof, and the like. 3 cleavable linker / TLR7 / 8 agonist moiety (-L) via a natural lysine residue using 2 -D) is conjugated.

[0300] In some embodiments, the tumor-targeting antibody F in Formula (I) is linked to a conjugation linker L well known to those skilled in the art, including maleimidocaproyl (MC), maleimidomethylcyclohexane-I-carboxylate (MCC), succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), derivatives thereof, and the like. 3 cleavable linker / self-eliminating linker / TLR7 / 8 agonist moiety (-L) via a native or engineered cysteine ​​residue using 2 -L 1 In some embodiments, L 1 is a bond, and the tumor-targeting antibody F in formula (I) can be linked to a conjugation linker L well known to those skilled in the art, including maleimidocaproyl (MC), maleimidomethylcyclohexane-1-carboxylate (MCC), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), derivatives thereof, and the like. 3 cleavable linker / TLR7 / 8 agonist moiety (-L) via a native or engineered cysteine ​​residue using 2 -D-) conjugated with dithiothreitol (D Methods for mildly reducing antibodies with tris(2-carboxyethyl)phosphine (TCEP) or tris(2-carboxyethyl)phosphine (TT) are well known to those skilled in the art. Partial reduction of antibody disulfide bonds allows conjugation to certain cysteine ​​residues without disrupting the secondary and tertiary structure of the protein and without causing aggregation (see, e.g., Sun, MC et al. 2005, Bioconjug Chem 16:1282-1290; Doronina, SO et al. 2003, Nature Biotechnology 21:778-784, etc.).

[0301] In some embodiments, the tumor-targeting antibody F in formula (I) may be modified to include a cleavable linker / self-eliminating linker / TLR7 / 8 agonist moiety (-L) by using protein engineering techniques that can control the number of positions on the antibody available for conjugation. 2 -L 1 In some embodiments, L 1 is a bond, and the tumor-targeting antibody F in formula (I) can be modified to include a cleavable linker / TLR7 / 8 agonist moiety (-L) by using protein engineering techniques that can control the number of positions on the antibody available for conjugation. 2 In some embodiments, the antibody F in formula (I) is conjugated to a conjugation linker L 3 These have been engineered to control the number of cysteine ​​residues available for conjugation with maleimide-based reagents such as cysteine-binding domains (e.g., Junutula, JR et al. 2008, Nat Biotechnol 26:925-932; Kung-Sutherland, MS et al. 2013, Blood 122:1455-1463; Puthenveetil, S. et al. 2017, PlosOne 12:e0178452). In some embodiments, antibody F in formula (I) is linked to an orthogonal conjugation linker (L in formula (I)). 3 ) have one or more unnatural amino acids engineered into their primary sequence to allow for chemical conjugation with, for example, Tian. See, e.g., F. et al. 2014, Proc Nat Acad Sci USA 111:1766-1771; Kato, A. et al. 2017, Bioconjug Chem 28:2099-2108. In some embodiments, The antibody F in formula (I) is a conjugation linker (L 3) have unnatural amino acid sequences engineered into their primary sequence to enable enzyme-assisted ligation. See, e.g., Agarwal, P. et al. 2013, Bioconjug Chem 24:846-851; Dorywalska, M. et al. 2015, Bioconjug Chem 26:650-659; Grunewald, J. et al. 2017, See, e.g., Bioconjug Chem 28:1906-1915. In some embodiments, the compound of formula (I) The antibody F in the formula (I) is linked to a conjugation linker (L 3 The N-glycan residues have been metabolically engineered, chemically oxidized, or glycoengineered to allow ligation to modified glycan residues of ribozymes (see, e.g., Okeley, NM et al. 2013, Bioconjug Chem 24:1650-1655; Zhou, Q. et al. 2013, Bioconjug Chem 25:510-520; Tang, F. et al. 2017, Nature Protocols 12:1702-1721).

[0302] In some embodiments, the ratio of antibody F in Formula (I) to TLR7 / 8 agonist moiety D in Formula (I) (x in Formula (I)) is an integer, indicating that the geometric mean of the population distribution average of D conjugated to F is between 1 and 10. In some embodiments, x is an integer, indicating that the geometric mean of the population distribution average of D conjugated to F is between 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, or 2 and 3. In some embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. III. Methods for preparing cleavable conjugates of TLR7 / 8 agonist compounds

[0303] The present disclosure further provides methods for preparing cleavable conjugates of the TLR7 / 8 agonist compounds described herein (i.e., compounds of formula (I) described herein or any variant thereof), as well as compositions and intermediates useful therefor. The compounds of the present disclosure can be prepared using methods described herein and methods well known to those skilled in the art, see, for example, Dubowchik, GM et al. 2002, Bioconjug. Chem. 13,855-869; Lyon, RP et al. 2014, Nat Biotechnol 32:1059-1062.

[0304] In one aspect, the present disclosure provides particle-based (i.e., nanoparticle or microparticle) conjugation moieties F comprising formula (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is a TLR7 / 8 agonist moiety; L 1 is a bond or a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10 is H or C1-C8 alkyl, x is an integer from 1 to 500; F is a particle-based conjugation moiety 1. A method for preparing a cleavable conjugate of a TLR7 / 8 agonist compound of formula (A): F-[WL 3a -Y 3a ] y (A) to a compound of formula (B): Y 3b -L3b -L 2 -L 1 -D (B) (wherein y is an integer of 1 to 500, and F, W, L 2 , L 1 and D are as defined for compounds of formula (I), and L 3a and L 3b are independently optional spacer fragments, and Y 3a and Y 3b react with each other to form the spacer fragment Y 3 is a precursor moiety that forms L 3a , Y 3 and L 3b Together, the linker L 3 to form Forming a compound of formula (I) The present invention provides a method comprising:

[0305] In some embodiments, the TLR7 / 8 agonist moiety D is IMDQ or meta-IMDQ, or a compound selected from any one of Compound Nos. 64-01 through 64-50, 64-58 through 64-69, 64-01a through 64-50a, and 64-58a through 64-69a. In some embodiments, the particle-based conjugation moiety is a branched copolymer of sucrose and epichlorohydrin (e.g., Ficoll® PM400 or Ficoll® PM70), and x is an integer between 3 and 500, 3 and 400, 3 and 300, or 3 and 200. In some preferred embodiments, x is an integer between 3 and 150, 3 and 100, or 3 and 75.

[0306] In some embodiments, the conjugation linker is made using "click" chemistry by reaction of an alkyne with an azide to form a [1,2,3]triazole moiety. 3a is an alkyne group (-C≡CH), and Y 3b is an azide group (-N3), and Y 3 is a 1,4-[1,2,3]triazolylene moiety.

[0307] In some embodiments, L 3a is an amide spacer fragment (e.g., -CHC(O)NHCH-), and L 3b is an acyl spacer fragment (e.g., -CHC(O)-) or a PEG-acyl spacer fragment (e.g., -CHCH(OCHCH) 12 C(O)-).

[0308] In some embodiments, L 3a teeth, [ka] is.

[0309] In some embodiments, L 3b is the expression: [ka] an acyl spacer fragment or of the formula: [ka] (wherein n is 0 to 200) In some embodiments, L is a PEG-acyl spacer fragment of 3b is the expression: [ka] or an acyl spacer fragment of the formula: [ka] (wherein n is 0 to 200) is a PEG-acyl spacer fragment of

[0310] In some embodiments, Y 3 teeth, [ka] is.

[0311] In some embodiments, L 3 is the following: [ka] is.

[0312] In some embodiments, W is O and F is a branched copolymer of sucrose and epichlorohydrin having an average molecular weight of about 50,000 to about 700,000 daltons, about 50,000 to about 80,000 daltons, about 200,000 to about 600,000 daltons, or about 300,000 to about 500,000 daltons.

[0313] In some embodiments, the TLR7 / 8 agonist is a derivative of 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (e.g., IMDQ or meta-IMDQ). In some embodiments, the TLR7 / 8 agonist is a compound selected from the group consisting of Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a.

[0314] In some embodiments, L 1 is a bond.

[0315] In another aspect, the present disclosure provides a compound of formula (I) comprising an antibody-based conjugation moiety F: F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is a TLR7 / 8 agonist moiety; L 1 is a bond or a self-eliminating linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 and R 10is H or C1-C8 alkyl, x is an integer from 1 to 50, F is an antibody-based conjugation moiety 1. A method for preparing a cleavable conjugate of a TLR7 / 8 agonist compound of formula (C): F-[W'] x (C) to a compound of formula (D): L 3 -L 2 -L 1 -D (D) (wherein x is an integer of 1 to 50, F, L 3 , L 2 , L 1 and D is a compound of formula (I) and W' is N, O, S, N3 or an alkyne, Forming a compound of formula (I) The present invention provides a method comprising:

[0316] In some embodiments, the compound of Formula (C) comprises an antibody-based conjugation moiety (F-[W'] x ), W' is S, and F is a recombinant antibody that targets compounds to the tumor microenvironment, optionally engineered to add and / or delete cysteine ​​residues. In some embodiments, the compound of formula (C) is an antibody-based conjugation moiety (F-[W'] x ), W' is N, O, S, N or alkyne, and F is a recombinant antibody that targets the compound of formula (I) to the tumor microenvironment, the recombinant antibody being engineered with unnatural amino acids or novel amino acid sequences with post-translational chemoenzymatic modifications. In some embodiments, the compound of formula (C) is an engineered antibody-based conjugation moiety (F-[W'] x), W' is N on a natural lysine residue, and F is a recombinant antibody that targets the compound of Formula (I) to the tumor microenvironment. In some embodiments, x is an integer from 1 to 50, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2.

[0317] In some embodiments, the compound of Formula (D) is 1 L 2 (ii) covalently attaching -L to D; 2 -L 1 and (iii) L 3 Ni-L 2 -L 1 -D is covalently attached to the compound of formula (D), 3 -L 2 -L 1 In some embodiments, L 1 is a bond, and the compound of formula (D) is 2 and (ii) L 3 Ni-L 2 -D is covalently attached to the compound of formula (D), 3 -L 2 In some embodiments, the linker L 3 is a maleimidocaproyl conjugation linker, a succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, an N-succinimidyl-4-(2-pyridyldithio)butanoate linker, an N-succinimidyl-4-(2-pyridyldithio)pentanoate linker, a hydrazone linker, etc. In some embodiments, the cleavable peptide linker L 2is the amino acid sequence AA1-AA2-AA3-AA4, where AA1 is absent, alanine, β-alanine, isoleucine, leucine, valine, or glycine, AA2 is absent, alanine, β-alanine, isoleucine, leucine, or valine, AA3 is alanine, β-alanine, isoleucine, leucine, or valine, and AA4 is arginine, serine, alanine, β-alanine, leucine, ornithine, or citrulline, etc. In some embodiments, the linker L 1 is a p-aminobenzyl carbamate self-eliminating linker, etc. In some embodiments, the TLR7 / 8 agonist moiety D is IMDQ, meta-IMDQ, or any one of Compound Nos. 64-01 through 64-50, 64-58 through 64-69, 64-01a through 64-50a, and 64-58a through 64-69a. IV. Use of Cleavable Conjugates of TLR7 / 8 Agonist Compounds A. Pharmaceutical Compositions

[0318] Pharmaceutical compositions comprising tumor-targeted or locally retained cleavable conjugates of the TLR7 / 8 agonist compounds of the present disclosure are also provided. The pharmaceutical compositions conventionally contain one or more pharmaceutically acceptable excipients. The pharmaceutical compositions of the present disclosure can be in the form of a solution or a lyophilized solid. The pharmaceutical compositions of the present disclosure are preferably sterile and preferably substantially endotoxin-free.

[0319] Pharmaceutically acceptable excipients of the present disclosure include, for example, solvents, bulking agents, emulsifiers / surfactants. In some embodiments, the pharmaceutical composition comprises a solution of 100 mg of 10 ... The pharmaceutical composition of the present disclosure may include excipients that function as one or more of a solvent, bulking agent, buffering agent, and tonicity agent (e.g., sodium chloride in saline can serve as both an aqueous vehicle and an isotonicity agent). The pharmaceutical composition of the present disclosure is suitable for parenteral administration. In some embodiments, the pharmaceutical composition of the present disclosure is not intended for enteral administration.

[0320] In some embodiments, the pharmaceutical composition comprises an aqueous vehicle as a solvent.Suitable vehicles include, for example, sterile water, saline solution, phosphate buffered saline and Ringer's solution.In some embodiments, the composition is isotonic or hypertonic.In some embodiments, the composition is sterile.

[0321] The pharmaceutical composition can include a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is lyophilized before administration. In some embodiments, the bulking agent is a lyoprotectant, which helps stabilize the active agent and prevent its degradation during lyophilization and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0322] The pharmaceutical composition may contain a buffering agent. The buffering agent controls the pH and inhibits degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffers include salts, such as acetate, citrate, phosphate, or sulfate. Other suitable buffers include amino acids, such as arginine, glycine, histidine, and lysine. The buffering agent may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a range of 4 to 9. In some embodiments, the pH is greater than a lower limit of 4, 5, 6, 7, or 8. In some embodiments, the pH is less than an upper limit of 9, 8, 7, 6, or 5. That is, the pH is in the range of about 4 to 9, where the lower limit is less than the upper limit.

[0323] The pharmaceutical composition may contain an isotonicity agent. Suitable isotonicity agents include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol.

[0324] The pharmaceutical composition may contain a surfactant or emulsifier. Suitable surfactants include, for example, anionic surfactants, cationic surfactants, nonionic surfactants, and zwitterionic surfactants. In some embodiments, the nonionic surfactant is polyoxyethylene (20) sorbitan monooleate (e.g., Tween 80® or Montanox 80®) or polyoxyethylene (20) sorbitan monolaurate (e.g., Tween 20® or Montanox 20®).

[0325] The pharmaceutical composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobial agents. In some embodiments, the pharmaceutical composition is prepared under sterile conditions and is in a single-use container, and therefore does not require the inclusion of a preservative.

[0326] The pharmaceutical compositions of the present disclosure are suitable for multiple uses, including stimulating an immune response in a mammalian subject in need thereof. Mammalian subjects include, but are not limited to, humans, non-human primates, rodents, pets, and livestock. In some embodiments, the pharmaceutical compositions are administered to a subject in an amount effective to achieve a particular outcome. B. Dosage and Mode of Administration

[0327] As with all pharmaceutical compositions, the effective amount and administration method can vary based on several factors that are obvious to those skilled in the art.Factors to consider include the efficacy of the modified 1H-imidazo[4,5-c]quinoline TLR7 / 8 agonist compound (i.e., the compound of formula (I)), the ability of the compound and pharmaceutical composition to facilitate retention of the agonist compound at the administration site, the route of administration, and whether the pharmaceutical composition is administered in combination with an antigen or other therapeutic agent for the treatment of cancer.Other factors to consider include the disease-modifying outcome to be achieved and the number / frequency of doses administered during the treatment regimen.

[0328] A suitable dosage range is one that achieves the desired clinical effect. The dosage can be determined by the amount of TLR7 / 8 agonist compound (i.e., compound of Formula (I)) in the pharmaceutical composition that needs to be administered to a subject to produce the desired therapeutic response with minimal adverse events. Exemplary dosage ranges for the TLR7 / 8 agonist compound administered in an amount delivered per subject's body weight include about 0.0001-75 mg / kg, about 0.0001-50 mg / kg, about 0.0001-25 mg / kg, about 0.0001-10 mg / kg, about 0.0001-8 mg / kg, about 0.0001-6 mg / kg, and about 0.0001-5 mg / kg. g / kg, approximately 0.0001~4mg / kg, approximately 0.0010~3mg / kg, approximately 0.0001~2mg / kg, approximately 0.0001~1mg / kg, approximately 0.0001~0. 5mg / kg, approx. 0.001~100mg / kg, approx. 0.01~100mg / kg, approx. 0.1~100mg / kg, approx. 0.5~100mg / kg, approx. 1~100mg / k g, approx. 2~100mg / kg, approx. 3~100mg / kg, approx. 4~100mg / kg, approx. 5~100mg / kg, approx. 6~100mg / kg, approx. 8~100mg / kg, approx. 10~100mg / kg, approx. 25~100mg / kg, approx. 50~100mg / kg, approx. 75~100mg / kg, approx. 0.01~10mg / kg, approx. 0.05~9mg / about 0.0001 to 100 mg / kg, such as about 0.1 to 8 mg / kg, about 0.2 to 7 mg / kg, about 0.3 to 6 mg / kg, about 0.4 to 5 mg / kg, about 0.5 to 4 mg / kg, about 0.6 to 3 mg / kg, about 0.7 to 2.5 mg / kg, about 0.8 to 2.2 mg / kg, about 0.9 to 2.1 mg / kg, or about 1 to 2 mg / kg. In some embodiments, the dosage is greater than (lower limit) about 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2, 5, 10, 25, 50, 75, or 90 mg / kg. In some embodiments, the dosage is less than about (upper limit) 100, 75, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, or 0.001 mg / kg, i.e., anywhere in the range of about 0.0001 to 100 mg / kg, where the lower limit is less than the upper limit.An exemplary dosage range for the TLR7 / 8 agonist administered in an amount delivered to a subject is about 0.0001 to 100 mg / kg.

