Alkyl chain modified imidazoquinoline TLR7 / 8 agonist compounds and uses thereof

Abstract

To provide alkyl chain modified imidazoquinoline TLR7 / 8 agonist compounds and uses thereof.SOLUTION: Disclosed are alkyl chain modified 1H-imidazoquinoline compounds of the formula (J), serving as Toll-like receptor-7 and -8 agonists for enhancing immune responses, and a salt thereof. Also provided are methods of making pharmaceutical compositions containing these compounds. The present disclosure also describes methods of use for these compounds and pharmaceutical compositions containing these compounds for the treatment of diseases such as infection and cancer in a subject. The present disclosure provides alkyl chain modified 1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7 / 8 agonists exhibiting balanced bioactivity against both receptors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 548,848, filed August 22, 2017, the entire disclosure of which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to alkyl chain-modified imidazoquinoline TLR7 / 8 agonist compounds for enhancing immune responses. The present disclosure also relates to pharmaceutical compositions comprising the alkyl chain-modified imidazoquinoline compounds, methods for preparing the same, methods for stimulating immune responses, and uses of the pharmaceutical compositions in treating diseases (e.g., infectious diseases and cancers) in subjects. [Background technology]

[0003] Background of the Invention Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize pathogen-derived and pathogen-specific structurally conserved molecules called pathogen-associated molecular patterns. Thus, TLRs function as frontline sensors of pathogen-associated molecular patterns in the mammalian immune system to detect the presence of invading pathogens (Takeuchi and Akira 2010 Cell 140:805-820). Engagement of TLRs on sentinel immune cells triggers the biosynthesis of selected cytokines (e.g., type I interferon), induction of costimulatory molecules, and enhanced antigen-presenting ability. These are important molecular mechanisms that activate innate and adaptive immune responses. Therefore, TLR agonists and antagonists are used to regulate immune responses. TLR agonists are typically used to stimulate immune responses, whereas TLR antagonists are typically used to inhibit immune responses (Gosu et al 2012 Molecules 17:13503-13529).

[0004] The human genome contains 10 known TLRs, of which TLR3, TLR7, TLR8, and TLR9 sense nucleic acids and their degradation products. The distribution of TLR7, TLR8, and TLR9 is restricted to the endolysosomal compartment of cells, and they are preferentially expressed in cells of the immune system. In the activated dimeric receptor configuration, TLR7 and TLR8 each recognize single-stranded RNA in one ligand-binding site and the ribonucleoside degradation products guanosine and uridine (and small molecule ligands with related structural motifs) in the second ligand-binding site (Zhang et al., 2016 Immunity 45:737-748; Tanji et al., 2015). Nat Struct Mol Biol 22:109-115). Engagement of TLR7 on plasmacytoid dendritic cells induces interferon-α / β, which plays an essential role in regulating adaptive immune responses (Bao and Liu 2013 Protein Cell 4:40-52). Engagement of TLR8 on myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells induces a prominent pro-inflammatory cytokine profile characterized by increased production of tumor necrosis factor-α, interleukin-12, and IL-18 (Eigenbrod et al 2015 J Immunol 195:1092-1099). Thus, virtually all major types of monocytes and dendritic cells can be activated by TLR7 and TLR8 agonists to become effective antigen-presenting cells, thereby effectively promoting innate and adaptive immune responses. Possesses potent agonist bioactivity for both TLR7 and TLR8 receptors, as most antigen-presenting cell types express only one of these two receptors Small molecules are potentially more effective immune adjuvants than agonists specific for only one of these TLRs. Thus, TLR7 / TLR8 (TLR7 / 8) small molecule agonists with balanced dual biological activity will elicit innate immune responses in a broader range of antigen-presenting cells and other important immune cell types, including plasmacytoid and myeloid dendritic cells, monocytes, and B cells (van Haren et al., 2016 J Immunol 197:4413-4424; Ganapathi et al., 2015 PLoS One 10:e0134640). Such potent dual TLR7 / 8 agonists may also be effective in stimulating effective anti-tumor immune responses in cancer (Singh et al 2014 J Immunol 193:4722-4731; Sabado et al 2015 Cancer Immunol Res 3:278-287; Spinetti et al 2016 Oncoimmunol 5:e1230578; Patil et al 2016 Mini Rev Med Chem 16:309-322).

[0005] Several small molecule structural classes are known to interact at the guanosine / uridine ligand binding site and have varying levels of TLR7 and / or TLR8 agonist biological activity (see, e.g., Lu et al. 2012 Clin Cancer Res 18:499-509; U.S. Patent Nos. 5,446,153, 6,194,425, 6,110,929, and 7,199,131), including derivatives of 1H-imidazo[4,5-c]quinoline that are TLR7 agonists or dual TLR7 / 8 agonists (see, e.g., Vasilakos and Tomai 2013 Expert Rev Vaccines 12:809-819; U.S. Patent No. 4,689,338). One such 1H-imidazo[4,5-c]quinoline is 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod), a TLR7-specific agonist approved in 1997 for the treatment of actinic keratosis, superficial basal cell carcinoma, and genital warts, and subsequently for the treatment of basal cell carcinoma (see, e.g., Hemmi et al 2002 Nat Immunol 3:196-200). Some 1H-imidazo[4,5-c]quinolines exhibit selective TLR7 or TLR8 agonist activity, while others exhibit dual TLR7 / 8 agonist activity. For example, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine was found to be a TLR7 agonist with little biological activity against TLR8 (Shukla et al 2010 J Med Chem 53:4450-4465). In contrast, 2-propyl[1,3]thiazolo[4,5-c]quinolin-4-amine was found to be a TLR8 agonist with little activity against TLR7 (Gorden et al 2005 J Immunol 174:1259-1268).1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ) and 1-(3-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (meta-IMDQ) have been found to be dual TLR7 / 8 agonists with potent agonist activity against both receptors (see, e.g., Shukla et al 2010 J Med Chem 53:4450-4465; Shukla et al 2010 Bioorg Med Chem Lett 10:6384-6386; U.S. Pat. No. 8,728,486; U.S. Pat. No. 9,441,005). However, the rapid systemic distribution of soluble 1H-imidazo[4,5-c]quinoline-based TLR7 / 8 agonists after subcutaneous, intratumoral, or intramuscular administration has been demonstrated to be highly toxic to patients (see, e.g., Vasilakos et al. 2013 Expert Rev Vaccines 12:809-819; Savage et al. 1996 Br J Cancer 74:1482-1486; Pockros et al. 2007 J Hepatol 47:174-182). Activation of TLRs in spleen and liver cells activates the systemic immune system. Injection of these compounds increases serum proinflammatory cytokine levels, which can cause influenza-like symptoms and other adverse events, limiting their use as human therapeutics to local administration routes. Thus, there remains a need for small molecule therapeutic agents that possess potent and balanced TLR7 / 8 agonist activity and also possess physiochemical properties that enable pharmaceutical compositions to promote compound retention at the injection site. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 5,446,153 [Patent Document 2] U.S. Patent No. 6,194,425 [Patent Document 3] U.S. Patent No. 7,199,131 [Non-patent literature]

[0007] [Non-Patent Document 1] Takeuchi and Akira 2010 Cell 140:805-820 [Non-patent document 2] Gosu et al 2012 Molecules 17:13503-13529 [Non-patent document 3] Zhang et al 2016 Immunity 45:737-748 [Non-patent document 4] Tanji et al 2015 Nat Struct Mol Biol 22:109-115 [Non-patent document 5] Bao and Liu 2013 Protein Cell 4:40-52 [Non-patent document 6] Eigenbrod et al 2015 J Immunol 195:1092-1099 Summary of the Invention [Means for solving the problem]

[0008] The present disclosure provides alkyl chain-modified 1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7 / 8 agonists that exhibit balanced biological activity against both receptors. In one embodiment, the compounds of formula (J): [ka] [In the formula, R 0 is a C4-C optionally substituted with 1 to 4 halogen atoms 21 is a hydrocarbyl; X is -NH- or -NH(C=O)-; R 1is 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 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R 3 are 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, or a salt thereof; Provided is a compound or a salt thereof, wherein the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-32); N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31); or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide (Compound No. 63-37). In some embodiments, R 0 is C4~C 14 It is a hydrocarbyl.

[0009] In some embodiments, X is -NH(C=O)-. In some embodiments, X is -NH-.

[0010] In some embodiments, R0 is branched C4 to C 14 Alkyl, -(CH2) z (C(CH3)2)R A , or -(CH2) m R A m is 0, 1, 2, or 3; z is 1 or 2; R A is a C3-C8 cycloalkyl optionally substituted with 1 to 4 groups independently selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene.

[0011] In some embodiments, R 0 Branched C4 to C 14 It is alkyl.

[0012] In some embodiments, R 0 Ha-(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0013] In some embodiments, R 0 Ha-(CH2) z (C(CH3)2)R A In one variation, z is 1 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0014] In some embodiments, R A is a C3-C8 cycloalkyl.

[0015] In some embodiments, R A is C3-C6 cycloalkyl optionally substituted with 1-3 groups independently selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2.

[0016] In some embodiments, R Ais cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, and m is 1 or 2.

[0017] In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0018] In some embodiments, R 0 is selected from the group consisting of: [ka]

[0019] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). In some embodiments, R 1 Ha-(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 Ha-(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 Ha-(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0020] In some embodiments, R 2 is NH2.

[0021] In some embodiments, q is 0. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0022] In some embodiments, R 4a and R 4bEach of is H.

[0023] In some embodiments, the compound is selected from the group consisting of compound numbers 63-33 to 63-36 and 63-38 to 63-49 in Table 1, or a salt thereof.

[0024] In another embodiment, a compound of formula (K): [ka] [In the formula, n is an integer from 4 to 21; X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b , or -(CH2) p R 1c where R 1a and R 1b are independently C1-C3 alkyl; R 1c is C3-C4 cycloalkyl; p is 1 or 2; R 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R 3 are 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, or a salt thereof; Provided is a compound or a salt thereof, wherein the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-32) or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31).

[0025] In some embodiments, X is -NH-. In one variation, n is an integer from 4 to 15. In one variation, n is 4, 5, 6, or 7.

[0026] In some embodiments, X is -NH(C=O)-. In one variation, n is 11, 12, 13, or 14.

[0027] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl). In some embodiments, R 1 Ha-(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 Ha-(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 Ha-(CH2) p R 1c In some embodiments, R 1c is cyclopropyl.

[0028] In some embodiments, R 2 is NH2.

[0029] In some embodiments, q is 0. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0030] In some embodiments, R 4a and R 4b Each of is H.

[0031] In some embodiments, the compound is selected from the group consisting of compound numbers 63-01 to 63-30 in Table 1, or a salt thereof.

[0032] Further provided are pharmaceutical compositions comprising (i) a compound of Formula (J) or (K) and (ii) one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition further comprises an antigen. In some embodiments, the pharmaceutical composition is comprised of pharmaceutically acceptable excipients including USP grade oils and organic modifiers (e.g., 95% sesame oil / 5% ethanol). In some embodiments, the pharmaceutical composition is comprised of pharmaceutically acceptable excipients that allow for oil-in-water nanoemulsion or liposomal formulations, examples of which are known to those skilled in the art. In some embodiments, the pharmaceutical composition can comprise a mixture of antigen(s), including, but not limited to, tumor-associated antigens or neoantigens.

[0033] The present disclosure also provides a method for stimulating an immune response in a mammalian subject in need thereof, comprising administering to the mammalian subject the pharmaceutical composition described above in an amount, frequency, and time frame sufficient to stimulate an immune response in the mammalian subject.In one embodiment, the immune response is a local immune response.In another embodiment, the immune response is a systemic immune response.

[0034] The present disclosure also provides methods for using the pharmaceutical compositions described above in mammalian subjects, such as human patients. In one embodiment, a method for treating cancer in a mammalian subject in need thereof is provided, comprising administering to the mammalian subject a pharmaceutical composition in an amount sufficient to treat the cancer in the mammalian subject. In another embodiment of the method, intratumoral delivery comprises injecting the pharmaceutical composition into at least one tumor lesion. In one embodiment of the method, an effective amount of a second therapeutic agent is further administered to the subject. In certain embodiments, the second therapeutic agent is a chemotherapeutic agent, an epigenetic modulator, an inducer of immunogenic cell death, or an antagonist of an inhibitory immune checkpoint molecule. In another embodiment, a method for inducing an antigen-specific antibody response in a mammalian subject in need thereof is provided, comprising administering to the mammalian subject a pharmaceutical composition in an amount sufficient to induce an antigen-specific antibody response and / or an antigen-specific T cell response in the mammalian subject. In one embodiment, there is provided a method of treating or preventing an infectious disease in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a pharmaceutical composition in an amount sufficient to treat or prevent the infectious disease in the mammalian subject. In one embodiment, there is provided a method of treating or preventing an IgE-related disorder in a mammalian subject, the method comprising administering to the mammalian subject a pharmaceutical composition in an amount sufficient to treat or prevent the IgE-related disorder in the mammalian subject.

[0035] Also provided herein are kits comprising the pharmaceutical compositions of the invention and instructions for use in treating infectious diseases and / or cancer. Methods for producing kits for use in treating infectious diseases and / or cancer are also provided. [Brief explanation of the drawings]

[0036] [Figure 1]1A-B show the time course of serum levels of IL-6 (FIG. 1A) and IL-12p40 (FIG. 1B) following a single subcutaneous injection of Compound Nos. 63-00 (black bars), 63-17 (white bars), and 63-10 (gray bars) into wild-type mice as described in Example B3. Group size = 3, + / - standard error of the mean.

[0037] [Figure 2-1] Figures 2A-C show tumor growth inhibition in syngeneic CT26 tumor-bearing wild-type mice after repeated intratumoral administration of compound Nos. 63-18 (Figure 2A), 63-33 (Figure 2B), or 63-10 (Figure 2C) as described in Example B4. Animals were administered either vehicle control (__●__), 20 μg of compound (__■__), 5 μg of compound (--■--), 0.5 μg of compound (--■--), or 50 μg of TLR9 agonist positive control (__◆__), as indicated. Group size = 5 for control, 8 for experimental condition, + / - standard error of the mean. [Figure 2-2] Figures 2A-C show tumor growth inhibition in syngeneic CT26 tumor-bearing wild-type mice after repeated intratumoral administration of compound Nos. 63-18 (Figure 2A), 63-33 (Figure 2B), or 63-10 (Figure 2C) as described in Example B4. Animals were administered either vehicle control (__●__), 20 μg of compound (__■__), 5 μg of compound (--■--), 0.5 μg of compound (--■--), or 50 μg of TLR9 agonist positive control (__◆__), as indicated. Group size = 5 for control, 8 for experimental condition, + / - standard error of the mean.

[0038] [Figure 3]Figures 3A-B show tumor growth inhibition over time in the injected tumor (Figure 3A) and distal tumor (Figure 3B) of CT26 tumor-bearing wild-type mice after repeated intratumoral administration of a pharmaceutical composition consisting of a squalene-based oil-in-water nanoemulsion vehicle control (__●), a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 (__■__), or a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 plus 50,000 ng of AH-1 class II peptide (__□__) as described in Example B5. Animals were dosed as described in Experiment 8, 12, 16, and 20. Group size = 8 for control and all experimental conditions. Data are expressed as mean tumor volume (in mm) + / - standard error of the mean. Differences in tumor volume between groups for injected tumors on day 27 or distant tumors on day 23 were analyzed using the Kruskall-Wallis test followed by Dunn's post hoc test for comparison of specific group pairs. ns indicates P≥0.050; * indicates P≤0.050.

[0039] [Figure 4]Figures 4A-B show tumor growth inhibition over time in the injected tumor (Figure 4A) and distant tumor (Figure 4B) of CT26 tumor-bearing wild-type mice following a single intratumoral administration on day 14 of a pharmaceutical composition consisting of a phosphate-buffered saline vehicle control (__●__), a phosphate-buffered saline vehicle control in combination with 250 μg of anti-PD-1 antibody (__○__), 5,000 ng of Compound No. 63-10 in 95% sesame oil / 5% ethanol (v / v) in combination with 250 μg of anti-PD-1 antibody (__■__), 5,000 ng of Compound No. 63-33 in 95% sesame oil / 5% ethanol (v / v) in combination with 250 μg of anti-PD-1 antibody (__▲__), or 5,000 ng of Compound No. 63-00 in phosphate-buffered saline in combination with 250 μg of anti-PD-1 antibody (__▼__) as described in Example B6. For all experimental groups using anti-PD-1 combinations, anti-PD-1 treatment was administered intraperitoneally on experimental days 12, 15, 19, 22, and 26. Group size = 10 for control and all experimental conditions, and data are expressed as mean tumor volume (in mm) + / - standard error of the mean. Differences in tumor volume between groups on experimental day 29 were analyzed using the Kruskall-Wallis test followed by Dunn's post-hoc test for comparisons of specific group pairs. ns indicates P > 0.050; * indicates P < 0.050; ** indicates P < 0.010. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure relates to alkyl-chain-modified 1H-imidazo[4,5-c]quinoline derivatives that are potent TLR7 / 8 agonists that exhibit balanced biological activity against both TLR7 / 8 receptors and have physiochemical properties that enable pharmaceutical compositions to promote retention of the compound at the injection site. The present disclosure also relates to pharmaceutical compositions containing the alkyl-chain-modified 1H-imidazo[4,5-c]quinoline compounds and methods for preparing them, uses of the pharmaceutical compositions to stimulate immune responses, and methods for treating diseases (e.g., infectious diseases and cancer) in subjects. I. General Methods and Definitions

[0041] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic chemistry, analytical chemistry, molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully described in the literature; see, e.g., Fiesers' Reagents for Organic Synthesis, 25 th edition(Ho,ed.,Wiley,2016);Comprehensive Organic Functional Group Transformations,2 nd edition(Katritsky and Taylor, eds., Elsevier, 2004);Comprehensive Organic Synthesis, version 1-8(Trost and Flemming, eds., Permagon Press, 1991);Beilsteins Handbuch der Organischen Chemie, 4 (Auflage, ed., Springer-Verlag, 1934);Animal Cell Culture, sixth edition (Freshney, Wiley-Blackwell, 2010); Current Protocols in Cell Biology (Bonifacino et al., eds., John Wiley & Sons, Inc., 1996 (including 2014 supplement)); Current Protocols in Immunology (Coligan et al., eds., John Wiley & Sons, Inc., 1991 (including 2014 supplement)); Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley & Sons, Inc., 1987 (including 2014 supplement)); Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russell, Cold Spring Harbor Laboratory Press, 2001); and Molecular Cloning: A Laboratory Manual, fourth edition (Green and Sambrook, Cold Spring Harbor Laboratory Press, 2012).

[0042] The terms "individual" and "subject" refer to mammals, including, but not limited to, humans, non-human primates (e.g., monkeys), farm animals, sport animals (e.g., horses), rodents (e.g., mice and rats), and pets (e.g., dogs and cats).

[0043] The term "antigen" refers to a substance that is specifically recognized and bound by an antibody or T cell antigen receptor. Antigens can include peptides, polypeptides, proteins, glycoproteins, polysaccharides, glycoconjugates, sugars, gangliosides, lipids, and phospholipids; portions thereof, and combinations thereof. When present in the compositions of the present disclosure, antigens can be synthetic or naturally isolated. Antigens suitable for administration in the methods of the present disclosure include any molecule that can induce an antigen-specific B cell response or an antigen-specific T cell response. Haptens are included within the scope of "antigen." A "hapten" is a low-molecular-weight compound that is not immunogenic by itself, but generally becomes immunogenic when conjugated to a larger immunogenic molecule.

[0044] "Polypeptide antigens" may include purified natural peptides, synthetic peptides, engineered peptides, recombinant peptides, crude peptide extracts, or partially purified or unpurified active peptides (such as peptides that are part of attenuated or inactivated viruses, microorganisms, or cells), or fragments of such peptides. Polypeptide antigens are preferably at least 6 amino acid residues in length, preferably 8 to 1800 amino acids in length, more preferably 9 to 1000 amino acids in length, or 10 to 100 amino acids in length. Similarly, in some embodiments, the polypeptide is about 9 to about 2000, about 9 to about 1000, about 9 to about 100, or about 9 to about 60 amino acids in length. In some embodiments, 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 is at most (upper limit) 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 150, 100, 50, or 25 amino acids in length. In some embodiments, the polypeptide antigen is 9 to 35 amino acids in length.

[0045] As used herein, the term "immunogenic" refers to an agent (e.g., a polypeptide antigen) that elicits an adaptive immune response when administered to a mammalian subject under appropriate conditions. The immune response can be a B cell (humoral) and / or a T cell (cellular)-mediated response.

[0046] "Adjuvant" refers to a substance that, when mixed with an immunogenic agent, such as an antigen, nonspecifically increases or enhances the immune response to the agent in a recipient exposed to the mixture.

[0047] The term "agonist" is used in the broadest sense and includes any molecule that activates receptor-mediated signal transduction. For example, a TLR7 agonist binds to Toll-like receptor 7 protein and activates the TLR7 signal transduction pathway; a TLR8 agonist binds to Toll-like receptor 8 protein and activates the TLR8 signal transduction pathway. A dual TLR7 / 8 agonist binds to both Toll-like receptor 7 protein and Toll-like receptor 8 protein and activates both the TLR7 signal transduction pathway and the TLR8 signal transduction pathway.

[0048] "Stimulation" of a response or parameter includes eliciting and / or increasing that response or parameter when compared to otherwise identical conditions other than the agent or molecule, or when compared to another condition (e.g., increased 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 the response.

[0049] An "effective amount" of an agent disclosed herein is an amount sufficient to accomplish a specifically stated purpose. An "effective amount" can be determined empirically and routinely for the stated purpose. An "effective amount" or "sufficient amount" of an agent is an amount appropriate to produce a desired biological effect (such as a beneficial result, including a beneficial clinical outcome). The term "therapeutically effective amount" refers to an amount of an agent (e.g., a TLR modulator) effective to "treat" a disease or disorder in a subject (e.g., a mammal such as a human).

