Cleavable conjugates of TLR7 / 8 agonist compounds, methods for their preparation and use.

Abstract

Provided are Toll-like receptor 7 / 8 agonist compounds that facilitate local release and / or local retention of the bioactive form of a TLR7 / 8 agonist and reduce an unwanted systemic inflammatory cytokine response. 【Solution means】 Formula (I): F-[W-L 3 -L 2 -L 1 -D]x(I) [wherein, D is a TLR7 / 8 agonist moiety, L 1 is a linking or self-detachable linker, L 2 is a cleavable linker, L 3 is a conjugation linker, W is O, S or NR 10 wherein, R 10 is H or C1-C8 alkyl, x is an integer from 1 to 500, F is a conjugation moiety, and the TLR7 / 8 agonist moiety is a 1H-imidazo[4,5-c]quinoline derivative]. Compounds are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 62 / 586,110, filed November 14, 2017, the entirety of which is incorporated herein by reference.

[0002] Field of Invention This disclosure relates to Toll-like receptor 7 / 8 agonist compounds that are covalently conjugated to a drug, such as a tumor-specific targeting agent or polymer nanoparticle agent, via a cleavable linker moiety, thereby facilitating local release and / or retention of the bioactive form of the TLR7 / 8 agonist, and reducing undesirable systemic pro-inflammatory cytokine responses. This disclosure also relates to methods for preparing the cleavable conjugate, its use for stimulating an effective immune response, and its use for the treatment of 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, known as pathogen-associated molecular patterns. Therefore, 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. Of 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 endolysosome compartment, and these receptors are preferentially expressed in antigen-presenting cells (APCs), a key cell type that modulates immune system activation.

[0004] In progenitor immune cells, TLRs are involved in key molecular mechanisms that activate innate and adaptive immune responses, including the biosynthesis of selected cytokines (e.g., interferon type I), induction of co-stimulatory molecules, and increased antigen-presenting ability via APCs. In plasmacytoid dendritic cells, TLR7 is involved in the induction of IFN-α / β, which plays an essential role in regulating adaptive immunity. TLR8 is expressed in bone marrow dendritic cells, monocytes, and monocyte-induced dendritic cells, and the involvement of TLR8 agonists induces a prominent pro-inflammatory cytokine profile characterized by increased production of tumor necrosis factor α (TNF-α), interleukin-12 (IL-12), and IL-18. Therefore, virtually all major types of monocytic and dendritic cells can be activated by either a TLR7 or TLR8 agonist to become highly effective antigen-presenting cells. Since 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 to 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 are promising as cancer treatments and vaccine adjuvants. It has been extensively studied as a stimulant of innate and adaptive immune responses, including its intended use (Sabado et al. 2015, Ca Immunol Res 3:278-287; Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819).

[0005] Several classes of small molecules are known to interact at the guanosine / uridine ligand binding site and possess varying levels of TLR7 and / or TLR8 agonist bioactivity, many of which are derivatives of the 1H-imidazo[4,5-c]quinoline-1H-imidazo[4,5-c]quinoline-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 efforts in medicinal chemistry have led to the creation of several derivatives with significant improvements in dual TLR7 / 8 agonist activity, most notably 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-yl)-2-methylpropan-2-ol (R848, Resiquimod), as well as 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (IMDQ, Figure 1) and 1-(3-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (meta-IMDQ, Figure 1; see, for example, Beesu, M. et al. 2015, J Med Chem, incorporated herein by reference). See 50:7833-7849; U.S. Patent Nos. 8,728,486 and 9,441,005). However, rapid systemic diffusion of these small molecule compounds after topical administration of pharmacologically relevant doses (e.g., subcutaneous [SC], intratumor [IT], or intramuscular [IM]) leads to an increased risk in humans of systemic induction of pro-inflammatory cytokine responses and adverse events (e.g., fever, discomfort, lymphopenia). See, for example, Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819; Smirnov, D. et al. 2011, Vaccine 29:5434-5442. Therefore, there is still a need for immunotherapeutic agents that possess 1) potent biological activity against both TLR7 / 8 agonist receptors and potent immunostimulatory activity that activates a larger subset of APCs than TLR7 or TLR8 agonists alone, 2) preferentially target the stable prodrug form of the TLR7 / 8 agonist in the tumor microenvironment, or locally maintain the stable prodrug form of the TLR7 / 8 agonist in the tumor microenvironment after SC or intravenous administration, and subsequently release the active form into the tumor microenvironment, and 3) physicochemical properties that restrict the subsequent distribution of the released active form of the TLR7 / 8 agonist from the tumor microenvironment (i.e., the unconjugated form). The present invention provides a conjugate of a TLR7 / 8 agonist compound containing a cleavable linker and an agent for tissue-specific targeting or local retention after administration, a method for preparing the same, its use for stimulating a local immune response and reducing undesirable systemic immune activation, and its 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 [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention This disclosure provides modified 1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7 / 8 agonists covalently conjugated to an agent for tumor-specific targeting or local retention after administration by a combination of auto-desorbing linkers, cleavable linkers, and conjugation linkers. These cleavable conjugates facilitate the local release and / or retention of the bioactive form of the TLR7 / 8 agonist and reduce undesirable systemic pro-inflammatory cytokine responses. This disclosure also relates to methods for preparing the cleavable conjugates, their use for stimulating effective immune responses, and their use for the treatment of cancer.

[0009] In one embodiment, equation (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is the TLR7 / 8 agonist section, L 1is a linking or self - detaching 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) A compound of is provided.

[0010] In one embodiment, D is of formula (D - 1):

Chemical formula

[0011] In one embodiment, D is of formula (D - 2):

Chemical formula

[0012] In another embodiment, D is formula (D-2a) or (D-2b): [ka] (In the formula, n is an integer between 4 and 21. R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1cis a C3-C4 cycloalkyl group, and p is 1 or 2. R 20 NHR 20a And R 20a is H, OH, NH2 or methyl, R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). A compound of formula (I) having is provided.

[0013] In another embodiment, D is given by equation (D-3): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, X is -NH- or -NH(C=O)-, R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). A compound of formula (I) having is provided.

[0014] In a further embodiment, D is formula (D-3a) or (D-3b): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 20 NHR 20a And R 20a is H, OH, NH2 or methyl, R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). A compound of formula (I) having is provided.

[0015] In yet another embodiment, D is given by equation (D-4): [Chemical formula] (wherein R 0 is a C4 - C 21 hydrocarbyl optionally substituted by 1 to 4 halogen atoms, L is X or -CH2-X-, X is -NH- or -NH(C=O)-, A is independently a C6 - C 14 arylene optionally substituted by from 1 to 4 groups selected from the group consisting of halogen and C1 - C8 alkyl optionally substituted by 1 to 8 halogen atoms, or independently a 5 - 14 member heteroarylene optionally substituted by from 1 to 4 groups selected from the group consisting of halogen and C1 - C8 alkyl optionally substituted by 1 to 8 halogen atoms, R 1 is C3 - C6 alkyl, -(CH2) p OR 1a -, -(CH2) p NHR 1b or -(CH2) p R 1c where R 1a and R 1b are independently C1 - C3 alkyl, R 1c is C3 - C4 cycloalkyl, 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 bonding point of D in formula (I)) There is provided a compound of formula (I) having

[0016] In another aspect, D is of formula (D-4a) or (D-4b):

Chemical formula

[0017] In one embodiment, L 1 However, equation (L-1): [ka] (In the formula, Y 1 It is S, O or NH, and Ar 1 R is an arylene that is substituted as needed, 11 and R 12 (These are independently H or C1-C8 alkyl groups which are substituted as needed.) A compound of formula (I) having is provided.

[0018] In another embodiment, L 2 However, equation (L-2): [ka] (In the formula, Y 2 , NR 30 , O or S, R 30 , R 31 , R 32 , R 33 and R 34 Compounds of formula (I) are provided, wherein H is independently a C1-C8 alkyl or C3-C8 cycloalkyl. In some embodiments, L 2 This is a peptide linker that can be cleaved 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. In some embodiments, L 2 It is a peptide linker that can be cleaved by endosomal cathepsins or pericellular type II transmembrane serine proteases.

[0019] In another embodiment, L 3 However, -L 3a -Y 3 -L 3b - and Y 3 , L 3aand L 3b However, a compound of formula (I) is provided, which is an independent spacer fragment as needed. In some embodiments, Y 3 teeth, [ka] In some embodiments, L 3a teeth, [ka] In some embodiments, L 3b The formula is: [ka] The acyl spacer fragment, or formula: [ka] The PEG-acyl spacer fragment is such that n is 0 to 200. In some embodiments, L 3b The formula is: [ka] The acyl spacer fragment, or formula: [ka] The PEG-acyl spacer fragment is such that n is 0 to 200. In some embodiments, L 3 The following: [ka] That is the case.

[0020] In another embodiment, -L 3 -L 2 -L 1 -Part D is as follows: [ka] A compound of formula (I) is provided.

[0021] In one embodiment, a compound of formula (I) is provided herein, wherein F is a tumor targeting agent. In some embodiments, the tumor targeting agent is an antibody or binding ligand that preferentially binds to tumor cell surface antigens, specific structural elements of the extracellular matrix of the tumor microenvironment, or specific structural elements of tumor blood vessels. In some embodiments, F has physical properties or surface chemical modifications designed to result in preferential distribution into the tumor microenvironment.

[0022] In another embodiment, compounds of formula (I) are provided herein, wherein F is an agent that facilitates the 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 polypeptide.

[0023] Further pharmaceutical compositions comprising a compound of formula (I) and pharmaceutically acceptable excipients are provided. In some embodiments of the pharmaceutical compositions, the excipients are solvents, fillers, emulsifiers / surfactants, buffers, isotonic agents and / or preservatives. In some embodiments of the pharmaceutical compositions, The form is either a solution or a freeze-dried solid.

[0024] In another embodiment, a method is provided for stimulating an immune response in a mammalian subject requiring stimulation of an immune response, the method comprising 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 for inducing an antigen-specific antibody response and / or antigen-specific T cell response in a mammalian subject where induction of an antigen-specific antibody response and / or antigen-specific T cell response is required, the method comprising the step of administering to the mammalian subject a pharmaceutical composition comprising a compound of formula (I) in an amount and dosing schedule sufficient to induce an antigen-specific antibody response and / or antigen-specific T cell response in the mammalian subject.

[0026] In mammalian subjects requiring treatment for cancer, the present invention also provides a plurality of methods for treating cancer, comprising the step of administering an effective amount of a pharmaceutical composition by a parenteral administration route, either as a monotherapy or in combination with other agents for treating cancer (e.g., chemotherapy, targeted therapy, and / or immunotherapy). The present invention also provides a kit comprising the pharmaceutical composition of the present invention and instructions for use in the treatment of cancer. [Brief explanation of the drawing]

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

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

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

[0030] [Figure 4] Figure 4 shows the in vitro release of IMDQ over time (as a percentage of total IMDQ) from compound numbers 64-53, 64-53a, 64-54, and 64-54a, or variants without a cleavable linker, after incubation with glutathione or 5 μM cathepsin B, respectively.

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

[0032] [Figure 5] Figure 5 shows the induction of interferon-related genes in the popliteal lymph nodes of BALB / c mice 24 hours after a single subcutaneous injection of compound number 64-53, compound number 64-54, or equimolar amounts of non-conjugate IMDQ.

[0033] [Figure 5A] Figure 5A shows the time course of interferon-related gene induction in subcutaneously implanted CT26 tumors in BALB / c mice after a single bolus intratumoral injection of compound no. 64-54a, compound no. 64-54b, or non-conjugate IMDQ of IMDQ equivalent mass. Data points are expressed as the ratio of gene expression to PBS-injected controls, with N=5 mice.

[0034] [Figure 5B] Figure 5B shows the time course of pro-inflammatory gene induction in subcutaneously implanted CT26 tumors of BALB / c mice after a single bolus intratumoral injection of compound no. 64-54a, compound no. 64-54b, or non-conjugate IMDQ in IMDQ equivalent mass. Data points are expressed as a ratio of gene expression to PBS-injected controls, with N=5 mice.

[0035] [Figure 6] Figure 6 shows the concentrations of cytokines ILs-12p40, IL-6, and TNFα in the serum of BALB / c mice two hours after a single subcutaneous injection into the paw of compound no. 64-53, compound no. 64-54, or equimolar concentrations of non-conjugated IMDQ or a PBS vehicle control.

[0036] [Figure 7]Figure 7 shows the induction of the mature marker CD86 in various antigen-presenting cells in the popliteal lymph nodes and spleen 24 hours after a single subcutaneous injection of compound no. 64-53, compound no. 64-54, equimolar amounts of non-conjugate IMDQ, or PBS vehicle control into the sole of the foot.

[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 a PBS vehicle control on tumor growth in BALB / c mice with a single subcutaneous tumor and syngeneic CT26 tumor. Panel A shows the effect on tumor volume over 27 days after tumor implantation. Panel B shows a scatter plot of tumor growth against treatment on day 27 (3 days after the last administration of the compound in three weekly doses). Data points are the mean + / - standard error for a group of 8 mice.

[0038] [Figure 9] Figure 9 shows the effect of weekly intratumoral administration of non-conjugate IMDQ, compound numbers 64-53a, 64-54a, 64-70, or PBS vehicle control in IMDQ-equivalent mass to tumor growth in BALB / c mice with syngeneic CT26 tumors and a single subcutaneous tumor. Time is the number of days after tumor implantation, and data points are the mean + / - standard error of the mean for a group of 8 mice.

[0039] [Figure 10] Figure 10 shows the effect on tumor growth of weekly intratumoral administration of compound number 64-54a (Panel A) or compound number 64-54b (Panel B) at 30, 125, or 500 ng of IMDQ equivalent mass, along with a PBS vehicle control, to BALB / c mice with a single subcutaneous tumor and syngeneic CT26 tumor. Time is the number of days after tumor implantation, and data points are the mean + / - standard error of the mean for a group of 10 mice.

[0040] [Figure 11] Figure 11 shows the effect of weekly intratumoral administration of 500 ng of IMDQ, 500 ng of IMDQ equivalent mass of compound 64-54a, or a PBS vehicle control, with or without twice-weekly immune checkpoint inhibitor (immunoblocker) administration, on tumor growth in BALB / c mice with syngeneic CT26 tumors, each having two subcutaneous tumors on the contralateral side. Time is the number of days after tumor implantation, and data points are the mean + / - standard error of the mean for a group of 10 mice.

[0041] [Figure 12] Figure 12 shows the synthesis scheme for preparing compound numbers 64-57.

[0042] [Figure 13] Figure 13 shows the synthesis scheme for preparing compounds 64-75.

[0043] [Figure 14] Figure 14 shows the synthesis scheme for preparing compounds 64-76.

[0044] [Figure 15] Figure 15 shows the synthesis scheme for preparing compounds 64-77. [Modes for carrying out the invention]

[0045] Detailed description of the invention This disclosure relates to a TLR7 / 8 agonist compound that exhibits potent bioactivity against both receptors of TLR7 / 8 agonists, and is covalently conjugated to a conjugation moiety that enables tumor-specific targeting or local retention after administration by a cleavable linker, thereby facilitating local release of the bioactive form of the TLR7 / 8 agonist and reducing undesirable systemic pro-inflammatory cytokine responses. The TLR7 / 8 agonist compound comprises a modified 1H-imidazo[4,5-c]quinoline derivative modified with an alkyl group or hydrocarbyl group, aryl group, 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 supramolecular, dendrimer or polypeptide. The cleavable linker moiety enables the release of the TLR7 / 8 agonist compound into the tumor microenvironment. This disclosure also relates to the use of cleavable conjugates to stimulate an immune response (e.g., an antigen-specific CD4+ / CD8+ T cell response), the use thereof to treat cancer, and methods for preparing cleavable conjugates. I. General Methods and Definitions

[0046] The implementation of this disclosure will utilize conventional techniques of organic chemistry, analytical chemistry, molecular biology, microbiology, cell biology, biochemistry, and immunology, unless otherwise specified, and these will be within the scope of the art. Such techniques are well described in the literature; see, for example, 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. “Mammals” include, but are not limited to, humans, non-human primates (e.g., monkeys), domesticated animals, athletic 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 to by an antibody or T cell antigen receptor. Antigens include peptides, polypeptides, proteins, glycoproteins, polysaccharides, This can include complex carbohydrates, sugars, gangliosides, lipids and phospholipids, their parts, and combinations thereof.

[0049] Antigens, if present in the compositions of the present disclosure, can be synthesized or isolated from nature. Suitable antigens for administration in the methods of the present disclosure include any molecule capable of eliciting an antigen-specific B-cell or T-cell response. Haptens are included within the scope of “antigens.” “Haptens” are low molecular weight compounds that are not immunogenic on their own but are generally made immunogenic when conjugated with larger immunogenic molecules.

[0050] The "polypeptide antigen" may include purified natural peptides, synthetic peptides, engineered peptides, recombinant peptides, crude peptide extracts, or partially purified active or unpurified active peptides (such as weakened or inactivated viruses, microorganisms, or peptides that are parts of cells), or fragments of such peptides. The polypeptide antigen is preferably at least 6 amino acid residues in length, preferably 8 to 1800 amino acids, more preferably 9 to 1000 amino acids, or 10 to 100 amino acids. Similarly, in some embodiments, the polypeptide is about 9 to 2000, about 9 to 1000, about 9 to 1000, or about 9 to 60 amino acids in length. In some embodiments, the polypeptide is 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 has a length of at most (upper limit) 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 50, or 25 amino acids. In some embodiments, the polypeptide antigen has a length of 9 to 35 amino acids.

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

[0052] An "adjuvant" is a substance that, when mixed with an immunogenic agent such as an antigen, nonspecifically enhances or activates the immune response to the immunogenic agent in a recipient upon exposure to the mixture.

[0053] The term "agonist," in its broadest sense, includes any molecule that activates signal transduction via a receptor. For example, a TLR7 agonist binds to the Toll-like receptor 7 protein and activates the TLR7 signaling pathway; a TLR8 agonist binds to the Toll-like receptor 8 protein and activates the TLR8 signaling pathway; and a dual TLR7 / 8 agonist binds to both the 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 a response or parameter when compared to other conditions that are otherwise identical except for the drug or molecule, or alternatively, when compared to another condition (e.g., increasing TLR signaling in the presence of a TLR agonist compared to the absence of a TLR agonist). For example, "stimulation" of an immune response means an increase in that response.

[0055] In this specification, the “effective dose” of a drug means a sufficient amount to perform the specifically stated purpose. The “effective dose” may be determined in an empirical and conventional manner in relation to the stated purpose. The “effective dose” or “sufficient dose” of a drug is an amount appropriate to produce the desired biological effect, such as beneficial results, including beneficial clinical outcomes. Yes, there is. The term "therapeutic dose" refers to the amount of a drug (e.g., a TLR modulator) that is effective in "treating" a disease or disorder in a subject (e.g., a mammal such as a human).

[0056] To “treat” a disease, or the term “treatment,” means to perform a protocol that may include administering one or more drugs to an individual (human or other) to alleviate the signs or symptoms of the disease. Therefore, “treating” or “treatment” does not require complete relief of the signs or symptoms, nor does it require a cure, and specifically includes protocols that only have a mitigating effect on the individual. As used herein and as is well understood in the art, “treatment” is a method for obtaining beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, relief or improvement of one or more symptoms, whether detectable or undetectable; reduction of disease severity; stabilization (i.e., no exacerbation) of the disease; prevention of disease spread; delay or slowing of disease progression; improvement or alleviation of the disease state; and remission (whether partial or comprehensive). “Treatment” can also mean an extension of survival compared to the expected survival of an individual that has not received treatment. To “alleviate” a disease or disability means that the severity of the disease or disability and / or undesirable clinical symptoms are reduced, and / or the progression of the disease or disability is slowed compared to the expected untreated outcome. In particular, in the context of cancer, alleviation can occur when a stable disease or remission of the disease is induced, resulting in an increased overall survival rate. Furthermore, alleviation does not need to occur with a single dose, but often occurs with a series of doses. Therefore, a sufficient amount to alleviate the response or disability may be administered in one or more doses.

[0057] As used herein, "alkyl" refers to a monovalent linear (i.e., unbranched) or branched saturated hydrocarbon chain, or a combination thereof. Specific alkyl groups have a specified number of carbon atoms, for example, 1 to 20 carbon atoms ("C1-C20"). 20 Alkyl) has 1 to 10 carbon atoms ("C1-C 10Alkyl groups are alkyl groups having 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, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0058] As used herein, "alkenyl" refers to a monovalent linear (i.e., unbranched) or branched unsaturated hydrocarbon chain or combination thereof having at least one olefinic unsaturated moiety (i.e., at least one C=C moiety). A specific alkenyl group has a specified number of carbon atoms, for example, 2 to 20 carbon atoms ("C2-C2"). 20 "Alkenyl" (having 2 to 10 carbon atoms) 10 Alkenyl groups are those having 2 to 8 carbon atoms ("C2-C8" alkenyls), 2 to 6 carbon atoms ("C2-C6" alkenyls), or 2 to 4 carbon atoms ("C2-C4" alkenyls). Alkenyl groups can have a "cis" or "trans" stereoconfiguration, or alternatively, an "E" or "Z" stereoconfiguration. Examples of alkenyl groups include, but are not limited to, ethenyl (or vinyl), propa-1-enyl, propa-2-enyl (or allyl), 2-methylpropa-1-enyl, buta-1-enyl, buta-2-enyl, buta-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, and their homologs and isomers.

[0059] As used herein, "alkynyl" refers to a monovalent linear (or monovalent) molecule having at least one acetylene unsaturated moiety (i.e., having at least one C≡C moiety). This refers to unbranched or branched unsaturated hydrocarbon chains or combinations thereof. Specifically, an alkynyl group has a specified number of carbon atoms, for example, a group with 2 to 20 carbon atoms ("C2-C2"). 20 Alkynyl) has 2 to 10 carbon atoms (C2-C2). 10 Alkynyl groups are those having 2 to 8 carbon atoms ("C2-C8 alkynyl"), 2 to 6 carbon atoms ("C2-C6 alkynyl"), or 2 to 4 carbon atoms ("C2-C4 alkynyl"). Examples of alkynyl groups include, but are not limited to, ethynyl (or acetylenyl), propa-1-inyl, propa-2-inyl (or propargyl), buta-1-inyl, buta-2-inyl, buta-3-inyl, and their homologs and isomers.

[0060] When used in this specification, "alkylene" refers to a residue that is the same as alkyl but has a divalent valency. Specific alkylene groups have 1 to 6 carbon atoms ("C1-C6 alkylene"), 1 to 5 carbon atoms ("C1-C5 alkylene"), 1 to 4 carbon atoms ("C1-C4 alkylene"), or 1 to 3 carbon atoms ("C1-C3 alkylene"). Examples of alkylene groups include, but are not limited to, methylene (-CH2- or =CH2), ethylene (-CH2CH2- or =CHCH3), propylene (-CH2CH2CH2- or =CHCH2CH3), and butylene (-CH2CH2CH2CH2- or =CHCH2CH2CH3).

[0061] As used herein, "cycloalkyl" refers to a monovalent, non-aromatic, saturated or unsaturated cyclic hydrocarbon structure. A specific cycloalkyl group has a specified number of annular (i.e., ring) carbon atoms, for example, a cycloalkyl group having 3 to 12 ring carbon atoms ("C3-C3"). 12A cycloalkyl group is a cyclic hydrocarbon 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, excluding aryl groups. Cycloalkyls containing more than one ring can be condensed, spiro-crosslinked, or in combination thereof. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, etc.

[0062] As used herein, "cycloalkylene" refers to a residue that is the same as cycloalkyl but has a divalent atom. Specifically, a cycloalkylene group has 3 to 12 ring carbon atoms ("C3-C"). 12 Cycloalkylenes are those having 3 to 8 ring carbon atoms ("C3-C8 cycloalkylenes") or 3 to 6 ring carbon atoms ("C3-C6 cycloalkylenes"). 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, etc.

[0063] When used herein, "hydrocarbyl" means having a specified number of carbon atoms (i.e., C1-C1). 20Hydrocarbyl groups refer to, and include, monovalent groups formed by removing hydrogen atoms from non-aromatic hydrocarbons (meaning 1 to 20 carbon atoms), which may be fully saturated, monounsaturated, or polyunsaturated. Hydrocarbyl groups may contain one or more linear, branched, or cyclic moieties, or combinations thereof. Alkyl, alkenyl, alkynyl, and cycloalkyl groups are specific subsets of hydrocarbyl groups. Hydrocarbyl groups may be further substituted by one or more cycloalkyl groups, alkyl groups, alkenyl groups, or alkynyl groups, and / or further substituted by one of more alkyl groups, alkenyl groups, and / or alkynyl groups. It may contain a cycloalkyl group. Examples of hydrocarbyl groups are, but are not limited to, the following: [ka] These include groups such as: Hydrocarbyl groups can 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] These include the following elements.