[0329] In some embodiments, when the pharmaceutical composition is further administered in combination with an antigen, the antigen dosage range delivered to a subject is about 1 μg to 500 μg. In some embodiments, the antigen dosage is about 1 μg to 50 μg. In some embodiments, the antigen dosage is greater than (lower limit) about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 400 μg. In some embodiments, the antigen dosage is less than (upper limit) about 500, 400, 300, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, or 10 μg. That is, the antigen dosage ranges anywhere from about 1 to 500 μg, with the lower limit being less than the upper limit. The optimal antigen dosage can be determined by experimental means for each individual antigen.

[0330] Similarly, a suitable administration route is one that produces the desired effect. Generally, the pharmaceutical compositions of the present disclosure are intended for parenteral administration (e.g., not oral administration or rectal administration). Suitable administration routes include injection, topical administration, and inhalation. In particular, the pharmaceutical compositions of the present disclosure can be administered by routes such as intratumoral, intramuscular, subcutaneous, transdermal, and inhalation. Suitable devices for administration by inhalation include, for example, atomizers, vaporizers, nebulizers, and dry powders. In some embodiments, the pharmaceutical composition is administered intratumorally and / or peritumorally (e.g., in and around a tumor lesion) when the pharmaceutical composition is intended to treat a solid tumor. In some embodiments, the pharmaceutical composition is administered intravenously.

[0331] A suitable dosing regimen for the TLR7 / 8 agonist compound formulated in the pharmaceutical composition is one that provides the desired effect in a prophylactic or therapeutic setting with minimal adverse events. The number of doses administered by the selected route can be one or more. Dosing frequencies can be weekly, every two weeks, monthly, every two months, or range from 3 to 12 months between doses. Exemplary dose frequencies for TLR7 / 8 agonist compounds are approximately once per week to once every 8 weeks. In some embodiments, the dose frequency is less than approximately once per 8, 6, 5, 4, 3, 2, or 1 week (at the upper end). In some embodiments, the dose frequency is more than approximately once per 7, 10, or 14 days (at the lower end). In some embodiments, the dose frequency is once per 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the dose frequency is once per 1, 2, 3, or 4 weeks. In some embodiments, the dose frequency is once every two, three, or four weeks. An exemplary dose frequency range for the TLR7 / 8 agonist compound delivered to a subject is approximately once per week to once every four weeks. In some embodiments, two doses are administered, with the second dose administered one to two months after the first dose. In some embodiments, three doses are administered, with the second dose administered one to two months after the first dose and the third dose administered one to five months after the second dose. In other embodiments, a series of doses may be administered over a 3- to 12-month treatment schedule, with the dose frequency being once per week, once every two weeks, once every three weeks, or once monthly. In other embodiments, shorter or longer periods may lapse between doses. In certain embodiments, the interval between successive doses may vary in terms of the number of weeks or months. In one embodiment, a series of 2, 3, 4, 5, or 6 weekly doses may be administered, followed by a second series of weekly doses at a later time point. One skilled in the art would be able to adjust the dosage regimen by measuring biological outcomes such as antigen-specific antibody responses, antigen-specific CD8+ T cell responses, or tumor regression.

[0332] In some embodiments, the pharmaceutical composition is administered intravenously to a subject over a period of about 5 to 120 minutes per infusion, hi some embodiments, the infusion time is about 5 to 120 minutes, about 5 to 90 minutes, about 5 to 60 minutes, about 5 to 45 minutes, about 5 to 30 minutes, about 5 to 15 minutes, about 5 to 10 minutes, about 10 to 120 minutes, about 15 to 120 minutes, about 30 to 120 minutes, about 45 to 120 minutes, about 60 to 120 minutes, about 90 to 120 minutes, about 10 to 45 minutes, or about 15 to 30 minutes. C. Stimulation of the immune response

[0333] In one aspect, the present disclosure provides a method of stimulating an immune response in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a pharmaceutical composition in an amount and frequency sufficient to stimulate an immune response in the mammalian subject. "Stimulating" an immune response means increasing the immune response, which can result from initiating an immune response de novo or enhancing an existing immune response. In some embodiments, stimulating the immune response includes stimulating IFNα production, stimulating interferon type 1 and / or type 2 production, stimulating IL-6 production, stimulating TNFα production, stimulating B lymphocyte proliferation, stimulating interferon pathway-related gene expression, stimulating chemoattractant-related gene expression, stimulating plasmacytoid dendritic cell (pDC) or myeloid dendritic cell (mDC) maturation and / or antigen presentation, and / or tumor antigen-specific CD4+ and / or CD8+ T cells. "Inducing" an antigen-specific T cell response refers to enhancing the number and functional characteristics of such lymphocytes, such as stimulating helper T lymphocytes and / or cytotoxic T lymphocytes to provide T cell help for an antibody response or generating cytotoxic T cells with anti-tumor activity. In embodiments where the pharmaceutical composition is further administered in combination with an antigen, stimulating an immune response includes inducing an antigen-specific antibody response. "Inducing" an antigen-specific antibody response refers to increasing the titer of an antigen-specific antibody above a threshold level, such as a baseline titer or seroprotective level prior to administration.

[0334] Analysis of immune responses (both qualitative and quantitative) can be performed by any method known in the art, including, but not limited to, measuring antigen-specific antibody production (including measurement of specific antibody subclasses), activation of specific populations of lymphocytes such as B cells and helper T cells, measuring the expression of sets of genes specific to specific immune cell types, production of cytokines such as IFNα, IL-6, IL-12, IL-18, TNFα, and / or histamine release. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assays (ELISAs). Cytokine production can also be measured by ELISA. Gene expression analysis can be performed by TaqMan® or nCounter® gene expression assays. Activation of specific populations of lymphocytes can be measured by proliferation assays and using fluorescence-activated cell sorting (FACS). Methods for measuring stimulation of immune responses are described in the biological examples of this disclosure. D. Cancer Treatment

[0335] The present disclosure provides methods of treating cancer in a mammalian subject in need thereof, comprising administering to the mammalian subject a pharmaceutical composition in an amount sufficient to treat the cancer in the mammalian subject. In some embodiments, the present disclosure provides methods of treating cancer in a mammalian subject in need thereof, comprising administering an effective amount of the pharmaceutical composition by intratumoral and / or peritumoral delivery. In some embodiments, intratumoral delivery comprises injecting the pharmaceutical composition into at least one tumor lesion. In some embodiments, treating cancer in a mammalian subject in need thereof comprises inducing accumulation of tumor antigen-specific T cells in the injected tumor in greater numbers than, for example, when the pharmaceutical composition is administered to an extratumoral site. In some embodiments, treating cancer in a mammalian subject in need thereof comprises eliciting a systemic tumor antigen-specific T cell response, including a greater magnitude of the systemic tumor antigen-specific T cell response than, for example, when the immunogenic composition is administered to an extratumoral site. In some embodiments, treating cancer in a mammalian subject in need thereof comprises eliciting a systemic tumor antigen-specific T cell response. In some embodiments, treating cancer in a mammalian subject in need thereof comprises reducing the number of CD4+FoxP3+ regulatory T cells in an injected tumor. In some embodiments, the subject has one or more non-injected tumors (primary or metastatic) in addition to the injected tumor, and treating cancer in the subject comprises one or more of the following: (a) reducing the number of non-injected tumors, (b) reducing the volume of the non-injected tumors, and (c) increasing the growth of the non-injected tumors. In some embodiments, treating cancer in a mammalian subject in need thereof comprises one or more of the following: (d) increasing the subject's survival time, (e) reducing the volume of the injected tumors, and (f) slowing the growth of the injected tumors. In some embodiments, if the cancer is a solid tumor, "treating" the cancer comprises reducing the size of the solid tumor and any metastatic lesions, or otherwise reducing the number of viable cancer cells.In other embodiments, when the cancer is a solid tumor, "treating" the cancer includes delaying the growth of the solid tumor and any metastatic lesions. In some aspects, treating cancer includes increasing progression-free survival or increasing the time to progression. In other embodiments, the method further comprises administering an effective amount of a second or additional therapeutic agent to the subject. "Treating" cancer means causing remission or causing a beneficial clinical outcome, such as prolonging survival compared to that otherwise expected in the absence of treatment. In some preferred embodiments, "treating cancer" includes assessing the patient's response to the immunogenic composition according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) described herein (see, e.g., Eisenhauer et al. 2009, Eur J Cancer 45:228-247). Response criteria for determining an objective anti-tumor response according to RECIST include complete response, partial response, progressive disease, and stable disease.

[0336] In some embodiments, the tumor is a sarcoma, carcinoma, or actinic keratosis. In some embodiments, the tumor is a lymphoma. In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, colorectal cancer, uterine cancer, bladder cancer, melanoma, head and neck cancer, non-Hodgkin's lymphoma, kidney cancer, ovarian cancer, pancreatic cancer, and thyroid cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is a primary cancer at a site selected from the group consisting of the oral cavity, digestive system, respiratory system, skin, breast, reproductive system, urinary tract system, ocular system, nervous system, endocrine system, and lymphoma.

[0337] In some embodiments, the method further comprises administering to the subject an effective amount of a second therapeutic agent. In some of these embodiments, the second therapeutic agent is selected from the group consisting of actinomycin, afatinib, alectinib, asparaginase, azacitidine, azathioprine, bicalutamide, binimetinib, bleomycin, bortezomib, camptothecin, carboplatin, capecitabine, carmustine, certinib, cisplatin, chlorambucil, cobimetinib, crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, encorafenib, erlotinib, epirubicin, epothilone, etoposide, fludarabine, flutamine, fluorouracil ... In some embodiments, the second therapeutic agent comprises a chemotherapeutic agent selected from the group consisting of rasil, gefitinib, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lapatinib, letrozole, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, octreotide, oxaliplatin, paclitaxel, pemetrexed, raltitrexed, sorafenib, sunitinib, tamoxifen, temozolomide, teniposide, thioguanine, topotecan, trametinib, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the second therapeutic agent comprises one or both of a BRAF inhibitor and a MEK inhibitor. In some embodiments, the second therapeutic agent is an HDAC inhibitor (see, e.g., voronistat [SAHA], romidepsin, entinostat, abexinostat, erinostat [CHR-3996], panobinostat, quisinostat [JNJ-26481585], 4SC-202, resminostat [SB939], pracinostat [CI-9940], and valproate), a DNA methyltransferase inhibitor (see, e.g., azacitidine, decitabine, zebularine, SGI-1027, RG-108, and sinfungin), and combinations thereof.

[0338] In some of these embodiments, the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule, e.g., an inhibitory immune checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 (CD152), LAG-3, TIM-3, TIGIT, IL-10, indoleamine 2,3-dioxygenase (IDO), P-selectin glycoprotein ligand-1 (PSGL-1), and TGF-beta. In some of these embodiments, the second therapeutic agent is an agonist of an immunostimulatory molecule. In some of these embodiments, the immunostimulatory molecule is selected from the group consisting of CD27, CD40, OX40 (CD134), GITR, 4-1BB CD137, CD28, and ICOS (CD278). In some of these embodiments, the second therapeutic agent comprises an antibody, fragment, or derivative thereof. In some of these embodiments, the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule, and the second therapeutic agent comprises an antibody, fragment, or derivative thereof.

[0339] In some embodiments, the method further comprises administering radiation therapy to the subject and / or administering an effective amount of a second therapeutic agent. In some of these embodiments, the effective amount of the immunogenic composition and the effective amount of the second therapeutic agent together provide an additive or better effect on the tumor. In some of these embodiments, the effective amount of the immunogenic composition and the effective amount of the second therapeutic agent together provide a synergistic effect on the tumor.

[0340] In some embodiments of the method, the treatment of the cancer does not result in the development of flu-like symptoms of such severity that repeated administration of the immunogenic composition is contraindicated, and the flu-like symptoms include one or more of the group consisting of fever, headache, chills, muscle aches and fatigue.

[0341] In some embodiments, the present disclosure provides kits that include a pharmaceutical composition (e.g., a compound of Formula (I), excipient(s), and optionally an antigen), and a set of instructions relating to the use of the composition for the methods described herein. The pharmaceutical composition of the kit is suitably packaged. If the pharmaceutical composition is a liquid, lyophilized form, or a suspension of nanoparticles, it may be packaged in a silicon dioxide vial (e.g., SCHOTT Type I) with a rubber stopper (e.g., Exxpro halobutyl elastomer) and an aluminum crimp top. plus®) is typically used as a container closure system. In some embodiments, the kit further comprises a device (e.g., a syringe and needle) for administering the pharmaceutical composition. In other embodiments, the kit further comprises a pre-filled syringe / needle system, an auto-injector, or a needleless device. Instructions associated with the use of the pharmaceutical composition generally include information regarding the dosage, schedule, and route of administration for the intended method of use. [Example]

[0342] V. Working Examples List of abbreviations: CDI: 1,1′-carbonyldiimidazole; DCM: dichloromethane DMF: N,N-dimethylformamide; DIPEA: N,N-diisopropylethylamine; DTT: dithiothreitol; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDTA: ethylenediaminetetraacetic acid; EMCS: N-ε-maleimidocaproyl-oxysuccinimide ester; Eq: equivalent Fmoc: 9-fluorenylmethoxycarbonyl; HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HCl: Hydrochloric acid K2CO3: Potassium carbonate LC-MS (LC / MS): Liquid chromatography-mass spectrometry; MALS: Multi-angle laser-light scattering; MeOH: methanol; Na2SO4: sodium sulfate; NMP: N-methylpyrrolidone; NMR: nuclear magnetic resonance (spectroscopy); PABC: p-aminobenzylcarbamate; PBS: 10 mM phosphate, 150 mM NaCl, pH 7.4; RP-HPLC: reversed-phase-high performance liquid chromatography; SEC: size exclusion chromatography; TEA: triethylamine; TFA: Trifluoroacetic acid THF: tetrahydrofuran. A. Synthesis Examples (Example S1) Preparation of Compounds Nos. 64-51 and 64-51a.

[0343] Using the procedures and schemes shown in Example S1 and Scheme S1-1, azido-disulfide-PABC-IMDQ compounds (-L in Formula (I)) bearing various alkyl disulfide groups (including, but not limited to, methyl-free disulfide (Formula (L-2a)), monomethyl disulfide (Formula (L-2b)), dimethyl disulfide (Formula (L-2c)), and cyclopropyl disulfide (Formula (L-2d))) can be prepared by replacing dimethylcysteamine hydrochloride with other amino-alkylthiol-containing compounds. 3 -L 2 -L 1-D) can be prepared. This example demonstrates the use of dimethylcysteamine hydrochloride to prepare Compound No. 64-51, an example of a general azido-disulfide-PABC-IMDQ structure. This example also describes the preparation of the monomethyl disulfide derivative Compound No. 64-51a. Furthermore, the TLR7 / 8 agonist moiety (D in Formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. IMDQ and meta-IMDQ can be prepared as described in U.S. Pat. Nos. 8,728,486 and 9,441,005. Scheme S1-1 [ka] General procedure for the preparation of compound No. 1.

[0344] To a solution of 4-mercaptobenzyl alcohol (700 mg, 5.0 mmol) in HO / THF (2 mL / 20 mL) was added 2,2'-dithiodipyridine (1.65 g, 7.5 mmol). The resulting clear yellow solution was heated to 40 °C and stirred for 4 h. The reaction solution was diluted with ethyl acetate (100 mL) and washed with 1 N HCl (100 mL) followed by water (150 mL). The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane, v / v, 1 / 2 to 1 / 1) to give compound No. 1 (990 mg, 80% yield) as a colorless oil. LC-MS: 250 [M+1] + . General procedure for the preparation of compound No. 2.