[0050] The term "treating" a disease or "treatment" of a disease refers to carrying out a protocol that may include administering one or more drugs to an individual (human or otherwise) with the goal of alleviating the signs or symptoms of the disease. Thus, "treating" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes protocols that only have a palliative effect on the individual. As used herein, and as is well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include the reduction or amelioration of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, a stabilized (i.e., not worsening) disease state, prevention of the spread of the disease, a delay or slowing of the progression of the disease, amelioration or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival for an individual not receiving treatment.

[0051] "Alleviating" a disease or disorder means lessening the severity and / or undesirable clinical symptoms of the disease or disorder, and / or slowing down the time course of progression of the disease or disorder, compared to the expected outcome of no treatment. In particular, in the context of allergy, alleviation may occur when a Th1 immune response is stimulated against an allergen(s). Furthermore, alleviation does not necessarily occur with a single administration, but often occurs with successive administrations. Thus, an amount sufficient to alleviate a response or disorder may be administered once or multiple times.

[0052] As used herein, "alkyl" refers to a saturated, straight-chain (i.e., unbranched) or branched monovalent hydrocarbon chain or combinations thereof. Particular alkyl groups include alkyl groups having a specified number of carbon atoms, e.g., 1 to 20 carbon atoms ("C1-C6"). 20 alkyl), 1 to 10 carbon atoms (C1 to C 10" alkyl"), 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, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers of, e.g., n-pentyl, n-hexyl, n-heptyl, and n-octyl.

[0053] As used herein, "alkenyl" refers to an unsaturated, linear (i.e., unbranched) or branched monovalent hydrocarbon chain, or combinations thereof, having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C). Particular alkenyl groups are alkenyl groups having a specified number of carbon atoms, e.g., 2 to 20 carbon atoms ("C-C"). 20 alkenyl), 2 to 10 carbon atoms ("C2-C 10 "C-C alkenyl"), alkenyl groups having 2 to 8 carbon atoms ("C-C alkenyl"), 2 to 6 carbon atoms ("C-C alkenyl"), or 2 to 4 carbon atoms ("C-C alkenyl"). Alkenyl groups can be in the "cis" or "trans" configuration, or in the "E" or "Z" configuration. Examples of alkenyl groups include, but are not limited to, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs, and isomers thereof.

[0054] As used herein, "alkynyl" refers to an unsaturated, linear (i.e., unbranched) or branched monovalent hydrocarbon chain, or combinations thereof, having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). Particular alkynyl groups are alkynyl groups having a specified number of carbon atoms, e.g., 2 to 20 carbon atoms ("C-C"). 20 alkynyl), 2 to 10 carbon atoms (C2 to C 10 Examples of alkynyl groups include alkynyl groups having 2 to 8 carbon atoms ("C-C alkynyl"), 2 to 6 carbon atoms ("C-C alkynyl"), or 2 to 4 carbon atoms ("C-C alkynyl"). Examples of alkynyl groups include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs, and isomers thereof.

[0055] As used herein, "alkylene" refers to the same residue as alkyl, but which is divalent. Particular alkylene groups include those containing 1 to 6 carbon atoms ("C1-C6 alkylene"), 1 to 6 carbon atoms ("C1-C6 alkylene"). Alkylene groups having 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, groups such as methylene (-CH2- or ═CH2), ethylene (-CH2CH2- or ═CHCH3), propylene (-CH2CH2CH2- or ═CHCH2CH3), and butylene (-CH2CH2CH2CH2- or ═CHCH2CH2CH3).

[0056] As used herein, "cycloalkyl" refers to a non-aromatic, saturated or unsaturated monovalent cyclic hydrocarbon structure. Particular cycloalkyl groups are cycloalkyl groups having a specified number of cyclic (i.e., ring) carbon atoms (e.g., cycloalkyl groups having 3 to 12 cyclic carbon atoms ("C3-C6"). 12Preferred cycloalkyls are cyclic hydrocarbons having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl") or 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). Cycloalkyls can consist of one ring (e.g., cyclohexyl) or multiple rings (e.g., adamantyl), but aryl groups are excluded. Cycloalkyls containing more than one ring can be fused, spiro, bridged, or combinations thereof. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and norbornyl.

[0057] As used herein, "cycloalkylene" refers to the same residue as cycloalkyl, but which is divalent. Particular cycloalkylene groups are those having 3 to 12 ring carbon atoms ("C3-C6"). 12 Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, 1,2-cyclohexenylene, 1,3-cyclohexenylene, 1,4-cyclohexenylene, cycloheptyl, and norbornyl. As used herein, "hydrocarbyl" refers to a cycloalkyl group having a specified number of carbon atoms (i.e., C1-C6 cycloalkylene), 3 to 8 ring carbon atoms ("C3-C8 cycloalkylene"), or 3 to 6 ring carbon atoms ("C3-C6 cycloalkylene"). Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, 1,2-cyclohexenylene, 1,3-cyclohexenylene, 1,4-cyclohexenylene, cycloheptyl, and norbornyl. As used herein, "hydrocarbyl" refers to a cycloalkyl group that may be fully saturated, monounsaturated, or polyunsaturated and has a specified number of carbon atoms (i.e., C1-C6). 20refers to and includes monovalent groups formed by the removal of hydrogen atoms from a non-aromatic hydrocarbon having 1 to 20 carbon atoms (wherein "hydrocarbyl" means 1 to 20 carbon atoms). Hydrocarbyl groups can contain one or more straight-chain, branched, or cyclic moieties, or combinations thereof. Alkyl, alkenyl, alkynyl, and cycloalkyl groups are specific subsets of hydrocarbyl groups. Hydrocarbyl groups can also include alkyl, alkenyl, or alkynyl groups further substituted with one or more cycloalkyl groups; and / or cycloalkyl groups further substituted with one or more alkyl, alkenyl, and / or alkynyl groups. Hydrocarbyl groups can be substituted in one or more positions with one or more halogen atoms (such as chlorine or fluorine). Examples of hydrocarbyl groups include, for example, the following: [ka] Examples include, but are not limited to, groups such as:

[0058] As used herein, "aryl" refers to an unsaturated, aromatic, carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), where one or more of the condensed rings may be non-aromatic. Particular aryl groups are aryl groups having 6 to 14 cyclic (i.e., ring) carbon atoms ("C6-C6"). 14 Aryl groups having more than one ring, wherein at least one ring is non-aromatic, can be attached to the parent structure at either an aromatic ring position or a non-aromatic ring position. In one variation, aryl groups having more than one ring, wherein at least one ring is non-aromatic, are attached to the parent structure at an aromatic ring position. Examples of aryl include, but are not limited to, groups such as phenyl, naphthyl, 1-naphthyl, and 2-naphthyl.

[0059] As used herein, "arylene" refers to the same residue as aryl, but which is divalent. Particular arylene groups are arylene groups having 6 to 14 ring carbon atoms ("C6-C 14 Examples of arylene include, but are not limited to, groups such as phenylene, o-phenylene (i.e., 1,2-phenylene), m-phenylene (i.e., 1,3-phenylene), p-phenylene (i.e., 1,4-phenylene), naphthylene, 1,2-naphthylene, 1,2-naphthylene, and 1,4-naphthylene.

[0060] "Halo" or "halogen" refers to elements in Group 17 of atomic number 9 to 85. Examples of halogen groups include fluoro, chloro, bromo, and iodo. If a residue is substituted with more than one halogen, the residue can be referred to using a prefix corresponding to the number of halogen moieties attached. For example, dihaloaryl, dihaloalkyl, and trihaloaryl refer to aryl and alkyl substituted with two ("di") or three ("tri") halo groups (where the halos can be, but are not necessarily, the same halo); thus, 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. An alkyl group in which each hydrogen is replaced with a halo group is called a "perhaloalkyl." A preferred perhaloalkyl group is trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" refers to an alkoxy group in which each H in the hydrocarbon making up the alkyl portion of the alkoxy group is replaced with a halogen. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

[0061] "Amino" refers to the group -NH2.

[0062] "Substituted amino" refers to the group -NR'R" where R' and R" are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one of R' and R" is not hydrogen.

[0063] "Optionally substituted," unless otherwise specified, means that a group may be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents listed for that group, which may be the same or different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has 1 to 2, 1 to 3, 1 to 4, or 1 to 5 substituents.

[0064] "Organic modifier," unless otherwise specified, means one of a group of solvents typically used to dissolve organic 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, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol (isopropanol), propyl acetate, and combinations thereof.

[0065] Further to the present disclosure herein, the term "substituted," when used to modify a designated group or radical, can also mean that one or more hydrogen atoms of the designated group or radical are each independently replaced with the same or different substituents as defined herein. In some embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0066] Unless a specific isotope of an element is indicated in a formula, the present invention includes all isotopic variations of the compounds disclosed herein, including deuterated derivatives of the compounds (wherein H is 2 H, i.e., it may be D). Isotopic substitutions may be made at any position in the structure. Any or all positions may be substituted with an isotope, or atoms may be present at natural abundance at any or all positions in a structure.

[0067] It is recognized that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of embodiments relating to chemical groups represented by variables are specifically included in the present invention, and to the extent such combinations encompass compounds that are stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity), each and every combination is disclosed herein as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of chemical groups listed in embodiments describing such variables are also specifically included in the present invention, and each and every subcombination of chemical groups is disclosed herein as if each and every such subcombination was individually and explicitly disclosed herein.

[0068] Aspects and embodiments described herein as "comprising" are understood to include the embodiments "consisting of" and "consisting essentially of."

[0069] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise indicated or clear from the context.

[0070] Unless expressly indicated otherwise, the term "about" is used to indicate that a value includes the standard deviation or standard error of the device or method being employed to determine the value. Reference to "about" a value or parameter herein includes (and describes) embodiments that relate to said value or parameter itself. For example, reference to "about X" includes reference to "X." II. Compounds

[0071] In one embodiment, the compound of formula (J): [ka] [In the formula, R 0 is a C4-C optionally substituted with 1 to 4 halogen atoms 21 is a hydrocarbyl; X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b , or -(CH2) p R 1c where R 1a and R 1b are independently C1-C3 alkyl; R 1c is C3-C4 cycloalkyl; p is 1 or 2; R 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R3 are 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, or a salt thereof; Provided is a compound or a salt thereof, wherein the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-32); N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31); or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide (Compound No. 63-37).

[0072] In some embodiments, R 0 is C4~C 21 In some embodiments, R 0 is C4~C 14 In some embodiments, R 0 is C5~C 10 In some embodiments, R 0 is C 10 ~C 14 In some embodiments, R 0 is a C5 to C7 hydrocarbyl.

[0073] In some embodiments, X is -NH(C=O)-. In other embodiments, X is -NH-.

[0074] In some embodiments, R 0 is branched C4 to C 14 Alkyl or -(CH2) m R Am is 0, 1, 2, or 3; R A is a C3-C8 cycloalkyl optionally substituted with 1 to 4 groups independently selected from the group consisting of C1-C4 alkyl and C1-C4 alkylene.

[0075] In some embodiments, R 0 Branched C4 to C 14 In some embodiments, R 0 Branches C5 to C 10 In some embodiments, R 0 is branch C 10 ~C 14 In some embodiments, R 0 is a branched C5-C7 alkyl.

[0076] In some embodiments, R 0 Ha-(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In another variation, m is 0 and R A is cyclobutyl, cyclopentyl, or cyclohexyl.

[0077] In some embodiments, R A is a C3-C8 cycloalkyl.

[0078] In some embodiments, R A is C3-C6 cycloalkyl optionally substituted with 1-3 groups independently selected from the group consisting of methyl and methylene. In one variation, m is 1 or 2.

[0079] In some embodiments, R A is cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, and m is 1 or 2.

[0080] In some embodiments, m is 0 or 1 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

[0081] In some embodiments, R A is a C3-C6 cycloalkyl optionally substituted with 1 to 4 halogen atoms. A is a C3-C6 cycloalkyl optionally substituted with 1-3 chlorine or fluorine atoms. A is a C3-C6 cycloalkyl optionally substituted with 1 to 2 chlorine or fluorine atoms. A is cyclobutyl optionally substituted with 1 to 2 fluorine atoms. In one variation, m is 1.

[0082] In some embodiments, R 0 Ha-(CH2) z (C(CH3)2)R A In one variation, z is 1 or 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In one variation, z is 1 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0083] In some embodiments, R 0 is selected from the group consisting of: [ka]

[0084] In some embodiments, R 0 is selected from the group consisting of: [ka] [ka]

[0085] In some embodiments, R 0 is selected from the group consisting of: [ka]

[0086] In some embodiments, X is —NH— and R 0 Ha-(CH2) m R A where m is 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0087] In some embodiments, X is —NH— and R 0 Ha-(CH2) z (C(CH3)2)R A where z is 1 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0088] In some embodiments, X is —NH— and R 0 Ha-(CH2) m R A where m is 0 and R A is cyclobutyl, cyclopentyl, or cyclohexyl.

[0089] In some embodiments, X is —NH(C═O)— and R 0 Ha-(CH2) m R A where m is 1 and R A is cyclopropyl, cyclobutyl, or cyclopentyl.

[0090] In some embodiments, R 1is C3-C6 alkyl (e.g., n-butyl). In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some preferred embodiments, R 1 is n-butyl. In some embodiments, R 1 is n-pentyl. In some embodiments, R 1 Ha-(CH2) p OR 1a (e.g., CH2OCH2CH3). In some embodiments, R 1 Ha-(CH2) p NHR 1b (e.g., CHNHCHCH). In some embodiments, R 1 Ha-(CH2) p R 1c In one variation, R 1c is cyclopropyl.

[0091] In some embodiments, R 2 is NHR 2a where R 2a is H, OH, NH, or methyl. In some embodiments, R 2 is NH. In some embodiments, R 2 is NHOH, NHNH2, or NHCH3.

[0092] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some embodiments, R 4a and R 4b Each of is H.

[0093] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0094] If present, X and R as described for formula (J) 0 The R described for formula (J) is intended to mean each and every variation of R as if each and every combination were specifically and individually described. 1 ,p,R 2 , q, R 3 , R 4a , and R 4b It is intended and understood that each and every variation of the above may be combined with any other variation.

[0095] In some embodiments, the compound of formula (J) has the formula (J-1): [ka] (In the formula, R 0 is C4~C 21 or a salt thereof. In some embodiments, R 0 Ha-(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In one variation, m is 2 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. In another variation, m is 0 and R Ais cyclobutyl, cyclopentyl, or cyclohexyl. In some embodiments, R 0 Ha-(CH2) z (C(CH3)2)R A In one variation, z is 1 and R A is cyclopropyl, cyclobutyl, or cyclopentyl. A is optionally substituted with 1 to 4 groups independently selected from the group consisting of methyl, methylene, and halogen.

[0096] In some embodiments, the compound of formula (J) has formula (J-2): [ka] (In the formula, R 0 is C4~C 21 or a salt thereof, wherein R is N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide (Compound No. 63-37). 0 Ha-(CH2) m R A In one variation, m is 1 or 2 and R A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In one variation, m is 1 and R A is cyclopropyl.

[0097] In another embodiment, a compound of formula (K): [ka] [In the formula, n is an integer from 4 to 21; X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a, -(CH2) p NHR 1b , or -(CH2) p R 1c where R 1a and R 1b are independently C1-C3 alkyl; R 1c is C3-C4 cycloalkyl; p is 1 or 2; R 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R 3 are 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, or a salt thereof; Provided is a compound or a salt thereof, wherein the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-32) or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31).

[0098] In some embodiments, X is -NH-. In some embodiments, X is -NH(C=O)-.

[0099] In some embodiments, X is -NH- and n is an integer from 4 to 15. In some preferred embodiments, X is -NH- and n is an integer from 4 to 12. In some preferred embodiments, X is -NH- and n is 4, 5, 6, or 7. In some embodiments, n is 8, 9, 10, 11, 12, 13, 14, or 15.

[0100] In some embodiments, X is —NH(C═O)— and n is an integer from 4 to 14. In some preferred embodiments, X is —NH(C═O)— and n is 11, 12, 13, or 14. In some embodiments, n is 4, 5, 6, 7, 8, 9, or 10.

[0101] In some embodiments, R 1 is C3-C6 alkyl. In some embodiments, R 1 is propyl, butyl, pentyl, or hexyl. In some preferred embodiments, R 1 is n-butyl. In some embodiments, R 1 is n-pentyl.

[0102] In some embodiments, R 1 Ha-(CH2) p OR 1a where p is 1 or 2, and R 1a is C1-C3 alkyl. In some embodiments, R 1 is -CH2OCH2CH3.

[0103] In some embodiments, R 1 Ha-(CH2) p NHR 1b where R 1b is C1-C3 alkyl. In some embodiments, R 1 is -CH2NHCH2CH3.

[0104] In some embodiments, R 1 Ha-(CH2) p R 1c where p is 1 or 2, and R 1c is cyclopropyl or cyclobutyl. In some embodiments, R 1 is -CH2-cyclopropyl or -CH2CH2-cyclopropyl.

[0105] In some embodiments, R 2 is NHR 2a where R 2a is H, OH, NH, or methyl. In some preferred embodiments, R 2 is NH. In some embodiments, R 2 is NHOH, NHNH2, or NHCH3.

[0106] In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is unsubstituted (i.e., q is 0). In some embodiments, the phenyl portion of the 1H-imidazo[4,5-c]quinoline core is substituted with 1, 2, 3, or 4 substituents independently selected from the group consisting of halogen, C1-C8 alkyl, —(C1-C7 alkylene)-NH2, and —CH2-phenylene-CH2NH2. In some embodiments, q is 1 and R 3 is a C1-C8 alkyl.

[0107] In some embodiments, R 4a and R 4b is independently H or C1-C8 alkyl. In some preferred embodiments, R 4a and R 4b Each of is H.

[0108] When present, each and every variation of X and n described for formula (K) is specifically and individually described as if each and every combination thereof were specifically and individually described. 1 , R 2 , q, R 3 , R 4a , and R 4b It is intended and understood that each of the following may be combined with any and all variations thereof. For example, In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl), and R 2is NH, q is 0, X is -NH-, and n is 4, 5, 6, or 7. In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl), and R 2 is NH, q is 0, X is —NH(C═O)—, and n is 11, 12, 13, or 14.

[0109] In some embodiments, the compound of formula (K) has the formula (K-1): [ka] (Wherein, R'' is a straight chain C4-C 21 or a salt thereof, with the proviso that the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-32). In some embodiments, R″ is a straight-chain C4-C 15 In some embodiments, R" is a straight chain C4 to C7 alkyl. In some embodiments, R" is a straight chain C8 to C 15 In some embodiments, R" is a straight chain C 17 ~C 21 It is alkyl.

[0110] In some embodiments, the compound of formula (K) has formula (K-2): [ka] (Wherein, R is a straight chain C4-C 21 or a salt thereof, with the proviso that said compound is other than N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31). In some embodiments, R is a straight-chain C4-C 14 In some embodiments, R is a straight chain C alkyl. 11 ~C 14In some embodiments, R is a straight chain C-C alkyl. 10 In some embodiments, R is a straight chain C alkyl. 16 ~C 21 It is alkyl.

[0111] Representative compounds of the present invention are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14]

[0112] Further compounds are listed in Table 2. [Table 2-1] [Table 2-2]

[0113] In some embodiments, the compounds of the present invention exclude the compounds listed in Table 2.

[0114] In some embodiments, there is provided a compound, or a salt thereof, selected from compound numbers 63-01 to 63-30, 63-33 to 63-36, and 63-38 to 63-49 in Table 1. In some embodiments, the compound is selected from the group consisting of one or more of compound numbers 63-01 to 63-30 in Table 1, or a salt thereof. In some embodiments, the compound is selected from the group consisting of one or more of compound numbers 63-33 to 63-36 and 63-38 to 63-49 in Table 1, or a salt thereof.

[0115] The present invention also includes all salts (e.g., pharmaceutically acceptable salts) of the compounds referred to herein. The present invention also includes any or all stereochemical forms of the described compounds, including any enantiomeric or diastereomeric forms, and any tautomeric or other forms. Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the foregoing structure or name is intended to encompass all possible stereoisomers of the depicted compound. Furthermore, when a specific stereochemical form is depicted, it is understood that other stereochemical forms are also included in the present invention. All forms of the compounds, such as crystalline or amorphous forms of the compounds, are also included in the present invention. Compositions comprising the compounds of the present invention, such as compositions of substantially pure compounds containing a specific stereochemical form of the compound, are also contemplated. Compositions comprising mixtures of the compounds of the present invention in any ratio (including mixtures of two or more stereochemical forms of the compounds of the present invention in any ratio), are also included in the present invention, including racemic mixtures, non-racemic mixtures, enantiomerically enriched mixtures, and scalamitic mixtures of enantiomers.

[0116] The compounds of the present disclosure are potent TLR7 / 8 agonists that exhibit balanced biological activity against both TLR7 and TLR8 receptors, and possess physiochemical properties (such as increased hydrophobicity). Compounds with potent agonist biological activity against both TLR7 and TLR8 receptors are potentially more effective immunoadjuvants than agonists specific to only one of these TLRs, and will promote innate immune responses in a wide range of antigen-presenting cells and other important immune cell types, including plasmacytoid and myeloid dendritic cells, monocytes, and B cells (see, e.g., Vasilakos et al. 2013 Expert Rev Vaccines 12:809-819). Compounds with physiochemical properties, such as increased hydrophobicity, are known to be compatible with oil-based formulation approaches and enable pharmaceutical compositions to promote compound retention at the injection site.

