Toll-like receptor 7 agonists as immunostimulators for innate antitumor immunity

Abstract

The present invention relates to compounds capable of specifically activating TLR7. The compounds of the present invention are useful because they can stimulate innate immunity. The compounds of the present invention can be used to treat diseases including cancer, viral infections, and skin lesions. The compounds can optionally be formulated for enhanced penetration after topical administration, and the compositions preferably initiate a localized, specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses. The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and, preferably, additional compounds such as imiquimod and / or resiquimod (R848). The present invention also relates to compositions comprising the compounds of the present invention and to compounds for use as pharmaceuticals. Other aspects, embodiments, advantages, and uses of the present invention will become apparent from the further description herein.

Description

[Technical Field]

[0001] The present invention relates to compounds capable of specifically activating TLR7. The compounds of the present invention are useful because they can stimulate innate immunity. The compounds of the present invention can be used to treat diseases including cancer, viral infections, and skin lesions. The compounds can optionally be formulated for enhanced penetration after topical administration, and the compositions preferably initiate a localized, specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses. The present invention also relates to pharmaceutical compositions comprising the compounds of the present invention and, preferably, additional compounds such as imiquimod and / or resiquimod (R848). The present invention also relates to compositions comprising the compounds of the present invention and to compounds for use as pharmaceuticals. Other aspects, embodiments, advantages, and uses of the present invention will become apparent from the further description herein. [Background technology]

[0002] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein found on the surface of T and B cells that downregulates the immune system's response to the body's own cells by promoting self-tolerance through suppressing T cell inflammatory activity. This prevents autoimmune diseases but may also prevent the immune system from killing cancer cells. The role of the human immune system in the anti-tumor field was underestimated for many years until the successful clinical application of immune checkpoint inhibitors (e.g., PD-1 / PD-L1 inhibitors). PD-1 and PD-L1 inhibitors are a group of checkpoint inhibitor drugs that block the activity of the PD-1 and PDL1 immune checkpoint proteins present on the surface of cells.

[0003] The immune system possesses multiple "brake" mechanisms that negatively downregulate the activation and function of T cells or other immune cells to protect the host's own healthy cells. Checkpoint inhibitors such as PD-1 / PD-L1 and CTLA-4 antagonists can selectively release the immune system's "brakes" to enhance so-called "adaptive immunity" and generate sustained antitumor responses. However, only a minority of cancer patients typically experience clinical benefit when using checkpoint inhibitors.

[0004] The human immune system is also designed to defend the body against foreign agents, such as viruses and bacteria, through systemic coordination among numerous cell types with highly selective immune functions, known as "innate immunity." These cell types include phagocytes, such as dendritic cells (DCs), macrophages, gamma delta T cells, and natural killer (NK) cells, which act as a barrier to foreign agents. The mechanism behind this is the use of conserved molecular patterns and expressed Fc receptors on pathogens to recognize and initiate rapid immune responses. Because B and T cells of the adaptive immune system can mount memory responses, DCs mediate communication between the innate and adaptive immune systems, initiating specific and sustained immune responses.

[0005] DCs are responsible for constantly sampling their environment through phagocytosis, receptor-mediated endocytosis, and micropinocytosis. With the help of pattern recognition receptors (PRRs), DCs can recognize countless foreign substances. Upon engagement of PRRs, DCs are activated and mature into more potent antigen-presenting cells (APCs), upregulating the cell surface expression of costimulatory molecules such as CD40 and CD86 for optimal T cell priming and activation.

[0006] Toll-like receptors (TLRs) are well-known members of the PRR family. Currently comprising a gene family of 10 receptors with different specificities, TLRs are part of the cellular pathogen pattern recognition system, which has evolved to defend against various infectious diseases (bacterial, viral, and fungal). TLR activation leads to cytokine responses, including the release of interferon and the activation of specific immune cells. The functional expression of selected TLRs in tissues varies significantly. Some receptors, such as TLR4 (stimulated by E. coli lipopolysaccharide (LPS)), are located on the cell surface, e.g., on epithelial cells, while TLRs 3, 7, 8, and 9 are located on the endosomal membrane in specific immune cells. All of the latter are activated by nucleic acids, but recognize various types of nucleic acids. For example, TLR9 is activated by single-stranded DNA containing CpG sequences, TLRs 7 and 8 are activated by single-stranded RNA, and TLR3 is activated by double-stranded RNA.

[0007] TLRs have also been implicated in a variety of autoimmune and inflammatory diseases, most notably the role played by TLR7 in the pathogenesis of systemic lupus erythematosus (Barrat and Coffman, Immunol Rev, 223:271-283, 2008). In addition, TLR8 polymorphisms are associated with rheumatoid arthritis (Enevold et al., J Rheumatol, 37:905-10, 2010).

[0008] Each TLR possesses a specific class of molecules for recognizing various surface and intracellular components of microorganisms, ranging from bacterial membrane lipids to viral single- or double-stranded RNA. Intracellularly expressed TLRs reside in endosomal compartments and include TLR3, TLR7, TLR8, TLR9, and TLR13. TLR7 and TLR8 are reported to be dimeric TLR receptors containing a leucine-rich repeat (LRR) motif as an ectodomain, a transmembrane domain, and a cytoplasmic domain with a Toll / interleukin-1 (IL-1) receptor (TIR) ​​signaling domain. TLR7 is reported to be expressed on B cells and plasmacytoid dendritic cells (PDCs), while TLR8 can be expressed on monocytes and myeloid dendritic cells (mDCs).

[0009] Agonist binding of TLR7 / 8 to degradation products such as guanosine from single-stranded RNA (ssRNA) results in rearrangement of the dimerization interface, including both protein-protein and ligand-mediated interfaces. Conformational changes in the TLR7 / 8 ectodomain lead to activation of the cytoplasmic TIR signaling domain configuration. This is followed by MyD88-dependent signaling, which in turn leads to a potent interferon response and proinflammatory cytokine production in DCs. Furthermore, activated DCs also upregulate the cell surface expression of costimulatory molecules such as CD40 and CD86. Costimulatory expression and engagement lead to optimal T cell priming and activation.

[0010] Furthermore, recent studies have shown that presentation of tumor-associated antigens (TAA) influences the priming and activation of T cells, generating robust and long-lasting antitumor immune responses. Studies have shown that antigen uptake and presentation by APCs is more efficient when the antigen is bound to an antibody-antigen complex, a so-called "immune complex." Activated DCs also upregulate the expression of major histocompatibility complexes (MHCs), so that antigens internalized during microbial encounters are digested and their peptides are displayed on the DC cell membrane by MHC. Activated T cells can recognize and kill tumor cells containing neoantigens bound to MHCs.

[0011] As mentioned above, only a minority of patients experience clinical benefit from checkpoint inhibitors. In cancer patients, one reason for this is that the presence and frequency of immune cells varies from tumor to tumor. A low T cell frequency in the tumor immune microenvironment (TIM) is considered "cold," while a high T cell frequency is considered "hot." Clinical studies have revealed that patients with hot tumors have better outcomes and longer disease-free survival than patients with cold tumors.

[0012] Although agonistic stimulators via the TLR7 / 8 pathway can activate DCs, which subsequently prime and activate T cells, small synthetic molecules such as imiquimod and resiquimod (R848) are only approved for local treatment, e.g., imiquimod for the treatment of basal cell carcinoma via intratumoral administration to avoid systemic inflammation. Such clinical treatments significantly limit TLR7 / 8 agonist therapy for solid tumors.

[0013] As cancerous lesions progress, they may become metastatic, and chemotherapy, radiation therapy, and immunotherapy may be used in addition to surgery. Resiquimod, a member of the imidazoquinoline family and structurally related to imiquimod, is an immune response modifier that acts as an agonist of Toll-like receptors 7 and 8. However, imiquimod signals exclusively through Toll-like receptor 7 (TLR7), making it distinct from imiquimod. Imiquimod is FDA-approved for the treatment of several skin diseases. Compared to imiquimod, resiquimod is a more potent inducer of TNF-α, IL-1, IL-6, IL-8, and IL-12. Resiquimod has been shown to promote cross-presentation of exogenous antigens and result in more efficient induction of antigen-specific CD8+ T cell responses in animal models. Animal studies have confirmed resiquimod's ability to activate dendritic cells, including inducing local activation of immune cells, stimulating the production of proinflammatory cytokines, and enhancing antigen presentation by dendritic cells, leading to the activation of effective cellular responses. Systemic delivery of resiquimod and irradiation primed durable antitumor immune responses in lymphoma models (Dovedi SJ 2013, Blood 121(2):251-9). Resiquimod has been used in clinical trials to treat actinic keratosis, cutaneous T-cell lymphoma, and herpes simplex virus with mixed results. Other prior uses of resiquimod include its administration as a vaccine adjuvant to treat various diseases, including metastatic melanoma, with inconsistent results. Resiquimod has been used as a vaccine adjuvant for the NYES0-1 protein vaccine in the treatment of melanoma (SaBado RL CanCeR Immunol Res. 2015).

[0014] There remains a need for alternative TLR7 agonists that are preferably more specific and effective. Summary of the Invention

[0015] The present invention aims to provide compounds that are effective TLR7 agonists that target innate immunity. The inventors have discovered novel imidazoquinoline-based TLR7 agonists that are effective in initiating specific and sustained immune responses against, for example, cancer.

[0016] Thus, in a first aspect, the present invention provides a compound according to formula I: [ka] or a pharmaceutically acceptable salt thereof, In the formula, X is an oxygen atom, a C1-C5-alkyl (preferably CH2) or NH; In the formula, R 2 and R 3 are each independently selected from the group consisting of hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably, R 2 and R 3 are both hydrogen; and wherein L 1 , L 2 , L 3 , and R 1 is as defined under (a), (b), or (c) below: (a) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or selected from the group consisting of hydrogen, C-C alkyl, and CHC(CH); and R 1 is absent or selected from the group consisting of hydrogen, NH2, OH, and SCH3; (b) L 1 is a C2-C6 alkyl; L 2 is selected from the group consisting of C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and R 1 is absent, methyl or hydrogen; (c) L 1 is C(O)CH2; L 2 is a six-membered heterocycle or NH; L 3 does not exist; and R 1 is methyl, hydrogen, or absent; and where L 3 If there is no L 2 is connected to R via a covalent bond 1 directly bonded to The compound or a pharmaceutically acceptable salt thereof is provided.

[0017] In a preferred embodiment of the first aspect, R 2 and R 3 is hydrogen. Preferably, X in the compounds of the present invention is an oxygen atom. In a further embodiment, the compound of the present invention has the structure set forth in Formula II: [ka] Formula II In the formula, L 1 , L 2 , L 3 , and R 1 is as defined under (d), (e), or (f) below: (d) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle (preferably triazole or piperazine), or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of hydrogen, NH2, OH, and SCH3; (e) L 1 is a C2-C3 alkyl; L 2 is selected from the group consisting of C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L 3 is selected from the group consisting of a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO2, and SO3H; and R 1 is absent, methyl or hydrogen; (f) L 1 is C(O)CH2; L 2 is a 6-membered heterocycle (preferably piperazine) or NH; L 3 does not exist; and R 1 is methyl, hydrogen, or absent.

[0018] In a preferred embodiment of the compounds of formula II of the present invention, L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of NH2, OH, and SCH3; In particularly preferred embodiments of the compounds of the present invention, the compounds have a structure selected from the group consisting of the structures designated as Examples 1 to 27 listed below. [ka]

[0019] Further preferred compounds of the present invention are X is NH; L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle; L 3 does not exist; and R 1 is methyl or hydrogen; and R 2 and R 3 are hydrogen. Also preferably, (i) activates TLR7 more strongly than TLR8; and / or (ii) induce the production of IL-6, IL1-β, and TNF-α; and / or (iii) induces upregulation of CD40 and / or CD86 in peripheral blood mononuclear cells (PBMCs); The compounds according to the present invention.

[0020] Also preferred are compounds according to the invention, which, when contacted with peripheral blood mononuclear cells (PBMC), induce TNFα secretion from said cells. Also preferred are compounds according to the invention, which have an EC50 for TLR7 of less than 10 μM, preferably less than 0.01 μM, when tested in the test system described in Example 6.2.1.

[0021] A further aspect of the present invention relates to pharmaceutical compositions comprising the compounds of the present invention.

[0022] The present invention also relates to pharmaceutical compositions comprising a compound of the present invention and an additional compound, preferably imiquimod and / or resiquimod (R848). A further aspect of the invention relates to the compounds of the invention for use as a pharmaceutical.

[0023] A further aspect of the invention relates to a compound of the invention for use in the treatment of a disease, wherein said compound is used in combination with the further compounds imiquimod and / or resiquimod (R848) for said treatment.

[0024] A preferred embodiment provides a compound of the invention for use in treating a condition selected from the group consisting of cancer, viral infection, non-cancerous skin lesions, pre-cancerous skin lesions, cancerous skin lesions, bladder cancer, viral-mediated skin disease, optionally formulated for enhanced penetration after topical administration, and preferably initiating a local-specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses.

[0025] In a preferred embodiment of the composition of the present invention, the compound of the present invention is contained in an amount of 0.01% to 1%.

[0026] A further aspect relates to a composition of the invention for use in treating bladder cancer, wherein the composition is formulated for intravesical administration.

[0027] In a preferred embodiment of the composition of the present invention, the present invention further comprises an additional component from any one of groups i to xiii outlined below.

[0028] i. oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1% wt / vol; or ii. isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; iii. oleic acid (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; iv. oleic acid (33%), isopropyl myristate (33%), and sodium lauryl sulfate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; v. isopropyl palmitate (50%) and sodium oleate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 2% wt / vol, or vi. Sodium lauryl sulfate (25%) and linoleic acid (75%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 1.0% wt / vol, or vii. Palmitic acid (50%) and isopropyl laurate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 2.0% wt / vol, or

[0029] viii. Oleic acid (50%) and linoleic acid (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1.5% wt / vol; or ix. Linoleic acid (25%), oleic acid (25%), and isopropyl linoleic acid (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol at a total concentration of 0.5% wt / vol; or x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 2.0% wt / vol; or xi. A solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xii. Oleic acid (10%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0030] A further aspect of the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a therapeutically effective amount of a compound of the present invention.

[0031] Moreover, a further aspect of the present invention provides a method for the treatment of skin tumor lesions and virus-induced skin diseases comprising topical administration of a composition of the present invention, wherein the composition is effective in reducing or eliminating the lesions or diseases while limiting adverse skin reactions, preferably selected from erythema and inflammation, and wherein the composition preferably reduces penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

[0032] Moreover, a further aspect of the present invention relates to a method for the treatment of bladder tumors, comprising intravesical administration of a composition of the present invention, wherein said composition preferably enhances penetration and delivery of a compound of the present invention to the bladder epithelium while limiting irritation, and wherein said composition preferably reduces tumor invasion into surrounding muscle tissue and metastasis to lymph nodes.

[0033] In a preferred embodiment of the methods of the present invention, said treatment further comprises systemic administration to said subject of at least one immune modulator selected from anti-PD1, anti-PD-L1, anti-CTLA-4 antibody, anti-CD137 antibody, agonistic CD40 antibody, CD134 (anti-OX40) agonist, and PLX3397.

[0034] In a preferred embodiment of the method of the present invention, said treatment further comprises systemic administration of interferon gamma to said subject. In a preferred embodiment of the method of the present invention, the treatment further comprises the administration of local radiation with or without systemic anti-PD1 antibodies. In a preferred embodiment of the method of the present invention, said treatment further comprises the administration of photodynamic therapy.

[0035] In a further aspect, the present invention provides a method of activating TLR7 and / or 8 in a biological sample comprising contacting the biological sample with a compound according to the invention. In a further aspect, the present invention provides a compound of the invention for use as a vaccine adjuvant or for use in the treatment of cancer in combination with an anti-cancer immunotherapeutic agent (preferably ipilimumab, nivolumab, or pembrolizumab).

[0036] Other aspects, advantages, applications and uses of the polypeptides and compositions will become apparent from the further disclosure herein. Throughout the text of this specification, several documents are cited. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1. Molecular bond between R848 and monkey TLR7 (PDB 5GMH), with the unburied hydrogen group of R848 highlighted by a black arrow. Top right: Chemical structure of R848.

[0038] [Figure 2] Chemical structures of recently reported TLR7 / 8 agonists.

[0039] [Figure 3] A library of highly potent and selective TLR7 agonists.

[0040] [Figure 4A]TLR7 agonists were tested in vitro. XY scatter plots were generated for each agonist / stimulator using Graph Pad Prism9 software by plotting the relationship between the concentration of agonist and the median fluorescence intensity of CD86 staining within cDC (Figure 4A), pDC (Figure 4B), and monocyte (Figure 4C) populations.

[0041] [Figure 4B-4C] TLR7 agonists were tested in vitro. XY scatter plots were generated for each agonist / stimulator using Graph Pad Prism9 software by plotting the relationship between the concentration of agonist and the median fluorescence intensity of CD86 staining within cDC (Figure 4A), pDC (Figure 4B), and monocyte (Figure 4C) populations. DETAILED DESCRIPTION OF THE INVENTION

[0042] Skin cancers are usually located superficially. In situ melanomas are still limited to the epidermis and typically grow slowly. It can take years for such lesions to progress to invasive melanoma. Invasive melanomas usually contain an epidermal component, but once melanoma cells invade the dermis, they are no longer limited to the epidermis. Metastatic cancers are usually located deeper. WO 2017 / 004421 (incorporated herein by reference) presents data demonstrating that resiquimod functions in multiple in vivo pigmented lesion transgenic mouse models and an autologous melanoma mouse model with an intact immune system. These data demonstrate that resiquimod is effective in inhibiting or killing melanocytic tumor cells in nevi, atypical intraepidermal melanocyte proliferation, and melanoma. Additionally, mice treated with resiquimod also had significantly reduced metastatic melanoma in lymph nodes, indicating that resiquimod can inhibit melanoma cell metastasis. Additionally, WO2017 / 004421 demonstrates that resiquimod, in combination with systemic anti-PD1 therapy, significantly enhances the therapeutic effect of anti-PD1 in melanoma models. Because the anti-tumor effect of resiquimod is mediated through the activation of CD8 T cells and the inhibition of myeloid-derived suppressor cells, the efficacy of resiquimod is not limited to melanocytic tumors. It is also effective in treating other epithelial cancers and cutaneous T-cell lymphoma.

[0043] However, and given the myriad types of cancers and tumors, there remains a need for additional compounds to treat cancer and other diseases.

[0044] Figure 1 shows a co-crystal structure analysis of TLR7 complexed with an agonist ligand, such as R848 (Shimizu et al., Cell Reports 2018, 25, 3371). Unexpectedly, the inventors identified potential modifications at the unburied hydrogen group of R848. The agonist binding site within the ectodomain of TLR7 is located within the cellular lysosome. Thus, the inventors discovered that any modifications with protonatable groups at low pH have the potential to enhance the retention time of agonist ligands within lysosomes. The inventors reasoned that this, in turn, would increase the binding between such agonist ligands and the TLR7 protein.

[0045] Therefore, we synthesized a large number of different imidazoquinoline-based TLR7 agonists. Further experiments outlined in the Examples section below confirmed that, consistent with our reasoning, this group of novel agonists performed extremely well in cell-based experiments.

[0046] Unexpectedly, compared with R484 and several recently reported TLR7 / 8 agonists (Fig. 2), these novel TLR7 agonists (Fig. 3) not only showed excellent activity in cellular TLR7 experiments, but also showed high selectivity between TLR7 and TLR8. Even more surprisingly, these novel highly selective TLR7 agonists initiated massive production of cytokines and chemokines (IL-6, IL1-b, and TNF-a) and induced strong upregulation of CD40 and CD86 in cellular monocyte experiments (Fig. 4).

[0047] Thus, in a first aspect, the present invention provides a compound according to formula I: [ka] or a pharmaceutically acceptable salt thereof, In the formula, X is an oxygen atom, a C1-C5-alkyl (preferably CH2) or NH; In the formula, R 2 and R3 are each independently selected from the group consisting of hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably, R 2 and R 3 are both hydrogen; and wherein L 1 , L 2 , L 3 , and R 1 is as defined under (a), (b), or (c) below: (a) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of hydrogen, NH2, OH, and SCH3; (b) L 1 is a C2-C6 alkyl; L 2 is selected from the group consisting of C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L 3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and R 1 is absent, methyl or hydrogen; (c) L 1 is C(O)CH2; L 2 is a six-membered heterocycle or NH; L 3 does not exist; and R 1 is methyl, hydrogen, or absent; and where L 3 If there is no L2 is connected to R via a covalent bond 1 directly bonded to The compound or a pharmaceutically acceptable salt thereof is provided.

[0048] In certain embodiments, the compounds of the present invention stimulate an immune response to treat cancer.

[0049] Before describing aspects and embodiments of the present invention in more detail, certain definitions that apply to the invention disclosed herein throughout the specification, including the claims, are provided below.

[0050] definition Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, as would be apparent to one of ordinary skill in the art. For example, Sambrook et al. (Molecular Cloning: A Laboratory Manual (2nd Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press, 1989), F. Ausubel et al. (Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York, 1987), Lewin (Genes II, John Wiley & Sons, New York, NY, 1985), Old et al. (Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd edition) University of California Press, Berkeley, CA, 1981); Roitt et al. (Immunology (6th Ed.) Mosby / Elsevier, Edinburgh, 2001), Roitt et al. (Roitt's Essential Immunology (10th Ed.) Blackwell Publishing, UK, 2001), and Janeway et al. (Immunobiology (6th Ed.) Garland Science Reference is made to standard handbooks such as "Theoretical and Applied Physics Handbook: A Guide to the Study of Electromagnetic Compatibility," IEEE Transactions on Electromagnetic Compatibility (IEEE Transactions on Electromagnetic Compatibility), ...

[0051] Unless otherwise indicated, all methods, steps, techniques and operations not specifically described can be and have been performed in a manner known per se, as will be apparent to those skilled in the art.

[0052] Unless otherwise indicated, the term "at least" preceding a series of elements is understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0053] The term "and / or" as used herein encompasses the meaning of "and", "or", and "all or any other combination of the elements connected by said term". As used herein, the term "about" or "approximately" means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range.