[0345] A solution of compound No. 1 (506 mg, 2.03 mmol) and dimethylcysteamine hydrochloride (375 mg, 2.65 mmol) in acetonitrile / water (6 mL / 6 mL) was stirred at room temperature for 18 hours. LC / MS showed the disappearance of compound No. 1. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with saturated aqueous NaHCO3 (100 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / MeOH / triethylamine, v / v, 1 / 0 / 0 to 100 / 10 / 3) to give compound No. 2 (460 mg, 93% yield) as a pale yellow solid. LC-MS: 244 [M+1] + . General procedure for the preparation of compound No. 3.

[0346] To a solution of 2-azidoacetic acid (285 mg, 2.82 mmol) in DMF (10 mL) was added HATU (1.14 g, 3.01 mmol) at 0 °C. After 10 min, a solution of Compound No. 2 (457 mg, 1.88 mmol) and DIPEA (0.66 mL, 3.76 mmol) in DMF (3 mL) was added to the above solution. The resulting solution was warmed to room temperature and stirred for 2 h. The mixture was stirred for 1 hour. LC / MS showed the disappearance of compound No. 2. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (ethyl acetate / hexane, v / v, 1 / 1 to 1 / 0) to give compound No. 3 (200 mg) as a colorless oil together with 505 mg of by-product compound 3B, which was treated with lithium hydroxide in THF / HO / MeOH to give compound No. 3. LC-MS: 325 [M-1] - . General procedure for the preparation of compound No. 4.

[0347] To a solution of compound No. 3 (310 mg, 0.95 mmol) in THF (5 mL) at 50 °C, CDI (232 mg, 1.43 mmol) in THF (5 mL) was added dropwise over 5 min. After 2 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane, v / v, 1 / 1 to 1 / 0) to give compound No. 4 (290 mg, 72% yield) as a white wax-like solid. LC-MS: 325 [M-1] - . General procedure for the preparation of compounds Nos. 64-51.

[0348] A solution of Cpd. No. 4 (183 mg, 0.435 mmol) and Cpd. No. 5 (IMDQ; 148 mg, 0.412 mmol) in DMF / THF (5 mL / 2 mL) was stirred at 50° C. for 18 hours. LC / MS showed that most of Cpd. No. 5 was consumed. The reaction mixture was cooled and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / MeOH, v / v, 15 / 1 to 8 / 1) to give Cpd. No. 64-51 (210 mg, 68% yield) as a white solid. LC-MS: 712 [M+1] + . 1 H NMR (300 MHz, CDCl3) δ 7.79 (d, 1H), 7.68 (d, 1H), 7.56 (d, 2H), 7.43 (td, 1H), 7.30 (d, 2H), 7.23 (d, 2H), 7.11 (td, 1H), 7.01 (d, 2H), 6.24 (t, 1H), 5.71 (s, 2H), 5.63 (bs, 2H), 5.14 (t, 1H), 5.08 (s, 2H), 4.34, (d, 2H), 3.86 (s, 2H), 3.32 (d, 2H), 2.86 (dd, 2H), 1.79 (p, 2H), 1.43 (h, 2H), 1.25 (s, 6H), 0.92 (t, 3H). General procedure for the preparation of compound No. 64-51a.

[0349] Compound No. 64-51a was prepared as shown in Scheme S1-1 for Compound No. 64-51, using HS(CH(CH))CHNH hydrochloride (i.e., 2-propanethiol, 1-amino, hydrochloride) instead of dimethylcysteamine hydrochloride (i.e., 2-propanethiol, 1-amino-2-methyl-, hydrochloride). The chemical structure of Compound No. 64-51a is shown in Scheme S4-1a. (Example S1a) Preparation of compounds Nos. 64-70.

[0350] Using the procedures and schemes shown in Example S1a and Scheme S1a-1, azido-thiol-PABC-IMDQ compounds can be prepared as non-cleavable conjugate controls. This example demonstrates the use of 4-mercaptobenzyl alcohol to prepare Compound No. 64-70, an example of a general azido-thiol-PABC-IMDQ structure. The TLR7 / 8 agonist moiety (D in Formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Scheme S1a-1 [ka] Procedure for the preparation of compound 21.

[0351] To a solution of 2-(Boc-amino)ethyl bromide (2.0 g, 8.9 mmol) in DMF (20 mL) was added 4-mercaptobenzyl alcohol (1.04 g, 7.4 mmol) and potassium carbonate (2.24 g, 16.2 mmol). The resulting cloudy solution was stirred at 40 °C for 18 h. The reaction solution was diluted with ethyl acetate (100 mL) and washed with 0.1 N HCl (100 mL) followed by water (100 mL). The organic layer was dried over NaSO and concentrated under reduced pressure. The residue was purified by flash column chromatography eluting with 1 / 2 to 1 / 1 (v / v) ethyl acetate / hexane to give 1.72 g of compound 21 as a colorless oil. The purity was 82% by RP-HPLC at 254 nm, and the mass of the desired compound, 549 daltons, was confirmed by LC-MS. Procedure for the preparation of compound 22.

[0352] To a solution of compound 21 (1.72 g, 6.0 mmol) in DCM (100 mL) was added TFA (8 mL). The resulting pale yellow solution was stirred at room temperature for 2 h. After concentration under reduced pressure, the mixture was co-evaporated three times with DCM (100 mL). The resulting residue was purified by silica gel flash column chromatography eluting with 1 / 0 / 0 to 100 / 10 / 2 (v / v) DCM / MeOH / trimethylamine to give 0.94 g of compound 22 as a pale yellow oil. The purity was 85% by RP-HPLC at 254 nm, and the mass of the desired compound, 183 Daltons, was confirmed by LC-MS. Procedure for the preparation of compound 23.

[0353] To a solution of 2-azidoacetic acid (625 mg, 6.18 mmol) in DMF (5 mL) was added O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (680 mg, 6.67 mmol) at room temperature. After 15 min, a solution of compound 22 (945 mg, 5.16 mmol) in DMF (5 mL) was added, followed by triethylamine (1.6 mL, 11.4 mmol). The resulting solution was stirred at room temperature for 2 h. The reaction mixture was then diluted with ethyl acetate (100 mL) and extracted with water (100 mL). The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 1 / 1 to 1 / 0 (v / v) ethyl acetate / hexane to give 0.5 g of compound 23 as a colorless oil. The purity was 36% by RP-HPLC at 254 nm, and the mass of the desired compound was 249 Daltons. was confirmed by LC-MS. Procedure for the preparation of compound 24.

[0354] To a solution of CDI (456 mg, 2.81 mmol) in THF (10 mL) was added compound 23 (500 mg, 1.87 mmol) in THF (3 mL) dropwise at 50 °C. After 3 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography eluting with 1 / 1 to 1 / 0 (v / v) ethyl acetate / hexane to give 325 mg of compound 24 as a colorless oil. The purity was 48% by RP-HPLC at 254 nm, and the mass of the desired compound, 360 Daltons, was confirmed by LC-MS. General procedure for the preparation of compounds Nos. 64-70.

[0355] To a solution of compound 24 (195 mg, 0.54 mmol) and IMDQ (compound 5, 97 mg, 0.27 mmol) in DMF (6 mL) was added TEA (6 drops). The resulting solution was stirred overnight at room temperature, and the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography eluting with 10 / 1 (v / v) dichloromethane / MeOH to give 52 mg of compounds Nos. 64-70 as a white solid. The purity was 99% by RP-HPLC at 254 nm, and the mass of the desired compound, 651.8 daltons, was confirmed by LC-MS at 300 MHz. 1 H NMR (CD3OD+CDCl3) δ 7.67 (td, 2H), 7.57 (s, 1H), 7.40 (td, 1H), 7.33-7.19 (m, 4H), 7.08 (td, 1H), 6.97-6.94 (m, 3H), 5.69 (s, 2H), 4.99 (s, 2H), 4.26 (s, 2H), 3.81 (s, 2H), 3.40 (t, 2H), 3.01 (t, 2H), 2.84 (t, 2H), Confirmed by 1.74 (m, 2H), 1.39 (m, 2H), 0.88 (t, 3H). (Example S2) Preparation of compounds Nos. 64-52, 64-52a, 64-52b, 64-52c, 64-52d, 64-52e, 64-52f and 64-52g.

[0356] Using the procedures and schemes shown in Example S2 and Schemes S2-1 to S2g-1, various conjugation linkers L in formula (I) can be prepared. 3 , the cleavable peptide sequence L in formula (I) 2 , the self-eliminating linker L in formula (I) 1 Various compounds of the present invention, -L in formula (I), have the following structure: 3 -L 2 -L 1-D can be prepared, and the TLR7 / 8 agonist compound D in formula (I) can be IMDQ or meta-IMDQ (see Figure 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), and any of compound numbers 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Compound number 64-52.

[0357] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To exemplify an embodiment of the present invention in which is an azido-PEG4 moiety, Compound Nos. 64-52 were prepared as shown in Scheme S2-1. Scheme S2-1 [ka] General procedure for the preparation of compound No. 7.

[0358] To a solution of compound No. 6 (465 mg, 0.61 mmol) and compound No. 5 (IMDQ; 190 mg, 0.53 mmol) in DMF (15 mL) was added triethylamine (0.13 mL, 0.94 mmol). The resulting clear yellow solution was stirred at room temperature for 15 hours. LC / MS showed mostly the desired product, compound No. 7, and that all of compound No. 5 had been consumed. The reaction mixture was used directly in the next step. LC-MS: 987 [M+1] + . General procedure for the preparation of compound No. 8.

[0359] To a crude solution of Cpd. No. 7 (approximately 0.528 mmol) in DMF (15 mL) was added piperidine (460 mg, 5.41 mmol), and the solution was stirred at room temperature for 6 h. LC / MS showed the disappearance of starting material Cpd. No. 7. The reaction mixture was diluted with 10% MeOH in dichloromethane (200 mL) and washed with water (200 mL × 2). The aqueous layer was extracted with ethyl acetate (150 mL). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / MeOH, v / v, 100 / 10 to 100 / 15) to give Cpd. No. 8 (304 mg, 65% yield for two steps) as a yellow solid. LC-MS: 76 5[M+1] + . General procedure for the preparation of compounds Nos. 64-52.

[0360] To a solution of Cpd. No. 8 (173 mg, 0.224 mmol) and Cpd. No. 9 (181 mg, 0.244 mmol) in DMF (5 mL) was added DIPEA (0.08 mL, 0.44 mmol). The resulting solution was stirred at room temperature for 16 h. LC / MS showed almost complete conversion to the desired product. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / MeOH, v / v, 100 / 10 to 100 / 15) to give Cpd. No. 64-52 (170 mg, 55% yield) as a white solid. LC-MS: 1390 [M+1] + . 1 H NMR (300 MHz, CDCl3) δ 7.68 (d, 1H), 6.62 (d, 1H), 7.52 (d, 2H), 7.38 (t, 1H), 7.20 (d, 2H), 7.18 (d, 2H), 7.07 (t, 1H), 6.92 (d, 2H), 5.66 (s, 2H), 4.95 (s, 2H), 4.46 (dd, 1H), 4.23 (s, 2H), 4.09 (d, 1H), 3.61-3.45 (m, 44H), 3.35-3.26 (m, 4H), 3.10 (t, 2H), 2.81 (t, 2H), 2.56-2.45 (m, 2H), 2.12-2.20 (1, 1H), 1.91-1.80 (m, 1H), 1.80-1.55 (m, 3H), 1.55-1.40 (m, 2H), 1.40-1.28 (m, 4H), 0.93-0.82 (m, 9H). Compound number 64-52a.

[0361] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To illustrate an embodiment of the present invention in which is an azido-PEG4 moiety, Compound No. 64-52a was prepared as shown in Scheme S2a-1. Scheme S2a-1 [ka] Preparation procedure for compound 7.

[0362] To an ice-cold solution of IMDQ (compound 5; 36 mg, 1 equiv.) in DMF (1.5 mL), DIPEA (0.1 mL) was added, and the solution was stirred for 10 minutes under an argon atmosphere. Compound 6 (77 mg, 1 equiv.) was added in two portions, and the resulting clear yellow solution was stirred at 0°C for 2 hours. The solution was slowly warmed to room temperature, and then stirring was continued for an additional 12 hours. Thin-layer chromatography analysis, developed with 10% (v / v) MeOH in DCM containing 1% (v / v) TEA, indicated that all of the IMQD starting material had been consumed. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the solid yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times. The off-white solid product was dried under reduced pressure to give 84 mg of compound 7. Preparation procedure for compound 8.

[0363] To an ice-cold solution of compound 7 (71 mg, 1 equiv.) in DMF (1.5 mL) was added DIPEA (0.1 mL), and the mixture was stirred overnight under an argon atmosphere, then slowly warmed to room temperature and stirred for an additional 12 hours. Reverse-phase HPLC analysis of the reaction indicated that the reaction had proceeded to completion. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate a pale yellow solid, and the supernatant was decanted. This process was repeated two more times, and the off-white solid was dried under reduced pressure. As a result, 50 mg of compound 8 was obtained. Preparation procedure for compound No. 64-52a.

[0364] Compound 8 (50 mg, 1.0 equiv.) and compound 25 (20.3 mg, 1.2 equiv.) were dissolved in DMF (1 mL), DIPEA (100 μL) was added, and the solution was stirred at room temperature for 1.5 h. RP-HPLC analysis of the reaction indicated that compound 12 was completely consumed. DMF was removed under reduced pressure, and the solid yellow residue was subjected to silica gel column chromatography eluting with 1–8% (v / v) MeOH in DCM containing 0.5% (v / v) ammonia to afford 34 mg of compound No. 64-52a as a white solid. Product purity was determined to be 95% by RP-HPLC at 254 nm, and LC-MS confirmed the mass of the desired compound, 1,035.5 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (CD3OD): δ 7.83 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.46 (t, J = 7.5,15.0 Hz, 1H), 7.25-7.31 (m, 1H), 7.14 (t, J = 7.5,15.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 5.88 (s, 2H), 5.03 (s, 2H), 4.48-4.55 (m, 1H), 4.20-4.26 (m, 3H), 3.73-3.76 (m, 2H), 3.55-3.68 (m, 14 H), 3.28, 3.38 (m, 4H), 3.0-3.28 (m, 2H), 2.97 (t, J = 7.5, 15.3 Hz, 2H), 2.55 (t, J = 6, 12 Hz, 2H), 1.72-2.18 (m, 5H), 1.58-1.70 (m, 2H), 1.43-1.55 (m, 2H), 0.88-1.01 (m, 9H). Compound number 64-52b.

[0365] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I) 1is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To illustrate an embodiment of the present invention in which is an azide moiety, Compound No. 64-52b was prepared as shown in Scheme S2b-1. Scheme S2b-1 [ka] Preparation procedure for compound No. 64-52b.

[0366] Compound 8 was prepared as described in Scheme S2a-1. To a solution of compound 8 (135 mg, 0.176 mmol, 1.0 equiv.) in DMF (1.0 mL) was added a DMF solution containing compound 26 (1.7 mL, 0.265 mmol, 1.5 equiv.). The resulting solution was stirred at room temperature for 2 h, by which time LC-MS analysis indicated that compound 8 had been consumed. The reaction was then diluted with MeOH (3 mL) and water (3 mL). The mixture was diluted with HCl and 1N NaOH (0.5 mL) was added. The resulting solution was stirred at room temperature for 24 hours and then directly purified by preparative RP-HPLC to give 62 mg of compound no. 64-52b as a white solid. Product purity was determined to be 96% by RP-HPLC at 254 nm, and LC-MS confirmed the mass of the desired compound, 847.4 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (acetone-d6): δ 8.49 (s, The molecular weight was confirmed by the following NMR spectra: 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.64-7.56 (m, 3H), 7.36-7.26 (m, 5H), 7.08 (d, J = 6.0 Hz, 2H), 5.88 (s, 2H), 5.05 (s, 2H), 4.50 (dd, J = 5.1 and 9.0 Hz, 1H), 4.29-4.25 (m, 3H), 4.05 (s, 2H), 3.19-3.13 (m, 1H), 3.01 (t, J = 7.8 Hz, 2H). Compound number 64-52c.

[0367] The TLR7 / 8 agonist D in formula (I) is IMDQ, and L in formula (I) 1 is a bond, and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 Azide-PEG 12 To exemplify a partial embodiment of the present invention, Compound No. 64-52c was prepared as shown in Scheme S2c-1. Scheme S2c-1 [ka] Procedure for the preparation of compound 28.