[0117] In some embodiments, compounds of Formula (J) or (K) can activate both TLR7 and TLR8. In some embodiments, compounds of Formula (J) or (K) have an EC 50 , and an EC of approximately 2000 nM or less for TLR8 50 where EC 50 Values ​​are as described in Example B1. In some embodiments, compounds of Formula (J) or (K) have an EC50 activity of about 50 nM or less for TLR7. 50 , and an EC of approximately 1000 nM or less for TLR8 50 In some embodiments, compounds of Formula (J) or (K) have an EC50 of about 200 nM, about 175 nM, about 150 nM, about 125 nM, about 100 nM, about 75 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 8 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, or about 0.5 nM for TLR7. 50 and an EC for TLR8 of about 2000 nM, 1500 nM, 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 1 nM, or 0.5 nM 50 It has.

[0118] The compound of formula (J-1) or (K-1) is an N-alkyl derivative of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ); while the compound of formula (J-2) or (K-2) is an N-acyl derivative of IMDQ. IMDQ (compound number 63-00) exhibits potent in vitro activity against both TLR7 and TLR8 receptors (see, for example, U.S. Pat. Nos. 8,728,486 and 9,441,005). The alkyl chain modification at the benzylamine of the compound of the present invention increases hydrophobicity, thereby enabling pharmaceutical compositions to promote retention of the compound at the injection site. However, while alkyl chain derivatives with increased carbon chain length may exhibit significantly increased hydrophobicity (e.g., as assessed by partition coefficient or cLogP calculations), these derivatives may also exhibit reduced agonist potency for both TLR7 and TLR8, or selectively reduced bioactivity for one of the two aforementioned receptors. For example, the N-octadecanoyl derivative (Compound No. 63-13) is 40-fold less potent against TLR7 and 26-fold less potent against TLR8 than its related parent, IMDQ, in the same in vitro human immune cell bioactivity assay. Unexpectedly, the N-tetradecanoyl derivative of IMDQ (Compound No. 63-10) is only 2-fold and 2.4-fold less potent than IMDQ for TLR7 and TLR8, respectively. These data demonstrate that optimal alkyl chain length results in potent and balanced TLR7 / 8 agonist bioactivity, and that increased hydrophobicity incorporated into the pharmaceutical composition promotes compound retention at the injection site.

[0119] Compounds of formula (J-1) or (K-1) are N-alkyl derivatives of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ). While alkyl chain derivatives with increasing carbon chain length may exhibit increased hydrophobicity (e.g., as assessed by partition coefficient or cLogP calculations), these derivatives may also exhibit decreased agonist potency for both TLR7 and TLR8, or selectively decreased biological activity for one of the two aforementioned receptors. For example, the 4-octadecylamino derivative (Compound No. 63-29) is 19-fold less potent against TLR7 and 12-fold less potent against TLR8 than its related parent, IMDQ, in the same in vitro human immune cell biological activity assay (see, e.g., Example B1). Interestingly, the 4-pentylamino derivative (compound no. 63-17) is only 2-fold less potent than IMDQ against TLR7 and twice as potent against TLR8, but is significantly less hydrophobic than compound no. 63-29. Unexpectedly, the 4-(2-cyclopropylethyl)amino derivative (compound no. 63-33) is only 4-fold less potent than IMDQ against TLR7 and 2.9-fold more potent than TLR8, with a linear 5-carbon barrier. The cLogP is increased compared to the control (compound no. 63-17). These data demonstrate that optimal alkyl chain length provides potent and balanced TLR7 / 8 agonist bioactivity and that increased hydrophobicity can be incorporated into pharmaceutical compositions to promote compound retention at the injection site.

[0120] Balanced dual-potency TLR7 / 8 agonist small molecules can also be synthesized and characterized as single active pharmaceutical ingredients, facilitating GMP manufacturing at lower cost and enabling simpler and more predictable regulatory pathways.

[0121] Compounds of formula (J) can be synthesized according to Scheme 1 and / or using methods known in the art. Scheme 1 [ka] (In the formula, R 1 , R 2 , q, and R 3 is as defined for formula (J), and R and R 0 is a hydrocarbyl group).

[0122] X is -NH- and R 0 Ga-(CH2) m R A where m is 1 or 2, and R A is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Preferred compounds of formula (J) can be prepared using the following carboxylic acids: cyclopropanecarboxylic acid, cyclopropylacetic acid, cyclobutanecarboxylic acid, cyclobutylacetic acid, cyclopentanecarboxylic acid, cyclopentylacetic acid, cyclohexanecarboxylic acid, and cyclohexaneacetic acid. 0 Ga-(CH2) m R A m is 1, 2, or 3; R A is cyclopropyl, 2-methylcyclopropyl, 2,2-dimethylcyclopropyl, 1-methylcyclopropyl, 1-methylcyclobutyl, 3-methylcyclobutyl, or 3-fluorocyclobutyl. Preferred compounds of formula (J) can be prepared using the following carboxylic acids: cyclopropanecarboxylic acid, 2-methylcyclopropanecarboxylic acid, 2,2-dimethylcyclopropanecarboxylic acid, 1-methylcyclopropanecarboxylic acid, 1-methylcyclobutanecarboxylic acid, 3-methylcyclobutanecarboxylic acid, or 3-fluorocyclobutanecarboxylic acid. 0 -(CH2)(C(CH3)2)R A and R A is cyclopropyl or cyclobutyl, preferred compounds of formula (J) can be prepared using the following carboxylic acids: cyclopropanecarboxylic acid or cyclobutanecarboxylic acid. A detailed description of the synthetic scheme for representative compounds Nos. 63-33 can be found in Example S3.

[0123] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl), and R 2 is NH2, X is -NH-, and R 0 Ha-(CH2) m R A where m is 0 and R A When R is cycloalkyl, the compound is synthesized according to Scheme 1-2. 1 is n-butyl, and R 2 is NH2, X is -NH-, and R 0 Ga-(CH2) m R A where m is 0 and R A is cyclohexyl, the compound of formula (J) The compound (compound no. 63-49) is prepared according to the synthesis described in Example S10. Scheme 1-2 [ka] (In the formula, R 1 , q, and R 3 is as defined for formula (J) and R is a cycloalkyl group).

[0124] Compounds of formula (K) can be synthesized according to Scheme 2 and / or using methods known in the art. Scheme 2 [ka] (In the formula, R 1 , R 2 , q, and R 3 is as defined for formula (K), and R and R″ are straight chain alkyl groups.

[0125] Preferred compounds of formula (K), where X is —NH(C═O)— and n is an integer from 4 to 21, with the proviso that the compound is other than N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide (Compound No. 63-31), can be prepared using the following carboxylic acids: pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, icosanoic, heneicosanoic, and docosanoic. Detailed descriptions of the synthetic schemes for representative Compounds Nos. 63-10 and 63-17 can be found in Examples S1 and S2, respectively.

[0126] In some embodiments, R 1 is C3-C6 alkyl (e.g., n-butyl), and R 2 When is NH and q is 0, the compound is synthesized according to Scheme 3 or 4. For a more detailed description of each reaction step useful in preparing Compound No. 63-00 (the starting compound in Schemes 3 and 4), see, for example, U.S. Pat. Nos. 8,728,486 and 9,441,005. Scheme 3 [ka] (Wherein R and R 0 is a hydrocarbyl group) Scheme 4 [ka] where R and R'' are straight chain alkyl groups.

[0127] Those skilled in the art will recognize that other synthetic routes (including different solvents, catalysts, reducing agents, temperatures, reaction times, and atmospheric conditions) can be used to synthesize compounds within the scope of the present invention.

[0128] The compounds of the present invention can be isolated using conventional separation and purification methods and techniques. Techniques may include high performance liquid chromatography (HPLC) using different matrices (e.g., see C18, C8, C4, etc.), chromatography using typical adsorbents (e.g., see silica gel, activated carbon, alumina, and zeolite, etc.), recrystallization, and differential extraction (e.g., liquid-liquid and solid phase, etc.). III. Pharmaceutical Compositions

[0129] Also provided is a pharmaceutical composition comprising the alkyl chain-modified 1H-imidazo[4,5-c]quinoline TLR7 / 8 agonist of the present disclosure. Pharmaceutical compositions typically include one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition further includes an antigen. The pharmaceutical compositions of the present disclosure are preferably sterile and preferably essentially endotoxin-free. excipients

[0130] Pharmaceutically acceptable excipients of the present disclosure include, for example, oils, lipids, solvents, bulking agents, surfactants, buffers, tonicity adjusters, and preservatives (see, e.g., Pramanick et al 2013 Pharma Times 45:65-77). In some embodiments, the pharmaceutical composition comprises an excipient that functions as one or more of a solvent, bulking agent, buffer, and tonicity adjuster (e.g., salt in saline). (Sodium chloride may serve as both an aqueous vehicle and a tonicity adjuster.) The pharmaceutical compositions of the present disclosure are suitable for parenteral administration, with intratumoral administration being preferred in some cases. In certain embodiments, the pharmaceutical compositions of the present disclosure are not intended for enteral administration.

[0131] In some embodiments, the pharmaceutical composition comprises an oil-based excipient to solubilize the TLR7 / 8 agonist compound to enable parenteral administration and promote retention of the compound at the injection site. Non-limiting examples of oil-based excipients are known to those skilled in the art and include pharmaceutical grade sesame oil, soybean oil, castor oil, corn oil, cottonseed oil, peanut oil, Miglyol®, and squalene oil. These oils can be purified or cleaned by chromatographic processes to reduce polar impurity levels, thereby producing a United States Pharmacopoeia-National Formulary / Japanese Pharmacopoeia / European Pharmacopoeia-grade product with consistent properties and impurity profile.

[0132] In some embodiments, the TLR7 / 8 agonist compound is first dissolved in 100% ethanol vehicle and then diluted to a final concentration of 2-20% ethanol with oil to facilitate solubilization of the compound in the oil. Suitable ethanol for use is ethanol that does not contain water or denaturants (e.g., see 200-proof ethanol, USP-grade dehydrated alcohol, etc.).

[0133] In some embodiments, the pharmaceutical composition contains a preservative. Suitable preservatives include, for example, antimicrobial agents and antioxidants. In preferred embodiments, the pharmaceutical composition is prepared under sterile conditions and packaged in a single-use container, eliminating the need for an antimicrobial agent. Those skilled in the art will recognize that pharmaceutical-grade antioxidants used to prevent color, odor, or peroxide formation, including, but not limited to, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), vitamin E, and propyl gallate, can be added to these oil-based formulations. In certain embodiments, the antioxidant concentration in the formulation is at least 10 ppm, 50 ppm, 100 ppm, 300 ppm, 500 ppm, and up to 1000 ppm to ensure stability of the oil-based formulation for up to one year when stored at temperatures between 5°C and 40°C.

[0134] In some embodiments, the pharmaceutical composition is an oil-in-water nanoemulsion (see, e.g., Dowling et al 2017 JCI Insight 2:e91020) or a liposome-based formulation (see, e.g., VanHoeven et al 2017 Sci Rep 7:46426). In one embodiment illustrating an oil-in-water nanoemulsion-based pharmaceutical composition, the TLR7 / 8 agonist compound of Formula (J) or (K) is dissolved in an oil phase composed of phospholipids (e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol); 1,2-dioleoyl-sn-glycero-3-phosphocholine; and L-α-phosphatidylcholine), triglyceride-based oils (see, e.g., sesame oil, soybean oil, castor oil, corn oil, cottonseed oil, peanut oil, Miglyol®, and squalene oil), and, optionally, an organic modifier (e.g., ethanol). Next, an aqueous phase containing appropriate buffers, isotonicity agents, emulsifiers, and optionally preservatives is added to the oil phase, and a coarse emulsion is formed by high-shear mixing (e.g., Polytron®) for 5-10 minutes. Finally, particles with a mean diameter in the range of 100-150 nm (assessed by dynamic light scattering) and a dispersity index in the range of 0.1-0.2 are obtained. A nanoemulsion containing is formed by processing the coarse emulsion through 10-15 passes through a high shear homogenizer (see, for example, Microfluidizer M110P) at 30,000 psi.

[0135] In another embodiment illustrating a liposome-based pharmaceutical composition, a TLR7 / 8 agonist compound of Formula (J) or (K) is dissolved in an appropriate mixture of phospholipids using various ratios (depending on the desired physiochemical properties of the liposomes) of uncharged, positively charged, negatively charged, and PEGylated phospholipids with various lipid tail lengths and cholesterol, as well as organic modifiers. The organic solvent is then removed using a rotary evaporator, and the lipid / compound film is redissolved in an aqueous buffer until the formulation is translucent with no visible particles. Finally, the resulting multilamellar liposomes are processed into unilamellar liposomes with an average diameter in the range of 100-150 nm (as assessed by dynamic light scattering) and a dispersity index in the range of 0.1-0.2 using either high-shear homogenization or a membrane extruder (e.g., Lipex®). These examples are non-limiting, and one of skill in the art will recognize that oil-in-water nanoemulsions and liposome-based pharmaceutical compositions can be formed by any of several different methods (see, e.g., Brito et al 2013 Seminar Immunol 25:130-145).

[0136] In some embodiments, the pharmaceutical composition comprises a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition must be lyophilized before administration. In some embodiments, the bulking agent is a lyoprotectant that helps stabilize and prevent degradation of the active agent during freeze-drying and / or storage. Suitable bulking agents are sugars (monosaccharides, disaccharides, and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose, and raffinose.

[0137] In some embodiments, the pharmaceutical composition includes a buffering agent. The buffering agent controls the pH to prevent degradation of the active agent during processing, storage, and, if necessary, reconstitution. Suitable buffering agents include, for example, salts including acetate, citrate, phosphate, or sulfate. Other suitable buffering agents include, for example, amino acids (e.g., arginine, glycine, histidine, and lysine). The buffering agent may further include hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within the range of 4 to 9. In some embodiments, the pH is greater than a lower limit of 4, 5, 6, 7, or 8. In some embodiments, the pH is less than an upper limit of 9, 8, 7, 6, or 5. That is, the pH is within the range of about 4.0 to 9.0, with the lower limit being less than the upper limit.

[0138] In some embodiments, the pharmaceutical composition comprises a tonicity adjuster. Suitable tonicity adjusters include, for example, dextrose, glycerol, sodium chloride, glycerin, and mannitol. antigen

[0139] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen. In some embodiments, the pharmaceutical composition comprises an alkyl chain-modified 1H-imidazo[4,5-c]quinoline TLR7 / 8 agonist, one or more excipients, and an antigen. In some of these embodiments, the antigen is a protein antigen. In some of these embodiments, the antigen is a polysaccharide antigen, preferably covalently attached to a carrier protein. In some embodiments, the antigen is a microbial antigen, an allergen, or a tumor-associated antigen. In some embodiments, the antigen is a viral antigen, a protozoan antigen, a bacterial antigen, or a fungal antigen. In some embodiments, the tumor antigen is an autoantigen or a neoantigen.

[0140] In some embodiments, the pharmaceutical composition comprises a microbial antigen selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, and a parasitic antigen. In some embodiments, the microbial antigen is derived from a microorganism that causes an infectious disease in a non-human mammalian subject. In some embodiments, the microbial antigen is derived from a microorganism that causes an infectious disease in a human subject. In some embodiments, the infectious disease is caused by a virus, a bacterium, a fungus, or a parasitic protozoan. Suitable microbial antigens include, for example, Adenovirus type 4, Adenovirus type 7, Bacillus anthracis (anthrax), Mycobacterium tuberculosis, Corynebacterium diphtheriae (e.g., diphtheria toxoid), Clostridium tetani (e.g., tetanus toxoid), Bordetella pertussis, Haemophilus influenzae type B, Hepatitis A virus, Hepatitis B virus (e.g., HBsAg), Human papillomavirus (types 6, 11, 16, 18, 31, 33, 45, 52, and 58), Influenza virus types A and B (e.g., hemagglutinin, neuraminadase), Influenza virus type B, Parainfluenza virus, Japanese encephalitis virus, Measles virus, Mumps virus, Rubella virus, Neisseria menigitidis (groups A, B, C, Y, and W-135), Streptococcus pneumoniae (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F), poliovirus, rabies virus, rotavirus, vaccinia virus, Salmonella typhi, varicella-zoster virus, and yellow fever virus antigens (see, e.g., Plotkin, SA, Orenstein, W., Offit, PA, Edwards KM (2017). Plotkin's Vaccines, 7th edition. Elsevier).In some embodiments, the microbial antigen is a viral antigen of herpes simplex virus type 1 or 2, human herpesvirus, human immunodeficiency virus type 1, and respiratory syncytial virus. In some embodiments, the microbial antigen is a fungal antigen of Candida albicans, Aspergillus flavus, Cryptococcus neoformans, Histoplasma capsulatum, and Pneumocystis carinii. In some embodiments, the microbial antigen is a parasitic antigen of Leishmania species, Plasmodium species, Schistosoma species, or Trypanosoma species.

[0141] In some embodiments, the pharmaceutical composition comprises an allergen. In some embodiments, the allergen is an environmental antigen (such as a mammalian, insect, plant, or mold allergen). In some embodiments, mammalian allergens include fur and dander. Suitable mammalian allergens include, for example, cat Fel d1, cow Bos d2, dog Can f I and Can f II, horse Equ c1, and mouse MUP. In some embodiments, insect allergens include insect feces and venom. Exemplary insect allergens include ant Sol i2, honeybee PLA and Hya, cockroach Bla g Bd9OK, Bla g4, GST, and Per a3, dust mite Der p2, Der f2, Der p10, and Tyr p2, hornet Dol m V, mosquito Aed a1, and hornet hyaluronidase and phospholipase. In some embodiments, plant allergens include grass, weed, and tree allergens (e.g., pollen).Suitable grass allergens include, for example, Kentucky bluegrass, tall fescue, orchard grass, bedgrass, perennial ryegrass, Japanese silvergrass, and timothy allergens.Exemplary plant allergens include barley Hor v9, birch Bet v1 and v2, cherry Pru a1, corn Zml3, grass Phl p1, 2, 4, 5, 6, 7, 11, and 12, Hol 15, Cyn Examples of allergens include d7 and d12, cedar Jun a2, Cry j1, and j2, juniper Jun o2, latex Hev b7, yellow mustard Sin a I, rape Bra r1, ragweed Amb a1, and rye Lol p1. In some embodiments, the mold allergen is an Aspergillus fumigatus allergen (such as Asp f 1, 2, 3, 4, and 6). In some embodiments, the allergen is a food allergen (such as a shellfish allergen, a legume allergen, a nut allergen, or a milk allergen). Exemplary food allergens include shrimp tropomyosin, peanut Ara h1, 2, 3, 8, and 9, walnut Jug r1 and 3, hazelnut Cor a1, 14, and 8 LTP, milk lactalbumin, casein, and lactoferrin.

[0142] In some embodiments, the pharmaceutical composition comprises a tumor antigen. In some embodiments, the tumor antigen comprises the amino acid sequence of a full-length protein or a fragment thereof (e.g., a polypeptide of about 10 to about 100 amino acids in length). In some embodiments, the tumor antigen is selected from the group consisting of WT1, MUC1, LMP2, HPV E6, HPV E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, p53, NY-ESO-1 (CTAG1), PSMA, CEA, MelanA / Mart1, Ras, gp100, proteinase 3, bcr-able, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint, EphA2, PAP, MP-IAP, AFP, EpCAM, ERG, NA17-A, PAX3, ALK, androgen receptor, cyclin B1, MYCN, PhoC, TRP-2, mesothelin, PSCA ... tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoint, EphA2, PAP, MP-IAP, AFP, EpCAM, ERG, NA17-A, PAX3, ALK, androgen receptor, cyclin B1, A1, CYP1B1, PLAC1, BORIS, ETV6-AML, NY-BR-1, RGS5, SART3, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7-H3, legumain, Tie2, Page4, VEGFR2, MAD-CT-1, FAP, PAP, PDGFR-beta, MAD-CT-2, CEA, TRP-1(gp75), BAGE1, BAGE2, BAGE3, BAGE4, ​​BAGE5, CAMEL, MAGE-A2, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, and Fos-related antigen 1. In some preferred embodiments, the tumor antigen comprises an amino acid sequence or a 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, PSA, Ras, survivin, TRP1 (gp75), TRP2, and tyrosinase. IV.How to use

[0143] The pharmaceutical compositions of the present disclosure are suitable for multiple uses, including stimulating an immune response in a mammalian subject in need thereof. Mammalian subjects include, but are not limited to, humans, non-human primates, rodents, pets, and livestock. In some embodiments, the pharmaceutical compositions are administered to the subject in an amount effective to achieve a particular result. Dosage and Mode of Administration

[0144] As with any pharmaceutical composition, the effective amount and the mode of administration can vary based on several factors that are obvious to those skilled in the art.Factors that should be considered include the efficacy of alkyl chain-modified 1H-imidazo[4,5-c]quinoline TLR7 / 8 agonist compounds, the ability of the compound and pharmaceutical composition to promote the retention of the agonist compound at the administration site, the route of administration, and whether the pharmaceutical composition contains an antigen.Other factors that should be considered include the disease modification results that should be achieved and the number / frequency of administrations given during the treatment regimen. .

[0145] An appropriate dosage range is one that achieves the desired clinical effect. The dosage can be determined by the amount of TLR7 / 8 agonist in the pharmaceutical composition that needs to be delivered to a subject to achieve the desired therapeutic response with minimal adverse events. Exemplary dosage ranges of TLR7 / 8 agonist compounds, expressed as the amount to be delivered based on the subject's body weight, include about 1-5,000 ng / kg (about 1-2,500 ng / kg, about 1-1,000 ng / kg, about 1-500 ng / kg, about 1-250 ng / kg, about 1-100 ng / kg, about 1-50 ng / kg, about 50-2,500 ng / kg, about 50-1,000 ng / kg, and about 50-500 ng / kg). g, about 100-5,000 ng / kg, about 100-2,500 ng / kg, about 100-1,000 ng / kg, about 100-500 ng / kg, about 500-5,000 ng / kg, about 1,000-5,000 ng / kg, about 2,000-5,000 ng / kg, about 2,500-5,000 ng / kg, about 3,000-5,000 ng / kg, or about 4,000-5,000 ng / kg, etc. In some embodiments, the dosage is greater than about (lower limit) 1, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 ng / kg. In some embodiments, the dosage is less than approximately (upper limit) 5000, 2000, 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, or 100 ng / kg. That is, the dosage is anywhere in the range of about 1 to 5000 ng / kg, with the lower limit being less than the upper limit. An exemplary dosage range for a TLR7 / 8 agonist, expressed as the amount to be delivered to a subject, is about 1 to 5000 ng.In some embodiments, the dosage may be even higher (e.g., about 2,500-500,000 ng / kg, about 5,000-500,000 ng / kg, about 2,500-150,000 ng / kg, about 2,500-100,000 ng / kg, about 2,500-50,000 ng / kg, about 2,500-25,000 ng / kg, about 2,500-10,000 ng / kg, about 10,000-500,000 ng / kg, about 25,000-500,000 ng / kg, about 50,000-500,000 ng / kg, about 100,000-500,000 ng / kg, or about 150,000-500,000 ng / kg).