[0054] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps. As used herein, the term "comprising" may be replaced with the terms "containing" or "including," or, as sometimes used herein, with the term "having."

[0055] "Agonist" refers to a compound that can combine with a receptor (e.g., a TLR) to induce a cellular activity. An agonist may be a ligand that binds directly to the receptor. Alternatively, an agonist may combine with the receptor indirectly, for example, by (a) forming a complex with another molecule that binds directly to the receptor, or (b) by other means that result in the modification of another compound so that it binds directly to the receptor. An agonist may be referred to as an agonist of a specific TLR (e.g., a TLR6 agonist) or a specific combination of TLRs (e.g., a TLR7 / 8 agonist—an agonist of both TLR7 and TLR8). "Amelioration" refers to a reduction in the extent, severity, frequency, and / or likelihood of symptoms or clinical signs characteristic of a particular disease.

[0056] "Cell-mediated immune activity" refers to a biological activity that is considered to be part of a cell-mediated immune response, such as, for example, increased production of at least one TH1 cytokine. "Immune cell" refers to a cell of the immune system, i.e., a cell that is directly or indirectly involved in generating or maintaining an immune response, whether the immune response is innate, acquired, humoral, or cell-mediated.

[0057] "Sign" or "clinical sign" refers to an objective physical finding associated with a particular condition that may be detectable by someone other than the patient. "Symptom" refers to subjective evidence of a disease or patient condition. "Treating" or variations thereof refers to reducing to any extent, limiting the progression of, ameliorating, or eliminating the symptoms or signs associated with a disease.

[0058] As used herein, "penetration enhancer" and "penetration enhancement" refer to increasing the permeability of a drug and a tissue to a drug, respectively, i.e., increasing the rate and extent to which a drug penetrates a tissue such as skin or a tumor. Enhanced penetration by the use of such enhancers can be observed, for example, by measuring the diffusion rate of a drug through animal or human skin or tumor tissue using a diffusion cell apparatus or in situ measurements. Diffusion cells are described by Merritt et al., Diffusion Apparatus for Skin Penetration, J. of Controlled Release, 1 (1984) pp. 161-162.

[0059] As used herein, "adjuvant" refers to the ability to affect nonspecific inflammation or specific immune responses caused by immune system activators. As used herein, the term "anti-cancer response" to treatment refers to any response of a cancer to treatment, preferably a change in tumor mass and / or volume after the initiation of treatment. Response to a hyperproliferative disorder may be assessed by comparing the size of the tumor after local or systemic intervention with the initial size and dimensions measured by CT, PET, mammogram, ultrasound, or palpation. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative form, such as the percentage change in tumor volume, or in a qualitative form, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of response to a hyperproliferative disorder may be performed early after the initiation of treatment, for example, within hours, days, weeks, or preferably months. A typical endpoint for response assessment is the end of chemotherapy or surgical removal of residual tumor cells and / or tumor bed, which is typically three months after the initiation of treatment.

[0060] As used herein, "reducing the size of a tumor" refers to a reduction in the size of a tumor. Such an effect can be achieved by reducing the number of proliferating tumor cells within the tumor (e.g., by reducing cell division of tumor cells) and / or by inducing cytotoxicity or cell death (apoptosis) of existing tumor cells. Thus, tumor growth is halted or prevented.

[0061] As used herein, the term "inhibiting cancer" or "inhibiting cancer cell growth" is intended to include the inhibition of unwanted or inappropriate cell growth. Inhibition is intended to include the inhibition of growth, including rapid proliferation. The term "inhibiting cancer cell growth" is also intended to encompass the inhibition of tumor growth, which includes preventing tumor growth in a subject or reducing the growth of an existing tumor in a subject. Inhibition may also be the inhibition of metastasis of a tumor from one site to another. Cancer is "inhibited" if at least one symptom of the cancer is alleviated, terminated, delayed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, delayed, postponed, or prevented.

[0062] "Therapeutic compound," "drug," and "therapeutic agent" are used interchangeably herein. As used herein, "a," "an," "the," "at least one," and "one or more" are used interchangeably. Thus, for example, "a pharmaceutical composition comprising an immune response modulator (IRM) compound" may be interpreted to mean a pharmaceutical composition that includes at least one IRM compound.

[0063] As used herein, the term "subject" means any animal, including, but not limited to, a human, a mouse, a rat, a rabbit, a non-human primate, or other mammal. In one embodiment, the subject is a primate. In another most preferred embodiment, the subject is a human.

[0064] Additionally, herein, the description of a numerical range using endpoints includes all numerical values ​​within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). The composition of the present invention may contain, for example, a compound of the present invention in an amount of 0.01% to 1% (vol / wt%).

[0065] As used herein, the term "synergistic" refers to a combination of therapeutic agents described herein that, when taken together, is more effective than the additive effects of the individual therapies. The synergistic effect of a therapeutic combination (e.g., a combination of therapeutic agents) may allow for the use of lower dosages of one or more therapeutic agents and / or less frequent administration of the agents to a subject with a disease or disorder, such as, for example, a proliferative disorder. The use of lower dosages of one or more therapeutic agents and / or less frequent administration of the therapeutic agents may reduce the toxicity associated with administering the agents to a subject without reducing the effectiveness of the treatment in treating the disease or disorder. In addition, the synergistic effect may improve the effectiveness of the agents in preventing, managing, or treating a disease or disorder, such as, for example, a proliferative disorder. Finally, the synergistic effect of a therapeutic combination may avoid or reduce adverse or undesirable side effects associated with the use of either therapeutic agent alone.

[0066] As used herein, the term "in combination" can refer to the use of more than one therapeutic agent. The use of the term "in combination" does not restrict the order in which therapeutic agents are administered to a subject with a disease or disorder, such as, for example, a proliferative disorder. A first therapeutic agent, such as a compound described herein, can be administered to a subject prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapeutic agent, such as an anticancer agent, to a subject with a disease or disorder, such as a proliferative disorder such as cancer.

[0067] Immune response modifiers (IRMs) include compounds that possess potent immunomodulatory activity, including, but not limited to, antiviral and antitumor activity. Certain IRMs regulate cytokine production and secretion. For example, certain IRM compounds induce the production and secretion of cytokines such as type I interferon, TNF-α, IL-1, IL-6, IL-8, IL-0, IL-12, MUM, and / or MCP-1. As another example, certain IRM compounds can inhibit the production and secretion of certain TH2 cytokines, such as IL-4 and IL-5.

[0068] In addition, some IRM compounds are said to suppress IL-1 and TNF (U.S. Pat. No. 6,518,265). Resiquimod (l-[4-amino-2-(ethoxymethyl)imidazo[4,5-C]quinolin-l-yl]-2-methylpropan-2-ol) is an immune response modifier (IRM) that acts by stimulating cells through Toll-like receptors (TLRs) 7 and 8.

[0069] As used herein, "effective amount," when used with respect to the compounds of the present invention and the drug combinations described herein, includes, but is not limited to, the amount of drug, or the amount of each drug in a combination, that produces a statistically significant desired effect on the disease or disorder being treated (e.g., cancer). Representative desired effects are described herein. For example, in the context of cancer treatment, the effect may be, but is not limited to, a reduction in tumor growth rate, cessation of tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, as measured by standard criteria such as Response Evaluation Criteria in Solid Tumors (RECIST), or a statistically significant increase in survival compared to treatment with an individual drug or subcombination of the combination alone. An effective amount may vary depending on factors such as the type of cell growth being treated or inhibited, the type of therapeutic agent employed, the specific therapeutic agent, the size of the subject, or the severity of the cancer cell growth or tumor. For example, the selection of each individual drug comprising the combination can affect what constitutes an "effective amount." One of skill in the art can study the aforementioned factors and make a determination regarding the effective amount of a therapeutic compound or therapeutic compound / drug combination.

[0070] For example, in vitro assays can be used to determine the "effective amount" of a therapeutic agent. One skilled in the art will select an appropriate amount of each individual agent in the combination for use in the aforementioned in vitro assay. The cell survival rate can be used to determine whether a selected amount was an "effective amount" for a particular combination of therapeutic agents. For example, the selected amount used in the assay should preferably result in killing at least 50% of the cells, more preferably 75%, and most preferably at least 95% of the cells. In a preferred embodiment, an effective dose of a therapeutic agent is a subtoxic dose. As used herein, the term "subtoxic dose" refers to a dosage that kills less than about 10% of the cells.

[0071] The administration regimen (e.g., sequence) can also affect what constitutes an effective amount. Furthermore, several divided doses, as well as staggered doses, can be administered locally daily or continuously, or the dosage can be continuously infused. Furthermore, the dosage can be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. As used herein, the phrase "pharmaceutically acceptable" is employed to refer to a therapeutic compound, or combination of therapeutic compounds / agents, materials, compositions, and / or dosage forms of the present invention, that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, and with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0072] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a chemical substance of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, sucrose, and the like; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) propolis. (11) glycols such as pyrene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0073] The compounds of the present invention include those generally described above and are further described by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of the present invention, chemical elements shall be defined as defined in the Elements, CAS version, Handbook of Chemistry and Physics, 75 thIn addition, the general principles of organic chemistry are identified in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the contents of which are incorporated herein by reference in their entireties.

[0074] As used herein, "alkyl" refers to a straight- or branched-chain hydrocarbon radical having the specified number of carbon atoms. The alkyl group may be unsubstituted or substituted with substituents that do not interfere with the specific function of the composition, and may be substituted once or twice with the same or different groups. Substituents may include, for example, alkoxy, hydroxy, mercapto, amino, alkyl-substituted amino, nitro, carboxy, carbonyl, carbonyloxy, cyano, methylsulfonylamino, or halogen. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and 3-methylpentyl. For example, C1-C2 alkyl encompasses methyl and ethyl radicals. As used herein, "alkyl" hydrocarbon encompasses monovalent and divalent radicals, i.e., alkylene radicals, provided the resulting structure containing the alkyl group is stable.

[0075] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic, or tricyclic hydrocarbon ring system having 3 to 14 carbon atoms and 0 heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, adamantyl, bicyclo[3.1.1]heptyl, cyclobutyl, cyclohexyl, cyclopentyl, and cyclopropyl.

[0076] The term "aryl," used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring systems having a total of 5 to 14 ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. The term "aryl" is used interchangeably with the term "aryl ring." In certain embodiments of the invention, "aryl" refers to an aromatic ring system. Exemplary aryl groups are phenyl, biphenyl, naphthyl, anthracyl, and the like, which optionally contain one or more substituents. Preferably, the aryl group contains no substituents. Also included within the scope of the term "aryl," as it is used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl.

[0077] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl" or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 pi electrons shared in the cyclic array; and having from 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen or sulfur and the quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroara-," as used herein, also include groups in which a heteroaromatic ring is fused with one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups are optionally mono- or bicyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include optionally substituted rings. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently may be optionally substituted.

[0078] As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety, which is either saturated or partially unsaturated and, in addition to carbon atoms, has one or more, preferably one to four, heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as found in N-substituted pyrrolidinyls).

[0079] A heterocycle can be attached to its pendant group(s) at a heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted (unsubstituted rings are preferred). Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and also include groups in which a heterocyclyl ring is fused with one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. Heterocyclyl groups are optionally monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions independently may be optionally substituted (but preferably are unsubstituted).

[0080] As described herein, certain compounds of the invention include "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. When more than one position in any given structure is substituted with more than one substituent selected from a specified group, the substituents may be either the same or different at every position. Combinations of substituents envisioned by the invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that remain substantially unchanged when subjected to conditions that permit their production, detection, and in some embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. A "stable" compound is usually not radical. Preferably, the compounds of the invention are not further substituted beyond those explicitly stated.

[0081] Unless otherwise indicated, reference to a compound (singular) and compounds (plural) of the invention may include the compound in any pharmaceutically acceptable form or pharmaceutically acceptable derivative, including any isomers (e.g., diastereomers or enantiomers), salts, esters, salts of esters, solvates, polymorphs, etc. In particular, if the compound is optically active, reference to the compound may include each of the compound's enantiomers, as well as racemic mixtures of the enantiomers. In addition, unless otherwise stated, structures and compounds described herein are meant to encompass compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the presented structure but including the replacement of a hydrogen with a deuterium or tritium, or the replacement of a carbon with a C- or C-enriched carbon, are within the scope of the invention. In some embodiments, a group contains one or more deuterium atoms. Preferably, "pharmaceutically acceptable salts, forms, or derivatives" encompass derivatives of the compounds of the invention that, upon administration to a recipient, are capable of directly or indirectly providing a compound of the invention or an agonist metabolite or residue thereof.

[0082] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (incorporated herein by reference). Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include those of adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bisulfate, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfate, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, hemisulfate, heptanoic acid, hexanoic acid, hydroiodic acid, 2-hydroxy-ethanesulfonic acid, and the like. Included are salts of sulfonic acid, lactobionic acid, lactic acid, lauric acid, lauryl sulfate, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, phosphoric acid, pivalic acid, propionic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecanoic acid, and valeric acid. These salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or by separately reacting the purified therapeutic agent in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts also include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19.)

[0083] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (Ci-4 alkyl) salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates, where appropriate.

[0084] Thus, in a first aspect, the present invention provides a compound according to formula I: [ka] or a pharmaceutically acceptable salt or derivative thereof, In the formula, X is an oxygen atom, a C1-C5-alkyl (preferably CH2) or NH; In the formula, R 2 and R 3are each independently selected from the group consisting of hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably, R 2 and R 3 are both hydrogen; and wherein L 1 , L 2 , L 3 , and R 1 is as defined under (a), (b), or (c) below: (a) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of hydrogen, NH2, OH, and SCH3; (b) L 1 is a C2-C6 alkyl; L 2 is selected from the group consisting of C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L 3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and R 1 is absent, methyl or hydrogen; (c) L 1 is C(O)CH2; L 2 is a six-membered heterocycle or NH; L 3 does not exist; and R 1 is methyl, hydrogen, or absent; and where L 3 If there is no L 2is connected to R via a covalent bond 1 directly bonded to The compound is a compound described above, or a pharmaceutically acceptable salt or derivative thereof.

[0085] In a preferred embodiment of the first aspect, R 2 and R 3 is hydrogen. Preferably, X in the compounds of the present invention is an oxygen atom. In a further embodiment, the compound of the present invention has the structure set forth in Formula II: [ka] Formula II In the formula, L 1 , L 2 , L 3 , and R 1 is as defined under (d), (e), or (f) below: (d) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle (preferably triazole or piperazine), or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of hydrogen, NH2, OH, and SCH3; (e) L 1 is a C2-C3 alkyl; L 2 is selected from the group consisting of C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L 3 is selected from the group consisting of a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO2, and SO3H; and R 1 is absent, methyl or hydrogen; (f) L 1 is C(O)CH2; L 2 is a 6-membered heterocycle (preferably piperazine) or NH; L 3 does not exist; and R 1 is methyl, hydrogen, or absent. In a more preferred embodiment of the compounds of the present invention having formula I or II, L 1 is C2-C4 alkyl;

[0086] In a preferred embodiment of the compounds of formula II of the present invention, L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or selected from the group consisting of hydrogen, C1-C2 alkyl, and CH2C(CH3)2; and R 1 is absent or selected from the group consisting of NH2, OH, and SCH3.

[0087] In particularly preferred embodiments of the compounds of the present invention, the compounds have a structure selected from the group consisting of the structures designated as Examples 1 to 27 listed below: [ka]

[0088] In a preferred embodiment of the invention, the compound of the invention is a compound as described in Example 13 or 14 outlined above, or as described in Example 26 or 27 outlined above.

[0089] In a preferred embodiment, the compound of the present invention is a compound selected from the group consisting of Examples 1-6 outlined above, or a pharmaceutically acceptable salt or derivative thereof. In a most preferred embodiment, the compound of the present invention is a compound selected from the group consisting of Examples 1, 3, 4, and 5 outlined above, or a pharmaceutically acceptable salt or derivative thereof.

[0090] Further preferred compounds of the present invention are X is NH; L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle; L 3 does not exist; and R 1 is methyl or hydrogen; and R 2 and R 3 are each hydrogen, Concerning compounds. Also preferably, (i) activates TLR7 more strongly than TLR8; and / or (ii) induce the production of IL-6, IL1-β, and TNF-α; and / or (iii) induces upregulation of CD40 and / or CD86 in peripheral blood mononuclear cells (PBMCs); The compounds according to the present invention.

[0091] In the above embodiments, one of ordinary skill can test whether a compound activates TLR7 more strongly than TLR8 by carrying out the method provided in Example 6.2.1 below. In the above embodiments, one of ordinary skill can test whether a compound induces TNF-α production by carrying out the method provided in Example 6.2.2 below. In the above embodiments, one of ordinary skill can test whether a compound induces upregulation of CD86 by carrying out the method provided in Example 6.2.3 below. Also preferred are compounds according to the invention, which, when contacted with peripheral blood mononuclear cells (PBMC), induce TNFα secretion from said spheres. Also preferred are compounds according to the invention, wherein the compound preferably has an EC50 for TLR7 of less than 10 μM, preferably less than 0.01 μM, when tested in the test system described in Example 6.2.1 below.

[0092] A further aspect of the present invention relates to pharmaceutical compositions comprising the compounds of the present invention. The present invention also relates to pharmaceutical compositions comprising a compound of the present invention and an additional compound, preferably imiquimod and / or resiquimod (R848). A further aspect of the present invention relates to a compound of the present invention for use as a pharmaceutical.

[0093] A further aspect of the invention relates to a compound of the invention for use in the treatment of a disease, wherein said compound is used for said treatment in combination with the further compounds imiquimod and / or resiquimod (R848).

[0094] A preferred embodiment provides a compound of the invention for use in treating a condition selected from the group consisting of cancer, viral infection, non-cancerous skin lesions, pre-cancerous skin lesions, cancerous skin lesions, bladder cancer, viral-mediated skin disease, optionally formulated for enhanced penetration after topical administration, and preferably initiating a local-specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses.

[0095] In a preferred embodiment of the composition of the present invention, the compound of the present invention is contained in an amount of 0.01% to 1% wt / vol. A further aspect relates to a composition of the invention for use in treating bladder cancer, wherein the composition is formulated for intravesical administration.

[0096] In a preferred embodiment of the composition of the present invention, the present invention further comprises an additional component of any one of groups i to xiii outlined below: i. oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1% wt / vol; or ii. isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; iii. oleic acid (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; iv. oleic acid (33%), isopropyl myristate (33%), and sodium lauryl sulfate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol, or; v. Isopropyl palmitate (50%) and sodium oleate (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, at a total concentration of 2% wt / vol, or vi. Sodium lauryl sulfate (25%) and linoleic acid (75%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 1.0% wt / vol, or vii. Palmitic acid (50%) and isopropyl laurate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 2.0% wt / vol, or viii. Oleic acid (50%) and linoleic acid (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 1.5% wt / vol; or ix. Linoleic acid (25%), oleic acid (25%), and isopropyl linoleic acid (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol at a total concentration of 0.5% wt / vol; or x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 2.0% wt / vol; or xi. A solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xii. Oleic acid (10%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0097] A further aspect of the present invention relates to a method for treating cancer, comprising administering to a subject suffering from cancer a therapeutically effective amount of a compound of the present invention. Moreover, a further aspect of the present invention provides a method for the treatment of cutaneous tumor lesions and virus-induced skin diseases, comprising topical administration of a composition of the present invention, wherein said composition is effective in reducing or eliminating said lesions or diseases while limiting adverse skin reactions, preferably selected from erythema and inflammation, and wherein said composition preferably reduces penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

[0098] Moreover, a further aspect of the present invention relates to a method for the treatment of bladder tumors, comprising intravesical administration of a composition of the present invention, wherein said composition preferably enhances penetration and delivery of resiquimod to the bladder epithelium while limiting irritation, and wherein said composition preferably reduces tumor invasion into surrounding muscle tissue and metastasis to lymph nodes.

[0099] In a preferred embodiment of the methods of the present invention, said treatment further comprises systemic administration to said subject of at least one immune modulator selected from anti-PD1, anti-PD-L1, anti-CTLA-4 antibody, anti-CD137 antibody, agonistic CD40 antibody, CD134 (anti-OX40) agonist, and PLX3397.

[0100] In a preferred embodiment of the method of the present invention, said treatment further comprises systemic administration of interferon gamma to said subject. In a preferred embodiment of the method of the present invention, the treatment further comprises the administration of local radiation with or without systemic anti-PD1 antibodies.

[0101] In a preferred embodiment of the method of the present invention, said treatment further comprises the administration of photodynamic therapy. In a further aspect, the present invention provides a method of activating TLR7 and / or 8 in a biological sample comprising contacting the biological sample with a compound according to the invention.

[0102] In a further aspect, the present invention provides a compound of the invention for use as a vaccine adjuvant or for use in the treatment of cancer in combination with an anti-cancer immunotherapeutic agent (preferably ipilimumab, nivolumab, or pembrolizumab).

[0103] preparation The TLR7 agonist compounds of the present invention can be further prepared according to the examples provided below. Alternative methods for synthesizing the compounds of the present invention can also be employed.

[0104] Uses, Formulation and Administration Pharmaceutically acceptable compositions The compounds of the present invention act as immune response modifiers and have antiviral and antitumor activity.For example, like resiquimod, a Toll-like receptor 7 (TLR7) agonist, the compounds of the present invention are expected to reduce hepatitis C virus (HCV) infection, as shown by resiquimod in clinical phase 2 studies.In a mouse model of allergic asthma, resiquimod (in, 20 μg / mouse) reduces allergen-induced airway reactivity and inflammation through reducing Nrf2 signaling (Int J Biochem Cell Biol. 2016 Apr;73:53-62. doi: 10.1016 / j.biocel.2016.02.004. Epub 2016 Feb 3). Resiquimod also modulates dendritic cells, increasing cytomegalovirus- and HIV-1-specific T cell responses (J Immunol. 2003 Oct 15;171(8):4320-8. doi: 10.4049 / jimmunol.171.8.4320). Resiquimod also induces the differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells, which can improve cancer immunotherapy by reducing immunosuppressive MDSCs (Arch Pharm Res. 2014;37(9):1234-40. doi: 10.1007 / s12272-014-0379-4. Epub 2014 Apr 19). Given these multiple medical benefits demonstrated for resiquimod as a TLR7 / 8 agonist, the compounds of the present invention, which are also TLR7 / 8 agonists, are expected to share the aforementioned medical uses and benefits.