[0064] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having a monocyclic (e.g., phenyl) or a multiple fused ring (e.g., naphthyl or anthryl) in which one or more of the fused rings cannot be aromatic. Specific aryl groups have 6 to 14 ring (i.e., ring) carbon atoms ("C6~C"). 14An aryl group is defined as having one or more rings, at least one of which is non-aromatic. An aryl group can be bonded to the parent structure either at the aromatic or non-aromatic ring position. In one variant, an aryl group having one or more rings, at least one of which is non-aromatic, is bonded to the parent structure at the aromatic ring position. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, 1-naphthyl, and 2-naphthyl groups.

[0065] "Arylene," as used herein, is the same as aryl but with a divalent atom. It refers to the residues it contains. Specifically, an arylene group has 6 to 14 ring carbon atoms ("C6~C 14 Arylenes are 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] As used herein, "heteroaryl" refers to an unsaturated aromatic cyclic group having 1 to 14 ring carbon atoms and at least one ring heteroatom, including, but not limited to, a heteroatom such as nitrogen, oxygen, and sulfur. Heteroaryl groups can have a monocyclic ring (e.g., pyridyl or imidazolyl) or a multiple fused ring (e.g., indolidinyl or pyrazolopyridazinyl), where at least one of the fused rings is aromatic. Specific heteroaryl groups include 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 heteroaryls"), 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 heteroaryls"), 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 heteroaryls"). In one variant, the heteroaryl includes a 5-, 6-, or 7-membered monocyclic aromatic ring having 1-6 ring carbon atoms and 1-4 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. In another variant, a heteroaryl group comprises a polycyclic aromatic ring having 1 to 12 ring carbon atoms and 1 to 6 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. A heteroaryl group having more than one ring in which at least one ring is non-aromatic can bond to the parent structure at either the aromatic or non-aromatic ring position. In one variant, a heteroaryl group having more than one ring in which at least one ring is non-aromatic is bonded to the parent structure at the aromatic ring position. Examples of heteroaryl groups include, but are not limited to, pyridyl, benzimidazolyl, benzotriazolyl, benzo[b]thienyl, quinolinyl, indolyl, and benzothiazolyl groups.

[0067] As used herein, "heteroarylene" refers to a residue that is the same as heteroaryl but has a divalent valency. Specific heteroarylene groups include 5-14 membered rings having 1-12 ring carbon atoms and 1-6 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroarylenes"), 5-10 membered rings having 1-8 ring carbon atoms and 1-4 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroarylenes"), or 5-, 6-, or 7-membered rings having 1-5 ring carbon atoms and 1-4 ring heteroatoms independently selected from nitrogen, oxygen, and sulfur ("5-7 membered heteroarylenes"). Examples of heteroarylenes include, but are not limited to, pyridylene, benzimidazoylene, benzotriazolylene, benzo[b]thienylene, quinolinylene, indolylene, and benzothiazoylene.

[0068] "Halo" or "halogen" refers to the elements of Group 17 series having atomic numbers 9-85. Preferred halo groups include fluoro, chloro, bromo, and iodine. When a residue is substituted with two or more halogens, it may be referred to using a prefix corresponding to the number of halogen moieties it is bonded to. For example, dihaloaryl, dihaloalkyl, and trihaloaryl refer to aryl and alkyl groups substituted with two ("di") or three ("tri") halo groups, and these can be the same halo, although this is not necessarily required. Thus, 4-chloro-3-fluorophenyl falls within the range of dihaloaryl. Alkyl groups in which each hydrogen is replaced by a halo group are referred to as "pe". These are referred to as "perhaloalkyl" groups. A preferred perhaloalkyl group is trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" refers to an alkoxy group in which the halogen replaces each H in the hydrocarbon that constitutes the alkyl portion of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

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

[0070] "Substituting amino" refers to the -NR'R" group, 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 may be unsubstituted or substituted with one or more substituents (e.g., one, two, three, four, or five) listed for that group, and that these substituents may be identical or different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In yet another embodiment, an optionally substituted group has three substituents. In yet another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has one to two, one to three, one to four, or one to five substituents.

[0072] Unless otherwise specified, "substituted alkyl" 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, aryloxy, azide, 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] Unless otherwise specified, "substituted alkenyl" 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, alkalyl, aryl, aryloxy, azide, 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] Unless otherwise specified, "substituted alkynyl" refers to acyloxy, hydroxy, mercapto, acyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, arylo This refers to an alkynyl group having one or more substituents (e.g., 1 to 5 substituents or 1 to 3 substituents) selected from xy, azide, 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] Unless otherwise specified, "substituted cycloalkyl" 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, aryloxy, azide, 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] Unless otherwise specified, "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, aryloxy, azide, 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.

[0077] Unless otherwise specified, "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, aryloxy, azide, 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.

[0078] Unless otherwise specified, "substituted heterocyclyl" refers to acyloxy, hydroxy, thiol, acyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, This 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, aryloxy, azide, 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 with respect to individual terms herein, one or more hydrogen atoms on a saturated carbon atom in a specified group or radical (any two hydrogen atoms on a single carbon atom are =O, =NR) 70 、=N-OR 70 The substituent to replace (which can be replaced by =N2 or =S) is -R unless otherwise specified. 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 R is selected from the group consisting of alkyl, cycloalkyl, heterocycloalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, which are substituted as needed. 70 Each of them independently consists of hydrogen or R 60 And R 80 Each of them is independent of R70 Alternatively, two R 80 These, together with the nitrogen atom to which they are bonded, form 3-membered, 4-membered, 5-membered, 6-membered, or 7-membered heterocycloalkyls, which may optionally contain 1 to 4 additional heteroatoms, the same or different, selected from the group consisting of O, N, and S, among which N may be -H, C1-C4 alkyl, or -C(O)C 1~4 Alkyl, -CO2C 1~4 Alkyl or -SO2C 1~4 It can have alkyl substitution, M + Each of these is a counterion with a net positive charge of 1. + Each of them is independent, for example, K + kaNa + Li + Alkaline ions such as; + N(R 60 ) Ammonium ions such as 4; or [Ca 2+ ] 0.5 [Mg 2+ ] 0.5 or [Ba 2+ ] 0.5 These can be alkaline earth ions such as (the subscript "0.5" means that one of the counterions for such divalent alkaline earth ions may be the ionized form of the compound of the embodiment, and the other may be a typical counterion such as a chloride, or that the two ionized compounds disclosed herein can act as counterions for such divalent alkaline earth ions, or that the biionized compound of this embodiment can act as a counterion for such divalent alkaline earth ions).

[0080] In addition to the disclosures herein, substituents on hydrogen atoms on unsaturated carbon atoms in “substituted” alkenes, alkynes, aryl and heteroaryl groups are, unless otherwise specified, -R 60 Hello, -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 + This is as previously defined, except in the case of substituted alkenes or alkynes, the substituent is -O - M + , -OR 70 , -SR 70 or -S - M + This is conditional on the fact that it is not the case.

[0081] In addition to the substituents disclosed with respect to the individual terms herein, substituents on hydrogen atoms on nitrogen atoms in "substituted" heterocycloalkyl groups and cycloalkyl groups are, unless otherwise specified, -R 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 + This is as previously defined.

[0082] It is understood that polymers achieved by defining substituents having further substituents on themselves in all of the substituted groups defined above (e.g., substituted aryls having a substituted aryl group as a substituent that is itself substituted by a substituted aryl group that is further substituted by a substituted aryl group, for example) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically intended herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0083] Unless otherwise specified, the naming of substituents not explicitly defined herein is achieved by naming the terminal portion of the functional group, and then the adjacent functional groups in the direction of the bond point. For example, the substituent "arylalkyloxycarbonyl" refers to the (aryl)-(alkyl)-OC(O)- group.

[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 to realize and / or synthetically impossible. Furthermore, the compounds in question include all stereochemical isomers resulting from the substitutions 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. Polyalkylene glycol subunits are single polyalkylene glycols. It is a cole unit. For example, examples of polyethylene glycol subunits are ethylene glycol, -[CH2-CH2-O]-; or propylene glycol, -[CH2-CH(CH3)-O]-, which is capped with hydrogen at the terminal chain. Other examples of poly(alkylene glycol) include, but are not limited to, PEG; PEG derivatives such as methoxypoly(ethylene glycol) (mPEG); poly(ethylene oxide); PPG; poly(tetramethylene ethylene glycol) (also known as poly(tetrahydrofuran) or polyTHF); poly(ethylene oxide-co-propylene oxide); or copolymers and combinations thereof.

[0086] Unless otherwise specified, "organic modifier" means one of the group of solvents commonly used to dissolve organic chemical compounds. This group may 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 (methylenthylketone), methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol (isopropanol), propyl acetate, and combinations thereof.

[0087] In addition to the disclosures herein, the term “substituted” can also mean, when used to modify a specified group or radical, that one or more hydrogen atoms of the specified group or radical are each independently replaced by identical or different substituents as defined herein. In some embodiments, the substituted group has one, two, three or four substituents, one, two or three substituents, one or two substituents, or one substituent.

[0088] In addition to the disclosures herein, in certain embodiments, the substituted group has one, two, three or four substituents, one, two or three substituents, one or two substituents, or one substituent.

[0089] Unless a specific isotope of an element is indicated in the formula, the present invention, for example, refers to deuterated derivatives of the compound (where H is 2This includes all isotope substitutions of the compounds disclosed herein, such as H (which can be replaced by D). An isotope substitution may have an isotopic substitution at any or all positions in the structure, or it may have an atom that is present in natural abundance at any or all positions in the structure.

[0090] A "solvate" refers to a complex formed by a combination of solvent molecules and 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] "Stereoisomers (singular)" and "stereoisomers (plural)" refer to compounds that have the same atomic connectivity but differ in the spatial arrangement of their atoms. Stereoiomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

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

[0093] For clarity, it is understood that certain features of the Invention 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 described in the context of a single embodiment may also be provided individually or in any preferred partial combination. All combinations of embodiments relating to the chemical groups represented by the variable groups are specifically encompassed by the Invention, and every conceivable combination is disclosed herein as if it were individually and expressly disclosed to the extent that such combinations encompass compounds that result in stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). Furthermore, all partial combinations of the chemical groups enumerated in embodiments describing such variable groups are also specifically encompassed by the Invention, and every conceivable partial combination of the chemical groups is disclosed herein as if it were individually and expressly disclosed herein.

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

[0095] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless otherwise indicated or is not clear from the context.

[0096] The term “approximately” is used to indicate, unless otherwise explicitly stated, that a value includes the standard deviation of error for the apparatus or method used to determine that value. When “approximately” a value or parameter is referred to herein, it includes (and describes) embodiments relating to that value or parameter itself. For example, any statement referring to “approximately X” includes any statement relating to “X”. II. Tumor-targeted, cleavable conjugates of TLR7 / 8 agonist compounds

[0097] This disclosure provides modified 1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7 / 8 agonists, which are covalently conjugated to agents for tumor-specific targeting or local retention after administration by a combination of autolytic, cleavable, and conjugation linkers, thereby 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 this disclosure are chemically cleaved, thereby facilitating the release of the TLR7 / 8 agonist in its most bioactive form (i.e., the unconjugated form). Thus, these locally released TLR7 / 8 agonists can induce an effective immune response. Furthermore, the systemic distribution of the covalently conjugated TLR7 / 8 agonist compounds of this disclosure may be restricted due to their chemical properties, thereby reducing undesirable systemic pro-inflammatory cytokine responses. This disclosure also relates to methods for preparing therapeutic agents or vaccine adjuvants comprising a covalent conjugate of a TLR7 / 8 agonist, their use for reducing systemic pro-inflammatory cytokine responses and stimulating immune responses, and their use as therapeutic agents for treating cancer.

[0098] In one embodiment, equation (I): F-[WL 3 -L 2 -L 1 -D] X (I) (In the formula, D is the TLR7 / 8 agonist part, L 1 is a binding or self-deactivating linker, L 2 It is a linker that can be cut, L 3 It is a conjugation linker, W is O, S or NR 10 And, R 10 is H or C1-C8 alkyl, x is an integer between 1 and 500. F is the conjugation part.) The compound is provided.

[0099] In another embodiment, equation (Ia): F-[WL 3 -L 2 -L 1 -D] X (Ia) (In the formula, D is the TLR7 / 8 agonist part, L 1 It is a self-deactivating linker, L 2 It is a linker that can be cut, L 3 It is a conjugation linker, W is O, S or NR 10 And, R 10 is H or C1-C8 alkyl, x is an integer between 1 and 500. F is the conjugation part.) The compound is provided. A.TLR7 / 8 Agonist

[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]quinoline-4-amine derivative.

[0101] In some embodiments, the TLR7 / 8 agonist portion of D in formula (I) is formula (D-1): [ka] (In the formula, R 1AThese are C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl. R 2 NHR 2a And R 2a is H or C1-C8 alkyl, R 35 Each of these is independently a halogen or a C1-C8 alkyl group. R 4a and R 4b These are independently H or C1-C8 alkyl groups. R 5 Each of these is independently a halogen or a C1-C8 alkyl group. p and q are independently 0, 1, 2, 3, or 4. The wavy line represents the connection point of D in equation (I). It holds.

[0102] In some embodiments, R 1A is a C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl which is substituted as needed. In one variant form, R 1A is a C1-C8 alkyl group that is substituted with hydroxyl as needed. In certain variants, R 1A is a C1-C8 alkyl group (e.g., n-butyl or isobutyl). In other specific variants, R 1A It is a C1-C8 hydroxyalkyl group. In one variant form, R 1A These are C3-C8 cycloalkyl groups.

[0103] In some embodiments, R 2 NH2 or NHR 2a And R 2a is an alkyl that is substituted as needed. In one variant, R 2 This is NH2. In another variant, R 2 NHR 2a And R 2a These are C1-C8 alkyl groups that are substituted as needed. In certain variants, R2 NHR 2a And R 2a These are C1-C8 alkyl groups.

[0104] In some embodiments, q is 0 (i.e., R 35 (It does not exist). In some embodiments, q is 1 and R 35 It is bonded to the 6th, 7th, 8th, or 9th position of the imidazo[4,5-c]quinoline core. In some embodiments, q is 1, and R 35 is an amino or substituted amino bonded to the 7th or 8th position of the imidazo[4,5-c]quinoline core. In some embodiments, q is 2, and there are two R 35 The groups are bonded to the 7th and 8th positions of the imidazo[4,5-c]quinoline core, and together with the carbons to which they are bonded, they form cycloalkyl, aryl, heteroaryl, or heterocyclyl groups. In some embodiments, q is 1 or 2, and R 35 Each of these is independently a halogen or a C1-C8 alkyl group.

[0105] In some embodiments, R 4a and R 4b These are H, respectively. In some embodiments, R 4a and R 4b These, together with the carbon atoms to which they are bonded, form C3-C8 cycloalkyl groups (e.g., cyclopropyl) which are substituted as needed. In some embodiments, p is 0 (i.e., R 5 (It does not exist). In some embodiments, p is 1 or 2, and R 5 Each of these is independently a halogen or a C1-C8 alkyl group. R is described in detail in this specification with respect to formula (D-1). 1A , R 2 , R 35 , R 4a , R 4b , R 5All variants of p and q are described in detail herein with respect to formula (D-1) as if every possible combination were described individually. 1A , R 2 , R 35 , R 4a , R 4b , R 5 It is intended and understood that, p and q can be combined with any other variants. For example, in some embodiments, D is R 1A However, R is a C1-C8 alkyl, C1-C8 hydroxyalkyl, or C3-C8 cycloalkyl, 2 However, NH2 or NHR 2a And R 2a However, it is a C1-C8 alkyl group, and R 35 Each of these is independently a halogen or a C1-C8 alkyl, and R 4a and R 4b However, independently, H or C1-C8 alkyl, R 5 The formula (D-1) is such that each element is independently a halogen or a C1-C8 alkyl group, 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 the p-aminomethylbenzyl-1H-imidazo[4,5-c]quinoline moiety of formula (D-1a) or the m-aminomethylbenzyl-1H-imidazo[4,5-c]quinoline moiety of formula (D-1b): [ka] (In the equation, the dashed line represents the connection point of D in equation (I)). In some embodiments, D has the formula (D-1a) or (D-1b), and R 1A It is butyl, and R 2 is NH2, and q is 0. In one variant form, R 4a and R 4b These are H, respectively.

[0106] In some embodiments, D is represented by formula (D-1a-1) or (D-1b-1): [ka] (In the equation, the dashed line represents the connection point of D in equation (I)) It holds.

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

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

[0109] In some embodiments, TLR7 / 8 agonists are incorporated herein by reference in their entirety, Shukla et al. J. Med. Chem. 2010, 53:4450-4465; and These are imidazoquinoline compounds or their aminomethyl derivatives, as described in WO2015 / 023958. For example, Shukla et al. J. Med. Chem. 2010, 53:4450-446. The compounds listed in Table 1 of 5, or their derivatives, are derived from the benzyl group. The conjugation moiety (i.e., part F in formula (I)) may be covalently linked (or conjugated) via an amino group or any other applicable part of the molecule. Similarly, the compounds of formula 11 or formula 11A described in WO2015 / 023958, or their derivatives, may be covalently linked (or conjugated) via an amino group derived from the benzyl group to the conjugation moiety (i.e., part F in formula (I)). [ka] Formulas 11 and 11A (wherein R 9 , R 10 and R 11 (This is as described in WO2015 / 023958).

[0110] In some embodiments, the TLR7 / 8 agonist portion of D is given by equation (D-2): [ka] (In the formula, n is an integer between 4 and 21. X is -NH- or -NH(C=O)-, R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4bThese are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

[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 a C3-C6 alkyl group. In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 is n-butyl. In some embodiments, R 1 It is n-pentyl.

[0115] In some embodiments, R 1 is, -(CH2) p Ure 1a And p is 1 or 2, R 1a is a C1-C3 alkyl group. In some embodiments, R 1 It is -CH2OCH2CH3.

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

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

[0118] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

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

[0120] Any variant of X and n described in relation to equation (D-2) is described in relation to R, if any, in the same way that any combination is described specifically and individually. 1 q, p, R 3 , R 4a and R 4b It is intended and understood that it can be combined with any and all variants of R. For example, in some embodiments, 1is a C3-C6 alkyl group (e.g., n-butyl), q is 0, X is -NH-, and n is 4, 5, 6, or 7. In some embodiments, R 1 is a C3-C6 alkyl group (e.g., 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 portion of D is expressed by formula (D-2a) or (D-2b): [ka] (In the formula, n is an integer between 4 and 21. R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 20 NHR 20a And R 20a is H, OH, NH2 or methyl, R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

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

[0123] In some embodiments, the TLR7 / 8 agonist portion of D has the formula (D-2a), where 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 portion of D has the formula (D-2a), where n is 16, 17, 18, 19, 20, or 21.

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

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

[0126] In some embodiments, R 1 is, -(CH2) p Ure 1a And p is 1 or 2, R 1a is a C1-C3 alkyl group. In some embodiments, R 1 It is -CH2OCH2CH3.

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

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

[0129] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

[0130] In some embodiments, R 20 NHR 20a And R 20a H, OH, NH2 or methyl. In some embodiments, R 20 is NH2. In some embodiments, R 20 These are 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 These are H, respectively.

[0132] Any variant of n described with respect to equation (D-2a) is described with respect to R, if any, in the same way that any combination is described specifically and individually with respect to equation (D-2a). 1 , R 20 q, p, R 3 , R 4a and R 4b It is intended and understood that it can be combined with any and all variants of . Similarly, any and all variants of n described with respect to formula (D-2b) are described with respect to R described with respect to formula (D-2b) in the same way that any and all combinations described specifically and individually are described. 1 , R 20 q, p, R 3 , R 4a and R 4b It is intended and understood that it can be combined with any and all variants of it. For example, in some embodiments of formula (D-2a), R 1 is a C3-C6 alkyl group (e.g., n-butyl), and R 20 is NH2, q is 0, and n is 4, 5, 6, or 7. In some embodiments of equation (D-2b), R 1 is a C3-C6 alkyl group (e.g., n-butyl), and R 20 is NH2, q is 0, and n is 11, 12, 13, or 14.

[0133] Representative compounds of formulas (D-2), (D-2a), and (D-2b) are listed in Table 1, where the wavy lines indicate the D bond points 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-hydrogen atoms that have hydrogen atoms in the positions indicated by the wavy lines. This is the chemical name of a 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 non-conjugate compounds, which can be synthesized in accordance with scheme D-2 and / or using methods known in the art. Scheme D-2 [ka] (In the formula, R 1 q and R 3 This is as defined with respect to equations (D-2), (D-2a), and (D-2b), and R 20 (where R is NH2, or as defined with respect to formulas (D-2a) and (D-2b), where R and R'' are linear alkyl groups).

[0135] In some embodiments, R 1 If is a C3-C6 alkyl (e.g., n-butyl), 0 is NH2, and q is 0, then this compound is synthesized according to scheme D-2-a. Individuals useful for preparing the starting compound (IMDQ) in scheme D-2-a For a more detailed description of the reaction process, see, for example, U.S. Patent Nos. 8,728,486 and 9,441,005. Scheme D-2-a [ka] (In the formula, R and R'' are linear alkyl groups).

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

[0137] In some embodiments, the TLR7 / 8 agonist portion of D is given by equation (D-3): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, X is -NH- or -NH(C=O)-, R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

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

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

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

[0141] In some embodiments, R 0 This is a branched C4~C 14 It is alkyl. In some embodiments, R 0 This is a branched C5~C 10 It is alkyl. In some embodiments, R 0 is a branched C 10 ~C 14 It is alkyl. In some embodiments, R 0 is a branched C5-C7 alkyl group. 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 variant form, m is 1 or 2, and R A These are cyclopropyl, cyclobutyl, or cyclopentyl.

[0143] In some embodiments, R A These are C3-C8 cycloalkyl groups.

[0144] In some embodiments, R A m is a C3-C6 cycloalkyl group that is independently and optionally substituted with 1-3 groups selected from the group consisting of methyl and methylene. In one variant, m is 1 or 2. In another variant, m is 0 and R AThese are cyclobutyl, cyclopentyl, or cyclohexyl.

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

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

[0147] In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with 1-4 halogen atoms. In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with 1-3 chlorine or fluorine atoms. In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with one or two chlorine or fluorine atoms. In some embodiments, R A This is cyclobutyl, which is optionally substituted with 1-2 fluorine atoms. In one variant, m is 1.

[0148] In some embodiments, R 0 is, -(CH2) z (C(CH3)2)R A In one variant form, z is 1 or 2, and R A is cyclopropyl, cyclobutyl or cyclo It is lopentyl. In one deformed form, z is 1 and R A These are cyclopropyl, cyclobutyl, or cyclopentyl.

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

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

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

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

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

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

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

[0156] In some embodiments, R 1 is a C3-C6 alkyl group (e.g., n-butyl). In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 is, -(CH2) p Ure 1a (For example, CH2OCH2CH3). In some embodiments, R 1 is, -(CH2) p NHR 1b (For example, CH2NHCH2CH3). In some embodiments, R 1 is, -(CH2) p R 1c In one modified form, R 1c It 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 moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

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

[0159] X and R described in relation to equation (D-3) 0 All variants of are described in relation to formula (D-3) in the same way as all combinations are described specifically and individually, if any exist. 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 it can be combined with any and all of its variants.

[0160] In some embodiments, the TLR7 / 8 agonist portion of D is expressed by formula (D-3a) or (D-3b): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 20 NHR 20a And R 20a is H, OH, NH2 or methyl, R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

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

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

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

[0164] In some embodiments, R 0 This is a branched C4~C 14 It is alkyl. In some embodiments, R 0 This is a branched C5~C 10 It is alkyl. In some embodiments, R 0 is a branched C 10 ~C 14 It is alkyl. In some embodiments, R 0 is a branched C5-C7 alkyl group. 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 variant form, m is 1 or 2, and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In another variant, m is 0 and R A These are cyclobutyl, cyclopentyl, or cyclohexyl.

[0166] In some embodiments, R A These are C3-C8 cycloalkyl groups.

[0167] In some embodiments, R A m is a C3-C6 cycloalkyl group that is independently and optionally substituted with 1-3 groups selected from the group consisting of methyl and methylene. In one variant, m is 1 or 2.

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

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

[0170] In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with 1-4 halogen atoms. In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with 1-3 chlorine or fluorine atoms. In some embodiments, R A is a C3-C6 cycloalkyl group which is optionally substituted with one or two chlorine or fluorine atoms. In some embodiments, R A This is cyclobutyl, which is optionally substituted with 1-2 fluorine atoms. In one variant, m is 1.

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

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

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

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

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

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

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

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

[0179] In some embodiments, R 1 is a C3-C6 alkyl group (e.g., n-butyl). In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 is, -(CH2) p Ure 1a (For example, CH2OCH2CH3). In some embodiments, R 1 is, -(CH2) p NHR 1b (For example, CH2NHCH2CH3). In some embodiments, R 1 is, -(CH2) p R 1c In one modified form, R 1c It is cyclopropyl.

[0180] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

[0181] In some embodiments, R 20 NHR20a And R 20a is H, OH, NH2, or methyl. In some embodiments, R 20 is NH2. In some embodiments, R 20 These are 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 These are H, respectively.

[0183] R described in relation to formula (D-3a) 0 All variants of are described in relation to formula (D-3a) in the same way that all combinations, if any, are described specifically and individually. 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 it can be combined with any and all variants of it. Similarly, R described with respect to formula (D-3b) 0 All variants of are described in relation to formula (D-3b) in the same way that all combinations, if any, are described specifically and individually. 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 it can be combined with any and all of its variants.