[0368] To a solution of compound 27 (100 mg, 0.401 mmol, 1.0 equiv.) and IMDQ (compound 5; 96 mg, 0.40 mmol, 1.0 equiv.) in DMF (3 mL) was added HATU (154 mg, 0.602 mmol, 1.5 equiv.), followed by DIPEA (89 μL, 0.80 mmol, 2.0 equiv.). The reaction was stirred at room temperature for 15 min and then purified by preparative RP-HPLC (95:5% (v / v) HO / acetonitrile + 0.1% (v / v) acetic acid, gradient from 95 / 5 to 0 / 100 within 30 min). Fractions containing the expected major peak with absorbance at 322 nm were collected and dried under reduced pressure to give 141 mg of compound 28 as a white solid. Procedure for the preparation of compound 29.

[0369] A solution of compound 28 (141 mg, 0.197 mmol, 1.0 equiv) in DCM (4 mL) was treated with TFA (1 mL) at room temperature for 1 h. The reaction was concentrated to dryness under reduced pressure, and the residue containing compound 29 was used without further purification. Preparation procedure for compound No. 64-52c.

[0370] To a solution of compound 29 (73 mg, 0.10 mmol, 1.0 equiv.) and compound 13 (74 mg, 0.10 mmol, 1.0 equiv.) in DMF (2 mL) was added DIPEA (0.1 mL), and the reaction was stirred at room temperature for 3 h. The mixture was purified by preparative RP-HPLC (95:5% (v / v) HO / acetonitrile + 0.1% (v / v) acetic acid, gradient from 95 / 5 to 0 / 100 within 30 min). Fractions containing the major peak with the expected absorbance at 322 nm were collected and dried under reduced pressure to give 79 mg of compound 64-52c as a white solid. Product purity was determined to be 97% by RP-HPLC at 254 nm, and LC-MS confirmed the mass of the desired compound, 1,241.5 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (CDCl3): δ 8.40 (t, 1H), 8.15 (d, 1H), 7.98 (d, 2H), 7.74 (d, 1H), 7.65 (t, 1H), 7.38 (t, 1H), 7.28 (d, 2H), 7.03 (d, 2H), 5.94 (s, 2H), 4.40-4.25 (m, 3H), 4.11 (t, 1H), 3.67-3.54 (m, 52H), 3.10-3.00 (m, 6H), 2.75-2.68 (m, 1H), 2.50-2.46 (m, 2H), 2.03-1.81 (m, 4H), 1.48-1.41 (m, 4H), 0.98-0.86 (m, 9H). Compound number 64-52d.

[0371] The TLR7 / 8 agonist D in formula (I) is IMDQ, and L in formula (I) 1 is a bond, and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To illustrate an embodiment of the present invention in which is an azido-PEG4 moiety, Compound No. 64-52d was prepared as shown in Scheme S2d-1. Scheme S2d-1 [ka] Preparation procedure for compound No. 64-52d.

[0372] Compound 29 (1.0 g, 1.0 equiv.) was added to a solution of compound 25 (1.1 equiv.) in DMF (10 mL) and the reaction was stirred at room temperature. The reaction progress was monitored by LC-MS, and after 1 h, compound 29 was completely consumed, so the solvent was removed under reduced pressure. The crude product was purified using flash chromatography on a Biotage Selekt eluting with MeOH (0-25%) in DCM to give 600 mg of compound 64-52d as a white solid. Product purity was determined to be 95% by RP-HPLC at 254 nm, and LC-MS confirmed the mass of the desired compound, 889.1 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (DMSO-d6): δ 9.10 (s, 1H), 8.36-8.30 (m, 1H), 7.95 (dd, J = 16.1, 8.0 Hz, 2H), 7.84 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.16 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 7.8 Hz, 2H), 5.91 (bs, 3H), 5.36 (bs, 1H), 4.24-4.11(m, 4H), 3.71-3.40 (m, 16H), 2.96-2.78 (m, 6H), 2.40-2.28 (m, 4H), 1.86-1.81 (m, 2H), 1.74-1.65 (m, 2H), 1.42-1.29 (m, 5H), 0.84 (t, J = 7.5 Hz, 3H), 0.71 (d, J = 6.8 Hz, 6H). Compound number 64-52e.

[0373] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is phenylalanine-citrulline, and the conjugation linker L 3 Azide-PEG 12 To exemplify a partial embodiment of the present invention, Compound No. 64-52e was prepared as shown in Scheme S2e-1. Scheme S2e-1 [ka] Procedure for the preparation of compound 30.

[0374] To a solution of compound 30 (0.27 g, 0.33 mmol, 1.2 equiv.) in DMF (10.0 mL) was added IMDQ (compound 5; 0.10 g, 0.28 mmol, 1.0 equiv.) and TEA (58 mg, 0.57 mmol, 2.0 equiv.). The mixture was stirred under nitrogen at room temperature for 16 hours, at which point LC-MS analysis indicated that all IMDQ had been consumed. The reaction mixture was used directly in the next step reaction without further purification. Procedure for the preparation of compound 31.

[0375] To the reaction mixture prepared above (approximately 0.28 mmol in DMF) was added piperidine (0.24 g, 2.8 mmol, 10.0 equivalents). The resulting mixture was stirred at room temperature for 6 hours, poured into diethyl ether (75 mL) and stirred. The yellow precipitate was collected by filtration and washed with ether. The crude product was purified by flash chromatography on a Biotage Select eluting with 20% MeOH in DCM containing 2% (v / v) TEA to give 140 mg of compound 31 as a yellow solid. Preparation procedure for compound No. 64-52e.

[0376] Compound 31 (73.4 mg, 0.09 mmol, 1.2 equiv) in DMF (3.0 mL) To a solution of 32 (3H, 2H, 2H, 2H, 2H) in DMF (0.3 mL) was added compound 13 (73.6 mg, 0.10 mmol, 1.1 equiv.) and DIPEA (23.3 mg, 0.18 mmol, 2.0 equiv.). The mixture was stirred under nitrogen at room temperature for 18 hours, at which point LC-MS analysis indicated that all of compound 32 had been consumed. The reaction mixture was purified by preparative RP-HPLC to give 54 mg of compound No. 64-52e as a white solid. Product purity was determined to be 98% by RP-HPLC at 254 nm, and LC-MS confirmed the mass of the desired compound, 1,438.7 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (DMSO-d6): δ 10.05 (s, 1H), 8.25 (d, 1H), 8.06 (d, 1H), 7.81-7.85 (m, 2H), 7.59-7.56 (m, 4H), 7.40-7.34 (m, 1H), 7.30-7.04 (m, 14H), 6.98-6.95 (m, 3H), 5.98 (t, 1H), 5.84 (s, 2H), 5.43 (s, 2H), 4.92 (s, 2H), 4.57 (bs, 1H), 4.40 (bs, 1H), 4.11 (d, 2H), 3.60-3.30 (m, 46 H), 3.08-2.8 (m, 8H), 2.78-2.70 (m, 1H), 2.28 (t, 2H), 1.75-1.65 (m, 4H), 1.50-1.30 (m, 4H), 0.84 (t, 3H). Compound number 64-52f.

[0377] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is glycine-isoleucine-valine-arginine, and the conjugation linker L 3 To exemplify an embodiment of the present invention in which is an azido-PEG4 moiety, Compound No. 64-52f was prepared as shown in Scheme S2f-1. Scheme S2f-1 [ka] General procedure for the preparation of compound 33.

[0378] To an ice-cold solution of IMDQ (compound 5; 1 equivalent) in DMF, DIPEA (0.1 mL) is added and the solution is stirred for 10 minutes under an argon atmosphere. Compound 33 (1 equivalent) is added in two portions, and the resulting clear yellow solution is stirred at 0°C for 2 hours. The solution is allowed to warm slowly to room temperature, and stirring is continued for an additional 12 hours, or until thin-layer chromatography analysis, developed with 10% (v / v) MeOH in DCM containing 1% (v / v) TEA, indicates that all of the IMDQ starting material has been consumed. DMF is removed under reduced pressure, and ethyl acetate is added to the solid yellow residue. The mixture is triturated to precipitate the pale yellow solid that forms, and the supernatant is decanted. This process is repeated two more times. The off-white solid product is dried under reduced pressure to give compound 34. General procedure for the preparation of compound 35.

[0379] A solution of compound 34 (1.0 equiv.) in DCM was treated with TFA (1.0 mL) at room temperature for 1 hour. The reaction was concentrated to dryness under reduced pressure, and the residue containing compound 35 was used without further purification. General procedure for the preparation of compound No. 64-52f.

[0380] Compound 35 (1.0 equiv.) and compound 25 (1.2 equiv.) were dissolved in DMF, DIPEA (0.1 mL) was added, and the solution was stirred at room temperature for 1.5 h. When RP-HPLC analysis of the reaction showed that compound 35 was completely consumed, DMF was removed under reduced pressure, and the yellow solid residue was subjected to silica gel column chromatography eluting with 1–8% (v / v) MeOH in DCM containing 0.5% (v / v) ammonia to obtain compound No. 64-52f as a white solid. Product purity was determined by RP-HPLC at 254 nm, the mass of the target compound was confirmed by LC-MS, and the structure of the target compound was confirmed by LC-MS. 1 Confirm by 1 H NMR. Compound number 64-52g.

[0381] The TLR7 / 8 agonist D in formula (I) is IMDQ, and the self-eliminating linker L in formula (I)1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L in formula (I) 2 is leucine-serine-glycine-arginine-serine-aspartic acid, and the conjugation linker L 3 To illustrate an embodiment of the present invention in which is an azido-PEG4 moiety, Compound No. 64-52g was prepared as shown in Scheme S2g-1. Scheme S2g-1 [ka] General procedure for the preparation of compound 37.

[0382] To an ice-cold solution of IMDQ (compound 5; 1 equivalent) in DMF, DIPEA (0.1 mL) is added and the solution is stirred for 10 minutes under an argon atmosphere. Compound 36 (1 equivalent) is added in two portions, and the resulting clear yellow solution is stirred at 0°C for 2 hours. The solution is allowed to warm slowly to room temperature, and stirring is continued for an additional 12 hours, or until thin-layer chromatography analysis, developed with 10% (v / v) MeOH in DCM containing 1% (v / v) TEA, indicates that all of the IMDQ starting material has been consumed. DMF is removed under reduced pressure, and ethyl acetate is added to the solid yellow residue. The mixture is triturated to precipitate the pale yellow solid that forms, and the supernatant is decanted. This process is repeated two more times. The off-white solid product is dried under reduced pressure to give compound 37. General procedure for the preparation of compound 38.

[0383] A solution of compound 37 (1.0 equiv.) in DCM was treated with TFA (1.0 mL) at room temperature for 1 h. The reaction was concentrated to dryness under reduced pressure to give a residue containing compound 38. was used without further purification. General procedure for the preparation of compound No. 64-52g.

[0384] Compound 38 (1.0 equiv.) and compound 25 (1.2 equiv.) were dissolved in DMF, DIPEA (0.1 mL) was added, and the solution was stirred at room temperature for 1.5 h. When RP-HPLC analysis of the reaction showed that compound 38 was completely consumed, DMF was removed under reduced pressure, and the yellow solid residue was subjected to silica gel column chromatography eluting with 1–8% (v / v) MeOH in DCM containing 0.5% (v / v) ammonia to obtain compound No. 64-52g as a white solid. Product purity was determined by RP-HPLC at 254 nm, and the mass and structure of the target compound were confirmed by LC-MS. 1 Confirm by 1 H NMR. Compound number 64-52h.

[0385] The TLR7 / 8 agonist D in formula (I) is 64-33, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety conjugated to the primary amine at position 4 of the imidazoquinoline ring of the TLR7 / 8 agonist (see Figure 1), and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To exemplify an embodiment of the present invention in which is an azido-PEG4 moiety, Compound No. 64-52h was prepared as shown in Scheme S2h-1. Scheme S2h-1 [ka] Procedure for the preparation of compound 52.

[0386] To an ice-cold solution of compound 64-33b (96 mg, 0.224 mmol, 1.0 equiv.) in DMF (5.0 mL) was added TEA (91 mg, 0.9 mmol), followed by di-tert-butyl dicarbonate (54 mg, 0.250 mmol, 1.1 equiv.). The solution was slowly warmed to room temperature and stirred overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 3–5% (v / v) MeOH / DCM containing 1% aqueous ammonia to give 94 mg of compound 52. Procedure for the preparation of compound 53.

[0387] Compound 52 (94 mg, 0.178 mmol, 1 equiv.) in DMF (1.5 mL) on ice To the cooled solution, DIPEA (0.2 mL) was added, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 6 (135 mg, 0.178 mmol, 1 equiv.) was added in one portion. The resulting clear yellow solution was stirred at 0°C for 1 hour, then slowly warmed to room temperature, and stirring was continued for 12 hours. Thin-layer chromatography analysis using 10% (v / v) MeOH in DCM containing 1% TEA indicated very little reaction had occurred. The mixture was then warmed to 70°C, stirring was continued, and the reaction progress was monitored by RP-HPLC. After 3 days, DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the off-white solid was dried under reduced pressure to give 84 mg of compound 53, which was used in the next reaction without further purification. Procedure for the preparation of compound 54.

[0388] To an ice-cold solution of compound 53 (84 mg, 0.072 mmol) in DMF (1.5 mL) was added diisopropylamine (0.1 mL) with stirring under an argon atmosphere. The resulting solution was then allowed to warm slowly to room temperature and continued stirring for 12 h. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the off-white solid was dried under reduced pressure to give 54 mg of crude compound 54. Procedure for the preparation of compound 55.

[0389] Compound 54 (54 mg, 0.057 mmol) and compound 25 (22 mg, 0.063 mmol) were dissolved in DMF (1.0 mL), DIPEA (0.1 mL) was added, and the solution was stirred for 2.5 h. DMF was removed under reduced pressure, and the yellow residue was subjected to silica gel column chromatography eluting with 1–8% (v / v) MeOH in DCM containing 0.5% ammonia to give 14 mg of compound 55 as an off-white solid. Preparation procedure for compound No. 64-52h.

[0390] To an ice-cold solution of compound 55 (12 mg, 1.0 equiv.) in DMF (1.5 mL) was added diisopropylamine (0.1 mL) while stirring under an argon atmosphere. The resulting solution was then slowly warmed to room temperature and continued stirring for 12 h. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the off-white solid was dried under reduced pressure to afford 10 mg of crude compound No. 64-52a as an off-white solid. Product purity was determined to be 30-40% by RP-HPLC at 254 nm, and LC-MS indicated a mass of the desired compound of 1,120.4 daltons. Compound number 64-52i.

[0391] The TLR7 / 8 agonist D in formula (I) is 64-10b, and the self-eliminating linker L in formula (I) 1 is a para-aminobenzyl carbamate moiety conjugated to the secondary amine in the benzylmethylamine of the TLR7 / 8 agonist (see FIG. 1 ), and the cleavable linker L in formula (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To illustrate an embodiment of the present invention in which D is an azido-PEG4 moiety, Compound No. 64-52i was prepared as shown in Scheme S2i-1. Additionally, the TLR7 / 8 agonist (D in Formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as any of Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Cut. Scheme S2i-1 [ka] General procedure for the preparation of compound No. 52.

[0392] To a solution of Compound No. 6 (1.1 equivalents) and Compound No. 64-10b (1.0 equivalents) in DMF, TEA (2.0 equivalents) is added and the resulting solution is stirred at room temperature. Once LC / MS shows that the majority is the desired product, Compound No. 52, and that all of Compound No. 64-10b has been consumed, the reaction mixture is used directly in the next step. General procedure for the preparation of compound No. 53.

[0393] To a crude solution of Compound No. 52 (1.0 eq.) in DMF, piperidine (approximately 10 eq.) is added and the solution is stirred at room temperature. Once LC / MS shows the disappearance of the starting material Compound No. 52, the reaction mixture is diluted with 10% MeOH in DCM and the resulting mixture is stirred at room temperature. The aqueous layer is extracted with ethyl acetate, and the combined organic layers are dried over NaSO and concentrated under reduced pressure. The residue is purified by column chromatography (100 / 10 to 100 / 15 (v / v) DCM / MeOH) to give Compound No. 53 as a yellow solid. General procedure for the preparation of compound No. 64-52i.