[0105] As shown in the examples, the compounds of the present invention, like resiquimod, are also agonists of Toll-like receptors 7 and 8. Thus, in a further aspect, the present invention provides compositions comprising a compound of the present invention, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In certain embodiments, the amount of the compound of the present invention in a composition of the present invention is an amount effective to measurably activate TLR7 / 8 or a mutant thereof in a biological sample or in a patient. In certain embodiments, a composition of the present invention is formulated for administration to a patient in need of such a composition.

[0106] The terms "patient" or "subject", as used herein, mean an animal, preferably a mammal, and most preferably a human.

[0107] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" includes a non-toxic carrier, adjuvant, or vehicle that does not impair the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or vehicles used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0108] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenteral," as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intracisternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present invention include aqueous or oleaginous suspensions. These suspensions are formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0109] For this purpose, any bland, fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, including natural pharmaceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, and other emulsifiers or bioavailability enhancers, commonly used in the production of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0110] In some embodiments, the pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form.Exemplary oral dosage forms are capsules, tablets, aqueous suspensions or solutions.In the case of tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also typically added.Useful diluents for oral administration in capsule form include lactose and dried corn starch.When aqueous suspension is required for oral use, the active ingredient can be combined with emulsifying and suspending agents.If desired, certain sweeteners, flavorings or coloring agents can also be optionally added.

[0111] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0112] The pharmaceutically acceptable compositions of this invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0113] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.

[0114] The pharmaceutically acceptable compositions of the present invention for topical application can be formulated into a suitable ointment containing the active compound of the present invention suspended or dissolved in one or more carriers.Exemplary carriers for topical administration of this compound include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, the pharmaceutically acceptable compositions can be formulated into a suitable lotion or cream containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0115] The pharmaceutically acceptable compositions of this invention are optionally administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorinated carbons, and / or other conventional solubilizing or dispersing agents.

[0116] As indicated, the pharmaceutically acceptable compositions of the present invention can also be formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0117] The amount of the compounds of the present invention that are optionally combined with a carrier material to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration, etc. Preferably, provided compositions can be formulated so that a dosage of between 0.01 and 100 mg / kg body weight / day of the compound can be administered to a patient receiving these compositions.

[0118] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, the patient's age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a compound of the invention in a composition will also depend on the particular compound in the composition.

[0119] Immunocompromised patients are highly susceptible to the development of severe infections, which often progress to the life-threatening disease of sepsis. Therefore, immunotherapy aimed at boosting the host's immune defenses is a highly attractive strategy for preventing infection and protecting patients. In recent years, increasing evidence has suggested that activation of the innate immune system can result in long-term functional reprogramming, allowing innate leukocytes to mount a more robust response upon secondary exposure to pathogens for more efficient clearance and host defense (referred to as trained immunity). Toll-like receptor (TLR) agonists are a class of drugs that have been shown to induce the phenomenon of trained immunity, resulting in significantly enhanced antimicrobial function through metabolic reprogramming and epigenetic modifications. Immunomodulatory TLR agonists are also highly useful as vaccine adjuvants. Furthermore, most cancer-associated antigens are self-antigens, requiring immune-stimulating adjuvants in addition to cancer-targeting strategies. Therefore, TLR7 / 8 agonists, such as the compounds of the present invention, are also useful for cancer treatment.

[0120] In one aspect, the present invention provides a method for treating a subject suffering from a TLR7 / 8-associated disorder, comprising administering to said subject an effective amount of a compound of the invention, preferably a compound of Formula II and related formulas.

[0121] The compounds of the present invention are useful as anti-cancer agents for cancers that respond to TLR7 activation.In some embodiments, cancers include but are not limited to cancers of breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands, esophagus, endometrium, germ cell, head and neck, kidney, liver, lung, larynx and hypopharynx, mesothelioma, sarcoma, ovary, pancreas, prostate, rectum, renal, small intestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva; hereditary cancer, retinoblastoma and Wilms' tumor; leukemia, lymphoma, non-Hodgkin's disease, chronic and acute myeloid leukemia, acute lymphoblastic leukemia, Hodgkin's disease, multiple myeloma and T-cell lymphoma; myelodysplastic syndrome, plasma cell neoplasm, paraneoplastic syndrome, cancer of unknown primary site and AIDS-related malignancies.

[0122] In some embodiments, the compounds of the present invention are used to treat cancers of the skin or kidney. The sensitivity of a given cancer to TLR7 activation can be assessed by, but is not limited to, measuring a reduction (minimal, partial, or complete) in primary or metastatic tumor load, an altered blood picture, altered hormone or cytokine blood levels, inhibition of further tumor load growth, stabilization of disease in a patient, assessment of a disease-associated biomarker or surrogate marker, prolonged overall survival in a patient, prolonged time to disease progression in a patient, prolonged progression-free survival in a patient, prolonged disease-free survival in a patient, improved quality of life in a patient, or modulation of disease comorbidities (such as, but not limited to, pain, cachexia, mobilization, hospitalization, altered blood picture, weight loss, wound healing, fever).

[0123] Compounds according to the present invention may also be useful as immune response modifiers, which may modulate the immune response from a number of different angles, making them useful in the treatment of a variety of disorders.

[0124] Provided herein are methods for activating an immune response in an individual using a compound as described herein, comprising administering to the individual an effective amount of an activator of TLR7 (e.g., a TLR7 activator). In some variations, the TLR activator activates a TLR7-dependent immune response. In some variations, the TLR activator activates a TLR7- and TLR8-dependent immune response. Unless otherwise specified, the term "TLR activator" refers to any one of the compounds of the invention disclosed herein. In some preferred embodiments, the individual is a human patient.

[0125] The present disclosure provides methods of immunomodulation, including methods of activating an immune response (including, but not limited to, an immune response). The present disclosure also provides methods for activating a TLR7- and / or TLR8-induced response (e.g., in vitro or in vivo). In some variations, the cell is contacted with an amount of a TLR activator effective to activate a response from a cell that contributes to the immune response. Activation of TLR7 and / or TLR8 is useful for treating and / or preventing a variety of diseases or disorders that are responsive to cytokines.

[0126] Provided herein are methods of activating an immune response in an individual, the method comprising administering to the individual at least one TLR activator as disclosed herein in an amount effective to activate an immune response in the individual.

[0127] Also provided herein are methods of treating a viral disease or disorder (e.g., infection with HIV), the methods comprising administering to an individual at least one compound of the invention in an amount effective to treat the viral disease or disorder.

[0128] In some embodiments of any of the methods involving administration of a compound of the present invention to a subject, the compound preferably has a therapeutically acceptable safety profile. The compound / TLR activator has a therapeutically acceptable histological profile, including, for example, acceptably low (if any) toxicity of the liver, kidney, pancreas, or other organs. In some embodiments, the safety profile includes assessment of toxicity, histological profile, and / or necrosis (e.g., liver, kidney, and / or heart). In some embodiments, the TLR activator has a therapeutically acceptable level of toxicity. In some embodiments, the TLR activator has a reduced level of toxicity compared to other TLR activators. In some embodiments, the TLR activator induces a therapeutically acceptable reduction in body weight compared to the treated individual's initial body weight. In some embodiments, the TLR activator induces a reduction in total body weight of less than 5%, 7.5%, 10%, 12.5, or 15%. In some embodiments, the TLR activator has a therapeutically acceptable histological profile. In some embodiments, the TLR activator has a better (e.g., lower severity score) histology profile, e.g., compared to a reference TLR activator. In some embodiments, the TLR activator has a better (e.g., lower severity score) histology profile, e.g., when evaluating the liver, kidney, and / or heart. In some embodiments, the TLR activator has a therapeutically acceptable necrosis score. In some embodiments, the TLR activator has reduced necrosis and / or a better (e.g., lower) necrosis score, e.g., compared to a reference TLR activator. In some embodiments, the TLR activator has a reduced kidney cell and / or liver cell necrosis score and / or a better kidney cell and / or liver cell necrosis score, e.g., compared to a reference TLR activator.

[0129] Accordingly, the present invention provides a method of activating TLR7 in an animal, particularly a mammal, preferably a human, comprising administering to the animal a compound of the present invention. Effective amounts of the compound will vary according to factors known in the art, but are expected to be doses of about 0.1-10 mg / kg, 0.5-10 mg / kg, 1-10 mg / kg, 0.1-20 mg / kg, 0.1-20 mg / kg, or 1-20 mg / kg.

[0130] The present invention also provides a method for treating a viral infection in an animal, comprising administering a compound of the present invention to the animal. An amount effective to treat or inhibit a viral infection is an amount that will cause a reduction in one or more symptoms of the viral infection, such as viral lesions, viral load, viral production rate, and mortality, compared to untreated control animals. The exact amount will vary according to factors known in the art, but is expected to be a dose as indicated above for activating TLR7, or from about 100 ng / kg to about 50 mg / kg, preferably from about 10 μg / kg to about 5 mg / kg.

[0131] The methods of the present invention can be carried out either in vitro or in vivo. The sensitivity of specific cells to treatment with the compounds of the present invention can be specifically determined by in vitro testing, whether in a research process or for clinical application. Typically, a culture of cells is combined with a compound of the present invention at various concentrations for a period of time sufficient for the active agent to inhibit TLR7 / 8 activity, usually between about one hour and one week. In vitro treatment can be carried out using cultured cells from a biopsy sample or a cell line.

[0132] The host or patient may belong to any mammalian species, such as a primate species, particularly humans; rodents, including mice, rats, and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are the subject of experimental investigation and provide models for the treatment of human diseases.

[0133] Moreover, the present invention also provides the use of compounds according to the invention and derivatives thereof for the production of a medicament for the prophylactic or therapeutic treatment of a disease caused, mediated, and / or propagated by insufficient TLR7 / 8 activity. In a particular embodiment, the present invention provides the use of a compound according to the invention, or a physiologically acceptable salt or derivative thereof, for the production of a medicament for the prophylactic or therapeutic treatment of a TLR7 / 8-mediated disorder.

[0134] The compounds of the present invention and / or their physiologically acceptable salts or derivatives may also be employed as intermediates for the preparation of further medicament active ingredients. The medicaments are preferably prepared in a non-chemical manner, for example by combining the active ingredient with at least one solid, liquid and / or semi-liquid carrier or excipient, optionally in combination with one or more other active substances in a suitable dosage form.

[0135] In some embodiments, the compounds of the present invention can act as a treatment by being administered once or several times before or after the onset of a disease. The compounds of use in the present invention are specifically used for therapeutic treatment. A therapeutically relevant effect is to alleviate one or more symptoms of a disorder to some extent, or to partially or completely restore one or more physiological or biochemical parameters associated with or contributing to a disease or pathological condition to normal. Monitoring is considered a type of treatment, provided that the compound is administered at distinct intervals, for example, to boost the response and completely eliminate the pathogen and / or symptoms of the disease. Either the same compound or different compounds can be applied. The methods of the present invention can also be used to reduce the likelihood of developing a disorder, or even to prevent the occurrence of a disorder associated with insufficient or reduced TLR7 / 8 activity (reduced compared to healthy subjects), or to treat existing and ongoing symptoms.

[0136] In the sense of the present invention, preventive treatment is advisable if the subject possesses any prerequisite for the aforementioned physiological or pathological conditions, such as a familial disposition, a genetic defect, or a pre-existing disease.

[0137] In a further aspect, the present invention relates to a medicament comprising at least one compound according to the invention and / or its pharmaceutically usable derivatives, salts, solvates and stereoisomers (including mixtures thereof in any ratio). In one embodiment, the present invention relates to a medicament comprising at least one compound according to the invention and / or its physiologically acceptable salt.

[0138] In various embodiments, the active ingredient may be administered alone or in combination with other treatments.Synergistic effects may be achieved by using more than one compound in a pharmaceutical composition, i.e., the compound of the present invention is combined with at least one other agent as an active ingredient (either another compound of the present invention or a compound of a different structural framework).The active ingredients may be used simultaneously or sequentially.

[0139] It has been shown in the literature that combining the Toll-like receptor 7 (TLR7) agonist R848 with radiation therapy (RT) leads to long-term tumor clearance in mice bearing T-cell and B-cell lymphoma. Thus, in a further aspect, the present invention provides a method of treating a subject suffering from lymphoma by administering a compound of the invention in combination with treating the subject with radiation therapy.

[0140] In a further aspect, the present invention provides a method for treating cancer in a patient in need of such treatment, the method comprising administering a therapeutically effective amount of a compound of the present invention and a therapeutically effective amount of (1) a taxane, (2) a platinum ligand compound, (3) an epidermal growth factor (EGF) inhibitor that is an antibody, (4) an EGF inhibitor that is a small molecule compound, (5) a vascular endothelial growth factor (VEGF) inhibitor that is an antibody, (6) a VEGF kinase inhibitor that is a small molecule compound, (7) an estrogen receptor antagonist or selective estrogen receptor modulator (SERM), (8) an antitumor nucleoside derivative, (9) an epothilone, (10) a topoisomerase inhibitor, (11) a vinca alkaloid, (12) an αVβ3 integrin that is an antibody; (13) a small molecule compound that inhibits αVβ3 integrin; (14) The method includes administering at least one different antitumor agent selected from the group consisting of folate antagonists, (15) ribonucleotide reductase inhibitors, (16) anthracyclines, (17) anticancer biologics, (18) thalidomide (or related imides), and (19) Gleevec. Examples of these antitumor compounds are disclosed in US20060183765, which is incorporated herein by reference.

[0141] The compounds of the present invention may be administered in combination with additional known therapeutic agents, including anti-cancer agents. As used herein, the term "anti-cancer agent" refers to any agent administered to a patient with cancer for the purpose of treating the cancer.

[0142] Anti-cancer treatment may be applied as a monotherapy or may involve, in addition to the compounds of the present invention disclosed herein, conventional surgery or radiation therapy or medicinal therapy. Such medicinal therapy (e.g., chemotherapy or targeted therapy) may include one or more, preferably one of the following anti-tumor agents: Alkylating agents:Altretamine, bendamustine, busulfan, carmustine, chlorambucil, chlormethine, cyclophosphamide, dacarbazine, ifosfamide, improsulfan, tosilate, lomustine, melphalan, mitobronitol, mitolactol, nimustine, ranimustine, temozolomide, thiotepa, treosulfan, mechlorethamine, carboquone; apaziquone, fotemustine, glufosfamide, palifosfamide, pipobroman, trofosfamide, uramustine, TH-302 4 , VAL-083 4 etc; Platinum compounds: Carboplatin, cisplatin, eptaplatin, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin; lobaplatin, nedaplatin, picoplatin, satraplatin, etc.; DNA modifying agents: Amrubicin, bisantrene, decitabine, mitoxantrone, procarbazine, trabectedin, clofarabine; amsacrine, brostallicin, pixantrone, laromustine, etc.; Topoisomerase inhibitors: Etoposide, irinotecan, razoxane, sobuzoxane, teniposide, topotecan; amonafide, belotecan, elliptinium acetate, voreloxin, etc.; Microtubule modifier: Cabazitaxel, docetaxel, eribulin, ixabepilone, paclitaxel, vinplastine, vincristine, vinorelbine, vindesine, vinflunine; fosbretabine, tesetaxel, etc. Antimetabolites: Asparaginase, azacitidine, levofolinate calcium, capecitabine, cladribine, cytarabine, enocitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, nelarabine, pemetrexed, pralatrexate, azathioprine, thioguanine, carmofur; doxifluridine, ellacitabine, raltitrexed, cepacitabine, tegafur, trimethotrexate, etc.; Anticancer antibiotics: Bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozocin, valrubicin, zinostatin, zorubicin, daunorubicin, plicamycin; aclarubicin, peplomycin, pirarubicin, etc.; Hormones / antagonists: Abarelix, abiraterone, bicalutamide, buserelin, calisthenol, chlorotonianic acid, degarelix, dexamethasone, estradiol, flutocortolone, fluoxymesterone, flutamide, fulvestrant, goserelin, histrelin, leuprorelin, megesterol, mitotane, nafarelin, nandrolone, nilutamide, octreotide, prednisolone, raloxifene, tamoxifen, thyrotropin alfa, toremifene, trilostane, triptorelin, diethylstilbestrol; acolbifene, danazol, deslorelin, epithiostanol, orteronel, enzalutamide, etc. Aromatase inhibitors: Aminoglutethimide, anastrozole, exemestane, fadrozole, letrozole, testolactone; formestane, etc.; Small molecule kinase inhibitors: Crizotinib, dasatinib, erlotinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, bosutinib, gefitinib, axitinib; afatinib, alisertib, dabrafenib, dacomitinib, dinaciclib, dovitinib, enzastaurin, nintedanib, lenvatinib, linifanib, linsitinib, masitinib , midostaurin, motesanib, neratinib, orantinib, perifosine, ponatinib, radotinib, rigosertib, tipifarnib, tivantinib, tivozanib, trametinib, pimasertib, brivanib alaninate, cediranib, apatinib, cabozantinib S-malate, ibrutinib, icotinib, buparlisib, cipatinib, cobimetinib, idelalisib, fedratinib, XL-647, etc.; Photosensitizers:Methoxsalen; porfimer sodium, talaporfin, temoporfin, etc.; antibody: Alemtuzumab, besilesomab, brentuximab vedotin, cetuximab, denosumab, ipilimumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, bevacizumab, pertuzumab 2,3 Catumaxomab, elotuzumab, epratuzumab, farletuzumab, mogamulizumab, necitumumab, nimotuzumab, obinutuzumab, ocaratuzumab, oregovomab, ramucirumab, rilotumumab, siltuximab, tocilizumab, zalutumumab, zanolimumab, matuzumab, dalotuzumab, onartuzumab, racotumomab, tabalumab, EMD-525797, nivolumab, etc. Cytokines: Aldesleukin, interferon alpha, interferon alpha 2a, interferon alpha 2b; cermoleukin, tasonermin, teceleukin, operelvekin, recombinant interferon beta-1a, etc.; Drug conjugates: Denileukin diftitox, ibritumomab tiuxetan, iobenguane I123, prednimustine, trastuzumab emtansine, estramustine, gemtuzumab, ozogamicin, aflibercept; syntredequin besudotox, edotreotide, inotuzumab ozogamicin, naptumomab estafenatox, oportuzumab monatox, technitium (99mTc) arcitumomab, vintafolide, etc. vaccine: sipuleucel; vitespen, emepepimt-S, oncoVAX, rindopepimt, troVax, MGN-1601, MGN-1703, etc.; and others: Alitretinoin, bexarotene, bortezomib, everolimus, ibandronate, imiquimod, lenalidomide, lentinan, metyrosine, mifamurtide, pamidronate, pegaspargase, pentostatin, sipuleucel 3, sizofiran, tamibarotene, temsirolimus, thalidomide, tretinoin, vismodegib, zoledronic acid, vorinostat; celecoxib, cilengitide, entinostat, etanidazole, ganetespib, idronoxyl, iniparib, ixazomib, lonidamine, nimorazole, parabinostat, peretinoin, plitidepsin, pomalidomide, procodazole (procod azol), ridaforolimus, tasquinimod, telotristat, simalfasin, tirapazamine, tosedostat, travedelsen, ubenimex, valspodar, gendicine, picibanil, leolysin, letaspimycin hydrochloride, trebananib, bilirudin, carfilzomib, endostatin, immucothel, belinstat, MGN-1703.

[0143] Accordingly, an aspect of the present invention relates to the treatment of a subject by administering a compound of the present invention in an appropriate amount together with one or more of the above-listed anti-tumor agents to achieve treatment of cancer disease in the subject.

[0144] In some embodiments, the combination of a TLR inhibitor with one or more additional therapeutic agents reduces the effective amount (including, but not limited to, dosage volume, dosage concentration, and / or total drug dose administered) of the TLR activator and / or one or more additional therapeutic agents administered to achieve the same result, compared to the effective amount administered when the TLR activator or additional therapeutic agents are administered alone.

[0145] TLR activators may also be useful as vaccine adjuvants for use in conjunction with any material that modulates either the humoral and / or cell-mediated immune response (e.g., live viral, bacterial, or parasitic immunogens; inactivated viral, tumor-derived, protozoan, organism-derived, fungal, or bacterial immunogens, toxoids, toxins; autoantigens; polysaccharides; proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines; recombinant proteins; glycoproteins; peptides; etc.). In some aspects, the combination treatments of the invention include, but are not limited to, the administration of a combination of a compound of the invention and a vaccine. In some aspects, the combination therapies of the present invention include, but are not limited to, the combination of a TLR activator with a vaccine for use in the treatment of infectious diseases.

[0146] A further aspect of the present invention pertains to kits comprising a compound of the invention as provided herein and instructions for use in a method of activating a TLR7- and / or TLR8-dependent immune response.

[0147] The kit may include one or more containers containing a compound of the present invention (or a formulation containing the compound) as described herein and a set of instructions, typically written instructions, although electronic storage media (e.g., magnetic diskettes or optical disks) containing instructions regarding the use and dosage of the compound of the present invention or a formulation containing the compound for an intended treatment (e.g., activating TLR7 and / or TLR8, treating viral infections, and / or treating and / or preventing one or more symptoms of a disease or disorder mediated by TLR7 and / or TLR8) are also acceptable. The instructions included in the kit generally include information about the dosage, dosing schedule, and route of administration for the intended treatment. The container for the TLR activator (or formulation containing the TLR activator) may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The kit may further include a container containing an adjuvant.

[0148] In another aspect, the present invention provides a kit consisting of separate packs of an effective amount of a compound according to the invention and / or its pharmaceutically acceptable salts, derivatives, solvates and stereoisomers (including mixtures thereof in any ratio), and optionally an effective amount of a further active ingredient. The kit comprises suitable containers such as boxes, individual bottles, bags or ampoules. The kit may, for example, comprise separate ampoules, each containing an effective amount of a compound according to the invention and / or its pharmaceutically acceptable salts, derivatives, solvates and stereoisomers (including mixtures thereof in any ratio), and optionally an effective amount of a further active ingredient, in dissolved or lyophilized form.