[0184] Representative compounds of formulas (D-3), (D-3a), and (D-3b) are listed in Table 2, with the wavy lines indicating the D bond point 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-hydrogen atoms that have hydrogen atoms in the positions indicated by the wavy lines. This is the chemical name 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 non-conjugate compounds, which can be synthesized in accordance with scheme D-3 and / or using methods known in the art. Scheme D-3 [ka] (In the formula, R 1 q and R 3 This is as defined with respect to equations (D-3), (D-3a), and (D-3b), and R 20 is either NH2, or as defined for formulas (D-3a) and (D-3b), and R and R 0 (These are hydrocarbyl groups that are substituted as needed.)

[0186] R 1 is a C3-C6 alkyl group (for example, n-butyl), and R 20In some embodiments, where is NH2 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. Patent Nos. 8,728,486 and 9,441,005. Scheme D-3-a [ka] (In the formula, R and R 0 (These are hydrocarbyl groups that are substituted as needed.)

[0187] In some embodiments of formula (D-3), X is -NH-, or R 1 is a C3-C6 alkyl group (for example, n-butyl), and R 0 ga-(CH2) m R A And m is 0, R A In some embodiments of formula (D-3a) where is a cycloalkyl group, the compound is synthesized according to scheme D-3-b. Scheme D-3-b [ka] (In the formula, R 1 q and R 3 This is as defined with respect to formulas (D-3) and (D-3a), where R is a cycloalkyl group.

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

[0189] In some embodiments, the TLR7 / 8 agonist portion of D is given by equation (D-4): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, L is X or -CH2-X-, X is -NH- or -NH(C=O)-, A is independently a C6-C alkyl group which is optionally substituted by halogens and C1-C8 alkyl groups which are optionally substituted by 1-8 halogen atoms. 14 It is an arylene, or independently a 5-14 member heteroarylene optionally substituted with a halogen and 1-4 groups selected from the group consisting of C1-C8 alkyl groups optionally substituted with 1-8 halogen atoms. R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

[0190] In some embodiments, A is independently a halogen and 1 to 8 halogen atoms C6-C alkyl groups are substituted as needed by 1-4 groups selected from the group consisting of C1-C8 alkyl groups. 10 It is arrine.

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

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

[0195] In some embodiments, A is naphthylene, independently and optionally substituted with halogens and 1 to 4 groups selected from the group consisting of C1 to C8 alkyl groups 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 C4~C 14 It is hydrocarbyl. In some embodiments, R 0 C5~C 10 It is hydrocarbyl. In some embodiments, R 0 C 10 ~C 14 It is hydrocarbyl. In some embodiments, R 0 These are C5-C7 hydrocarbyls. In some embodiments, R 0 C 15 ~C 21 It is hydrocarbyl. In some embodiments, R 0 C4-C is substituted with 1-4 halogen atoms. 21 It is hydrocarbyl. In some embodiments, R 0 C4-C is substituted with 1-4 halogen atoms. 14 It is hydrocarbyl. In some embodiments, R 0 C4-C is substituted with 1-2 halogen atoms. 10It is hydrocarbyl. In some embodiments, R 0 C is substituted with 1-2 halogen atoms. 10 ~C 14 Hydrocal It is a building. In some embodiments, R 0 This is a C4-C7 hydrocarbyl substituted with 1-2 halogen atoms. In some embodiments, R 0 is a C4-C7 hydrocarbyl substituted with one halogen atom. In some embodiments, R 0 C is substituted with 1 to 4 halogen atoms. 15 ~C 21 It is hydrocarbil.

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

[0201] In some embodiments, R 0 The following: [Chemical] selected from the group consisting of.

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

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

[0204] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). In some embodiments, R 1 is propyl, butyl, pentyl or hexyl. In some embodiments, R 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., CH2NHCH2CH3). In some embodiments, R 1 is -(CH2) p R 1c In one variant, 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 These are H, respectively.

[0206] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

[0207] A, L, and R described in relation to formula (D-4) 0 All variants of R, if any, are described with respect to formula (D-4) in the same way as all combinations are described specifically and individually. 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 it can be combined with any and all variants of R. In some embodiments, 1 is n-butyl, q is 0, and R 4a and R 4b Each of the following is H, A is 1,4-naphthylene, L is -CH2-X-, X is -NH-, and R 0 is, -(CH2) m R A And m is 1 or 2, R A These are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0208] In some embodiments, the TLR7 / 8 agonist portion of D is expressed by formula (D-4a) or (D-4b): [ka] (In the formula, R 0 C4-C is substituted as needed by 1-4 halogen atoms. 21 It is hydrocarbil, A is independently a C6-C alkyl group which is optionally substituted by halogens and C1-C8 alkyl groups which are optionally substituted by 1-8 halogen atoms. 14 It is an arylene, or independently a 5-14 member heteroarylene optionally substituted with halogens and 1-4 groups independently selected from the group consisting of C1-C8 alkyl groups optionally substituted with 1-8 halogen atoms. R 1 C3-C6 alkyl, -(CH2) p Ure 1a ,-(CH2) p NHR 1b or -(CH2) p R 1c And R 1a and R 1b These are independently C1-C3 alkyl groups, and R 1c is a C3-C4 cycloalkyl group, and p is 1 or 2. R 20 NHR 20a And R 20a is H, OH, NH2 or methyl, R 3 Each of these is independently a halogen, a C1-C8 alkyl, a -(C1-C7 alkylene)-NH2, or a -CH2-phenylene-CH2NH2. q is 0, 1, 2, 3, or 4. R 4a and R 4b These are independently H or C1-C8 alkyl groups. The wavy line represents the connection point of D in equation (I). It holds.

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

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

[0211] In some embodiments, A is phenylene optionally substituted independently with 1 to 4 groups selected from the group consisting of halogens and C1 to C8 alkyl groups optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted independently with 1 to 4 groups selected from the group consisting of halogens and C1 to C8 alkyl groups optionally substituted with 1 to 8 halogen atoms. In some embodiments, A is 1,4-phenylene optionally substituted independently with 1 to 4 groups 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, independently, 1,3-phenylene 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.

[0214] In some embodiments, A is, independently, 5- to 10-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.

[0215] In some embodiments, A is, independently, naphthylene 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. In some embodiments, A is , 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 formula (D-4a). In some embodiments, the TLR7 / 8 agonist moiety of D has formula (D-4b).

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

[0219] In some embodiments, R 0 is, -(CH2) m R A And m is 0, 1, 2 or 3, R A is a C3-C8 cycloalkyl group that is independently and optionally substituted with 1-4 groups selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene groups. In one variant, m is 1 or 2, and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In some embodiments, R A m is a C3-C6 cycloalkyl group that is independently and optionally substituted with 1-3 groups selected from the group consisting of methyl and methylene. In one variant, m is 1 or 2. In some embodiments, R Am is a cyclopropyl which is independently and optionally substituted with 1 to 3 groups selected from the group consisting of methyl and methylene, where m is 1 or 2. In some embodiments, m is 0 or 1, and R A It is a cyclohexyl molecule that is independently and optionally substituted with 1 to 3 groups selected from the group consisting of methyl and methylene.

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

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

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

[0223] In some embodiments, R 1 is a C3-C6 alkyl group (e.g., n-butyl). In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some embodiments, R 1 is n-butyl. In some embodiments, R 1is n-pentyl. In some embodiments, R 1 is, -(CH2) p Ure 1a (For example, CH2OCH2CH3). In some embodiments, R 1 is, -(CH2) p NHR 1b (For example, CH2NHCH2CH3). In some embodiments, R 1 is, -(CH2) p R 1c In one modified form, R 1c It is cyclopropyl.

[0224] In some embodiments, R 20 NHR 20a And R 20a is H, OH, NH2, or methyl. In some embodiments, R 20 is NH2. In some embodiments, R 20 These are 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 These are H, respectively.

[0226] In some embodiments, the phenyl moiety of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl moiety 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 halogens, C1-C8 alkyls, -(C1-C7 alkylene)-NH2 and -CH2-phenylene-CH2NH2. In some embodiments, q is 1, and R 3 These are C1-C8 alkyl groups.

[0227] A and R described in relation to formula (D-4a) 0All variants of are described in relation to formula (D-4a) in the same way as all combinations are described specifically and individually, if any exist. 1 , R 1a , R 1b , R 1c , R 20 , p, R A , m, q, R 3 , R 4a and R 4b It is intended and understood that it can be combined with any and all variants of it. Similarly, A and R described with respect to formula (D-4b) 0 All variants of are described in relation to formula (D-4b) in the same way as all combinations are described specifically and individually, if any exist. 1 , R 1a , R 1b , R 1c , R 20 , p, R A , m, q, R 3 , R 4a and R 4b It is intended and understood that it can be combined with any and all variants of it. For example, in some embodiments of formula (D-4a), R 1 It is n-butyl, and R 20 is NH2, q is 0, and R 4a and R 4b These are H, A is 1,4-naphthylene, and R 0 is, -(CH2) m R A And m is 1 or 2, R A These are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0228] Representative compounds of formulas (D-4), (D-4a), and (D-4b) are listed in Table 3, where the wavy lines indicate the D bond points in formula (I). [Table 3-1] [Table 3-2] [Table 3-3] 1 The listed chemical names are the corresponding non-hydrogen atoms that have hydrogen atoms in the positions indicated by the wavy lines. This is the chemical name 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 non-conjugate compounds, which can be synthesized in accordance with 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 is defined with respect to equations (D-4), (D-4a), and (D-4b), and R 20 is either NH2 or as defined with respect to formulas (D-4a) and (D-4b), and R and R 0 (These are hydrocarbyl groups that are substituted as needed.)

[0230] Those skilled in the art will recognize that the TLR7 / 8 agonist moieties of formulas (D-4), (D-4a), and (D-4b) described herein can be synthesized using other synthetic routes, including various solvents, catalysts, reducing agents, temperatures, reaction times, and atmospheric conditions. B. Self-destructive 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 bioactivity when stably conjugated, and have greater TLR7 / 8 agonist bioactivity 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 this disclosure, the self-destructive linker portion (L in formula (I)) 1 ) consists of a TLR7 / 8 agonist compound (D in formula (I)) and a cleavable linker moiety (L in formula (I)). 2 ) is used to connect by covalent bond. Self-detaching linker L in formula (I) 1 The presence of the linker L allows the TLR7 / 8 agonist portion D to remain relatively inactive. 2 After hydrolysis, this self-determinating linker L 1 This undergoes a spontaneous chemical rearrangement, detaching the above-mentioned portion from the TLR7 / 8 agonist conjugate and releasing the unconjugated chemical form of the TLR7 / 8 agonist.

[0232] The amino group in the TLR7 / 8 agonist moiety (D) of formula (I) can act as a reactive chemical group capable of making the chemical conjugate a self-leaving linker. In some embodiments, the primary amine on the N1-benzyl group of 1-(aminomethylbenzyl)-1H-imidazo[4,5-c]quinoline-4-amine (see, for example, the compound of formula (D-1a-1) or (D-1b-1)) is L 1 It 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]quinoline-4-amine derivatives (see, for example, compound numbers 64-01 to 64-50 and 64-58 to 64-69 in Tables 1 to 3) is L1 It 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]quinoline-4-amine derivatives (see, for example, compound numbers 64-01a to 64-50a and 64-58a to 64-69a in Tables 1 to 3) is L 1 This can be used to conjugate D in equation (I). However, derivatization of these amino groups may result in a decrease in TLR7 and / or TLR8 agonist activity. Therefore, it is desirable to have a linker that, once the conjugate is cleaved, releases the TLR7 / 8 agonist moiety in its original form (i.e., the unconjugated form; i.e., as a compound with a free primary amine group). The self-leaving linker of this disclosure realizes a system in which cleavage of a particular bond triggers a series of 1,6- and / or 1,4-elimination reactions, resulting 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-destructive linker L in formula (I) 1 This is a moiety that can be eliminated from the compound of formula (I) when a specific bond is cleaved by one or more 1,6- and / or 1,4-elimination reactions.

[0234] In some embodiments, the autoeliminating linker L in the compound of formula (I) 1 Equation (L-1): [ka] (Y 1 It is S, O or NH, and Ar 1 R is an arylene that is substituted as needed, 11 and R 12 (These are, independently, H or an alkyl group which may be substituted as needed.) It holds.

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

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

[0237] In some embodiments, Ar 1 is phenylene, which is substituted as needed. In some embodiments, Ar 1 is 1,4-phenylene, which is substituted as needed. In some embodiments, Ar 1 is 1,2-phenylene, which is substituted as needed. In some embodiments, Ar 1 is naphthylene which is substituted as needed. In some embodiments, Ar 1 This is substituted as necessary, 1,4-naphthylene, substituted as necessary, 1,2-naphthylene, or substituted as necessary, 2,6-naphthylene. In some embodiments, Ar 1 is 1,4-phenylene. In some embodiments, Ar 1 It is 1,2-phenylene.

[0238] In some embodiments, Ar 1 It is 1,4-phenylene, and R 11 and R 12 These are H and L respectively. 1 Equation (L-1a): [ka] It holds.

[0239] In some embodiments, Ar 1 is 1,2-phenylene, and R11 and R 12 These are H and L respectively. 1 Equation (L-1b): [ka] It holds.

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

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

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

[0243] The self-leaving linker portion of the present invention imparts two important properties to the cleavable conjugate of the TLR7 / 8 agonist compound. First, when the self-leaving linker portion is connected to the TLR7 / 8 agonist, the linker portion maintains the TLR7 / 8 agonist in a relatively inert state. Second, the leaching linker portion is equivalent to the cleavable linker L in formula (I). 2During hydrolysis, it undergoes autolysis to produce its original (i.e., non-conjugated) TLR7 / 8 agonist moiety. Therefore, in some embodiments, L in formula (I) 1 -D is expressed in formulas (L1-D-1), (L1-D-2), (L1-D-3), or (L1-D-4): [ka] [ka] It holds.

[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 holds.

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

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

[0247] In some embodiments, L in the compound of formula (I) 1 This is a bond, and the compound of formula (I) is F-[WL 3 -L 2 -D] x In some embodiments, L in formula (I) 1 -D is as follows: [ka] (L 1 (This is a combination.) C. Cuttable linker

[0248] The severable linker portion L in equation (I) 2 This allows for the subsequent release of the original (i.e., non-conjugated) form of the TLR7 / 8 agonist moiety D upon localization or retention of the compound of formula (I) into the tumor microenvironment. In some embodiments, L 1 This is a self-detaching linker, consisting of a conjugation portion F and a conjugation linker portion WL. 3 (That is, FWL in equation (I)) 3 ) is the cuttable linker portion L 2 Through the TLR7 / 8 agonist portion D and the self-detaching linker L 1 (That is, -L in equation (I)) 1 -D) is conjugated by covalent bond. 1 This is a conjugation, consisting of a conjugation portion F and a conjugation linker portion WL. 3 (That is, FWL in equation (I)) 3 ) is the cuttable linker portion L 2 The TLR7 / 8 agonist moiety D is covalently conjugated via [WL], and the compound of formula (I) is F-[WL]. 3 -L 2 -D] x That is the case.

[0249] Those skilled in the art have used cleavable linker systems in the development of ADCs for antibody targeting of highly potent chemotherapeutic agents (e.g., Beck et al. 2017, Nature Reviews 16:315-337). (See reference below), it will be recognized that this addresses the narrow therapeutic range observed when increasingly potent cytotoxic agents are used as cancer treatments. The cleavable linkers used in the construction of ADCs generally fall into three classes: 1) linkers based on enzyme-unstable peptides, 2) disulfide linkers cleavable by glution reduction, and 3) linkers susceptible to hydrolysis under acidic conditions (Nolting, B. 2013, Methods Mol Biol 1045:71-100).

[0250] In some embodiments, the severable linker portion L in formula (I) 2 L is a peptide-based cleavable linker. In some embodiments, the cleavable linker L of the Disclosure 2 The amino acid molecule can be cleaved by proteolytic enzymes such as cathepsin B. Including f, an unstable intermediate is produced that self-desorbs in a manner that yields the original (i.e., non-conjugated) TLR7 / 8 agonist moiety D (see, for example, Figure 2). In such embodiments, L 2 This is a peptide, such as a peptide that can be cleaved by a proteolytic enzyme (e.g., cathepsin B). In some embodiments, L 2 This is a peptide linker consisting of 2-10, 2-8, 2-6, or 2-4 amino acid residues. In some embodiments, L 2 L is a dipeptide. In some embodiments, L 2 It is a peptide consisting of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0251] The amino acid residues of this disclosure include protein-constituting 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, selenocysteine, norleucine ("Nle"), norvaline ("Nva"), beta-alanine, L- or D-naphthoalanine, and 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 endolysosome 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). See 21:778-784). In some embodiments, L 2This is a peptide linker expressed by cells in the tumor microenvironment that can be cleaved by one or more endosomal or lysosomal peptidases or proteases, or one or more pericellular peptidases or proteases (see, for example, 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 This is a peptide linker that can be cleaved by endosomal cathepsins, comprising cathepsins B, C, D, H, L, Z and / or S. In some embodiments, L 2 It is a peptide linker that can be cleaved by pericellular proteases, comprising urokinase-type plasminogen activator (uPA), membrane-bound serine protease 1 (matryptase), matryptase-2, and / or regmine.

[0254] In some embodiments, L 2 L is a peptide linker that is cleaved by cathepsin B. In some embodiments, L 2 AA4 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 The following: [ka] [ka] That is the case.

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

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

[0258] In some embodiments, the severable linker portion L in formula (I) 2 This is the self-destructive linker L in equation (I). 1 This can form a disulfide linker (-SS-) and may produce an unstable intermediate that auto-desorbs in a manner that yields the original (i.e., non-conjugated) TLR7 / 8 agonist moiety D upon reductive thiolysis by high levels of reducing agents within the tumor microenvironment (see, for example, Figure 3). Preferential disulfide in tumors Glutathione cleavage depends on relatively high levels of reduced glutathione found in the tumor microenvironment and tumor cell cytosol compared to lower levels of free cysteine ​​in plasma (see, for example, 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 is understood by those skilled in the art that the balance between stability and tumor cleavage (relatively reductive thiorysis) can be regulated by the absence or presence of adjacent alkyl groups (see, for example, 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 detachable linker L in formula (I) 2 Equation (L-2): [ka] (In the formula, Y 2 , NR 30 , O or S, R 30 , R 31 , R 32 , R 33 and R 34 Independently, H, optionally substituted alkyl or optionally substituted cycloalkyl, or R 30 , R 31 , R 32 , R 33 and R 34 Two of these atoms, together with the atom(s) they are bonded to, form a cycloalkyl or heterocycline, which may be substituted as needed. It has. The sulfur atom in formula (L-2) is the same as the L in formula (I). 1 It forms a disulfide bond with the sulfur atom of the portion, and Y of (L-2) 2 is a conjugation linker L 3 Combine.

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

[0261] In some embodiments, L 2 This is expressed by equations (L-2a), (L-2b), (L-2c), or (L-2d): [ka] [ka] It holds.

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

[0263] Cuttable linker L 2 This is the self-destructive linker L in equation (I). 1 It is conjugated by a covalent bond (i.e., -L in equation (I)) 2 -L 1 -). In some embodiments, L 2 This is a self-destructive linker L 1 It is conjugated by a covalent bond, L 1 Equation (L-1): [ka] (In the formula, Y 1 It is S, O or NH, and Ar 1 R is an arylene that is substituted as needed, 11 and R12 (These are independently H or C1-C8 alkyl groups which are substituted as needed.) It holds.

[0264] In some embodiments, L 2 It is a peptide linker, Y 1 NH is -L 2 -L 1 -The part is equation (L-2-L-1): [ka] (In the formula, Ar 1 , R 11 and R 12 is either as defined for formula (L-1) or any variant described herein, and AA 1 and AA 2 (These are, independently, amino acid residues.) It has. In some embodiments, AA 1 It comprises an amino acid residue selected from the group consisting of lysine, lysine protected by acetyl or formyl, arginine, arginine protected by a tosyl or nitro group, histidine, ornithine, ornithine protected by acetyl or formyl, and citrulline (e.g., lysine or citrulline), AA 2 This includes 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). In some embodiments, AA 1 This is a citrulline residue (Cit), and AA 2 This is a valine residue (Val).

[0265] In some embodiments, L 2 This is the self-destructive linker L of equation (I). 1 It forms a disulfide bond with Y. 1 is S, and -L 2 -L 1 - is equation (L-2a-L-1a): [ka] (In the formula, Ar 1 , R 11 and R 12 is either as defined with respect to formula (L-1), or any variant described herein, and Y 2 , R 31 , R 32 , R 33 and R 34 (This is either as defined with respect to formula (L-2), or any variant described herein.) It holds.

[0266] Since those skilled in the art will recognize that other severable linkers may be functionally equivalent, the description of severable linkers provided herein is not intended to limit the scope of the invention.

[0267] In some embodiments, L 1 L is a linkage, and is detachable. 2 It is directly coupled to the TLR7 / 8 agonist portion D. D. Conjugation section and conjugation linker

[0268] Those skilled in the art may use several conjugation moieties / linkers (i.e., FWLs in formula (I)) to facilitate the preferential accumulation of the compound of formula (I) within the tumor microenvironment or to facilitate the retention of the compound of formula (I) at the site of local administration. 3It is recognized that -) exists. 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) consists 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 tumor perivascular retention. In some embodiments, the conjugation moiety / linker of the compound of formula (I) consists 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). Nano / microparticle-based or antibody-based conjugation moiety (FWL in formula (I)) 3 -) facilitates the 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 portion L in formula (I) to be cleaved. 2 During cutting and self-detachable linker L 1 Upon autolysis, the target tissue becomes capable of releasing the original (i.e., unconjugated) form of the TLR7 / 8 agonist portion D in equation (I). Particle-based conjugation portion

[0269] In some embodiments, the conjugation portion F in formula (I) is a micro / nanoparticle-based conjugation portion 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 portion F of formula (I) has physical properties (e.g., charge, size, shape, hydrophobicity, etc.) or surface chemical modifications (e.g., tumor cell-specific binding ligand) 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 portion F leads to the cleavable linker L 2 During the subsequent detachment, and the self-detaching linker L 1 During self-deactivation, the target of the TLR7 / 8 agonist portion D This makes it possible for release to occur within the organization.

[0270] In some embodiments, the particle-based conjugation moiety is a nanoparticle polymer (e.g., a branched copolymer of sucrose and epichlorohydrin, more specifically, 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(hydroxypropyl methacrylamide)). 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 bound to one TLR7 / 8 agonist moiety (e.g., a compound of formula (I) where x is 1). In some embodiments, the conjugated portion is polyvalent, meaning that each conjugation portion can bind to multiple TLR7 / 8 agonist subformula (I) compounds, for example, x is greater than 1 (e.g., up to 500). Macromolecules, supramolecules, nanoparticles, microparticles

[0271] Macromolecules and supramolecules, particularly those having molecular weights of approximately 5,000 to 2,000,000 daltons, that preferentially target TLR7 / 8 agonist compounds to the tumor microenvironment or enhance local retention at desired anatomical sites, 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(hydroxypropyl methacrylamide)), are useful as conjugation moieties for the cleavable conjugates of this disclosure.

[0272] In some embodiments, the macromolecular or supramolecular conjugation moiety is a nanoparticle polymer with a diameter of ≥10 nm. In some embodiments, the nanoparticle polymer has a diameter of 10 to 150 nm. In some embodiments, the macromolecular or supramolecular is a branched copolymer of sucrose and epichlorohydrin, or epichlorohydrin-crosslinked sucrose, for example, a branched copolymer of sucrose and epichlorohydrin sold as 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(hydroxypropyl methacrylamide)).

[0273] Polysaccharides derivatized to be linkable to a TLR7 / 8 agonist moiety can be used as conjugation moieties for the cleavable conjugates of this disclosure. Suitable polysaccharides may be naturally occurring or synthetic. Exemplary polysaccharides include, for example, dextran, mannin, chitosan, agarose, and starch. In some embodiments, the polysaccharides are crosslinked.

[0274] In some embodiments, the disclosure provides a cleavable conjugate of formula (I) in which the conjugation portion F is a macromolecule or supramolecular. In some embodiments, the macromolecule or supramolecular 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 via ether linkage, L 3 It is bonded to the ether linkage, which is derived from the hydroxyl group of sucrose in the copolymer. In some embodiments, it is a macromolecular or supramolecular conjugate of F. The conjugation portion is a branched copolymer of sucrose and epichlorohydrin having a molecular weight greater than (lower limit) approximately 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 portion F is a branched copolymer of sucrose and epichlorohydrin having a molecular weight less than (upper limit) approximately 1,000,000, 900,000, 800,000, 700,000, 600,000, 500,000, 400,000, 300,000, or 200,000 Daltons. In other words, the molecular weight of the macromolecular or supramolecular conjugation portion F can be any size in the range of about 50,000 to about 1,000,000 daltons, in which case the lower limit is smaller than the upper limit. In some embodiments, the macromolecular or supramolecular conjugation portion of 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 portion 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 linked to L via an ether linkage derived from a hydroxyl group in the polysaccharide. 3The conjugation moiety F is a polysaccharide (e.g., dextran) having a molecular weight of approximately 5,000 to approximately 2,000,000 daltons. In some embodiments, the conjugation moiety F is a polysaccharide (e.g., dextran) having a molecular weight greater than (lower limit) approximately 5,000, 10,000, 25,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 daltons. In some embodiments, the conjugation moiety F is a polysaccharide (e.g., dextran) having a molecular weight less than (upper limit) approximately 2,000,000, 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 daltons. In other words, the molecular weight of a polysaccharide can be anywhere in the size range of approximately 5,000 to 2,000,000 Daltons, in which case the lower limit is smaller than the upper limit.

