Toll-like receptor 7 agonists as immunostimulators to elicit innate anti-tumor immunity

Abstract

The present invention relates to compounds that can specifically activate TLR7. The compounds of the present invention are useful as they can stimulate innate immunity. The compounds of the invention may be used to treat conditions including cancer, viral infections, and skin lesions. These compounds may optionally be formulated to enhance penetration following topical administration, which compositions preferably elicit topically specific inflammatory cytokine responses while limiting undesirable erythema and other inflammatory responses. The invention also relates to pharmaceutical compositions comprising a compound of the invention and preferably further compounds such as imiquimod and / or requinimod (R848). The invention also relates to a composition comprising the compounds of the invention and to compounds for use as medicaments. Other aspects, embodiments, advantages, and applications of the invention will become clear from further description herein.

Description

Technical Field

[0001] The present invention relates to compounds that can specifically activate 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 conditions including cancer, viral infections and skin lesions. These compounds can optionally be formulated to enhance penetration after topical application, and the composition preferably triggers a local specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory reactions. 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 a composition comprising the compounds of the present invention and a compound used as a medicament. Other aspects, embodiments, advantages and applications of the present invention will become clear from the further description herein. Background Art

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

[0003] The immune system possesses multiple "brake" mechanisms that negatively regulate the activation and function of T cells and other immune cells, thereby protecting the host's own healthy cells. Checkpoint inhibitors such as PD-1 / PD-L1 and CTLA-4 antagonists can selectively release these immune system "brakes," leveraging so-called "adaptive immunity" and generating durable anti-tumor responses. However, only a small percentage of cancer patients typically experience clinical benefit from checkpoint inhibitors.

[0004] The human immune system is also designed to protect the body from foreign entities such as viruses and bacteria through systematic coordination between a large number of cell types with highly selective immune functions, the so-called "innate immunity". Cell types including phagocytes such as dendritic cells (DCs), macrophages, γδT cells and natural killer (NK) cells act as a barrier against foreign entities. The mechanism behind this is to use the conserved molecular patterns and expressed Fc receptors of pathogens to recognize and trigger a rapid immune response. Because B cells and T cells from the adaptive immune system are able to initiate memory responses, the communication between the innate and adaptive immune systems mediated by DCs can trigger specific and long-lasting immune responses.

[0005] DCs are responsible for continuously sampling their environment through phagocytosis, receptor-mediated endocytosis, and micropinocytosis. With the help of pattern recognition receptors (PRRs), DCs are able to recognize a wide range of foreign entities. Once PRRs are engaged, DCs are activated and mature into stronger antigen-presenting cells (APCs) and upregulate the cell surface expression of co-stimulatory molecules such as CD40 and CD86 for optimal T cell priming and activation.

[0006] As a member of the PRR family, Tool-like receptors (TLRs) are well-known. Toll-like receptors (TLRs) currently comprise 10 gene families of receptors with different specificities, which are part of the cell pathogen pattern recognition system that has evolved to defend against a variety of infections (bacteria, viruses, fungi). The activation of TLRs can lead to cytokine responses, such as the release of interferon and the activation of specific immune cells. The functional expression of selected TLRs in tissues varies greatly. Some receptors are located on the cell surface, such as TLR4 (stimulated by E. coli lipopolysaccharide LPS) on epithelial cells, or TLR3, 7, 8 and 9 at the endosomal membrane in specific immune cells. The latter are all activated by nucleic acids, but recognize different types of nucleic acids. For example, TLR9 is activated by single-stranded DNA comprising CpG subsequences, TLR7 and TLR8 are activated by single-stranded RNA, and TLR3 is activated by double-stranded RNA.

[0007] TLRs have also been implicated in various autoimmune and inflammatory diseases, the most notable example being the role of TLR7 in the pathogenesis of systemic lupus erythematosus (Barrat and Coffman, Immunol Rev, 223:271-283, 2008). Additionally, TLR8 polymorphisms have been associated with rheumatoid arthritis (Enevold et al., J Rheumatol, 37:905-10, 2010).

[0008] Each TLR has a specific class of molecules that recognize different surface and intracellular components of microorganisms, ranging from bacterial membrane lipids to viral single-stranded or double-stranded RNA. TLRs expressed intracellularly are in endosomal compartments and include TLRs such as TLR3, TLR7, TLR8, TLR9, and TLR13. TLR7 and TLR8 are reported to be dimeric TLR receptors that contain a leucine-rich repeat (LRR) motif as an extracellular domain, a transmembrane domain, and a cytoplasmic domain that possesses the Toll / interleukin-1 (IL-1) receptor (TIR) signaling domain. TLR7 has been reported to be expressed in B cells and plasmacytoid dendritic cells (pDCs), while TLR8 can be expressed in monocytes and myeloid dendritic cells (mDCs).

[0009] When TLR7 / 8 is agonist-bound by degradation products such as single-stranded RNA (ssRNA)-derived guanosine, the dimerization interface undergoes rearrangements, including the protein-protein interface and the ligand-mediated interface. Conformational changes in the TLR7 / 8 extracellular domain lead to the activation configuration of the cytoplasmic TIR signaling domain. Subsequently, MyD88-dependent signaling is observed, followed by a robust interferon response and proinflammatory cytokine production in DCs. In addition, activated DCs 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] More recently, it has been discovered that the presentation of tumor-associated antigens (TAAs) affects the priming and activation of T cells, resulting in a robust, long-term anti-tumor immune response. Studies have shown that when antigens are bound to antibody-antigen complexes (so-called "immune complexes"), the uptake and presentation of antigens by APCs will be more efficient. Since activated DCs also upregulate the expression of major histocompatibility complexes (MHC), antigens internalized during contact with microorganisms will be digested and their peptides will be displayed on the cell membrane of DCs by MHC. Activated T cells can recognize and kill tumor cells containing MHC-bound neoantigens.

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

[0012] Although agonist stimulation via the TLR7 / 8 pathway can activate DCs and subsequently prime and activate T cells, synthetic small molecules such as imiquimod and resiquimod (R848) have only been approved for use as local therapeutic agents, for example, imiquimod is used intratumorally to treat basal cell carcinoma to avoid systemic inflammation. Such clinical treatments have largely limited the application of TLR7 / 8 agonist therapy to solid tumors.

[0013] When cancerous lesions progress, metastasis may occur, and in addition to surgery, chemotherapy, radiation therapy, and immunotherapy may be used. Resiquimod, a member of the imidazoquinoline family and structurally related to imiquimod, is an immune response modifier that acts as an agonist for Toll-like receptors 7 and 8. However, it differs significantly from imiquimod in that imiquimod signals exclusively through toll-like receptor 7 (TLR7). Imiquimod is FDA-approved for the treatment of various skin conditions. 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, leading to the efficient induction of antigen-specific CD8+ T-cell responses in animal models. Results from animal studies have demonstrated the ability of resiquimod to activate dendritic cells, including inducing localized 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. In a lymphoma model, systemic delivery of resiquimod plus radiation triggered a durable anti-tumor immune response (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 art uses of resiquimod include administration as a vaccine adjuvant to treat various diseases, including metastatic melanoma, but with inconsistent results. Resiquimod has been used as a vaccine adjuvant for the NYESO-1 protein vaccine to treat melanoma (Sabado RL Cancer Immunol Res. 2015).

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

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

[0016] Therefore, in a first aspect of the present invention, there is provided a compound according to formula I,

[0017]

[0018] or a pharmaceutically acceptable salt thereof,

[0019] wherein X is an oxygen atom, a C1-C5-alkyl group (preferably CH2) or NH;

[0020] where R 2 and R 3 Each is independently selected from hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably R 2 and R 3 are each hydrogen;

[0021] And where L 1 , L 2 , L 3 and R 1 is as defined below under (a) or (b) or (c):

[0022] (a)L 1 is a C2-C6 alkyl group;

[0023] L 2 is a 5- or 6-membered heterocyclic ring or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0024] R 1 Absent or selected from hydrogen, NH2, OH and SCH3;

[0025] (b)L 1 is a C2-C6 alkyl group;

[0026] L 2 selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2;

[0027] L 3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and

[0028] R 1 Absent or methyl or hydrogen;

[0029] (c)L 1 is C(O)CH2;

[0030] L 2 is a 6-membered heterocyclic ring or NH2;

[0031] L 3 does not exist; and

[0032] R 1is methyl or hydrogen or is absent;

[0033] And if L 3 If it does not exist, then L 2 Through covalent bond with R 1 Direct combination.

[0034] In a preferred embodiment of the first aspect, R 2 and R 3 For hydrogen.

[0035] Preferably, X in the compounds of the present invention is an oxygen atom.

[0036] In a further embodiment, the compounds of the present invention have a structure according to Formula II:

[0037]

[0038] Among them L 1 、L 2 、L 3 and R 1 as defined below under (d), (e) or (f)

[0039] (d)L 1 is a C2-C6 alkyl group;

[0040] L 2 is a 5- or 6-membered heterocycle (preferably, triazole or piperazine) or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0041] R 1 Absent or selected from hydrogen, NH2, OH and SCH3;

[0042] (e)L 1 is a C2-C3 alkyl group;

[0043] L 2 selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2;

[0044] L 3 is selected from a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO2 and SO3H; and

[0045] R 1 Absent or methyl or hydrogen;

[0046] (f)L 1 is C(O)CH2;

[0047] L 2is a 6-membered heterocycle (preferably piperazine) or NH2;

[0048] L 3 does not exist; and

[0049] R 1 is methyl or hydrogen or is absent.

[0050] In a preferred embodiment of the compound of formula II of the present invention

[0051] L 1 is a C2-C6 alkyl group;

[0052] L 2 is a 5- or 6-membered heterocyclic ring or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0053] R 1 Absent or selected from NH2, OH and SCH3.