[0394] To a solution of Cpd. No. 53 (1.0 equiv.) and Cpd. No. 25 (1.1 equiv.) in DMF, DIPEA (0.2 mL) was added, and the resulting solution was stirred at room temperature. Once LC / MS showed majority conversion to the desired product, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (100 / 10 to 100 / 15 (v / v) DCM / MeOH) to give Cpd. No. 64-52i as a white solid. (Example S3) Preparation of propargylamine-carboxymethylated Ficoll.

[0395] Carboxymethylated Ficoll (CM-Ficoll) was prepared at 37.9 mg / mL in 0.2 M sodium chloride as described elsewhere (e.g., Inman, JK (See J Immunol 114:704-709, 1975). Propargylamine-carboxymethylated Ficoll (PACM-Ficoll) was prepared as shown in Scheme S3-1. Briefly, 1.2 g of propargylamine hydrochloride was dissolved in 10 mL of CM-Ficoll, and then 150 mg of EDC was added to the mixture in 25 mg increments over 10 min with mixing. The pH of the resulting solution was adjusted to 4.7 with 1N HCl, and the reaction was allowed to proceed for 3.5 h at ambient temperature (22-24°C) with mixing. The pH of the solution was checked periodically throughout the reaction and adjusted to 4.7 with 1N HCl if necessary. The resulting product was purified by tangential flow filtration in PBS (pH 7.5) using a 100 kDa molecular weight cutoff membrane. The final PACM-Ficoll contained 22.2 mg / mL Ficoll and was stored at -80°C. The propargyl content in PACM-Ficoll was determined by conjugation of a fluorophore (Alexa Fluor™ 594 azide, Thermo Scientific, Rockford IL, catalog number A10270) with subsequent quantification of fluorescence compared to a standard curve. The batch of PACM-Ficoll was shown to have approximately 250 moles of propargyl groups / mole of Ficoll. Scheme S3-1 [ka] (Example S4) Preparation of compounds Nos. 64-53, 64-53a, 64-54, 54-54a, 64-54b and 64-54c.

[0396] -L in formula (I) 3 -L 2 -L 1Azido-disulfide-PABC-IMDQ (compound numbers 64-51 and 64-51a) and azido-dipeptide-PABC-IMDQ (compound numbers 64-52, 54-54a, 64-54b, and 64-52c), corresponding to -D, were synthesized by PACM-Ficoll synthesis according to Scheme S4-1, Scheme S4-1a, Scheme S4-2, Scheme S4-2a, Scheme S4-2b, and Scheme S4-2c. to give Ficoll-azide-disulfide-PABC-IMDQ compounds (compound numbers 64-53 and 64-53a) or Ficoll-azide-dipeptide-PABC-IMDQ compounds (compound numbers 64-54, 64-54a, 64-54b, and 64-54c). Compound numbers 64-53, 64-53a, 64-54, 64-54a, 64-54b, and 64-54c are representative examples of compounds of formula (I), i.e., F-[WL 3 -L 2 -L 1 -D] x is. Scheme S4-1 [ka] Preparation of Compound Nos. 64-53.

[0397] Compound No. 64-51 was dissolved in DMF at 5 mg / mL. PACM-Ficoll was prepared at 22.2 mg / mL in PBS (pH 7.5) buffer. Copper(II) sulfate pentahydrate (CuSO4) and L-ascorbic acid were each prepared at 5 mg / mL in pure water. PACM-Ficoll (0.79 mL, 0.044 μmol) was mixed with 0.97 mL of ascorbic acid solution, 0.27 mL of CuSO4 solution, 1.65 mL of 400 mM sodium acetate (pH 5.5) buffer, and 0.95 mL of DMF. Compound No. 64-51 (0.62 mL, 4.4 μmol) was then added in 0.2 mL increments with mixing. The reaction was allowed to proceed at ambient temperature (22-24 °C) for 18 hours with stirring. The next day, the reaction mixture was dialyzed in 8 liters of PBS (pH 7.5) using a 10,000 dalton molecular weight cutoff dialysis cassette. Aliquots of purified Compound No. 64-53 were taken and stored at -80°C. Scheme S4-1a [ka] Preparation of Compound No. 64-53a.

[0398] Compound No. 64-53a was prepared in a manner similar to that described above for Compound No. 64-53, using Compound No. 64-51a instead of Compound No. 64-51. The synthesis of Compound No. 64-53a is outlined in Scheme S4-1a. Scheme S4-2 [ka] Preparation of Compound Nos. 64-54.

[0399] Compound No. 64-52 was dissolved in DMF at 5 mg / mL. PACM-Ficoll was prepared at 22.2 mg / mL in PBS (pH 7.5). Copper(II) sulfate pentahydrate (CuSO4) and L-ascorbic acid were each prepared at 5 mg / mL in pure water. PACM-Ficoll (0.79 mL, 0.044 μmol) was mixed with 0.97 mL of ascorbic acid solution, 0.27 mL of CuSO4 solution, 2.6 mL of 400 mM sodium acetate (pH 5.5) buffer, and 0.22 mL of DMF. Compound No. 64-52 (0.61 mL, 4.4 μmol) was then added in 0.2 mL increments while mixing. The reaction was allowed to proceed at ambient temperature (22-24°C) for 18 hours while mixing. The following day, The reaction mixture (approximately 4.9 mL) was dialyzed in 8 L of PBS (pH 7.5) using a 10,000 dalton molecular weight cutoff dialysis cassette. Aliquots of purified Compound No. 64-54 were taken and stored at -80°C. Scheme S4-2a [ka] Preparation of Compound No. 64-54a.

[0400] Compound No. 64-54a was prepared in a manner similar to that described above for Compound No. 64-54, using Compound No. 64-52a instead of Compound No. 64-52. The synthesis of Compound No. 64-54a is outlined in Scheme S4-2a. Preparation of Compound No. 64-54b.

[0401] Compound No. 64-54b was prepared in a manner similar to that described above for Compound No. 64-54, using Compound No. 64-52c instead of Compound No. 64-52. The synthesis of Compound No. 64-54b is outlined in Scheme S4-2b. Scheme S4-2b [ka] Preparation of Compound No. 64-54c.

[0402] Compound No. 64-54c was prepared in a manner similar to that described above for Compound No. 64-54, using Compound No. 64-52b instead of Compound No. 64-52. The synthesis of Compound No. 64-54c is outlined in Scheme S4-2c. Scheme S4-2c [ka] (Example S4-a) Preparation of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide (Compound No. 64-10b).

[0403] Part A. Nitric acid (125 mL) was added to a slurry of quinoline-2,4-diol (50 g, 0.3 mol) in acetic acid (500 mL), and the reaction mixture was heated to 65° C. for 3 hours. The mixture was then cooled to 5-10° C., and the solid material was collected by filtration and rinsed with cold water. The resulting solid was then recrystallized from methanol and dried under vacuum to give 58 g of 3-nitro-2,4-quinolinediol as a yellow solid.

[0404] Part B. Under an argon atmosphere, phosphorus oxychloride (150 mL) was added to 3-nitro-2,4-quinolinediol (58 g) and heated to 95° C. for 4 hours. The mixture was then cooled to room temperature and poured onto crushed ice with constant stirring. The precipitated product was collected by filtration, washed with water, and dried under vacuum. The crude solid was purified by flash chromatography on silica gel using hexane / ethyl acetate to give 35 g of 2,4-dichloro-3-nitroquinoline.

[0405] Part C. To a solution of 2,4-dichloro-3-nitroquinoline (35 g, 0.15 mol, 1.0 eq) in anhydrous dichloromethane (400 mL) and trimethylamine (16.0 g, 0.16 mol, 1.1 eq), tert-butyl (4-(aminomethyl)benzyl)carbamate (37.3 g, 0.16 mol, 1.1 eq) was added and stirred at room temperature overnight. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel using hexane / ethyl acetate to give 52 g of tert-butyl (4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0406] Part D. A solution of tert-butyl (4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate (52 g, 0.12 mol) in ethyl acetate (250 mL) was hydrogenated in the presence of 5% platinum on carbon (2.0 g) and sodium sulfate (52 g) at 60 psi for 12 hours using a Parr hydrogenation apparatus. The platinum catalyst and sodium sulfate were removed by filtration through a pad of Celite®, and the filtrate was concentrated under reduced pressure. The product was further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give 32 g of tert-butyl (4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0407] Part E. To a solution of tert-butyl (4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate (32 g, 77.6 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (350 mL) and pyridine (30 mL) at 0–5° C. was slowly added pentanoyl chloride (9.7 mL, 81.5 mmol, 1.05 equiv.). The reaction mixture was then warmed to room temperature and stirred for 12 h. The solvent was removed under reduced pressure, and the solid was then redissolved in ethyl acetate (400 mL), washed successively with water and saturated sodium bicarbonate (150 mL), and finally dried over anhydrous magnesium sulfate. The product was further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give 22 g of tert-butyl (4-(((3-butylamido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0408] Part F. To a solution of tert-butyl (4-(((3-butylamido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate (22 g, 44.2 mmol, 1.0 equiv) in ethanol (320 mL) was added water (80 mL), followed by potassium carbonate (12.2 g, 88.4 mmol, 2.0 equiv), and the mixture was heated to 55° C. with vigorous stirring for 16 hours. The reaction mixture was concentrated, and the residue was partitioned between ethyl acetate (500 mL) and water (250 mL). The ethyl acetate layer was then washed with water (100 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The product was further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give 15.4 g of tert-butyl (4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

[0409] Part G. tert-Butyl (4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate (15.4 g, 32.2 mmol, 1 equiv.) was dissolved in anhydrous dimethylformamide (125 mL), and then sodium azide (8.4 g, 128.6 mmol, 4 equiv.) was added to the solution. The resulting suspension was degassed and stirred at 110–115 °C under an argon atmosphere, and the progress of the reaction was monitored by reverse-phase HPLC analysis. After 18 h, the reaction mixture was cooled to room temperature, poured into cold water (500 mL), and extracted with ethyl acetate (3 × 100 mL). The combined extracts were washed with water (2 × 75 mL), dried over magnesium sulfate, filtered through Celite, and concentrated under reduced pressure to give an off-white solid. This solid was worked up by recrystallization with 1:1 ethyl / hexane to give 12.5 g of tert-butyl (4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

[0410] Part H. tert-Butyl (4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate (12.5 g, 25.7 mmol) was added to concentrated hydrochloric acid (65 mL), and 10% platinum on carbon (3.0 g) was added to the suspension. The reaction mixture was subjected to hydrogenation at 65 psi, and the reaction progress was monitored by reverse-phase HPLC analysis. After 6 days, the catalyst was filtered off, and the filter cake was washed with water (2 × 25 mL). The cake was cooled in an ice bath, and ice-cold 1 N sodium hydroxide was added dropwise with vigorous stirring until the pH reached 8.5, and the material was extracted with dichloromethane containing 5% methanol (4 × 75 mL). The combined extracts were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column using 8% methanol / dichloromethane containing 1% aqueous ammonia to give 5.3 g of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0411] Part I. To a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (34 mg, 0.1 mmol, 1.0 equiv) in anhydrous dimethylformamide (2 mL) was added myristic acid (27.4 mg, 0.12 mmol, 1.2 equiv) and trimethylamine (0.2 mL). The slurry was mixed for 5 minutes, and then HBTU (47.4 mg, 0.125 mmol, 1.25 equiv) was added. The reaction mixture was stirred for an additional 2 hours under an argon atmosphere. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (30 mL) and washed with water (2 × 10 mL), then dried over magnesium sulfate and concentrated in vacuo. The product was purified using column chromatography (6% methanol / dichloromethane) to yield 35 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide (Compound No. 64-10a). Product purity was determined to be approximately 98% by reverse-phase HPLC, and LC / MS confirmed the mass of the desired compound, 569.8, and the structure of the desired compound was confirmed by 300 MHz proton NMR (CDCl): δ 7.98 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 8.4 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H). Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s, 2H), 3.74 (s, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.20 (t, J = 7.5 Hz, 2H), 1.82-1.9 (m, 2H), 1.42-1.70 (m, 4H), 1.26-1.48 (m, 20H), 0.85-1.05 (m, 6H). (Example S4-b) Preparation of 2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 64-33b).

[0412] Parts AH were the same as Example S4-a.

[0413] Part I. To a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (100 mg, 0.28 mmol, 1.0 equiv.) in anhydrous dimethylformamide (2 mL) was added cyclopropylacetic acid (33 mg, 0.33 mmol, 1.2 equiv.) and trimethylamine (140 mg, 1.39 mmol, 5.0 equiv.), the slurry was mixed for 5 minutes, and then HBTU (131 mg, 0.34 mmol, 1.25 equiv.) was added. The reaction mixture was stirred for an additional 2 hours under an argon atmosphere. The reaction was diluted with ethyl acetate (100 mL) and washed with water (3×30 mL), then dried using magnesium sulfate and concentrated in vacuo. The crude residue was dissolved in ethyl acetate and methanol and purified using column chromatography (6% methanol / dichloromethane) to afford 140 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide.

[0414] Part J. To a solution of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide (123 mg, 0.28 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (5 mL) was added a solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL, excess) at room temperature, and the reaction mixture was heated to reflux for 12 hours. The mixture was cooled to ambient temperature, quenched with 3 N HCl (1 mL), and stirred for 4 hours. The pH of the reaction mixture was made alkaline by adding 2 N sodium hydroxide, and the product was extracted with dichloromethane (20 mL × 10). The combined organic layers were concentrated under reduced pressure, and the residue was purified by flash chromatography using 6% methanol / dichloromethane as eluent to give 28 mg of 2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 64-33b). Product purity was determined to be 97% by reverse-phase HPLC, and LC / MS confirmed the mass of the desired compound, 427.6, and the structure of the desired compound was confirmed by 400 MHz NMR. 1 H NMR (CDCl3): δ 7.80 (dd, J = 8.5, 1.0 Hz, 1H), 7.70 (dd, J = 8.3, 1.1 Hz, 1H), 7.42 (m, J = 8.4. 7.0, 1.4 Hz, 1H), 7.29 (as, 1H), 7.25 (as, 1H), 7.10 (m, J = 8.2, 7.1, 1.3 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 5.92 (bs, 2H), 5.69 (s, 2H), 3.75 (s, 2H), 2.86 (dd, The identity was confirmed by the following affinities: J = 8.0 Hz, 2H), 2.68 (dd, J = 8.0 Hz, 2H), 1.82-1.74 (m, 2H), 1.45-1.36 (m, 4H), 0.91 (t, J = 7.8 Hz, 3H), 0.91-0.85 (m, 1H), 0.68-0.60 (m, 1H), 0.42-0.37 (m, 2H), 0.04-0.00 (m, 2H). (Example S4-c) Preparation of 2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 64-60b).

[0415] Part A. To a solution of 1-methylcyclobutanecarboxylic acid (57 mg, 0.5 mmol) and pentafluorophenol (94 mg, 0.52 mmol) in dichloromethane (3 mL), N,N-diisopropylcarbodiimide (76 mg, 0.6 mmol) was added in the presence of a catalytic amount of N,N-dimethylaminopyridine (6 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. The crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 126 mg of the desired (2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutanecarboxylate product.

[0416] Part B. To a solution of (2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutanecarboxylate (72 mg, 0.26 mmol) in dichloromethane (3 mL) was added IMDQ (84 mg, 0.23 mmol) in the presence of triethylamine (48 mg, 0.47 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexanes. The residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl, then water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to provide 88 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamide as an off-white solid.

[0417] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamide (88 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by adding ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. After recrystallization using 9:1 ethyl acetate / hexanes, the reaction yielded 32 mg of 2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 64-60b). Product purity was determined to be 96% pure by reverse-phase HPLC at 254 nm, and LC / MS confirmed the mass of the desired compound, 441.3 daltons, and the structure of the desired compound was confirmed by 300 MHz 1H NMR (CDCl3): δ 7.80 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 7.5, 15.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.14 (t, J = 7.5, 15.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 5.71 (s, 2H), 5.52 (s, 2H), 3.78 (s, 2H), 2.89 (t, J = 7.5, 15.6 Hz, 2H), 2.52 (s, 2H), The molecular weight was confirmed by the following peaks: 1.6-1.88 (m, 8H), 1.35-1.60 (m, 2H), 1.12 (s, 3H), 0.94 (t, J = 7.5, 14.7 Hz, 3H). (Example S4-d) Preparation of 2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound Nos. 64-66b).