[0149] The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.As used herein, the expression "dosage unit form" refers to a physically discrete unit of agent appropriate for the patient to be treated.However, it will be understood that the total daily use amount of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment.The specific dosage level effective for any specific patient or organism will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific compound being used; the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific compound being used; the duration of treatment; drugs used in combination or simultaneously with the specific compound being used, and similar factors well known in the medical arts.

[0150] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, or drops), buccally, as an oral or nasal spray, or the like, depending on the severity of the infection being treated. In some embodiments, the compounds of the present invention are administered orally or parenterally at dosage levels of from about 0.01 mg / kg to about 100 mg / kg of body weight (of the subject) per day, preferably from about 1 mg / kg to about 50 mg / kg of body weight (of the subject), one or more times per day to achieve the desired therapeutic effect.

[0151] In certain embodiments, the therapeutically effective amount of the compounds of the present invention and related formulas, as well as other active ingredients, will depend on numerous factors, including, for example, the age and weight of the animal, the exact disease state and its severity, the nature of the formulation, and the method of administration, and will ultimately be determined by the treating physician or veterinarian. However, an effective amount of the compound will generally be in the range of 0.1 to 100 mg / kg body weight (of the recipient (mammal)) per day, and more specifically, typically in the range of 1 to 10 mg / kg body weight per day. Thus, the actual daily dose for an adult mammal weighing 70 kg will usually be between 70 mg and 700 mg, where this amount can be administered as individual doses per day, or in a set of partial doses (e.g., 2, 3, 4, 5, 6, etc.) per day so that the total daily dose is the same. An effective amount of a salt or solvate, or an effective amount of a physiologically functional derivative thereof, can be determined as a fraction of the effective amount of the compound itself.

[0152] In some embodiments, pharmaceutical preparations can be administered in the form of dosage units, each containing a predetermined amount of active ingredient. Such units can contain, for example, 0.5 mg to 1 g, preferably 1 mg to 700 mg, particularly preferably 5 mg to 100 mg of the compound according to the present invention, depending on the disease state to be treated, the method of administration, and the age, weight, and condition of the patient. Alternatively, pharmaceutical preparations can be administered in the form of dosage units, each containing a predetermined amount of active ingredient. Preferred dosage unit preparations are those containing the daily dose or partial dose indicated above, or a corresponding fraction thereof, of the active ingredient. Furthermore, pharmaceutical preparations of this type can be prepared using processes commonly known in the pharmaceutical arts.

[0153] The liquid dosage form of the compound of the present invention for oral administration includes, but is not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active compound, the liquid dosage form optionally contains an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.In addition to the inert diluent, the oral composition also contains adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and coloring agents.

[0154] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents.Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol.Among acceptable vehicles and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution.In addition, sterile fixed oils are conventionally used as solvents or suspending media.For this purpose, any bland fixed oil that can be used includes synthetic mono- or diglycerides.In addition, fatty acids such as oleic acid are used in injectable preparations.

[0155] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0156] To prolong the effect of a compound of the present invention, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of poorly soluble crystalline or amorphous material. The rate of absorption of the compound then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form can be achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the compound-to-polymer ratio and the nature of the specific polymer employed, the compound release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0157] Compositions for rectal or vaginal administration are preferably suppositories which may be prepared by mixing a compound of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, and the like, or a suppository wax which is solid at ambient temperature but liquid at body temperature and which melts in the rectum or vaginal cavity to release the active compound.

[0158] The solid dosage form of the compound of the present invention for oral administration comprises capsule, tablet, pill, powder and granule.In this solid dosage form, active compound is at least one pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) filler or extender, such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binder, such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidinone, sucrose and gum acacia, c) humectant, such as glycerol, d) agar, calcium carbonate, potato or tapioca starch, alginic acid, They are mixed with disintegrating agents such as certain silicates and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form also optionally comprises buffering agents.

[0159] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0160] The compounds of the present invention may also be formulated in microencapsulated form with one or more excipients as noted above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active compounds of the present invention may be admixed with at least one inert diluent, such as sucrose, lactose, or starch. Conventional dosage forms may also contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms also optionally contain buffering agents. They may optionally contain opacifying agents and be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and polymeric waxes.

[0161] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers. Ophthalmic formulations, ear drops, and eye drops are also contemplated as being within the scope of the present invention. Additionally, the present invention contemplates the use of transdermal patches, in preferred embodiments, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0162] According to one aspect, the present invention relates to a method of activating TLR7 / 8 activity in a biological sample, said method comprising the step of contacting said biological sample with a compound of the present invention or a composition comprising said compound.

[0163] According to another aspect, the present invention relates to a method for activating TLR7 / 8, or a mutant thereof, activity in a biological sample, said method comprising the step of contacting said biological sample with a compound of the present invention or a composition comprising said compound.

[0164] The compounds of the present invention are useful in vitro as unique tools for understanding the biological role of TLR7 / 8, including evaluation of the numerous factors thought to affect and be affected by TLR7 / 8 production and TLR7 / 8 interaction.

[0165] The present compounds are also useful for the development of other compounds that interact with TLR7 / 8, because they provide important structure-activity relationship (SAR) information that facilitates their development. The compounds of the present invention that bind to TLR7 / 8 can be used as reagents for detecting TLR7 / 8, preferably from live cells, fixed cells, biological fluids, tissue homogenates, purified natural biological materials, etc. For example, cells expressing TLR7 / 8 can be identified by detectably labeling the compounds of the present invention. In addition, based on their ability to bind to TLR7 / 8, the compounds of the present invention can be used in enzyme purification, such as in-situ staining, FACS (fluorescence-activated cell sorting), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ELISA (enzyme-linked immunosorbent assay), or to purify cells expressing TLR7 / 8 within permeabilized cells. The compounds of the present invention can also be utilized as commercial research reagents for various medical research and diagnostic uses. Such uses include, but are not limited to, the following: use as a calibration standard to quantify the activity of candidate TLR7 / 8 activators in various functional assays; use as an activating agent in random screening of compounds, i.e., in searching for new families of TLR7 / 8 ligands; use in co-crystallization with TLR7 / 8, i.e., compounds of the present invention will form crystals of the compound bound to TLR7 / 8, allowing the determination of the enzyme / compound structure by X-ray crystallography; other research and diagnostic applications, where TLR7 / 8 is preferably activated, or such activation is conveniently calibrated against a known quantity of, e.g., a TLR7 / 8 activator; use in assays as a probe to determine the expression of TLR7 / 8 in cells; and developing assays to detect compounds that bind to the same site as TLR7 / 8-binding ligands.

[0166] The compounds of the present invention can be applied either by themselves and / or in combination with physical measurements for the diagnosis of treatment efficacy. Pharmaceutical compositions containing the compounds and their use to treat TLR7 / 8-mediated conditions are promising new approaches to broad-spectrum treatments that cause direct and immediate improvement in the state of health, whether in humans or animals. The new orally bioavailable and active chemical entities of the present invention improve convenience for patients and compliance for physicians.

[0167] The compounds of the present invention, their salts, isomers, tautomers, enantiomeric forms, diastereomers, racemates, derivatives, prodrugs and / or metabolites are characterized by high specificity and stability, low production costs and convenient handling. These features form the basis for reproducible action with a lack of cross-reactivity and a reliable and safe interaction with target structures. As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof, biopsies or extracts thereof obtained from mammals, and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. Modulation of the activity of TLR7 / 8 or mutants thereof in biological samples is useful for a variety of purposes known to those of skill in the art, including, but not limited to, blood transfusion, organ transplantation, biological specimen storage, and biological assays.

[0168] Further Uses In a further aspect, the present invention provides pharmaceutical compositions comprising a compound of the present invention described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the pharmaceutical composition further comprises a therapeutically effective amount of a chemotherapeutic agent. In one embodiment, the present invention provides methods for stimulating an immune response in a subject. The method comprises administering a therapeutically effective amount of a compound of the present invention described herein under conditions effective to stimulate an immune response. In some embodiments, the method is performed on a subject with cancer. In other embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach cancer, testicular cancer, biliary tract cancer, colon cancer, endometrial cancer, head / neck cancer, medullary thyroid cancer, renal cancer, eye cancer, neuroblastoma, mycosis fungoides, glioma, other brain tumors, spinal cord tumors, liver cancer, leukemia, lymphoma, and any combination thereof. In certain embodiments, the compound of the present invention can be comprised in a liquid pharmaceutical composition. The liquid compositions of the present invention can be administered intratumorally (e.g., intratumorally (IT)) to preferably induce innate and cellular immune responses against tumor antigens (e.g., shrink or stabilize tumors). In a further embodiment, the compounds of the present invention are conjugated to peptides. Peptide-containing conjugates are not necessarily antigens or immunogens, but rather reduce the solubility of TLR7 and / or TLR8 agonists, creating a depot that is retained at the site of administration, such as within a tumor or the tumor microenvironment. The conjugated TLR7 and / or TLR8 agonists of the present invention can stimulate immunosuppressive cells and induce an immune response against antigens present within a tumor. Furthermore, the recruitment of immunosuppressive cells can induce an immune response not only against the tumor at the administration site, but also against peripheral, nearby, and / or distant tumors. In one embodiment, a method for stimulating an anti-tumor immune response in a subject is provided, wherein the method comprises administering a liquid form of the pharmaceutical composition of the present invention to a subject intratumorally or peritumorally, and the anti-tumor immune response is preferably effective at a site distant from the administration site of the pharmaceutical composition.In a further aspect, the present invention also provides a method for inducing an anti-tumor immune response in a subject. The method comprises administering a therapeutically effective amount of a compound of the present invention described herein under conditions effective to induce an anti-tumor immune response. In some embodiments, the method is performed on a selected subject having a tumor. In some embodiments, the tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cyst gland carcinoma, and the like. The tumor or abnormal cell growth is selected from the group consisting of cancer, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. In a further aspect, the present invention provides a method for treating a tumor or abnormal cell growth in a subject. The method comprises administering a therapeutically effective amount of a compound of the present invention described herein under conditions effective to treat the tumor or abnormal cell growth. In some embodiments, the tumor or abnormal cell growth is cancer. In some embodiments, the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer, lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, stomach cancer, testicular cancer, biliary tract cancer, colon cancer, endometrial cancer, head and neck cancer, medullary thyroid cancer, kidney cancer, eye cancer, neuroblastoma, mycosis fungoides, glioma, other brain tumors, spinal cord tumors, liver cancer, leukemia, lymphoma, and any combination thereof. In yet another aspect, the present invention provides a method for treating an infectious disease in a subject. The method comprises administering a therapeutically effective amount of a compound of the present invention described herein under conditions effective to treat the infectious disease.In some embodiments, the infectious disease is a viral infection, a bacterial infection, a fungal infection, or any combination thereof. In some embodiments, the infectious disease is a viral infection, and the infectious disease is preferably selected from the group consisting of coronavirus (including but not limited to severe acute respiratory syndrome (SARS), SARS-CoV-2 (COVID-19), Middle East respiratory syndrome (MERS), and the common cold), Ebola hemorrhagic fever, influenza, hepatitis, Hib disease, human immunodeficiency virus (HIV), human papillomavirus (HPV), meningococcal disease, pneumococcal disease, measles, mumps, norovirus, polio, respiratory syncytial virus (RSV), rotavirus, rubella virus, shingles, West Nile virus, rabies virus, enterovirus, cytomegalovirus, herpes virus, chickenpox, yellow fever, Zika virus, and any combination thereof. In some embodiments, the infection is a bacterial infection, and the infection is preferably selected from the group consisting of streptococcal infection, staphylococcal infection, diphtheria, meningococcal infection, tetanus, whooping cough, pneumococcal infection, bacterial food poisoning, sexually transmitted diseases, tuberculosis, Lyme disease, botulism, or any combination thereof. In some embodiments, the infection is a fungal infection, and the infection is candidiasis, histoplasmosis, dermatophytosis, tinea pedis, aspergillosis, cryptococcal meningitis, coccidioidomycosis, and any combination thereof.

[0169] Topical, intracystic and intratumoral compositions for the treatment of diseases comprising compounds of the present invention The compounds of the present invention can be formulated with other compounds, such as chemical penetration enhancers. Chemical penetration enhancers are often used in topical and transdermal formulations to enhance the absorption, uptake, and delivery of active pharmaceutical ingredients (drugs or drug substances) to the skin. Such formulations containing the compounds of the present invention may be useful, for example, in the treatment of skin cancer. Suitable formulations may also be employed to supplement further penetration within the local tissue compartment and / or to enhance immune-related responses in the tissue to which they are delivered. To enhance the penetration and uptake of the compounds of the present invention into the skin / tumor, the formulation may include, but is not limited to, a combination of one or more chemical enhancers, one or more solvents or vehicles that improve the distribution of the drug and chemical enhancer into the skin / tumor, and a gelling agent or matrix for incorporation into a topical or intratumoral formulation. Several examples of chemicals belonging to each category have been previously disclosed in the literature. However, the specific combination in which each component is employed to match the physicochemical properties of the drug and improve skin penetration is not trivial or solely dependent on the specific properties of the individual chemicals. It is important to note that the effect of each component on the skin / tumor depends on the concentration employed in the formulation, and synergistic effects are expected from their combination in a single formulation. These effects may be additive, positively synergistic, or negatively synergistic. The rate of penetration of the drug into and across the skin / tumor and the kinetics of the cumulative effect depend, in turn, on the specific formulation employed. Therefore, the physiological, biological, and therapeutic endpoints of a drug are largely determined by the formulation in addition to the drug's properties. These endpoints include, but are not limited to, pharmacokinetics / pharmacodynamics (PK / PD), cumulative absorption as determined by area under the curve (AUC), bioequivalence, and therapeutic index (TI), to name a few. Furthermore, the specific formulation of a drug not only influences its efficacy but also its tolerability and safety on the skin upon application. This may include, but is not limited to, adverse effects such as irritation, skin toxicity, and erythema.

[0170] The beneficial effect of chemical penetration enhancers is to enhance the penetration of the compounds of the invention throughout tissues or within local tissue compartments such as tumors, as well as to provide an inflammatory effect that is influential in directing immune responses important in, for example, tumor therapy. Thus, incorporation of the agent into topical, intracystic, and / or injectable formulations can achieve additive or more than additive effects on the therapeutic endpoints of tumor treatment.

[0171] Formulations suitable for administration include creams, ointments, solutions, gels, lotions, pastes, patches, foams, or spray formulations containing carriers known in the art.Dosage forms for local, intracystic, or intratumoral administration of the compounds of the present invention and optionally other drugs include powders, sprays, ointments, pastes, creams, ointment lotions, gels, solutions, and patches.The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, preservatives, buffers, or propellants.Ointments, pastes, creams, lotions, solutions, foams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof, in addition to the therapeutic agent. Injectable formulations containing the compounds of the present invention can also be achieved by using refined oils (soybean oil, safflower oil, triolein, castor oil, fractionated coconut oil, miglyol 810, 812, Neobee MS, Captex 300) and FDA-approved dermal fillers (hydroxylapatite, hyaluronic acid, poly-L-lactic acid, collagen, hydrogels) as components of the injectable formulation. Topical, intracystic, and intratumoral formulations containing the compounds of the present invention can be optimized by selecting specific components of the formulation, including enhancers, vehicles, and matrices (Karande 2004 Nature Biotechnology, Karande 2005 PNAS, and related references within these studies and citations). A specific formulation may have a significant impact on the efficacy and target indication of the drug. The physicochemical properties of the active compound can be derived from its structure and chemical composition. Chemical descriptors for penetration enhancers can be estimated to balance efficacy and safety. Based on this, chemical penetration enhancer combinations can be identified that contain chemicals from the following list, in total concentrations ranging from 0-2% wt / vol and weight fractions ranging from 0-100%: Formulations may contain two or three individual chemicals in a solvent to create the formulation.

[0172] The compounds of the invention may be combined in formulations containing one or more of the following additives: sodium octyl sulfate, sodium decyl sulfate, sodium dodecyl sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate, sodium eicosyl sulfate, sodium laureth sulfate, nicotine sulfate, sodium taurocholate, dimethyl sulfoxide, sodium tridecyl phosphate, ChemBetaine CAS, ChemBetaine Oleyl, ChemBetaine C, hexadecyldimethylammoniopropanesulfonic acid, decyldimethylammoniopropanesulfonic acid, dodecyldimethylammoniopropanesulfonic acid, myristyldimethylammoniopropanesulfonic acid, benzylpyridinium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, benzyldimethyldodecylammonium chloride, benzyldimethylmyristylammonium chloride, benzyldimethylstearylammonium chloride, octyltrimethylammonium bromide, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, myristyltrimethylammonium chloride, cetyltrimethylammonium bromide, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, Brij 97, Brij 30, Brij 56, TritonX-100, hexanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid, palmitic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, cholic acid, methyl hexanoate, ethyl undecanoate, methyl laurate, methyl tridecanoate, methyl myristate, isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethyl sebacate, tetracaine, glyceryl monolaurate, glyceryl monooleate, ethylpiperazine carboxylate, N-lauryl sarcosine, sodium caprylate, sodium decanoate Sodium, sodium palmitate, octylamine, decylamine, dodecylamine, tetradecylamine, oleylamine, urea, methylpyrrolidone, cyclohexylpyrrolidone, octylpyrrolidone, decylpyrrolidone, decylmethylpyrrolidone, methylpiperazine, phenylpiperazine, octanamide, hexadecanamide, caprolactam, carveol, pinene oxide, limonene, menthol, pulegone, carvacrol pinene, menthone, terpineol, cineole, fenchone, triacetin, trimethoxypropylene methylbenzene, linalool geraniol, octyldodecanol. Others include sulfoxides, alcohols, polyols, alkanes, fatty acids, esters, amines and amides, terpenes, surfactants, cyclodextrins, C2 or C3 or higher alcohols, C3 or C4 or higher diols, DMSO, DMF, DMA and related solvents, ln-dodecylcyclazacycloheptan-2-one, N-methylpyrrolidone and N-(2-hydroxyethyl)pyrrolidone, and broader classes of azones, and mixtures (binary, ternary, or higher).

[0173] The compounds of the invention can be formulated with chemical enhancers that can improve the delivery of the compounds. Optional chemical enhancers include long chain hydrocarbons with polar head groups, such as surfactants, fatty acids, and fatty esters.

[0174] Unsaturation (single or double) in the hydrocarbon chain can further aid in fluidizing the skin lipid bilayer and the penetration of the compounds of the present invention. Examples of fatty acids include oleic acid, linoleic acid, linoleic acid, palmitic acid, and myristic acid. Examples of fatty acid esters include esters of small alkyl groups such as methyl, ethyl, propyl, and butyl with fatty acids such as palmitic acid, myristic acid, oleic acid, and linoleic acid. Examples of surfactants include salts of fatty acid esters such as sodium lauryl sulfate and sodium oleate. Examples of solvents include short-chain alcohols such as ethanol, isopropanol, butanol, and hexanol. When the compounds of the present invention are formulated as topical preparations, the combination of chemicals and solvents can significantly enhance the penetration of the compounds of the present invention across the skin. Such example combinations are viable formulations with varying PK / PD profiles of API compounds of the present invention, as well as varying efficacy and safety.

[0175] Examples of formulations embodying the above chemical entities and combinations thereof are as follows: the compounds of the invention at concentrations of 0.01, 0.02, 0.03, 0.04 to 1 wt / vol %, wherein the compounds may be preferably combined with one or more, preferably all, of the following components listed under i, ii, iii, iv, v, vi, vii, viii, ix, x, xi, xii or xiii: i. oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1% wt / vol; ii. isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 0.5% wt / vol; iii. Oleic acid (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, for a total concentration of 0.5% wt / vol; iv. oleic acid (33%), isopropyl myristate (33%), and sodium lauryl sulfate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 0.5% wt / vol; v. Isopropyl palmitate (50%) and sodium oleate (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, at a total concentration of 2% wt / vol; vi. Sodium lauryl sulfate (25%) and linoleic acid (75%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1.0% wt / vol; vii. Palmitic acid (50%) and isopropyl laurate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 2.0% wt / vol; viii. Oleic acid (50%) and linoleic acid (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1.5% wt / vol; ix. Linoleic acid (25%), oleic acid (25%) and isopropyl linoleic acid (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 0.5% wt / vol; x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 2.0% wt / vol; xi. A solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol; xii. Oleic acid (10%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol; xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0176] Pharmaceutical and Further Uses of the Compounds and Compositions of the Invention As mentioned above, aspects of the present invention include the provision of a composition according to the invention for use as a medicament. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage per patient will depend on many factors, including the patient's size, weight, body surface area, age, the specific compound being administered, the activity of the compound employed, the time and route of administration, general health, and combination with other therapies or procedures. Proteinaceous pharmaceutically active substances can be present in amounts of 1 g to 100 mg / kg body weight per dose; however, doses below or above this exemplary range are also contemplated. If the regimen is a continuous infusion, the dose may range from 1 pg to 100 mg per minute per kilogram of body weight.

[0177] The specific amount or dose to be administered can again be determined by a clinician based on the factors cited above. Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art, see, for example, US 4,938,949.

[0178] Generally, the clinician will be able to determine appropriate dosages depending on the particular disease, disorder or condition being treated, the potency of the particular compound of the invention, the particular route of administration, and the particular pharmaceutical formulation or composition used.

[0179] The desired dosage can be conveniently presented as a single dose or as divided doses that are administered at appropriate intervals, for example, as two, three, four or more subdoses per day.The subdose itself can be further divided into a number of administrations that are, for example, loosely spaced apart.Preferably, the dose is administered once a week, or even less frequently, for example, once every two weeks, once every three weeks, once a month, or even once every two months.

[0180] The administration regimen can include long-term treatment. "Long-term" means a period of at least two weeks, preferably several weeks, months, or years. Necessary modifications in this dosage range can be determined by one skilled in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA. In the event of any complications, the dosage can also be adjusted by the individual physician.

[0181] Typically, a compound of the present invention is used in the above methods, however, it is also within the scope of the present invention to use two or more different compounds of the present invention in combination, or to use a compound of the present invention in combination with an additional pharmaceutical agent. Again, the clinician will be able to select such additional compounds or agents, and appropriate combined treatment regimens, based on the factors cited above and their professional judgment.

[0182] When two or more substances or components are used as part of a combined treatment regimen, they can be administered via the same route of administration or different routes of administration, at essentially the same time or at different times (e.g., essentially simultaneously, sequentially, or according to an alternating schedule). When substances or components are administered simultaneously via the same route of administration, they can be administered in different pharmaceutical formulations or compositions or as part of a combined pharmaceutical formulation or composition, as will be apparent to those skilled in the art.