[0054] In a particularly preferred embodiment of the compounds of the present invention, the compounds have a structure selected from the structures listed below designated as Examples 1 to 27:

[0055]

[0056] A more preferred compound of the present invention relates to a compound wherein

[0057] X is NH;

[0058] L 1 is a C2-C6 alkyl group;

[0059] L 2 is a 5- or 6-membered heterocyclic ring;

[0060] L 3 does not exist; and

[0061] R 1 is methyl or hydrogen; and

[0062] R 2 and R 3 Each is hydrogen.

[0063] Also preferred are compounds of the present invention wherein the compound

[0064] (i) activates TLR7 more strongly than TLR8; and / or

[0065] (ii) induces the production of IL-6, IL1-b and TNF-α; and / or

[0066] (iii) Inducing upregulation of CD40 and / or CD86 in peripheral blood mononuclear cells (PBMCs).

[0067] Preference is also given to compounds according to the invention, wherein, if the compound is contacted with peripheral blood mononuclear cells (PBMC), the compound induces the secretion of TNFα from said cells.

[0068] Also preferred are compounds according to the invention, wherein said compounds have an EC50 for TLR7 of less than 10 μM and preferably less than 0.01 μM when tested in the test system described in Example 6.2.1.

[0069] Yet another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention.

[0070] The present invention also relates to a pharmaceutical composition comprising a compound of the present invention and preferably an additional compound such as imiquimod and / or resiquimod (R848).

[0071] Yet another aspect of the present invention relates to a compound of the present invention for use as a medicament.

[0072] Yet another aspect of the present invention relates to a compound of the present invention for use in the treatment of a disease, wherein said compound is used in said treatment in combination with the additional compounds imiquimod and / or resiquimod (R848).

[0073] A preferred embodiment provides a compound of the present invention for use in treating a condition selected from the group consisting of cancer, viral infection, noncancerous skin lesions, precancerous skin lesions, cancerous skin lesions, bladder cancer, virally mediated skin diseases, the composition optionally being formulated to enhance penetration upon topical administration, the composition preferably eliciting a localized specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses.

[0074] In a preferred embodiment of the composition of the present invention, the compound of the present invention is contained therein in an amount between 0.01% and 1%.

[0075] Yet another aspect relates to a composition of the present invention for use in treating bladder cancer, wherein the composition is formulated for intravesical administration.

[0076] In a preferred embodiment of the composition of the present invention, the present invention further comprises an additional component selected from one of the following groups i to xiii:

[0077] i. a total concentration of 1% wt / vol oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0078] ii. a total concentration of 0.5% wt / vol isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0079] iii. oleic acid (50%) and sodium lauryl sulfate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0080] iv. a total concentration of 0.5% wt / vol 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, or

[0081] v. isopropyl palmitate (50%) and sodium oleate (50%) at a total concentration of 2% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0082] vi. sodium lauryl sulfate (25%) and linoleic acid (75%) at a total concentration of 1.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0083] vii. Palmitic acid (50%) and isopropyl laurate (50%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0084] viii. oleic acid (50%) and linoleic acid (50%) at a total concentration of 1.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0085] ix. linoleic acid (25%), oleic acid (25%), and isopropyl linoleate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0086] x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0087] xi. A solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, or

[0088] xii. Oleic acid (10%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, or

[0089] xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0090] Yet another 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.

[0091] Yet another aspect of the present invention provides a method for treating skin tumor lesions and viral-induced skin diseases, comprising topically administering a composition of the present invention, which is preferably effective in reducing or eliminating the lesions or diseases while limiting adverse skin reactions selected from erythema and inflammation, and which preferably reduces the penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

[0092] Yet another aspect of the present invention relates to a method for treating bladder tumors, comprising intravesical administration of a composition of the present invention, wherein the composition preferably enhances the penetration and delivery of the compound of the present invention into the bladder epithelium while limiting irritation, and the composition preferably reduces tumor infiltration into surrounding muscle tissue and metastasis to lymph nodes.

[0093] In a preferred embodiment of the method of the present invention, the treatment further comprises systemically administering to the subject at least one immunomodulator selected from anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-CD137 antibodies, agonist CD40 antibodies, CD134 (anti-OX40) agonists and PLX3397.

[0094] In a preferred embodiment of the method of the present invention, said treatment further comprises systemically administering interferon gamma to said subject.

[0095] In a preferred embodiment of the method of the present invention, the treatment further comprises administration of localized radiation with or without systemic anti-PD1 antibodies.

[0096] In a preferred embodiment of the method of the present invention, the treatment further comprises administering photodynamic therapy.

[0097] In a further aspect, the present invention provides a method of activating TLR 7 and / or 8 in a biological sample, comprising contacting the biological sample with a compound according to the present invention.

[0098] In yet another aspect, the present invention provides a compound of the present invention for use as a vaccine adjuvant or in combination with an anti-cancer immunotherapeutic agent (preferably ipilimumab, nivolumab or pembrolizumab) for treating cancer.

[0099] Other aspects, advantages, applications and uses of the polypeptides and compositions will become clear from the further disclosure herein. Several references are cited throughout this specification. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 : Molecular binding between R848 and monkey TLR7 (PDB 5GMH), where the unburied hydrogen groups of R848 are highlighted with black arrows. Top right: Chemical structure of R848.

[0101] Figure 2 : Chemical structures of recently reported TLR7 / 8 agonists.

[0102] Figure 3 : A library of highly potent and selective TLR7 agonists.

[0103] Figure 4: In vitro testing of TLR7 agonists. The effect of agonist concentration on cDC population ( Figure 4A ), pDC population ( Figure 4B ) and monocytes ( Figure 4C ) to generate an XY scatter plot for each agonist / stimulator. DETAILED DESCRIPTION

[0104] Skin cancer is usually located in superficial parts. Melanoma in situ is still confined to the epidermis and usually grows slowly. Such lesions may take several years to develop into invasive melanoma. Invasive melanoma usually has an epidermal component, but as melanoma cells invade the dermis, they are no longer confined to the epidermis. Metastatic cancer is usually located deep. WO 2017 / 004421 (incorporated herein by reference) shows data showing that resiquimod is effective in a variety of in vivo transgenic mouse models of pigmented lesions and in a syngeneic melanoma mouse model with a complete immune system. These data show that resiquimod is effective in inhibiting or killing melanocyte tumor cells in nevus, atypical intraepidermal melanocyte proliferation, and melanoma. In addition, the number of metastatic melanomas in the lymph nodes of mice treated with resiquimod was also significantly reduced, indicating that resiquimod can inhibit the metastasis of melanoma cells. Furthermore, WO 2017 / 004421 demonstrates that the combination of resiquimod and systemic anti-PD-1 therapy significantly enhanced the efficacy of anti-PD-1 therapy in a melanoma model. Because resiquimod's anti-tumor effects are mediated by activating CD8 T cells and inhibiting myeloid-derived suppressor cells, its effects are not limited to melanocytic tumors. It is also effective in treating other epithelial cancers and cutaneous T-cell lymphomas.

[0105] However, given the wide variety of cancers and tumors, more compounds are still needed to treat cancer and other diseases.

[0106] Figure 1 Shown is a co-crystal structure analysis of TLR7 complexed with agonist ligands such as R848 (Shimizu et al., Cell Reports 2018, 25, 3371). Unexpectedly, the present inventors have discovered the possibility of modification at the unburied hydrogen group of R848. The agonist binding site in the extracellular domain of TLR7 is located in the cell lysosome. Therefore, the present inventors have found that any modification with a protonable group at low pH should have the potential to extend the residence time of the agonist ligand in the lysosome. The present inventors infer that this, in turn, will increase the binding of such agonist ligands to TLR7 proteins.

[0107] Therefore, the present inventors have synthesized a large number of different TLR7 agonists based on imidazoquinolines. According to further experiments listed in the Examples section below, which are consistent with the inventors' assertions, it was confirmed that this group of novel agonists performs very well in cell-based assays.

[0108] Unexpectedly, compared with R484 and some recently reported TLR7 / 8 agonists ( Figure 2 ) compared to these novel TLR7 agonists ( Figure 3 ) not only showed good activity in cell-based TLR7 assays but also exhibited high selectivity between TLR7 and TLR8. Even more surprisingly, these novel, highly selective TLR7 agonists triggered high production of cytokines and chemokines (IL-6, IL1-b, and TNF-a) and induced strong upregulation of CD40 and CD86 in cell-based monocyte assays (Figure 4).

[0109] Therefore, the present invention provides in a first aspect a compound according to formula I,

[0110]

[0111] or a pharmaceutically acceptable salt thereof,

[0112] wherein X is an oxygen atom, a C1-C5-alkyl group (preferably CH2) or NH;

[0113] where R 2 and R 3 Each is independently selected from hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably R 2 and R 3 are each hydrogen;

[0114] And where L1 , L 2 , L 3 and R 1 is as defined below under (a) or (b) or (c):

[0115] (a)L 1 is a C2-C6 alkyl group;

[0116] L 2 is a 5- or 6-membered heterocycle (preferably, triazole or piperazine) or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0117] R 1 Absent or selected from hydrogen, NH2, OH and SCH3;

[0118] (b)L 1 is a C2-C6 alkyl group;

[0119] L 2 selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2;

[0120] L 3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and

[0121] R 1 Absent or methyl or hydrogen;

[0122] (c)L 1 is C(O)CH2;

[0123] L 2 is a 6-membered heterocyclic ring or NH2;

[0124] L 3 does not exist; and

[0125] R 1 is methyl or hydrogen or is absent;

[0126] And if L 3 If it does not exist, then L 2 Through covalent bond with R 1 Direct combination.

[0127] In certain embodiments, compounds of the invention stimulate an immune response to treat cancer.

[0128] Before describing aspects and embodiments of the present invention in greater detail, certain definitions applicable to the invention disclosed throughout the specification herein, including the claims, are provided below.

[0129] definition

[0130] Unless otherwise noted or defined, all terms used have their ordinary meanings in the art that are clear to those skilled in the art. For example, reference is made to standard manuals such as 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 ed.) 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 Publishing / Churchill Livingstone, New York, 2005); and the general background art cited herein.