[0146] In some embodiments, when the pharmaceutical composition further comprises an antigen, the antigen dosage range, expressed as the amount to be delivered to a subject, is about 1 μg to 500 μg. In some embodiments, the antigen dosage is about 1 μg to 50 μg. In some embodiments, the antigen dosage is about (lower limit) 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or greater than 400 μg. In some embodiments, the antigen dosage is about (upper limit) less than 500, 400, 300, 200, 100, 50, 45, 40, 35, 30, 25, 20, 15, or 10 μg. That is, the antigen dosage is anywhere in the range of about 1 to 500 μg, with the lower limit being less than the upper limit. The optimal antigen dosage can be determined by experimental means for a particular antigen.

[0147] Similarly, an appropriate route of administration is one that achieves the desired effect. Generally, the pharmaceutical compositions of the present disclosure are intended for parenteral administration (e.g., not oral or rectal administration). Suitable routes of administration include injection, topical, and inhalation. In particular, the pharmaceutical compositions of the present disclosure can be administered by routes such as intratumoral, intramuscular, subcutaneous, intravenous, epidermal (gene gun), transdermal, and inhalation. Suitable devices for administration by inhalation include, for example, atomizers, vaporizers, nebulizers, and dry powder inhalation delivery devices. In some embodiments, when the pharmaceutical composition is intended to treat a solid tumor, the composition is administered intratumorally. In one embodiment, the intratumor administration is by injection into at least one tumor lesion.

[0148] An appropriate dosing regimen for a TLR7 / 8 agonist formulated in a pharmaceutical composition may be used for prophylaxis. A dosing regimen is one that produces the desired effect with minimal adverse events in a clinical or therapeutic setting. The number of doses administered by the selected route can be one or more than one. The dosing frequency can range from weekly, biweekly, monthly, bimonthly, or between 3 and 12 months. An exemplary dosing frequency of a TLR7 / 8 agonist is from approximately once per week to once every 8 weeks. In some embodiments, the dosing frequency is greater than about once per 8, 6, 4, 2, or 1 week (higher limits). In some embodiments, the dosing frequency is less than about once per 7, 10, or 14 days (lower limits). An exemplary dosing frequency range for a TLR7 / 8 agonist to be delivered to a subject is from approximately once per week to once every 4 weeks. In some embodiments, two doses are administered, with the second dose being administered 1 to 2 months after the first dose. In some embodiments, three doses are administered, with the second dose administered 1-2 months after the first dose and the third dose administered 1-5 months after the second dose. In other embodiments, a series of doses can be administered over a 3-12 month treatment schedule, with the administration frequency being weekly, biweekly, every three weeks, or monthly. In other embodiments, shorter or longer periods can elapse between doses. In certain embodiments, the interval between successive doses can vary in weeks or months. In one embodiment, a series of 2, 3, 4, 5, or 6 doses per week can be administered, followed at a later time by a second series of doses administered several times per week. One skilled in the art will be able to adjust the dosing regimen by measuring biological outcomes (such as antigen-specific antibody responses or tumor regression). Stimulating the immune response

[0149] In one aspect, the present disclosure provides a method for stimulating an immune response in a mammalian subject in need thereof, the method comprising administering to the mammalian subject a pharmaceutical composition in an amount and frequency sufficient to stimulate an immune response in the subject. "Stimulating" an immune response refers to an increase in the immune response, which can occur by inducing a new immune response (e.g., as a result of an initial vaccination regimen) or by enhancing an existing immune response (e.g., as a result of a booster vaccination regimen). In some embodiments, stimulating the immune response includes one or more of the following: stimulating the production of IFNα, stimulating the production of type 1 and / or type 2 interferon, stimulating the production of IL-6, stimulating the production of TNFα, stimulating the proliferation of B lymphocytes, stimulating the expression of genes associated with the interferon pathway, stimulating the expression of genes associated with chemoattractants, and stimulating the maturation of plasmacytoid dendritic cells (pDCs) or myeloid dendritic cells (mDCs). Methods for measuring stimulation of an immune response are known in the art and are described in the Biological Examples of this disclosure. In embodiments where the pharmaceutical composition further comprises an antigen, stimulating an immune response comprises inducing an antigen-specific antibody response.

[0150] For example, in some embodiments where the pharmaceutical composition further comprises an antigen, the present disclosure provides a method of inducing an antigen-specific antibody response and / or a T cell response in a mammalian subject in need thereof by administering the pharmaceutical composition in an amount sufficient to induce an antigen-specific antibody response and / or a T cell response in the subject. "Inducing" an antigen-specific antibody response means raising the titer of an antigen-specific antibody above a threshold level (such as a baseline titer or a seroprotective level before administration). "Inducing" an antigen-specific T cell response means stimulating antigen-specific cytotoxic T lymphocytes, generating antigen-specific T cells present at non-immunized tumor sites, and generating T cells that are less exhausted and / or have an enhanced immune response to additional tumor antigens due to epitope expansion.

[0151] Analysis of the immune response (both qualitative and quantitative) can be by any method known in the art, including measurement of antigen-specific antibody production (specific antibody subpopulations). These include, but are not limited to, measuring the expression of specific gene sets for specific immune cell types, measuring cytokine production (e.g., IFNα, IL-6, IL-12, IL-18, TNFα, etc.), measuring activation of specific populations of lymphocytes (e.g., B cells and helper T cells), measuring the expression of specific gene sets for specific immune cell types, measuring cytokine production (e.g., IFNα, IL-6, IL-12, IL-18, TNFα, etc.), and / or measuring histamine release from basophils or mast cells. Methods for measuring antigen-specific antibody responses include enzyme-linked immunosorbent assays (ELISAs). Cytokine production can also be measured by ELISA. Gene expression analysis can be performed by TaqMan® Gene Expression Assays or nCounter® Gene Expression Assays. Activation of specific populations of lymphocytes can be measured by proliferation assays and fluorescence-activated cell sorting (FACS).

[0152] Preferably, a Th1-type immune response is stimulated (i.e., induced or augmented). In the context of the present disclosure, stimulation of a Th1-type immune response can be determined in vitro or ex vivo by measuring cytokine production from cells treated with an active agent (a TLR7 / 8 agonist alkyl chain derivative of 1H-imidazo[4,5-c]quinoline) of the present disclosure compared to control cells not treated with the active agent. Examples of "Th1-type cytokines" include, but are not limited to, IL-2, IL-12, IFNγ, and IFNα. In contrast, "Th2-type cytokines" include, but are not limited to, IL-4, IL-5, and IL-13. Cells useful for determining immunostimulatory activity include cells of the immune system (such as antigen-presenting cells, lymphocytes, preferably macrophages, and T cells). Suitable immune cells include primary cells isolated from a mammalian subject, such as peripheral blood mononuclear cells, including pDCs, monocytes, mDCs, and B cells, or splenocytes.

[0153] Stimulation of a Th1-type immune response can also be determined by measuring the levels of IL-2, IL-12, and interferon in mammalian subjects treated with an active agent of the present disclosure before and after administration, or compared with control subjects not treated with the active agent. Stimulation of a Th1-type immune response can also be determined by measuring the ratio of Th1-type antibody titers to Th2-type antibody titers. "Th1-type" antibodies include human IgG1 and IgG3 and mouse IgG2a. In contrast, "Th2-type" antibodies include human IgG2, IgG4, and IgE, and mouse IgG1 and IgE. Disease Treatment

[0154] The present disclosure further provides a method of preventing an infectious disease in a mammalian subject in need thereof, the method comprising administering a pharmaceutical composition in an amount sufficient to prevent the infectious disease in the subject. That is, in some embodiments, the present disclosure provides a prophylactic vaccine. In some embodiments, the mammalian subject is at risk of exposure to an infectious agent. "Preventing" an infectious disease means protecting the subject from developing the infectious disease. In some embodiments, preventing an infectious disease further comprises protecting the subject from infection with the infectious agent (e.g., protecting the subject from developing an acute or chronic infection). Furthermore, the present disclosure provides a method of ameliorating the symptoms of an infectious disease in a mammalian subject in need thereof, the method comprising administering a pharmaceutical composition in an amount sufficient to ameliorate the symptoms of the infectious disease in the subject. That is, in some embodiments, the present disclosure provides a therapeutic vaccine. In some embodiments, the subject is acutely or chronically infected with an infectious agent. The infectious disease can be a viral (e.g., hepatitis virus, herpes virus, or human papilloma virus), bacterial, fungal, or parasitic disease. In some embodiments, the pharmaceutical composition further comprises a viral, bacterial, fungal, or parasitic antigen. "Ameliorating" the symptoms of an infectious disease means improving the symptoms of the disease, preferably reducing the extent of the disease.

[0155] Furthermore, the present disclosure provides a method for ameliorating symptoms of an IgE-related disorder in a mammalian subject in need thereof, the method comprising administering a pharmaceutical composition in an amount sufficient to ameliorate the symptoms of the IgE-related disorder in the subject. In some preferred embodiments, the IgE-related disorder is an allergy. Allergies include, but are not limited to, allergic rhinitis (hay fever), sinusitis, eczema, and hives. In some embodiments, the pharmaceutical composition further comprises an allergen. "Ameliorating" the symptoms of an IgE-related disorder means improving the symptoms of the disorder, preferably reducing the extent of the disorder. For example, if the IgE-related disorder is allergic rhinitis, ameliorating the symptoms means reducing swelling of the nasal mucosa, reducing rhinorrhea (runny nose), and / or reducing sneezing.

[0156] Furthermore, the present disclosure provides methods for treating cancer in a mammalian subject in need thereof, comprising administering a pharmaceutical composition in an amount sufficient to treat the cancer in the subject. In certain embodiments, the present disclosure provides a method for treating cancer in a mammalian subject in need thereof, comprising administering an effective amount of a pharmaceutical composition by intratumoral delivery. In another aspect of the method, the intratumoral delivery comprises injecting the pharmaceutical composition into at least one tumor lesion. In other aspects, treating cancer comprises, for example, inducing the accumulation of tumor antigen-specific T cells in the injected tumor in greater numbers than when the pharmaceutical composition is administered to an extratumoral site. In other aspects, treating cancer comprises inducing a systemic tumor antigen-specific T cell response (e.g., including a higher systemic tumor antigen-specific T cell response than when the immunogenic composition is administered to an extratumoral site). In other aspects, treating cancer comprises inducing a systemic tumor antigen-specific T cell response. In other aspects, treating cancer comprises reducing the number of CD4+FoxP3+ regulatory T cells in the injected tumor. In other embodiments, the subject has one or more uninjected tumors (primary or metastatic lesions) in addition to the aforementioned injected tumor, and treating the cancer includes one or more of the following: (a) reducing the number of uninjected tumors; (b) reducing the volume of uninjected tumors; and (c) slowing the growth of uninjected tumors. In some embodiments, treating the cancer includes one or more of the following: (d) extending the survival time of the subject; (e) reducing the volume of injected tumors; and (f) slowing the growth of injected tumors. In some embodiments, if the cancer is a solid tumor, "treating" the cancer includes reducing the size of the solid tumor and any metastatic lesions or reducing the number of viable cancer cells. In other embodiments, if the cancer is a solid tumor, "treating" the cancer includes slowing the growth of the solid tumor and any metastatic lesions.In some embodiments, treating cancer includes extending progression-free survival or extending the time to progression. In other embodiments, the method further includes administering an effective amount of a second or additional therapeutic agent to the subject. "Treating" cancer means producing a beneficial clinical outcome (such as causing remission or extending survival compared to expected survival in the absence of treatment). In some preferred embodiments, "treating cancer" includes assessing the patient's response to the immunogenic composition according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) as described (see, e.g., Eisenhauer et al. 2009 Eur J Cancer 45:228-247). Response criteria for determining an objective anti-tumor response according to RECIST include complete response, partial response, progressive disease, and stable disease.

[0157] 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 In some embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, colorectal cancer, uterine cancer, bladder cancer, melanoma, head and neck cancer, non-Hodgkin's lymphoma, kidney cancer, ovarian cancer, pancreatic cancer, and thyroid cancer. In some embodiments, the cancer is a primary cancer at a site selected from the group consisting of the oral cavity, digestive system, respiratory system, skin, breast, reproductive system, urinary system, ocular system, nervous system, endocrine system, and lymphoma.

[0158] In some embodiments, the method further comprises administering to the subject an effective amount of a second therapeutic agent. In some of these embodiments, the second therapeutic agent is actinomycin, afatinib, alectinib, asparaginase, azacitidine, azathioprine, bicalutamide, binimetinib, bleomycin, bortezomib, camptothecin, carboplatin, capecitabine, carmustine, certinib, cisplatin, chlorambucil, cobimetinib, crizotinib, cyclophosphamide, cytarabine, dabrafenib, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, encorafenib, erlotinib, epirubicin, epothilone, etoposide, fludarabine, flutamine, flumethicone, flucloxin, fluoxetine ... In some embodiments, the second therapeutic agent comprises a chemotherapeutic agent selected from the group consisting of fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lapatinib, letrozole, mechlorethamine, mercaptopurine, methotrexate, mitomycin, mitoxantrone, octreotide, oxaliplatin, paclitaxel, pemetrexed, raltitrexed, sorafenib, sunitinib, tamoxifen, temozolomide, teniposide, thioguanine, topotecan, trametinib, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the second therapeutic agent comprises one or both of a BRAF inhibitor and a MEK inhibitor.In some embodiments, the second therapeutic agent comprises an epigenetic modulator selected from the group consisting of an HDAC inhibitor (e.g., voronistat [SAHA], romidepsin, entinostat, abexinostat, elinostat [CHR-3996], panobinostat, quisinostat [JNJ-26481585], 4SC-202, resminostat [SB939], pracinostat [CI-9940], and valproate), a DNA methyltransferase inhibitor (see, e.g., azacitidine, decitabine, zebularine, SGI-1027, RG-108, and sinfungin)), and combinations thereof.

[0159] In some of these embodiments, the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule, e.g., 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 immune stimulatory molecule. In some of these embodiments, the immune stimulatory molecule is selected from the group consisting of CD27, CD40, OX40 (CD134), GITR, 4-1BB (CD137), CD28, and ICOS (CD278). In some of these embodiments, the second therapeutic agent comprises an antibody, fragment, or derivative thereof. In some of these embodiments, the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule, and the second therapeutic agent comprises an antibody, fragment, or derivative thereof. In some embodiments, the method further comprises administering radiation therapy to the subject and / or administering an effective amount of the second therapeutic agent. In some of these embodiments, the effective amount of the immunogenic composition and the effective amount of the second therapeutic agent together provide a therapeutic effect against the tumor. In some of these embodiments, an effective amount of the immunogenic composition and an effective amount of the second therapeutic agent together produce a synergistic effect against the tumor.

[0160] In some embodiments of the methods, treating the infectious disease or cancer does not result in the development of flu-like symptoms of such severity that repeated administration of the immunogenic composition is contraindicated, the flu-like symptoms comprising one or more of the group consisting of fever, headache, chills, muscle aches and fatigue.

[0161] In some embodiments, the present disclosure provides a kit comprising a pharmaceutical composition (e.g., a TLR7 / 8 agonist compound of Formula (K), excipient(s), and optionally an antigen) and a set of instructions for using the composition for the methods described herein. The pharmaceutical composition of the kit is suitably packaged. When the pharmaceutical composition is a liquid or nanoparticle suspension, a silicon dioxide vial (e.g., SCHOTT Type I Plus®) with a rubber stopper (e.g., Exxpro halobutyl elastomer) and an aluminum crimp top is typically used as the container closure system. In some embodiments, the kit further comprises a device for administering the pharmaceutical composition (e.g., a syringe and needle). In other embodiments, the kit further comprises a pre-filled syringe / needle system, an autoinjector, or a needleless device. Instructions for using the pharmaceutical composition generally include information regarding the dosage, schedule, and route of administration for the intended method of use. V. Working Examples

[0162] Although this disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the following synthetic and biological examples should not be construed as limiting the scope of the present disclosure, which is delineated by the appended claims. Synthesis Example Example S1: Synthesis of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide (Compound No. 63-10)

[0163] Part A. Nitric acid (125 mL) was added to a slurry of quinoline-2,4-diol (50 g, 0.3 mol) in acetic acid (500 mL), and the reaction mixture was heated to 65 °C for 3 h. The mixture was then cooled to 5-10 °C, and the solid material was collected by filtration, washed with cold water, and air-dried. The resulting solid was then recrystallized from methanol and dried under vacuum to give 58 g of 3-nitro-2,4-quinolinediol as a yellow solid.

[0164] Part B. Phosphorus oxychloride (150 mL) was added to 3-nitro-2,4-quinolinediol (58 g) under an argon atmosphere and heated to 95° C. for 4 hours. The mixture was then cooled to room temperature and poured onto crushed ice with constant stirring. The precipitated product was collected by filtration, washed with water, and dried under vacuum. The crude solid was purified by flash chromatography on silica gel using hexane / ethyl acetate to give 35 g of 2,4-dichloro-3-nitroquinoline.

[0165] Part C. tert-Butyl (4-(aminomethyl)benzyl)carbamate (37.3 g, 0.16 mole, 1.1 equiv.) was added to a solution of 2,4-dichloro-3-nitroquinoline (35 g, 0.15 mole, 1.0 equiv.) in anhydrous dichloromethane (400 mL) and trimethylamine (16.0 g, 0.16 mole, 1.1 equiv.) and stirred overnight at room temperature. The solvent was removed under reduced pressure and the crude product was purified by hexane / ethyl acetate. Purification by flash chromatography on silica gel gave 52 g of tert-butyl (4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0166] Part D. A solution of tert-butyl (4-(((2-chloro-3-nitroquinolin-4-yl)amino)methyl)benzyl)carbamate (52 g, 0.12 mol) in ethyl acetate (250 mL) was hydrogenated in the presence of 5% platinum on carbon (2.0 g) and sodium sulfate (52 g) using a Parr hydrogenation apparatus at 60 psi for 12 hours. 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 eluting with hexane / ethyl acetate to give 32 g of tert-butyl (4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0167] Part E. Pentanoyl chloride (9.7 mL, 81.5 mmol, 1.05 equiv.) was added slowly to a solution of tert-butyl (4-(((3-amino-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate (32 g, 77.6 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (350 mL) and pyridine (30 mL) at 0–5° C. The reaction mixture was then warmed to room temperature and stirred for 12 h. The solvent was removed under reduced pressure, and the solid was then redissolved in ethyl acetate (400 mL), washed successively with water and saturated sodium bicarbonate (150 mL), and finally dried over anhydrous magnesium sulfate. The product was further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give 22 g of tert-butyl (4-(((3-butylamido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate.

[0168] Part F. Water (80 mL) was added to a solution of tert-butyl (4-(((3-butylamido-2-chloroquinolin-4-yl)amino)methyl)benzyl)carbamate (22 g, 44.2 mmol, 1.0 equiv) in ethanol (320 mL), followed by potassium carbonate (12.2 g, 88.4 mmol, 2.0 equiv) and the mixture was heated to 55° C. with vigorous stirring for 16 hours. The reaction mixture was concentrated, and the residue was partitioned between ethyl acetate (500 mL) and water (250 mL). The ethyl acetate layer was then washed with water (100 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The product was further purified by flash chromatography on silica gel eluting with hexane / ethyl acetate to give 15.4 g of tert-butyl (4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

[0169] Part G. tert-Butyl (4-((2-butyl-4-chloro-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate (15.4 g, 32.2 mmol, 1 equiv.) was dissolved in anhydrous dimethylformamide (125 mL), and then sodium azide (8.4 g, 128.6 mmol, 4 equiv.) was added to the solution. The resulting suspension was degassed and stirred at 110–115 °C under an argon atmosphere, and the progress of the reaction was monitored by reverse-phase HPLC analysis. After 18 h, the reaction mixture was cooled to room temperature, poured into cold water (500 mL), and extracted with ethyl acetate (3 × 100 mL). The combined extracts were washed with water (2 × 75 mL), dried over magnesium sulfate, filtered through Celite, and concentrated under reduced pressure to give an off-white solid. This solid was further worked up by recrystallization in 1:1 ethyl / hexane to give 12.5 g of tert-butyl (4-((4-azido-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)carbamate.