[0418] Part A. To a solution of cyclobutanecarboxylic acid (106 mg, 0.93 mmol) and pentafluorophenol (175 mg, 0.97 mmol) in dichloromethane (3 mL) was added N,N-diisopropylcarbodiimide (127 mg, 1.0 mmol) in the presence of a catalytic amount of N,N-dimethylaminopyridine (12 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. The crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 126 mg of the desired (2,3,4,5,6-pentafluorophenyl)cyclobutanecarboxylate product.

[0419] Part B. To a solution of (2,3,4,5,6-pentafluorophenyl)cyclobutanecarboxylate (50 mg, 0.18 mmol) in dichloromethane (3 mL), IMDQ (62 mg, 0.17 mmol) was added in the presence of triethylamine (35 mg, 0.34 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexane. The residue was diluted with dichloromethane (15 mL). L), washed with 1 M HCl then water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 85 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamide as an off-white solid.

[0420] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamide (85 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by adding ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. After recrystallization using 9:1 ethyl acetate / hexanes, the reaction yielded 21 mg of 2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound Nos. 64-66b). Product purity was determined to be 95% pure by reverse-phase HPLC at 254 nm, and LC / MS confirmed the desired product mass of 427.3 daltons and the structure of the desired product by 300 MHz 1H NMR (CDCl3): δ 7.80 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.5, 15.0 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.15 (t, J = 7.5, 15.0 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 5.72 (s, 2H), 5.51 (s, 2H), 3.74 (s, 2H), 2.89 (t, J = 7.5, 15.6 Hz, 2H), 2.62 (d, J = 7.2 Hz, 2H), 2.40-2.58 (m, The molecular weights were confirmed by the following: 1H), 1.75-2.15 (m, 7H), 1.60-1.70 (m, 2H), 1.35-1.50 (m, 2H), 0.94 (t, J = 7.5, 14.7 Hz, 3H). (Example S5) Determination of percent free IMDQ in compound numbers 64-53, 64-53a, 64-54, 64-54a, 64-54b and 64-54c.

[0421] To detect free (i.e., unconjugated) IMDQ, compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, and 64-54c were analyzed by RP-HPLC. Aliquots of compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, or 64-54c were loaded onto a Zobrax Eclipse C18 column (column size: 1.4 mL; temperature: 23 °C; detection wavelength: 322 nm) and eluted with a water / acetonitrile gradient (5 to 95% acetonitrile). For quantification, the free IMDQ peak (retention time 4.5 min) was integrated, and the response area was compared to an IMDQ standard curve. Percent free IMDQ was calculated using the following formula: Calculations were made using the formula: % Free IMDQ = (concentration of unconjugated IMDQ) x 100 / (total IMDQ concentration), where total IMDQ concentration is determined as described in Example S6 for Compound Nos. 64-53 and 54-53a, or as described in Example S8 for Compound Nos. 64-54, 64-54a, 64-54b, and 64-54c. (Example S6) Determination of total IMDQ concentration in compounds Nos. 64-53 and 64-53a.

[0422] The total IMDQ concentration in Compound Nos. 64-53 and 64-53a was determined by quantification of the released IMDQ after complete thiolysis. Briefly, Compound Nos. 64-53 or 64-53a was incubated with 10 mM dithiothreitol (DTT) in PBS (pH 7.5) / EDTA buffer at room temperature for 18 hours. The resulting samples were analyzed by RP-HPLC, as described in Example S5, and the area of ​​the released IMDQ peak was quantified against an IMDQ standard curve. (Example S7) Determination of Ficoll concentration in compounds 64-53, 64-53a, 64-54a, and 64-54.

[0423] Ficoll concentrations in compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, or 64-54c were determined using the sodium periodate method (Glycoprotein Carbohydrate Estimation Kit, Thermo Scientific, Rockford IL, catalog number 23260) as described by the manufacturer, except that a standard curve was generated using Ficoll PM400 (GE Healthcare, Pittsburgh PA, catalog number 17-0300-50). (Example S8) Determination of total IMDQ concentrations in compounds nos. 64-54, 64-54a, 64-54b, and 64-54c.

[0424] The total IMDQ concentration in Compound Nos. 64-54, 64-54a, 64-54b, and 64-54c was determined by quantification of IMDQ released after complete proteolysis. Briefly, Compound Nos. 64-54, 64-54a, 64-54b, or 64-54c was incubated with 3.7 μM cathepsin B in acetic acid / EDTA (pH 5.5) buffer in the presence of 3.9 mM DTT at 37°C for 18 hours. The resulting samples were analyzed by RP-HPLC, as described in Example S5, and the area of ​​the released IMDQ peak was quantified against an IMDQ standard curve. (Example S9) Determination of particle size for compound nos. 64-53, 64-53a, 64-54, 64-54a, 64-54b or 64-54c.

[0425] The mean particle size (Z-average) of compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, and 64-54c was measured by dynamic light scattering using a Malvern Zetasizer (Malvern Instruments, Malvern, UK). Samples were diluted to a Ficoll concentration of 0.5 mg / mL in PBS (pH 7.5) buffer and measured under the specified instrument settings. 50 nm polystyrene nanospheres (Thermo Scientific, Rockford, IL, Cat. No. 3050A) were included in this analysis as a reference standard control and had a defined particle size of 49 ± 6 nm. (Example S10) Chemical reaction conditions and characterization data for the synthesis of compounds 64-53, 64-53a, 64-54, and 64-54a

[0426] A summary of the reaction conditions used for the synthesis of compounds 64-53, 64-53a, 64-54, and 64-54a is presented in Table S10-1. Assays for determining unconjugated IMDQ, total IMDQ, and Ficoll concentrations were as described in Examples S5-S9. The total IMDQ amount was calculated by multiplying the total IMDQ concentration by the volume of the solution. [Table S10-1] (Example S11) Preparation of Compounds Nos. 64-55 and 64-56.

[0427] Using the procedures and synthetic schemes shown in Example S11 and Schemes S11-1 and S11-2, maleimidocaproyl-dipeptide-PABC-TLR7 / 8 agonist compounds (-L in Formula (I)) bearing various TLR7 / 8 agonist moieties were synthesized. 3 -L 2 -L 1 -D) can be prepared. These examples demonstrate the use of TLR7 / 8 agonist Compound Nos. 64-10a and 64-33a to prepare Compound Nos. 64-55 (Scheme S11-1) and 64-56 (Scheme S11-2) as examples of general maleimidocaproyl-dipeptide-PABC-TLR7 / 8 agonist structures. Furthermore, the TLR7 / 8 agonist (D in Formula (I)) can be IMDQ or meta-IMDQ (see FIG. 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Scheme S11-1 [ka] General procedure for the preparation of compound No. 10.

[0428] To a solution of 2,4-dichloro-3-nitroquinoline (1.0 equivalent) in anhydrous dichloromethane (400 mL) and trimethylamine (1.1 equivalent) is added tert-butyl(4-(aminomethyl)benzyl)carbamate (1.1 equivalents), followed by stirring at room temperature overnight. The solvent is removed under reduced pressure, and the crude product is purified by flash chromatography on silica gel using hexane / ethyl acetate to obtain Compound No. 10 (tert-butyl(4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate). General procedure for the preparation of compound No. 11.

[0429] A solution of Compound No. 10 (0.12 mol) in ethyl acetate (250 mL) is hydrogenated in the presence of 5% platinum on carbon (2.0 g) and sodium sulfate (52 g) at 60 psi for 12 hours using a Parr hydrogenation apparatus. The platinum catalyst and sodium sulfate are removed by filtration through a pad of Celite®, and the filtrate is concentrated under reduced pressure. The product is further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give Compound No. 11 (tert-butyl(4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate). General procedure for the preparation of compound No. 12.

[0430] To a solution of Compound No. 11 (1.0 equiv.) in anhydrous tetrahydrofuran (350 mL) and pyridine (30 mL) at 0-5° C., pentanoyl chloride (1.05 equiv.) is slowly added. The reaction mixture is then warmed to room temperature and stirred for 12 hours. The solvent is removed under reduced pressure, and the solid is then redissolved in ethyl acetate (400 mL), washed successively with water and saturated sodium bicarbonate (150 mL), and finally dried over anhydrous magnesium sulfate. The product is further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give the intermediate tert-butyl (4-(((3-butylamido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate. To a solution of the intermediate (1.0 equivalent) in 4 volumes of ethanol is added water, followed by potassium carbonate (2.0 equivalents), and the mixture is heated to 55° C. for 16 hours with vigorous stirring. The reaction mixture is then concentrated, and the residue is partitioned between ethyl acetate and water. The ethyl acetate layer is then washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The product is further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give Compound No. 12 (tert-butyl (4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate). General procedure for the preparation of compound No. 13.

[0431] Compound No. 12 (1.0 equiv.) was dissolved in anhydrous dimethylformamide, and then sodium azide (4.0 equiv.) was added to the solution. The resulting suspension was degassed and stirred at 110-115 °C under an argon atmosphere, and the reaction progress was monitored by reverse-phase HPLC analysis. After 18 h, the reaction mixture was cooled to room temperature, poured into cold water, and extracted with ethyl acetate. The combined extracts were washed with water, dried over magnesium sulfate, filtered through Celite, and concentrated under reduced pressure to give an off-white solid. This solid was further worked up by recrystallization from 1:1 ethyl acetate / hexane to give Compound No. 13 (tert-butyl (4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate). General procedure for the preparation of compound No. 5 (IMDQ).

[0432] Compound No. 13 (25.7 mmol) is added to concentrated hydrochloric acid. 10% platinum on carbon (3.0 g) is added to the suspension. The reaction mixture is subjected to hydrogenation at 65 psi, and the progress of the reaction is monitored by reverse-phase HPLC analysis. After 6 days, the catalyst is filtered off, and the filter cake is washed with water. The cake is cooled in an ice bath, and ice-cold 1N sodium hydroxide is added dropwise with vigorous stirring until the pH reaches 8.5, and the material is extracted with dichloromethane containing 5% methanol. The combined extracts are dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue is purified on a silica gel column using 8% methanol / dichloromethane containing 1% aqueous ammonia to give compound No. 5 (1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine). General procedure for the preparation of compound No. 15.

[0433] To a solution of Compound No. 5 (1.0 equivalent) in anhydrous dimethylformamide, myristic acid (1.2 equivalents) and trimethylamine are added, the slurry is mixed for 5 minutes, and then HBTU (1.25 equivalents) is added. The reaction mixture is further stirred for 2 hours under an argon atmosphere. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The product is purified using column chromatography (6% methanol / dichloromethane) to give Compound No. 15 (N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide). General procedure for the preparation of compound No. 16.

[0434] To a solution of compound No. 15 (1.0 equivalent) in anhydrous DMF, 9-fluorenylmethyloxycarbonyl-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate (1.2 equivalents) and trimethylamine are added, the slurry is mixed for 5 minutes, and then HBTU (1.25 equivalents) is added. The reaction mixture is further stirred at room temperature for 15 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The product is purified using column chromatography (6% methanol / dichloromethane) to give compound No. No. 16 ((9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((2-butyl-1-(4-(tetradecanamidomethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate) is obtained. General procedure for the preparation of compounds Nos. 64-55.

[0435] Compound No. 16 (1.2 equivalents) is dissolved in NMP and then treated with diethylamine at room temperature for 2 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The intermediate 4-((S)-2-((S)-2-(12-azanyl)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-butyl-1-(4-(tetradecanamidomethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamate (1.0 equivalents) and N-(e-maleimidocaproyloxy)succinide ester (1.0 equivalents) are obtained. 0.1 equivalent) is dissolved in NMP and stirred at room temperature for 0.5 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The product is purified using column chromatography (6% methanol / dichloromethane) to give Compound No. 64-55 (4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-butyl-1-(4-(tetradecanamidomethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamate). Scheme S11-2 [ka] General procedure for the preparation of compound No. 17.

[0436] To a solution of Compound No. 5 (IMDQ; 1.0 equiv.) in anhydrous DMF, cyclopropylacetic acid (1.2 equiv.) and trimethylamine are added, and the slurry is mixed for 5 minutes, after which HBTU (1.25 equiv.) is added. The reaction mixture is stirred under an argon atmosphere for 2 hours. and further stirring. The reaction is diluted with ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The crude residue is dissolved in ethyl acetate and MeOH and purified using column chromatography (6% methanol / dichloromethane) to give the intermediate N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide. To a solution of the intermediate (1.0 equivalent) in anhydrous tetrahydrofuran is added a solution of excess borane-dimethylsulfide complex at room temperature, and the reaction mixture is heated to reflux for 12 hours. The mixture is cooled to ambient temperature, quenched with 3N HCl, and stirred for an additional 4 hours. The pH of the reaction mixture is made alkaline by adding 2N sodium hydroxide, and the product is extracted with dichloromethane. The combined organic layers are concentrated under reduced pressure and the residue is purified by flash chromatography using 6% methanol / dichloromethane as eluent to give compound no. 17 (2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine). General procedure for the preparation of compound No. 18.

[0437] To a solution of Compound No. 17 (1.0 equivalent) in anhydrous DMF, 9-fluorenylmethyloxycarbonyl-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate (1.2 equivalents) and trimethylamine are added, the slurry is mixed for 5 minutes, and then HBTU (1.25 equivalents) is added. The reaction mixture is further stirred at room temperature for 15 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated in vacuo. The product is purified using column chromatography (6% MeOH / dichloromethane) to give compound number 18 ((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate). General procedure for the preparation of compounds Nos. 64-56.

[0438] Compound No. 18 (1.2 equivalents) is dissolved in NMP and then treated with diethylamine at room temperature for 2 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate, and concentrated in vacuo. The intermediate 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamate (1.0 equivalents) and N-(e-maleimidocaproyloxy)succinide ester (1.1 equivalents) are dissolved in DMF and then stirred at room temperature for 0.5 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate, and concentrated in vacuo. The product is purified using column chromatography (6% methanol / dichloromethane) to give compound number 64-56 (4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)carbamate). (Example S11a) Preparation of compounds Nos. 64-71, 64-72, 64-73 and 64-74.

[0439] Using the procedures and synthetic schemes shown in Example S11a and Schemes S11a-1, S11a-2, S11a-3, and S11a-4, various TLR7 / 8 amino acids were synthesized. AzidoPEG4-dipeptide-PABC-TLR7 / 8 agonist compound having an agonist moiety (-L in formula (I)) 3 -L 2 -L 1These examples demonstrate the use of TLR7 / 8 agonist Compound Nos. 64-33b, meta-IMDQ, 64-60b, and 64-66b to prepare Compound Nos. 64-71 (Scheme S11a-1), 64-72 (Scheme S11b-2), 64-73 (Scheme S11c-3), and 64-74 (Scheme S11d-4) as examples of general azidoPEG4-dipeptide-PABC-TLR7 / 8 agonist structures. Additionally, the TLR7 / 8 agonist (D in Formula (I)) can be IMDQ or meta-IMDQ (see FIG. 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as any of Compound Nos. 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Scheme S11a-1 [ka] Procedure for the preparation of compound 21.

[0440] To a solution of pentafluorophenol ester of cyclopropanecarboxylic acid (280 mg; 1.2 equiv.) in dichloromethane (14 mL) in the presence of triethylamine (105 mg; 0.3 mmol), IMDQ (compound 5; 350 mg; 1.0 equiv.) was added and stirred overnight at room temperature. After 18 h of reverse-phase HPLC analysis of the solution, the reaction was complete. The solution was concentrated under reduced pressure and purified by flash chromatography using 5-10% methanol / dichloromethane containing 1% aqueous ammonia to give 350 mg of compound 39. Preparation procedure for compound No. 64-33b

[0441] Compound 39 (300 mg, 1 equiv.) in THF (5.0 mL) was reduced with borane dimethyl sulfide complex (3.5 equiv.) for 12 h at 55° C. The reaction was cooled to room temperature, carefully quenched with 2 M hydrochloric acid (excess), and stirred at 55° C. for an additional 6 h. The reaction was then cooled to room temperature, diluted with water (15 mL), and extracted with dichloromethane (15 mL) to remove impurities. The pH was adjusted to 8.0 by adding cold 1 M NaOH solution, and re-extracted with dichloromethane (3 × 10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. Recrystallization with ethyl acetate yielded 114 mg of Compound No. 64-33b as a white solid. Procedure for the preparation of compound 40.