[0131] Unless otherwise stated, all methods, steps, techniques and operations not explicitly described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person.

[0132] Unless otherwise indicated, the term "at least" preceding a series of elements should be 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 the present invention.

[0133] Wherever used herein, the term "and / or" includes the meanings of "and," "or," and "all or any other combinations of the elements linked by the term."

[0134] 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.

[0135] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprise" may be replaced by the term "contains" or "includes," or sometimes, when used herein, by the term "having."

[0136] "Agonist" refers to a compound that can be combined with a receptor (e.g., TLR) to induce cellular activity. An agonist can be a ligand that binds directly to a receptor. Alternatively, an agonist can be indirectly combined with a receptor, for example, by (a) forming a complex with another molecule that binds directly to the receptor, or (b) otherwise causing the modification of another compound so that the other compound binds directly to the receptor. An agonist can be referred to as an agonist of a specific TLR (e.g., a TLR6 agonist) or an agonist of a specific combination of TLRs (e.g., a TLR7 / 8 agonist--an agonist of both TLR7 and TLR8). "Improvement" refers to any reduction in the degree, severity, frequency, and / or likelihood of symptoms or clinical signs characteristic of a particular condition.

[0137] "Cell-mediated immune activity" refers to a biological activity that is considered part of a cell-mediated immune response, such as, for example, an increase in the production of at least one TH1 cytokine.

[0138] "Immune cell" refers to a cell of the immune system, ie, a cell that is directly or indirectly involved in the generation or maintenance of an immune response, whether innate, acquired, humoral, or cell-mediated.

[0139] "Signs" or "clinical signs" refer to objective physical findings associated with a particular condition that can be detected by someone other than the patient.

[0140] "Symptom" refers to any subjective indication of a disease or patient condition.

[0141] "Treat," "treat," or variations thereof, means to alleviate, limit the progression, ameliorate, or eliminate to any extent the symptoms or signs associated with a condition.

[0142] As used herein, "permeation enhancer" and "permeation enhancement" refer to agents that increase the permeability of tissue to a drug and an increase in the permeability of tissue to a drug, respectively, i.e., to increase the rate and extent of drug penetration through tissues such as skin or tumors. The permeation enhancement achieved by using such enhancers can be observed, for example, by measuring the diffusion rate of the drug through animal or human skin or tumor tissue using a diffusion cell apparatus or in situ measurement methods. Merritt et al., Diffusion Apparatus for Skin Penetration, J. of Controlled Release, 1 (1984) pp. 161-162 describe a diffusion cell.

[0143] As used herein, "adjuvancy" refers to the ability to affect nonspecific inflammatory or specific immune responses elicited by an immune system activator.

[0144] As used herein, the term "anti-cancer response" for therapy refers to any response of cancer to therapy, preferably refers to changes in tumor mass and / or volume after therapy begins. Hyperproliferative disorder responses can be assessed by comparing the size of the tumor after local or systemic intervention with the initial size and dimensions, such as by CT, PET, mammography, ultrasound, or palpation. Responses can also be assessed by measuring the tumor with a caliper after biopsy or surgical resection or by performing a pathological examination of the tumor. Responses can be recorded quantitatively, such as percentage change in tumor volume, or qualitatively, such as "pathological complete remission" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical disease progression" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder responses can be performed early after therapy begins, such as a few hours, a few days, a few weeks, or preferably a few months later. Typical endpoints for response assessment are when chemotherapy terminates or when residual tumor cells and / or tumor beds are surgically removed. This is typically three months after treatment begins.

[0145] As used herein, "reducing tumor size" is defined as a decrease in the size of a tumor. This effect can be achieved by reducing the number of proliferating tumor cells in 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 suppressed or arrested.

[0146] As used herein, the terms "inhibiting cancer" or "inhibiting cancer cell growth" are intended to include inhibiting unwanted or inappropriate cell growth. Inhibition is intended to include inhibiting proliferation, including rapid proliferation. The term "inhibiting cancer cell growth" is also intended to encompass inhibiting tumor growth, which includes preventing the growth of a tumor in a subject or reducing the growth of a pre-existing tumor in a subject. Inhibition can also be inhibiting the metastasis of a tumor from one site to another. Cancer is "inhibited" if at least one symptom of the cancer is alleviated, stopped, slowed, or prevented. As used herein, cancer is also "inhibited" if the recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

[0147] "Therapeutic compound," "agent," and "therapeutic agent" are used synonymously herein.

[0148] 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 modifier (IRM) compound can be interpreted to mean that the pharmaceutical composition comprises at least one IRM compound.

[0149] As used herein, the term "subject" shall refer to any animal, including but not limited to humans, mice, rats, rabbits, non-human primates, or any other mammals. In one embodiment, the subject is a primate. In another and most preferred embodiment, the subject is a human.

[0150] Also herein, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). The compositions of the present invention may, for example, contain a compound of the present invention in an amount of 0.01%-1% (vol / wt%).

[0151] 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 effect of a single therapy. The synergistic effect of a conjoint therapy (e.g., a therapeutic combination) allows the use of lower doses of one or more therapeutic agents and / or the administration of one or more therapeutic agents at a lower frequency to a subject suffering from a disease or condition (e.g., a proliferative disorder). The ability to utilize lower doses of one or more therapeutic agents and / or the administration of therapeutic agents at a lower frequency will reduce the toxicity associated with administering the agent to the subject without reducing the efficacy of the therapy in the treatment of the disease or condition. In addition, a synergistic effect can result in an improved efficacy of the agent in preventing, controlling, or treating a disease or condition (e.g., a proliferative disorder). Finally, the synergistic effect of a conjoint therapy can avoid or reduce the adverse or undesirable side effects associated with the use of any therapeutic agent alone.

[0152] As used herein, the term "combination" may refer to the use of more than one therapeutic agent. The use of the term "combination" does not limit the order in which therapeutic agents are applied to the subject suffering from a disease or illness (for example, a proliferative disorder). The first therapeutic agent (such as a compound described herein) can be before the administration of a second therapeutic agent (such as an anticancer agent) (for example, 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 or afterwards (for example, 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) applied to the subject suffering from a disease or illness (for example, a proliferative disorder, such as cancer).

[0153] Immune response modifiers ("IRMs") include compounds with potent immunomodulatory activity (including but not limited to antiviral and antitumor activity). Certain IRMs regulate the production and secretion of cytokines. For example, certain IRM compounds induce the production and secretion of cytokines such as, for example, type I interferon, TNF-α, IL-1, IL-6, IL-8, IL-0, IL-12, MUM, and / or MCP-1. For another example, certain IRM compounds can inhibit the production and secretion of certain TH2 cytokines such as IL-4 and IL-5.

[0154] Additionally, some IRM compounds are claimed to inhibit IL-1 and TNF (U.S. Pat. No. 6,518,265). Resiquimod (1-[4-amino-2-(ethoxymethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol) is an immune response modifier (IRM) that acts by stimulating cells through toll-like receptors (TLRs) 7 and 8.

[0155] As used herein, when used in combination with respect to the compounds of the present invention and with respect to the medicaments described herein, "effective amount" includes but is not limited to the amount of each medicament in the medicament or combination that provides statistically significant expected effects for the disease or condition to be treated (e.g., cancer). Representative expected effects are described herein. For example, in the context of cancer treatment, the effect can be a slowing down of tumor growth rate, a cessation of tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume (as measured by standard metrics such as, but not limited to, solid tumor response evaluation criteria (RECIST)), a statistically significant improvement in survival rate relative to treatment with a separate medicament or a sub-combination of the combination alone. The effective amount may vary with factors such as the type of cell growth to be treated or suppressed, the type of therapeutic agent employed, a specific therapeutic agent, the (physique) size of the subject, or the severity of cancer cell growth or tumor. For example, the selection of each separate medicament constituting the combination can affect the composition of an "effective amount". One of ordinary skill in the art will be able to study the above factors and determine the effective amount of a combination of a therapeutic compound or a therapeutic compound / agent.

[0156] For example, an "effective amount" of a therapeutic agent can be determined using in vitro assays. One of ordinary skill will select the appropriate amount of each individual agent in the combination for use in the above-described in vitro assays. The cell survival fraction can be used to determine whether the selected amount is an "effective amount" for a particular combination of therapeutic agents. For example, the selected amount used in the assay should preferably result in the killing of at least 50% of the cells, more preferably 75%, and most preferably at least 95% of the cells. In a preferred embodiment, the effective dose of the therapeutic agent is a subtoxic dose. As used herein, the term "subtoxic dose" refers to a dose that results in the killing of less than about 10% of the cells.

[0157] The administration scheme (e.g., the order of administration) also can affect the composition of the effective amount. In addition, several divided doses and staggered doses can be applied topically daily or in sequence, or the dose can be continuously infused. In addition, the administration can be increased or decreased proportionally according to the needs of the treatment situation.

[0158] The expression "pharmaceutically acceptable" is used herein to refer to those therapeutic compounds of the invention or combinations of therapeutic compounds / agents, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0159] As used herein, the expression "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle involved in carrying or delivering the subject chemical from one organ or part of the body to another, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not causing harm to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0160] For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th edition, eds.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0161] As used herein, "alkyl" refers to a straight or branched hydrocarbon group with a specified number of carbon atoms. The alkyl group can be unsubstituted or substituted with a substituent that does not interfere with the specified function of the composition, and can be substituted once or twice by the same or different groups. The substituent can include, for example, alkoxy, hydroxyl, sulfhydryl, amino, alkyl-substituted amino, nitro, carboxyl, carbonyl, carbonyloxy, cyano, methylsulfonylamino or halogen. Examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, 3-methylpentyl, etc. For example, C1-C2 alkyl includes methyl and ethyl groups. As used herein, "alkyl" hydrocarbons include monovalent and divalent groups, i.e., include alkylene groups, if the resulting structure containing the alkyl group is stable.