[0170] Part H. tert-Butyl (4-((4-azido-2-butyl-1H-imidazo[4 ,5-c]quinolin-1-yl)methyl)benzyl)carbamate (12.5 g, 25.7 mmol) was added to concentrated hydrochloric acid (65 mL), and 10% platinum on carbon (3.0 g) was added to the suspension. The reaction mixture was subjected to hydrogenation at 65 psi, and the reaction progress was monitored by reverse-phase HPLC analysis. After 6 days, the catalyst was filtered off, and the filter cake was washed with water (2 × 25 mL). The cake was cooled in an ice bath, and ice-cold 1 N sodium hydroxide was added dropwise with vigorous stirring until a pH of 8.5 was reached, and the material was extracted with 5% methanol in dichloromethane (4 × 75 mL). The combined extracts were dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified on a silica gel column using 8% methanol / 1% aqueous ammonia in dichloromethane to give 5.3 g of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0171] Part I. Myristic acid (27.4 mg, 0.12 mmol, 1.2 equiv.) and trimethylamine (0.2 mL) were added to a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (34 mg, 0.1 mmol, 1.0 equiv.) in anhydrous dimethylformamide (2 mL). The slurry was mixed for 5 minutes, and then HBTU (47.4 mg, 0.125 mmol, 1.25 equiv.) was added. The reaction mixture was further stirred for 2 hours under an argon atmosphere. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (30 mL), washed with water (2 × 10 mL), dried over magnesium sulfate, and concentrated in vacuo. The product was purified using column chromatography (6% methanol / dichloromethane) to yield 35 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)tetradecanamide (compound number 63-10). The purity of the product was estimated to be approximately 98% by reverse-phase HPLC, and the intended compound mass of 569.8 was confirmed by LC / MS, and the intended compound structure was confirmed by 300 MHz proton NMR (CDCl): δ 7.98 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 8.4 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H). Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s, 2H), 3.74 (s, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.20 (t, J = 7.5 Hz, 2H), 1.82-1.9 (m, 2H), 1.42-1.70 (m, 4H), 1.26-1.48 (m, 20H), 0.85-1.05 (m, 6H). Example S2: Synthesis of 2-butyl-1-(4-((pentylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-17)

[0172] Parts AH were identical to Example S1.

[0173] Part I. Valeric acid (34 mg, 0.33 mmol, 1.2 equiv.) and trimethylamine (140 mg, 1.39 mmol, 5.0 equiv.) were added to a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (100 mg, 0.28 mmol, 1.0 equiv.) in anhydrous dimethylformamide (2 mL). The slurry was mixed for 5 minutes, and then HBTU (131 mg, 0.34 mmol, 1.25 equiv.) was added. The reaction mixture was further stirred for 2 hours under an argon atmosphere. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water (3 × 30 mL), dried using magnesium sulfate, and concentrated in vacuo. The crude residue was taken up in ethyl acetate and methanol and purified using column chromatography (6% methanol / dichloromethane) to give 160 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentanamide.

[0174] Part J. A solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL, excess) was added to a solution of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pentanamide (127 mg, 0.28 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (5 mL) at room temperature, and the reaction mixture was heated to 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 the addition of 2N sodium hydroxide, and the product was extracted with dichloromethane (20 mL × 10). The combined organic layers were concentrated under reduced pressure, and the residue was purified by flash chromatography using 6% methanol / dichloromethane as eluent to give 24 mg of 2-butyl-1-(4-((pentylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-17). The purity of the product was estimated to be approximately 96% by reverse-phase HPLC, the intended compound mass of 429.6 was confirmed by LC / MS, and the intended compound structure was confirmed by 300 MHz proton NMR (CDCl3): δ 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4 Hz, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 2H). = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.2-1.75 (m, 8H), 0.65-1.0 (m, 6H). Example S3: Synthesis of 2-butyl-1-(4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-33)

[0175] Parts AH were identical to Example S1.

[0176] Part I. Cyclopropylacetic acid (33 mg, 0.33 mmol, 1.2 equiv.) and trimethylamine (140 mg, 1.39 mmol, 5.0 equiv.) were added to a solution of 1-(4-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (100 mg, 0.28 mmol, 1.0 equiv.) in anhydrous dimethylformamide (2 mL). The slurry was mixed for 5 minutes, and then HBTU (131 mg, 0.34 mmol, 1.25 equiv.) was added. The reaction mixture was further stirred for 2 hours under an argon atmosphere. The reaction mixture was diluted with ethyl acetate (100 mL), washed with water (3 × 30 mL), dried using magnesium sulfate, and concentrated in vacuo. The crude residue was taken up in ethyl acetate and methanol and purified using column chromatography (6% methanol / dichloromethane) to give 140 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide (compound number 63-34).

[0177] Part J. A solution of borane-dimethyl sulfide complex (2.0 M, 1.5 mL, excess) was added to a solution of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropylacetamide (123 mg, 0.28 mmol, 1.0 equiv) in anhydrous tetrahydrofuran (5 mL) at room temperature, and the reaction mixture was heated to reflux for 12 hours. The mixture was cooled to ambient temperature, quenched with 3 N HCl (1 mL), and stirred for 4 hours. The pH of the reaction mixture was made alkaline by the addition of 2 N sodium hydroxide, and the product was extracted with dichloromethane (20 mL × 10). The combined organic layers were concentrated under reduced pressure, and the residue was purified by flash chromatography using 6% methanol / dichloromethane as the eluent to give 28 mg of 2-butyl-1- (4-(((2-cyclopropylethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-33) was obtained. The purity of the product was assessed to be 97% by reverse phase HPLC, and the intended compound mass of 427.6 was confirmed by LC / MS, indicating that the intended compound structure was identical to that of the 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, J = 8.0 Hz, 2H), 2.68 (dd, J = 8.0 Hz, 2H), 1.82-1.74 (m, 2H), 1.45-1.36 (m, 4H), 0.91 (t, J = 7.8 Hz, 3H), 0.91-0.85 (m, 1H), 0.68-0.60 (m, 1H), 0.42-0.37 (m, 2H), 0.04-0.00 (m, 2H). Example S4: Synthesis of other exemplary compounds

[0178] Exemplary N-alkyl compounds of formula (J-1) were synthesized using procedures similar to Compound Nos. 63-33. Using techniques known to those skilled in the art, the calculated partition coefficients (cLogP) of exemplary compounds were determined using the Molecular Descriptors algorithm in Molecular Operating Environment software (e.g., Labute P, The Derivation and Applications of Molecular Descriptors Based Upon (Approximate) Surface Area; Chemoinformatics: Concepts, Methods, and Tools for Drug Discovery). (See Discovery, J. Bajorath ed. 2003). 1 H NMR and mass spectrometry data are detailed below for certain compounds of the invention, and Table 3 provides purity data and cLogP values. [Table 3] ND=Not determined.

[0179] Compound No. 63-33: 1 H NMR (CDCl3, 400 MHz): 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, J = 8.0 Hz, 2H), 2.68 (dd, J = 8.0 Hz, 2H), 1.82-1.74 (m, 2H), 1.45-1.36 (m, 4H), 0.91 (t, J = 7.8 Hz, 3H), 0.91-0.85 (m, 1H), 0.68-0.60 (m, 1H), 0.42-0.37 (m, 2H), 0.04-0.00 (m, 2H). Mass Spec: m / z 428.6 (M+1).

[0180] Compound No. 63-35: 1 H NMR (CDCl3, 300 MHz): δ 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.72 (s, 2H), 3.85 (s, 2H), 2.89 (t, J =7.6 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.20-2.35 (m, 1H), 1.8-2.1(m, 2H), 1.65-1.8 (m, 6H), 1.4-1.55 (m, 4H), 0.94 (t, J = 7.3 Hz, 3H). Mass Spec: m / z 442.9 (M+1).

[0181] Compound No. 63-36: 1H NMR (CDCl3, 300 MHz): δ 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.72 (s, 2H), 3.80 (br s, 4H), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.62-1.84 (m, 7H), 1.0-1.20 (m, 1H), 0.94 (t, J = 7.3 Hz, 3H). Mass Spec: m / z 456.4 (M+1).

[0182] Compound No. 63-39: 1 H NMR (CD3OD, 300 MHz): δ 7.80 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.30-7.48 (m, 3H), 7.05-7.14 (m, 3H), 5.88 (s, 2H), 3.81 (s, 2H), 2.89 (t, J = 7.5, 15.6 Hz, 2H), 2.44-2.55 (m, 2H), 1.70-1.85 (m, 2H), 1.35-1.55 (m,3H), 1.02 (s, J = 6 Hz, 3H), 0.94 (t, J = 7.5, 14.7 Hz, 3H), 0.50-0.75 (m, 2H), 0.20-0.35 (m,2H). Mass Spec: m / z 428.4 [M+1].

[0183] Compound No. 63-40: 11H NMR (CDCl3, 300 MHz): δ 7.82 (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), 5.73 (s, 2H), 5.54 (s, 2H), 3.78 (s, 2H), 2.50 - 2.70 (m, 2H), 1.75 - 1.80 (m, 2H), 1.40 - 1.55 (m, 2H), 1.04 (s, 3H), 1.02 (s, 3H), 0.94 (t, J = 7.5, 14.7 Hz, 3H), 0.7 - 0.8 (m, 1H), 0.4 - 0.5 (m, 1H), 0.1 - 0.05 (m, 1H). Mass Spec: m / z 442.5 [M + 1].

[0184] Compound No. 63 - 42: 1 1H NMR (CDCl3, 300 MHz): 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), 3.76 (s, 2H), 2.89 (t, J = 7.5, 15.6 Hz, 2H), 2.70 (t, J = 7.2, 15.0 Hz, 2H), 1.77 - 1.92 (m, 2H), 1.70 - 1.84 (m, 4H), 0.98 (s, 3H), 0.94 (t, J = 7.5, 14.7 Hz, 3H), 0.18 - 0.35 (m, 4H ). Mass Spec: m / z 442.5 [M+1].

[0185] Compound No. 63-43: 1 H NMR (CDCl3, 300 MHz): 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), 7.25-7.31 (m, 2H), 7.14 (t, J = 7.5, 15.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 2H), 5.71 (s, 2H), 5.59(s, 2H), 3.75 (s, 2H), 2.88 (br t, 2H), 2.63 (t, J = 7.2, 14.1 Hz, 3H), 0.55-0.72 (m, 1H), 0.4 (br d, 2H), 0.01 (d, J = 4.5 Hz, 2H). Mass Spec: m / z 442.5 [M+1].

[0186] Compound No. 63-46: 1 H NMR (CDCl3, 300 MHz): δ 7.81 (d, J = 8.1 Hz, 1H), 7.74 (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), 5.73 (s, 2H), 5.51 (s, 2H), 3.74 (s, 2H), 2.86 (t, J = 7.5, 15.6 Hz, 2H), 2.60-2.7 (m, 2H), 2.1-2.4 (m, 3H), 1.6-1.85 (m, 4H), 1.40-1.55 (m, 2H), 0.95-1.15 (m, 5H), 0.94 (t, J = 7.5, 14.7 Hz, 3H). Mass Spec: m / z 442.4 [M+1]. Example S5: Synthesis of 2-butyl-1-(4-(((cyclopropylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-38).

[0187] Part A. N,N-Diisopropylcarbodiimide (272 mg, 2.15 mmol) was added to a solution of cyclopropanecarboxylic acid (172 mg, 2.0 mmol) and pentafluorophenol (387 mg, 2.1 mmol) in dichloromethane (4 mL) in the presence of a catalytic amount of N,N-dimethylaminopyridine (12 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 440 mg of the desired (2,3,4,5,6-pentafluorophenyl)-cyclopropanecarboxylate product.

[0188] Part B. Compound No. 63-00 (80 mg, 0.22 mmol) was added to a solution of (2,3,4,5,6-pentafluorophenyl)-cyclopropanecarboxylate (61 mg, 0.24 mmol) in dichloromethane (3 mL) in the presence of triethylamine (45 mg, 0.44 mmol) and stirred at room temperature for 2 h. The reaction mixture was then concentrated under reduced pressure and purified by flash chromatography eluting with 3–8% methanol / 1% aqueous ammonia in dichloromethane to give 78 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclopropanecarboxamide as an off-white solid.

[0189] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclopropanecarboxamide (78 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was cooled to room temperature, carefully quenched with 1 M HCl (excess), and stirred at 55 °C for an additional 3 h. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by the addition of ice-cold 1 M NaOH solution, extracted with dichloromethane (3 × 10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. Upon recrystallization with 9:1 ethyl acetate / hexanes, the reaction gave 27 mg of 2-butyl-1-(4-(((cyclopropylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (Compound No. 63-38). The purity of the product was assessed to be 96% pure by reversed-phase HPLC at 254 nm, and the intended synthetic mass of 413.3 daltons was confirmed by LC / MS, with the intended synthetic structure at 300 MHz. 1 H NMR (CD3OD): δ 7.80 (d, J = 8.1 Hz, 1H), 7.67 (d, The molecular weights were confirmed by the following indices: J = 8.4 Hz, 1H), 7.38-7.48 (m, 3H), 7.07-7.20 (m, 3H), 5.92 (s, 2H), 3.97 (s, 2H), 3.0 (t, J = 7.8, 15.3 Hz, 2H), 2.66 (2, J = 7.2 Hz, 2H), 1.78-1.90 (m, 2H), 1.40-1.60 (m, 2H), 1.0 (t, J = 7.5 Hz, 3H), 0.5-0.6 (m, 2H), 0.2-0.3 (m, 2H). Example S6: Synthesis of 2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-45).

[0190] Part A. N,N-Diisopropylcarbodiimide (76 mg, 0.6 mmol) was added to a solution of 1-methylcyclobutanecarboxylic acid (57 mg, 0.5 mmol) and pentafluorophenol (94 mg, 0.52 mmol) in dichloromethane (3 mL) in the presence of a catalytic amount of N,N-dimethylaminopyridine (6 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 126 mg of the desired (2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutanecarboxylate product.

[0191] Part B. Compound No. 63-00 (84 mg, 0.23 mmol) was added to a solution of (2,3,4,5,6-pentafluorophenyl)-1-methylcyclobutanecarboxylate (72 mg, 0.26 mmol) in dichloromethane (3 mL) in the presence of triethylamine (48 mg, 0.47 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexanes. The residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl followed by water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 88 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamide as an off-white solid.

[0192] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-1-methylcyclobutane-1-carboxamide (88 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by the addition of ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. Upon recrystallization from 9:1 ethyl acetate / hexane, the reaction gave 32 mg of 2-butyl-1-(4-((((1-methylcyclobutyl)methyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 63-45). Product purity was assessed to be 96% pure by reverse-phase HPLC at 254 nm, and the intended mass of 441.3 daltons was confirmed by LC / MS at 300 MHz. 1 H 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), 1.6-1.88 (m, 8H), 1.35-1.60 (m, 2H), 1.12 (s, 3H), 0.94 (t, J = 7.5, 14.7 Hz, 3H). Example S7: Synthesis of 2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-44).

[0193] Part A. N,N-Diisopropylcarbodiimide (127 mg, 1.0 mmol) was added to a solution of cyclobutanecarboxylic acid (106 mg, 0.93 mmol) and pentafluorophenol (175 mg, 0.97 mmol) in dichloromethane (3 mL) in the presence of a catalytic amount of N,N-dimethylaminopyridine (12 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 126 mg of the desired (2,3,4,5,6-pentafluorophenyl)cyclobutanecarboxylate product.

[0194] Part B. Compound No. 63-00 (62 mg, 0.17 mmol) was added to a solution of (2,3,4,5,6-pentafluorophenyl)cyclobutanecarboxylate (50 mg, 0.18 mmol) in dichloromethane (3 mL) in the presence of triethylamine (35 mg, 0.34 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate / hexanes. The residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl followed by water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 85 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamide as an off-white solid.

[0195] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)cyclobutanecarboxamide (85 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by the addition of ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. After recrystallization from 9:1 ethyl acetate / hexane, the reaction gave 21 mg of 2-butyl-1-(4-(((cyclobutylmethyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 63-44). The purity of the product was confirmed by reverse-phase HPLC at 254 nm. The purity was assessed to be 95% by LC / MS, the intended synthetic mass of 427.3 Daltons was confirmed by LC / MS, and the intended synthetic structure was confirmed by 300 MHz. 1 H 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, 1H), 1.75-2.15 (m, 7H), 1.60-1.70 (m, 2H), 1.35-1.50 (m, 2H), 0.94 (t, J = 7.5, 14.7 Hz, 3H). Example S8: Synthesis of 2-butyl-1-(4-(((2-cyclobutyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-47).

[0196] Part A. N,N-Diisopropylcarbodiimide (113 mg, 0.89 mmol) was added to a solution of 3-cyclobutyl-3-methyl-butan-2-one (70 mg, 0.5 mmol) and pentafluorophenol (108 mg, 0.59 mmol) in dichloromethane (3 mL) in the presence of a catalytic amount of N,N-dimethylaminopyridine (11 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 141 mg of the desired (2,3,4,5,6-pentafluorophenyl)-2-cyclobutyl-2-methyl-propanoate product. Part B. Compound No. 63-00 (60 mg, 0.17 mmol) was added to a solution of (2,3,4,5,6-pentafluorophenyl)-2-cyclobutyl-2-methyl-propanoate (54 mg, 0.18 mmol) in dichloromethane (3 mL) in the presence of triethylamine (34 mg, 0.34 mmol) and stirred at reflux for 3 days. The reaction mixture was then cooled to room temperature, concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate in hexanes. The washed residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl followed by water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 67 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclobutyl-2-methylpropanamide as an off-white solid.

[0197] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclobutyl-2-methylpropanamide (67 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by the addition of ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. Upon recrystallization from 9:1 ethyl acetate / hexane, the reaction gave 13 mg of 2-butyl-1-(4-(((2-cyclobutyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 63-47). Product purity was assessed to be 92% pure by reverse-phase HPLC at 254 nm, and the intended mass of 469.3 daltons was confirmed by LC / MS at 300 MHz. 1 H NMR (CDCl3): δ 7.80 (d, J = 8.1 H z, 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), 6.60 (br s, 1H), 5.73 (s, 2H), 3.74 (br s, 4H), 2.90 (t, J = 7.5, 15.6 Hz, 2H), 2.35 (s, 2H), 1.20-1.8 (m, 10H), 0.94 (t, J = 7.5, 14.7 Hz, 3H), This was confirmed by 0.90 (s, 6H). Example S9: Synthesis of 2-butyl-1-(4-(((2-cyclopropyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-41).

[0198] Part A. N,N-Diisopropylcarbodiimide (127 mg, 1.0 mmol) was added to a solution of 2-cyclopropyl-2-methyl-propanoic acid (106 mg, 0.93 mmol) and pentafluorophenol (175 mg, 0.97 mmol) in dichloromethane (3 mL) in the presence of a catalytic amount of N,N-dimethylaminopyridine (12 mg) and stirred overnight at room temperature. The mixture was then diluted with ether (20 mL), the precipitated urea was removed by filtration, and the filtrate was concentrated to give the crude product. This crude product was suspended in 1% ethyl acetate / hexane, and any remaining precipitated urea was again removed by filtration. The resulting filtrate was concentrated under reduced pressure to give 130 mg of the desired (2,3,4,5,6-pentafluorophenyl)-2,2-dimethylcyclopropanecarboxylate product.

[0199] Part B. Compound No. 63-00 (62 mg, 0.17 mmol) was added to a solution of (2,3,4,5,6-pentafluorophenyl)-2,2-dimethylcyclopropanecarboxylate (50 mg, 0.18 mmol) in dichloromethane (3 mL) in the presence of triethylamine (35 mg, 0.34 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure, and the residue was washed with 5% ethyl acetate in hexanes. The washed residue was dissolved in dichloromethane (15 mL), washed with 1 M HCl followed by water (20 mL), dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give 85 mg of N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropyl-2-methylpropanamide as an off-white solid.

[0200] Part C. N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-2-cyclopropyl-2-methylpropanamide (85 mg) was reduced with borane dimethyl sulfide complex (3.5 equivalents) at 55° C. for 12 hours. The reaction was then cooled to room temperature, carefully quenched with 2 M HCl (excess), and stirred at 55° C. for an additional 3 hours. The reaction was cooled to room temperature, diluted with water (10 mL), and then extracted with dichloromethane (10 mL) to remove impurities. The pH of the reaction mixture was adjusted to 8.0 by the addition of ice-cold 1 M NaOH solution, extracted with dichloromethane (3×10 mL), dried over MgSO4, and concentrated under reduced pressure to give an off-white solid. Upon recrystallization from 9:1 ethyl acetate / hexane, the reaction gave 14 mg of 2-butyl-1-(4-(((2-cyclopropyl-2-methylpropyl)amino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound no. 63-41). Product purity was assessed to be 94% pure by reverse-phase HPLC at 254 nm, and the intended mass of 455.3 daltons was confirmed by LC / MS at 300 MHz. 1 H NMR (CDCl3): δ 7.82 (d, J = 8.1 Hz, 1H), 7.75 (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, The cleavage was confirmed by the following NMR spectra: 5.62 (s, 2H), 3.79 (s, 2H), 2.90 (t, J = 7.5, 15.6 Hz, 2H), 2.87 (s, 2H), 1.75-1.90 (m, 2H), 1.40-1.55 (m, 2H), 0.94 (t, J = 7.5, 14.7 Hz, 3H), 0.75 (s, 6H), 0.6-0.75 (m, 1H), 0.15-0.3 (m, 4H). Example S10: Synthesis of 2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine (compound number 63-49).

[0201] Part A. tert-Butyldimethylsilyl chloride (3.31 g, 22 mmol) was added to a solution of 4-cyanobenzyl alcohol (2.66 g, 20 mmol) in N,N-dimethylformamide (20 mL) in the presence of imidazole (2.72 g, 40 mmol) at room temperature and stirred for 4 hours. The reaction mixture was poured into water (150 mL) and extracted with 10% ethyl acetate / hexane (3 × 75 mL). The combined organic extracts were washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting product was further purified by flash chromatography eluting with hexane to give 4.84 g of 4-(((tert-butyldimethylsilyl)oxy)methyl)benzonitrile.

[0202] Part B. A solution of 4-(((tert-butyldimethylsilyl)oxy)methyl)benzonitrile (4.84 g) in methanol (200 mL) was hydrogenated in the presence of Raney nickel (1.0 g slurry in water) under 60 psi of hydrogen using a Parr hydrogenation apparatus for 4 hours. The reaction was filtered, and the filtrate was concentrated under reduced pressure. The resulting product, (4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanamine (4.8 g), was used without purification.