[0276] For conjugates where 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 between 3 and 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 approximately 3 to 500, in which case the lower limit is smaller 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 approximately 120 ± 30 or approximately 70 ± 20.

[0277] The loading level of the TLR7 / 8 agonist moiety D in the 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), for example, the number of TLR7 / 8 agonist moieties D per 100,000 daltons (i.e., 100 kDa) of the compound of formula (I) ("relative loading ratio"). In some embodiments, the compound of formula (I) contains TLR7 / 8 agonist moieties D between about 1 and about 200 per 100 kDa of the molecular weight of the conjugation moiety F, i.e., a relative loading ratio between about 1 and about 200 D per 100 kDa of F. The relative loading ratio is approximately 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 F. In some embodiments, the relative loading ratio is (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 F. In some embodiments, the relative loading ratio is D less than 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 be in the range of D from approximately 1 to 200 per 100 kDa of F, in which case the lower limit is smaller than the upper limit. For example, in some embodiments, the relative loading ratio is approximately 5 to 200 per 100kDa F, approximately 10 to 180 per 100kDa F, approximately 10 to 50 per 100kDa F, approximately 10 to 20 per 100kDa F, approximately 20 to 200 per 100kDa F, approximately 20 to 100 per 100kDa F, approximately 20 to 50 per 100kDa F, approximately 30 to 150 per 100kDa F, approximately 40 to 100 per 100kDa F, and 100kDa The D values ​​are approximately 50 to 150 per F, approximately 50 to 100 per F for 100kDa, approximately 80 to 200 per F for 100kDa, approximately 80 to 160 per F for 100kDa, approximately 80 to 120 per F for 100kDa, approximately 100 to 200 per F for 100kDa, approximately 100 to 150 per F for 100kDa, approximately 120 to 200 per F for 100kDa, or approximately 150 to 200 per F for 100kDa. 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 disclosure provides compounds of formula (I) in which the conjugation moiety F is a dendrimer and the TLR7 / 8 agonist moiety D is covalently linked to the terminal functional group of the dendrimer via a cleavable linker.

[0279] In some embodiments, the dendrimer is a dendrimer derived from tris(2-aminoethyl)amine, and this dendrimer is conjugated via a terminal amino group L 3 It is coupled to 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 with respect to formula (I), or any variant thereof as detailed herein.) The compound is provided.

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

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

[0282] In some embodiments, the conjugation moiety F of formula (I) is bonded to the TLR7 / 8 agonist D via an ether linkage derived from a hydroxyl group, as in formula (F-2): [ka] (In the formula, n is between 100 and 1000, L 3 , L 2 , L 1 (and D is as defined with respect to formula (I), or any variant thereof as detailed herein, and x is 4 to 32) This is a poly(ethylene glycol)-based bis-MPA dendrimer. 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 portion F is a polyamidoamine (PAMAM)-based dendrimer, for example, scheme F-3: [ka] (In the formula, L 3 , L 2 , L 1D is as defined with respect to formula (I) or any variant thereof as detailed herein, and x is the core structure shown in (16, or larger if the core structure is further extended, up to 4000), and this dendrimer may be bonded to the TLR7 / 8 agonist moiety D via ether linkage derived, for example, from a surface hydroxyl group in the PAMAM molecule. Compounds of formula (I), in which the conjugation moiety F is a dendrimer, can be prepared using methods known in the art and methods described herein. For example, the surface hydroxyl group on the dendrimer described herein can be converted to a propargylamine-carboxymethyl (PACM) group for reaction with an azide-linked IMDQ compound using click chemistry similar to the method described herein for linking IMDQ to a surface hydroxyl group of Ficoll® (e.g., Kolb, HC See et al. 2001, Angew Chem Int Ed Engl 40:2004-2021; Kolb, HC and Sharpless, KB 2003, Drug Discov Today 8:1128-1137). Polypeptide

[0284] In some embodiments, the disclosure provides a compound of formula (I) in which the conjugation moiety F is, for example, a polypeptide comprising at least nine amino acid residues. In some embodiments, the polypeptide conjugation moiety of F is a polypeptide antigen. In some embodiments, the polypeptide of F is a cancer antigen. In some embodiments, the polypeptide of F is a viral antigen, a bacterial antigen, or an allergen antigen. In some embodiments, the polypeptide of F contains polyalanine. In some embodiments, the polypeptide of F contains polyglutamic acid. In some embodiments, the polypeptide of F is not an antigen.

[0285] TLR7 / 8 agonist D conjugated to an antigen can enhance the immunological response to the antigen (e.g., cancer antigen). In the case of antigen conjugation, typically 1 to 30 TLR7 / 8 agonist moieties D can be conjugated to each of the polypeptide molecules F (as detailed herein for applicable -WL). 3 -L 2 -L 1 - Via any of the linkers (i.e., x is an integer from 1 to 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 or a fragment thereof of at least one full-length protein. Preferred tumor antigens are described in the art (e.g., Cheever et al.). , 2009, Clinical Cancer Research, 15:5323-5337; and Caballero and Chen, See Cancer Science, 100:2014-2021 (2009). 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, survivorbin, 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, and MAGE This includes 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, Regmine, 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 and 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 derived 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 the tumor. In some embodiments, the mammalian antigen is either a neoantigen or encoded by a gene containing a mutation in a gene present in normal cells derived from a mammalian subject. Neoantigens are thought to be particularly useful in enabling T cells to distinguish between cancer cells 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 See and Wang, 2017, Cell Research 27:11-37.

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

[0288] In some embodiments, the conjugation portion 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 methods described herein. The polypeptide or VLP can be conjugated to the TLR7 / 8 agonist-containing moiety of the compound of formula (I) by using a hydroxyl group, thiol group, amino group, or carboxyl group present in the amino acid residues of the polypeptide or VLP (-L 3 -L 2 -L 1 -D). For example, a hydroxyl group can be converted to an ether linkage using the method described herein for conjugating Ficoll® via a hydroxyl group. A thiol group can be converted to a thioether linkage or succinimide spacer by reaction with a maleimide compound.

[0290] It is intended and understood that any variant of F detailed herein with respect to the compound of formula (I) can be combined with any variant of D detailed herein with respect to the compound of formula (I), as if every possible combination were described individually. For example, in some embodiments, formula (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is the TLR7 / 8 agonist part, L 1 is a binding or self-deactivating linker, L 2 It is a linker that can be cut, L 3 It is a conjugation linker, W is O, S or NR 10 And, R 10 is H or C1-C8 alkyl, x is an integer between 3 and 300. F is a branched copolymer of sucrose and epichlorohydrin with a molecular weight of approximately 50,000 to 700,000 daltons (e.g., GE Healthcare's FICOLL® PM400). D is a 1H-imidazo[4,5-c]quinoline derivative. The compound is provided.

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

[0292] In some embodiments, the compound of formula (I) is a cleavable linker L 2 Self-detaching linker L 1 Conjugation Linker L 3 , and via the connecting part W (-WL 3 -L 2 -L 1 -As) a polymer prodrug containing a TLR7 / 8 agonist portion D linked to a conjugation portion F, L 1 It is a self-deactivating linker, L 2 It is a linker that can be cut, L 3 is a conjugation linker, and W is O, S or NR 10 And R 10 L is H or C1-C8 alkyl. 1 In some embodiments where the bond is a linker L, the compound of formula (I) is a cleavable linker L 2 Conjugation Linker L 3 , and via the connecting part W (-WL 3 -L 2 -As) a polymer prodrug containing a TLR7 / 8 agonist portion D linked to a conjugation portion F, L 2 It is a linker that can be cut, L 3 W is a conjugation linker, and W is O, S or NR 10 And R 10 It is either H or a C1-C8 alkyl group. antibody-based conjugation portion

[0293] In some embodiments, the conjugation moiety F in formula (I) is a tumor-targeting antibody conjugation moiety that causes the preferential accumulation of the compound of formula (I) in the tumor microenvironment by the antibody's ability to preferentially bind to tumor cell surface antigens, specific structural elements of the extracellular matrix of the tumor microenvironment, or specific structural elements of tumor blood vessels. This allows for the cleavable linker L 2 During the subsequent hydrolysis of, and L 1 However, in the case of a self-deactivating linker, self-deactivating linker L 1 During self-desorption, local release of the TLR7 / 8 agonist portion becomes possible. In some embodiments, L 1 This is a bond, and local release of the TLR7 / 8 agonist portion is a cleavable linker L 2 This occurs during subsequent hydrolysis.

[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, for example, Beck, A. et al. 2017, Nature Rev Drug Discovery 16:315-337; Sau, S. et al. 2017, Drug Discovery Today 22:1547-1556). Conjugation to tumor-targeting antibodies can affect the tumor microenvironment. By preferentially accumulating ADCs, the therapeutic range of these highly potent cytotoxic agents is improved, and then, in this tumor microenvironment, the original (i.e., non-conjugated) cytotoxic agent is released by preferential cleavage of cleavable linkers. Currently, more than 60 ADC therapies are either approved by the U.S. Food and Drug Administration (FDA) or are currently in clinical trials. The key properties that contribute to the antitumor efficacy of ADCs are These are tumor-targeting antibodies, and research in this field has shown that a wide range of antibody targets are currently under investigation (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; See also Wagh, A. et al. 2018, MABS 10:222-243.

[0295] Another important property of ADCs is the range and homogeneity of the loading amount of cleavable linker / cytotoxic agent onto the antibody, commonly referred to as the antibody-to-drug ratio. Studies have shown that ADCs with a controlled, homogeneous stoichiometry (generally, the antibody-to-drug ratio is between 2 and 6-8 for highly hydrophobic cytotoxic agents) exhibit superior plasma half-lives, which are interpreted as 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 degree and homogeneity of the loading amount of cleavable linker / cytotoxic agent onto the antibody is achieved through the specific conjugate chemistry used, including the selection of synthetic linkers and reactive moieties for the antibody. The initial ADC constructs included a conjugate of a cleavable linker-cytotoxic agent into the primary sequence of the antibody, targeting native lysine and cysteine ​​amino acids (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, the cleavable linker-cytotoxic agent conjugate... More recent developments in injury agent conjugate chemistry, along with the more stable chemical properties of linkers in plasma, allow for more precise control of the antibody-to-drug ratio by creating a more precisely defined number of reaction sites in the antibody (e.g., Agarwal, P. et al. 2013). See 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 portion F in formula (I) is an antibody or antibody derivative. In some embodiments, F is an antibody having a mouse sequence, a "humanized" mouse sequence, or a fully human sequence. In some embodiments, F is an antibody of the IgG1, 2, or 4 class, including but not limited to bivalent monospecific antibodies, bivalent bispecific antibodies, etc., or derivatives or fragments thereof, or derivatives without an Fc region containing a single-chain variable fragment (scFv), and derivatives such as tandem bivalent scFv, diabody, tandem trivalent scFv, tribody, bispecific tandem bivalent scFv, etc. (see, for example, Weidle, UH et al. 2014, Seminars in Oncology 41:653-660). In some embodiments, the IgG scaffold is manipulated to modulate the effector function of the molecule (see, for example, Warnkke, M. et al. 2012, J Immunol 188:4405-4411; Jacobsen, FW et al. 2017, J Biol Chem 292:1865-1875). .

[0297] The descriptions of ADCs provided herein are not intended to limit the scope of the invention, as those skilled in the art will recognize that other ADCs may be functionally equivalent.

[0298] In some embodiments, the antibody, or its derivative or fragment, specifically binds to tumor microenvironment-specific antigens. In some embodiments, the antibody, or its derivative or fragment, binds to tumor cell surface markers. In some embodiments, the antibody, or its derivative or fragment, binds to HER2, transmembrane glycoprotein NMB (gpNMB), EGFR, EGFRvIII, glutamate carboxypeptidase II (GCPII / PSMA), CD11b, CD16A, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD71 (transferrin), CD74, CD79b, CD103, CD117 (KIT), CD123, CD138, CD142, 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, It 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, ROR-1, etc. In some embodiments, the antibody is a bispecific antibody, or a derivative thereof, or a fragment thereof, which 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 a conjugation linker L well known to those skilled in the art, which includes N-succinimidyl-4-(2-pyridyldthio) butanoate (SPDB), N-succinimidyl-4-(2-pyridyldthio)-2-butanoate (sulfo-SPDB), maleimidomethylcyclohexane-I-carboxylate (MCC), 4-(4-acetylphenoxy)butanoic acid (AcBut), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldthio)pentanoate (SPP), and their derivatives. 3 Using a cleavable linker / self-leaving linker / TLR7 / 8 agonist moiety (-L) via a native lysine residue. 2 -L 1 -D) is conjugated with L 1 is a conjugation, and the tumor-targeting antibody F in formula (I) is a conjugation linker L well known to those skilled in the art, which includes 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-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), and their derivatives. 3 Using the natural lysine residue, the cleavable linker / TLR7 / 8 agonist moiety (-L 2 It is conjugated with -D).

[0300] In some embodiments, the tumor-targeting antibody F in formula (I) is replaced with a conjugation linker L well known to those skilled in the art, which includes maleimidocaproyl (MC), maleimidomethylcyclohexane-I-carboxylate (MCC), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), and derivatives thereof. 3 Using a cleavable linker / self-leaving linker / TLR7 / 8 agonist moiety (-L) via a native or manipulated cysteine ​​residue. 2 -L 1 It is conjugated with -D-). 1 is a conjugation, and the tumor-targeting antibody F in formula (I) is a conjugation linker L that is well known to those skilled in the art, including maleimidocaproyl (MC), maleimidomethylcyclohexane-I-carboxylate (MCC), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), and its derivatives. 3 Using a cleavable linker / TLR7 / 8 agonist moiety (-L) via a natural or manipulated cysteine ​​residue. 2 It is conjugated with -D-). Dithiothreitol (D Methods for mildly reducing antibodies with TT or tris(2-carboxyethyl)phosphine (TCEP) are well known to those skilled in the art. Partial reduction of the antibody's disulfide bonds allows for conjugation to specific cysteine ​​residues without disrupting the protein's secondary and tertiary structures, and without causing aggregation (see, for example, Sun, MC et al. 2005, Bioconjug Chem 16:1282-1290; Doronina, SO et al. 2003, Nature Biotechnology 21:778-784).

[0301] In some embodiments, the tumor-targeting antibody F in formula (I) is modified by using protein engineering techniques that allow control over the number of positions on the antibody available for the conjugate, thereby enabling the cleavable linker / auto-detachable linker / TLR7 / 8 agonist moiety (-L 2 -L 1 -D) is conjugated. In some embodiments, L 1 The linker is a conjugate, and the tumor-targeting antibody F in formula (I) has a cleavable linker / TLR7 / 8 agonist moiety (-L) by using protein engineering techniques that allow control over the number of positions on the antibody available for the conjugate. 2 -D) is conjugated. In some embodiments, antibody F in formula (I) is conjugated to conjugation linker L 3 It is engineered to control the number of cysteine ​​residues available for conjugation with maleimide-based reagents (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, (e.g., PlosOne 12:e0178452). In some embodiments, antibody F in formula (I) is an orthogonal conjugation linker (L in formula (I)). 3 It has one or more non-natural amino acids that have been manipulated into a primary sequence to enable chemical conjugation using ). For example, Tian. See F. et al. 2014, Proc Nat Acad Sci USA 111:1766-1771; Kato, A. et al. 2017, Bioconjug Chem 28:2099-2108, etc. In some embodiments, see F. et al. 2014, Proc Nat Acad Sci USA 111:1766-1771; Kato, A. et al. 2017, Bioconjug Chem 28:2099-2108, etc. In formula (I), antibody F is a conjugation linker (L in formula (I)). 3It has a non-natural amino acid sequence that has been manipulated into the primary sequence to enable enzyme-assisted ligation. For example, 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 Bioconjug Chem 28:1906-1915, etc. In some embodiments, formula (I) The antibody F inside is the conjugation linker (L in formula (I)). 3 N-glycan residues are subjected to metabolic engineering, chemical oxidation, or sugar engineering to enable ligation to modified glycan residues. See, for example, 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 portion D in formula (I) (x in formula (I)) is an integer, indicating that the geometric mean of the population distribution mean 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 mean 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. Method for preparing cleavable conjugates of TLR7 / 8 agonist compounds

[0303] This disclosure further provides methods for preparing cleavable conjugates of the TLR7 / 8 agonist compounds detailed herein (i.e., compounds of formula (I) or any variant thereof as detailed herein), as well as compositions and useful intermediates therefor. The compounds of this disclosure can be prepared using the methods detailed 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 embodiment, the present disclosure relates to a particle-based (i.e., nanoparticle or microparticle) conjugation moiety F of formula (I): F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is the TLR7 / 8 agonist part, L 1 is a binding or self-deactivating linker, L 2 It is a linker that can be cut, L 3 It is a conjugation linker, W is O, S or NR 10 And, R 10 is H or C1-C8 alkyl, x is an integer between 1 and 500. F is the particle-based conjugation portion. A method for preparing a cleavable conjugate of a TLR7 / 8 agonist compound, wherein the compound of formula (A): F-[WL 3a -Y 3a ] y (A) Compound of formula (B): Y 3b -L3b -L 2 -L 1 -D (B) (In the formula, y is an integer between 1 and 500, and F, W, L 2 , L 1 And D is as defined for the compound of formula (I), and L 3a and L 3b These are independent spacer fragments as needed, Y 3a and Y 3b They react with each other, spacer fragment Y 3 This is the precursor portion that forms L 3a , Y 3 and L 3b Together, Linker L 3 (to form) and react with Steps to form the compound of formula (I) This provides a method that includes [something].

[0305] In some embodiments, the TLR7 / 8 agonist moiety D is IMDQ or meta-IMDQ, or a compound selected from any one of compound numbers 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 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), where 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 prepared using "click" chemistry, in which the reaction of an alkyne with an azide forms a [1,2,3]triazole moiety. In some embodiments, Y 3a This is an alkyne group (-C≡CH), and Y 3b This is an azide group (-N3), and Y 3 This is the 1,4-[1,2,3]triazolylene moiety.

[0307] In some embodiments, L 3a is an amide spacer fragment (e.g., -CH2C(O)NHCH2-), L 3b This is an acyl spacer fragment (e.g., -CH2C(O)-) or a PEG-acyl spacer fragment (e.g., -CH2CH2(OCH2CH2)). 12 It is C(O)-).

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

[0309] In some embodiments, L 3b The formula is: [ka] acyl spacer fragment or formula: [ka] (In the formula, n is between 0 and 200) This is a PEG-acyl spacer fragment. In some embodiments, L 3b The formula is: [ka] The acyl spacer fragment, or formula: [ka] (In the formula, n is between 0 and 200) This is a PEG-acyl spacer fragment.

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

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

[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]quinoline-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 numbers 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 It is a combination.

[0315] In another embodiment, the present disclosure relates to formula (I) including an antibody-based conjugation moiety F: F-[WL 3 -L 2 -L 1 -D] x (I) (In the formula, D is the TLR7 / 8 agonist part, L 1 is a binding or self-deactivating linker, L 2 It is a linker that can be cut, L 3 It is a conjugation linker, W is O, S or NR 10 And, R 10is H or C1-C8 alkyl, x is an integer between 1 and 50. F is the antibody-based conjugation portion. A method for preparing a cleavable conjugate of a TLR7 / 8 agonist compound, wherein the compound of formula (C): F-[W'] x (C) Compound of formula (D): L 3 -L 2 -L 1 -D (D) (In the formula, x is an integer between 1 and 50, and F, L 3 , L 2 , L 1 And D is the chemical of equation (I) As defined for the compound, W' reacts with N, O, S, N3 or alkyne. Steps to form the compound of formula (I) This provides a method that includes [something].

[0316] In some embodiments, the compound of formula (C) has an antibody-based conjugation moiety (F-[W'] x The compound of formula (C) is produced by limited reduction of a natural cysteine ​​residue in (F-[W']), where W' is S and F is a recombinant antibody that targets the compound in the tumor microenvironment and is modified as necessary to add and / or delete cysteine ​​residues. In some embodiments, the compound of formula (C) is an antibody-based conjugation moiety (F-[W'] x ) where W' is N, O, S, N3 or alkyne, and F is a recombinant antibody that targets the compound of formula (I) to the tumor microenvironment, and is a recombinant antibody manipulated with a novel amino acid sequence that is non-natural or post-translational chemoenzyme modified. In some embodiments, the compound of formula (C) is a conjugation moiety (F-[W']) based on the manipulated antibody. x) where 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 between 1 and 50, 1 and 25, 1 and 20, 1 and 15, 1 and 10, 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, or 1 and 2.

[0317] In some embodiments, the compound of formula (D) is (i)L 1 L 2 (ii) To join by covalent bond, (ii) D to -L 2 -L 1 (iii)L 3 ni-L 2 -L 1 -D is covalently bonded, and L is an example of a compound of formula (D). 3 -L 2 -L 1 -It is produced by synthesizing D. In some embodiments, L 1 This is a bond, and the compound of formula (D) is (i)D with L 2 (ii)L 3 ni-L 2 -D is covalently bonded, and L is an example of a compound of formula (D). 3 -L 2 -It is produced by synthesizing D. In some embodiments, linker L 3 These include maleimidocaproyl conjugation linkers, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linkers, N-succinimidyl-4-(2-pyridyldithio)butanoate linkers, N-succinimidyl-4-(2-pyridyldithio)pentanoate linkers, hydrazone linkers, etc. In some embodiments, cleavable peptide linkers L 2The amino acid sequence is AA1-AA2-AA3-AA4, where AA1 is absent or alanine, β-alanine, isoleucine, leucine, valine, or glycine; AA2 is absent or 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, linker L 1 These include p-aminobenzylcarbamate auto-leaving linkers. In some embodiments, the TLR7 / 8 agonist moiety D is IMDQ, meta-IMDQ, or one of compound numbers 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. IV. Use of cleavable conjugates of TLR7 / 8 agonist compounds A. Pharmaceutical Compositions

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

[0319] The pharmaceutically acceptable excipients of this disclosure include, for example, solvents, fillers, emulsifiers / surfactants. This includes buffering agents, isotonic agents, and preservatives (see, for example, Pramanick et al. Pharma Times, 45:65-77, 2013). In some embodiments, the pharmaceutical composition is dissolved The pharmaceutical compositions of this disclosure include excipients that function as one or more of a medium, filler, buffer, and isotonic agent (for example, sodium chloride in physiological saline can act as both an aqueous vehicle and an isotonic agent). The pharmaceutical compositions of this disclosure are suitable for parenteral administration. In some embodiments, the pharmaceutical compositions of this disclosure are not intended for intravenous administration.

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

[0321] The pharmaceutical composition may contain fillers. Fillers are particularly useful when the pharmaceutical composition is freeze-dried before administration. In some embodiments, the filler is a lyoprotectant that helps stabilize the activator and prevent its degradation during freeze-drying and / or storage. Suitable fillers are sugars (monosaccharides, disaccharides, and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose, and raffinose.

[0322] The pharmaceutical composition may contain a buffering agent. The buffering agent controls the pH to inhibit the degradation of the activator during processing, storage, and, if necessary, reconstitution. Suitable buffers include, for example, salts containing acetates, citrates, phosphates, or sulfates. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 4 to 9. In some embodiments, the pH is higher than (lower limit) 4, 5, 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, 7, 6, or 5. That is, the pH is in the range of about 4 to 9, in which case the lower limit is less than the upper limit.

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

[0324] This 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] This pharmaceutical composition may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. In some embodiments, this pharmaceutical composition is prepared under sterile conditions and is contained in single-use containers, and therefore does not require the inclusion of preservatives.

[0326] The pharmaceutical compositions of this disclosure are suitable for a number of uses, including stimulating an immune response, in mammalian subjects that require stimulation of an immune response. 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 the subject in an amount effective to achieve a specific outcome. B. Dosage and Mode of Administration

[0327] As with all pharmaceutical compositions, the effective dose and mode of administration may be modified based on several factors that are apparent 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 the 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 other therapeutic agents for the treatment of an antigen or cancer. Other factors to consider include the disease modification outcome to be achieved and the number / frequency of doses administered during the treatment regimen.