[0183] Furthermore, when two or more active substances or ingredients are used as part of a combined treatment plan, each of the substances or ingredients can be administered in the same amount and according to the same schedule as when the compound or ingredient is used alone, and such combined use may or may not lead to a synergistic effect. However, if the combined use of two or more active substances or ingredients leads to a synergistic effect, it may be possible to reduce the amount of one, several, or all of the substances or ingredients administered while still achieving the desired therapeutic effect. This may be useful, for example, to avoid, limit, or reduce undesirable side effects associated with the use of one or more substances or ingredients when used in normal amounts, while still achieving the desired medicinal or therapeutic effect.

[0184] The efficacy of a treatment regimen used in accordance with the present disclosure can be determined and / or assessed in any manner known per se for the disease, disorder, or condition involved, as would be apparent to a clinician, who may also modify or alter, where appropriate and on a case-by-case basis, a particular treatment regimen to achieve a desired therapeutic effect and avoid, limit, or reduce undesirable side effects, and / or to achieve an appropriate balance between achieving a desired therapeutic effect, on the one hand, and avoiding, limiting, or reducing undesirable side effects, on the other hand. Generally, a treatment regimen is continued until the desired therapeutic effect is achieved and / or the desired therapeutic effect is maintained, which can also be determined by the clinician.

[0185] Thus, in a further aspect, the present invention relates to a pharmaceutical composition comprising at least one compound of the present invention, at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a compound according to the present invention (preferably at least one compound selected from Examples 1 to 27 set out in the claims, more preferably a compound selected from Examples 1 to 6 of the list set out in the claims), at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.

[0186] The subject to be treated is any warm-blooded animal, but particularly a mammal, and more particularly a human.In veterinary applications, the subject to be treated includes any animal that is commercially bred or kept as a pet.As will be apparent to those skilled in the art, the subject to be treated is particularly a person suffering from or at risk of the diseases, disorders and conditions described herein.Therefore, in a preferred embodiment of the present invention, the pharmaceutical composition comprising the compound of the present invention is for use in medicine or diagnostics.Preferably, the pharmaceutical composition is for use in human medicine, but can also be used for veterinary purposes. In such pharmaceutical compositions, one or more compounds of the present invention may also be suitably combined with one or more other active ingredients, as described herein.

[0187] The present invention also relates to pharmaceutical compositions for use either in vitro (e.g., in vitro or cellular assays) or in vivo (e.g., in a single cell or a multicellular organism, particularly a mammal, more particularly a human, e.g., a human at risk of or suffering from a disease, disorder or condition of the invention). Unless expressly stated otherwise, references to treatment will be understood to include both treatment of established symptoms and prophylactic treatment.

[0188] Generally, for pharmaceutical use, the compounds of the present invention can be formulated as pharmaceutical preparations or compositions containing at least one compound used in the present invention, at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. By way of non-limiting example, such formulations may be in a form suitable for oral administration, parenteral administration (such as intravenous, intramuscular, or subcutaneous injection or intravenous infusion), topical administration (such as intra-articular administration), inhalation, skin patch, implant, suppository, etc., with intra-articular administration being preferred. Such suitable dosage forms (which may be solid, semi-solid, or liquid depending on the method of administration) and methods and carriers for use in their preparation will be apparent to those skilled in the art and are further described herein. Such pharmaceutical preparations or compositions are generally referred to herein as "pharmaceutical compositions."

[0189] Disintegrants, binders, fillers, and lubricants can be mentioned as exemplary excipients.Examples of disintegrants include agar, algin, calcium carbonate, cellulose, colloidal silicon dioxide, gum, magnesium aluminum silicate, methylcellulose, and starch.Examples of binders include microcrystalline cellulose, hydroxymethylcellulose, hydroxypropylcellulose, and polyvinylpyrrolidone.Examples of fillers include calcium carbonate, calcium phosphate, tricalcium sulfate, calcium carboxymethylcellulose, cellulose, dextrin, dextrose, fructose, lactitol, lactose, magnesium carbonate, magnesium oxide, maltitol, maltodextrin, maltose, sorbitol, starch, sucrose, sugar, and xylitol. Examples of lubricants include agar, ethyl oleate, ethyl laurate, glycerin, glyceryl palmitostearate, hydrogenated vegetable oils, magnesium oxide, stearates, mannitol, poloxamer, glycol, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. As pharmaceutical adjuvants, conventional stabilizers, preservatives, wetting agents and emulsifiers, consistency improvers, flavor improvers, salts for varying osmotic pressure, buffer substances, solubilizers, diluents, emollients, colorants, masking agents, and antioxidants come into consideration.

[0190] Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, water, alcohol, polyol, glycerol, and vegetable oils.

[0191] In general, the compounds of the present invention can be formulated and administered in any suitable manner known per se, for example as described in the general background art cited above (in particular WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867, and WO 08 / 020079), and in Remington's Pharmaceutical Sciences, 18 th Reference may be made to standard handbooks such as Remington, The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see, e.g., pages 252-255).

[0192] In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a compound according to the present invention, further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharmaceutically active compounds. Suitable formulations and methods for their preparation will be apparent to those skilled in the art and include, for example, preferred formulations suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intracavity, intraarterial, epidural, intranasal or intrabronchial administration) as well as for topical administration (e.g., intraarticular, transdermal or intradermal).

[0193] Preparations for local or parenteral administration may be, for example, sterile solutions, suspensions, dispersions or emulsions suitable for infusion or injection. Suitable carriers or diluents for such preparations include, for example, those described on page 143 of WO08 / 020079. Usually, aqueous solutions or suspensions are preferred. The present invention also provides, in a further aspect, a kit comprising at least one compound according to the invention. It is contemplated that the kit may be provided in a variety of forms. The present invention is further described by the following non-limiting preferred aspects, examples and figures.

[0194] 6 Examples The following examples illustrate the methods and products of the present disclosure.

[0195] As outlined in more detail below, a series of novel pharmaceutical TLR7 agonists have been designed, chemically prepared, and biologically evaluated. Unexpectedly, compared with prior art compounds such as R484 and several recently reported TLR7 / 8 agonists (see Figure 2), several novel TLR7 agonists not only exhibited low nanomolar activity in cellular TLR7 experiments, but also demonstrated high selectivity between TLR7 and TLR8. Even more surprisingly, these novel highly selective TLR7 agonists were able to initiate massive production of cytokines and chemokines (IL-6, IL1-b, TNF-a) and induce potent upregulation of CD40 and CD86 in cellular monocyte experiments.

[0196] 6.1 Chemical synthesis of TLR7 agonists Chemical synthesis of Example 1 [ka] Step 1 A stirred solution of R848 (750.00 mg, 2.36 mmol, 1.00 eq.) in acetonitrile (15.00 ml, 20.00 V) was added to triethylamine (0.93 ml, 7.09 mmol, 3.00 eq.) under a nitrogen atmosphere, followed by the addition of chloro-(diphenyl)methylbenzene (1.33 g, 4.72 mmol, 2.00 eq.). The resulting suspension was irradiated in a microwave reactor at 100 °C for 1.15 h. The reaction was monitored by TLC, and the solvent was then removed under vacuum to give the crude product. The crude product was purified by flash column chromatography on 100-200 silica gel with ethyl acetate and hexane. The desired product was eluted with 25-30% ethyl acetate and hexane, and the eluate was concentrated to give compound 1 (1.30 g, 2.31 mmol, 97.8%, white solid, purified product). Yield: 1.30 g (2.31 mmol), % yield: 97.8. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O; B: ACN; Flow rate: 1.5ml / min RT(min):2.13;Purity:98.94%;M+H:557.10.

[0197] Step 2: To a stirred solution of sodium hydride (179.63 mg, 4.49 mmol, 2.50 eq.) in DMF (10.00 ml) was added a solution of compound 1 (1.00 g, 1.80 mmol, 1.00 eq.) dissolved in DMF (3 ml). The reaction mixture was stirred at 0° C. under a nitrogen atmosphere for 30 minutes, followed by the addition of tert-butyl 1,2,3-oxathiazolidine-3-carboxylate 2,2-dioxide (441.12 mg; 1.98 mmol; 1.10 eq.). The reaction mixture was stirred at RT for 12 hours, and the progress of the reaction was monitored by LCMS. Upon completion, the reaction mixture was quenched with cold water and extracted with ethyl acetate (2×100 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography. The desired product was eluted with 60% ethyl acetate in petroleum ether. The combined pure fractions were concentrated under reduced pressure to give compound 2 (550.00 mg, 0.75 mmol, 41.9%, off-white gum, purified product). Yield: 550.00mg (0.75mmol), % yield: 41.9. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O; B: ACN; Flow rate: 1.5ml / min RT (min):2.60;Purity:95.70%;M+H:700.00.

[0198] Step 3: To a stirred solution of compound 2 (550.00 mg; 0.75 mmol; 1.00 eq.) in DCM (10.00 ml) was added TFA (0.08 ml; 1.03 mmol; 1.37 eq.) at 0° C. The reaction mixture was stirred at RT for 3 h. The reaction progress was monitored by LCMS. Upon completion, the reaction mixture was concentrated under reduced pressure. The crude product was washed with diethyl ether (2×25 ml) and dried under reduced pressure to give compound 3 as a trifluoroacetate salt (360.00 mg; 0.73 mmol; 97.4%; off-white solid; purified product). Yield: 360.00mg (0.73mmol), % yield: 97.4. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O; B: ACN; Flow rate: 1.5ml / min RT(min):0.94;Purity:95.90%;M+H:358.00.

[0199] Step 4: To a stirred solution of Compound 3 trifluoroacetate (200.00 mg; 0.41 mmol; 1.00 eq.) and bis(2-chloroethyl)(methyl)amine hydrochloride (0.16 g; 0.81 mmol; 2.00 eq.) in DCM (10.00 ml) was added potassium carbonate (112.45 mg; 0.81 mmol; 2.00 eq.). The reaction mixture was heated to 100 °C and stirred for 16 hours. The reaction progress was monitored by LCMS. Upon completion, the reaction mixture was partitioned between water and ethyl acetate. The separated organic layer was washed with brine solution, filtered, and the filtrate was concentrated under reduced pressure. The crude residue was purified by preparative HPLC using 0.1% aqueous NHOAc and acetonitrile as eluents, and the desired fractions were lyophilized to give Example 1 (4.50 mg, 0.01 mmol, 2.4%, brown gummy solid, purified product). Yield: 4.50 mg (0.01 mmol), % yield: 2.4. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O; B: ACN; Flow rate: 1.5ml / min RT (min): 1.76; Purity: 95.81%; M+H: 441.30. HPLC: Column: XBridge C8, 3.5 μm, 4.6 × 50 mm; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. RT(min):1.72;Purity:96.98%(max);Purity:88.09%(220nm). 1H NMR (400 MHz, DMSO-d6): 9.12 (s, 2H), 8.55 (d, J = 8.00 Hz, 1H), 7.80 (d, J = 8.00 Hz, 1H), 7.71 (t, J = 8.00 Hz, 1H), 7.54 (t, J = 7.20 Hz, 1H), 4.95 (s, 4H), 3.53 (m, 4H), 3.36 (m, 4H), 2.73-3.29 (m, 4H), 2.51 (s, 3H), 1.22 (s, 6H), 1.16 (t, J = 6.80 Hz, 3H).

[0200] Chemical synthesis of Example 2 [ka] Step 1: To a stirred solution of sodium hydride (60%) (158.00 mg, 3.95 mmol, 2.59 eq.) in DMSO (9.00 ml, 10.00 V) under a nitrogen atmosphere at 0° C., a solution of compound 1 (900.00 mg, 1.53 mmol, 1.00 eq.) in DMSO (5 ml) was added dropwise, and the reaction mixture was stirred at RT for 30 min. Then, (3-bromopropoxy)(tert-butyl)dimethylsilane (0.78 ml, 4.77 mmol, 3.12 eq.) was added at 0° C. and stirred at RT for 3 h under a nitrogen atmosphere. The reaction mixture was monitored by LCMS. Upon completion, the reaction mixture was quenched with ice water at 0° C. and diluted with ethyl acetate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by silica gel chromatography. The desired product was eluted with 15% ethyl acetate in hexane to give compound 4 (215.00 mg; 0.28 mmol; 18.4%; gum; purified product). Yield: 215.00 mg (0.28 mmol), % yield: 18.4. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5 ml / min RT(min):3.59;Purity:95.53%;M+H:729.30.

[0201] Step 2: To a stirred solution of compound 4 (180.00 mg, 0.24 mmol, 1.00 eq.) in THF (10.00 ml, 55.56 V) was added TBAF (1.0 M in THF) (1.00 ml, 1.00 mmol, 4.24 eq.) under a nitrogen atmosphere at 0° C. The reaction mixture was stirred at RT for 3 hours, and TLC confirmed the completion of the reaction. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was collected and concentrated in vacuo to give compound 5 (150.00 mg; 0.24 mmol; 100%, gum; purified product). Yield: 150.00 mg (0.24 mmol), % yield: 100. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):2.22;Purity:99.51%;M+H: 615.00.

[0202] Step 3: To a solution of compound 5 (160.00 mg; 0.26 mmol; 1.00 eq.) in DCM (3.30 ml; 20.63 V), 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (0.34 g; 0.77 mmol; 3.00 eq.) was added at 0 °C, and the reaction mixture was stirred at RT for 30 min. Completion of the reaction was confirmed by TLC. The reaction mixture was washed with 10% NaHCO solution. The organic layer was collected and concentrated in vacuo to give compound 6 (170.00 mg; 0.21 mmol; 81.8%; colorless liquid; crude product). Yield: 170.00 mg (0.21 mmol), % yield: 81.8. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):2.36;Purity:75.96%;M+H:613.00.

[0203] Step 4: To a stirred solution of compound 6 (170.00 mg; 0.21 mmol; 1.00 eq.) in 1,2-dichloroethane (10.00 ml; 58.82 V) was added 1-methylpiperazine (0.10 ml; 0.96 mmol; 4.55 eq.) at RT. The reaction mixture was stirred at RT for 10 minutes, and sodium triacetoxyborohydride (300.00 mg, 1.34 mmol, 6.38 eq.) was added to the reaction mixture at 0° C. The reaction mixture was stirred at RT for 30 minutes. Completion of the reaction was confirmed by TLC. The reaction mixture was quenched with water and extracted with DCM. The organic layer was collected and concentrated in vacuo to give compound 7 (140.00 mg; 0.15 mmol; 71.7%; gum; crude product). Yield: 140.00 mg (0.15 mmol), % yield: 71.7. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):1.75;Purity:75.26%;M+H:697.00.

[0204] Step 5: To a solution of compound 7 (100.00 mg; 0.11 mmol; 1.00 eq.) in DCM (5.00 ml; 50.00 V) was added TFA (0.90 ml; 11.60 mmol; 9.00 V) at 0° C. The resulting solution was warmed to room temperature and stirred for 3 hours. The reaction was monitored by LCMS, and then the solvent was removed under reduced pressure below 35° C. to obtain the crude product. The crude product was purified by preparative HPLC in the HCOOH method, and the desired fractions were lyophilized to give Example 2 as the formate salt (32.91 mg, 0.07 mmol, 60.7%, off-white gum, purified product). Yield: 32.91 mg (0.07 mmol) % yield: 60.7. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):1.03;M+H:455.20. HPLC: Column: XBridge C8, 3.5 μm, 4.6 × 50 mm; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. RT(min):9.52;Purity:99.83%(220nm). 1 HNMR (400 MHz, DMSO-d6): 8.36 (dd, J = 8.00, Hz, 1H), 8.15 (s, 1H), 7.62 (dd, J = 0.80, 8.20 Hz, 1H), 7.45 (t, J = 7.20 Hz, 1H), 7.25 (t, J = 6.80 Hz, 1H), 6.91 (s, 2H), 4.72 (s, 4H), 3.54-3.53 (m, 2H), 3.24-3.22 (m, 2H), 2.76-2.75 (m, 2H), 2.52-2.50 (m, 11H), 2.10 (m, 2H), 1.42-1.38 (m, 2H), 1.24-1.12 (m, 9H).

[0205] Chemical synthesis of Example 3 [ka] Step 1: To a stirred solution of sodium hydride (60%) (0.70 g, 17.50 mmol, 2.41 eq.) in DMSO (40.00 ml, 9.76 V) was added compound 1 (4.10 g, 7.26 mmol, 1.00 eq.) in DMSO (7 ml) at RT under a nitrogen atmosphere. The reaction mixture was stirred at RT for 30 min. 1,4-Dibromobutane (4.83 ml, 39.46 mmol, 5.43 eq.) was added at RT, and the reaction mixture was stirred at RT under a nitrogen atmosphere for 3 h. The progress of the reaction was monitored by LCMS. The reaction mixture was quenched with ice water at 0 °C. The reaction mixture was diluted with ethyl acetate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude product. The crude residue was purified by silica gel chromatography. The product was eluted with 15% ethyl acetate in hexane to give compound 8 (145.00 mg; 0.14 mmol; 2.0%; white gum; purified product), and the unreacted starting material was eluted with 45% ethyl acetate in petroleum ether to give compound 1 (3.20 g; 5.12 mmol; 70.4%; white solid; purified product). Yield: 145 mg (0.14 mmol), % yield: 2.0. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5 ml / min RT(min):2.75;Purity:68.10%;M+H: 690.90.

[0206] Step 2: To a solution of compound 8 (470.00 mg; 0.67 mmol; 1.00 eq.) and 1-methylpiperazine (0.08 ml; 0.74 mmol; 1.10 eq.) in acetonitrile (14.10 ml; 30.00 V) at RT was added potassium carbonate (189.73 mg; 1.35 mmol; 2.00 eq.). The suspension was heated at 50° C. for 16 h, and the reaction was monitored by TLC. Upon completion, the solvent was removed to give a residue. The residue was dissolved in water and extracted with DCM (8 mL). The organic layer was washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated to give the crude product. This material was purified by silica gel column chromatography. The desired product was eluted with 7% MeOH in DCM to give compound 9 (400.00 mg, 0.55 mmol, 82.1%, off-white powder, purified product). Yield: 400 mg (0.55 mmol), % yield: 82.1. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 1.5 ml / min. RT(min):1.79;Purity:98.20%.

[0207] Step 3: To a solution of compound 9 (95.00 mg; 0.11 mmol; 1.00 eq.) in DCM (4.75 ml; 50.00 V) was added TFA (0.36 ml; 4.58 mmol; 40.00 eq.) at 0° C. The resulting solution was warmed to room temperature and stirred for 3 hours. The reaction was monitored by LCMS. Upon completion, the solvent was removed under reduced pressure below 35° C. to give the crude product. The crude product was purified by preparative HPLC in the HCOOH method, and the desired fractions were lyophilized to give Example 3 as the formate salt (44.00 mg, 0.08 mmol, 74.1%, off-white gum, purified product). Yield: 44.00 mg (0.08 mmol), % yield: 74.1. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 1.5 ml / min. RT(min):0.99;Purity:99.05%;M+H:469.10. HPLC: Column: Phenomenex Gemini C18 (150*4.6) mm, 3.0 μm; Solvent A: water + 10 mM ammonium acetate; Solvent B: acetonitrile; Flow rate: 1.0 ml / min. RT(min):7.96;Purity:99.22%(max);Purity:97.91%(220nm). 1 HNMR (400 MHz, DMSO-d6): 8.33 (d, J = 8.40 Hz, 1H), 7.59 (d, J = 8.40 Hz, 1H), 7.42 (t, J = Hz, 1H), 7.22 (t, J = Hz, 1H), 6.67 (s, 2H), 4.73 (s, 2H), 3.49-3.54 (m, 6H), 3.20 (t, J = 9.20 Hz, 4H), 2.078-2.335 (m, 11H), 1.13-1.26 (m, 14H), (s, H), (s, H), (s, H), (s, H).

[0208] Chemical synthesis of Example 4 [ka] Step 1: To a stirred solution of sodium hydride (104.78 mg, 2.62 mmol, 3.00 eq.) in DMSO (10.00 ml, 20.00 V) was added a solution of compound 1 (500.00 mg, 0.87 mmol, 1.00 eq.) in DMSO (5 ml) at 0 °C under a nitrogen atmosphere. The reaction mixture was stirred for 30 minutes, then 1,5-dibromopentane (614.64 mg; 2.62 mmol; 3.00 eq.) was added, and the resulting solution was stirred at RT for 16 hours. The reaction was monitored by LCMS. Upon completion, the reaction mixture was quenched with cold water and extracted with ethyl acetate (2 × 100 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give the crude material. The crude material was purified by silica gel column chromatography. The desired product was eluted with 16% ethyl acetate in petroleum ether. The combined pure fractions were concentrated under reduced pressure to give compound 10 (150.00 mg, 0.21 mmol, 23.7%, off-white gum, purified product). Yield: 150.00 mg (0.21 mmol), % yield: 23.7. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):3.32;Purity:97.45%;M+H:705.30.

[0209] Step 2: To a stirred solution of compound 10 (150.00 mg, 0.21 mmol, 1.00 eq.) in acetonitrile (3.00 ml, 20.00 V) at RT was added potassium carbonate (87.63 mg, 0.62 mmol, 3.00 eq.) and 1-methylpiperazine (0.03 ml, 0.31 mmol, 1.50 eq.). The suspension was heated to 50° C. and stirred for 16 hours. The reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated under reduced pressure to give a crude residue. The residue was dissolved in DCM (100 ml) and washed with brine (2×50 ml). The organic layer was concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography. The desired product was eluted with 7% MeOH in DCM. The combined pure fractions were concentrated under reduced pressure to give compound 11 (70.00 mg; 0.09 mmol; 41.1%; off-white gum; purified product). Yield: 70.00 mg (0.09 mmol), % yield: 41.1. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):1.72;Purity:88.13%;M+H:725.20.