[0162] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic, or tricyclic hydrocarbon ring system having three to fourteen carbon atoms and zero heteroatoms. Representative examples of cycloalkyl groups include, but are not limited to, adamantyl, bicyclo[3.1.1]heptyl, cyclobutyl, cyclohexyl, cyclopentyl, and cyclopropyl.

[0163] 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 five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" is used interchangeably with the term "aryl ring." In certain embodiments of the present invention, "aryl" refers to an aromatic ring system. Exemplary aryl groups include phenyl, biphenyl, naphthyl, anthracenyl, 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 used herein, are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthalimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0164] The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety such as "heteroaralkyl" or "heteroaralkoxy", refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; 6, 10, or 14 shared π electrons in the cyclic array; and one to five heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, as well as any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. As used herein, the terms "heteroaryl" and "heteroaryl-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic or heterocyclic rings, wherein the linker or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, o-naphthyridinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, benzoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl and pyrido [2,3-b] -1,4-oxazine-3 (4H) -one. The heteroaryl group is optionally monocyclic or bicyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl, wherein the alkyl and heteroaryl portions are independently optionally substituted.

[0165] As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic group," and "heterocyclic ring" are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more (preferably one to four) heteroatoms as defined above. The term "nitrogen" when used in conjunction with a ring atom of a heterocycle includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen is N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).

[0166] The heterocyclic ring may be attached to its side group (or groups) at any heteroatom or carbon atom which results in a stable structure and any ring atom may be optionally substituted (preferably an unsubstituted ring). Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolane, diazepine, oxazolidinyl, pyrrolidino ... Oxazolin thiazolinone The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic radical" are used interchangeably herein and also include groups in which the heterocyclyl ring is fused to one or more aryl, heteroaryl or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl, wherein the linker or point of attachment is on the heterocyclyl ring. The heterocyclyl group is optionally monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl moieties are independently optionally substituted (but preferably unsubstituted).

[0167] As described herein, certain compounds of the present invention contain "optionally substituted" moieties. In general, the term "substituted", whether or not preceded by the term "optionally", refers to that one or more hydrogens of the designated moiety are replaced by suitable substituents. When more than one position in any given structure is substituted by more than one substituent selected from a specified group, at each position, the substituents are the same or different. The combinations of substituents contemplated by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that do not substantially change when subjected to conditions that allow their production, detection, and in certain embodiments, for one or more purposes disclosed herein, conditions that allow their recovery, purification, and use. "Stable" compounds are generally not free radicals. Preferably, the compounds of the present invention are not further substituted except for those substitutions explicitly noted.

[0168] Unless otherwise indicated, references to compounds and compounds of the present invention may include compounds in any pharmaceutically acceptable form or pharmaceutically acceptable derivative, including any isomers (e.g., diastereomers or enantiomers), salts, esters, salts of esters, solvates, polymorphs, and the like. In particular, if a compound is optically active, references to the compound may include each of the enantiomers of the compound and racemic mixtures of these enantiomers. Additionally, unless otherwise indicated, the structures and compounds depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the present invention, including replacement of hydrogen with deuterium or tritium or replacement of carbon with 13C- or 14C-rich carbon, are within the scope of the present invention. In some embodiments, the group comprises one or more deuterium atoms. Preferably, "pharmaceutically acceptable salts, forms, or derivatives" include derivatives of the compounds of the present invention that, upon administration to a recipient, are capable of providing, directly or indirectly, a compound of the present invention or an agonist metabolite or residue thereof.

[0169] As used herein, the term "pharmaceutically acceptable salt" refers to salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. 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 include salts formed of amino groups 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 salts formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. These salts can be prepared in situ during the final isolation and purification of the therapeutic agent, or by reacting the purified therapeutic agent in its free base form with a suitable organic or inorganic acid alone and isolating the salt thus formed. Representative salts also include hydrobromate, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate, and laurylsulfonate. (See, for example, Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19).

[0170] Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C1-4 alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates.

[0171] Therefore, in a first aspect of the present invention, there is provided a compound according to formula I,

[0172]

[0173] or a pharmaceutically acceptable salt or derivative thereof,

[0174] wherein X is an oxygen atom, a C1-C5-alkyl group (preferably CH2) or NH;

[0175] where R 2 and R 3 Each is independently selected from hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably R 2 and R 3 are each hydrogen;

[0176] And where L 1 、L 2 、L 3 and R 1 is as defined below under (a) or (b) or (c):

[0177] (a)L 1 is a C2-C6 alkyl group;

[0178] L 2 is a 5- or 6-membered heterocyclic ring or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0179] R 1 Absent or selected from hydrogen, NH2, OH and SCH3;

[0180] (b)L 1 is a C2-C6 alkyl group;

[0181] L 2 selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2;

[0182] L 3 is selected from the group consisting of a 6-membered heterocycle, OH, C(O)O, S, SO2, and SO3H; and

[0183] R 1 Absent or methyl or hydrogen;

[0184] (c)L 1 is C(O)CH2;

[0185] L 2 is a 6-membered heterocyclic ring or NH2;

[0186] L 3 does not exist; and

[0187] R 1 is methyl or hydrogen or is absent;

[0188] And if L 3 If it does not exist, then L 2 Through covalent bond with R 1 Direct combination.

[0189] In a preferred embodiment of the first aspect, R 2 and R 3 For hydrogen.

[0190] Preferably, X in the compounds of the present invention is an oxygen atom.

[0191] In a further embodiment, the compounds of the present invention have a structure according to Formula II:

[0192]

[0193] Among them L 1 、L 2 、L 3 and R 1 as defined below under (d), (e) or (f)

[0194] (d)L 1 is a C2-C6 alkyl group (e.g., ethyl, propyl, butyl, pentyl or hexyl);

[0195] L 2 is a 5- or 6-membered heterocycle (preferably, triazole or piperazine) or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0196] R 1 Absent or selected from hydrogen, NH2, OH and SCH3;

[0197] (e)L 1 is a C2-C3 alkyl group;

[0198] L 2selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2;

[0199] L 3 is selected from a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO2 and SO3H; and

[0200] R 1 Absent or methyl or hydrogen;

[0201] (f)L 1 is C(O)CH2;

[0202] L 2 is a 6-membered heterocycle (preferably piperazine) or NH2;

[0203] L 3 does not exist; and

[0204] R 1 is methyl or hydrogen or is absent;

[0205] In a more preferred embodiment of the present invention, the compound of formula I or II

[0206] L 1 It is a C2-C4 alkyl group.

[0207] In a preferred embodiment of the compound of formula II of the present invention

[0208] L 1 is a C2-C6 alkyl group (preferably a C2-C4 alkyl group);

[0209] L 2 is a 5- or 6-membered heterocycle (preferably, triazole or piperazine) or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and

[0210] R 1 Not present or selected from NH2, OH and SCH3;

[0211] In a particularly preferred embodiment of the compounds of the present invention, the compounds have a structure selected from the structures listed below designated as Examples 1 to 27:

[0212]

[0213]

[0214] In a preferred embodiment of the present invention, the compound of the present invention is a compound according to Example 13 or 14 as listed above or according to Example 26 or 27 as listed above.

[0215] In a preferred embodiment, the compound of the present invention is a compound selected from the compounds of Examples 1 to 6 as listed 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 compounds of Examples 1, 3, 4 and 5 as listed above, or a pharmaceutically acceptable salt or derivative thereof.

[0216] A more preferred compound of the present invention relates to a compound wherein

[0217] X is NH;

[0218] L 1 is a C2-C6 alkyl group;

[0219] L 2 is a 5- or 6-membered heterocyclic ring;

[0220] L 3 does not exist; and

[0221] R 1 is methyl or hydrogen; and

[0222] R 2 and R 3 Each is hydrogen.

[0223] Also preferred are compounds of the present invention wherein the compound

[0224] (i) activates TLR7 more strongly than TLR8; and / or

[0225] (ii) induces the production of IL-6, IL1-b and TNF-α; and / or

[0226] (iii) Inducing upregulation of CD40 and / or CD86 in peripheral blood mononuclear cells (PBMCs).

[0227] In the above embodiments, one of ordinary skill can test whether a compound activates TLR7 more strongly than TLR8 by performing the method provided below in Example 6.2.1.

[0228] In the above embodiments, one of ordinary skill can test whether a compound induces TNF-α production by performing the method provided below in Example 6.2.2.

[0229] In the above embodiments, one of ordinary skill can test whether a compound induces upregulation of CD86 by performing the method provided below in Example 6.2.3.

[0230] Preference is also given to compounds according to the invention, wherein, if the compound is contacted with peripheral blood mononuclear cells (PBMC), the compound induces the secretion of TNFα from said cells.

[0231] Also preferred are compounds according to the invention which have an EC50 for TLR7 of less than 10 μM and preferably less than 0.01 μM, preferably when tested in the test system described in Example 6.2.1 hereinafter.

[0232] Yet another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention.

[0233] The present invention also relates to a pharmaceutical composition comprising a compound of the present invention and preferably an additional compound such as imiquimod and / or resiquimod (R848).

[0234] Yet another aspect of the present invention relates to a compound of the present invention for use as a medicament.

[0235] Yet another aspect of the present invention relates to a compound of the present invention for use in the treatment of a disease, wherein said compound is used in said treatment in combination with the additional compounds imiquimod and / or resiquimod (R848).

[0236] A preferred embodiment provides a compound of the present invention for use in treating a condition selected from the group consisting of cancer, viral infection, noncancerous skin lesions, precancerous skin lesions, cancerous skin lesions, bladder cancer, virally mediated skin diseases, the composition optionally being formulated to enhance penetration upon topical administration, the composition preferably eliciting a localized specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses.

[0237] In a preferred embodiment of the composition of the present invention, the compound of the present invention is contained therein in an amount between 0.01% and 1% wt / vol.