[0328] The preferred dosage range is one that achieves the desired clinical effect. The dosage may be determined by the amount of the TLR7 / 8 agonist compound (i.e., the compound of formula (I)) in the pharmaceutical composition that needs to be administered to the subject to produce the desired therapeutic response with minimal adverse events. Exemplary dosage ranges of the TLR7 / 8 agonist compound administered in amounts delivered per subject's body weight are approximately 0.0001–75 mg / kg, approximately 0.0001–50 mg / kg, approximately 0.0001–25 mg / kg, approximately 0.0001–10 mg / kg, approximately 0.0001–8 mg / kg, approximately 0.0001–6 mg / kg, and approximately 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 / The doses range from approximately 0.0001 to 100 mg / kg, such as approximately 0.1-8 mg / kg, approximately 0.2-7 mg / kg, approximately 0.3-6 mg / kg, approximately 0.4-5 mg / kg, approximately 0.5-4 mg / kg, approximately 0.6-3 mg / kg, approximately 0.7-2.5 mg / kg, approximately 0.8-2.2 mg / kg, approximately 0.9-2.1 mg / kg, or approximately 1-2 mg / kg. In some embodiments, the dose is greater than (lower limit) approximately 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 dose is less than (upper limit) approximately 100, 75, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, or 0.001 mg / kg. That is, the dose is in the range of approximately 0.0001 to 100 mg / kg, in which case the lower limit is less than the upper limit.The exemplary dose range for TLR7 / 8 agonists administered in amounts delivered to the target population is approximately 0.0001 to 100 mg / kg.

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

[0330] Similarly, a preferred route of administration is one that produces the desired effect. Generally, the pharmaceutical compositions of this disclosure are intended for parenteral administration (e.g., neither oral nor rectal administration). Preferred routes of administration include injection, topical, and inhalation. In particular, the pharmaceutical compositions of this disclosure may be administered by routes such as intratumor, intramuscular, subcutaneous, transdermal, and inhalation. Suitable devices for administration by inhalation include, for example, nebulizers, vaporizers, and dry powders. A device for end-inhalation delivery is included. In some embodiments, if the pharmaceutical composition is intended to treat a solid tumor, the composition is administered intratumorally and / or peritumorally (e.g., within and around the tumor lesion). In some embodiments, the pharmaceutical composition is administered intravenously.

[0331] A preferred administration regimen for the TLR7 / 8 agonist compound formulated in this pharmaceutical composition is one that delivers the desired effect in a prophylactic or therapeutic situation with minimal adverse events. The number of doses administered via the selected route may be one or more. The frequency of administration may be weekly, every two weeks, monthly, every two months, or with intervals between doses ranging from 3 to 12 months. An exemplary dose frequency for the TLR7 / 8 agonist compound is approximately once per week to once every 8 weeks. In some embodiments, the dose frequency is less than (upper limit) approximately once every 8 weeks, 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week. In some embodiments, the dose frequency is more than (lower limit) approximately once every 7 days, 10 days, or 14 days. In some embodiments, the dose frequency is once every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In some embodiments, the dose frequency is once every 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the dose frequency is once every two, three, or four weeks. The exemplary dose frequency range for the TLR7 / 8 agonist compound delivered to the subject is approximately once every week to once every four weeks. In some embodiments, two doses are administered, with the second dose given 1 to 2 months after the first dose. In some embodiments, three doses are administered, with the second dose given 1 to 2 months after the first dose and the third dose given 1 to 5 months after the second dose. In other embodiments, a series of doses may be administered over a treatment schedule of 3 to 12 months, in which case the dose frequency is once every week, once every two weeks, once every three weeks, or once a month. In other embodiments, shorter or longer periods may elapse between doses. In certain embodiments, the interval between consecutive doses may vary in terms of weeks or months. In one embodiment, a series of doses every two, three, four, five, or six weeks may be administered, followed at a later point in time by a second series of weekly doses. Those skilled in the art may adjust the dosage regimen by measuring biological outcomes such as antigen-specific antibody response, antigen-specific CD8+ T cell response, or tumor regression.

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

[0333] In one embodiment, the present disclosure provides a method for stimulating an immune response in a mammalian subject requiring stimulation of an immune response, the method comprising the step of 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 de novo induction of an immune response or enhancement of an existing immune response. In some embodiments, stimulating an immune response involves 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 cells (pDCs) or myeloid dendritic cells (mDCs) maturation and / or antigen presentation, and / or tumor antigen-specific CD4+ and / or CD8+ T cells. This includes one of the group comprising inducing the production of antigen-specific T cells. "Inducing" an antigen-specific T cell response means enhancing the number and functional properties of the lymphocytes, such as stimulating helper T lymphocytes and / or cytotoxic T lymphocytes to enable T cells to assist in an antibody response, or to generate cytotoxic T cells having antitumor activity. In embodiments in which the pharmaceutical composition is combined with an antigen and further administered, stimulating an immune response includes inducing an antigen-specific antibody response. "Inducing" an antigen-specific antibody response means increasing the titer of an antigen-specific antibody beyond a threshold level, such as a baseline titer or serum protective level before administration.

[0334] Analysis of the immune response (both qualitative and quantitative) can be performed by any method known in the art, but is 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 (ELISA). 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 the stimulation of the immune response are described in the biological examples of this disclosure. D. Cancer treatment

[0335] This disclosure provides a plurality of methods for treating cancer in mammalian subjects requiring treatment of cancer, the methods comprising the step of administering a pharmaceutical composition to a mammalian subject in an amount sufficient to treat cancer in the mammalian subject. In some embodiments, this disclosure provides a method for treating cancer in mammalian subjects requiring treatment of cancer, the methods comprising the step of administering an effective amount of the pharmaceutical composition by intratumor and / or peritumor delivery. In some embodiments, intratumor delivery comprises injecting the pharmaceutical composition into at least one tumor lesion. In some embodiments, treating cancer in a mammalian subject requiring treatment of cancer comprises inducing an accumulation of tumor antigen-specific T cells in the injected tumor in a larger number than, for example, the pharmaceutical composition being administered to an peritumor site. In some embodiments, treating cancer in a mammalian subject requiring treatment of cancer comprises eliciting a systemic tumor antigen-specific T cell response, including, for example, a systemic tumor antigen-specific T cell response of a larger magnitude than that of an immunogenic composition being administered to an peritumor site. In some embodiments, treating cancer in a mammalian subject requiring treatment includes inducing a systemic tumor antigen-specific T cell response. In some embodiments, treating cancer in a mammalian subject requiring treatment includes reducing the number of CD4+FoxP3+ regulatory T cells in the injected tumor. In some embodiments, the subject has one or more non-injected tumors (primary or metastatic lesions) in addition to the injected tumor, and treatment of cancer in the subject includes one or more of the following: (a) reduction of the number of non-injected tumors, (b) reduction of the volume of non-injected tumors, and (c) growth of non-injected tumors. In some embodiments, treatment of cancer in a mammalian subject requiring treatment includes one or more of the following: (d) increase the survival time of the subject, (e) reduction of the volume of injected tumors, and (f) delay of the growth of injected tumors. In some embodiments, if the cancer is a solid tumor, “treating” cancer includes reducing the size of the solid tumor and any metastatic lesions, or, if not, reducing the number of viable cancer cells.In other embodiments, if the cancer is a solid tumor, “treating” the cancer includes delaying the growth of the solid tumor and any metastatic lesions. In some embodiments, treating the cancer includes increasing progression-free survival or increasing the time to progression. In other embodiments, the method further includes the step of administering an effective amount of a second or additional therapeutic agent to the subject. “Treating” cancer means causing a beneficial clinical outcome, such as inducing remission or extending survival compared to the survival expected without other treatment. In some preferred embodiments, “treating cancer” includes assessing the patient’s response to the immunogenic composition in accordance with the described diagnostic criteria for evaluating response in solid tumors (RECIST version 1.1) (see, for example, Eisenhauer et al. 2009, Eur J Cancer 45:228-247). The diagnostic criteria for determining an objective antitumor 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 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 of a site selected from the group consisting of the oral cavity, digestive system, respiratory system, skin, breast, reproductive system, urinary tract, ocular system, nervous system, and endocrine system, and lymphoma.

[0337] In some embodiments, the method further includes the step of administering an effective amount of a second therapeutic agent to a subject. In some of these embodiments, the second therapeutic agent is actinomycin, afatinib, alectinib, asparaginase, azacitidine, azathioprine, bicalutamide, binimetinib, bleomycin, bortezomib, camptothecin, carboplatin, capecitabine, carmustine, cerutinib, cisplatin, chlorambucil, cobimetinib, crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, encorafenib, erlotinib, epirubicin, epothiron, etoposide, fludarabine, flutamine, fluoro The chemotherapeutic agent is selected from the group consisting of racil, gefitinib, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lapatinib, letrozole, mechloretamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, octreotide, oxaliplatin, paclitaxel, pemetrexed, larcitrexed, sorafenib, sunitinib, tamoxifen, temozolomide, teniposide, thioguanine, topotecan, trametinib, barrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the second therapeutic agent includes one or both of a BRAF inhibitor and a MEK inhibitor. In some embodiments, the second therapeutic agent is an HDAC inhibitor (see, for example, voronistat [SAHA], romidepsin, entinostat, avexinostat, erinostat [CHR-3996], panobinostat, cuidinostat [JNJ-26481585], 4SC-202, resminostat [SB939], prasinostat [CI-9940], and valproate), a DNA methyltransferase inhibitor (see, for example, azacitidine, decitabine, zebralin, SGI-1027, RG-108, and synefungin), This includes epigenetic modulators selected from a group consisting of combinations thereof.

[0338] In some of these embodiments, the second therapeutic agent is an antagonist of 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 includes an antibody, a fragment thereof, or a 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 includes an antibody, a fragment thereof, or a derivative thereof.

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

[0340] In some embodiments of this method, cancer treatment does not result in the development of influenza-like symptoms of a severity that would contraindicate repeated administration of the immunogenic composition, and these influenza-like symptoms include one or more of the following: fever, headache, chills, muscle pain, and malaise.

[0341] In some embodiments, the Disclosure provides a kit comprising a pharmaceutical composition (e.g., a compound of formula (I), one or more excipients, 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 appropriately packaged. If the pharmaceutical composition is a liquid, it may be in lyophilized form, or a suspension of nanoparticles, in a silicon dioxide vial with a rubber stopper (e.g., Exxpro halobutyl elastomer) and an aluminum crimped top (e.g., SCHOTT Type I plus® is typically used as the container sealing system. In some embodiments, the kit further includes a device for administering the pharmaceutical composition (e.g., a syringe and needle). In other embodiments, the kit further includes a pre-filled syringe / needle system, an auto-injector, or a needleless device. Instructions relating to the use of the pharmaceutical composition generally include information regarding dosage, schedule, and route of administration for the intended method of use. [Examples]

[0342] V. 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 method; 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: Reverse-phase high-performance liquid chromatography; SEC: Size exclusion chromatography; TEA: Triethylamine; TFA: Trifluoroacetic acid THF: Tetrahydrofuran. A. Synthesis Examples (Example S1) Preparation of compounds 64-51 and 64-51a.

[0343] By replacing dimethylcysteamine hydrochloride with other amino-alkylthiol-containing compounds using the procedures and schemes shown in Example S1 and Scheme S1-1, 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))) are obtained for azido-disulfide-PABC-IMDQ compounds (formula (I) -L 3 -L 2 -L 1-D) can be prepared. This example demonstrates the use of dimethylcysteamine hydrochloride to prepare compound number 64-51, which is an example of a common azido-disulfide-PABC-IMDQ structure. This example also describes the preparation of the monomethyl disulfide derivative compound number 64-51a. Furthermore, the TLR7 / 8 agonist moiety (D in formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 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. Patents No. 8,728,486 and No. 9,441,005. Scheme S1-1 [ka] General procedure for the preparation of compound number 1.

[0344] To a solution of 4-mercaptobenzyl alcohol (700 mg, 5.0 mmol) in H2O / THF (2 mL / 20 mL), 2,2'-dithiodipyridine (1.65 g, 7.5 mmol) was added. The resulting clear yellow solution was heated to 40°C and stirred for 4 hours. This reaction solution was diluted with ethyl acetate (100 mL) and washed with 1N HCl (100 mL), then with water (150 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / hexane, v / v, 1 / 2 to 1 / 1) to obtain compound number 1 (990 mg, 80% yield) as a colorless oil. LC-MS: 250[M+1] + . General procedure for the preparation of compound number 2.

[0345] A solution of compound number 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 number 1. This reaction mixture was diluted with ethyl acetate (100 mL) and washed with saturated NaHCO3 aqueous solution (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 / 0~100 / 10 / 3) to obtain compound number 2 (460 mg, 93% yield) as a pale yellow solid. LC-MS: 244[M+1] + . General procedure for the preparation of compound number 3.

[0346] To a solution of 2-azidoacetic acid (285 mg, 2.82 mmol) in DMF (10 mL), HATU (1.14 g, 3.01 mmol) was added at 0°C. After 10 minutes, solutions of compound number 2 (457 mg, 1.88 mmol) and DIPEA (0.66 mL, 3.76 mmol) in DMF (3 mL) were added to the above solution. The resulting solution was warmed to room temperature and heated for 2 hours. The mixture was stirred. LC / MS showed the disappearance of compound number 2. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (ethyl acetate / hexane, v / v, 1 / 1~1 / 0) to obtain compound 3 (200 mg) as a colorless oil, together with 505 mg of by-product compound 3B. This by-product compound was treated with lithium hydroxide in THF / H2O / MeOH to obtain compound number 3. LC-MS: 325[M-1] - . General procedure for the preparation of compound number 4.

[0347] To a solution of compound number 3 (310 mg, 0.95 mmol) in THF (5 mL), CDI (232 mg, 1.43 mmol) in THF (5 mL) was added dropwise over 5 minutes at 50°C. After 2 hours, 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 / 1~1 / 0) to obtain compound number 4 (290 mg, 72% yield) as a white waxy solid. LC-MS:325[M-1] - . General procedure for the preparation of compound number 64-51.

[0348] Solutions of compound number 4 (183 mg, 0.435 mmol) and compound number 5 (IMDQ; 148 mg, 0.412 mmol) in DMF / THF (5 mL / 2 mL) were stirred at 50°C for 18 hours. LC / MS showed that most of compound number 5 had been consumed. The reaction mixture was cooled and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / MeOH, v / v, 15 / 1~8 / 1) to obtain compounds 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 number 64-51a.

[0349] Compound number 64-51a was prepared using HS(CH(CH3))CH2NH2 hydrochloride (i.e., 2-propantheol,1-amino,hydrochloride) instead of dimethylcysteamine hydrochloride (i.e., 2-propantheol,1-amino,hydrochloride), as shown in scheme S1-1 for compound number 64-51. The chemical structure of compound number 64-51a is shown in scheme S4-1a. (Example S1a) Preparation of compounds 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 shows the use of 4-mercaptobenzyl alcohol to prepare compound numbers 64-70, which are examples of a common azido-thiol-PABC-IMDQ structure. The TLR7 / 8 agonist moiety (D in formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for representations of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 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] Preparation procedure for compound 21.

[0351] To a solution of 2.0 g, 8.9 mmol of 2-(Boc-amino)ethyl bromide in DMF (20 mL), 1.04 g, 7.4 mmol of 4-mercaptobenzyl alcohol and 2.24 g, 16.2 mmol of potassium carbonate were added. The resulting cloudy solution was stirred at 40°C for 18 hours. This reaction solution was diluted with ethyl acetate (100 mL) and washed with 0.1 N HCl (100 mL) and then water (100 mL). The organic layer was dried over Na₂SO₄ 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 obtain 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 target compound, 549 daltons, was confirmed by LC-MS. Preparation procedure for compound 22.

[0352] To a solution of compound 21 (1.72 g, 6.0 mmol) in DCM (100 mL), TFA (8 mL) was added. The resulting pale yellow solution was stirred at room temperature for 2 hours. 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 / 0~100 / 10 / 2 (v / v)DCM / MeOH / trimethylamine to obtain 0.94 g of compound 22 as a pale yellow oil. The purity was 85% according to RP-HPLC at 254 nm, and the mass of the target compound, 183 daltons, was confirmed by LC-MS. Preparation procedure for compound 23.

[0353] To a solution of 2-azidoacetic acid (625 mg, 6.18 mmol) in DMF (5 mL), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (680 mg, 6.67 mmol) was added at room temperature. After 15 minutes, 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 hours. Next, this reaction mixture was 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 obtain 0.5 g of compound 23 as a colorless oil. The purity is 36% according to RP-HPLC at 254 nm, and the mass of the target compound is 249 daltons. This was confirmed by LC-MS. Preparation procedure for compound 24.

[0354] Compound 23 (500 mg, 1.87 mmol) from THF (3 mL) was added dropwise to a solution of CDI (456 mg, 2.81 mmol) in THF (10 mL) at 50°C. After 3 hours, 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 obtain 325 mg of compound 24 as a colorless oil. The purity was 48% by RP-HPLC at 254 nm, and the mass of the target compound, 360 daltons, was confirmed by LC-MS. General procedure for the preparation of compounds 64-70.

[0355] Six drops of TEA were added to a solution of compound 24 (195 mg, 0.54 mmol) and IMDQ (compound 5, 97 mg, 0.27 mmol) in DMF (6 mL). 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 obtain 52 mg of compound numbers 64-70 as a white solid. The purity was 99% by RP-HPLC at 254 nm, and the mass of the target compound, 651.8 daltons, was confirmed by LC-MS. The structure of the target compound was determined 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), This was confirmed by values ​​of 1.74 (m, 2H), 1.39 (m, 2H), and 0.88 (t, 3H). (Example S2) Preparation of compounds 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) are used. 3 , the cleavable peptide sequence L in formula (I) 2 , the self-destructive linker L in equation (I) 1 The various compounds of the present invention that have -L in formula (I) 3 -L 2 -L 1-D can be prepared, in which the TLR7 / 8 agonist compound D in formula (I) can be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as 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] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is 1 The para-aminobenzylcarbamate moiety is 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 the azide-PEG4 portion is present, compound number 64-52 was prepared as shown in scheme S2-1. Scheme S2-1 [ka] General procedure for the preparation of compound number 7.

[0358] Triethylamine (0.13 mL, 0.94 mmol) was added to a solution of compound number 6 (465 mg, 0.61 mmol) and compound number 5 (IMDQ; 190 mg, 0.53 mmol) in DMF (15 mL). The resulting clear yellow solution was stirred at room temperature for 15 hours. LC / MS showed that the solution was almost entirely compound number 7, the desired product, and that all of compound number 5 had been consumed. This reaction mixture was used directly in the next step. LC-MS: 987[M+1] + . General procedure for the preparation of compound number 8.

[0359] To a crude solution of compound number 7 (approximately 0.528 mmol) in DMF (15 mL), piperidine (460 mg, 5.41 mmol) was added, and the solution was stirred at room temperature for 6 hours. LC / MS showed the disappearance of the starting material, compound number 7. This reaction mixture was diluted with 10% MeOH in dichloromethane (200 mL) and washed with water (200 mL x 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 / 10~100 / 15) to obtain compound number 8 (304 mg, 65% yield in total over two steps) as a yellow solid. LC-MS:76 5[M+1] + . General procedure for the preparation of compound number 64-52.

[0360] To a solution of compound number 8 (173 mg, 0.224 mmol) and compound number 9 (181 mg, 0.244 mmol) in DMF (5 mL), DIPEA (0.08 mL, 0.44 mmol) was added. The resulting solution was stirred at room temperature for 16 hours. LC / MS showed that it was almost completely converted to the desired product. This reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / MeOH, v / v, 100 / 10~100 / 15) to obtain compound numbers 64-52 (170 mg, 55% yield) as a white solid. LC-MS: 1390 [M+1] + . 1 1H 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] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is 1 The para-aminobenzylcarbamate moiety is 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 the azide-PEG4 portion is present, compound number 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 equivalent) in DMF (1.5 mL), DIPEA (0.1 mL) was added, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 6 (77 mg, 1 equivalent) was added in two portions, and the resulting clear yellow solution was stirred at 0°C for 2 hours. The solution was then slowly warmed to room temperature, and stirring was continued for a further 12 hours. Thin-layer chromatography analysis using 10% (v / v) MeOH in DCM containing 1% (v / v) TEA showed that all of the IMQD starting material was consumed. The DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the resulting solid yellow residue. The mixture was pulverized to precipitate the resulting pale yellow solid, and the supernatant was decanted. This process was repeated two more times. The off-white solid product was dried under reduced pressure to obtain 84 mg of compound 7. Preparation procedure for compound 8.

[0363] A cold solution of compound 7 (71 mg, 1 equivalent) in DMF (1.5 mL) was mixed with DIPEA (0.1 mL), stirred overnight under an argon atmosphere, then slowly warmed to room temperature and stirred for a further 12 hours. Reverse-phase HPLC analysis of the reaction showed that it had proceeded and completed. The DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate a pale yellow solid. The supernatant was decanted. This process was repeated two more times, and the off-white solid was dried under reduced pressure. Thus, 50 mg of compound 8 was obtained. Preparation procedure for compound number 64-52a.

[0364] Compound 8 (50 mg, 1.0 equivalent) and Compound 25 (20.3 mg, 1.2 equivalents) were dissolved in DMF (1 mL), DIPEA (100 μL) was added, and the solution was stirred at room temperature for 1.5 hours. RP-HPLC analysis of this reaction showed that Compound 12 was completely consumed. The 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 obtain 34 mg of Compound No. 64-52a as a white solid. The product purity was determined to be 95% by RP-HPLC at 254 nm, and the mass of the target compound, 1,035.5 daltons, was confirmed by LC-MS. The structure of the target compound was determined 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 This was confirmed by (t, J = 6, 12 Hz, 2H), 1.72-2.18 (m, 5H), 1.58-1.70 (m, 2H), 1.43-1.55 (m, 2H), and 0.88-1.01 (m, 9H). Compound number 64-52b.

[0365] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is 1The para-aminobenzylcarbamate moiety is 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 the azide portion is present, compound number 64-52b was prepared as shown in scheme S2b-1. Scheme S2b-1 [ka] Preparation procedure for compound number 64-52b.

[0366] Compound 8 was prepared as described in scheme S2a-1. A solution of compound 8 (135 mg, 0.176 mmol, 1.0 equivalent) in DMF (1.0 mL) was added to a DMF solution containing compound 26 (1.7 mL, 0.265 mmol, 1.5 equivalents). The resulting solution was stirred at room temperature for 2 hours, during which time LC-MS analysis showed that compound 8 had been consumed. Next, this reaction product was mixed with MeOH (3 mL) and then water (3 mL) The solution was diluted and 1N NaOH (0.5 mL) was added. The resulting solution was stirred at room temperature for 24 hours, and then purified directly by preparative RP-HPLC to obtain 62 mg of compound number 64-52b as a white solid. The product purity was determined to be 96% by RP-HPLC at 254 nm, and the mass of the target compound, 847.4 daltons, was confirmed by LC-MS. The structure of the target compound was determined at 300 MHz. 1 ¹H NMR (acetone-d6): δ 8.49 (s, This was confirmed by the following values: 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), and 3.01 (t, J=7.8 Hz, 2H). Compound number 64-52c.

[0367] The TLR7 / 8 agonist D in equation (I) is IMDQ, and L in equation (I) 1 The link is a bond, and the cleavable linker L in equation (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 Azido-PEG 12 To illustrate a partial embodiment of the present invention, compound number 64-52c was prepared as shown in scheme S2c-1. Scheme S2c-1 [ka] Preparation procedure for compound 28.

[0368] To a solution of compound 27 (100 mg, 0.401 mmol, 1.0 equivalent) and IMDQ (compound 5; 96 mg, 0.40 mmol, 1.0 equivalent) in DMF (3 mL), HATU (154 mg, 0.602 mmol, 1.5 equivalents) was added, followed by DIPEA (89 μL, 0.80 mmol, 2.0 equivalents). The reaction mixture was stirred at room temperature for 15 minutes and then purified by preparative RP-HPLC (95:5% (v / v) H2O / acetonitrile + 0.1% (v / v) acetic acid, gradient from 95 / 5 to 0 / 100 within 30 minutes). The fraction containing the major peak with absorbance at the expected 322 nm was collected and dried under reduced pressure to obtain 141 mg of compound 28 as a white solid. Preparation procedure for compound 29.

[0369] A solution of compound 28 (141 mg, 0.197 mmol, 1.0 equivalent) in DCM (4 mL) was treated with TFA (1 mL) at room temperature for 1 hour. The reaction product was concentrated to dryness under reduced pressure, and the residue containing compound 29 was used without further purification. Preparation procedure for compound number 64-52c.

[0370] A solution of compound 29 (73 mg, 0.10 mmol, 1.0 equivalent) and compound 13 (74 mg, 0.10 mmol, 1.0 equivalent) in DMF (2 mL) was mixed with DIPEA (0.1 mL), and the reaction mixture was stirred at room temperature for 3 hours. The mixture was purified by preparative RP-HPLC (95:5% (v / v) H2O / acetonitrile + 0.1% (v / v) acetic acid, gradient from 95 / 5 to 0 / 100 within 30 minutes). The fraction containing the major peak with the expected absorbance at 322 nm was collected and dried under reduced pressure to obtain 79 mg of compound 64-52c as a white solid. The product purity was determined to be 97% by RP-HPLC at 254 nm, and the mass of the target compound, 1,241.5 daltons, was confirmed by LC-MS. The structure of the target compound was determined 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 This was confirmed by the following measurements: (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), and 0.98-0.86 (m, 9H). Compound number 64-52d.