[0203] Part C. A solution of 4-chloro-3-nitroquinoline (4.16 g, 20 mmol) in dichloromethane (100 mL) was slowly added to a solution of 4-(tert-butyldimethylsiloxymethyl)benzylamine (4.8 g, 19 mmol) and diisopropylethylamine (DIPEA) (3.87 g, 30 mmol) in dichloromethane (100 mL) and stirred for 12 hours. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate (200 mL), washed with water (2 × 100 mL), and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure, and the resulting product was purified by flash chromatography eluting with 1:1 ethyl acetate / hexane to give 4.67 g of (4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)quinolin-3-yl)(oxo)-λ 4 -Azanol was obtained.

[0204] Part D. (4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)-quinolin-3-yl)(oxo)-λ 4 A solution of -azanol (4.67 g) in ethyl acetate (250 mL) was hydrogenated in the presence of palladium on carbon (10%, 1.0 g) at 60 psi for 4 hours. The catalyst was removed by filtration and the solvent was removed from the filtrate under reduced pressure to give the product N 4 -(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)quinoline-3,4-diamine (4.5 g) was used without further purification.

[0205] Part E. A solution of valeryl chloride (1.50 g, 12.46 mmol, 1.05 equiv) in dichloromethane (30 mL) was dissolved in N 4 The solution was added dropwise to a solution of 4-(4-(((tert-butyldimethylsilyl)oxy)methyl)-benzyl)quinoline-3,4-diamine (4.5 g, 11.86 mmol) in dry pyridine (20 mL) at 0°C. After addition, the reaction mixture was warmed to room temperature and stirred for 4 hours. The solvent and pyridine were removed under reduced pressure. The residue was dissolved in dichloromethane (300 mL) and then washed successively with water, saturated sodium bicarbonate solution (100 mL), saturated copper sulfate solution (3 × 50 mL), and water (100 mL), then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The product, N-(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)quinolin-3-yl)pentanamide (5.5 g), was used without further purification.

[0206] Part F. A solution of N-(4-((4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)amino)-quinolin-3-yl)pentanamide (5.5 g, 11.87 mmol) in a mixture of ethanol / water (8:2 v / v, 150 mL) was heated to 60° C. for 18 hours in the presence of potassium carbonate (2.5 g, 18.11 mmol, 1.5 equiv). The solvent was removed under reduced pressure. The residue was partitioned between ethyl acetate (200 mL) and water (100 mL), the ethyl acetate layer was separated and dried over anhydrous magnesium sulfate, and the residue was concentrated under reduced pressure. Further purification by flash chromatography eluting with 20% ethyl acetate / hexanes afforded 3.43 g of 2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinoline.

[0207] Part G. meta-Chloroperbenzoic acid (60-70%, 2.5 g) was added to a solution of 2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinoline (3.43 g, 7.4 mmol) in dichloromethane (200 mL) at room temperature and stirred for 6 hours. The reaction was quenched by the addition of saturated sodium sulfite solution (20 mL). The organic layer was separated, washed sequentially with saturated sodium bicarbonate solution (50 mL) and then water (50 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Further purification by flash chromatography using 25% ethyl acetate / hexane elution gave 3.1 g of 2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)-benzyl)-1H-5λ. 4 -imidazo[4,5-c]quinolin-5-ol was obtained.

[0208] Part H. 2-Butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-5λ 4 A solution of 4-imidazo[4,5-c]quinolin-5-ol (3.1 g, 6.5 mmol) in dichloromethane (50 mL) was added to tri-n-butylamine (2.4 g, 13 mmol) and phthalimide (1.91 g, 13 mmol), and the reaction mixture was cooled to 0 °C. A solution of benzoyl chloride (1.82 g, 13 mmol) in dichloromethane (10 mL) was slowly added to the reaction, and the mixture was warmed to room temperature and stirred for 30 minutes. The reaction was diluted with dichloromethane (100 mL), washed sequentially with saturated aqueous ammonium chloride (100 mL) and then water (100 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting product was isolated by flash chromatography eluting with 10% ethyl acetate / hexane to give 2.85 g of 2-(2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione.

[0209] Part I. 1 M tetrabutylammonium fluoride solution (6 mL) was added to a solution of 2-(2-butyl-1-(4-(((tert-butyldimethylsilyl)oxy)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione (2.85 g, 4.7 mmol) in dry tetrahydrofuran (10 mL) at 0° C. After the addition, the reaction mixture was warmed to room temperature and further stirred for 6 hours. The reaction was quenched by the addition of saturated ammonium chloride (20 mL) and ethyl acetate (100 mL). ), washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Further purification by flash chromatography eluting with dichloromethane / hexane (1:1) gave 1.57 g of 2-(2-butyl-1-(4-(hydroxymethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione.

[0210] Part J. A solution of DMSO (2.5 g, 32 mmol) in CHCl (10 mL) was added to a solution of oxalyl chloride (2.0 g, 16 mmol) in dichloromethane (10 mL) and 3 Å molecular sieves in CHCl (20 mL) at −78° C. under argon. After 15 min, a solution of 2-(2-butyl-1-(4-(hydroxymethyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione (1.57 g, 3.2 mmol) in CHCl (3 mL) was added dropwise slowly. After 30 min, EtN (4.5 g, 45 mmol) was added dropwise, and the reaction was stirred at −78° C. for 30 min and then allowed to warm slowly to room temperature. After stirring at room temperature for an additional hour, the reaction mixture was quenched by the addition of saturated ammonium chloride solution. The organic layer was separated, washed with water (25 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. Further purification by flash chromatography afforded 1.15 g of 4-((2-butyl-4-(1,3-dioxoisoindolin-2-yl)-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzaldehyde.

[0211] Part K. A solution of 4-((2-butyl-4-(1,3-dioxoisoindolin-2-yl)-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzaldehyde (2.33 mmol) and cyclohexylamine (7 mmol) in dichloromethane is heated to reflux for 12 hours in the presence of a catalytic amount of p-toluenesulfonic acid. The reaction mixture is concentrated under reduced pressure. The crude product, 2-(2-butyl-1-(4-((cyclohexylimino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione, is used without further purification.

[0212] Part L. Sodium borohydride (10 mmol) is added to a solution of 2-(2-butyl-1-(4-((cyclohexylimino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione in methanol at room temperature and stirred for 2 hours. The reaction mixture is quenched with saturated ammonium chloride solution and extracted with dichloromethane (3 × 30 mL). The combined organic layers are washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The product, 2-(2-butyl-1-(4-((cyclohexylamino)methyl)-benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione, is purified by flash chromatography.

[0213] Part M. Hydrazine (100 mg) is added to a solution of 2-(2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-yl)isoindoline-1,3-dione (0.38 mmol) in methanol and stirred for 12 hours. The solvent is removed under reduced pressure, and the resulting product, 2-butyl-1-(4-((cyclohexylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine, is purified by flash chromatography. Example S11: Synthesis of exemplary N-acyl compounds of formula (J-2)

[0214] Exemplary N-acyl compounds of formula (J-2) were synthesized using procedures similar to those in Part I of Compound Nos. 63-33. 1 H NMR and mass spectrometry data are detailed below for certain compounds of the invention, and Table 4 provides purity data and cLogP values. [Table 4]

[0215] Compound No. 63-34: 1 H NMR (CDCl3, 400 MHz): δ 7.75 (dd, J = 8.3, 0.9 Hz, 1H), 7.67 (dd, J = 8.3, 1.1 Hz, 1H), 7.40 (m, J = 8.3, 7.1, 1.4 Hz, 1H), 7.22 (d, J = 8.2 Hz, 2H), 7.11 (m, J = 8.2, 7.1, 1.2 Hz, 1H), 6.98 (d, J = 8.2 Hz, 2H), 6.30 (at, 1H), 5.68 (s, 2H), 4.42 (d, J = 6.0 Hz, 2H), 2.84 (add, 2H), 2.16 (dd, J = 7.2 Hz, Mass Spec: m / z 442.6 (M+1). Example S12: Synthesis of exemplary compounds of formula (K)

[0216] An exemplary N-acyl compound of formula (K-2) was synthesized using a procedure similar to compound No. 63-10. 1 H NMR and mass spectrometry data are detailed below for certain compounds of the invention, and Table 5 lists the compounds along with purity data and cLogP values.

[0217] Exemplary N-alkyl compounds of formula (K-1) were synthesized using procedures similar to compound No. 63-17. 1 H NMR and mass spectrometry data are detailed below for certain compounds of the invention, and Table 6 provides purity data and cLogP values.

[0218] Compound No. 63-02: 1 H NMR (CDCl3, 400 MHz): δ 7.80 (dd, J = 8.4, 1.2 Hz, 1H), 7.64 (dd, J = 8.2, 1.0 Hz, 1H), 7.40 (m, J = 8.4, 7.0, 1.0 Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 7.09 (m, J = 8.2, 7.1, 1.3 Hz, 1H), 6.96 (d, J = 8.2 Hz, 2H), 6.28 (bs, 2H), 6.02 (bt, J = 5.7 Hz, 1H), 5.65 (s, 2H), 4.37 (d, J = 5.8 Hz, 2H), 2.83 (dd, J = 7.8, 7.8 Hz, 2H), 2.15 (dd, J = 7.6, 7.6 Hz, 2H), 1.81-1.73 (m, 2H), 1.64-1.56 (m, 2H), 1.42 (dq, J = 15.0, 7.4 Hz, 2H), 1.32-1.21 (m, 4H), 0.91 (t, J= 7.3 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H). Spec: m / z 468.2 (M+1).

[0219] Compound No. 63-05: 1 H NMR (CDCl3, 300 MHz): 7.89 (d, J = 8.1 Hz, 1H),7.86 (d, J = 8.1 Hz, 1H), 7.69(t, 1H), 7.74 (t, J = 15.6, 8.4, 1H), 7.47 (t, J = 15.5, 7.5 Hz, 1H), 7.38 (m, 4H), 7.14(bs, 1H) 7.08 (d, J=8.1 Hz) 5.85 (s, 2H), 4.53 (d, J=6Hz, 2H), 2.99 (t, J = 7.8 Hz, 2H), 2.30 (t, J=7.5 Hz, 2H), 1.90-1.97 (m, 4H), 1.52-1.75 (m, 4H), 1.26-1.48 (m, 14 H), 0.90-1.10 (m, 6H). Mass Spec: m / z 500.8 (M+1).

[0220] Compound No. 63-06: 1 H NMR (CDCl3, 300 MHz): 8.05 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.74 (t, J = 8.4 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.14(d, J = 8.1 Hz, 2H), 6.04 (s, 2H), 4.42 (s, 2H), 3.74 (s, 2H), ), 3.10 (t, J = 7.8 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.90-1.97 (m, 2H), 1.52-1.75 (m,4H), 1.26-1.48 (m, 12 H), 0.90-1.10 (m, 6H). Mass Spec: m / z 514.5 (M+1).

[0221] Compound No. 63-07: 11H NMR (CDCl3, 300 MHz): 7.88 (d, J = 8.1 Hz, 1H), 7.69(d, J = 8.4 Hz, 1H), 7.50 (t, J = 15.6, 8.4, 1H),7.29 (d, J = 8.1 Hz, 2H), 7.20 (t, J = 15.5, 7.5 Hz, 1H), 7.06(d, J = 8.1 Hz, 2H), 5.90 (s, 2H), 4.33 (s, 2H), 3.74 (s, 2H), ), 3.0 (t, J = 15.0, 7.8 Hz, 2H), 2.21 (t, J = 14.7, 7.5 Hz, 2H), 1.90 - 1.97 (m, 2H), 1.42 - 1.70 (m,4H), 1.26 - 1.48 (m, 14 H), 0.85 - 1.05 (m, 6H). Mass Spec: m / z 528.6 (M+1).

[0222] Compound No. 63 - 08: 1 1H NMR (CDCl3, 400 MHz): δ 7.74 (d, J = 8.2 Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.37 (dd, J = 7.4, 7.4 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.07 (dd, J = 7.5, 7.5 Hz, 1H), 6.95 (d, J = 8.0 Hz, 2H), 5.98 (bt, J = 5.6 Hz, 1H), 5.89 (bs, 2H), 5.63 (s, 2H), 4.35 (d, J = 5.8 Hz, 2H), 2.81 (dd, J = 8.0, 8.0 Hz, 2H), 2.14 (dd, J = 7.6, 7.6 Hz, 2H), 1.80 - 1.71 (m, 2H), 1.61 - 1.56 (m, 2H), 1.39 (dq, J = 15.0, 7.4 Hz, 2H), 1.32 - 1.21 (m, 18H), 0.90 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H). Mass Spec: m / z 542.4 ( M+1).

[0223] Compound No. 63-09: 1 H NMR (DMSO-d6, 300 MHz): 8.18 (t, 1H), 7.8 (d, J = 8.4 Hz, 1H), 7.6 (d, J = 8.5 Hz, 1H), 7.34 (t, J = 8.4, 1H), 7.18 (d, J = 8.5 Hz, 2H), 6.9-7.12 (m, 3H),6.55 (s, 2H), 5.8 (s, 2H),4.2 (d, 2H), 2.90 (t, J =7.6 Hz, 2H), 2.2 (t, J = 7.2 Hz, 2H), 1.65-1.80 (m, 2H), 1.3-1.75 (m, 4H), 1.25-1.38 (m, 10 H), 0.65-1.0 (m, 6H). Mass Spec: m / z 556.9 (M+1).

[0224] Compound No. 63-10: 1 H NMR (CDCl3, 300 MHz): 7.98 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 8.4 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 8.1 Hz, 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, 20 H), 0.85-1.05 (m, 6H). Mass Spec: m / z 570.8 (M+1).

[0225] Compound No. 63-11: 1H NMR (CDCl3, 300 MHz): 7.98 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 8.4 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.30 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 5.95 (s, 2H), 4.33 (s, 2H), 3.74 (s, 2H), 3.03 (t, J = 7.8 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.82-1.9 (m, 2H), 1.42-1.70 (m,4H), 1.26-1.48 (m, 22 H), 0.85-1.05 (m, 6H). Mass Spec: m / z 585.2 (M+1).

[0226] Compound No. 63-31: 1 H NMR (CD3OD, 300 MHz): 7.82 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.42 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.9 (s, 2H), 4.39 (s, 2H), ), 3.0 (t, J =7.6 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 1.42-1.90 (m, 6H), 1.2-1.4 (m, 13H), 0.65-1.0 (m, 6H). Mass Spec: m / z 598.7 (M+1).

[0227] Compound No. 63-13: 11H NMR (CDCl3, 400 MHz): δ 7.77 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.41 (dd, J = 7.4, 7.4 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.11 (dd, J = 7.5, 7.5 Hz, 1H), 6.98 (d, J = 8.0 H z, 2H), 5.87 (bt, J = 5.6 Hz, 1H), 5.87 (bs, 2H), 5.68 (s, 2H), 4.39 (d, J = 5.8 Hz, 2H), 2.85 (dd, J = 8.0, 8.0 Hz, 2H), 2.17 (dd, J = 7.6, 7.6 Hz, 2H), 1.83 - 1.75 (m, 2H), 1.65 - 1.58 (m, 2H), 1.43 (dq, J = 15.0, 7.4 Hz, 2H), 1.32 - 1.21 (m, 30H), 0.92 (t, J = 7.3 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). Mass Spec: m / z 625.5 (M+1).

[0228] Compound No. 63 - 00: 1 1H NMR (MeOHd4, 400 MHz): δ 7.70 (dd, J = 8.4, 1.0 Hz, 1H), 7.63 (dd, J = 8.4, 0.8 Hz, 1H), 7.36 (m, J = 8.4, 7.0, 1.4 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.03 (m, J = 8.3, 7.0, 1.3 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H), 5.72 (s, 2H), 3.72 (s, 2H), 2.88 (dd, J = 7.6, 7.6 Hz, 2H), 1.77 - 1.69 (m, 2H), 1.45 - 1.35 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). Mass Spec: m / z 360.2 (M+1).

[0229] Compound No. 63-17: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.2-1.75 (m, 8H), 0.65-1.0 (m, 6H). Mass Spec: m / z 430.3 (M+1).

[0230] Compound No. 63-18: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.72 (br s, 4H), 3.76 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.35 (br s, 4H), 0.65-1.0 (m, 6H). Mass Spec: m / z 444.6 (M+1).

[0231] Compound No. 63-19: 1H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H),1.4-1.6 (m, 4H),1.2-1.35 ( b, s, 8H), 0.65-1.0 (m, 6H). Mass Spec: m / z 458.4 (M+1).

[0232] Compound No. 63-20: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 10H), 0.65-1.0 (m, 6H). Mass Spec: m / z 472.4 (M+1).

[0233] Compound No. 63-21: 1H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 12H), 0.65-1.0 (m, 6H). Mass Spec: m / z 486.5 (M+1).

[0234] Compound No. 63-22: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.80 (s, 2H), ), 2.89 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.84 (m, 2H), 1.4-1.6 (m, 4H), 1.2-1.35 (b, s, 14H), 0.65-1.0 (m, 6H). Mass Spec: m / z 500.5 (M+1).

[0235] Compound No. 63-24: 1 H NMR (CDCl3, 300 MHz): 7.78 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 8.4, 1H), 7.28 (d, J = 8.5 Hz, 2H), 7.10 (t, J = 8.4 Hz, 1H), 6.98 (d, J = 8.3 Hz, 2H), 5.70 (br s, 4H), 3.73 (s, 2H), ), 2.87 (t, J =7.6 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.71-1.84 (m, 2H), 1.38-1.55 (m, 4H), 1.10-1.35 (m, 18H), 0.65-1.0 (m, 6H). Mass Spec: m / z 528.7 (M+1).

[0236] Compound No. 63-32: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.42 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.73 ( s, 2H), 5.5 (br s, 2H) 3.75 (s, 2H), ), 2.90 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.75-1.85 (m, 4H), 1.4-1.6 (m, 4H), 1.25-1.38 (m, 15 H), 0.65-1.0 (m, 6H). Mass Spec: m / z 584.9 (M+1).

[0237] Compound No. 63-29: 1 H NMR (CDCl3, 300 MHz): 7.8 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.42 (t, J = 8.4, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.3 Hz, 2H), 5.72 (s, 2H), 3.80 (s, 2H), 3.78 (s, 2H), 2.90 (t, J =7.6 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.65-1.85 (m, 2H),1.35-1.6 (m, 4H), 1.25-1.38 (m, 15H), 0.65-1.0 (m, 6H). Mass Spec: m / z 612.8 (M+1). [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] Example S13: Synthesis of further exemplary N-alkyl compounds of formula (K-1)

[0238] Exemplary N-alkyl Compounds Nos. 63-16, 63-23, 63-25, 63-26, 63-27, 63-28, and 63-30 of Formula (K-1) are synthesized using a procedure similar to that described for Compound No. 63-17 (Example S2). In Part I, the following carboxylic acids are used in place of the valeric acid used for Compound No. 63-17: n-butanoic acid (Compound No. 63-16); undecanoic acid (Compound No. 63-23); tridecanoic acid (Compound No. 63-25); tetradecanoic acid (Compound No. 63-26); pentadecanoic acid (Compound No. 63-27); heptadecanoic acid (Compound No. 63-28); and nonadecanoic acid (Compound No. 63-30). The resulting N-acyl derivatives are then reduced to the final N-alkyl compounds according to Part J described for Example S2. Example S14: Synthesis of further exemplary N-acyl compounds of formula (K-2)

[0239] Exemplary N-acyl Compound Nos. 63-01, 63-03, 63-04, 63-12, 63-14, and 63-15 of Formula (K-2) are synthesized using procedures similar to those described through Part H of Compound No. 63-10 (Example S1). In Part I, the following carboxylic acids are used in place of the myristic acid used for Compound No. 63-10 to form the N-acyl product: pentanoic acid (Compound No. 63-01), heptanoic acid (Compound No. 63-03), octanoic acid (Compound No. 63-04), heptadecanoic acid (Compound No. 63-12), nonadecanoic acid (Compound No. 63-14), and arachidic acid (Compound No. 63-15). Biological Examples Example B1. In vitro biological assays and results method

[0240] Plasmacytoid dendritic cell (pDC)-enriched peripheral blood mononuclear cells (PBMCs) were prepared from the blood of a series of human donors (3-5 donors per experiment). PBMCs were isolated using Ficoll-Paque Premium® (GE Healthcare, Chicago, IL) using methods well known to those skilled in the art. pDCs were magnetically isolated from the collected total PBMC population using CD304 (BDCA-4 / Neuropilin-1) microbeads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Isolated pDCs were then diluted to 1 x 10 cells to achieve relative enrichment for this cell type. 8 and 2×10 8 Duplicate cultures of pDC-enriched PBMCs (2.5 x 10 in RPMI-1640 medium + 10% fetal bovine serum) were then established. 6Cells (cells / mL, cultured in 96-well plates) were incubated for 24 hours with 10 serially diluted concentrations covering the range of 0.1 nM to 400 nM of Compounds 63-02, 63-05, 63-11, 63-13, 63-31, 63-34, 63-38, and 63-47, and their unmodified analog, 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (Compound 63-00). Culture supernatants were collected, and interferon-alpha (IFNα) protein levels were measured by ELISA (MabTech, Cincinnati, OH) according to the manufacturer's instructions.