[0238] Yet another aspect relates to a composition of the present invention for use in treating bladder cancer, wherein the composition is formulated for intravesical administration.

[0239] In a preferred embodiment of the composition of the present invention, the present invention further comprises an additional component selected from one of the following groups i to xiii:

[0240] i. a total concentration of 1% wt / vol oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0241] ii. a total concentration of 0.5% wt / vol isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0242] iii. oleic acid (50%) and sodium lauryl sulfate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0243] iv. a total concentration of 0.5% wt / vol 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, or

[0244] v. isopropyl palmitate (50%) and sodium oleate (50%) at a total concentration of 2% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0245] vi. sodium lauryl sulfate (25%) and linoleic acid (75%) at a total concentration of 1.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0246] vii. Palmitic acid (50%) and isopropyl laurate (50%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0247] viii. oleic acid (50%) and linoleic acid (50%) at a total concentration of 1.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0248] ix. linoleic acid (25%), oleic acid (25%), and isopropyl linoleate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0249] x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or

[0250] xi. A solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, or

[0251] xii. Oleic acid (10%) in a solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol, or

[0252] xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0253] Yet another 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.

[0254] Yet another aspect of the present invention provides a method for treating skin tumor lesions and viral-induced skin diseases, comprising topically administering a composition of the present invention, which is preferably effective in reducing or eliminating the lesions or diseases while limiting adverse skin reactions selected from erythema and inflammation, and which preferably reduces the penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

[0255] Yet another aspect of the present invention relates to a method for treating bladder tumors, comprising intravesical administration of a composition of the present invention, wherein the composition preferably enhances the penetration and delivery of resiquimod into the bladder epithelium while limiting irritation, and the composition preferably reduces tumor infiltration into surrounding muscle tissue and metastasis to lymph nodes.

[0256] In a preferred embodiment of the method of the present invention, the treatment further comprises systemically administering to the subject at least one immunomodulator selected from anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-CD137 antibodies, agonist CD40 antibodies, CD134 (anti-OX40) agonists and PLX3397.

[0257] In a preferred embodiment of the method of the present invention, said treatment further comprises systemically administering interferon gamma to said subject.

[0258] In a preferred embodiment of the method of the present invention, the treatment further comprises administration of localized radiation with or without systemic anti-PD1 antibodies.

[0259] In a preferred embodiment of the method of the present invention, the treatment further comprises administering photodynamic therapy.

[0260] In a further aspect, the present invention provides a method of activating TLR 7 and / or 8 in a biological sample, comprising contacting the biological sample with a compound according to the present invention.

[0261] In yet another aspect, the present invention provides a compound of the present invention for use as a vaccine adjuvant or in combination with an anti-cancer immunotherapeutic agent (preferably ipilimumab, nivolumab or pembrolizumab) for treating cancer.

[0262] preparation

[0263] The TLR7 agonist compounds of the present invention can be prepared according to the examples provided further below.Alternative methods for synthesizing the compounds of the present invention can also be employed.

[0264] Use, formulation and administration

[0265] Pharmaceutically acceptable compositions

[0266] The compounds of the present invention will be used as immune response modulators and will have antiviral and antitumor activity. For example, similar to the toll-like receptor 7 (TLR7) agonist resiquimod, the compounds of the present invention are expected to reduce hepatitis C virus (HCV) infection, as shown in a Phase 2 clinical study of resiquimod. In a murine model of allergic asthma, resiquimod (in, 20 μg / mouse) reduces allergen-induced airway responsiveness and inflammation via 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 regulates dendritic cells to enhance 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 and can improve cancer immunotherapy by reducing immunosuppressive MDSCs (see Arch Pharm Res. 2014; 37(9): 1234-40. doi: 10.1007 / s12272-014-0379-4. Epub 2014 Apr 19.). In view of these multiple medical benefits exhibited by resiquimod as a TLR7 / 8 agonist, it is expected that the compounds of the present invention, which are also TLR7 / 8 agonists, will also enjoy the above-mentioned medical uses and benefits.

[0267] 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 yet another aspect, the present invention provides a composition 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 the composition of the present invention is such that it is effective to measurably activate TLR7 / 8 or a mutant thereof in a biological sample or in a patient. In certain embodiments, the composition of the present invention is formulated for administration to a patient in need of such a composition.

[0268] As used herein, the term "patient" or "subject" means an animal, preferably a mammal, most preferably a human.

[0269] The term "pharmaceutically acceptable carrier, adjuvant or vehicle" includes non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compound formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles for use in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0270] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally, or intravenously. Sterile injectable formulations of the compositions of the present invention include aqueous or oily suspensions. These suspensions are formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in parenterally acceptable non-toxic diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are generally used as solvents or suspending media. Non-volatile oils that can be used include synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used to prepare injections, as can pharmaceutically acceptable natural oils such as olive oil or castor oil (especially their polyoxyethylated forms). These oil solutions or suspensions also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the preparation 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 manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0271] In one embodiment, the pharmaceutically acceptable compositions of the present invention can be orally administered in any orally acceptable dosage form. Exemplary oral dosage forms include 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. For oral administration in capsule form, available diluents include lactose and dry corn starch. When an aqueous suspension is required for oral administration, the active ingredient can be combined with an emulsifier and a suspending agent. If desired, certain sweeteners, flavorings, or coloring agents are optionally added.

[0272] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These suppository forms can be prepared by mixing the medicament with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0273] The pharmaceutically acceptable compositions of this invention can also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0274] Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.Topical transdermal patches may also be used.

[0275] For topical application, the pharmaceutically acceptable compositions of the present invention provided can be formulated into suitable ointments containing the active compound of the present invention suspended or dissolved in one or more carriers. Exemplary carriers for topical application of the compound include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutically acceptable compositions provided can be formulated into suitable lotions or creams containing the active compound 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 esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0276] The pharmaceutically acceptable compositions of the present invention are optionally administered by nasal aerosol or inhalation. Such compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons and / or other conventional solubilizing agents or dispersants. As mentioned, the pharmaceutically acceptable compositions of the present invention can also be formulated for oral administration. Such preparations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are not administered with food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0277] The amount of the compound of the invention optionally combined with a carrier material to produce a single dosage form of the composition will vary with the host being treated, the particular mode of administration. Preferably, provided compositions can be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the compound can be administered to a patient receiving these compositions.

[0278] It will 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 specific compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, concomitant medications, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of the compound of the invention in the composition will also depend on the specific compound in the composition.

[0279] Patients with compromised immune function are very susceptible to developing severe infections, which often develop into life-threatening conditions such as sepsis. Therefore, immunotherapy aimed at enhancing host immune defense is an attractive strategy for resisting infection and protecting patients. Recently, increasing evidence has shown that activating the innate immune system can confer long-term functional reprogramming, whereby innate leukocytes initiate a more robust response after secondary exposure to pathogens, thereby more efficiently removing (pathogens) and protecting the host, which is referred to as trained immunity. Toll-like receptor (TLR) agonists are a class of agents that have been shown to trigger trained immune phenomena through metabolic reprogramming and epigenetic modification, which will drive a significant enhancement of antimicrobial function. Immunomodulatory TLR agonists are also highly beneficial as vaccine adjuvants. In addition, most cancer-associated antigens are self-antigens, and in addition to cancer targeting strategies, immunostimulatory adjuvants are also needed. Therefore, TLR7 / 8 agonists such as the compounds of the present invention will also be useful for treating cancer.

[0280] In one aspect, the present invention provides a method of treating a subject suffering from a TLR7 / 8-related disorder, comprising administering to the subject an effective amount of a compound of the present invention, preferably a compound of Formula II and related formulae.

[0281] The compounds of the present invention can be used as anticancer agents for cancers that respond to TLR7 activation. In certain embodiments, the cancers include, but are not limited to, breast cancer, bladder cancer, bone cancer, brain cancer, central and peripheral nervous system cancer, colon cancer, endocrine gland cancer, esophageal cancer, endometrial cancer, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, laryngeal cancer and hypopharyngeal cancer, mesothelioma, sarcoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, small intestine cancer, soft tissue cancer, testicular cancer, stomach cancer, skin cancer, ureteral cancer, vaginal cancer and vulvar cancer; hereditary cancers, retinoblastoma and Wilms' tumor; leukemia, lymphoma, non-Hodgkin's disease, chronic and acute myeloid leukemia, acute lymphocytic leukemia, Hodgkin's disease, multiple myeloma and T-cell lymphoma; myelodysplastic syndrome, plasma cell neoplasms, paraneoplastic syndromes, cancers of unknown primary site and AIDS-related malignancies.

[0282] In certain embodiments, the compounds of the present invention are used to treat skin cancer or renal cancer. The sensitivity of a given cancer to TLR7 activation can be assessed by, but is not limited to, measuring a reduction in primary or metastatic tumor burden (mild, partial, or complete regression), changes in blood counts, changes in blood hormone or cytokine concentrations, inhibition of further increase in tumor burden, stabilization of the patient's disease, assessment of disease-related biomarkers or surrogate markers, prolongation of the patient's overall survival, prolongation of the patient's time to disease progression, prolongation of the patient's progression-free survival, prolongation of the patient's disease-free survival, improvement in the patient's quality of life, or modulation of disease comorbidities (such as, but not limited to, pain, cachexia, mobilization, hospitalization, blood count changes, weight loss, wound healing, fever).

[0283] The compounds according to the invention are also useful as immune response modifiers, which may modulate the immune response in a variety of different ways, making them useful in the treatment of a variety of conditions.

[0284] Provided herein is a method for activating an individual immune response, comprising administering to an individual an effective amount of an activator of TLR7 (e.g., a TLR7 activator) using a compound as described herein. In some variations, the TLR activator activates a TLR7-dependent immune response. In some variations, the TLR activator activates TLR7 and TLR8-dependent immune responses. Unless otherwise indicated, the term "TLR activator" refers to any one of the compounds of the present invention disclosed herein. In some preferred embodiments, the individual is a human patient.