[0371] The TLR7 / 8 agonist D in equation (I) is IMDQ, and L in equation (I) 1 The link is a bond, and the cleavable linker L in equation (I) 2 is valine-citrulline, and the conjugation linker L in formula (I) 3 To illustrate an embodiment of the present invention in which the azide-PEG4 portion is present, compound number 64-52d was prepared as shown in scheme S2d-1. Scheme S2d-1 [ka] Preparation procedure for compound number 64-52d.

[0372] Compound 29 (1.0 g, 1.0 equivalent) was added to a solution of Compound 25 (1.1 equivalents) in DMF (10 mL), and the reaction mixture was stirred at room temperature. The progress of the reaction was monitored by LC-MS, and after 1 hour, Compound 29 was completely consumed, so the solvent was removed under reduced pressure. The crude product was purified using flash chromatography with Biotage Selekt, eluting with MeOH (0-25%) in DCM, to obtain 600 mg of compound 64-52d as a white solid. The product purity was determined to be 95% by RP-HPLC at 254 nm, and the mass of the target compound, 889.1 daltons, was confirmed by LC-MS. The structure of the target compound was determined 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 This was confirmed by the following values: (m, 2H), 1.74-1.65; (m, 2H), 1.42-1.29; (m, 5H), 0.84 (t, J = 7.5 Hz, 3H); and 0.71 (d, J = 6.8 Hz, 6H). Compound number 64-52e.

[0373] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is 1 The para-aminobenzylcarbamate moiety is the cleavable linker L in formula (I). 2 L is phenylalanine-citrulline, and the conjugation linker L in formula (I) 3 Azido-PEG 12 To illustrate a partial embodiment of the present invention, compound number 64-52e was prepared as shown in scheme S2e-1. Scheme S2e-1 [ka] Preparation procedure for compound 30.

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

[0375] To the reaction mixture prepared above (approximately 0.28 mmol in DMF), piperidine (0.24 g, 2.8 mmol, 10.0 equivalents) was added. The resulting mixture was stirred at room temperature for 6 hours, then poured into diethyl ether (75 mL) and stirred. The yellow precipitate was filtered and washed with ether. The crude product was purified by flash chromatography using Biotage Selekt, eluting with 20% MeOH in DCM containing 2% (v / v) TEA, to obtain 140 mg of compound 31 as a yellow solid. Preparation procedure for compound number 64-52e.

[0376] Compound 31 (73.4 mg, 0.09 mmol, 1.2 equivalents) in DMF (3.0 mL) Compound 13 (73.6 mg, 0.10 mmol, 1.1 equivalents) and DIPEA (23.3 mg, 0.18 mmol, 2.0 equivalents) in DMF (0.3 mL) were added to a solution of ). The mixture was stirred under nitrogen at room temperature for 18 hours, at which point LC-MS analysis showed that all of compound 32 had been consumed. This reaction mixture was purified by preparative RP-HPLC to obtain 54 mg of compound number 64-52e as a white solid. The product purity was determined to be 98% by RP-HPLC at 254 nm, and the mass of the target compound, 1,438.7 daltons, was confirmed by LC-MS. The structure of the target compound was determined 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, This was confirmed by the following measurements: 2H), 1.75-1.65 (m, 4H), 1.50-1.30 (m, 4H), and 0.84 (t, 3H). Compound number 64-52f.

[0377] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is 1 The para-aminobenzylcarbamate moiety is the cleavable linker L in formula (I). 2 The conjugation linker L in formula (I) is glycine-isoleucine-valine-arginine. 3 To illustrate an embodiment of the present invention in which the azide-PEG4 portion is present, compound number 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, add DIPEA (0.1 mL) and stir the solution under an argon atmosphere for 10 minutes. Add compound 33 (1 equivalent) in two portions and stir the resulting clear yellow solution at 0°C for 2 hours. Slowly warm the solution to room temperature and continue stirring for a further 12 hours, or until thin-layer chromatography analysis using 10% (v / v) MeOH in DCM containing 1% (v / v) TEA indicates that all of the IMDQ starting material has been consumed. Remove the DMF under reduced pressure, add ethyl acetate to the solid yellow residue, and grind the mixture to precipitate the resulting pale yellow solid. Decant the supernatant. Repeat this process two more times. Dry the off-white solid product under reduced pressure to obtain compound 34. General procedure for the preparation of compound 35.

[0379] A solution of compound 34 (1.0 equivalent) in DCM was treated with TFA (1.0 mL) at room temperature for 1 hour. The reaction product 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 number 64-52f.

[0380] Compound 35 (1.0 equivalent) and compound 25 (1.2 equivalents) are dissolved in DMF, DIPEA (0.1 mL) is added, and the solution is stirred at room temperature for 1.5 hours. RP-HPLC analysis of this reaction indicates that compound 35 has been completely consumed. The DMF is then removed under reduced pressure, and the yellow solid residue is subjected to silica gel column chromatography, eluting it with 1-8% (v / v) MeOH in DCM containing 0.5% (v / v) ammonia, to obtain compound numbers 64-52f as a white solid. Product purity is determined by RP-HPLC at 254 nm, the mass of the target compound is confirmed by LC-MS, and the structure of the target compound is determined. 1 Confirmation is performed by 1H NMR. Compound number 64-52g.

[0381] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L in equation (I) is1 The para-aminobenzylcarbamate moiety is the cleavable linker L in formula (I). 2 The conjugation linker L in formula (I) is leucine-serine-glycine-arginine-serine-aspartic acid. 3 To illustrate an embodiment of the present invention in which the azide-PEG4 portion is present, compound number 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, add DIPEA (0.1 mL) and stir the solution under an argon atmosphere for 10 minutes. Add compound 36 (1 equivalent) in two portions and stir the resulting clear yellow solution at 0°C for 2 hours. Slowly warm the solution to room temperature and continue stirring for a further 12 hours, or until thin-layer chromatography analysis using 10% (v / v) MeOH in DCM containing 1% (v / v) TEA shows that all of the IMDQ starting material has been consumed. Remove the DMF under reduced pressure, add ethyl acetate to the solid yellow residue, and grind the mixture to precipitate the resulting pale yellow solid. Decant the supernatant. Repeat this process two more times. Dry the off-white solid product under reduced pressure to obtain compound 37. General procedure for the preparation of compound 38.

[0383] A solution of compound 37 (1.0 equivalent) in DCM is treated with TFA (1.0 mL) at room temperature for 1 hour. The reaction product is concentrated to dryness under reduced pressure, and the residue containing compound 38 is further processed. It was used without purification. General procedure for the preparation of compound number 64-52g.

[0384] Compound 38 (1.0 equivalent) and compound 25 (1.2 equivalents) are dissolved in DMF, DIPEA (0.1 mL) is added, and the solution is stirred at room temperature for 1.5 hours. RP-HPLC analysis of this reaction shows that compound 38 has been completely consumed. The DMF is then removed under reduced pressure, and the yellow solid residue is subjected to silica gel column chromatography, eluting with 1-8% (v / v) MeOH in DCM containing 0.5% (v / v) ammonia, to obtain compound numbers 64-52 g as a white solid. Product purity is determined by RP-HPLC at 254 nm, the mass of the target compound is confirmed by LC-MS, and the structure of the target compound is determined. 1 Confirmation is performed by 1H NMR. Compound number 64-52h.

[0385] In equation (I), the TLR7 / 8 agonist D is 64-33, and the self-deactivating linker L in equation (I) 1 However, it is the para-aminobenzylcarbamate 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 illustrate an embodiment of the present invention in which the azide-PEG4 portion is present, compound number 64-52h was prepared as shown in scheme S2h-1. Scheme S2h-1 [ka] Preparation procedure for compound 52.

[0386] To an ice-cold solution of compound 64-33b (96 mg, 0.224 mmol, 1.0 equivalent) in DMF (5.0 mL), TEA (91 mg, 0.9 mmol) was added, followed by di-tert-butyl dicarbonate (54 mg, 0.250 mmol, 1.1 equivalents). This 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 obtain 94 mg of compound 52. Preparation procedure for compound 53.

[0387] Ice of compound 52 (94 mg, 0.178 mmol, 1 equivalent) in DMF (1.5 mL) 0.2 mL of DIPEA was added to the cold solution, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 6 (135 mg, 0.178 mmol, 1 equivalent) was added all at once. The resulting clear yellow solution was stirred at 0°C for 1 hour, then slowly warmed to room temperature and stirred for 12 hours. Thin-layer chromatography analysis in 10% (v / v) MeOH in DCM containing 1% TEA showed that only a very slight reaction had occurred. Next, the mixture was warmed to 70°C, stirred, and the progress of the reaction was monitored by RP-HPLC. After 3 days, the DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate the resulting 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 to obtain 84 mg of compound 53, which was used in the next reaction without further purification. Preparation procedure for compound 54.

[0388] To an ice-cold solution of compound 53 (84 mg, 0.072 mmol) in DMF (1.5 mL), diisopropylamine (0.1 mL) was added while stirring under an argon atmosphere. The resulting solution was then slowly warmed to room temperature, and stirring was continued for 12 hours. The DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was pulverized to precipitate the resulting 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 to obtain 54 mg of crude compound 54. Preparation procedure for 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), and DIPEA (0.1 mL) was added. The solution was stirred for 2.5 hours. The 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 obtain 14 mg of compound 55 as an off-white solid. Preparation procedure for compound number 64-52h.

[0390] Under an argon atmosphere, a chilled solution of compound 55 (12 mg, 1.0 equivalent) in DMF (1.5 mL) was added with stirring, to which diisopropylamine (0.1 mL) was added. Next, the resulting solution was slowly warmed to room temperature, and stirring was continued for 12 hours. The DMF was removed under reduced pressure, and ethyl acetate (3 mL) was added to the yellow residue. The mixture was pulverized to precipitate the resulting 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 to obtain 10 mg of crude compound number 64-52a as an off-white solid. The product purity was determined to be 30-40% by RP-HPLC at 254 nm, and the mass of the target compound was given as 1,120.4 Daltons by LC-MS. Compound number 64-52i.

[0391] In equation (I), the TLR7 / 8 agonist D is 64-10b, and the self-deactivating linker L in equation (I) is 1 However, this is the para-aminobenzylcarbamate moiety conjugated to the secondary amine in the TLR7 / 8 agonist benzylmethylamine (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 illustrate an embodiment of the present invention in which is the azide-PEG4 moiety, compound number 64-52i was prepared as shown in scheme S2i-1. Furthermore, the TLR7 / 8 agonist (D in formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 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 number 52.

[0392] Add 2.0 equivalents of TEA to a solution of compound number 6 (1.1 equivalents) and compound number 64-10b (1.0 equivalent) in DMF, and stir the resulting solution at room temperature. Once LC / MS shows that the majority is the desired product, compound number 52, and that all of compound number 64-10b is consumed, use this reaction mixture directly in the next step. General procedure for the preparation of compound number 53.

[0393] To a crude solution of compound number 52 (1.0 equivalent) in DMF, add piperidine (approximately 10 equivalents) and stir the solution at room temperature. Once LC / MS shows the disappearance of the starting material, compound number 52, dilute this reaction mixture with 10% MeOH in DCM and water. Wash twice. Extract the aqueous layer with ethyl acetate, dry the combined organic layer over Na2SO4, and concentrate under reduced pressure. Purify the residue by column chromatography (100 / 10~100 / 15 (v / v) DCM / MeOH) to obtain compound number 53 as a yellow solid. General procedure for the preparation of compound number 64-52i.

[0394] To a solution of compound number 53 (1.0 equivalent) and compound number 25 (1.1 equivalent) in DMF, add DIPEA (0.2 mL) and stir the resulting solution at room temperature. Once LC / MS shows that most of the product has been converted to the desired product, concentrate the reaction mixture under reduced pressure. Purify the residue by column chromatography (100 / 10~100 / 15 (v / v) DCM / MeOH) to obtain compound numbers 64-52i as a white solid. (Example S3) Preparation of propargylamine-carboxymethylated Ficoll.

[0395] Carboxymethylated Ficoll (CM-Ficoll) was prepared at a concentration of 37.9 mg / mL in 0.2 M sodium chloride as described elsewhere (e.g., Inman, JK). See 1975, J Immunol 114:704-709). 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 this mixture in 25 mg increments over 10 minutes while mixing. The pH of the resulting solution was adjusted to 4.7 with 1N HCl, and the reaction was allowed to proceed at ambient temperature (22-24°C) for 3.5 hours while mixing the reaction product. The pH of the solution was checked periodically during this time 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 of Ficoll and was stored at -80°C. The propargyl group content in PACM-Ficoll was determined by conjugation of a fluorophore (Alexa Fluor® 594 azide, Thermo Scientific, Rockford IL, catalog number A10270) by subsequent fluorescence quantification compared to a standard curve. Each batch of PACM-Ficoll was shown to contain approximately 250 moles of propargyl groups / mol Ficoll. Scheme S3-1 [ka] (Example S4) Preparation of compounds 64-53, 64-53a, 64-54, 54-54a, 64-54b, and 64-54c.

[0396] -L in equation (I) 3 -L 2 -L 1Azide-disulfide-PABC-IMDQ (compound numbers 64-51 and 64-51a) and azide-dipeptide-PABC-IMDQ (compound numbers 64-52, 54-54a, 64-54b and 64-52c), corresponding to -D, are PACM-Ficoll in accordance with schemes S4-1, S4-1a, S4-2, S4-2a, S4-2b and S4-2c. By covalent conjugation, Ficoll-azido-disulfide-PABC-IMDQ compounds (compound numbers 64-53 and 64-53a) or Ficoll-azido-dipeptide-PABC-IMDQ compounds (compound numbers 64-54, 64-54a, 64-54b, and 64-54c) were obtained. 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 That is the case. Scheme S4-1 [ka] Preparation of compound numbers 64-53.

[0397] Compound number 64-51 was dissolved in DMF at a concentration of 5 mg / mL. PACM-Ficoll at a concentration of 22.2 mg / mL was prepared in PBS (pH 7.5) buffer. Copper(II) sulfate pentahydrate (CuSO4) and L-ascorbic acid were each prepared in pure water at a concentration of 5 mg / mL. 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 number 64-51 (0.62 mL, 4.4 micromol) 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 with stirring. The following day, this reaction mixture was dialyzed in 8 liters of PBS (pH 7.5) using a molecular weight cutoff dialysis cassette with a value of 10,000 Daltons. A fixed amount of the purified compound number 64-53 was taken and stored at -80°C. Scheme S4-1a [ka] Preparation of compound number 64-53a.

[0398] Compound number 64-53a was prepared using compound number 64-51a instead of compound number 64-51, in a manner similar to that described above for compound number 64-53. The synthesis of compound number 64-53a is outlined in scheme S4-1a. Scheme S4-2 [ka] Preparation of compound number 64-54.

[0399] Compound number 64-52 was dissolved in DMF at a concentration of 5 mg / mL. PACM-Ficoll was prepared in PBS (pH 7.5) at a concentration of 22.2 mg / mL. Copper(II) sulfate pentahydrate (CuSO4) and L-ascorbic acid were each prepared in pure water at a concentration of 5 mg / mL. PACM-Ficoll (0.79 mL, 0.044 micromoles) 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. Next, compound number 64-52 (0.61 mL, 4.4 micromoles) was added in increments of 0.2 mL while mixing. The reaction was allowed to proceed at ambient temperature (22-24°C) for 18 hours while mixing. On that day, this reaction mixture (approximately 4.9 mL) was dialyzed in 8 liters of PBS (pH 7.5) using a molecular weight cutoff dialysis cassette with a value of 10,000 Daltons. A fixed amount of the purified compound number 64-54 was taken and stored at -80°C. Scheme S4-2a [ka] Preparation of compound number 64-54a.

[0400] Compound number 64-54a was prepared using compound number 64-52a instead of compound number 64-52, in a manner similar to that described above for compound number 64-54. The synthesis of compound number 64-54a is outlined in scheme S4-2a. Preparation of compound number 64-54b.

[0401] Compound number 64-54b was prepared using compound number 64-52c instead of compound number 64-52, in a manner similar to that described above for compound number 64-54. The synthesis of compound number 64-54b is outlined in scheme S4-2b. Scheme S4-2b [ka] Preparation of compound number 64-54c.

[0402] Compound number 64-54c was prepared using compound number 64-52b instead of compound number 64-52, in a manner similar to that described above for compound number 64-54. The synthesis of compound number 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]quinoline-1-yl)methyl)benzyl)tetradecanamide (compound number 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 this reaction mixture was heated to 65°C for 3 hours. Next, the mixture was cooled to 5-10°C, and the solid substance was collected by filtration and rinsed with cold water. The solid was washed and air-dried. Next, the obtained solid was recrystallized from methanol and dried under vacuum to obtain 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-quinoline diol (58 g) and heated to 95°C for 4 hours. Next, the mixture was cooled to room temperature and poured onto crushed ice while constantly 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 obtain 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 equivalent) in anhydrous dichloromethane (400 mL) and trimethylamine (16.0 g, 0.16 mol, 1.1 equivalent), tert-butyl(4-(aminomethyl)benzyl)carbamate (37.3 g, 0.16 mol, 1.1 equivalent) was added and stirred overnight at room temperature. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography on silica gel using hexane / ethyl acetate to obtain 52 g of tert-butyl(4-(((2-chloro-3-nitroquinoline-4-yl)amino)methyl)benzyl)carbamate.

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

[0407] Part E. To a solution of tert-butyl(4-(((3-amino-2-chloroquinoline-4-yl)amino)methyl)benzyl)carbamate (32 g, 77.6 mmol, 1.0 equivalent) in anhydrous tetrahydrofuran (350 mL) and pyridine (30 mL), pentanoyl chloride (9.7 mL, 81.5 mmol, 1.05 equivalent) was slowly added at 0–5°C. The reaction mixture was then warmed to room temperature and stirred for 12 hours. The solvent was removed under reduced pressure, and the solid was then redissolved in ethyl acetate (400 mL), washed sequentially 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 eluted with hexane / ethyl acetate to obtain 22 g of tert-butyl(4-(((3-butylamide-2-chloroquinoline-4-yl)amino)methyl)benzyl)carbamate.

[0408] Part F. To a solution of tert-butyl(4-(((3-butylamido-2-chloroquinoline-4-yl)aminomethyl)benzyl)carbamate (22 g, 44.2 mmol, 1.0 equivalent) in ethanol (320 mL), water (80 mL) was added, followed by potassium carbonate (12.2 g, 88.4 mmol, 2.0 equivalents). The mixture was heated at 55°C for 16 hours with vigorous stirring. 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 eluted with hexane / ethyl acetate to obtain 15.4 g of tert-butyl(4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)carbamate.

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

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

[0411] Part I. To a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine (34 mg, 0.1 mmol, 1.0 equivalent) in anhydrous dimethylformamide (2 mL), myristic acid (27.4 mg, 0.12 mmol, 1.2 equivalents) and trimethylamine (0.2 mL) were added, and the slurry was mixed for 5 minutes, after which HBTU (47.4 mg, 0.125 mmol, 1.25 equivalents) was added. The reaction mixture was stirred under an argon atmosphere for a further 2 hours. The solvent was removed under reduced pressure, the residue was dissolved in ethyl acetate (30 mL) and washed with water (2 × 10 mL), then dried using magnesium sulfate and concentrated under vacuum. This product was purified using column chromatography (6% methanol / dichloromethane) to obtain 35 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)tetradecanamide (compound number 64-10a). The product purity was determined to be approximately 98% by reverse-phase HPLC, and the mass of the target compound, 569.8, was confirmed by LC / MS. The structure of the target compound was determined by 300 MHz proton NMR (CDCl3): 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 This was confirmed by the following values: 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), and 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]quinoline-4-amine (compound number 64-33b).

[0412] Parts A through H were the same as in Example S4-a.

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

[0414] Part J. To a solution of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)-2-cyclopropylacetamide (123 mg, 0.28 mmol, 1.0 equivalent) in anhydrous tetrahydrofuran (5 mL), a solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL, excess) was added at room temperature, and the reaction mixture was heated under reflux for 12 hours. The mixture was cooled to ambient temperature, quenched with 3N HCl (1 mL), and stirred for 4 hours. The pH of the reaction mixture was made alkaline by adding 2N sodium hydroxide, and the product was extracted with dichloromethane (20 mL × 10⁻⁶). The combined organic layers were concentrated under reduced pressure, and the residue was purified by flash chromatography using 6% methanol / dichloromethane as the eluent to obtain 28 mg of 2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine (compound number 64-33b). The product purity was determined to be 97% by reverse-phase HPLC, and the mass of the target compound, 427.6, was confirmed by LC / MS. The structure of the target compound was determined at 400 MHz. 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, This was confirmed by the following values: 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), and 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]quinoline-4-amine (compound number 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 the mixture was stirred overnight at room temperature. Next, the mixture was diluted with ether (20 mL), and the precipitated urea was removed by filtration. The filtrate was concentrated to obtain the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any residual precipitated urea was removed again by filtration. The resulting filtrate was concentrated under reduced pressure to obtain 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-methylcyclobutane carboxylate (72 mg, 0.26 mmol) in dichloromethane (3 mL), IMDQ (84 mg, 0.23 mmol) was added in the presence of triethylamine (48 mg, 0.47 mmol), and the mixture was stirred at room temperature for 2 hours. Next, the reaction mixture was concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexane. The residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl, then with water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 88 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-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]quinoline-1-yl)methyl)benzyl)-1-methylcyclobutan-1-carboxamide (88 mg) was reduced with boranedimethyl sulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction mixture was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred for a further 3 hours at 55°C. The reaction mixture 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 obtain an off-white solid. Recrystallization using 9:1 ethyl acetate / hexane yielded 32 mg of 2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine (compound numbers 64-60b). Product purity was determined to be 96% by reverse-phase HPLC at 254 nm. The mass of the target compound, 441.3 daltons, was confirmed by LC / MS, and the structure of the target compound was determined 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), This was confirmed by the following values: 1.6-1.88 (m, 8H), 1.35-1.60 (m, 2H), 1.12 (s, 3H), and 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]quinoline-4-amine (compound number 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), N,N-diisopropylcarbodiimide (127 mg, 1.0 mmol) was added in the presence of a catalytic amount of N,N-dimethylaminopyridine (12 mg), and the mixture was stirred overnight at room temperature. Next, the mixture was diluted with ether (20 mL), and the precipitated urea was removed by filtration. The filtrate was concentrated to obtain the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any residual precipitated urea was removed again by filtration. The resulting filtrate was concentrated under reduced pressure to obtain 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 the mixture was stirred at room temperature for 2 hours. Next, the reaction mixture was concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexane. The residue was then mixed with dichloromethane (15 mL). The solution was dissolved in (L), washed with 1M HCl, then with water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 85 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)cyclobutanecarboxamide as an off-white solid.

[0420] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)cyclobutanecarboxamide (85 mg) was reduced with boranedimethyl sulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction mixture was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55°C for a further 3 hours. The reaction mixture 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 obtain an off-white solid. Recrystallization using 9:1 ethyl acetate / hexane yielded 21 mg of 2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine (compound numbers 64-66b). Product purity was determined to be 95% by reverse-phase HPLC at 254 nm, and the mass of the target compound, 427.3 daltons, was confirmed by LC / MS. The structure of the target compound was determined 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, This was confirmed by the following measurements: 1H), 1.75-2.15 (m, 7H), 1.60-1.70 (m, 2H), 1.35-1.50 (m, 2H), and 0.94 (t, J = 7.5, 14.7 Hz, 3H). (Example S5) Determination of percentage free IMDQ for compound numbers 64-53, 64-53a, 64-54, 64-54a, 64-54b, and 64-54c.

[0421] To detect free (i.e., non-conjugated) IMDQ, compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, and 64-54c were analyzed by RP-HPLC. A fixed amount of compound 64-53, 64-53a, 64-54, 64-54a, 64-54b, or 64-54c was 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 (5-95% acetonitrile). For quantification, the free IMDQ peak (retention time 4.5 minutes) was integrated, and the response area was compared to the IMDQ standard curve. Percent free IMDQ is expressed by the following formula: % Free IMDQ was calculated using the formula: % Free IMDQ = (Concentration of unconjugated IMDQ) × 100 / (Total IMDQ concentration) (wherein the formula, the total IMDQ concentration is determined as described in Example S6 for compound numbers 64-53 and 54-53a, or as described in Example S8 for compound numbers 64-54, 64-54a, 64-54b, and 64-54c). (Example S6) Determination of the total IMDQ concentration in compounds 64-53 and 64-53a.

[0422] The total IMDQ concentration in compounds 64-53 and 64-53a was determined by quantification of IMDQ released after complete thiolysis. Briefly, compound 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. As described in Example S5, the resulting samples were analyzed by RP-HPLC, and the area of ​​the released IMDQ peak was quantified relative to the IMDQ standard curve. (Example S7) Determination of Ficoll concentrations in compounds 64-53, 64-53a, 64-54a, and 64-54.

[0423] The Ficoll concentrations in compound numbers 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 created using Ficoll PM400 (GE Healthcare, Pittsburgh PA, catalog number 17-0300-50). (Example S8) Determination of total IMDQ concentrations in compounds 64-54, 64-54a, 64-54b, and 64-54c.