[0210] Step 3: To a solution of compound 11 (70.00 mg; 0.08 mmol; 1.00 eq.) in DCM (3.50 ml; 50.00 V) was added TFA (0.26 ml; 3.32 mmol; 40.00 eq.) at 0 °C. The resulting solution was stirred for 3 hours. The reaction was monitored by LCMS. Upon completion, the solvent was removed under reduced pressure below 35 °C to give the crude product. This material was purified by preparative HPLC in the HCOOH method. The desired fractions were lyophilized to give Example 4 as the formate salt (32.00 mg; 0.06 mmol; 72.6%; pale yellow gum; purified product). Yield: 32.00 mg (0.06 mmol), % yield: 72.6. Analysis data: LCMS: カラム:Atlantis dC18 (50x4.6mm) 3.5μm; solvent A; 0.1%HCOOH in H2O:ACN (95:5), solvent B; ACN; flow rate: 1.5ml / min. RT(min): 1.09; Purity: 99.24%; M+H: 483.10. HPLC: カラム: Phenomenex Gemini C18 (150*4.6)mm, 3.0μm; solvent A: 0.1% TFA in H2O:ACN (95:5), solvent B: ACN; flow rate: 1.0 ml / min. RT (min): 8.82; Purity: 99.42% (max); Purity: 98.26% (220nm). 1HNMR (400 MHz, DMSO-d6): δ 8.57 (d, J = 7.60 Hz, 1H), 8.37 (s, 1H), 7.71-7.79 (m, 1H), 7.67-7.71 (m, 1H), 7.52-7.56 (m, 1H), 4.90 (s, 4H), 3.64 (s, 4H), 3.28-3.33 (m, 8H), 2.86 (s, 3H), 2.43-2.47 (m, 2H), 1.21-1.32 (m, 12H), 0.96 (d, J = 4.40 Hz, 2H).

[0211] Chemical synthesis of Example 5

change

[0212] Step 2: To a stirred solution of compound 12 (200.00 mg, 0.21 mmol, 1.00 eq.) and 1-methylpiperazine (0.05 ml, 0.52 mmol, 2.50 eq.) in acetonitrile (2.00 ml, 10.00 V) at RT was added potassium carbonate (88.64 mg, 0.63 mmol, 3.00 eq.). The suspension was heated to 50° C. and stirred for 16 h. The reaction was monitored by LCMS. Upon completion, the solvent was removed to give a residue. The residue was dissolved in water and extracted with DCM (20 ml). The organic layer was washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated to give a crude residue. The residue was purified by silica gel chromatography. The desired product was eluted with 8% MeOH in DCM to give compound 13 (110.00 mg; 0.14 mmol; 67.6%; white solid; purified product). Yield: 110.00 mg (0.14 mmol), % yield: 67.6. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH in H2O, B: ACN; Flow rate: 1.5ml / min RT(min):1.79;Purity:95.17%;M+H:739.00.

[0213] Step 3: To a stirred solution of compound 13 (110.00 mg; 0.14 mmol; 1.00 eq.) in DCM (5.50 ml; 50.00 V) was added TFA (0.44 ml; 5.67 mmol; 40.00 eq.) at 0° C. The resulting solution was stirred for 3 hours. The reaction progress was monitored by LCMS. Upon completion, the solvent was removed under reduced pressure below 35° C. to give the crude product. The crude product was purified by preparative HPLC with HCOOH method. The desired fractions were lyophilized to give Example 5 as the formate salt (60.00 mg; 0.11 mmol; 77.1%; white solid; purified product). Yield: 60.00 mg (0.11 mmol), % yield: 77.1. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 1.5ml / min. RT(min):1.17;Purity:98.84%;M+H:497.10. HPLC: Column: XBridge C8, 3.5 μm, 4.6 × 50 mm; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. RT(min):5.39;Purity:99.49%(max);Purity:99.54%(220nm). 1 HNMR (400 MHz, MeOD): δ 8.60 (d, J = 8.40 Hz, 1H), 7.80-7.71 (m, 2H), 7.61-7.57 (m, 1H), 4.87-4.84 (m, 6H), 3.67-3.62 (m, 2H), 3.34-3.15 (m, 10H), 2.84-2.79 (m, 5H), 1.53-1.49 (m, 2H), 1.31-1.22 (m, 11H), 1.16-1.09 (m, 4H).

[0214] Chemical synthesis of Example 6 [ka] Step 1: To a stirred solution of benzyl alcohol (1.00 g, 9.20 mmol, 1.00 eq.) in 10% aqueous sodium hydroxide (0.92 g, 23.00 mmol, 2.50 eq.) dissolved in water (20.00 ml, 20.00 V) at 0 °C, tetrabutylammonium hydrogen sulfate (64.42 mg, 0.18 mmol, 0.02 eq.) was added, followed by 1,4-dibromobutane (3.99 g, 18.4 mmol, 2 eq.). The resulting mixture was stirred at 70 °C for 4 h, and the reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give the crude material. This material was purified by flash column chromatography on 100-200 silica gel using ethyl acetate and hexanes. The desired product was eluted with 1-5% ethyl acetate and hexanes, and the eluate was concentrated to give compound 14 (1.00 g; 4.05 mmol; 44.0%; colorless oil; purified product). Yield: 1 g (4.05 mmol), % yield: 44. Analysis data: 1 HNMR (400 MHz, DMSO-d6): δ 7.40-7.29 (m, 5H), 4.53 (s, 2H), 3.53 (t, J = -12.40 Hz, 2H), 3.46 (t, J = 6.80 Hz, 2H), 2.04-1.79 (m, 4H).

[0215] Step 2: To a stirred suspension of sodium hydride (60%) (179.63 mg, 4.49 mmol, 2.50 eq.) in DMSO (20.00 mL, 20.00 V) under a nitrogen atmosphere at 0° C., a solution of compound 1 in DMSO (5 mL) was added dropwise, and the reaction mixture was stirred at RT for 30 min. Then, [(4-bromobutoxy)methyl]benzene (882.33 mg, 3.59 mmol, 2.00 eq.) was added at 0° C., and the reaction mixture was stirred at RT for 16 h under a nitrogen atmosphere. The reaction mixture was monitored by TLC. Upon completion, the reaction mixture was quenched with ice water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to give the crude material. This material was purified by flash column chromatography on 100-200 silica gel with ethyl acetate and hexanes. The desired product was eluted with 10-15% ethyl acetate and hexanes, and the eluate was concentrated to give compound 15 (330.00 mg; 0.45 mmol; 24.8%; off-white gum; purified product). Yield: 330 mg (0.45 mmol), % yield: 24.8. Analysis data: LCMS: Column: XBridge C8 (50x4.6mm) 3.5µm; Solvent A: 0.1% TFA in H2O:ACN (95:5), Solvent B: ACN; Flow rate: 1.5ml / min. RT(min):3.35;Purity:97.21%;M+H:719.3.

[0216] Step 3: To a stirred solution of compound 15 (100.00 mg; 0.14 mmol; 1.00 eq.) in ethanol (5.00 ml) and AcOEt (5.00 ml) was added palladium on carbon (100.00 mg; 0.09 mmol; 0.70 eq.) at RT. The resulting reaction mixture was stirred in an autoclave under 5 kg of hydrogen pressure at RT. Upon completion, the reaction mixture was filtered through Celite. The filtrate was concentrated under reduced pressure to obtain a crude material. This material was purified by preparative HPLC using 0.1% aqueous HCOOHA and ACN as eluents. The combined pure fractions were lyophilized to obtain Example 6 as a formate salt (20.00 mg, 0.05 mmol, 37.9%, off-white solid, purified product). Yield: 20 mg (0.05 mmol), % yield: 37.9. Analysis data: LCMS: Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 1.5ml / min. RT(min):1.46;Purity:99.69%;M+H:387.1 HPLC: Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN RT(min):7.01;Purity(max):99.86%;Purity(220nm):99.32%. 1 HNMR (400 MHz, DMSO-d6): δ 0.00 (s, 1H), 7.58-7.60 (m, 1H), 7.39-7.43 (m, 1H), 7.20-7.24 (m, 1H), 4.77 (s, 5H), 3.52 (d, J = 7.20 Hz, 2H), 3.35 (t, J = 6.00 Hz, 2H), 3.21 (s, 1H), 1.32 (q, J = 2.00 Hz, 4H), 1.26 (q, J = 10.80 Hz, 4H), 1.14 (q, J = 7.20 Hz, 4H).

[0217] Chemical synthesis of Example 7 [ka] Step 1: To a suspension of sodium hydride (60%) (0.21 g, 5.31 mmol, 2.30 eq.) in DMSO (13.00 ml, 10.00 V) was added dropwise a solution of compound 1 (1.30 g, 2.31 mmol, 1.00 eq.) in DMSO (13.00 ml, 10.00 V) under a nitrogen atmosphere at 0 °C. The mixture was then stirred at rt for 30 min. 1,4-Dibromobutane (0.76 g, 3.46 mmol, 1.50 eq.) was added at 0 °C, and the reaction mixture was stirred at rt for an additional 1 h. After completion, the reaction mixture was quenched with ice water and diluted with ethyl acetate. The organic layer was separated, dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The crude residue was purified by flash chromatography (15% ethyl acetate in hexanes) to provide compound 16 (120.00 mg; 0.17 mmol; 7.3%; white solid; purified product). Yield: 120.00 mg (0.17 mmol), % yield: 7.3. Analysis data: LCMS-Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA in H2O:ACN (95:5); Mobile phase B: 0.1% TFA in CAN; Flow rate: 1.5 ml / min. RT(min):3.28;Purity(max):97.18%;M+H:691.20

[0218] Step 2: Potassium carbonate (35.67 mg; 0.25 mmol; 3.00 eq.) was added to a solution of compound 16 (3, 60.00 mg; 0.08 mmol; 1.00 eq.) and morpholine (22.26 mg; 0.25 mmol; 3.00 eq.) in acetonitrile (1.80 ml; 30.00 V) at RT. The suspension was then heated to 70 °C and stirred for 20 hours, and the reaction progress was monitored by TLC. After completion, the solvent was evaporated, and the residue was dissolved in water. It was then extracted with dichloromethane. The organic layer was washed with water and dried over sodium sulfate, and the solvent was then evaporated under reduced pressure. The crude product was purified by flash chromatography (60% ethyl acetate in petroleum ether) to give compound 17 (60.00 mg; 0.08 mmol; 97.8%; gum; purified product). Yield: 60.00 mg (0.08 mmol), % yield: 97.8. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase A: 0.1% HCOOH in water; B: 0.1% HCOOH in ACN; Flow rate: 0.8ml / min. RT(min):1.88;Purity(max):95.95%;M+H:698.00

[0219] Step 3: To a stirred solution of compound 17 (5, 60.00 mg; 0.08 mmol; 1.00 eq.) in dichloromethane (3.00 ml; 50.00 V) was added TFA (0.20 ml; 2.58 mmol; 3.33 V) at 0 °C. The resulting solution was warmed to rt and stirred for an additional 3 h. The reaction progress was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column chromatography (10% methanol in dichloromethane) to give Example 7 (34.00 mg, 0.07 mmol, 89.5%, off-white gum, purified product). Yield: 34.00 mg (0.07 mmol), % yield: 89.5. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase A: 0.1% HCOOH in water; B: 0.1% HCOOH in ACN; Flow rate: 0.8ml / min. RT(min):1.15;Purity(max):98.94%;M+H:456.10. HPLC-Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase A: 0.1% TFA in MilliQ water; Mobile phase B: acetonitrile; Flow rate: 1.0ml / min. RT(min):9.25;Purity(max):95.25%;M+H:456.10;Purity(220nm):96.41%.

[0220] Chemical synthesis of Example 8 [ka] Step 1: To a stirred solution of compound 16 (60.00 mg, 0.08 mmol, 1.00 eq.) and piperidine (18.54 mg, 0.21 mmol, 2.50 eq.) in acetonitrile (1.80 ml, 30.00 V) was added potassium carbonate (30.09 mg, 0.21 mmol, 2.50 eq.) at RT. The suspension was heated to 70 °C with stirring for 20 h. The reaction progress was monitored by TLC. After completion, the solvent was evaporated and the residue was subjected to flash chromatography (5% MeOH in dichloromethane) to give compound 18 (40.00 mg; 0.06 mmol, 66.9%; gum, purified product). Yield: 40.00 mg (0.06 mmol), % yield: 66.9. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: 0.1% HCOOH in A:H2O:ACN (95:5), B:ACN: Flow rate: 1.5ml / min. RT(min):2.01;Purity(max):99.36%;M+H:696.1

[0221] Step 2: To a stirred solution of compound 18 (40.00 mg, 0.06 mmol, 1.00 eq.) in dichloromethane (2.00 ml, 50.00 V) was added TFA (0.15 ml, 1.93 mmol, 3.75 V) at 0 °C. The reaction mixture was warmed to RT and stirred for an additional 3 h. The reaction progress was monitored by TLC. After completion of the reaction, it was concentrated under reduced pressure and the crude product was purified by flash column chromatography (15% methanol in dichloromethane) to give Example 8 (16.00 mg, 0.03 mmol, 61.0%, light brown gum, purified product). Yield: 16.00 mg (0.03 mmol), % yield: 61.0. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: 0.1% HCOOH in A:H2O:ACN (95:5), B:ACN: Flow rate: 1.5ml / min. RT(min):1.24;Purity(max):98.78%;M+H:454.10.

[0222] Chemical synthesis of Example 9 [ka] Step 1: To a stirred solution of compound 16 (60.00 mg, 0.09 mmol, 1.00 eq.) and pyrrolidine (19.17 mg, 0.26 mmol, 3.00 eq.) in acetonitrile (1.20 ml, 20.00 V) was added potassium carbonate (35.39 mg, 0.26 mmol, 3.00 eq.) at RT. The suspension was heated to 70 °C and stirred for 20 h, and the reaction was monitored by TLC. After completion of the reaction, the solvent was removed, and the residue was dissolved in water and extracted with DCM. The organic layer was washed with water and dried over sodium sulfate. The solvent was then evaporated under reduced pressure, and the crude product was purified by flash chromatography (60% EtOAc in petroleum ether) to give compound 19 (60.00 mg; 0.09 mmol; 99.8%; gum; purified product). Yield: 60.00 mg (0.09 mmol), % yield: 99.8. Analysis data: LCMS-Column: Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase: A: 0.1% HCOOH in H2O:ACN (95:5), B: CAN; Flow rate: 1.5ml / min. RT(min):1.99;Purity(max):96.78%;M+H:682.00.

[0223] Step 2: To a stirred solution of compound 19 (60.00 mg; 0.08 mmol; 1.00 eq.) in dichloromethane (3.00 ml; 50.00 V) was added TFA (0.15 ml; 1.93 mmol; 2.50 V) at 0 °C. The resulting solution was warmed to RT and stirred for 3 h. The reaction was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography (10% methanol in dichloromethane) to give Example 9 (20.00 mg, 0.05 mmol, 54.3%, off-white gum, purified product). Yield: 20.00 mg (0.05 mmol), yield: 54.3. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase: A: 0.1% HCOOH in H2O:ACN (95:5), B:ACN; Flow rate: 1.5ml / min. RT(min):1.20;Purity(max):99.56%;M+H:440.10. HPLC-Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase A: 0.1% TFA in MilliQ water; Mobile phase B: acetonitrile; Flow rate: 1.0ml / min. RT(min):5.76;Purity(max):99.55%;M+H:456.10;Purity(220 nm):99.42%

[0224] Chemical synthesis of Example 10 [ka] Step 1: To a stirred solution of compound 16 (50.0 mg, 0.07 mmol, 1.00 eq.) and piperazine-1-carboxylic acid tert-butyl ester (34.0 mg, 0.18 mmol, 2.50 eq.) in acetonitrile (1.50 ml, 30.00 V) was added potassium carbonate (25.23 mg, 0.18 mmol, 2.50 eq.) at RT. The suspension was heated to 70 °C and stirred for 20 h. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was removed, and the residue was redissolved in water and then extracted with dichloromethane. The organic layer was washed with water and then dried over anhydrous sodium sulfate. The solvent was concentrated under reduced pressure, and the crude product was purified by flash column chromatography (60% EtOAc in petroleum ether) to give compound 20 (45 mg, 0.06 mmol, 78.5%, gum, purified product). Yield: 45.00 mg (0.06 mmol), % yield: 78.5. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6mm, 5µm), +ve mode; 0.1% HCOOH in water, B: ACN; Flow rate: 1.5ml / min. RT(min):2.07;Purity(max):99.44%;M+H:797.00

[0225] Step 2: To a solution of compound 20 (3, 45.00 mg; 0.06 mmol; 1.00 eq.) in dichloromethane (2.25 ml; 50.00 V) was added TFA (0.15 ml; 1.93 mmol; 3.33 V) at 0 °C. The resulting solution was warmed to RT and stirred for 3 h. The reaction was monitored by LCMS. After completion, the solvent was removed under reduced pressure below 35 °C, and the crude product was purified by preparative HPLC. The product fractions were lyophilized to give Example 10 as the formate salt (25.00 mg, 0.05 mmol, 88.1%, off-white gum, purified product). Yield: 25.00 mg (0.05 mmol), % yield: 88.1. Analysis data: LCMS-Column: Atlantis C18 (50x4.6 mm, 5 μm); Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min. RT(min):9.46;Purity(max):98.13%;M+H:455.30. HPLC column: Atlantis dC18 (50x4.6 mm, 5 μm), +ve mode; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. Flow rate: 0.8 ml / min. RT(min):8.47;Purity(max):99.05%;Purity(220nm):98.46%. 1 H NMR (400 MHz, MeOD): □□8.56 (d, J = 8.40 Hz, 1H), 8.44 (s, 1H), 7.78 (d, J = 0.52 Hz, 1H), 7.69-7.65 (m, 1H), 7.53-7.49 (m, 1H), 4.91 (m, 4H), 3.67-3.61 (m, 2H), 3.18-3.15 (m, 4H), 2.50 (m, 4H), 2.15-2.06 (m, 2H), 1.32 (m, 6H), 1.28-1.25 (m, 5H), 1.14-1.11 (m, 2H).

[0226] Chemical synthesis of Example 11 [ka] Step 1: To a stirred solution of compound 16 (145.00 mg, 0.14 mmol, 1.00 eq.) and (2-piperazin-1-yl-ethyl)-carbamic acid tert-butyl ester (136.40 mg, 0.57 mmol, 4.00 eq.) in acetonitrile (4.35 ml, 30.00 V) was added potassium carbonate (60.40 mg, 0.43 mmol, 3.00 eq.) at RT. The suspension was heated to 80 °C and stirred for 16 h. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography in methanol and DCM. The desired product was eluted with 4-8% methanol and DCM. The fractions were concentrated to give compound 21 (105.00 mg; 0.12 mmol; 84.4%; off-white powder; purified product). Yield: 105.00 mg (0.12 mmol), % yield: 84.4 Analysis data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: A: 0.1% HCOOH in H2O B: ACN; Flow rate: 1.5ml / min RT(min):2.04;Purity:96.44%;M+H:840.10

[0227] Step 2: To a solution of compound 21 (100.00 mg; 0.11 mmol; 1.00 eq.) in DCM (5.00 ml; 50.00 V) was added TFA (0.36 ml; 4.59 mmol; 40.00 eq.) at 0 °C. The resulting solution was warmed to RT and stirred for 3 h. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure, and the crude reaction mixture was purified by preparative HPLC using 0.1% HCOOH in HO and ACN. The product fractions were lyophilized to give Example 11 as the formate salt (49.00 mg, 0.09 mmol, 77.9%, brown gum, purified product). Yield: 49 mg (0.09 mmol), % yield: 77.9 Analysis data: LCMS: Color: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA in H2O, B: ACN; Flow rate: 1.5 ml / min. RT (min): 0.188; Purity: 99.42%; M+H: 498.30 HPLC: Color: ATLANTIS dC18 (50 x 4.6 mm) 5 μm; Solvent A: Water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gravity: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B; RT (min): 4.09; Purity (max): 99.14%; Purity (220 nm): 99.53%. 1 HNMR (400 MHz, DMSO-d6): δ 8.44 (d, J = 8.40 Hz, 1H), 8.16 (s, 1H), 7.51-7.67 (m, 5H), 7.34 (t, J = 7.60 Hz, 1H), 4.75 (s, 4H), 3.52 (t, J = 6.80 Hz, 3H), 3.38 (s, 2H), 3.21 (s, 2H), (s, 3H), 2.66 (s, 2H), 2.51 (d, J = 2.00 Hz, 3H), 2.46 (d, J = 36.00 Hz, 3H), 1.13-1.20 (m, 13H), (s, H).

[0228] Chemical synthesis of Example 12

change

[0229] Step 2: To a solution of compound 22 (45.00 mg; 0.06 mmol; 1.00 eq.) in DCM (2.25 ml; 50.00 V) was added TFA (0.18 ml; 2.35 mmol; 40.00 eq.) at 0 °C. The resulting solution was warmed to room temperature and stirred for 3 hours. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC using 0.1% HCOOH in HO and ACN. The product fractions were lyophilized to give Example 12 as the formate salt (19.00 mg, 0.03 mmol, 56.5%, off-white gum, purified product). Yield: 19 mg (0.03 mmol), % yield: 56.5 Analysis data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: A: 0.1% HCOOH in H2O B: ACN; Flow rate: 1.5ml / min RT(min):0.98;Purity:97.32%;M+H:499.10 HPLC: Column: ATLANTIS dC18 (50x4.6mm) 5µm; Solvent A: Water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2ml / min; Gradient: 0min: 5% B, 8min: 100% B, 8.1min: 100% B, 8.5min: 5% B, 10min: 5% B. RT (min): 9.14; Purity (max): 95.04%; Purity (220nm): 92.88%. 1 HNMR (400 MHz, DMSO-d6): δ 8.54 (d, J = 0.80 Hz, 2H), 7.76 (d, J = 0.80 Hz, 1H), 7.65-7.74 (m, 1H), 7.45-7.49 (m, 1H), 3.77 (t, J = 11.20 Hz, 2H), 3.65 (t, J = 14.00 Hz, 3H), 2.81-2.89 (m, 6H), 2.69 (s, 4H), 2.36 (s, 2H), 2.00 (s, 3H), 1.15-1.33 (m, 14H), (s, H), (s, H), (s, H),

[0230] Chemical synthesis of Example 13 [ka] Step 1: To a stirred solution of compound 16 (180.00 mg; 0.25 mmol; 1.00 eq.) and sodium azide (20.00 mg; 0.30 mmol; 1.20 eq.) in DMF (3.60 ml; 20.00 V). The suspension was stirred at RT for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in water and extracted with DCM (8 mL). The organic layer was washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated to give crude compound 23 (150.00 mg; 0.15 mmol; 57.7%; off-white powder; crude). Yield: 150 mg (0.15 mmol), % yield: 57.7 Analysis data: LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA in H2O; B: ACN; Flow rate: 1.5 ml / min RT(min):3.24;Purity:63.86%;M+H:654.30.