[0285] The present disclosure provides immunomodulatory methods and includes those that activate immune responses, including but not limited to immune responses. The present disclosure also provides methods (e.g., in vitro or in vivo) for activating TLR7 and / or TLR8 induction responses. In some variations, cells are contacted with a TLR activator effective in activating an amount of a response that contributes to an immune response.

[0286] Activation of TLR7 and / or TLR8 can be used to treat and / or prevent a variety of cytokine-responsive diseases or conditions.

[0287] Provided herein are methods of activating an immune response in a subject, the methods comprising administering to the subject at least one TLR activator as disclosed herein in an amount effective to activate the immune response in the subject.

[0288] Also provided herein are methods of treating a viral disease or condition (eg, HIV infection), comprising administering to a subject at least one compound of the invention in an amount effective to treat the viral disease or condition.

[0289] In some embodiments of any method involving administering a compound of the present invention to a subject, the compound preferably has a therapeutically acceptable safety profile. For example, the compound / TLR activator may have therapeutically acceptable histological characteristics, including acceptably low toxicity (if any) to the liver, kidney, pancreas or other organs. In some embodiments, safety characteristics include evaluation of toxicity, histological characteristics 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, such as compared to another TLR activator. In some embodiments, the TLR activator induces therapeutically acceptable weight loss, such as compared to the initial weight of the individual receiving treatment. 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 therapeutically acceptable histological characteristics. In some embodiments, for example, as compared to a reference TLR activator, the TLR activator has better (e.g., a lower severity score) histological characteristics. In some embodiments, for example, when evaluating the liver, kidneys and / or heart, the TLR activator has better (e.g., a lower severity score) histological characteristics. In some embodiments, the TLR activator has a therapeutically acceptable necrosis score. In some embodiments, for example, as compared to a reference TLR activator, the TLR activator has reduced necrosis and / or a better (e.g., lower) necrosis score. In some embodiments, for example, as compared to a reference TLR activator, the TLR activator has reduced kidney and / or liver cell necrosis and / or a better kidney and / or liver cell necrosis score.

[0290] Therefore, the present invention provides a method for activating TLR7 in an animal (especially a mammal, preferably a human), comprising administering to the animal an effective amount of a compound of the present invention. The effective amount of the compound will vary with factors known in the art, but is expected to be about 0.1 to 10 mg / kg, 0.5 to 10 mg / kg, 1 to 10 mg / kg, 0.1 to 20 mg / kg, 0.1 to 20 mg / kg, or 1 to 20 mg / kg.

[0291] The present invention also provides a method for treating viral infection in animals, comprising administering an effective amount of a compound of the present invention to an animal. The amount effective for treating or inhibiting viral infection is an amount that will result in a reduction in one or more manifestations of viral infection (such as viral lesions, viral load, viral production rate, and mortality) compared to untreated control animals. The precise amount will vary with factors known in the art, but it is expected to be a dosage as indicated above for the activation of TLR7, or a dosage of about 100 ng / kg to about 50 mg / kg, preferably about 10 μg / kg to about 5 mg / kg.

[0292] The methods of the present invention can be performed in vitro or in vivo. The sensitivity of specific cells to treatment with a compound according to the present invention can be determined, in particular, by in vitro testing, whether during research or clinical application. Typically, a culture of cells is combined with a compound according to the present invention at various concentrations for a period of time sufficient for the active agent to inhibit TLR7 / 8 activity, typically between about one hour and one week. In vitro treatment can be performed using cultured cells from a biopsy sample or a cell line.

[0293] The host or patient can be of any mammalian species, such as primate species, particularly humans; rodents, including mice, rats, and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of great significance for experimental research, as they provide models for the treatment of human diseases.

[0294] In addition, the present invention also provides the use of compounds according to the present invention and derivatives thereof for the production of medicaments for the preventive or therapeutic treatment of diseases caused, mediated and / or propagated by insufficient TLR7 / 8 activity. In certain embodiments, the present invention provides the use of compounds according to the present invention or physiologically acceptable salts or derivatives thereof for the production of medicaments for the preventive or therapeutic treatment of TLR7 / 8-mediated conditions. In addition, the compounds of the present invention and / or physiologically acceptable salts or derivatives thereof can be used as intermediates for the preparation of other active pharmaceutical ingredients. The drug is preferably prepared in a non-chemical manner, for example, by combining the active ingredient with at least one solid, fluid and / or semi-fluid carrier or excipient, and optionally combined with a single or multiple other active substances in an appropriate dosage form.

[0295] In one embodiment, the compounds of the present invention can be administered once or several times as treatment before or after the onset of the disease. These compounds are particularly useful for therapeutic treatment. The treatment-related effects will alleviate one or more symptoms of the disease to a certain extent, or partially or completely restore one or more physiological or biochemical parameters associated with or causing the disease or pathological state to normal. If the compound is administered at different time intervals, for example, to enhance the response and completely eradicate the pathogen and / or symptoms of the disease, monitoring is considered a treatment. The same compound or different compounds can be applied. The method of the present invention can also be used to reduce the likelihood of the occurrence of the disease or even prevent in advance the occurrence of the disease associated with insufficient or reduced TLR7 / 8 activity (reduced compared to healthy subjects), or for treating the symptoms that occur and persist.

[0296] In the meaning of the present invention, a preventive treatment is recommended if the subject has any preconditions for the above mentioned physiological or pathological conditions such as a familial predisposition, a genetic defect or a pre-existing disease.

[0297] In addition, the present invention relates in yet another aspect to a medicament comprising at least one compound according to the invention and / or its pharmaceutically acceptable derivatives, salts, solvates and stereoisomers, including mixtures thereof in all ratios. In certain embodiments, the present invention relates to a medicament comprising at least one compound according to the invention and / or its physiologically acceptable salts.

[0298] In various embodiments, the active ingredients can be administered alone or in combination with other therapies. Synergistic effects may be achieved by using more than one compound in a pharmaceutical composition, i.e., combining a compound of the invention with at least one other active agent, which is another compound of the invention or a compound with a different structural scaffold. The active ingredients can be administered simultaneously or sequentially.

[0299] Literature has shown that the toll-like receptor 7 (TLR7) agonist R848 combined with radiation therapy (RT) will lead to long-term clearance of tumors in mice bearing T- and B-cell lymphomas. Therefore, in another aspect, the present invention provides a method for treating a subject suffering from lymphoma by administering a compound of the present invention in combination with radiation therapy to the subject.

[0300] In yet another aspect, the present invention provides a method for treating cancer in a patient in need of such treatment, comprising administering a therapeutically effective amount of a compound of the present invention and administering a therapeutically effective amount of at least one different anti-tumor agent selected from the group consisting of: (1) taxanes, (2) platinum coordination compounds, (3) epidermal growth factor (EGF) inhibitors that are antibodies, (4) EGF inhibitors that are small molecule compounds, (5) vascular endothelial growth factor (VEGF) inhibitors that are antibodies, (6) VEGF kinase inhibitors that are small molecule compounds, (7) estrogens, Hormone receptor antagonists or selective estrogen receptor modulators (SERMs), (8) antitumor nucleoside derivatives, (9) epothilones, (10) topoisomerase inhibitors, (11) vinca alkaloids, (12) antibodies that are inhibitors of αVβ3 integrin; (13) small molecule compounds that inhibit αVβ3 integrin; (14) folic acid antagonists; (15) ribonucleotide reductase inhibitors; (16) anthracyclines; (17) anticancer biological agents; (18) thalidomide (or related imides); and (19) Gleevec. Examples of these antitumor compounds are disclosed in US20060183765, which is incorporated herein by reference.

[0301] The compounds of the present invention may be administered in combination with other known therapeutic agents, including anticancer agents.As used herein, the term "anticancer agent" relates to any agent that is administered to a patient suffering from cancer for the purpose of treating the cancer.

[0302] Anticancer treatment may be applied as a monotherapy or may involve conventional surgery or radiation therapy or drug therapy in addition to the compounds of the invention disclosed herein. Such drug therapy, such as chemotherapy or targeted therapy, may include one or more of the following antineoplastic agents, but preferably one:

[0303] Alkylating agents : such as altretamine, bendamustine, busulfan, carmustine, chlorambucil, nitrogen mustard, cyclophosphamide, dacarbazine, ifosfamide, improsulfan, p-toluenesulfonic acid, lomustine, melphalan, dibromomannitol, dibromodulcitol, nimustine, ranimustine, temozolomide, thiotepa, thiosulfate, mechloretamine, carboquinone, apaziquinone, fotemustine, glufosfamide, palivamide, pipobroman, trofosfamide, uracil, TH-302, VAL-083;

[0304] Platinum compounds : such as carboplatin, cisplatin, epazote, miriplatin hydrate, oxaliplatin, lobaplatin, nedaplatin, picoplatin, satraplatin; lobaplatin, nedaplatin, picoplatin, satraplatin;

[0305] DNA-altering agents: such as amrubicin, bisantrene, decitabine, mitoxantrone, procarbazine, trabectedin, clofarabine; amsacrine, brostallicin, pixantrone, laromustine;

[0306] Topoisomerase inhibitors : Such as etoposide, irinotecan, razoxane, sobuzoxane, teniposide, topotecan; amifide, belotecan, elixirsium acetate, voreloxin;

[0307] microtubule modifiers : such as cabazitaxel, docetaxel, eribulin, ixabepilone, paclitaxel, vinblastine, vincristine, vinorelbine, vindesine, vinflunine; fosbretabulin, tesetaxel;

[0308] Antimetabolites : For example, asparaginase, azacitidine, leucovorin, capecitabine, cladribine, cytarabine, enocitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, nelarabine, pemetrexed, pralatrexate, azathioprine, thioguanine, carmofur; doxifluridine, elcitabine, raltitrexed, sapacitabine, tegafur, trimetrexate;

[0309] anticancer antibiotics : such as bleomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, levamisole, miltefosine, mitomycin C, romidepsin, streptozotocin, valrubicin, zenastatin, daunorubicin, plicamycin; aclarubicin, peplomycin, pirarubicin;