[0424] The total IMDQ concentrations in compounds 64-54, 64-54a, 64-54b, and 64-54c were determined by quantification of IMDQ released after complete proteolysis. Briefly, compounds 64-54, 64-54a, 64-54b, or 64-54c were 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. As described in Example S5, the obtained samples were analyzed by RP-HPLC, and the area of ​​the released IMDQ peak was quantified relative to the IMDQ standard curve. (Example S9) Determination of particle sizes for compound numbers 64-53, 64-53a, 64-54, 64-54a, 64-54b, or 64-54c.

[0425] The mean particle size (Z-mean) of compounds 64-53, 64-53a, 64-54, 64-54a, 64-54b, or 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 specified instrument settings. A 50 nm polystyrene nanosphere (Thermo Scientific, Rockford IL, catalog number 3050A) was 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] Table S10-1 summarizes the reaction conditions used for the synthesis of compounds 64-53, 64-53a, 64-54, and 64-54a. Assays for determining non-conjugate IMDQ, total IMDQ, and Ficoll concentration are described in Examples S5-S9. The amount of total IMDQ was calculated by multiplying the total IMDQ concentration by the volume of the solution. [Table S10-1] (Example S11) Preparation of compounds 64-55 and 64-56.

[0427] Using the procedures and synthesis schemes shown in Example S11 and Schemes S11-1 and S11-2, various maleimidocaproyl-dipeptide-PABC-TLR7 / 8 agonist compounds having different TLR7 / 8 agonist moieties (in formula (I) -L 3 -L 2 -L 1 -D) can be prepared. These examples show that TLR7 / 8 agonist compounds 64-10a and 64-33a are used to prepare compound numbers 64-55 (scheme S11-1) and 64-56 (scheme S11-2) as examples of a common maleimidocaproyl-dipeptide-PABC-TLR7 / 8 agonist structure. Furthermore, the TLR7 / 8 agonist (D in formula (I)) can be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 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 number 10.

[0428] To a solution of 2,4-dichloro-3-nitroquinoline (1.0 equivalent) in anhydrous dichloromethane (400 mL) and trimethylamine (1.1 equivalents), tert-butyl(4-(aminomethyl)benzyl)carbamate (1.1 equivalents) is added, and the mixture is stirred overnight at room temperature. 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 number 10 (tert-butyl(4-(((2-chloro-3-nitroquinoline-4-yl)amino)methyl)benzyl)carbamate). General procedure for the preparation of compound number 11.

[0429] Using a Parr hydrogenation apparatus, a solution of compound number 10 (0.12 mol) in ethyl acetate (250 mL) is hydrogenated for 12 hours at 60 psi in the presence of 5% carbon-supported platinum (2.0 g) and sodium sulfate (52 g). The platinum catalyst and sodium sulfate are removed by filtration through a Celite® pad, and the filtrate is concentrated under reduced pressure. The product is further purified by flash chromatography on silica gel eluted with hexane / ethyl acetate to obtain compound number 11 (tert-butyl(4-(((3-amino-2-chloroquinoline-4-yl)amino)methyl)benzyl)carbamate. General procedure for the preparation of compound number 12.

[0430] To a solution of compound number 11 (1.0 equivalent) in anhydrous tetrahydrofuran (350 mL) and pyridine (30 mL), pentanoyl chloride (1.05 equivalent) is slowly added at 0-5°C. 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 redissolved in ethyl acetate (400 mL). The mixture is then sequentially washed 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 eluted with hexane / ethyl acetate to obtain the intermediate tert-butyl(4-(((3-butylamido-2-chloroquinoline-4-yl)aminomethyl)benzyl)carbamate. Water is added to a solution of the intermediate (1.0 equivalent) in 4 × volume of ethanol, then potassium carbonate (2.0 equivalents) is added, and the mixture is heated at 55°C for 16 hours with vigorous stirring. Next, the reaction mixture is concentrated, and the residue is partitioned between ethyl acetate and water. Next, the ethyl acetate layer is washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The product is further purified by flash chromatography on silica gel eluted with hexane / ethyl acetate to obtain compound number 12 (tert-butyl(4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)carbamate). General procedure for the preparation of compound number 13.

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

[0432] Compound number 13 (25.7 mmol) was added to concentrated hydrochloric acid. 10% carbon-supported platinum (3.0 g) was added to this suspension. The reaction mixture was hydrogenated at 65 psi, and the progress of the reaction was monitored by reverse-phase HPLC analysis. After 6 days, the catalyst was filtered off, and the filter cake was washed with water. The cake was cooled in an ice bath, and ice-cold 1N sodium hydroxide was added dropwise with vigorous stirring until the pH reached 8.5. The substance was extracted with dichloromethane containing 5% methanol. The combined extracts were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column using 8% methanol / dichloromethane containing 1% aqueous ammonia to obtain compound number 5 (1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinoline-4-amine). General procedure for the preparation of compound number 15.

[0433] To a solution of compound number 5 (1.0 equivalent) in anhydrous dimethylformamide, myristic acid (1.2 equivalents) and trimethylamine are added, and the slurry is mixed for 5 minutes, after which HBTU (1.25 equivalents) is added. The reaction mixture is stirred further for 2 hours under an argon atmosphere. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The product is purified by column chromatography (6% methanol / dichloromethane) to obtain compound number 15 (N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)tetradecanamide). General procedure for the preparation of compound number 16.

[0434] To a solution of compound number 15 (1.0 equivalent) in anhydrous DMF, 9-fluorenylmethyloxycarbonyl-valyl-citrullyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate (1.2 equivalents) and trimethylamine are added, and the slurry is mixed for 5 minutes, then HBTU (1.25 equivalents) is added. The reaction mixture is stirred further at room temperature for 15 hours. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The product is purified by column chromatography (6% methanol / dichloromethane) to obtain compound number 15. We obtain No. 16 ((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((((2-butyl-1-(4-(tetradecanamidemethyl)benzyl)-1H-imidazo[4,5-c]quinoline-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 compound numbers 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, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The intermediates are 4-((S)-2-((S)-2-(12-azanyl)-3-methylbutanamide)-5-ureidopentanamide)benzyl(2-butyl-1-(4-(tetradecanamidemethyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-yl)carbamate (1.0 equivalent) and N-(e-maleimidocaproyloxy)succinide ester (1 Dissolve 0.1 equivalent in NMP and stir at room temperature for 0.5 hours. Remove the solvent under reduced pressure, dissolve the residue in ethyl acetate and wash with water, then dry with magnesium sulfate and concentrate under vacuum. Purify the product using column chromatography (6% methanol / dichloromethane) to obtain compound no. 64-55 (4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl(2-butyl-1-(4-(tetradecanamidemethyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-yl)carbamate). Scheme S11-2 [ka] General procedure for the preparation of compound number 17.

[0436] To a solution of compound number 5 (IMDQ; 1.0 equivalent) in anhydrous DMF, add cyclopropylacetic acid (1.2 equivalents) and trimethylamine. Mix this slurry for 5 minutes, then add HBTU (1.25 equivalents). Allow this reaction mixture to stand under an argon atmosphere for 2 hours. The mixture is then stirred. The reaction product 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 by column chromatography (6% methanol / dichloromethane) to obtain the intermediate N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)methyl)benzyl)-2-cyclopropylacetamide. A solution of the intermediate (1.0 equivalent) in anhydrous tetrahydrofuran is added to a solution of borane-dimethyl sulfide complex in excess at room temperature, and the reaction mixture is heated under reflux for 12 hours. The mixture is cooled to ambient temperature, quenched with 3N HCl, and stirred for a further 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 were concentrated under reduced pressure, and the residue was purified by flash chromatography using 6% methanol / dichloromethane as the eluent to obtain compound number 17 (2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-amine). General procedure for the preparation of compound number 18.

[0437] To a solution of compound number 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 stirred further at room temperature for 15 hours. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. This product was purified using column chromatography (6% MeOH / dichloromethane) to obtain 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]quinoline-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 compound number 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, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. The intermediates 4-((S)-2-((S)-2-amino-3-methylbutanamide)-5-ureidopentanamide)benzyl(2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-yl)carbamate (1.0 equivalent) 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, the residue is dissolved in ethyl acetate and washed with water, then dried using magnesium sulfate and concentrated under vacuum. This product was purified using column chromatography (6% methanol / dichloromethane) to obtain compound number 64-56 (4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)-5-ureidopentanamide)benzyl(2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinoline-4-yl)carbamate). (Example S11a) Preparation of compounds 64-71, 64-72, 64-73, and 64-74.

[0439] Various TLR7 / 8 agors can be synthesized using the procedures and synthesis schemes shown in Example S11a and Schemes S11a-1, S11a-2, S11a-3 and S11a-4. Azide PEG4-dipeptide-PABC-TLR7 / 8 agonist compound having a rist moiety (in formula (I), -L 3 -L 2 -L 1-D) can be prepared. These examples show that TLR7 / 8 agonist compounds 64-33b, meta-IMDQ, 64-60b, and 64-66b can be used to prepare compound numbers 64-71 (scheme S11a-1), 64-72 (scheme S11b-2), 64-73 (scheme S11c-3), and 64-74 (scheme S11d-4) as examples of common azide PEG4-dipeptide-PABC-TLR7 / 8 agonist structures. Furthermore, the TLR7 / 8 agonist (D in formula (I)) may be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 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] Preparation procedure for compound 21.

[0440] To a solution of cyclopropanecarboxylic acid pentafluorophenol ester (280 mg; 1.2 equivalents) in dichloromethane (14 mL), IMDQ (compound 5; 350 mg; 1.0 equivalent) was added in the presence of triethylamine (105 mg; 0.3 mmol), and the mixture was stirred overnight at room temperature. After 18 hours 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 obtain 350 mg of compound 39. Preparation procedure for compound number 64-33b

[0441] Compound 39 (300 mg, 1 equivalent) in THF (5.0 mL) was reduced with boranedimethyl sulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction mixture was cooled to room temperature, carefully quenched with 2 M hydrochloric acid (excess), and stirred at 55°C for a further 6 hours. Next, the reaction mixture was 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 a cold 1 M NaOH solution, and the mixture was re-extracted with dichloromethane (3 × 10 mL). The mixture was dried over MgSO4 and concentrated under reduced pressure to obtain an off-white solid. Recrystallization with ethyl acetate yielded 114 mg of compound number 64-33b as a white solid. Preparation procedure for compound 40.

[0442] To a solution of compound number 64-33b (33 mg, 1.0 equivalent) in DMF (1.5 mL), diisopropylethylamine (0.1 mL) was added, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 10 (59 mg, 1 equivalent) was added to this solution 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 showed that all of compound number 64-33b had been consumed. The DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate the resulting pale yellow solid. The supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to obtain 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 and slowly warmed to room temperature, with stirring continued for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicated that the Fmoc moiety had been completely removed. DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate the resulting pale yellow solid. The supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to obtain 47 mg of compound 40. Preparation procedure for compound numbers 64 and 71.

[0443] Compound 40 (47 mg, 1.0 equivalent) and Compound 25 (24 mg, 1.2 equivalents) 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 that the reaction was complete. The DMF was removed under reduced pressure, and silica gel column chromatography was performed on the yellow residue, eluting with 4-10% methanol in dichloromethane containing 0.5% ammonia, to obtain 22.2 mg of compound numbers 64-71 as a white solid. The purity was determined to be 72% pure by reverse-phase HPLC at 254 nm, and the mass of the target compound, 1,105.6 daltons, was confirmed by HRMS. The structure of the target compound was determined 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] Preparation procedure for compound 41.

[0444] To a solution of meta-IMDQ (46 mg, 1.0 equivalent) in DMF (1.5 mL), diisopropylethylamine (0.2 mL) was added, and the solution was stirred under an argon atmosphere for 10 minutes. Compound 10 (98 mg, 1.0 equivalent) was added to this solution in two portions. The resulting clear yellow solution was stirred at 0°C for 2 hours, then slowly warmed to room temperature and stirred for 12 hours. Thin-layer chromatography analysis using 10% methanol in dichloromethane containing 1% trimethylamine showed that all of the meta-IMDQ had been consumed. The DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate the resulting pale yellow solid. The supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to obtain 102 mg of an off-white solid. An 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 slowly warmed to room temperature, with stirring continued for 12 hours. Reverse-phase HPLC analysis of the reaction mixture showed that the Fmoc moiety had been completely removed. The DMF was removed under reduced pressure, ethyl acetate (3 mL) was added to the yellow residue, and the mixture was pulverized to precipitate the resulting pale yellow solid. The supernatant was decanted. This process was repeated two more times, and the product was dried under reduced pressure to obtain 68 mg of compound 41. Preparation procedure for compound numbers 64-72.

[0445] Compound 41 (68 mg, 1.0 equivalent) and Compound 25 (38 mg, 1.2 equivalents) were dissolved in DMF (1 mL). Diisopropylethylamine (0.1 mL) was added to this solution, and the mixture was stirred at room temperature for 1.5 hours. Reverse-phase HPLC analysis confirmed the completion of the reaction. The DMF was removed under reduced pressure, and the yellow residue contained 0.5% ammonia. 47 mg of compound numbers 64-72 was obtained as a white solid by silica gel column chromatography eluting with 1-8% methanol in dichloromethane. Purity was determined to be 96% by reverse-phase HPLC at 254 nm, and the mass of the target compound, 1,037.8 daltons, was confirmed by HRMS. The structure of the target compound was determined at 300 MHz. 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), This was confirmed by the following values: 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), and 0.88-1.01 (m, 9H). Scheme S11a-3 [ka] General procedure for the preparation of compound 42.

[0446] To a solution of 1-methylcyclobutanecarboxylic acid pentafluorophenol ester (1.2 equivalents) in DCM, IMDQ (compound 5; 1.0 equivalent) was added in the presence of TEA and stirred overnight at room temperature. After 18 hours, reverse-phase HPLC analysis of the solution demonstrated that the reaction was complete. This 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 obtain compound 42. General procedure for the preparation of compound number 64-60b.

[0447] Compound 42 (1 equivalent) in THF is reduced with borane-dimethyl sulfide complex (3.5 equivalents) at 55°C for 12 hours. The reaction mixture is cooled to room temperature, carefully quenched with 2M hydrochloric acid (excess), and stirred at 55°C for a further 6 hours. Next, the reaction mixture is cooled to room temperature, diluted with water, and extracted with DCM to remove impurities. The pH is adjusted to 8.0 by adding a cold 1M NaOH solution, re-extracted with DCM, and the solution is dried over MgSO4 and concentrated under reduced pressure to obtain an off-white solid. Recrystallization with ethyl acetate yields compound number 64-60b as a white solid. General procedure for the preparation of compound 43.

[0448] Add DIPEA to a solution of compound number 64-60b (1.0 equivalent) in DMF, and stir the solution under an argon atmosphere for 10 minutes. Add compound 10 (1.0 equivalent) in two portions to this solution, and stir the resulting clear yellow solution at 35°C for 2 hours. Thin-layer chromatography analysis using 10% (v / v) methanol in DCM containing 1% (v / v) TEA shows that compound number 64-60b has been completely consumed. Remove the DMF under reduced pressure, add ethyl acetate to the yellow residue, and grind the mixture to precipitate the resulting pale yellow solid. Decant the supernatant. Repeat this process two more times, and dry the product under reduced pressure to obtain an off-white solid. Dissolve the off-white solid in ice-cold DMF, and add diisopropylamine. Stir the resulting solution under an argon atmosphere and slowly warm it to room temperature while continuing to stir for 12 hours. Reverse-phase HPLC analysis of the reaction mixture indicates that the Fmoc moiety has been completely removed. Next, DMF is removed under reduced pressure, ethyl acetate is added to the yellow residue, and the mixture is pulverized to precipitate the resulting pale yellow solid, and the supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to obtain compound 43. General procedure for the preparation of compounds number 64-73.

[0449] Compound 43 (1.0 equivalent) and compound 25 (1.2 equivalents) are dissolved in DMF. DIPEA is added, and the solution is stirred at room temperature for 2.5 hours. Reverse-phase HPLC analysis indicates that the reaction is complete. The DMF is removed under reduced pressure, and the yellow residue is subjected to silica gel column chromatography, eluting with 4-10% (v / v) methanol in dichloromethane containing 0.5% (v / v) ammonia, to obtain compounds 64-73 as white solids. Product purity is determined by RP-HPLC at 254 nm, the mass of the target compound is confirmed by LC-MS, and the structure of the target compound is determined. 1 Confirmation is performed by 1H NMR. Scheme S11a-4 [ka] General procedure for the preparation of compound 44.

[0450] To a solution of cyclobutanecarboxylic acid pentafluorophenol ester (1.2 equivalents) in DCM, IMDQ (compound 5; 1.0 equivalent) was added in the presence of TEA and stirred overnight at room temperature. After 18 hours, reverse-phase HPLC analysis of the solution demonstrated that the reaction was complete. This 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 obtain compound 44. General procedure for the preparation of compound number 64-66b.

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

[0452] To a solution of compound numbers 64-66b (1.0 equivalent) in DMF, DIPEA is added, and the solution is stirred under an argon atmosphere for 10 minutes. Compound 10 (1.0 equivalent) is added to this solution in two portions, and the resulting clear yellow solution is stirred at 35°C for 2 hours. Thin-layer chromatography analysis using 10% (v / v) methanol in DCM containing 1% (v / v) TEA shows that compound numbers 64-66b are completely consumed. DM F is removed under reduced pressure, ethyl acetate is added to the yellow residue, and the mixture is pulverized to precipitate the resulting pale yellow solid. The supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to obtain 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 slowly warmed to room temperature while continuing to stir for 12 hours. Reverse-phase HPLC analysis of the reaction mixture shows that the removal of the Fmoc moiety is complete. Next, the DMF is removed under reduced pressure, ethyl acetate is added to the yellow residue, and the mixture is pulverized to precipitate the resulting pale yellow solid. The supernatant is decanted. This process is repeated two more times, and the product is dried under reduced pressure to obtain compound 45. General procedure for the preparation of compounds 64-74.

[0453] Compound 45 (1.0 equivalent) and compound 25 (1.2 equivalents) are dissolved in DMF. DIPEA is added, and the solution is stirred at room temperature for 2.5 hours. Reverse-phase HPLC analysis indicates that the reaction is complete. The DMF is removed under reduced pressure, and the yellow residue is subjected to silica gel column chromatography, eluting with 4-10% (v / v) methanol in dichloromethane containing 0.5% (v / v) ammonia, to obtain compounds 64-74 as white solids. Product purity is determined by RP-HPLC at 254 nm, the mass of the target compound is confirmed by LC-MS, and the structure of the target compound is determined. 1 Confirmation is performed by 1H NMR. (Example S12) Preparation of compounds numbered 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 maleimidocaproil or NHS-PEG4-triazole-PEG4 conjugation. Compound numbers 64-57, 64-75, 64-76, and 64-77 were prepared as examples of compounds of formula (I) in which the conjugation portion F is an antibody, using trastuzumab, an anti-HER2 antibody, together with a linker, a valine-citrulline cleavable linker, a PABC auto-desorbing linker, and compound numbers 64-10a and 64-66a or IMDQ as the TLR7 / 8 agonist portion (the agonist:antibody ratio is 4 on average (i.e., 4 TLR7 / 8 agonist compounds per antibody)). Furthermore, the TLR7 / 8 agonist moiety (D in formula (I)) may be IMDQ or meta-IMDQ (see Figure 1 for the representation of the chemical structures of IMDQ and meta-IMDQ), as well as compound numbers 64-01 to 64-50, 64-58 to 64-69, 64-01a to 64-50a, and 64-58a to 64-69a. Those skilled in the art will understand that the general procedures described herein for using trastuzumab also apply to other recombinant antibodies and their derivatives. General procedure for limited reduction of anti-HER2 antibody and preparation of compound number 19.

[0455] Trastuzumab is a humanized IgG1 monoclonal antibody that targets the HER2 receptor (also known as HER2 / neu, human epidermal growth factor receptor 2), 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, which has a manipulated S228P mutation in the hinge region, is commercially available from InvivoGen, San Diego CA, catalog number her2tra-mab14, or MedChem Express, Monmouth. The antibody is obtained from Junction NJ (catalog number HY-P9907). This antibody is generated by recombinant DNA technology and purified by affinity chromatography using methods well known to those skilled in the art (e.g., Kuner, R. and Reinhart, D. 2016, Appl). (See Microbiol Biotechnol 100:3451-3461). The trastuzumab antibody is mixed with 2.5 molar equivalents of Tris- in PBS (pH 7.5), 1 mM diethylenetriaminepentaacetic acid. Compound number 19 is produced by limited reduction using 2-carboxyethylphosphine at 37°C for 2 hours. General procedure for the preparation of compound number 20.

[0456] Compound number 19 (1.0 equivalent) in PBS (pH 7.5) and 1 mM diethylenetriaminepentaacetic acid is reacted with compounds number 64-56 (8-10 equivalents) in PBS (pH 7.5) containing 10% (v / v) dimethylacetamide at 0°C for 0.5 hours, and this reaction is quenched with 1 mM cysteine. Next, compound number 20 is purified using G-25 size exclusion chromatography (GE Healthcare, Pisquataway NJ) equilibrated in PBS (pH 7.5) and the buffer is exchanged to PBS (pH 7.5). General procedure for the preparation of compound number 64-57.

[0457] The ring hydrolysis of succinimide-thioether produces a bond with greater plasma stability; therefore, using tangential flow filtration, compound number 20 is buffered with 50 mM borate (pH 9.2), heated at 45°C for 48 hours, then cooled to room temperature and buffered with PBS (pH 7.5). Compounds 64-57 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any residual impurities and aggregates and ensure complete buffer exchange. The synthesis scheme for preparing compound numbers 64-57 is shown in Figure 12. General procedure for the preparation of compound number 46

[0458] Compound No. 45 (1.0 equivalent) and N-(ε-maleimidocaproyloxy) succinide ester (1.1 equivalents) are dissolved in DMF, DIPEA is added, and the reaction mixture is stirred at room temperature for 2.5 hours. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate and washed with water, then dried using MgSO4 and concentrated under vacuum. The product is purified by column chromatography (6% methanol / dichloromethane) to obtain compound No. 46. General procedure for the preparation of compound number 47.

[0459] Compound number 19 (1.0 equivalent) in PBS (pH 7.5) and 1 mM diethylenetriaminepentaacetic acid is reacted with compound number 46 (8-10 equivalents) in PBS (pH 7.5) containing 10% (v / v) dimethylacetamide at 0°C for 0.5 hours, and this reaction is quenched with 1 mM cysteine. Next, compound number 47 is purified using G-25 size exclusion chromatography (GE Healthcare, Pisquataway NJ) equilibrated in PBS (pH 7.5) and the buffer is exchanged to PBS (pH 7.5). General procedure for the preparation of compounds number 64-75.

[0460] The ring hydrolysis of succinimide-thioether produces a bond with greater plasma stability; therefore, using tangential flow filtration, compound number 47 is buffered to 50 mM borate (pH 9.2), heated at 45°C for 48 hours, then cooled to room temperature and buffered to PBS (pH 7.5). Compound numbers 64-75 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any residual impurities and aggregates and ensure complete buffer exchange. The synthesis scheme for preparing compound numbers 64-75 is shown in Figure 13. General procedure for the preparation of compound number 48.

[0461] Compound No. 12 (1.0 equivalent) and N-(ε-maleimidocaproyloxy) succinide ester (1.1 equivalents) are dissolved in DMF, DIPEA is added, and the reaction mixture is prepared 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 number 48. General procedure for the preparation of compound number 49.

[0462] Compound number 19 (1.0 equivalent) in PBS (pH 7.5) and 1 mM diethylenetriaminepentaacetic acid is reacted with compound number 48 (8-10 equivalents) in PBS (pH 7.5) containing 10% (v / v) dimethylacetamide at 0°C for 0.5 hours, and this reaction is quenched with 1 mM cysteine. Next, compound number 49 is purified using G-25 size exclusion chromatography (GE Healthcare, Pisquataway NJ) equilibrated in PBS (pH 7.5) and the buffer is exchanged to PBS (pH 7.5). General procedure for the preparation of compounds number 64-76.

[0463] The ring hydrolysis of succinimide-thioether produces a bond with greater plasma stability; therefore, using tangential flow filtration, compound number 49 is buffered to 50 mM borate (pH 9.2), heated at 45°C for 48 hours, then cooled to room temperature and buffered to PBS (pH 7.5). Compounds 64-76 are subjected to size exclusion chromatography equilibrated in PBS (pH 7.5) to remove any residual impurities or aggregates and ensure complete buffer exchange. The synthesis scheme for preparing compound numbers 64-76 is shown in Figure 14. Preparation procedure for compound number 51.

[0464] To a solution of trastuzumab antibody (compound 50; 10 mg, 1 equivalent) in PBS (pH 7.4) (1 mL), dibenzocyclooctin-PEG4-N-hydroxysuccinimidyl ester (4.0 mg, 10 equivalents) in PBS (pH 7.4) and 1.5% (v / v) DMSO (1 mL) was added, 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) and 0.05% (v / v) Tween® 80. The protein concentration of the purified compound 51 was determined by spectrophotometric measurement at 280 nm using compound 50 as a calibration standard. Preparation procedure for compound numbers 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) and 5% (v / v) DMSO (0.5 mL). This reaction was allowed to proceed at room temperature for 2 hours, followed by a further 3 hours at 4°C. 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 64-77 was determined to be 96% by analytical SEC analysis, and the drug-to-antibody ratio was calculated to be 3.7 using a UV / Vis absorbance spectrometer. The synthesis scheme for preparing compound 64-77 is shown in Figure 15. General procedure for characterizing compounds number 64-57, 64-75, 64-76, and 64-77.