[0442] To a solution of Compound (64-33b) (33 mg, 1.0 equiv.) in DMF (1.5 mL) was added diisopropylethylamine (0.1 mL), and the solution was stirred for 10 minutes under an argon atmosphere. Compound (10) (59 mg, 1 equiv.) was added in two portions, and the resulting clear yellow solution was stirred at 35°C for 2 hours. Thin-layer chromatography analysis using 10% methanol in dichloromethane containing 1% trimethylamine indicated that all of Compound (64-33b) had been consumed. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to give 73 mg of an off-white solid. The off-white solid (70 mg, 0.066 mmol) was dissolved in ice-cold DMF (1.5 mL), and diisopropylamine (0.1 mL) was added. The resulting solution was stirred under an argon atmosphere, allowed to warm slowly to room temperature, and continued stirring for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicated complete removal of the Fmoc moiety. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to give 47 mg of compound 40. Preparation procedures for compounds 64-71.

[0443] Compound 40 (47 mg, 1.0 equiv.) and compound 25 (24 mg, 1.2 equiv.) were dissolved in DMF (1 mL). Diisopropylethylamine (0.1 mL) was added, and the solution was stirred at room temperature for 2.5 hours. Reverse-phase HPLC analysis indicated the reaction was complete. DMF was removed under reduced pressure, and the yellow residue was subjected to silica gel column chromatography eluting with 4-10% methanol in dichloromethane containing 0.5% ammonia to afford 22.2 mg of compounds Nos. 64-71 as a white solid. Purity was determined to be 72% pure by reverse-phase HPLC at 254 nm. HRMS confirmed the mass of the desired compound, 1,105.6 daltons, and the structure of the desired compound was confirmed by HPLC at 300 MHz. 1 H NMR (CD3OD): δ 7.83 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.50-7.65 (m, 3H), 6.95-7.50 (m, 7H), 5.89 (s, 2H), 5.05 (brs, 2H), 4.42-4.60 (m, 3H), 4.20 (d, 1H), 3.73-3.76 (m, 2H), 3.55-3.68 (m, 14 H), 3.28, 3.38 (m, 4H), 3.0-3.28 (m, 2H), 2.97 (t, J = 7.5, 15.3 Hz, 2H), 2.55 (t, J = 6, 12 Hz, 2H), 1.72-2.18 (m, 7H), 1.58-1.70 (m, 2H), 1.43-1.55 (m, 2H), 0.88-1.01 (m, 9H), 0.30-0.50 (m, 3H), Confirmed by 0.10-0.08 (m, 2H). Scheme S11a-2 [ka] Procedure for the preparation of compound 41.

[0444] To a solution of meta-IMDQ (46 mg, 1.0 equiv.) in DMF (1.5 mL) was added diisopropylethylamine (0.2 mL), and the solution was stirred under an argon atmosphere for 10 minutes. Compound 10 (98 mg, 1.0 equiv.) was added in two portions. The resulting clear yellow solution was stirred at 0°C for 2 hours, then slowly warmed to room temperature, and stirring was continued for 12 hours. Thin-layer chromatography analysis using 10% methanol in dichloromethane containing 1% trimethylamine indicated that all of the meta-IMDQ had been consumed. DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to give 102 mg of an off-white solid. The off-white solid (99 mg, 0.1 mmol) was dissolved in ice-cold DMF (1.5 mL) and diisopropylamine (0.1 mL) was added. The resulting solution was stirred under an argon atmosphere and allowed to slowly warm to room temperature. Stirring was continued for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicated complete removal of the Fmoc moiety. DMF was evaporated under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to give 68 mg of compound 41. Preparation procedures for compounds 64-72.

[0445] Compound 41 (68 mg, 1.0 equiv.) and compound 25 (38 mg, 1.2 equiv.) were dissolved in DMF (1 mL). Diisopropylethylamine (0.1 mL) was added to the solution, which was stirred at room temperature for 1.5 hours. Reverse-phase HPLC analysis indicated the reaction was complete. DMF was removed under reduced pressure, and the yellow residue was treated with 0.5% ammonia. Silica gel column chromatography eluting with 1-8% methanol in dichloromethane afforded 47 mg of Compound Nos. 64-72 as a white solid. The purity was determined to be 96% pure by reverse-phase HPLC at 254 nm, and HRMS confirmed the mass of the desired compound, 1,037.8 daltons. The structure of the desired compound was confirmed by 300 MHz HPLC. 1 H NMR (CD3OD): δ 7.79 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.1 Hz, 2H), 7.41 (t, J = 7.5, 15.0 Hz, 1H), 7.21-7.37 (m, 3H), 7.05-7.29 (m, 2H), 7.03 (s, 1H), 6.93 (d, J = 7.5 Hz, 1H), 5.82 (s, 2H), 4.96 (s, 2H), 4.48-4.55 (m, 1H), 4.20-4.26 (m, 3H), 3.73-3.76 (m, 2H), 3.55-3.68 (m, 14 H), 3.28, 3.38 (m, 4H), The NMR spectrum was confirmed by the following NMR spectra: 3.0-3.28 (m, 2H), 2.97 (t, J = 7.5, 15.3 Hz, 2H), 2.55 (t, J = 6, 12 Hz, 2H), 1.72-2.18 (m, 5H), 1.58-1.70 (m, 2H), 1.43-1.55 (m, 2H), 0.88-1.01 (m, 9H). Scheme S11a-3 [ka] General procedure for the preparation of compound 42.

[0446] To a solution of the pentafluorophenol ester of 1-methylcyclobutanecarboxylic acid (1.2 equiv.) in DCM, IMDQ (compound 5; 1.0 equiv.) was added in the presence of TEA and stirred overnight at room temperature. After 18 h, reverse-phase HPLC analysis of the solution demonstrated the reaction was complete. The solution was concentrated under reduced pressure and purified by flash chromatography eluting with 5-10% methanol containing 1% (v / v) aqueous ammonia in DCM to give compound 42. General procedure for the preparation of compounds Nos. 64-60b.

[0447] Compound 42 (1 equivalent) in THF is reduced with borane-dimethylsulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction is cooled to room temperature, carefully quenched with 2M hydrochloric acid (excess), and stirred at 55°C for an additional 6 hours. The reaction is then cooled to room temperature, diluted with water, and extracted with DCM to remove impurities. The pH is adjusted to 8.0 by adding cold 1M NaOH solution, and re-extracted with DCM. The solution is dried over MgSO4 and concentrated under reduced pressure to give an off-white solid. Upon recrystallization with ethyl acetate, the reaction affords compound no. 64-60b as a white solid. General procedure for the preparation of compound 43.

[0448] DIPEA was added to a solution of Compound Nos. 64-60b (1.0 equiv.) in DMF, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 10 (1.0 equiv.) was added to the solution in two portions, and the resulting clear yellow solution was stirred at 35°C for 2 hours. Thin-layer chromatography analysis, developed with 10% (v / v) methanol in DCM containing 1% (v / v) TEA, indicated that Compound Nos. 64-60b had been completely consumed. DMF was removed under reduced pressure, and ethyl acetate was added to the yellow residue. The mixture was triturated to precipitate the pale yellow solid that formed, and the supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to obtain an off-white solid. The off-white solid was dissolved in ice-cold DMF, and diisopropylamine was added. The resulting solution was stirred under an argon atmosphere and allowed to slowly warm to room temperature with continued stirring for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicates complete removal of the Fmoc moiety. The DMF is then removed under reduced pressure, and ethyl acetate is added to the yellow residue. The mixture is triturated to precipitate the pale yellow solid that forms, and the supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to give compound 43. General procedure for the preparation of compounds Nos. 64-73.

[0449] Compound 43 (1.0 equiv.) and compound 25 (1.2 equiv.) were dissolved in DMF. DIPEA was added, and the solution was stirred at room temperature for 2.5 h. Reverse-phase HPLC analysis indicated the reaction was complete. DMF was removed under reduced pressure, and the yellow residue was subjected to silica gel column chromatography eluting with 4–10% (v / v) methanol in dichloromethane containing 0.5% (v / v) ammonia to give compounds Nos. 64–73 as white solids. Product purity was determined by RP-HPLC at 254 nm, and the masses and structures of the target compounds were confirmed by LC-MS. 1 Confirm by 1 H NMR. Scheme S11a-4 [ka] General procedure for the preparation of compound 44.

[0450] To a solution of the pentafluorophenol ester of cyclobutanecarboxylic acid (1.2 equiv.) in DCM, IMDQ (compound 5; 1.0 equiv.) was added in the presence of TEA and stirred overnight at room temperature. After 18 h, reverse-phase HPLC analysis of the solution demonstrated the reaction was complete. The solution was concentrated under reduced pressure and purified by flash chromatography eluting with 5-10% methanol containing 1% (v / v) aqueous ammonia in DCM to give compound 44. General procedure for the preparation of compounds Nos. 64-66b.

[0451] Compound 44 (1 equivalent) in THF is reduced with borane-dimethylsulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction is cooled to room temperature, carefully quenched with 2M hydrochloric acid (excess), and stirred at 55°C for an additional 6 hours. The reaction is then cooled to room temperature, diluted with water, and extracted with DCM to remove impurities. The pH is adjusted to 8.0 by adding cold 1M NaOH solution, and re-extracted with DCM. The solution is dried over MgSO4 and concentrated under reduced pressure to give an off-white solid. Upon recrystallization with ethyl acetate, the reaction affords compound nos. 64-66b as a white solid. General procedure for the preparation of compound 45.

[0452] To a solution of Compounds Nos. 64-66b (1.0 equiv.) in DMF, DIPEA was added, and the solution was stirred for 10 minutes under an argon atmosphere. Compound 10 (1.0 equiv.) was added in two portions, and the resulting clear yellow solution was stirred at 35° C. for 2 hours. Thin-layer chromatography analysis, developed with 10% (v / v) methanol in DCM containing 1% (v / v) TEA, indicated that all of Compounds Nos. 64-66b had been consumed. F is removed under reduced pressure, ethyl acetate is added to the yellow residue, the mixture is triturated to precipitate the pale yellow solid that forms, and the supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to give an off-white solid. The off-white solid is dissolved in ice-cold DMF, and diisopropylamine is added. The resulting solution is stirred under an argon atmosphere and allowed to slowly warm to room temperature with continued stirring for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicates complete removal of the Fmoc moiety. Next, DMF is removed under reduced pressure, ethyl acetate is added to the yellow residue, the mixture is triturated to precipitate the pale yellow solid that forms, and the supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to give compound 45. General procedure for the preparation of compounds Nos. 64-74.

[0453] Compound 45 (1.0 equiv.) and compound 25 (1.2 equiv.) were dissolved in DMF. DIPEA was added, and the solution was stirred at room temperature for 2.5 h. Reverse-phase HPLC analysis indicated the reaction was complete. DMF was removed under reduced pressure, and the yellow residue was subjected to silica gel column chromatography eluting with 4–10% (v / v) methanol in dichloromethane containing 0.5% (v / v) ammonia to give compounds Nos. 64–74 as white solids. Product purity was determined by RP-HPLC at 254 nm, and the masses and structures of the target compounds were confirmed by LC-MS. 1 Confirm by 1 H NMR. (Example S12) Preparation of compounds Nos. 64-57, 64-75, 64-76 and 64-77.

[0454] Compounds of formula (I) can be prepared using the general procedures and schemes described in Examples S12, S12a, S12b, and S12c and Schemes S12-1, S12a-1, S12b-1, and S12c-1. These examples use maleimidocaproyl or NHS-PEG4-trizole-PEG4 conjugation. Compounds Nos. 64-57, 64-75, 64-76, and 64-77 are prepared as examples of compounds of formula (I) where the conjugation moiety F is an antibody, using the anti-HER2 antibody trastuzumab together with the linker, a valine-citrulline cleavable linker, a PABC self-eliminating linker, and compound Nos. 64-10a and 64-66a or IMDQ as the TLR7 / 8 agonist moiety (agonist:antibody ratio is an average of 4 (i.e., 4 TLR7 / 8 agonist compounds per antibody)). Additionally, the TLR7 / 8 agonist moiety (D in Formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for a representation of the chemical structures of IMDQ and meta-IMDQ), as well as any of Compound Nos. 64-01 through 64-50, 64-58 through 64-69, 64-01a through 64-50a, and 64-58a through 64-69a. Those skilled in the art will understand that the general procedures described herein using trastuzumab also apply to other recombinant antibodies and derivatives thereof. General procedure for limited reduction of anti-HER2 antibodies and preparation of compound No. 19.

[0455] Trastuzumab is a humanized IgG1 monoclonal antibody that targets the HER2 receptor (human epidermal growth factor receptor 2, also known as HER2 / neu), which is found on the cell membrane of epithelial cells and is severely overexpressed in certain breast cancer cells. A humanized IgG4 variant of the anti-HER2 antibody trastuzumab, with an engineered S228P mutation in the hinge region, is available from commercial sources (InvivoGen, San Diego CA, catalog number her2tra-mab14 or MedChem Express, Monmouth). Junction NJ, Catalog No. HY-P9907). The antibody is produced by recombinant DNA technology and purified by affinity chromatography using methods well known to those skilled in the art (see, e.g., Kuner, R. and Reinhart, D. 2016, Appl. J. Immunol. 2016, 14:147-152). (See Microbiol Biotechnol 100:3451-3461.) Trastuzumab antibody was diluted with 2.5 molar equivalents of Tris- in PBS (pH 7.5), 1 mM diethylenetriaminepentaacetic acid. Limited reduction with 2-carboxyethylphosphine at 37° C. for 2 hours yields compound no. 19. General procedure for the preparation of compound No. 20.

[0456] Compound No. 19 (1.0 equiv.) in 1 mM diethylenetriaminepentaacetic acid (PBS, pH 7.5) was reacted with Compounds Nos. 64-56 (8-10 equiv.) in 10% (v / v) dimethylacetamide (PBS, pH 7.5) at 0°C for 0.5 h, and the reaction was quenched with 1 mM cysteine. Compound No. 20 was then purified / buffer exchanged into PBS, pH 7.5, using a G-25 size-exclusion column (GE Healthcare, Pisquataway, NJ) equilibrated in PBS, pH 7.5. General procedure for the preparation of compounds Nos. 64-57.

[0457] Ring hydrolysis of the succinimide-thioether results in a linkage with greater plasma stability; therefore, using tangential flow filtration, Compound No. 20 is buffer exchanged into 50 mM borate (pH 9.2), heated to 45° C. for 48 hours, then cooled to room temperature and buffer exchanged into PBS (pH 7.5). Compounds Nos. 64-57 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any remaining impurities, aggregates, and ensure complete buffer exchange. The synthetic scheme for preparing Compounds Nos. 64-57 is shown in FIG. 12. General procedure for the preparation of compound No. 46

[0458] Compound No. 45 (1.0 equivalent) and N-(ε-maleimidocaproyloxy)succinide ester (1.1 equivalent) are dissolved in DMF, DIPEA is added, and the reaction is stirred at room temperature for 2.5 hours. The solvent is removed under reduced pressure, and the residue is dissolved in ethyl acetate and washed with water, then dried using MgSO and concentrated under vacuum. The product is purified using column chromatography (6% methanol / dichloromethane) to give Compound No. 46. General procedure for the preparation of compound No. 47.

[0459] Compound No. 19 (1.0 equiv.) in 1 mM diethylenetriaminepentaacetic acid (PBS, pH 7.5) was reacted with Compound No. 46 (8–10 equiv.) in 10% (v / v) dimethylacetamide (PBS, pH 7.5) for 0.5 h at 0°C, and the reaction was quenched with 1 mM cysteine. Compound No. 47 was then purified / buffer exchanged into PBS, pH 7.5, using a G-25 size-exclusion chromatography column (GE Healthcare, Pisquataway, NJ) equilibrated in PBS, pH 7.5. General procedure for the preparation of compounds Nos. 64-75.

[0460] Ring hydrolysis of the succinimide-thioether results in a bond with greater plasma stability; therefore, using tangential flow filtration, Compound No. 47 is buffer exchanged into 50 mM borate (pH 9.2), heated to 45° C. for 48 hours, then cooled to room temperature and buffer exchanged into PBS (pH 7.5). Compounds Nos. 64-75 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any remaining impurities, aggregates, and ensure complete buffer exchange. The synthetic scheme for preparing Compounds Nos. 64-75 is shown in FIG. 13. General procedure for the preparation of compound No. 48.