[0310] Hormones / antagonists : such as abarelix, abiraterone, bicalutamide, buserelin, carlosterone, chlorethoxyquin, degarelix, dexamethasone, estradiol, fluocortolone, fluoxymesterone, flutamide, fulvestrant, goserelin, histrelin, leuprorelin, megestrol acetate, mitotane, nafarelin, nandrolone, nilutamide, octreotide, prednisolone, raloxifene, tamoxifen, thyrotropin alfa, toremifene, trilostane, triptorelin, diethylstilbestrol; acolbifene, danazol, deslorelin, cyclothiocarbamate, orteronel, enzalutamide;

[0311] Aromatase inhibitors : such as aminoglutethimide, anastrozole, exemestane, fadrozole, letrozole, testolactone, formestane;

[0312] Small molecule kinase inhibitors: such as crizotinib, dasatinib, erlotinib, imatinib, lapatinib, nilotinib, pazopanib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, bosutinib, gefitinib, axitinib; afatinib, alectinib, dabrafenib, dacomitinib, dinaciclib, dovitinib, enzastaurin, nintedanib, lenvatinib, linifanib, linsitinib, masitinib, midostaurin Lin, motesanib, neratinib, olantinib, perifosine, ponatinib, radotinib, reglucosidase, tipifarnib, tivantinib, tivozanib, trametinib, pimasertib, brivanib alanine, cediranib, apatinib, cabozantinib S-malate, ibrutinib, icotinib, buparicilib, sipatinib, cobimetinib, idelalisib, fuzetinib, XL-647;

[0313] photosensitizers : such as methoxsalen; porfimer sodium, talaporfin, temoporfin;

[0314] Antibody : such as alemtuzumab, bexoluzumab, brentuximab, cetuximab, denosumab, ipilimumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, bevacizumab, pertuzumab; catumaxomab, elotuzumab, epratuzumab, fatuzumab, moglizumab, necituzumab, nimotuzumab, obtuzumab, okatuzumab, ogavuzumab, ramucirumab, rituximab, siltuximab, tocilizumab, zalutumumab, zalumab, matuzumab, dalotuzumab, onatuzumab, rituximab, tabciruciumab, abiraterone, nivolumab;

[0315] cytokines : such as aldesleukin, interferon α, interferon α2a, interferon α2b; simoleukin, tasonermin, tesileukin, oprelewkin, recombinant interferon beta-1a;

[0316] Drug conjugates : such as denileukin-diftitox, ibritumomab tiuxetan, iodobenzylguanidine I123, prednimustine, trastuzumab emtansine, estramustine, gemtuzumab tuzumab, ozogamicin, aflibercept; cintredekinbesudotox, edotreotide, ozogamicin, etotreotide, oportuzumab monatox, technetium (99mTc) acitumomab, vintafolide;

[0317] vaccine : such as sipuleucel; vitespe, emepepimut-S, oncoVAX, rindopepimut, troVax, MGN-1601, MGN-1703; and

[0318] Miscellaneous : alitretinoin, bexarotene, bortezomib, everolimus, ibandronic acid, imiquimod, lenalidomide, lentinan, methyltyrosine, mifatide, pamidronate, pegaspargase, pentostatin, sipuleucel, sizoran, tamibarotene, temsirolimus, thalidomide, tretinoin, vimodegib, zoledronic acid, vorinostat; celecoxib, cilengitide, entinostat, etanidazole, ganetespib, idronoxil, iniparib, ixazomib, lonidamine, nimorazole, panobinostat, peretinoin, plitidep sin, pomalidomide, procodazol, lidaformolimus, tasquinimod, telotristat, thymalfasin, tirapazamine, tosedostat, trabedersen, ubenimex, gendicine4, picibanil4, reolysin4, retamycin hydrochloride 1, trebananib, virulizin, carfilzomib, endostatin, immucothel, belinostat, MGN-1703.

[0319] Thus, aspects of the present invention relate to treating a subject by administering a compound of the present invention in a suitable amount together with one or more of the anti-tumor agents listed above to achieve treatment of a cancer disease in the subject.

[0320] In some embodiments, the combination of a TLR inhibitor and one or more additional therapeutic agents reduces the effective amount of the TLR activator and / or additional therapeutic agent that needs to be administered to achieve the same result (including but not limited to dose volume, dose concentration, and / or total drug dose administered), as compared to the effective amount administered when the TLR activator or the one or more additional therapeutic agents are administered alone.

[0321] TLR activators can also be used as vaccine adjuvants in combination with any substance that modulates humoral and / or cell-mediated immune responses, such as, for example, live viruses, bacteria, or parasite immunogens; inactivated viruses, tumor-derived, protozoan, organism-derived, fungal or bacterial immunogens, toxoids, toxins; autoantigens; polysaccharides; proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines; recombinant proteins; glycoproteins; peptides; and the like. In some aspects, the combination therapies of the present invention include, but are not limited to, administering a combination of a compound of the present invention and a vaccine. In some aspects, the combination therapies of the present invention include, but are not limited to, administering a combination of a compound of the present invention and a vaccine. In some aspects, the combination therapies of the present invention include, but are not limited to, using a combination of a compound of the present invention and a vaccine in the treatment of an infectious disease.

[0322] Yet another aspect of the present invention relates to a kit comprising a compound of the present invention as provided herein and instructions for use in a method of activating a TLR7- and / or TLR8-dependent immune response.

[0323] The kit may include one or more containers containing a compound of the invention as described herein (or a preparation containing the compound) and a set of instructions, which are generally written instructions, but electronic storage media (e.g., magnetic or optical disks) containing instructions for the use and dosage of the compound of the invention or the preparation containing the compound for the 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 condition mediated by TLR7 and / or TLR8) are also acceptable. The instructions included in the kit typically include information such as the dosage, dosing schedule, and route of administration for the intended treatment. The container of the TLR activator (or preparation containing the TLR activator) may be a unit dose, bulk package (e.g., multi-dose package) or subunit dose. The kit may also include a container containing an adjuvant.

[0324] In another aspect, the invention provides a kind of test kit, it is made up of independent packaging, independent packaging contains the compound according to the present invention and / or its pharmaceutically acceptable salt, derivative, solvate and stereoisomer (including its all ratios mixture), and optionally an effective amount of other active ingredient. Test kit includes suitable container, such as box, separate bottle, bag or ampoule. Test kit can, for example, include separate ampoule, each of which contains an effective amount of the compound according to the present invention and / or its pharmaceutically acceptable salt, derivative, solvate and stereoisomer (including its all ratios mixture) in dissolved or lyophilized form, and an effective amount of other active ingredient.

[0325] The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and maintaining uniform dosage. As used herein, the expression "dosage unit form" refers to physically discrete units of a medicament suitable for the patient to be treated. However, it should be understood that the total daily dosage 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 effective dosage level for any particular patient or organism will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound being employed; the specific composition being employed; 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 employed; the duration of treatment; drugs used in combination with or concurrently with the specific compound being employed, and similar factors well known in the medical field.

[0326] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (e.g., by powder, ointment, or drops), buccally, as an oral or nasal spray, etc., depending on the severity of the infection being treated. In certain embodiments, the compounds of the present invention are administered orally or parenterally at a dosage level of about 0.01 mg / kg to about 100 mg / kg, preferably about 1 mg / kg to about 50 mg / kg of subject body weight per day, once or more daily to achieve the desired therapeutic effect. In certain embodiments, the therapeutically effective amount of the compounds of the present invention and related formulas, as well as other active ingredients, may depend on many factors, including, for example, the age and weight of the animal, the exact disease condition to be treated and its severity, the nature of the formulation, and the method of administration, and is ultimately determined by the treating physician or veterinarian. However, the effective amount of the compound is typically in the range of 0.1 to 100 mg / kg of recipient (mammal) body weight per day, and in particular, typically in the range of 1 to 10 mg / kg body weight per day. Thus, the actual daily amount for an adult mammal weighing 70 kg is generally between 70 and 700 mg, which amount can be administered as a single dose daily, or generally in a series of divided doses (e.g., two, three, four, five or six divided doses) daily to give the same total daily dose. The effective amount of a salt or solvate or physiologically functional derivative thereof can be determined as a fraction of the effective amount of the compound itself.

[0327] In certain embodiments, pharmaceutical preparation can be used in dosage unit form, and each dosage unit comprises the active component of predetermined amount.Such unit can comprise the compound according to the present invention of for example 0.5mg to 1g, preferably 1mg to 700mg, particularly preferably 5mg to 100mg, specifically depending on the disease condition treated, the method for applying and the age, body weight and the patient's condition, or, pharmaceutical preparation can be used in dosage unit form, and each dosage unit comprises the active component of predetermined amount.Preferred dosage unit preparation is those comprising the daily dose as described above or divided dose or its corresponding part of active component.In addition, pharmaceutical preparation of this type can be prepared using methods known in the pharmaceutical field.

[0328] Liquid dosage forms for oral administration of the compounds of the present invention include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form optionally contains inert diluents commonly used in the art (such as, for example, water or other solvents), solubilizers and emulsifiers (such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide), oils (particularly 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 inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, and perfuming agents.

[0329] Injectable preparations, such as sterile aqueous or oily suspensions for injection, can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions or emulsions in parenterally acceptable non-toxic diluents or solvents, for example as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution USP and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are generally used as solvents or suspending media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injections.

[0330] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0331] In order to prolong the effect of the compound of the present invention, it is generally desirable to slow down the absorption of the compound injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of crystalline or amorphous materials with poor water solubility. The absorption rate of the compound then depends on its dissolution rate, which in turn can depend on the crystal size and crystalline form. Alternatively, delayed absorption of the compound form administered parenterally can be achieved by dissolving or suspending the compound in an oil vehicle. Injectable reservoir forms can be prepared by forming a microcapsule matrix of the compound in a biodegradable polymer such as polylactide-polyglycolide. The rate of compound release can be controlled according to the ratio of the compound to the polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable reservoir preparations can also be prepared by embedding the compound in liposomes or microemulsions compatible with body tissues.