[0241] Monocytes were prepared as described above and magnetically isolated from PBMCs after labeling with CD14 microbeads (Miltenyi Biotec, San Diego, CA) according to the manufacturer's instructions. Duplicate cultures of monocytes (1 × 10 in duplicate in RPMI-1640 + 10% fetal bovine serum) were cultured. 6 Cells / mL, cultured in 96-well plates) were incubated with 10 serially diluted concentrations covering the range of 2 nM to 40 μM of compounds Nos. 63-02, 63-05 to 63-11, 63-13, 63-31, 63-34, 63-38 to 63-47, and their unmodified congeners, 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5- c]quinolin-4-amine (Compound No. 63-00) for 24 hours. Culture supernatants were collected, and tumor necrosis factor alpha (TNFα) protein levels were measured by ELISA (MabTech, Cincinnati, Ohio) according to the manufacturer's instructions. result

[0242] i) a compound of formula (K-2) wherein R is a linear alkyl chain having 5 (Compound No. 63-02), 8 (Compound No. 63-05), 9 (Compound No. 63-06), 10 (Compound No. 63-07), 11 (Compound No. 63-08), 12 (Compound No. 63-09), 13 (Compound No. 63-10), 14 (Compound No. 63-11), 15 (Compound No. 63-31), or 17 (Compound No. 63-13) carbons added to the amide group on the benzylmethyl moiety, or ii) R 0 The modifying effect of one of the compounds of formula (J-2), in which a cyclopropylmethyl moiety has four carbons added to the amide group on the benzylmethyl moiety (Compound No. 63-34), on the in vitro agonist bioactivity of TLR7 (induction of IFNα protein in pDC-enriched PBMC cultures) and TLR8 (induction of TNFα protein in monocyte cultures) was evaluated. Table B1-1 summarizes the relationship between structure, calculated cLogP, and TLR7 / 8 agonist bioactivity for Compound Nos. 63-00, 63-02, 63-05 to 63-11, 63-13, 63-31, and 63-34. The agonist potencies for TLR7 and TLR8 are shown in Table B1-1 and are expressed in nanomolar as effective concentrations for 50% of the maximal response (EC 50These alkyl chain modifications of Compound 63-00 generally resulted in a 2- to 3-fold reduction in TLR7 agonist potency, except for the 5-carbon variant (Compound 63-02) and the 17-carbon variant (Compound 63-13), which had 11- and 41-fold reductions in TLR7 agonist potency, respectively. In contrast, the 5-carbon variant (Compound 63-02), 8-carbon variant (Compound 63-05), 9-carbon variant (Compound 63-06), 10-carbon variant (Compound 63-07), 15-carbon variant (Compound 63-31), and 17-carbon variant (Compound 63-13) had less than 10- to 26-fold reductions in TLR8 agonist potency compared to unmodified Compound 63-00. Unexpectedly, variants with carbon chain lengths of 11 (Compound No. 63-08), 12 (Compound No. 63-09), 13 (Compound No. 63-10), and 14 (Compound No. 63-11) showed less reduced agonist activity, with Compound No. 63-10 demonstrating only a 2.4-fold reduction in TLR8 agonist potency compared to unmodified Compound No. 63-00. 0 Compound No. 63-34, in which is a cyclopropylmethyl group, demonstrated an 8-fold reduction in TLR7 agonist potency and a 7-fold reduction in TLR8 agonist potency compared to unmodified Compound No. 63-00. These data are comparable to the reduction in TLR7 and TLR8 agonist potency observed with the linear 5-carbon alkyl chain modified Compound No. 63-02. [Table B1-1]

[0243] i) a modification of a compound of formula (K-1) where R is a linear alkyl chain with 5 (Compound No. 63-17), 6 (Compound No. 63-18), 7 (Compound No. 63-19), 8 (Compound No. 63-20), 9 (Compound No. 63-21), 10 (Compound No. 63-22), 12 (Compound No. 63-24), 16 (Compound No. 63-32), or 18 (Compound No. 63-29) carbons added to the amine group on the benzylmethyl moiety; or ii) R 0The effect of modifying the compound of formula (J-1) by adding five carbons (compound number 63-33) to the amide group (cyclopropylethyl), six carbons (compound number 63-35) to the cyclobutylethyl, or seven carbons (compound number 63-36) to the cyclopentylethyl moiety was evaluated on the in vitro agonistic bioactivity of TLR7 (induction of IFNα protein in pDC-enriched PBMC cultures) and TLR8 (induction of TNFα protein in monocyte cultures). Table B1-2 summarizes the relationship between structure, calculated cLogP values, and TLR7 / 8 agonistic bioactivity for compounds numbered 63-00, 63-17 to 63-22, 63-24, 63-29, 63-32, 63-33, 63-35, and 63-36. Agonist potencies for TLR7 and TLR8 are shown in Table B1-2 and are expressed as effective concentrations for 50% of the maximal response (EC ) in nanomolar. 50 , reported in nM). Modification of 63-00 with linear alkyl chains demonstrated a greater decrease in TLR7 / 8 agonist potency with increasing carbon number, while the five (63-17) and six (63-18) carbon variants demonstrated slightly improved TLR8 agonist potency. Modification of 63-00 with three hydrocarbyl groups (cyclopropylethyl, cyclobutylethyl, and cyclopentylethyl) resulted in a 4- to 12-fold decrease in TLR7 agonist bioactivity but a 2- to 3-fold increase in agonist potency. [Table B1-2]

[0244] R 0However, the (cyclopropyl)ethyl moiety (Compound No. 63-33), (cyclobutyl)ethyl moiety (Compound No. 63-35), (cyclopentyl)ethyl moiety (Compound No. 63-36), (cyclopropyl)methyl moiety (Compound No. 63-38), (2-methylcyclopropyl)methyl moiety (Compound No. 63-39), (2,2-dimethylcyclopropyl)methyl moiety (Compound No. 63-40), (2-cyclopropyl)-(2,2-dimethyl)ethyl moiety (Compound No. 63-41), (1-methylcyclopropyl)ethyl moiety (Compound No. 63-42), (3-cyclopropyl)propyl The modifying effects of compounds of formula (J-1) containing a propyl moiety (compound number 63-43), a (cyclobutyl)methyl moiety (compound number 63-44), a (1-methylcyclobutyl)methyl moiety (compound number 63-45), a (3-methylcyclobutyl)methyl moiety (compound number 63-46), or a (2-cyclobutyl)-(2,2-dimethyl)ethyl moiety (compound number 63-47) on the in vitro agonistic bioactivity of TLR7 (induction of IFNα protein in pDC-enriched human PBMC cultures) and TLR8 (induction of TNFα protein in human monocyte cultures). Table B1-3 summarizes the TLR7 / 8 agonistic bioactivity relationships for compounds numbered 63-00, 63-33, 63-35, 63-36, and 63-38 to 63-47. The TLR7 and TLR8 agonist potencies shown in Table B1-3 are reported as a percentage of the 50% effective compound concentration of the maximal response determined for Compound No. 63-00. The EC for a given compound was 0.01 due to variability among human blood donors in cytokine levels secreted from purified immune cells used to assess the potency of TLR7 and TLR8 agonist compounds. 50 The calculated absolute values ​​of potency fluctuated slightly; to normalize for this effect, the data in Table B1-3 were compared to the EC determined for the unmodified imidazoquinoline-based chemical structure (Compound No. 63-00). 50 The values ​​are expressed as a percentage of the total.

[0245] As shown in Table B1-3, modification of the chemical structure of Compound No. 63-00 with various cycloalkyl moieties to generate compounds of formula (J-1) resulted in up to an 11-fold reduction in TLR7 agonist potency. The (cyclobutyl)methyl variant (Compound No. 63-44) and the (1-methylcyclobutyl)methyl variant (Compound No. 63-45) demonstrated the least reduction in TLR7 agonist activity (1.2- and 1.9-fold reduction in potency, respectively), whereas the (cyclopentyl)ethyl variant (Compound No. 63-36) and the (2-cyclobutyl)-(2,2-dimethyl)ethyl variant (Compound No. 63-47) demonstrated the greatest reduction in TLR7 agonist activity (10.9- and 9.0-fold reduction in potency, respectively). Unexpectedly, identical sets of structural modifications to the chemical structure of Compound No. 63-00 demonstrated similar or even greater TLR8 agonist potency. The (cyclobutyl)methyl variant (Compound No. 63-44) and the (1-methylcyclobutyl)methyl variant (Compound No. 63-45) demonstrated the greatest increases in TLR8 agonist potency (5.9-fold and 7.7-fold, respectively). In contrast, the (1-methylcyclopropyl)ethyl variant (Compound No. 63-42) and the (2-cyclobutyl)-(2,2-dimethyl)ethyl variant (Compound No. 63-47) demonstrated slightly improved to slightly inferior TLR8 agonist activity compared to Compound No. 63-00. TLR7 / 8 agonist small molecules with more closely matched TLR7 and TLR8 agonist potencies are more likely to achieve comparable activation of the two-receptor system upon administration of a therapeutic dose of the compound in a given pharmaceutical composition, and therefore are more likely to activate a broader range of relevant immune cell types. Compounds with balanced dual potency also allow for the synthesis and characterization of single active pharmaceutical ingredients, facilitating GMP manufacturing at lower costs and enabling simpler and more predictable regulatory pathways. [Table B1-3] Example B2. Preparation of pharmaceutical compositions

[0246] Example B2-1. Preparation of sesame oil-based pharmaceutical compositions. Compounds Nos. 63-17, 63-18, 63-10, and 63-33 were formulated for in vivo administration in 95% sesame oil / 5% ethanol (v / v) as follows: Ultra-Refined® sesame oil was obtained from Croda Inc. (Edison, NJ), and ethanol (200 proof, USP grade) was obtained from Pharmaco-AAPER (Brookfield, CT). The compounds were placed in glass vials, and 100% ethanol was added to create a 2.75 mg / mL suspension. The solution was solubilized by vortexing for 30 seconds and then placed in an ultrasonic water bath set at 50°C for 30 minutes. 1 mL of this solution was then transferred to a 20 mL glass vial containing 16.0 g of sesame oil, mixed on a rotary mixer at ambient temperature for 20 minutes, and then transferred to a 90°C water bath for 2 hours to ensure complete solubilization. The formulated compounds were sterilized by 0.2 micron filtration after cooling to 37° C. The formulated compounds were stored in sterile glass vials with rubber stoppers at 2-8° C. The concentrations of the components in the final formulation were as follows: 0.1 mg / mL (w / v) compound in 95% sesame oil and 5% ethanol (v / v).

[0247] Example B2-2. Preparation of squalene oil-in-water nanoemulsion-based pharmaceutical compositions. Compounds Nos. 63-17, 63-18, 63-10, and 63-33 were formulated for in vivo administration in squalene oil-in-water nanoemulsions as follows: Squalene (≥98%, liquid), Tween® 80 (Polysorbate 80), glycerol, and trisodium citrate dihydrate were purchased from Sigma-Aldrich (S 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was obtained from Avanti Polar Lipids (Alabaster, AL). Cell culture grade water (sterile water for injection) was obtained from Corning Obtained from Life Sciences (Tewksbury, MA). To form the oil phase, DOPC (175.6 mg) was added to squalene oil (1.4 mL) in a 4 mL glass vial. The mixture was then incubated in an ultrasonic water bath at 70 °C for 45 minutes, with brief vortexing every 15 minutes until the lipids were dissolved. The indicated compound (13.7 mg) was added to the squalene / DOPC solution and vortexed vigorously for 1 minute. The solution was then incubated in an ultrasonic water bath at 70 °C for 30 minutes, with brief vortexing every 10 minutes. The mixture was further incubated in a 90 °C water bath for 2 hours, with brief vortexing every 15 minutes, to create a clear solution as needed. To form the separate aqueous phase, Tween® 80 (70 mg) and glycerol (315 mg) were mixed with 100 mM sodium citrate (pH 6.5) solution (3.5 mL) and water (29.8 mL) in a 50 mL polypropylene tube.

[0248] An oil-in-water emulsion was formed by combining the squalene oil / DOPC / compound-containing oil phase and the Tween® 80 / glycerol / sodium citrate-containing aqueous phase and then high-shear mixing using a Polytron® mixer (Kinematica, Luzern, CH) at 24,000 rpm for 5 minutes. The crude emulsion was then subjected to high-pressure homogenization using a Microfluidics M-110P Microfluidizer® (Westwood, MA) at approximately 30,000 psi for eight passes. Analysis by dynamic light scattering (Malvern NanoS®, Malvern, UK) showed a mean oil droplet diameter of 150-175 nm with a dispersion index of less than 0.15. The compound-containing nanoemulsion formulation was then sterile filtered using a 0.2-micron sterile syringe filter and stored at 2-8°C in sterile glass vials with rubber stoppers.

[0249] The concentrations of the various components in the final nanoemulsion formulation were as follows: 0.4 mg / mL compound (w / v), 4% squalene oil (v / v), 0.5% DOPC (w / v), 0.2% Tween® 80 (w / v), 0.9% glycerol (w / v), and 10 mM sodium acetate. Pharmaceutical preparations for in vivo administration of compounds in squalene oil-in-water nanoemulsion formulations were prepared before use by diluting 1:1 with Dulbecco's phosphate-buffered saline with gentle inversion mixing. Example B3. In vivo systemic immune activation assay

[0250] Small molecule TLR7 and TLR7 / 8 agonists derived from 1H-imidazo[4,5-c]quinoline privileged templates (e.g., imiquimod, resiquimod) are known to rapidly distribute to systemic compartments after intratumoral, subcutaneous, or intramuscular injection. The widespread systemic distribution of these agonist compounds in wild-type mice induces TLR7-dependent cytokine responses primarily in spleen and liver cells, which can subsequently be detected in serum within 3–6 h. Rapid increases in serum cytokine biomarkers (e.g., IL-6 and IL-12p40) can be used to assess the kinetics of systemic distribution of locally administered TLR agonists.

[0251] The distribution kinetics of pharmaceutical compositions composed of Compound Nos. 63-00, 63-17, or 63-10 formulated in 95% sesame oil / 5% ethanol (v / v) were evaluated after a single subcutaneous injection in wild-type mice. All in vivo procedures were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols. Animals were maintained at the Association for They were housed in an Accreditation and Laboratory Animal Care (AALAC, Frederick, MD)-accredited facility. Wild-type female BALB / c mice (15–20 gm) were obtained from Envigo (Hayward, CA) and allowed to acclimate for 2–3 days before use.

[0252] Pharmaceutical compositions were prepared using the three compounds at a final concentration of 200 μg / mL in a manner similar to that described in Example B2-1. At T=0, groups of three mice were anesthetized with 1% isoflurane and subcutaneously injected with 25 μL of 95% sesame oil / 5% ethanol (v / v) containing 5 μg of each of the three compounds or vehicle control into the right footpad. Then, at T=3, 6, and 24 hours, three animals in each group were anesthetized with 1% isoflurane to facilitate blood collection by cardiac puncture. Samples were processed into serum and stored at -20°C for further analysis.

[0253] Serum levels of IL-6 and IL-12p40 were quantified from each animal by ELISA to determine whether compounds derivatized with longer alkyl chains exhibited slower systemic distribution. As shown in Figure 1, compound 63-00 without alkyl chain modifications induced elevated serum levels of IL-6 and IL-12p40 at 3 hours (IL-6 = 772 ± 141 pg / mL, IL-12p40 = 139,767 ± 31,024 pg / mL) and 6 hours (IL-6 = 160 ± 19 pg / mL, IL-12p40 = 142,359 ± 22,350 pg / mL), which returned to baseline by 24 hours (IL-6 = 32 ± 2 pg / mL, IL-12p40 = 7,796 ± 1,545 pg / mL). The pentylamino variant (compound no. 63-17) induced similar magnitude and kinetics of initial cytokine production in the serum compartment at 3 hours (IL-6 = 853 ± 539 pg / mL, IL-12p40 = 84,731 ± 32,530 pg / mL) and 6 hours (IL-6 = 414 ± 105 pg / mL, IL-12p40 = 258,645 ± 19,982 pg / mL), with similar recovery to baseline levels by 24 hours (IL-6 = 33 ± 4 pg / mL, IL-12p40 = 19,546 ± 2,116 pg / mL). The tetradecanamide variant (Compound No. 63-10), which had substantially higher cLogP values, did not demonstrate detectable increases in serum cytokines at 3, 6, or 24 hours (3 hours: IL-6 = 49 ± 32 pg / mL, IL-12p40 = 1,012 ± 246 pg / mL; 6 hours: IL-6 = 33 ± 4 pg / mL, IL-12p40 = 2,179 ± 597 pg / mL; and 24 hours: IL-6 = 32 ± 2 pg / mL, IL-12p40 = 2,436 ± 237 pg / mL). These data, combined with the fact that all three compounds demonstrate equivalent TLR7 agonist bioactivity in vitro (see Tables B1-1 and B1-2), are consistent with the interpretation that the highly hydrophobic nature of Compound No. 63-10 and the sesame oil / ethanol pharmaceutical composition formulation promotes retention of the molecule at the injection site. Example B4. Antitumor efficacy of alkyl chain-modified TLR7 / 8 agonists in CT26 colon carcinoma-bearing wild-type mice

[0254] The effect of repeated weekly administration of intratumorally delivered pharmaceutical compositions consisting of Compound Nos. 63-10, 63-18, or 63-33 formulated in 95% sesame oil / 5% ethanol (v / v) on tumor growth inhibition was evaluated in Balb / c mice bearing syngeneic CT26 colon carcinoma. All in vivo procedures were performed in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols. Animals were housed in an Association for Accreditation and Laboratory Animal Care (AALAC, Frederick, MD)-accredited facility. Wild-type female Balb / c mice (15-20 gm) were obtained from Envigo (Hayward, CA) and allowed to acclimate for 2-3 days before use.

[0255] Pharmaceutical compositions were prepared using three compounds at final concentrations of 5, 50, and 200 μg / mL in a manner similar to that described in Example B2-1. A TLR9 CpG agonist, whose efficacy has previously been demonstrated in this mouse tumor model, was used as a positive control (Wang et al. 2016 PNAS 113:E7240-E7249). On day 0, mice were anesthetized with 1% isoflurane and injected subcutaneously with 80,000 CT26 tumor cells in 200 μL of RMPI-1640 culture medium + 2.5% fetal bovine serum into the right flank. Tumors were grown to approximately 35 mm 3The tumors were allowed to grow until the tumor size reached 100 μL, at which point the animals were assigned to groups for treatment initiation. Mice were intratumorally injected with 100 μL of a pharmaceutical composition containing 20, 5, or 0.5 μg of Compound No. 63-10, or 20 μg or 5 μg of Compound No. 63-18, or 63-33 formulated in 95% sesame oil / 5% ethanol (v / v) once a week for 4 weeks, or with a vehicle control twice a week for 3 weeks (experimental days 9, 12, 16, 19, 23, and 26). TLR9 CpG agonists were intratumorally injected with 100 μL of a pharmaceutical composition consisting of 50 μg of the compound formulated in phosphate-buffered saline, using the same dosing schedule. Tumor size was measured twice a week from day 8 to day 30 using a caliper, and tumor volume was calculated using the following formula: major axis × minor axis × minor axis / 2.

[0256] Compounds Nos. 63-10, 63-18, and 63-33 demonstrated potent control of CT26 tumor growth across the dose range tested, compared with vehicle control ( FIG. 2 ). The levels of tumor growth control observed for Compounds Nos. 63-10, 63-18, and 63-33 were comparable to TLR9 CpG. These data demonstrate that the TLR7 / 8 agonists of the present invention have potent antitumor effects in this syngeneic mouse tumor growth model system, which are more effective in controlling tumor growth compared with vehicle control (and comparable to tumor growth inhibition by TLR9 CpG). These data are consistent with the interpretation that tumor growth inhibition by Compounds Nos. 63-10, 63-18, and 63-33 correlates with their TLR7 agonist bioactivity in vitro (see, e.g., Tables B1-1 and B1-2) and is independent of the modified alkyl chain length. Example B5. Antitumor efficacy of alkyl chain-modified TLR7 / 8 agonists co-administered with tumor-associated antigens in wild-type mice bearing CT26 colon carcinoma in two flanks

[0257] The effect of weekly repeated administration of intratumorally delivered pharmaceutical compositions consisting of Compound No. 63-10 co-formulated with the CT-26 tumor-associated AH-1 class II peptide (an immunodominant epitope sequence derived from the endogenous retroviral gene product gp70; see, e.g., Rice J, Buchan S, and Stevenson F 2002 J Immunol 169:3908-3913) in a squalene-based oil-in-water nanoemulsion on growth inhibition of injected and distant tumors was evaluated in CT26 colon carcinoma-bearing Balb / c mice bearing tumors in two flanks. All in vivo procedures were performed in accordance with approved IACUC protocols. Animals were housed in an AALAC-accredited facility. Wild-type female Balb / c mice (15-20 g) were obtained from Envigo (Hayward, CA) and allowed to acclimate for 2-3 days before use.

[0258] Pharmaceutical compositions consisting of squalene-based oil-in-water nanoemulsions were prepared generally as described in Example B2-2. In addition to the control squalene-based oil-in-water nanoemulsion, additional nanoemulsions were prepared incorporating either Compound No. 63-10 alone at a final concentration of 500 ng / mL or Compound No. 63-10 at a final concentration of 500 ng / mL plus AH-1 class II peptide at a final concentration of 500,000 ng / mL. For the latter pharmaceutical composition, the AH-1 peptide was first dissolved at 2x concentration in phosphate-buffered saline and then formulated into the squalene-based oil-in-water nanoemulsion during a final mixing step to yield a nanoemulsion at a final concentration of 500,000 ng / mL. On day 0, mice were anesthetized with 1% isoflurane and incubated in a 2.5% fetal bovine serum containing 1% isoflurane. 80,000 CT26 tumor cells in 200 μL of RMPI-1640 culture medium were injected subcutaneously into both the right and left flanks. Tumors were allowed to grow until day 8, when the average tumor size reached approximately 50 mm. 3When the tumor volume reached 100 μL, the mice were randomized into groups and injected intratumorally into the right flank tumor with 100 μL of a squalene-based oil-in-water nanoemulsion vehicle control, a nanoemulsion containing 50 ng of Compound 63-10, or a nanoemulsion containing 50 ng of Compound 63-10 plus 50,000 ng of the AH-1 tumor antigen peptide. These three pharmaceutical compositions were further injected into the right flank tumor on experimental days 12, 16, and 20. The right (injection) and left (distal) tumor volumes were then measured twice weekly from day 8 to day 30 using calipers, and tumor volume was calculated using the following formula: major axis × minor axis × minor axis / 2.