[0461] Compound No. 12 (1.0 equiv.) and N-(ε-maleimidocaproyloxy)succinide ester (1.1 equiv.) were dissolved in DMF, DIPEA was added, and the reaction was stirred at room temperature. Stir for 2.5 hours. Remove the solvent under reduced pressure, dissolve the residue in ethyl acetate and wash with water, then dry using magnesium sulfate and concentrate under vacuum. Purify the product using column chromatography (6% methanol / dichloromethane) to obtain Compound No. 48. General procedure for the preparation of compound No. 49.

[0462] Compound No. 19 (1.0 equiv.) in 1 mM diethylenetriaminepentaacetic acid (PBS, pH 7.5) was reacted with Compound No. 48 (8–10 equiv.) in 10% (v / v) dimethylacetamide (PBS, pH 7.5) at 0°C for 0.5 h, and the reaction was quenched with 1 mM cysteine. Compound No. 49 was then purified / buffer-exchanged into PBS, pH 7.5, using a G-25 size-exclusion chromatography column (GE Healthcare, Pisquataway, NJ) equilibrated in PBS, pH 7.5. General procedure for the preparation of compounds Nos. 64-76.

[0463] Ring hydrolysis of the succinimide-thioether results in a bond with greater plasma stability; therefore, using tangential flow filtration, Compound No. 49 is buffer exchanged into 50 mM borate (pH 9.2), heated to 45° C. for 48 hours, then cooled to room temperature and buffer exchanged into PBS (pH 7.5). Compounds Nos. 64-76 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any remaining impurities, aggregates, and ensure complete buffer exchange. The synthetic scheme for preparing Compounds Nos. 64-76 is shown in FIG. 14. Preparation procedure for compound No. 51.

[0464] To a solution of trastuzumab antibody (compound 50; 10 mg, 1 equivalent) in PBS (pH 7.4) (1 mL) was added dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (4.0 mg, 10 equivalents) in PBS (pH 7.4), 1.5% (v / v) DMSO (1 mL), and the reaction was allowed to proceed at room temperature for 2 hours. The resulting derivatized antibody was purified by size-exclusion chromatography using a Sephadex G-25 column equilibrated with PBS (pH 7.2), 0.05% (v / v) Tween® 80. The protein concentration of purified compound 51 was determined spectrophotometrically at 280 nm using compound 50 as a calibration standard. Preparation procedures for compounds 64-77.

[0465] Compound 51 (0.5 mg, 1 equivalent) in PBS (pH 7.4) (1.0 mL) was reacted with compound 64-52a (0.013 mg, 4 equivalents) in PBS (pH 7.5), 5% (v / v) DMSO (0.5 mL). The reaction proceeded at room temperature for 2 hours and then at 4°C for an additional 3 hours. Compounds 64-77 were purified by size-exclusion chromatography using a Sephadex G-25 Fine column equilibrated with PBS (pH 7.2). The purity of compound Nos. 64-77 was determined to be 96% by analytical SEC analysis, and the drug-to-antibody ratio was calculated to be 3.7 using UV / Vis absorption spectroscopy. The synthetic scheme for preparing compound Nos. 64-77 is shown in Figure 15. General procedure for characterization of compounds Nos. 64-57, 64-75, 64-76, and 64-77.

[0466] The concentrations of Compound Nos. 64-57, 64-75, 64-76, and 64-77 are determined by UV absorbance at 280 nm and / or amino acid analysis. The integrity of the polypeptide backbone is determined by SDS-PAGE and N-terminal sequencing. Purity is determined by analytical SEC by independent monitoring of the wavelengths at the absorbance maxima of both the antibody and the TLR7 / 8 agonist. Aggregation state is determined by analytical SEC-UPLC-MALS. The TLR7 / 8 agonist-to-antibody ratio is determined using UV / Vis absorption spectroscopy (see, e.g., Examples S5 and S8) and / or TOF-LC-MS analysis (with or without proteolysis). These analytical methods for characterizing antibody-drug conjugates are well known to those skilled in the art (e.g., Doronina, SO et al. 2003, Nature Biotechnol 21:778-784; Kim, MT, et al. 2014, Bioconjugate Chem 25:123-1232). B. Biological Examples

[0467] The functionality of the cleavable linkers in Compounds Nos. 64-53, 64-53a, 64-54, 64-54a, 64-53b, and 64-54c was confirmed by in vitro incubation with glutathione or purified human cathepsin B, as appropriate, followed by assessment of the release of the parent (i.e., unconjugated) TRL7 / 8 agonist moiety IMDQ from the compounds over time. In vivo bioactivity (pharmacodynamic response) of Compounds Nos. 64-53 and 64-54 was assessed in wild-type mice following a single subcutaneous (footpad) injection, along with assessment of local immune responses measured as induction of TLR7-induced gene expression in draining lymph node tissue and maturation marker expression on antigen-presenting cells (APCs), and systemic responses measured as TLR7-induced gene expression in spleen tissue. Along with evaluation of local immune responses measured as TLR7-induced gene expression, in vivo bioactivity (pharmacodynamic response) in tumors after a single intratumoral injection of Compounds Nos. 64-54a and 64-54b was also assessed. The antitumor efficacy of Compounds Nos. 64-53a, 64-54a, and 64-54b was assessed by measuring tumor growth inhibition over time after repeated intratumoral administration in Balb / c mice bearing syngeneic CT26 tumors. Example B1 In vitro cleavage of compounds 64-53, 64-52a, 64-54, and 64-54a by cathepsin B and glutathione. method

[0468] Compounds 64-53 and 64-53a (80 μM) and a noncleavable control compound containing only a single S atom instead of a disulfide (compound no. 64-70) were incubated with 5 mM glutathione in PBS (pH 7.5) at 37° C. Aliquots were taken from the reactions at the indicated times, and the amount of IMDQ released was assessed as described in Example S6.

[0469] Purified cathepsin B (5 μM; R&D Systems, Minneapolis, MN, catalog no. 953-CY) in 10 mM acetate (pH 5) buffer was activated with 4 mM DTT / EDTA by incubation at 37° C. for 15 minutes. Compounds 64-54 and 64-54a were then added to make an 80 μM solution, and the reaction was allowed to proceed at 37° C. Aliquots were taken from the reaction at the indicated times, and the amount of IMDQ released was assessed as described in Example S8. result

[0470] While the non-cleavable control compound demonstrated low levels of IMDQ release over the 26-hour incubation period, Compound No. 64-53, which contains a dimethyl disulfide cleavable linker, demonstrated a maximum of approximately 60% IMDQ release in the presence of excess glutathione over the same time frame (Figure 4). Compound No. 64-53a, which contains the monomethyl disulfide variant, demonstrated a maximum of approximately 80% IMDQ release under similar treatment conditions. Compounds Nos. 64-54 and 64-54a demonstrated a maximum of approximately 80% IMDQ release in the presence of excess cathepsin B by the 6-hour incubation time point. These data support the efficacy of these constructs. We demonstrate that the dipeptide- and disulfide-based cleavable linker moieties function in the predicted manner (e.g., release of unmodified IMDQ). Example B1a In vitro cleavage of compounds 64-54a, 64-54b, and 64-54c by cathepsin B. method

[0471] Purified cathepsin B (30 nM; R&D Systems, Minneapolis, MN, catalog no. 953-CY) in 10 mM acetate (pH 5) buffer was activated with 4 mM DTT / 1 mM EDTA by incubation at 37° C. for 15 minutes. Compounds nos. 64-54a, 64-54b, and 64-54c were then prepared as 6 μM solutions, and the reaction was allowed to proceed at 37° C. At the indicated times, aliquots were removed, and the amount of IMDQ released was assessed as described in Example S5. result

[0472] In formula (I), the TLR7 / 8 agonist D is IMDQ and the self-eliminating linker L 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L 2 is a valine-citrulline dipeptide, and the conjugation linker L 3 One embodiment of the present invention, Compound No. 64-54a, in which D is a triazole-PEG4 moiety and F is an amine-modified Ficoll nanoparticle, demonstrated that the unmodified chemical form of IMDQ resulted in the highest release level (63%) when incubated with cathepsin B enzyme for 23 hours (FIG. 4A). 1 is a bond and a cleavable linker L 2 is a valine-citrulline dipeptide, and the conjugation linker L 3 However, triazole-PEG 12Compound No. 64-54b, one embodiment of the present invention, wherein the TLR7 / 8 agonist D is IMDQ and the self-eliminating linker L is an amine-modified Ficoll nanoparticle, demonstrated that the unmodified chemical form of IMDQ had the lowest release level (14%) when incubated with cathepsin B enzyme for 23 hours. 1 is a para-aminobenzyl carbamate moiety, and the cleavable linker L 2 is a valine-citrulline dipeptide, and the conjugation linker L 3 One embodiment of the present invention, Compound No. 64-54c, in which L is a triazole moiety and F is an amine-modified Ficoll nanoparticle, demonstrated that the unmodified chemical form of IMDQ resulted in an intermediate release level (48%) when incubated with cathepsin B enzyme for 23 hours. These data support the use of L in the Ficoll-conjugated compound of formula (I). 1 or L 3 demonstrate that varying the chemical structure of can result in variations in the rate of in vitro release of the unmodified chemical form of IMDQ (TLR7 / 8 agonist D). (Example B2) Local versus systemic immune activation after a single subcutaneous injection in wild-type mice. method

[0473] For serum cytokine and gene expression assays, BALB / c mice (n=3 / group) were subcutaneously injected into the right hind footpad with IMDQ, Compound No. 64-53, Compound No. 64-54, or PBS (pH 7.5) as a vehicle control at IMDQ molar equivalents of 0.04, 0.2, 1, or 5 μg. Blood was drawn two hours later, serum samples were prepared, and IL-6, IL-12p40, and TNFα protein levels were measured by ELISA. The lower limit of quantitation (LLOQ) for each of these assays was 31.3 pg / mL; the dotted lines on the graphs represent the LLOQ multiplied by the serum dilution factor. Popliteal lymph nodes (draining the injection site) were removed six hours later, tissue homogenized, and gene expression assays were performed using TaqMan® (Applied Biosystems, Foster City, CA) with a StepOnePlus Real-Time PCR System. Prior to isolation of RNA for the current analysis, it was stored in RNAlater (Qiagen, Hilden DE). Gene expression data are expressed as fold increase in gene induction relative to PBS controls (mean ± standard error).

[0474] For flow cytometry assays, BALB / c mice (n=3 / group) were injected into the footpads of both hind paws with either IMDQ, Compound No. 64-53, Compound No. 64-54, or PBS control (n=6 / group for controls) at a molar equivalent of 1.8 μg IMDQ. Popliteal lymph nodes and spleens were harvested 24 hours later, and single-cell suspensions were prepared and subsequently stained and analyzed by flow cytometry using antibodies against cell surface markers to identify distinct APC and lymphocyte cell populations. The biological effects of IMDQ, Compound No. 64-53, and Compound No. 64-54 were assessed by measuring their effect on the APC maturation marker CD86. In vivo gene expression results

[0475] Wild-type mice, of course, lack functional TLR8, so only TLR7-mediated gene induction is measured by this readout. These in vivo data demonstrated that both Compound No. 64-53 and Compound No. 64-54 have relatively similar potencies to IMDQ for inducing interferon-related genes in the popliteal lymph node (FIG. 5), but the response to Compound No. 64-53 was absent at the lowest dose (0.04 μg), while both IMDQ and Compound No. 64-54 induced gene expression at that dose. In vivo serum cytokine results

[0476] Small molecule TLR7 / 8 agonists, such as IMDQ, are known to rapidly distribute to systemic compartments from the injection site. In this case, the agonist rapidly induces TLR7-dependent proinflammatory cytokine responses in spleen and liver cells, resulting in increased levels of certain cytokines detected in serum. Plasma was collected and serum was prepared 2 hours after footpad injection of different doses of Compound No. 64-53, Compound No. 64-54, or IMDQ in BALB / c mice. As expected, injection of 5 μg of IMDQ rapidly induced elevated serum levels of IL-12p40, IL-6, and TNFα (Figure 6). This response was dose-dependent, demonstrating that only IL-12p40 reached levels above background following injection of 1 μg of IMDQ. Compound No. 64-54 at a dose of 5 μg also induced IL-12p40, although at lower levels than IMDQ. However, in contrast to IMDQ, Compound No. 64-54 did not induce IL-6 or TNFα above background levels at either dose. Compound No. 64-53 did not induce significant levels of serum cytokines above background at either dose. These data suggest that conjugation of the TLR7 / 8 agonist moiety IMDQ to the nanoparticle conjugation moiety Ficoll prevented the rapid systemic distribution of IMDQ and the associated increase in systemic cytokine levels compared to an equimolar amount of IMDQ. In vivo antigen-presenting cell maturation marker expression levels

[0477] Compound #64-53, Compound #64-54, and IMDQ all induced cell activation / maturation in the popliteal lymph node 24 hours after subcutaneous footpad injection, as measured by increased expression levels of the APC maturation marker CD86, indicating comparable TLR7-mediated biological effects in lymph nodes proximal to the injection site (Figure 7). However, in the spleen (i.e., distal to the injection site), CD86 expression levels were comparable in mice injected with IMDQ or Compound #64-54, but significantly lower on APCs from mice injected with Compound #64-53. This finding is consistent with the difference in serum cytokine responses between the two compounds at 2 hours post-injection and suggests that compound No. 64-54 may be cleaved, at least in part, extracellularly, thus allowing some systemic distribution of the IMDQ molecule. A similar trend was observed with CD80 expression (data not shown). Example B2a Local immune activation in the tumor after a single intratumoral injection in wild-type mice bearing syngeneic CT26 tumors. method

[0478] Changes in TLR7-mediated interferon-related gene expression and proinflammatory gene expression in subcutaneous tumors following a single intratumoral administration of IMDQ or compounds 64-54a and 64-54b in wild-type BALB / c mice bearing syngeneic CT26 colon carcinoma were assessed by RT-PCR. All in vivo procedures were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols. Animals were housed in a facility certified by the Association for Accreditation of Laboratory Animal Care International (AALAC, Frederick, MD). Wild-type female Balb / c mice (15–20 gm) were obtained from Envigo (Hayward, CA) and allowed to acclimate for 2–3 days before use.

[0479] Pharmaceutical compositions were prepared by dissolving 200 μg of the indicated compound in 1 mL of PBS. The pharmaceutical compositions were sterilized by 0.2 μm filtration and demonstrated to be endotoxin-free by Limulus amebocyte lysate assay (EndoSafe MCS; Charles River, Wilmington MA). On day 0, mice were anesthetized with 1% isoflurane and injected subcutaneously into the right flank with 80,000 CT26 tumor cells in 200 μL of RMPI-1640 culture medium and 2.5% fetal bovine serum. Tumors were 100–200 mm 3 The mice were allowed to grow until they reached a mass of 1000 μg, at which point the animals were assigned to groups and treatment was initiated (N=5 per group). Mice were then administered a single intratumoral injection of 25 μL of each pharmaceutical composition containing 5,000 μg of IMDQ, Compound Nos. 64-54a and 64-54b, each with an IMDQ equiva...

Claims

1. below: In the formula, IMDQ is That is, A compound selected from the group consisting of:

2. The compound is In the formula, IMDQ is 2. The compound of claim 1, wherein:

3. The compound is In the formula, IMDQ is 2. The compound of claim 1, wherein:

4. The compound is In the formula, IMDQ is 2. The compound of claim 1, wherein:

5. The compound is In the formula, IMDQ is 2. The compound of claim 1, wherein:

6. The compound is In the formula, IMDQ is 2. The compound of claim 1, wherein:

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. 1. A pharmaceutical composition, wherein the compound is capable of stimulating cytokine production by mammalian leukocytes, the composition comprising: stimulating the production of IFNα by human peripheral blood mononuclear cells; Stimulating the production of IL-6 and TNFα by human monocytes; and stimulating the production of one or both of IL-12p40 and IL-6 by mouse spleen cells; 8. The pharmaceutical composition of claim 7, comprising one or more of the group consisting of:

9. 10. A pharmaceutical composition for use in a method of stimulating an immune response in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7 or 8, in an amount sufficient to stimulate an immune response in the mammalian subject.

10. 10. A pharmaceutical composition for use in a method of inducing an antigen-specific antibody or antigen-specific T-cell response in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7 or 8, in an amount sufficient to induce an antigen-specific antibody response and / or an antigen-specific T-cell response in the mammalian subject.

11. 10. A pharmaceutical composition for use in a method of treating cancer in a mammalian subject in need thereof, comprising administering to said mammalian subject a compound of any one of claims 1-6, or a pharmaceutical composition of claim 7 or 8, in an amount sufficient to treat cancer in said mammalian subject.

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