[0231] Step 2: To a solution of compound 23 (70.00 mg; 0.07 mmol; 1.00 eq.) and prop-2-yn-1-ol (3.91 mg; 0.07 mmol; 1.00 eq.) in THF (1.40 ml; 20.00 V), copper(I) iodide (7.90 mg; 0.04 mmol; 0.60 eq.) and ethyldiisopropylamine (0.01 ml; 0.07 mmol; 1.00 eq.) were added. The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with ethyl acetate and washed with water and brine solution. The organic layer was dried over sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by column chromatography using methanol / DCM as the eluent, and the desired product was eluted with 8% methanol in DCM. The desired fractions were evaporated to give compound 24 (40.00 mg; 0.05 mmol; 70.6%; white solid; purified product). Yield: 40 mg (0.05 mmol), % yield: 70.6 Analysis data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: A: 0.1% HCOOH in H2O B: ACN; Flow rate: 1.5ml / min. RT(min):2.23;Purity:85.71%;M+H:710.00

[0232] Step 3: To a stirred solution of compound 24 (40.00 mg; 0.05 mmol; 1.00 eq.) in DCM (2.00 ml; 50.00 V) was added TFA (0.30 ml; 3.87 mmol; 7.50 V) at 0° C. The resulting solution was warmed to room temperature and stirred for 3 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography using methanol / DCM as the eluent, and the desired product was eluted with 15% methanol in DCM. The desired fractions were evaporated to give Example 13 (18.00 mg; 0.04 mmol; 76.5%; pale brown gum; purified product). Yield: 18 mg (0.04 mmol), % yield: 76.5. Analysis data: LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA in H2O B: ACN; Flow rate: 1.5ml / min RT(min):2.10;Purity:97.88%;M+H: 468.20 HPLC: Column: Phenomenex Gemini C18 (150*4.6) mm, 3.0 μm; Mobile phase A: 10 mM ammonium acetate in milli-q water; Mobile phase B: acetonitrile; Flow rate: 1.0 ml / min. RT(min):8.10;Purity(max):95.26%;Purity(220nm):90.72%

[0233] Chemical synthesis of Example 14 [ka] Step 1: To a solution of compound 23 (90.00 mg; 0.13 mmol; 1.00 eq.) and tert-butyl N-(prop-2-yn-1-yl)carbamate (20.99 mg; 0.13 mmol; 1.00 eq.) in THF (1.80 ml; 20.00 V), copper(I) iodide (15.30 mg; 0.08 mmol; 0.60 eq.) and ethyldiisopropylamine (0.05 ml; 0.27 mmol; 2.00 eq.) were added. The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was diluted with ethyl acetate and washed with water and brine solution. The organic layer was dried over sodium sulfate, filtered, and concentrated to obtain the crude compound. The crude product was purified by column chromatography using methanol / DCM as the eluent, and the desired product was eluted with 8% methanol in DCM. The desired fractions were evaporated to give Compound 25 (50.00 mg; 0.06 mmol; 45.2%; white solid; purified product). Yield: 50 mg (0.06 mmol), % yield: 45.2. Analysis data: LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase A: 0.1% HCOOH in water; B: 0.1% HCOOH in ACN; Flow rate: 0.8ml / min. RT(min):2.51;Purity:96.95%;M+H:809.00

[0234] Step 2: To a stirred solution of compound 25 (50.00 mg; 0.07 mmol; 1.00 eq.) in DCM (3.00 ml; 60.00 V) was added TFA (0.30 ml; 3.87 mmol; 6.00 V) at 0 °C. The resulting solution was warmed to RT and stirred for 3 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure to obtain the crude product. This crude product was purified by column chromatography using methanol / DCM as the eluent, and the desired product was eluted with 15% methanol in DCM. The desired fractions were evaporated to give Example 14 (15.00 mg; 0.03 mmol; 46.2%; off-white gum; purified product). Yield: 15 mg (0.03 mmol), % yield: 46.2 Analysis data: LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA in H2O:ACN (95:5); Mobile phase B: 0.1% TFA in ACN; Flow rate: 1.5 ml / min. RT(min):1.98;Purity:99.94%;M+H:467.20 HPLC: Column: ATLANTIS dC18 (50 x 4.6 mm) 5 mm; Mobile phase A: 0.1% TFA in MilliQ; Mobile phase B: acetonitrile; Flow rate: 1.0 ml / min. RT(min):5.42;Purity(max):99.86%;Purity(220nm):99.30%.

[0235] Chemical synthesis of Example 15 [ka] Step 1: To a stirred solution of Example 6 (5, 50.00 mg, 0.13 mmol, 1.00 eq.) in acetone (2.00 ml, 40.00 V) was added sulfuric acid and chromium trioxide (0.10 ml) at 0 °C. The resulting reaction mixture was stirred at RT for 1 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting crude was washed with diethyl ether (10 mL) to give compound 26 (45.00 mg; 0.10 mmol; 81.1%; blue gum; crude product). The crude was used directly for the next step. Yield: 45.00 mg (0.10 mmol), % yield: 81.1. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6 mm) 3.5 m; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 1.5 mL / min. RT(min):1.47;Purity(max):92.18%;M+H:401.10.

[0236] Step 2: To a stirred solution of compound 26 (40.00 mg; 0.09 mmol; 1.00 eq.) in DMF (0.40 ml; 10.00 V) was added [dimethylamino-([1,2,3]triazolo[4,5-B]pyridin-3-yloxy)methylene]dimethylammonium; hexafluorophosphate (67.06 mg; 0.17 mmol; 2.00 eq.). Ethyldiisopropylamine (0.05 ml; 0.26 mmol; 3.00 eq.) and 1-methylpiperazine (0.02 ml; 0.17 mmol; 2.00 eq.) were then added at 0° under a nitrogen atmosphere. The reaction mixture was then stirred at RT for an additional 2 hours. The progress of the reaction was monitored by LCMS. Upon completion, the reaction mixture was lyophilized, and the crude product was purified by preparative HPLC. The product fractions were lyophilized to give Example 15 as the formate salt (13.00 mg, 0.02 mmol, 26.6%, brown gum, purified product). Yield: 13.00 mg (0.02 mmol), % yield: 26.6. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 0.8 mL / min. RT(min):8.48;Purity(max):94.47%;M+H:483.20. HPLC-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% TFA in HO:ACN (95:5), Solvent B: ACN, Flow rate: 0.8 mL / min. RT(min):5.21;Purity(max):94.95%;Purity(220nm):92.87%. 1H-NMR (400 MHz, CD3OD-d6): δ 8.83 (d, J = 8.00 Hz, 1H), 7.72 (d, J = 8.40 Hz, 1H), 7.58 (t, J = 7.60 Hz, 1H), 7.41 (t, J = 7.20 Hz, 1H), 4.88 (s, 4H), 3.65 (t, J = 6.80 Hz, 2H), 3.61 (s, 2H), 3.50 (d, J = 3.20 Hz, 2H), 3.15 (d, J = 4.00 Hz, 2H), 2.34 (s, 6H), 1.91 (t, J = 6.80 Hz, 2H), 1.58 (q, J = 6.00 Hz, 2H), 1.27 (q, J = 7.20 Hz, 11H).

[0237] Chemical synthesis of Example 16 [ka] Step 1: (4-Methylpiperazin-1-yl)acetic acid (10,000 mg; 1.00 eq.) was dissolved in DMF (1,000 ml) and cooled to 0 ° C. Then, 4-methylmorpholine (17,374 μl; 2.50 eq.), [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium; hexafluorophosphate (26,439 mg; 1.10 eq.), and finally compound 3 (23,222 mg; 1.00 eq.) were added. The reaction mixture was warmed to RT and stirred for 1 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give Example 16 as the trifluoroacetate salt (31.00 mg, 0.050 mmol, 79%) as a clear solid. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):0.98;Purity(max):98.3%;M+H:498.6.

[0238] Chemical synthesis of Example 17 [ka] Step 1: R848 (20,00 mg; 0.062 mmol; 1.0 eq.), 2-(4-methylpiperazin-1-yl)acetic acid (29,588 mg; 0.187 mmol; 3.0 eq.), and 4-(pyrrolidin-1-yl)pyridine (13,860 mg; 0.094 mmol; 1.5 eq.) were combined with dichloromethane (2,000 ml) to give a white suspension. N,N'-Diisopropylcarbodiimide (0.029 ml; 0.187 mmol; 3.0 eq.) was added. The reaction mixture was heated at 50°C overnight. Upon completion, the solution was directly injected onto a preparative HPLC column eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give Example 17 as the trifluoroacetate salt (8.5 mg; 22.5%, clear oil, purified product). Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):0.97;Purity(max):94.0%;M+H=455

[0239] Chemical synthesis of Example 18 [ka] Step 1: To a solution of 4-chloro-3-nitroquinoline (4.50 g; 21.14 mmol; 1.00 eq.) dissolved in DCM (45.00 ml; 10.00 V) was added triethylamine (9.00 ml; 63.42 mmol; 3.00 eq.) at RT, followed by 1-amino-2-methylpropan-2-ol (3.81 g; 42.28 mmol; 2.00 eq.). The reaction mixture was refluxed for 3 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated in vacuo, water was added to form a solid, and the solid was filtered under vacuum to give compound 27 (5.30 g; 19.93 mmol; 94.3%; yellow solid; purified product). Yield: 5.30 g (19.93 mmol), % yield: 94.3. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 0.8 mL / min. RT(min):0.436;Purity(max):98.27%;M+H:260.20.

[0240] Step 2: To a solution of compound 27 (5.30 g, 20.28 mmol, 1.00 eq.) in a solvent of water (4.58 ml, 0.86 V) and methanol (50.00 ml, 9.43 V), nickel(II) chloride hexahydrate (0.19 g, 0.80 mmol, 0.04 eq.) was added, followed by sodium borohydride (1.57 g, 40.67 mmol, 2.00 eq.) at 0 °C. The reaction mixture was stirred at RT for 2 h. The reaction was monitored by TLC. After completion, the black solution was filtered through Celite, and the filtrate was concentrated to obtain the crude product. The crude product was then purified by flash column chromatography (3% MeOH in DCM) to obtain compound 28 (4.20 g, 16.55 mmol, 81.6%, orange powder, purified product). Yield: 4.20 g (16.55 mmol), % yield: 81.6. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% HCOOH in HO: ACN (95:5); Solvent B: ACN; Flow rate: 0.8 mL / min. RT(min):0.37;Purity(max):91.11%;M+H:232.20.

[0241] Step 3: To a solution of compound 28 (4.20 g; 18.16 mmol; 1.00 eq.) in DMF (42.00 ml; 10.00 V) was added 2-{[(tert-butoxy)carbonyl](ethyl)amino}acetic acid (5.59 g; 27.24 mmol; 1.50 eq.), [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium, hexafluorophosphate (20.92 g; 54.48 mmol; 3.00 eq.), triethylamine (7.14 ml; 54.48 mmol; 3.00 eq.), and 4-dimethylaminopyridine (0.22 g; 1.82 mmol; 0.10 eq.). The reaction mixture was stirred at RT for 3 hours. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under vacuum to obtain a residue. The residue was diluted with DCM and washed with water. The organic layer was collected and concentrated under vacuum to obtain an intermediate, which was dissolved in ethanol (84.00 ml; 20.00 V) and sodium hydroxide (1.02 g; 2.54 mmol; 0.14 eq.) in water (2.10 ml; 0.50 V) was added at RT. The cyclization reaction was refluxed for 6 hours and the reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel chromatography using 7% MeOH in DCM to give compound 29 (4.90 g, 9.27 mmol, 51.0%, brown light solid, purified product). Yield: 4.90 g (9.27 mmol), % yield: 51.0. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: HO:ACN (95:5) 0.1% HCOOH; Solvent B: ACN; Flow rate: 0.8mL / min. RT(min):0.67;Purity(max):82.73%;M+H:399.20.

[0242] Step 4: To a stirred solution of compound 29 (0.80 g, 1.92 mmol, 1.00 eq.) in acetonitrile (24.00 ml, 30.00 V) under a nitrogen atmosphere, triethylamine (1.36 ml, 9.58 mmol, 5.00 eq.) was added, followed by [chloro(diphenyl)methyl]benzene (1.08 g, 3.83 mmol, 2.00 eq.). The resulting suspension was heated to 100° C. and stirred for 16 hours. The reaction was monitored by TLC. Upon completion, the solvent was removed to give the crude product. The crude product was purified by flash column chromatography on 60-120 silica gel with ethyl acetate and petroleum ether. The product was eluted with 40-100% ethyl acetate and petroleum ether, and the eluate was concentrated to give compound 30 (800.00 mg, 1.22 mmol, 63.6%, white solid, purified product). Yield: 800.00 mg (1.22 mmol), % yield: 63.6. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6mm) 3.5µm; Solvent A: HO:ACN (95:5) 0.1% HCOOH; Solvent B: ACN; Flow rate: 0.8mL / min. RT(min):1.14;Purity(max):93.25%;M+H: 656.20.

[0243] Step 5: To a stirred solution of NaH (60%) (0.05 g; 1.19 mmol; 1.50 eq.) in DMSO (5.60 ml; 10.00 V) under a nitrogen atmosphere at 0 °C, a solution of compound 30 (0.56 g; 0.80 mmol; 1.00 eq.) in DMSO (5.60 ml; 10.00 V) was added dropwise and stirred at RT for 30 min. 1,4-Dibromobutane (0.53 g; 2.39 mmol; 3.00 eq.) was then added at 0 °C and stirred at RT for 1 h under a nitrogen atmosphere. The reaction was monitored by TLC. Upon completion, the reaction mixture was quenched with ice water at 0 °C. The reaction mixture was diluted with ethyl acetate, and the organic layer was separated, dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography, eluting with 15% ethyl acetate in hexane. The desired fractions were evaporated to give Compound 31 (23.00 mg; 0.03 mmol; 3.6%; white gum; purified product). Yield: 23.00 mg (0.03 mmol), % yield: 3.6. Analysis data: HPLC-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% TFA in HO:ACN (95:5), Solvent B: ACN, Flow rate: 0.8 mL / min. RT(min):3.09;Purity(max):96.95%; M+H:790.30.

[0244] Step 6: To a stirred solution of compound 31 (80.00 mg; 0.08 mmol; 1.00 eq.) and 1-methylpiperazine (0.01 ml; 0.09 mmol; 1.10 eq.) in acetonitrile (2.40 ml; 30.00 V) at RT was added potassium carbonate (23.91 mg; 0.17 mmol; 2.00 eq.). The suspension was heated to 50° C. and stirred for 16 hours. The reaction was monitored by LCMS. Upon completion, the solvent was removed to give the crude product. The crude product was dissolved in water and extracted with DCM (8 mL). The organic layer was washed with water. The organic layer was dried over sodium sulfate, filtered, and concentrated to give compound 32 (55.00 mg; 0.06 mmol; 67.4%; off-white gum; purified product). Yield: 55.00 mg (0.06 mmol), % yield: 67.4. Analysis data: LCMS-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% HCOOH in HO:ACN (95:5); Solvent B: ACN; Flow rate: 0.8 mL / min. RT(min):1.70;Purity(max):84.18%;M+H:810.40.

[0245] Step 7: To a solution of compound 32 (55.00 mg; 0.04 mmol; 1.00 eq.) in DCM (2.75 ml; 50.00 V) was added TFA (0.13 ml; 1.70 mmol; 40.00 eq.) at 0 °C. The resulting solution was warmed to RT and stirred for 3 h. The reaction was monitored by LCMS. Upon completion, the solvent was removed under reduced pressure below 35 °C to give the crude product. The crude product was purified by preparative HPLC with TFA method, and the product fractions were lyophilized to give Example 18 as the trifluoroacetate salt (13.10 mg; 0.02 mmol; 51.6%; white gum; purified product). Analysis data: HPLC-Column: Atlantis dC18 (50x4.6 mm) 3.5 μm; Solvent A: 0.1% TFA in HO:ACN (95:5), Solvent B: ACN, Flow rate: 0.8 mL / min. RT(min):8.32;Purity(max):97.35%;M+H:468.30. 1 H-NMR (400 MHz, DMSO-d6): δ 9.23 (s, br, 2H), 8.99 (s, br, 2H), 8.61 (d, J = 8.00 Hz, 1H), 7.83 (d, J = 8.00 Hz, 1H), 7.75 (t, J = 15.20 Hz, 1H), 7.55 (t, J = 15.20 Hz, 1H), 4.68 (s, br, 6H), 3.19- 2.22 (m, 15H), 1.20-1.31 (m, 13H).

[0246] Chemical synthesis of Example 19 [ka] Step 1: R848 (18.00 mg; 0.057 mmol; 1.0 eq.) was added to toluene (2.00 ml) to give a white suspension. 1,4-Diazabicyclo[2.2.2]octane (0.017 ml; 0.170 mmol; 3.0 eq.) and phthaloyl dichloride (0.010 ml; 0.068 mmol; 1.2 eq.) were added. The reaction mixture was stirred at 110° C. for 2 hours and monitored by LCMS. Upon completion, the mixture was cooled, diluted with ethyl acetate, and washed with 1N HCl. The organic phase was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated in vacuo to give crude compound 33 (25.20 mg, 0.070 mmol, white gum; crude), which was used in the next reaction without further purification. Analysis data: HPLC-MS: Column: Chromolith HR C18 5,0μm; 50-4.6mm; Method information (Chromolith): A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow: 3,3ml / min|MS:100-2000amu positive|1%->100% B:0->2,0min|100%B:2,0->2,5min RT(min):1.50;Purity(max):30.81%;M+H=445

[0247] Step 2: Compound 33 (18.00 mg; 0.040 mmol; 1.0 eq.) as crude material obtained in Step 1, (1,3-dioxo-1,3-dihydro-isoindol-2-yl)acetic acid (24.93 mg; 0.121 mmol; 3.0 eq.), and 4-(pyrrolidin-1-yl)pyridine (9.00 mg; 0.061 mmol; 1.5 eq.) were combined with DCM (2.00 ml) to give a white suspension. N,N'-Diisopropylcarbodiimide (0.019 ml; 0.121 mmol; 3.0 eq.) and molecular sieves were added. The reaction mixture was heated to 50 °C overnight and monitored by LCMS. After completion, the reaction mixture was purified by preparative RP-HPLC eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give compound 34 (16.70 mg, 0.02 mmol, 61.0%, white powder, purified product). Yield: 16.70 mg (0.02 mmol), % yield: 61.0. Analysis data: HPLC-MS: Column: Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.725;Purity(max):93.5%;M+H=631.7.

[0248] Step 3: Compound 34 (16.700 mg; 0.026 mmol; 1.0 eq.) was combined with THF (2.00 ml) to give a white suspension. To this suspension was added aqueous hydrazine (35%) (0.057 ml, 0.624 mmol, 23.6 eq.), and the solution was stirred at RT for 4 hours and monitored by LCMS. Upon completion, the reaction mixture was directly injected into a preparative HPLC column eluted with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give Example 19 as a trifluoroacetate salt (3.70 mg, 0.01 mmol, 29.0%, white powder, purified product). Yield: 3.70 mg (0.01 mmol), % yield: 29.0. Analysis data: HPLC-MS: Column: Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):0.902;Purity(max):100%;M+H=371.9.

[0249] Chemical synthesis of Example 20 [ka] Glycolic acid (6.482 mg; 1.00 eq.) was dissolved in DMF (2.00 ml), 4-methylmorpholine (0.023 ml; 2.50 eq.) and [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium hexafluorophosphate (35.294 mg; 1.10 eq.) were added, and the mixture was stirred for 10 minutes. Finally, compound 3 (31.00 mg; 1.00 eq.) was added, and the mixture was stirred at room temperature overnight and monitored by LCMS. After overnight reaction, the starting material was not completely converted to the product, so a solution of glycolic acid (6.482 mg, 1.00 eq.), [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)methylene]dimethylammonium hexafluorophosphate (35,294 mg, 1.10 eq.), and 4-methylmorpholine (0.023 mL, 2.50 eq.) in DMF (0.500 mL) was added to the mixture, which was again stirred overnight and monitored by LCMS. Upon completion, the reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give Example 20 (17.10 mg, 0.041 mmol, 48.8%, off-white solid, purified product). Yield: 17.10 mg (0.041 mmol), % yield: 48.8. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.10;Purity(max):99.9%;M+H=416.1.

[0250] Chemical synthesis of Example 21 [ka] 2-Hydroxyisobutyric acid (8.675 mg; 1.00 eq.) was dissolved in DMF (2.000 ml), 4-methylmorpholine (0.018 ml; 2.00 eq.) and [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium; hexafluorophosphate (31.050 mg; 1.00 eq.) were added, and the mixture was stirred for 10 minutes. Finally, compound 3 (30.000 mg; 1.00 eq.) was added, and the mixture was stirred at room temperature overnight and monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined and lyophilized overnight to give Example 21 as the trifluoroacetate salt (29.000 mg, 0.052 mmol, 63.7%, white solid, purified product). Yield: 29.000 mg (0.052 mmol), % yield: 63.7. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.103;Purity(max):100%;M+H=444.0.

[0251] Chemical synthesis of Example 22 [ka] Malonic acid monomethyl ester (14.248 μL; 1.00 eq.) was dissolved in DMF (2,000 ml), 4-methylmorpholine (0.030 ml; 2.00 eq.) and [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium; hexafluorophosphate (51.750 mg; 1.00 eq.) were added, and the mixture was stirred for 10 minutes. Finally, compound 3 (50.000 mg; 1.00 eq.) was added, and the mixture was stirred at room temperature overnight and monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). Fractions containing the product were combined and lyophilized overnight to give Example 22 as the trifluoroacetate salt (52.300 mg, 0.084 mmol, 61.9%, clear glassy solid, purified product). Yield: 52.300 mg (0.084 mmol), % yield: 61.9. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.11;Purity(max):92.0%;M+H=458.0.