[0259] A pharmaceutical composition composed of a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 demonstrated a trend toward greater tumor growth inhibition of the injected (right) tumor compared to the nanoemulsion vehicle control (Figure 3A); however, this tumor growth inhibition was not significantly different from the vehicle control at day 27. In contrast, a pharmaceutical composition composed of a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 plus 50,000 ng of AH-1 tumor-associated peptide demonstrated significantly greater tumor growth inhibition of the injected (right) tumor compared to the nanoemulsion vehicle control at day 27. Furthermore, a pharmaceutical composition composed of a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 demonstrated a trend toward greater tumor growth inhibition of the distal (left) tumor compared to the nanoemulsion vehicle control (Figure 3B); however, this tumor growth inhibition was not significantly different from the vehicle control on day 23. In contrast, a pharmaceutical composition composed of a squalene-based oil-in-water nanoemulsion containing 50 ng of Compound No. 63-10 plus 50,000 ng of AH-1 tumor-associated peptide demonstrated significantly greater tumor growth inhibition of the distal (left) tumor compared to the nanoemulsion vehicle control on day 23. These data are consistent with the interpretation that growth inhibition of injected and distal tumors by Compound No. 63-10 is superior when the compound is delivered to antigen-presenting cells in the tumor microenvironment along with exogenously added CT26 tumor-associated antigen. Example B6. Antitumor efficacy of alkyl chain-modified TLR7 / 8 agonists in combination with immune checkpoint blockade in wild-type mice bearing bilateral CT26 colon carcinomas

[0260] The effect of intratumoral delivery of a pharmaceutical composition consisting of Compound No. 63-10 or 63-33 formulated in 95% sesame oil / 5% ethanol (v / v) or Compound No. 63-00 formulated in phosphate-buffered saline, combined with intraperitoneal delivery of an anti-mouse PD-1 (CD279) antibody (immune checkpoint inhibitor; BioX Cell, Lebanon, NH) on tumor growth inhibition was evaluated in CT26 colon carcinoma-bearing Balb / c mice bearing tumors in two flanks. All in vivo procedures were performed in accordance with approved IACUC protocols. Animals were housed in an AALAC-accredited facility. Wild-type female Balb / c mice (15–20 g) were obtained from Envigo (Hayward, CA) and allowed to acclimate for 2–3 days before use.

[0261] Pharmaceutical compositions were prepared using Compound No. 63-10 or 63-33 at a final concentration of 50,000 ng / mL in a manner similar to that described in Example B2-1. A pharmaceutical composition of Compound No. 63-00 was prepared using phosphate-buffered saline at a final concentration of 50,000 ng / mL. On day 0, mice were anesthetized with 1% isoflurane and 80,000 CT26 tumor cells in 200 uL of RMPI-1640 culture medium + 2.5% fetal bovine serum were injected subcutaneously into both the right and left flanks. Tumors (left and right) were allowed to grow until day 8, when tumor sizes on the right and left flanks reached approximately 35 mm. 3 When the PD-1 antibody dose reached 100 mg / kg, mice were then treated with 250 μg of anti-PD-1 antibody formulated in phosphate-buffered saline or phosphate-buffered saline. Saline vehicle control was injected intraperitoneally. Anti-PD-1 treatment was repeated on experimental days 12, 15, 19, 22, and 26. On experimental day 14, right (injected) and left (distal) flank tumors grew to approximately 100 mm 3Upon reaching a tumor size of 100 μL, mice were randomized into treatment groups. Only right flank tumors in the anti-PD-1+ treatment group received an additional intratumoral injection of 100 μL of a pharmaceutical composition containing 5,000 ng of Compound No. 63-00 in phosphate-buffered saline, 5,000 ng of Compound No. 63-10 in 95% sesame oil / 5% ethanol (v / v), or 5,000 ng of Compound No. 63-33 in 95% sesame oil / 5% ethanol (v / v). Tumor size was measured twice weekly from day 14 to day 29 using calipers, and tumor volume was calculated using the following formula: major axis × minor axis × minor axis / 2.

[0262] Pharmaceutical compositions composed of 5,000 ng of Compound No. 63-00, 63-10, or 63-33 in combination with anti-PD-1 treatment demonstrated greater tumor growth inhibition of the injected tumor (right flank) compared to anti-PD-1 treatment alone (Figure 4A). This tumor growth inhibition was significantly different from the anti-PD-1-treated control at day 29 for Compound Nos. 63-33 and 63-00. Furthermore, pharmaceutical compositions composed of 5,000 ng of Compound Nos. 63-00 or 63-33 in combination with anti-PD-1 treatment demonstrated a trend toward greater tumor growth inhibition of the distal tumor (left flank) compared to anti-PD-1 treatment alone (Figure 4B), although this tumor growth inhibition did not reach statistical significance from the anti-PD-1-treated control at day 29. Pharmaceutical compositions composed of 5,000 ng of Compound No. 63-10 in combination with anti-PD-1 treatment did not demonstrate improved distal tumor growth inhibition compared to anti-PD-1 treatment alone. These data are consistent with the interpretation that compound #63-33 in combination with the immune checkpoint inhibitor anti-PD-1 is superior in controlling both injected and distal CT26 tumor growth compared to treatment with vehicle control plus anti-PD-1.

[0263] All publications, including patents, patent applications, and scientific articles, mentioned in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including a patent, patent application, or scientific article, was specifically and individually indicated to be incorporated by reference.

[0264] Although the foregoing invention has been described in some detail for purposes of illustration and example to facilitate understanding, it will be apparent to those skilled in the art that certain minor changes and modifications will occur in light of the above teachings. Accordingly, the foregoing description and examples should not be construed as limiting the scope of the invention. In one embodiment, for example, the following items are provided: (Item 1) Formula (J): [ka] [In the formula, R 0 is a C4-C optionally substituted with 1 to 4 halogen atoms 21 is a hydrocarbyl; X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b , or -(CH2) p R 1c where R 1a and R 1b are independently C1-C3 alkyl; R 1c is C3-C4 cycloalkyl; p is 1 or 2; R 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R 3 are 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, or a salt thereof; The compound or a salt thereof is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine; N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide; or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide. (Item 2) R 0 C4~C 14 Item 1, wherein the compound or salt thereof is hydrocarbyl. (Item 3) 3. The compound or salt thereof according to item 1 or 2, wherein X is —NH(C═O)—. (Item 4) 3. The compound or salt thereof according to item 1 or 2, wherein X is —NH—. (Item 5) R 0 is branched C4 to C 14 Alkyl, -(CH2) z (C(CH3)2)R A , or -(CH2) m R A m is 0, 1, 2, or 3; z is 1 or 2; R A is a C3-C8 cycloalkyl optionally substituted with 1 to 4 groups independently selected from the group consisting of C1-C4 alkyl, C1-C4 alkylene, and halogen; or a salt thereof. (Item 6) R 0 Branched C4~C 14 6. The compound or salt thereof according to any one of items 1 to 5, wherein R is alkyl. (Item 7) R 0 Ga-(CH2) m R A 6. The compound or salt thereof according to any one of items 1 to 5, wherein (Item 8) 8. The compound or salt thereof according to item 7, wherein m is 2. (Item 9) R 0 Ga-(CH2) z (C(CH3)2)R A 6. The compound or salt thereof according to any one of items 1 to 5, wherein (Item 10) Item 10. The compound or salt thereof according to item 9, wherein z is 1. (Item 11) R A 11. The compound or salt thereof according to any one of items 7 to 10, wherein is cyclopropyl, cyclobutyl, or cyclopentyl. (Item 12) R A is a C3-C6 cycloalkyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl, methylene, and halogen; or a salt thereof. (Item 13) Item 6. The compound or salt thereof according to item 5, wherein m is 1 or 2. (Item 14) R A Item 14. The compound or salt thereof according to item 13, wherein is C3-C8 cycloalkyl. (Item 15) R A is cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, or a salt thereof. (Item 16) m is 0 and R A is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, or a salt thereof. (Item 17) R 0 but, [ka] Item 1. The compound according to item 1, or a salt thereof, selected from the group consisting of: (Item 18) 2. The compound or salt thereof according to item 1, wherein the compound is selected from the group consisting of compound numbers 63-33 to 63-36 and 63-38 to 63-49 in Table 1. (Item 19) Formula (K): [ka] [In the formula, n is an integer from 4 to 21; X is -NH- or -NH(C=O)-; R 1 is C3-C6 alkyl, -(CH2) p OR 1a , -(CH2) p NHR 1b , or -(CH2) p R 1c where R 1a and R 1b are independently C1-C3 alkyl; R 1c is C3-C4 cycloalkyl; p is 1 or 2; R 2 is NHR 2a where R 2a is H, OH, NH2, or methyl; Each R 3 are 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, or a salt thereof; The compound or salt thereof is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide. (Item 20) Item 20. The compound or salt thereof according to item 19, wherein X is —NH—. (Item 21) 21. The compound or salt thereof according to item 19 or 20, wherein n is an integer of 4 to 15. (Item 22) 22. The compound or salt thereof according to any one of items 19 to 21, wherein n is 4, 5, 6, or 7. (Item 23) 20. The compound according to item 19, or a salt thereof, wherein X is —NH(C═O)—. (Item 24) 24. The compound or salt thereof according to item 23, wherein n is 11, 12, 13, or 14. (Item 25) R 1 25. The compound or salt thereof according to any one of items 19 to 24, wherein is C3 to C6 alkyl. (Item 26) R 1 26. The compound according to item 25, wherein is n-butyl. (Item 27) R 1 Ga-(CH2) p OR 1a 25. The compound or salt thereof according to any one of items 19 to 24, wherein (Item 28) R 1 Ga-(CH2) p NHR 1b 25. The compound or salt thereof according to any one of items 19 to 24, wherein (Item 29) R 1 Ga-(CH2) p R 1c The compound according to any one of items 19 to 24, is the salt. (Item 30) R 2 30. The compound or salt thereof according to any one of items 19 to 29, wherein is NH2. (Item 31) 31. The compound or salt thereof according to any one of items 19 to 30, wherein q is 0. (Item 32) q is 1 and R 3 31. The compound or salt thereof according to any one of items 19 to 30, wherein is C1 to C8 alkyl. (Item 33) R 4a and R 4b 33. The compound or salt thereof according to any one of items 19 to 32, wherein each of (Item 34) 20. The compound or salt thereof according to item 19, wherein the compound is selected from the group consisting of compound numbers 63-01 to 63-30 in Table 1. (Item 35) A pharmaceutical composition comprising: (i) a compound according to any one of items 1 to 34 or a salt thereof; and (ii) a pharmaceutically acceptable excipient. (Item 36) 36. The pharmaceutical composition of item 35, further comprising an antigen. (Item 37) 37. The pharmaceutical composition according to item 35 or 36, wherein the pharmaceutically acceptable excipient comprises an oil. (Item 38) 38. The pharmaceutical composition according to any one of items 35 to 37, wherein the pharmaceutical composition is a squalene-based oil-in-water nanoemulsion. (Item 39) Item 40. The pharmaceutical composition according to Item 35 or 36, wherein the pharmaceutical composition is a liposome formulation. Item 41. A method for stimulating an immune response in a mammalian subject in need thereof, comprising administering to the mammalian subject an amount of the pharmaceutical composition according to any one of items 35 to 39 sufficient to stimulate the immune response in the mammalian subject. 40. A method for inducing an antigen-specific antibody response in a mammalian subject in need thereof, the method comprising administering to the mammalian subject the pharmaceutical composition according to any one of items 35 to 39 in an amount sufficient to induce the antigen-specific antibody response and / or antigen-specific T cell response in the mammalian subject. (Item 42) 40. A method of treating an infectious disease in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition according to any one of items 35 to 39 in an amount sufficient to treat said infectious disease in said mammalian subject. (Item 43) 40. A method of preventing an infectious disease in a mammalian subject in need thereof, comprising administering to said mammalian subject the pharmaceutical composition according to any one of items 35 to 39 in an amount sufficient to prevent said infectious disease in said mammalian subject. (Item 44) 40. A method of treating an IgE-related disorder in a mammalian subject in need thereof, comprising administering to said mammalian subject an amount of the pharmaceutical composition according to any one of items 35 to 39 sufficient to treat said IgE-related disorder in said mammalian subject. (Item 45) 40. A method for preventing an IgE-related disorder in a mammalian subject in need thereof, comprising administering to said mammalian subject an amount of the pharmaceutical composition according to any one of items 35 to 39 sufficient to prevent said IgE-related disorder in said mammalian subject. (Item 46) 40. A method of treating cancer in a mammalian subject in need thereof, comprising administering to said mammalian subject the pharmaceutical composition of any one of items 35 to 39 in an amount sufficient to treat cancer in said mammalian subject. (Item 47) 47. The method of claim 46, wherein the pharmaceutical composition is administered by intratumoral injection. (Item 48) 48. The method of item 46 or 47, further comprising administering to the subject an effective amount of a second therapeutic agent. (Item 49) 49. The method of item 48, wherein the second therapeutic agent is a chemotherapeutic agent. (Item 50) 49. The method of claim 48, wherein the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule. (Item 51) 49. The method of item 48, wherein the second therapeutic agent is an epigenetic modulating agent. (Item 52) 49. The method of item 48, wherein the second therapeutic agent is an inducer of immunogenic cell death. (Item 53) 51. The method of claim 50, wherein the inhibitory immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 (CD152), LAG-3, TIM-3, TIGIT, IL-10, and TGF-beta.

Claims

1. Formula (J): 【Chemistry 20】 [In the formula, R ０ is a C optionally substituted with 1 to 4 halogen atoms ４ ~C ２１ is a hydrocarbyl; X is —NH— or —NH(C═O)—; R １ is C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ , -(CH ２ ) ｐ NHR １ｂ , or -(CH ２ ) ｐ R １ｃ where R １ａ and R １ｂ are independently １ ~C ３ alkyl; R １ｃ is C ３ ~C ４ cycloalkyl; p is 1 or 2; R ２ is NHR ２ａ where R ２ａ is H, OH, NH ２ or methyl; Each R ３ are independently halogen, C １ ~C ８ Alkyl, -(C １ ~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 are independently H or C １ ~C ８ or a salt thereof, With the proviso that the compound is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine; N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide; or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)pent-4-ynamide, or a salt thereof.

2. R ０ is C ４ ~C １４ 2. The compound of claim 1 or a salt thereof, which is hydrocarbyl.

3. 3. The compound or salt thereof according to claim 1 or 2, wherein X is -NH(C=O)-.

4. 3. The compound or salt thereof according to claim 1 or 2, wherein X is -NH-.

5. R ０ is branch C ４ ~C １４ Alkyl, -(CH ２ ) ｚ (C(CH ３ ) ２ ) R Ａ , or -(CH ２ ) ｍ R Ａ m is 0, 1, 2, or 3; z is 1 or 2; R Ａ is C １ ~C ４ Alkyl, C １ ~C ４ C optionally substituted with 1 to 4 groups independently selected from the group consisting of alkylene, and halogen. ３ ~C ８ The compound or salt thereof according to any one of claims 1 to 4, which is cycloalkyl.

6. R ０ is branch C ４ ~C １４ The compound or salt thereof according to any one of claims 1 to 5, wherein the aryl group is alkyl.

7. R ０ Ga-(CH ２ ) ｍ R Ａ The compound according to any one of claims 1 to 5, or a salt thereof,

8. 8. The compound or salt thereof according to claim 7, wherein m is 2.

9. R ０ Ga-(CH ２ ) ｚ (C(CH ３ ) ２ ) R Ａ The compound according to any one of claims 1 to 5, or a salt thereof,

10. 10. The compound or salt thereof according to claim 9, wherein z is 1.

11. R Ａ The compound or salt thereof according to any one of claims 7 to 10, wherein is cyclopropyl, cyclobutyl, or cyclopentyl.

12. R Ａ optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl, methylene, and halogen; ３ ~C ６ The compound or salt thereof according to any one of claims 7 to 10, which is cycloalkyl.

13. 6. The compound or salt thereof according to claim 5, wherein m is 1 or 2.

14. R Ａ is C ３ ~C ８ 14. The compound of claim 13, or a salt thereof, which is cycloalkyl.

15. R Ａ is cyclopropyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene, or a salt thereof.

16. m is 0, and R Ａ The compound or salt thereof according to claim 5, wherein is cyclohexyl optionally substituted with 1 to 3 groups independently selected from the group consisting of methyl and methylene.

17. R ０ but, 【Chemical 21】 2. The compound of claim 1, or a salt thereof, selected from the group consisting of:

18. The compound or salt thereof according to claim 1, wherein the compound is selected from the group consisting of compound numbers 63-33 to 63-36 and 63-38 to 63-49 in Table 1.

19. Formula (K): 【Chemical 22】 [In the formula, n is an integer from 4 to 21; X is —NH— or —NH(C═O)—; R １ is C ３ ~C ６ Alkyl, -(CH ２ ) ｐ OR １ａ , -(CH ２ ) ｐ NHR １ｂ , or -(CH ２ ) ｐ R １ｃ where R １ａ and R １ｂ are independently １ ~C ３ alkyl; R １ｃ is C ３ ~C ４ cycloalkyl; p is 1 or 2; R ２ is NHR ２ａ where R ２ａ is H, OH, NH ２ or methyl; Each R ３ are independently halogen, C １ ~C ８ Alkyl, -(C １ ~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 are independently H or C １ ~C ８ or a salt thereof, The compound or salt thereof is other than 2-butyl-1-(4-((hexadecylamino)methyl)benzyl)-1H-imidazo[4,5-c]quinolin-4-amine or N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)palmitamide.

20. 20. The compound or salt thereof according to claim 19, wherein X is -NH-.

21. 21. The compound or salt thereof according to claim 19 or 20, wherein n is an integer of 4 to 15.

22. The compound or salt thereof according to any one of claims 19 to 21, wherein n is 4, 5, 6, or 7.

23. 20. The compound or salt thereof according to claim 19, wherein X is -NH(C=O)-.

24. 24. The compound of claim 23, or a salt thereof, wherein n is 11, 12, 13, or 14.

25. R １ is C ３ ~C ６ The compound or salt thereof according to any one of claims 19 to 24, which is alkyl.

26. R １ The compound of claim 25, wherein is n-butyl.

27. R １ Ga-(CH ２ ) ｐ OR １ａ The compound according to any one of claims 19 to 24, or a salt thereof,

28. R １ Ga-(CH ２ ) ｐ NHR １ｂ The compound according to any one of claims 19 to 24, or a salt thereof,

29. R １ Ga-(CH ２ ) ｐ R １ｃ The compound according to any one of claims 19 to 24, or a salt thereof,

30. R ２ NH ２ The compound according to any one of claims 19 to 29, or a salt thereof,

31. The compound or salt thereof according to any one of claims 19 to 30, wherein q is 0.

32. q is 1 and R ３ is C １ ~C ８ The compound or salt thereof according to any one of claims 19 to 30, which is alkyl.

33. R ４ａ and R ４ｂ The compound according to any one of claims 19 to 32, or a salt thereof, wherein each of

34. 20. The compound or salt thereof according to claim 19, wherein the compound is selected from the group consisting of compound numbers 63-01 to 63-30 in Table 1.

35. A pharmaceutical composition comprising: (i) a compound according to any one of claims 1 to 34 or a salt thereof; and (ii) a pharmaceutically acceptable excipient.

36. 36. The pharmaceutical composition of claim 35, further comprising an antigen.

37. 37. The pharmaceutical composition of claim 35 or 36, wherein the pharmaceutically acceptable excipient comprises an oil.

38. 38. The pharmaceutical composition of any one of claims 35 to 37, wherein the pharmaceutical composition is a squalene-based oil-in-water nanoemulsion.

39. 37. The pharmaceutical composition of claim 35 or 36, wherein the pharmaceutical composition is a liposomal formulation.

40. 40. A method of stimulating an immune response in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition of any one of claims 35 to 39 in an amount sufficient to stimulate said immune response in said mammalian subject.

41. 40. A method of inducing an antigen-specific antibody response in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition according to any one of claims 35 to 39 in an amount sufficient to induce said antigen-specific antibody response and / or antigen-specific T cell response in said mammalian subject.

42. 40. A method of treating an infectious disease in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition of any one of claims 35-39 in an amount sufficient to treat said infectious disease in said mammalian subject.

43. 40. A method of preventing an infectious disease in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition of any one of claims 35 to 39 in an amount sufficient to prevent said infectious disease in said mammalian subject.

44. 40. A method of treating an IgE-related disorder in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition according to any one of claims 35 to 39 in an amount sufficient to treat said IgE-related disorder in said mammalian subject.

45. 40. A method of preventing an IgE-related disorder in a mammalian subject in need thereof, comprising administering to said mammalian subject an amount of the pharmaceutical composition of any one of claims 35 to 39 sufficient to prevent said IgE-related disorder in said mammalian subject.

46. 40. A method of treating cancer in a mammalian subject in need thereof, comprising administering to said mammalian subject a pharmaceutical composition of any one of claims 35-39 in an amount sufficient to treat cancer in said mammalian subject.

47. 47. The method of claim 46, wherein the pharmaceutical composition is administered by intratumoral injection.

48. 48. The method of claim 46 or 47, further comprising administering to the subject an effective amount of a second therapeutic agent.

49. 49. The method of claim 48, wherein the second therapeutic agent is a chemotherapeutic agent.

50. 49. The method of claim 48, wherein the second therapeutic agent is an antagonist of an inhibitory immune checkpoint molecule.

51. 49. The method of claim 48, wherein the second therapeutic agent is an epigenetic modulating agent.

52. 49. The method of claim 48, wherein the second therapeutic agent is an inducer of immunogenic cell death.

53. 51. The method of claim 50, wherein the inhibitory immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4 (CD152), LAG-3, TIM-3, TIGIT, IL-10, and TGF-beta.

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