[0466] The concentrations of compounds 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 with separate wavelength monitoring at the maximum absorption of both the antibody and the TLR7 / 8 agonist. Aggregation status is determined by analytical SEC-UPLC-MALS. The TLR7 / 8 agonist versus antibody ratio is determined using UV / Vis absorbance spectrometer (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). See Chem 25:123-1232. B. Biological Examples

[0467] The functional groups of cleavable linkers in compounds 64-53, 64-53a, 64-54, 64-54a, 64-53b, and 64-54c were identified, as appropriate, by in vitro incubation with glutathione or purified human cathepsin B, and the release of the original (i.e., non-conjugated) TRL7 / 8 agonist moiety IMDQ from the above compounds over time was assessed. In vivo bioactivity (pharmacodynamic response) of compounds 64-53 and 64-54 after a single subcutaneous (paw) injection was assessed in wild-type mice, along with evaluation of local immune responses, measured as induction of TLR7-induced gene expression and maturation marker expression on antigen-presenting cells (APCs) in influx lymph node tissue, and evaluation of systemic responses, measured as TLR7-induced gene expression in spleen tissue. Along with evaluating the local immune response as measured by TLR7-induced gene expression, we also assessed the in vivo biological activity (pharmacodynamic response) in tumors after a single intratumoral injection of compounds 64-54a and 64-54b. The antitumor efficacy of compounds 64-53a, 64-54a, and 64-54b was assessed by measuring the time-dependent inhibition of tumor growth after repeated intratumoral administration in Balb / c mice with syngeneic CT26 tumors. (Example B1) In vitro cleavage of compounds 64-53, 64-52a, 64-54, and 64-54a with cathepsin B and glutathione. method

[0468] Compounds 64-53 and 64-53a (80 μM), as well as a non-cleavable control compound (compound number 64-70) containing only a single sulfur atom instead of a disulfide, were incubated in PBS (pH 7.5) with 5 mM glutathione at 37°C. At the indicated time, a fixed amount was taken from the reaction, 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) was activated with 4 mM DTT / EDTA by incubation in 10 mM acetate (pH 5) buffer at 37°C for 15 minutes. Next, an 80 μM solution was prepared using compounds no. 64-54 and 64-54a, and this reaction was carried out at 37°C. At the indicated time, a fixed amount was taken from the reactant, and the amount of IMDQ released was assessed as described in Example S8. result

[0470] The non-cleavable control compound demonstrated low levels of IMDQ release over a 26-hour incubation period, while compound numbers 64-53, containing a dimethyl disulfide-cleavable linker, demonstrated up to approximately 60% higher IMDQ release over the same timeframe in the presence of excess glutathione (Figure 4). Compound number 64-53a, containing a monomethyl disulfide variant, demonstrated up to approximately 80% higher IMDQ release under similar treatment conditions. Compound numbers 64-54 and 64-54a demonstrated up to approximately 80% higher IMDQ release in the presence of excess cathepsin B by the 6-hour incubation time point. These data are relevant to these constructs. The cleavable linker moieties, based on dipeptides and disulfides, have been shown to perform their functions in the expected manner (e.g., release of unmodified IMDQ). (Example B1a) In vitro cleavage of compounds 64-54a, 64-54b, and 64-54c using cathepsin B. method

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

[0472] In equation (I), the TLR7 / 8 agonist D is IMDQ, and the self-deactivating linker L 1 This is the para-aminobenzylcarbamate portion, and the linker L is cleavable. 2 However, it is a valine-citrulline dipeptide and conjugation linker L 3 Compound number 64-54a, an embodiment of the present invention in which the triazole-PEG4 moiety is a...

Claims

1. Equation (I): F-[W-L ３ -L ２ -L １ -D] ｘ (I) [In the formula, D is the TLR7 / 8 agonist section, L １ is a binding or self-deactivating linker, L ２ It is a linker that can be cut, L ３ It is a conjugation linker, W is O, S or NR １０ And, R １０ is H or C １ ~C ８ alkyl, x is an integer between 1 and 500. F is the conjugation part, The TLR7 / 8 agonist moiety is a 1H-imidazo[4,5-c]quinoline derivative. A compound of [unclear].

2. D is given by equation (D-1): 【Chemical Engineering 102】 [In the formula, R １Ａ C １ ~C ８ Alkyl, C １ ~C ８ Hydroxyalkyl or C ３ ~C ８ It is a cycloalkyl, R ２ NHR ２ａ And R ２ａ is H or C １ ~C ８ It is alkyl, R ３５ Each of these is independently halogen or C １ ~C ８ It is alkyl, R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, R ５ Each of these is independently halogen or C １ ~C ８ It is alkyl, p and q are independently 0, 1, 2, 3, or 4. The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

3. D is equation (D-2): 【Chemistry 103】 [In the formula, n is an integer between 4 and 21. X is -NH- or -NH(C=O)-, R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ３ Each of these is independently halogen and C １ ~C ８ Alkyl, -(C １ ~C ７ Alkilen)-NH ２ or -CH ２ -Phenylene-CH ２ NH ２ And, q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

4. D is given by equation (D-2a) or (D-2b): 【Chemical 104】 [In the formula, n is an integer between 4 and 21. R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ２０ NHR ２０ａ And R ２０ａ H, OH, NH ２ or methyl, R ３ is each, independently, halogen, C １ to C ８ alkyl, -(C １ to C ７ alkylene)-NH ２ or -CH ２ -phenylene-CH ２ NH ２ and q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

5. The compound according to claim 3, wherein X is -NH-.

6. The compound according to any one of claims 3 to 5, wherein n is an integer from 4 to 15.

7. The compound according to any one of claims 3 to 6, wherein n is 4, 5, 6, or 7.

8. The compound according to claim 3, wherein X is -NH(C=O)-.

9. The compound according to claim 4 or 8, wherein n is 11, 12, 13, or 14.

10. R １ However, C ３ ~C ６ A compound according to any one of claims 3 to 9, wherein it is alkyl.

11. R １ The compound according to claim 10, wherein n-butyl is present.

12. R １ ga- (CH ２ ) ｐ OR １ａ The compound according to any one of claims 3 to 9.

13. R １ However, - (CH ２ ) ｐ NHR １ｂ The compound according to any one of claims 3 to 9.

14. R １ However, - (CH ２ ) ｐ R １ｃ The compound according to any one of claims 3 to 9.

15. The compound according to any one of claims 3 to 14, wherein q is 0.

16. q is 1, R ３ C １ ~C ８ The compound according to any one of claims 3 to 14, wherein it is alkyl.

17. R ４ａ and R ４ｂ The compound according to any one of claims 3 to 16, wherein each of them is H.

18. D is equation (D-3): 【Chemistry 105】 [In the formula, R ０ C is substituted as needed by 1 to 4 halogen atoms. ４ ~C ２１ It is hydrocarbil, X is -NH- or -NH(C=O)-, R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ３ Each of these is independently halogen and C １ ~C ８ Alkyl, -(C １ ~C ７ Alkilen)-NH ２ or -CH ２ -Phenylene-CH ２ NH ２ And, q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

19. D is given by equation (D-3a) or (D-3b): 【Chemistry 106】 [In the formula, R ０ C is substituted as needed by 1 to 4 halogen atoms. ４ ~C ２１ It is hydrocarbil, R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ２０ NHR ２０ａ And R ２０ａ H, OH, NH ２ or methyl, R ３ Each of these is independently halogen and C １ ~C ８ Alkyl, -(C １ ~C ７ Alkilen)-NH ２ or -CH ２ -Phenylene-CH ２ NH ２ And, q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

20. The compound according to claim 18, wherein X is -NH(C=O)-.

21. The compound according to claim 18, wherein X is -NH-.

22. R ０ However, C is substituted as needed by one or two halogen atoms. ４ ~C １４ A compound according to any one of claims 18 to 21, wherein the compound is hydrocarbyl.

23. R ０ However, branched C ４ ~C １４ Alkyl, -(CH ２ ) ｚ (C(CH ３ ) ２ ) R Ａ or - (CH ２ ) ｍ R Ａ And m is 0, 1, 2 or 3, z is 1 or 2, R Ａ However, independently, C １ ~C ４ Alkyl, C １ ~C ４ C is optionally substituted with 1 to 4 groups selected from the group consisting of alkylenes and halogens. ３ ~C ８ The compound according to any one of claims 18 to 22, wherein it is a cycloalkyl compound.

24. R ０ However, branched C ４ ~C １４ The compound according to any one of claims 18 to 23, wherein it is alkyl.

25. R ０ However, - (CH ２ ) ｍ R Ａ The compound according to any one of claims 18 to 23.

26. The compound according to claim 25, wherein m is 2.

27. The compound according to claim 25, wherein m is 1.

28. R ０ However, - (CH ２ ) ｚ (C(CH ３ ) ２ ) R Ａ The compound according to any one of claims 18 to 23.

29. The compound according to claim 28, wherein z is 1.

30. R Ａ The compound according to any one of claims 25 to 29, wherein the compound is cyclopropyl, cyclobutyl, or cyclopentyl.

31. R Ａ However, C is independently substituted as needed with one to three groups selected from the group consisting of methyl, methylene, and halogen. ３ ~C ６ The compound according to any one of claims 25 to 29, wherein it is a cycloalkyl compound.

32. R Ａ However, C ３ ~C ８ The compound according to any one of claims 25 to 29, wherein it is a cycloalkyl compound.

33. R Ａ The compound according to claim 32, wherein the compound is a cyclopropyl that is independently and optionally substituted with one to three groups selected from the group consisting of methyl and methylene.

34. m is 0, R Ａ The compound according to claim 25, wherein the compound is a cyclohexyl which is independently and optionally substituted with one to three groups selected from the group consisting of methyl and methylene.

35. R ０ but, 【Chemistry 107】 A compound according to claim 18 or 19, selected from the group consisting of the following.

36. D is equation (D-4): 【Chemistry 108】 [In the formula, R ０ C is substituted as needed by 1 to 4 halogen atoms. ４ ~C ２１ It is hydrocarbil, L is X or -CH ２ -X-, X is -NH- or -NH(C=O)-, A is independently a halogen, and C is optionally substituted with 1 to 8 halogen atoms. １ ~C ８ C is optionally substituted with 1 to 4 groups selected from the group consisting of alkyl groups. ６ ~C １４ The C is either arylene or, independently, a halogen and, as needed, substituted with 1 to 8 halogen atoms. １ ~C ８ A 5-14 member heteroarylene which is optionally substituted with 1-4 groups selected from the group consisting of alkyl groups, R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ３ Each of these is independently halogen and C １ ~C ８ Alkyl, -(C １ ~C ７ Alkilen)-NH ２ or -CH ２ -Phenylene-CH ２ NH ２ And, q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

37. D is given by equation (D-4a) or (D-4b): 【Chemistry 109】 [In the formula, R ０ C is substituted as needed by 1 to 4 halogen atoms. ４ ~C ２１ It is hydrocarbil, A is independently a halogen, and C is optionally substituted with 1 to 8 halogen atoms. １ ~C ８ C is optionally substituted with 1 to 4 groups selected from the group consisting of alkyl groups. ６ ~C １４ The C is either arylene or, independently, a halogen and, as needed, substituted with 1 to 8 halogen atoms. １ ~C ８ A 5-14 member heteroarylene which is optionally substituted with 1-4 groups selected from the group consisting of alkyl groups, R １ C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ ,-(CH ２ ) ｐ NHR １ｂ or - (CH ２ ) ｐ R １ｃ And R １ａ and R １ｂ Independently, C １ ~C ３ It is alkyl, R １ｃ C ３ ~C ４ It is a cycloalkyl group, and p is 1 or 2. R ２０ NHR ２０ａ And R ２０ａ H, OH, NH ２ or methyl, R ３ Each of these is independently halogen and C １ ~C ８ Alkyl, -(C １ ~C ７ Alkilen)-NH ２ or -CH ２ -Phenylene-CH ２ NH ２ And, q is 0, 1, 2, 3, or 4. R ４ａ and R ４ｂ H or C １ ~C ８ It is alkyl, The wavy line indicates the connection point of D in equation (I). The compound according to claim 1, having the following characteristics.

38. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ C is optionally substituted with 1 to 4 groups selected from the group consisting of alkyl groups. ６ ~C １０ The compound according to claim 36 or 37, which is arylene.

39. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ The compound according to any one of claims 36 to 38, wherein the phenylene is optionally substituted with one to four groups selected from the group consisting of alkyl groups.

40. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ The compound according to any one of claims 36 to 39, which is 1,4-phenylene that is optionally substituted with one to four groups selected from the group consisting of alkyl groups.

41. A independently consists of F, Cl, and CF. ３ The compound according to any one of claims 36 to 40, which is 1,4-phenylene, optionally substituted with one to four groups selected from the group consisting of and methyl.

42. 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; The compound according to claim 41, which is 2,3,5,6-tetramethyl-1,4-phenylene; or 2,3,5,6-tetrafluoro-1,4-phenylene.

43. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ The compound according to any one of claims 36 to 39, which is 1,3-phenylene that is optionally substituted with one to four groups selected from the group consisting of alkyl groups.

44. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ The compound according to claim 36 or 37, which is a 5- to 10-membered heteroarylene optionally substituted with 1 to 4 groups selected from the group consisting of alkyl groups.

45. A is independently substituted with halogens and C as needed with 1 to 8 halogen atoms. １ ~C ８ The compound according to any one of claims 36 to 38, which is a naphthylene optionally substituted with one to four groups selected from the group consisting of alkyl groups.

46. The compound according to claim 45, wherein A is 1,4-naphthylene, 1,3-naphthylene, 2,6-naphthylene, or 2,7-naphthylene.

47. The compound according to claim 46, wherein A is 1,4-naphthylene.

48. The compound according to any one of claims 36, 37, or 44, wherein A is 4,7-benzo[b]thiophene.

49. The compound according to any one of claims 36, 37, or 44, wherein A is 2,5-1H-benzo[d]imidazole.

50. L is -CH ２ A compound according to claim 36 or any one of claims 38 to 49, wherein the compound is -X-.

51. The compound according to claim 36, or any one of claims 38 to 49, wherein L is X.

52. The compound according to claim 50 or 51, wherein X is -NH-.

53. The compound according to claim 50 or 51, wherein X is -NH(C=O)-.

54. R ０ The compound according to any one of claims 36 to 53, wherein the compound is (cyclopropyl)methyl, 2-(cyclopropyl)ethyl, 2-(cyclobutyl)ethyl, 2-(cyclopentyl)ethyl, or 2-(cyclohexyl)ethyl.

55. The compound according to claim 1, wherein D is selected from the compounds listed in Tables 1 to 3.

56. L １ However, equation (L-1): 【Chemical 110】 [In the formula, Y １ is S, O or NH, and Ar １ R is an arylene that is substituted as needed, １１ and R １２ This is independently H or C which is substituted as needed. １ ~C ８ It is alkyl. A compound according to any one of claims 1 to 55, having the following characteristics.

57. Ar １ The compound according to claim 56, wherein the compound is substituted 1,4-phenylene or substituted 1,2-phenylene as needed.

58. R １１ and R １２ The compound according to claim 56 or 57, wherein each of them is H.

59. Y １ The compound according to any one of claims 56 to 58, wherein is NH.

60. L １ but, 【Chemistry 111】 The compound according to claim 59.

61. Y １ The compound according to any one of claims 56 to 58, wherein is S.

62. L １ but, 【Chemistry 112】 The compound according to claim 61.

63. L １ The compound according to any one of claims 1 to 55, wherein the compound is bonded.

64. L １ -D is, 【Chemistry 113】 【Chemistry 114】 The compound according to claim 1.

65. L １ -D is, 【Chemical 115】 The compound according to claim 1.

66. L １ -D is, 【Chemistry 116】 【Chemistry 117】 The compound according to any one of claims 1, 64, or 65.

67. L ２ However, equation (L-2): 【Chemistry 118】 [In the formula, Y ２ NR ３０ , O or S, R ３０ , R ３１ , R ３２ , R ３３ and R ３４ These are H and C, independently. １ ~C ８ Alkyl or C ３ ~C ８ The compound according to any one of claims 1 to 66, having [being cycloalkyl].

68. L ２ but, 【Chemical 119】 The compound according to claim 67.

69. L ２ The compound according to any one of claims 1 to 66, wherein the compound is a peptide linker.

70. L ２ The compound according to claim 69, wherein the compound is a peptide linker that can be cleaved 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.

71. L ２ The compound according to claim 70, wherein the compound is a peptide linker that can be cleaved by an endosomal cathepsin or a pericellular protease.

72. The compound according to claim 71, wherein the endosomal cathepsin or pericellular protease is selected from the group consisting of cathepsin B, urokinase-type plasminogen activator (uPA), membrane-bound serine protease 1 (matryptase), matryptase-2, and regmine.

73. The peptide linker that can be cleaved by cathepsin B has the amino acid sequence AA １ -AA ２ -AA ３ -AA ４ This includes, here AA １ However, it is either absent, or is alanine, β-alanine, isoleucine, leucine, valine, or glycine. AA ２ However, it is either absent, or is alanine, β-alanine, isoleucine, leucine, or valine. AA ３ However, these are alanine, β-alanine, isoleucine, leucine, or valine. AA ４ The compound according to claim 72, wherein the compound is arginine, serine, alanine, β-alanine, leucine, ornithine, or citrulline.

74. L ２ but, 【Chemical 120】 The compound according to claim 73.

75. L ２ The compound according to claim 72, wherein the compound is a peptide linker that can be cleaved by urokinase-type plasminogen activator (uPA), membrane-bound serine protease 1 (matryptase), matryptase-2 and / or legmine.

76. L ２ but, 【Chemistry 121】 【Chemistry 122】 The compound according to claim 75.

77. L ３ However, -L ３ａ -Y ３ -L ３ｂ - and Y ３ , L ３ａ and L ３ｂ The compound according to any one of claims 1 to 76, which is independently a spacer fragment as needed.

78. Y ３ but, 【Chemical 123】 The compound according to claim 77.

79. Y ３ but, 【Chemistry 124】 The compound according to claim 77.

80. Y ３ but, 【Chemistry 125】 The compound according to claim 77.

81. Y ３ but, 【Chemistry 126】 The compound according to claim 77.

82. L ３ａ A compound according to any one of claims 77 to 81, wherein the compound is not present.

83. L ３ａ but, 【Chemistry 127】 The compound according to any one of claims 77 to 81.

84. L ３ａ but, 【Chemistry 128】 The compound according to any one of claims 77 to 81.

85. L ３ａ but, 【Chemistry 129】 The compound according to any one of claims 77 to 81.

86. L ３ｂ However, the formula is: 【Chemistry 130】 The acyl spacer fragment, or formula: 【Chemistry 131】 [In the formula, n is between 0 and 200.] The compound according to any one of claims 77 to 85, which is a PEG-acyl spacer fragment.

87. L ３ｂ However, the formula is: 【Chemistry 132】 The acyl spacer fragment or formula: 【Chemistry 133】 [In the formula, n is between 0 and 200.] The compound according to claim 86, which is a PEG-acyl spacer fragment.

88. L ３ but, 【Chemistry 134】 The compound according to claim 77.

89. L ３ but, 【Chemistry 135】 The compound according to claim 77.

90. L ３ but, 【Transformation 136】 The compound according to claim 77.

91. L ３ but, 【Chemistry 137】 The compound according to claim 77.

92. -L ３ -L ２ -L １ - Part D is 【Chemistry 138】 The compound according to claim 1.

93. -L ３ -L ２ -L １ - Part D is 【Chemistry 139】 The compound according to claim 1.

94. -L ３ -L ２ -L １ - Part D is [Chemical 140] The compound according to claim 1.

95. -L ３ -L ２ -L １ - Part D is 【Chemistry 141】 The compound according to claim 1.

96. A compound according to any one of claims 1 to 95, wherein F is a tumor targeting agent.

97. The compound according to claim 96, wherein the tumor targeting agent is an antibody or binding ligand that preferentially binds to tumor cell surface antigens, specific structural elements of the extracellular matrix of the tumor microenvironment, or specific structural elements of tumor blood vessels.

98. The compound according to claim 96 or 97, wherein F has physical properties or surface chemical modifications designed to result in preferential distribution to and / or retention within the tumor microenvironment.

99. The compound according to any one of claims 96 to 98, wherein F is an antibody of the IgG1, IgG2, or IgG4 class, or a derivative or fragment thereof.

100. F is a derivative that does not contain an Fc region selected from the group consisting of a bivalent monospecific antibody, a bivalent bispecific antibody, or a single-chain variable fragment (ScFv), a tandem bivalent scFv, a diabody, a tandem trivalent scFv, a triabody, and a bispecific tandem bivalent scFv, and the formula is ( I) The compound according to claim 99, wherein x is an integer between 1 and 10.

101. The compound according to any one of claims 1 to 95, wherein F is an agent that facilitates the local retention of the compound of formula (I).

102. The compound according to claim 101, wherein F is a liposome, virus-like particle, nanoparticle, microparticle, macromolecule or supramolecular, dendrimer, or polypeptide.

103. The compound according to claim 101 or 102, wherein F is a branched copolymer of sucrose and epichlorohydrin having a molecular weight of about 100,000 to about 700,000 daltons.

104. The compound according to claim 103, wherein the molecular weight is approximately 300,000 to approximately 500,000 daltons.

105. The compound according to claim 103 or 104, wherein in formula (I), W is O and x is an integer from 3 to 500.

106. The compound according to claim 105, wherein x is an integer between approximately 30 and 150.

107. (i) a compound according to any one of claims 1 to 106, and (ii) a pharmaceutically acceptable excipient, comprising a pharmaceutical composition.

108. The compound is a pharmaceutical composition that can stimulate cytokine production by mammalian leukocytes, and the following: Stimulating IFNα production by human peripheral blood mononuclear cells To stimulate the production of IL-6 and TNFα by human monocytes, and Stimulating the production of one or both IL-12p40 and IL-6 by mouse splenocytes. The pharmaceutical composition according to claim 107, comprising one or more of the group consisting of the following.

109. A method for stimulating an immune response in a mammalian subject requiring stimulation of an immune response, comprising the step of administering to the mammalian subject an amount sufficient to stimulate an immune response in the mammalian subject of the pharmaceutical composition according to claim 107 or 108.

110. A method for inducing an antigen-specific antibody or antigen-specific T cell response in a mammalian subject that requires induction of an antigen-specific antibody or antigen-specific T cell response, comprising the step of administering to the mammalian subject a pharmaceutical composition according to claim 107 or 108 in an amount sufficient to induce an antigen-specific antibody response and / or an antigen-specific T cell response in the mammalian subject.

111. A method for treating cancer in a mammalian subject requiring treatment, comprising the step of administering to the mammalian subject a pharmaceutical composition according to claim 107 or 108 in an amount sufficient to treat cancer in the mammalian subject.

112. Compound of formula (A): F-[W-L ３ａ -Y ３ａ ] ｙ (A) The compound of formula (B): 9 ３ｂ -8 ３ｂ -8 ２ -8 １ -X (B) [In the formula, y is an integer between 1 and 500, and W and L are integers.] ２ , L １ and D are defined in claim 1 As stated, F is a particle-based conjugation part, L ３ａ and L ３ｂ These are spacer fragments as needed, Y ３ａ and Y ３ｂ They react with each other, spacer fragment Y ３ This is the precursor portion that forms L ３ａ , Y ３ and L ３ｂ Together, L ３ [to form] and react with The step of forming the compound of formula (I) A method for preparing the compound according to claim 1, comprising:

113. Y ３ａ However, it is an alkyne group, Y ３ｂ However, it is an azide group, Y ３ The method according to claim 112, wherein the moiety is 1,4-[1,2,3]triazolylene.

114. L ３ａ However, it is an amide spacer fragment, L ３ｂ The method according to claim 112, wherein the object is an acyl spacer fragment or a PEG-acyl spacer fragment.

115. Compound of formula (C): F-[W’] ｘ (C) The compound of formula (D): L ３ -8 ２ -8 １ -D (D) [In the formula, x is an integer from 1 to 50, L ３ , L ２ , L １ And D are as defined for the compound of formula (I), F is an antibody-based conjugation moiety, and W' is N, O, S, N ３ Or react with an alkyne] The step of forming the compound of formula (I) A method for preparing the compound according to claim 1, comprising:

116. Equation (I-a): F-[W-L ３ -L ２ -L １ -D] ｘ (I-a) [In the formula, D is the TLR7 / 8 agonist section, L １ It is a self-deactivating linker, L ２ It is a linker that can be cut, L ３ It is a conjugation linker, W is O, S or NR １０ And, R １０ is H or C １ ~C ８ It is alkyl, x is an integer between 1 and 500. F is the conjugation part, The TLR7 / 8 agonist portion is a 1H-imidazo[4,5-c]quinoline derivative. A compound of [unclear].

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