[0252] Chemical synthesis of Example 23 [ka] Methylthioacetic acid (8.203 μL; 1.00 eq.) was dissolved in DMF (2.000 ml). N-ethyldiisopropylamine (79.306 μL; 5.00 eq.) and [dimethylamino-([1,2,3]triazolo-[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium hexafluorophosphate (70.927 mg; 2.00 eq.) were added and stirred for 20 minutes. Subsequently, compound 3 (33.339 mg; 1.00 eq.) was added and the reaction mixture was stirred at room temperature for 2 hours and monitored by LCMS. After completion, the reaction mixture was purified on a Sunfire column by preparative HPLC eluting with water / acetonitrile (0.1% TFA). The product-containing fractions were combined, concentrated, and lyophilized to give Example 23 as the trifluoroacetate salt (16.500 mg, 0.028 mmol, 30.3%, beige solid, purified product). Yield: 16.500 mg (0.028 mmol), % yield: 30.3. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.177;Purity(max):95.9%;M+H=446.2.

[0253] Chemical synthesis of Example 24 [ka] Methanesulfonylacetic acid (12.058 mg; 0.087 mmol; 1.2 eq.) was dissolved in DMF (2.000 ml). 4-Methylmorpholine (15.994 μl; 0.145 mmol; 2.0 eq.) and [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium; hexafluorophosphate (33.188 mg; 0.087 mmol; 1.2 eq.) were added and stirred for 10 minutes, followed by the addition of compound 3 (26.000 mg; 0.073 mmol; 1.0 eq.). The reaction mixture was stirred overnight and monitored by LCMS. After completion, the reaction mixture was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). Fractions containing the product were collected and lyophilized overnight to give Example 24 as the trifluoroacetate salt (9.10 mg, 0.01 mmol, 20.5%, pale beige solid, purified product). Yield: 9.10 mg (0.01 mmol), % yield: 20.5. Analysis data: HPLC-MS: Column: Chromolith HR C18 5,0μm;50-4.6mm; Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.14;Purity(max):97.0%;M+H=478.1.

[0254] Chemical synthesis of Example 25 [ka] 2-Sulfoacetic acid (12.740 mg; 0.087 mmol; 1.2 eq.) was dissolved in DMF (2,000 ml). 4-Methylmorpholine (15.994 μL; 0.145 mmol; 2.0 eq.) and [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium; hexafluorophosphate (33.188 mg; 0.087 mmol; 1.2 eq.) were added and stirred for 10 minutes, followed by the addition of compound 3 (26.000 mg; 0.073 mmol; 1.0 eq.). The reaction mixture was stirred overnight at room temperature and monitored by LCMS. After completion, the reaction mixture was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). Fractions containing the product were collected and lyophilized overnight to give Example 25 as the trifluoroacetate salt (26.3 mg, 0.04 mmol, 60.3%, white powder, purified product). Yield: 26.3 mg (0.04 mmol), % yield: 60.3. Analysis data: HPLC-MS:Column:Chromolith HR C18 5,0μm;50-4.6mm;Method information (Chromolith):A:H2O+0,05%HCOOH|B:MeCN+0,04%HCOOH|T:40℃|Flow:3,3ml / min|MS:100-2000amu positive|1%->100%B:0->2,0min|100%B:2,0->2,5min RT(min):1.108;Purity(max):99%;M+H=480.1.

[0255] Chemical synthesis of Example 26 [ka] Step 1: To a solution of compound 16 (200.00 mg; 0.29 mmol; 1.00 eq.) and 1-[2-(methylsulfanyl)ethyl]piperazine (69.52 mg; 0.43 mmol; 1.50 eq.) in acetonitrile (4.000 ml), potassium carbonate (122.33 mg; 0.87 mmol; 3.00 eq.) was added and stirred at 60 °C overnight. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated, and the crude residue was purified by silica gel chromatography using 10% methanol:DCM to give compound 35 (152.73 mg, 0.19 mmol, 67.3%, purified product). Yield: 152.73 mg (0.19 mmol), % yield: 67.3. Analysis data: HPLC: Column: XBridge C8, 3.5 μm, 4.6 × 50 mm; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. RT(min):2.59;Purity(max):98.2%;M+H=771.40.

[0256] Step 2: To a solution of compound 35 (175.00 mg; 0.23 mmol; 1.00 eq.) in DCM (1.75 ml), trifluoroacetic acid (0.18 ml; 2.27 mmol; 10.00 eq.) was added dropwise and stirred at RT overnight. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a crude residue, which was purified by silica gel chromatography using 10% methanol and DCM to give Example 26 (21.4 mg; 0.04 mmol; 17.5%; purified product). Yield: 21.4 mg (0.04 mmol), % yield: 17.5. Analysis data: HPLC: Column: XBridge C8, 3.5 μm, 4.6 × 50 mm; Solvent A: water + 0.1% TFA; Solvent B: ACN + 0.1% TFA; Flow rate: 2 ml / min; Gradient: 0 min: 5% B, 8 min: 100% B, 8.1 min: 100% B, 8.5 min: 5% B, 10 min: 5% B. RT(min):1.96;Purity(max):97.4%;M+H=529.20. 1 H-NMR (400 MHz, DMSO-d6): δ 8.58 (d, J = 8.00 Hz, 1H), 7.81 (d, J = 7.60 Hz, 1H), 7.71 (t, J = 15.60 Hz, 1H), 7.54 (t, J = 15.60 Hz, 1H), 5.03 (s, br, 3H), 4.70 (s, br, 3H), 3.55-3.57 (m, 15H), 3.20 (s, br, 4H), 2.51-2.52 (m, 3H), 2.11 (s, 3H), 1.14-1.22 (m, 9H).

[0257] Chemical synthesis of Example 27 [ka] To a solution of Example 10 (100.00 mg, 0.22 mmol, 1.00 eq.) dissolved in acetonitrile (2.00 ml), 1-chloro-2-methyl-2-(methylsulfanyl)propane (91.50 mg, 0.66 mmol, 3.00 eq.) and potassium carbonate (46.53 mg, 0.33 mmol, 1.50 eq.) were added and stirred at 50 °C overnight. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with ethyl acetate and washed with water, and the organic layer was dried over sodium sulfate, filtered, and reduced under pressure to give a crude residue. The crude residue was purified by preparative HPLC using 0.1% TFA% in water and ACN to give Example 27 (5.80 mg; 0.01 mmol; 4.7%; purified product). Yield: 5.80 mg (0.01 mmol), % yield: 4.7. Analysis data: LCMS-Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: 0.1% HCOOH in A:H2O:ACN (95:5), B:ACN Flow rate: 1.5ml / min. RT(min):2.00;Purity(max):99.48%;M+H:557.40.

[0258] 6.2. Biological Evaluation of TLR7 Agonists 6.2.1 TLR7 / 8 reporter assay Materials and General Procedures HEK-Blu™ TLR7 and TLR8 cell lines were obtained from InvivoGen (San Diego, CA, USA). HEK-Blue cells were cultured in DMEM medium high glucose (Gibco-10569010) supplemented with 10% HI-FBS (Australia) (Gibco-10100-147) and 1% penicillin / streptomycin (Gibco-15140-122-100ml) according to the manufacturer's recommendations. In addition, a mixture of specific antibiotics was added to the cultures. For HEK-Blue™ hTLR7 cells, 100 μg / ml Zeocin (11006-33-0), 10 μg / ml Blasticidin (ant-bl-05), and 50 μg / ml Normocin (ANT-NR-1-500MG) were used; for HEK-Blue™ hTLR8 cells, 100 μg / ml Zeocin, 30 μg / ml Blasticidin, and 50 μg / ml Normocin (all from InvivoGen).

[0259] General procedure for in vitro agonist screening All compounds were prepared in 100% DMSO (Sigma-D2650). Compounds were serially diluted (3-fold) in 100% DMSO starting from 30 μM to create a 10-point dose-response curve. The final concentration of DMSO in the plate was 0.3%. Resiquimod and TL8-506 (InvivoGen-TLRl-tl8506) were used as controls. 0.3% DMSO and 10 μM resiquimod were used as the minimum and maximum values, respectively, and the % activation was calculated using the following formula:

[0260] %activation:{100-(maximum absorbance-absorbance) / maximum absorbance-minimum absorbance)}x100. For stimulation with TLR7 / 8 agonists, cells were resuspended in complete medium, and on day 1, 40 μl of the cell suspension (10,000 cells per well) was seeded into a 384-well assay plate (Thermo Scientific™ Nunc-16468) with compound pre-dispensed using an Echo Liquid Handler. The plate was spun at 100 g for 1 minute to pellet the cells and incubated at 37°C and 5% CO2 for 24 hours. To measure SEAP production after 24 hours of incubation, 10 μl of Quanti-Blue™ solution (InvivoGen rep-qbs2) prepared according to the manufacturer's instructions was added to the assay plate in the dark and incubated at 37°C and 5% CO2 for 30 minutes. After incubation, absorbance at 620 nm was measured using a Tecan spark (Spark control Magellan). EC 50 was determined using Graph Pad Prism Statistical software (Version 7.05), and each data point represents the absorbance value of a duplicate. Table 1 shows the EC<0.01 μM. 50 Compounds of the present disclosure having a value are described as "A" activity, those between 0.01 μm and 0.1 μm as "B" activity, those between 0.1 μm and 1 μm as "C" activity, those between 1 μm and 10 μm as "D" activity, and those above 10 μm as "E" activity. Table 1: In vitro screening of TLR7 / 8 agonists in HEKBlue cells [Table 1]

[0261] In general, most of our examples have much higher potency against TLR7 than the benchmarks.

[0262] 6.2.2 TNFα secretion from PBMCs Materials and General Procedures Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human volunteers and stored in liquid nitrogen. A vial of frozen PBMCs was thawed in a 37°C water bath for less than 2 minutes. The contents of the tube were aseptically transferred to prewarmed complete medium containing RPMI basal medium (Gibco-21875034) supplemented with 10% HI-FBS (Australia) (Gibco-10100-147) and 1% penicillin / streptomycin (Gibco-15140-122-100ml), followed by centrifugation. Cells were resuspended in complete medium (3ml) and counted using a hemocytometer.

[0263] General procedure for in vitro agonist screening All selected compounds (five benchmarks and 15 example molecules) were prepared in 100% DMSO (Sigma-D2650) (10 mM stock). Compounds were serially diluted 4-fold in 100% DMSO to an 8-point DRC in a U-bottom 96-well plate (Cellstar-650180). 3 μl of this serially diluted DRC was mixed with 97 μl of complete medium (1:33.33) to obtain an intermediate dilution (300 μM) of 3% DMSO. 11 μl of the intermediate dilution was added to the PBMC plate (1:10) so that the final DMSO concentration in the culture plate was 0.3% and the starting compound concentration was 30 μM. Resiquimod (30 μM) and DMSO (0.3%) were used as positive and negative controls, respectively.

[0264] Plates were incubated at 37°C with 5% CO for 18 hours. After incubation, plates were spun at 2000 rpm for 5 minutes, and the supernatant was gently aspirated and collected into a low-protein binding V-bottom 96-well plate (Thermo Fisher-249944). Supernatant plates were sealed, appropriately labeled, and stored at -80°C until cytokine analysis (<1 month). For the measurement of TNF-alpha, the MILLIPLEX MAP Human Cytokine / Chemokine Magnetic Bead Panel kit (HCYTOMAG-60K) was used according to the kit's recommendations.

[0265] EC 50 was determined using Graph Pad Prism Statistical software (Version 7.05), and each data point represents the absorbance value of a duplicate. Table 2 shows the EC<0.1 μM. 50 Compounds of the present disclosure having a value are described as "A" active, those between 0.1 μm and 1 μm as "B" active, those between 1 μm and 10 μm as "C" active, and those above 10 μm as "D" active. Table 2: In vitro screening of selected TLR7 / 8 agonists in PBMCs [Table 2]

[0266] 6.2.3 Expression of the activation marker CD86 in PBMCs Materials and General Procedures PBMCs were commercially procured from HemaCare Corporation and cultured in RPMI (Gibco-11875093) containing 10% HI-FBS. For stimulation, 50 μl of cell suspension containing 2×10^5 cells was seeded into a 96-well assay plate with a flat bottom and white lid (Greiner Bio one-655180). Agonists were screened using freshly cultured PBMCs. For flow cytometry staining, FITC anti-human CD14 (Bioscience, 11-0149-42), PE anti-human CD123 (Biolegend, 396704), BV421 anti-human CD11C (Biolegend, 301628), and APC anti-human CD86 (Biolegend, 374208) antibodies were used.

[0267] General Procedure for In Vitro Testing of Agonists All compounds were made at 2x higher concentrations to generate an intermediate stock (2 μM) using culture medium. Compounds were serially diluted in culture medium using 10-fold dilutions for a total of 4-point DRCs (2-0.002 μM). The intermediate stock (50 μl) and serially diluted stocks were added to the assay plate to achieve final concentrations of 1-0.001 μM. Unstimulated cells or cells stimulated with 0.02% DMSO were used as controls. Plates were incubated at 37°C, 5% CO2 for 21 hours.

[0268] The next day, cells were harvested using ice-cold FACS buffer (DPBS without cations containing 2% HI-FBS) and transferred to separate 96-well V-bottom plates (Greiner, 650201). Cells were washed once with FACS buffer by spinning at 1400 rpm for 2 minutes at 4°C. Subsequent washes with PBS were followed by live-dead staining of cells using Zombie Yellow stain (Biolegend, 423103, 1:100) diluted in PBS according to the manufacturer's protocol.

[0269] Cells were washed twice with FACS buffer and stained with anti-CD14 FITC, anti-CD11C BV421, anti-CD123 PE, and anti-CD86 APC antibodies (2 μl / test of each antibody) for 1 hour at 4°C in the dark. Following staining, cells were washed twice with FACS buffer at 1400 rpm for 2 minutes at 4°C. Cells suspended in FACS buffer (100 μl / well) were analyzed using an ACEA NovoCyte flow cytometer after setting up a compensation matrix using compensation beads other than live-dead staining using PBMCs stained with Zombie Yellow dye.

[0270] PBMCs were gated using FSC-H and SSC-H, then further gated for single cells using FSC-H and FSC-A. Singlets were gated for viability using live-dead staining captured in the Pacific Orange channel. Viable singlets were gated for CD14+ (monocyte), CD14-CD11C+ (cDC), and CD14-CD11C-CD123+ (pDC) populations. Each population was further assessed for CD86+ expression in the respective channel. Negative and positive gates were assigned using the respective FMO controls using NovoExpress Software. XY scatter plots were generated for each stimulator by plotting the concentration of CD86 staining versus median fluorescence intensity within the cDC (Figure 4A), pDC (Figure 4B), and monocyte (Figure 4C) populations using GraphPad Prism9 software.

Claims

1. A compound according to formula I, 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, In the formula, X is an oxygen atom, a C1-C5 alkyl (preferably CH 2 ) or NH; In the formula, R 2 and R 3 are each independently selected from the group consisting of hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably, R 2 and R 3 are each hydrogen; and wherein L 1 , L 2 , L 3 , and R 1 is as defined under (a), (b), or (c) below: (a) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or is selected from hydrogen, C1-C2 alkyl, and CH 2 C(CH 3 ) 2 selected from the group consisting of: R 1 is absent or is hydrogen, NH 2 , O.H., and S.C.H. 3 selected from the group consisting of: (b) L 1 is a C2-C6 alkyl; L 2 is C(O), NHC(O)CH 2 and NHC(O)C(CH 3 ) 2 selected from the group consisting of: L 3 is a six-membered heterocycle, OH, C(O)O, S, SO 2 , and SO 3 H; and R 1 is absent, methyl or hydrogen; (c) L 1 is C(O)CH 2 and L 2 is a six-membered heterocycle or NH 2 and L 3 is not present; and R 1 is methyl, hydrogen, or absent; and where L 3 If there is no L 2 is connected to R via a covalent bond 1 directly bonded to The compound or a pharmaceutically acceptable salt thereof.

2. R 2 and R 3 The compound of claim 1, wherein is hydrogen.

3. 3. The compound according to claim 1, wherein X is an oxygen atom.

4. 4. The compound of claim 3 having the structure according to formula II: 【Chemistry 2】 Formula II In the formula, L 1 , L 2 , L 3 , and R 1 is as defined under (d), (e), or (f) below: (d) L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle (preferably triazole or piperazine), or OH; L 3 is absent or is selected from hydrogen, C1-C2 alkyl, and CH 2 C(CH 3 ) 2 selected from the group consisting of: R 1 is absent or is hydrogen, NH 2 , O.H., and S.C.H. 3 selected from the group consisting of: (e) L 1 is a C2-C3 alkyl; L 2 is C(O), NHC(O)CH 2 and NHC(O)C(CH 3 ) 2 selected from the group consisting of: L 3 is a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO 2 , and SO 3 H; and R 1 is absent, methyl or hydrogen; (f) L 1 is C(O)CH 2 and L 2 is a 6-membered heterocycle (preferably piperazine) or NH 2 and L 3 is not present; and R 1 is methyl, hydrogen, or absent.

5. 5. The compound of claim 4, wherein: L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle or OH; L 3 is absent or is selected from hydrogen, C1-C2 alkyl, and CH 2 C(CH 3 ) 2 selected from the group consisting of: R 1 is absent or NH 2 , O.H., and S.C.H. 3 The compound is selected from the group consisting of:

6. 10. The compound of any one of the preceding claims, having a structure selected from the group consisting of the structures designated as Example 1 to Example 27 listed below. 【Transformation 3】

7. 10. The compound of claim 1, wherein: X is NH; L 1 is a C2-C6 alkyl; L 2 is a 5- or 6-membered heterocycle; L 3 is not present; and R 1 is methyl or hydrogen; and R 2 and R 3 are each hydrogen.

8. A compound according to any one of the preceding claims, (i) activates TLR7 more strongly than TLR8; and / or (ii) induce the production of IL-6, IL1-β, and TNF-α; and / or (iii) induces upregulation of CD40 and / or CD86 in peripheral blood mononuclear cells (PBMCs); The compound.

9. 10. A compound according to any one of the preceding claims, wherein when the compound contacts peripheral blood mononuclear cells (PBMCs), the compound induces TNFα secretion from the cells.

10. A compound according to any one of the preceding claims, which has an EC50 for TLR7 of less than 10 μM, preferably less than 0.01 μM, when tested preferably in the test system described in Example 6.2.

1.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10.

12. A compound according to any one of claims 1 to 10 for use as a medicament.

13. 11. A compound according to any one of claims 1 to 10 for use in the treatment of a disease, used in treatment in combination with the further compounds imiquimod and / or resiquimod (R848).

14. 11. A composition comprising a compound of any one of claims 1 to 10 for use in treating a condition selected from the group consisting of cancer, viral infection, non-cancerous skin lesions, pre-cancerous skin lesions, cancerous skin lesions, bladder cancer, and viral-mediated skin diseases, optionally formulated for enhanced penetration after topical administration, wherein the composition preferably initiates a local-specific inflammatory cytokine response while limiting undesired erythema and other inflammatory responses.

15. 15. The composition of claim 11 or 14, comprising 0.01% to 1% (wt / vol) of a compound according to any one of claims 1 to 10.

16. 16. The composition of claim 14 or 15 for use in the treatment of bladder cancer, formulated for intravesical administration.

17. 17. The composition of any one of claims 11, 14, 15, or 16, further comprising one additional component from groups i to xiii outlined below: i. oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1% wt / vol; or ii. isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, for a total concentration of 0.5% wt / vol, or; iii. oleic acid (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, for a total concentration of 0.5% wt / vol, or; iv. oleic acid (33%), isopropyl myristate (33%), and sodium lauryl sulfate (33%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, for a total concentration of 0.5% wt / vol, or; v. Isopropyl palmitate (50%) and sodium oleate (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, at a total concentration of 2% wt / vol, or vi. Sodium lauryl sulfate (25%) and linoleic acid (75%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 1.0% wt / vol, or vii. Palmitic acid (50%) and isopropyl laurate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol at a total concentration of 2.0% wt / vol, or viii. Oleic acid (50%) and linoleic acid (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, at a total concentration of 1.5% wt / vol; or ix. Linoleic acid (25%), oleic acid (25%), and isopropyl linoleic acid (50%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol at a total concentration of 0.5% wt / vol; or x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, for a total concentration of 2.0% wt / vol; or xi. A solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xii. Oleic acid (10%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

18. 19. A method for treating cancer, comprising administering to a subject suffering from cancer a therapeutically effective amount of a compound according to any one of claims 1 to 10, or a composition according to any one of claims 11 and 14 to 17.

19. 18. A method for the treatment of skin tumor lesions and virus-induced skin diseases, comprising topical administration of a composition according to any one of claims 11 and 14 to 17, wherein the composition is effective in reducing or eliminating the lesions or diseases while limiting adverse skin reactions, preferably selected from erythema and inflammation, and wherein the composition preferably reduces penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

20. 18. A method for the treatment of bladder tumors, comprising intravesical administration of a composition according to any one of claims 11 and 14 to 17, wherein the composition preferably enhances penetration and delivery of the composition to the bladder epithelium while limiting irritation, and wherein the composition preferably reduces tumor invasion into surrounding muscle tissue and metastasis to lymph nodes.

21. 21. The method of any one of claims 18 to 20, wherein the treatment further comprises systemic administration of at least one immune modulator selected from anti-PD1, anti-PD-L1, anti-CTLA-4 antibody, anti-CD137 antibody, agonistic CD40 antibody, CD134 (anti-OX40) agonist and PLX3397.

22. 21. The method of any one of claims 18 to 20, further comprising systemic administration of interferon gamma.

23. 21. The method of any one of claims 18 to 20, further comprising administration of local radiation with or without systemic anti-PD1 antibody.

24. 21. The method of any one of claims 18 to 20, further comprising the administration of photodynamic therapy.

25. A method of activating TLR7 and / or 8 in a biological sample, comprising contacting the biological sample with a compound according to any one of claims 1 to 10.

26. 11. A compound according to any one of claims 1 to 10 for use as a vaccine adjuvant or for use in the treatment of cancer in combination with an anti-cancer immunotherapeutic agent (preferably ipilimumab, nivolumab, or pembrolizumab).

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