[0332] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity to release the active compound.

[0333] Solid dosage forms for oral administration of the compounds of the invention include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and gum arabic, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) dissolution retardants such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form optionally also comprises buffering agents.

[0334] Also can adopt similar type solid composition as the filler in the soft or hard filled gelatin capsule using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol etc.Solid dosage form tablets, dragees, capsules, pills and granules can be prepared as having coating and shell, such as other coatings known in the field of enteric coating and pharmaceutical formulation.They optionally contain opacifiers, and can also be compositions that release active ingredient only in or preferentially in a certain part of intestinal tract in a delayed manner.The example of operable embedded composition includes polymeric substances and wax.Also adopt similar type solid composition as the filler in the soft and hard filled gelatin capsule using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol etc.

[0335] The compounds of the present invention can also be formulated into microencapsulated forms together with one or more excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlled coatings, and other coatings known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound of the present invention can be mixed with at least one inert diluent such as sucrose, lactose, or starch. As a general practice, in addition to inert diluents, such dosage forms can also include other substances, 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 form optionally includes a buffer. They optionally contain an emulsifier and can also be a composition that releases the active ingredient only in 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.

[0336] The dosage form for topical or transdermal administration of the compound of the present invention includes ointment, paste, cream, lotion, gel, powder, solution, spray, inhalant or patch. As needed under sterile conditions, active ingredient is mixed with a pharmaceutically acceptable carrier and any required preservative or buffer. Ophthalmic preparations, ear drops and eye drops are also encompassed within the scope of the present invention. In addition, 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 prepared by dissolving or distributing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. Rate control can be achieved by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0337] According to one embodiment, the present invention relates to a method for activating TLR7 / 8 activity in a biological sample, comprising the step of contacting the biological sample with a compound of the present invention or a composition comprising the compound. According to another embodiment, the present invention relates to a method for activating TLR7 / 8 or a mutant thereof in a biological sample, comprising the step of contacting the biological sample with a compound of the present invention or a composition comprising the compound.

[0338] The compounds of the present invention can be used as a unique tool for understanding the biological effects of TLR7 / 8 in vitro, including evaluating factors believed to affect the generation of TLR7 / 8 and the interaction of TLR7 / 8, as well as factors affected by the generation of TLR7 / 8 and the interaction of TLR7 / 8. The compounds of the present invention can also be used to develop other compounds that interact with TLR7 / 8, because the compounds of the present invention provide important structure-activity relationship (SAR) information that contributes to this development. The compounds of the present invention that bind to TLR7 / 8 can be preferably used as reagents for detecting TLR7 / 8 in living cells, fixed cells, biological fluids, tissue homogenates, purified natural biological materials, etc. For example, by detectably labeling the compounds of the present invention, cells expressing TLR7 / 8 can be identified. In addition, based on their ability to bind to TLR7 / 8, the compounds of the present invention can be used for in situ staining, FACS (fluorescence activated cell sorting), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ELISA (enzyme-linked immunosorbent assay), etc., enzyme purification, or for purifying cells expressing TLR7 / 8 in permeabilized cells. The compounds of the present invention can also be used as commercial research reagents for various medical research and diagnostic purposes. Such uses include, but are not limited to: as calibration standards to quantify the activity of candidate TLR7 / 8 activators in various functional assays; as activation reagents in random compound screening (i.e., looking for new TLR7 / 8 ligand families); for co-crystallization with TLR7 / 8, i.e., the compounds of the present invention will allow the formation of crystals of compounds bound to TLR7 / 8, enabling the determination of enzyme / compound structure by x-ray crystallography; other research and diagnostic applications, wherein TLR7 / 8 is preferably activated or such activation is conveniently calibrated relative to a known amount of TLR7 / 8 activator, etc.; used as probes for determining the expression of TLR7 / 8 in cells in assays; and developing assays for detecting compounds bound to the same site as TLR7 / 8 binding ligands. The compounds of the present invention can be used by themselves and / or in combination with physical measurements for diagnosing the effectiveness of treatment. Pharmaceutical compositions containing the compounds and the use of the compounds for treating TLR7 / 8-mediated conditions are promising new broad-spectrum therapies that result in direct and immediate improvements in health in both humans and animals. The orally bioavailable and active novel chemical entities of the present invention will improve patient convenience and physician compliance.

[0339] 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 manufacturing costs and convenient handling. These features form the basis for reproducible action (including lack of cross-reactivity) and reliable and safe interaction with target structures.

[0340] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof; biopsy material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears or other body fluids or extracts thereof.

[0341] Modulation of TLR7 / 8 or its mutant activity in biological samples can be used for a variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ transplantation, biological sample storage, and bioassays.

[0342] Other Uses

[0343] In yet another aspect, the present invention provides a pharmaceutical composition comprising the compounds 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 aspect, the present invention provides a method of stimulating an immune response in a subject. The method includes administering a therapeutically effective amount of the compounds of the present invention described herein under conditions effective to stimulate an immune response. In some embodiments, the method is performed on a subject suffering from cancer. In other embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, kidney cancer (kidney cancer), lung cancer, esophageal cancer, ovarian cancer, prostate cancer, pancreatic cancer, skin cancer, gastric cancer, testicular cancer, bile duct cancer, colorectal cancer, endometrial cancer, head / neck cancer, medullary thyroid cancer, kidney 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 may be included in a liquid pharmaceutical composition. The liquid composition of the present invention can be administered to a tumor (e.g., intratumoral (IT) administration) to preferably induce an innate immune response and a cell-mediated immune response (e.g., to reduce or stabilize a tumor) against tumor antigens. In another embodiment, the compound of the present invention is conjugated to a peptide. The conjugate comprising a peptide is not necessarily an antigen or immunogen, but a mechanism for reducing the dissolution of a TLR7 and / or TLR8 agonist, thereby creating a reservoir trapped at the site of administration (such as within a tumor or in a tumor microenvironment). The conjugated TLR7 and / or TLR8 agonist of the present invention can stimulate immunosuppressive cells and can induce an immune response against antigens present in the tumor. In addition, the mobilization of immunosuppressive cells can not only induce an immune response against the tumor at the site of administration, but can also induce an immune response 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 the subject in an intratumoral or peritumoral manner, wherein the anti-tumor immune response is preferably effective at a site away from the site of administration of the pharmaceutical composition. In another 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 invention as 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, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, cystadenocarcinoma , medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma. In yet another aspect, the present invention provides a method for treating a tumor or abnormal cell proliferation in a subject. The method includes administering a therapeutically effective amount of a compound of the present invention described herein under conditions effective for treating a tumor or abnormal cell proliferation. In some embodiments, the tumor or abnormal cell proliferation is cancer. In some embodiments, the cancer is selected from 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, colorectal cancer, endometrial cancer, head and 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 yet another aspect, the present invention also 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 as described herein under conditions effective to treat an 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 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, 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, herpes zoster, West Nile virus, rabies virus, enterovirus, cytomegalovirus, herpes virus, chickenpox, yellow fever, Zika virus and any combination thereof.In some embodiments, the infectious disease is a bacterial infection, and the infectious disease is preferably selected from streptococcal disease, staphylococcal disease, diphtheria, meningococcal disease, tetanus, pertussis, pneumococcal disease, bacterial food poisoning, sexually transmitted infections, tuberculosis, Lyme disease, botulism, or any combination thereof. In some embodiments, the infectious disease is a fungal infection, and the infectious disease is candidiasis, histoplasmosis, dermatophytosis, tinea pedis, aspergillosis, cryptococcal meningitis, coccidioidomycosis, and any combination thereof.

[0344] Topical, intravesicular and intratumoral compositions comprising compounds of the invention for treating disease

[0345] The compounds of the present invention may be formulated with other compounds such as chemical penetration enhancers. Chemical penetration enhancers are commonly used in topical and transdermal formulations to enhance the absorption, uptake, and delivery of active pharmaceutical ingredients (drugs or drug substances) into the skin. Such formulations containing the compounds of the present invention may be used, for example, to treat skin cancer. Suitable formulations may also be used to supplement further penetration within the local compartment of the tissue and / or 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 ratio of drug and chemical enhancer to skin / tumor penetration; and a gelling agent or matrix for their incorporation into topical or intratumoral formulations. Several examples of chemicals within 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 its skin permeability is neither trivial nor particularly 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 can be expected from their combination in a single formulation. These effects can be additive, positively synergistic, or negatively synergistic. This means that the skin / tumor penetration kinetics and cumulative effects of drugs delivered from these formulations into and through the skin / tumor depend on the specific formulation used. Therefore, it can be seen that, in addition to the drug properties, the physiology, biology, and therapeutic endpoints of the drug also depend to a large extent on the formulation. These endpoints include, but are not limited to, pharmacokinetics / pharmacodynamics (PK / PD), cumulative absorption as determined by the area under the curve (AUC), bioequivalence, therapeutic index (TI), to name a few. In addition, the specific formulation of the drug not only affects its efficacy, but also affects the tolerance / safety of the skin when used. This may include, but is not limited to, adverse effects such as irritation, skin toxicity, erythema, etc.

[0346] In addition to enhancing the penetration of the compounds of the invention through tissues or into local tissue compartments such as tumors, the beneficial effect of chemical penetration enhancers is to provide an inflammatory effect, which is important in directing important immune responses, such as in tumor treatment. Therefore, the incorporation of penetration enhancers into topical, intrathecal and / or injectable formulations can achieve an additive or more than additive effect toward the therapeutic endpoint of tumor treatment.

[0347] The dosage form suitable for use includes creams, ointments, solutions, gels, lotions, pastes, patches, foams or spray formulations, which contain suitable carriers as known in the art. The dosage form for locally, intracapsularly or intratumorally administering the compound of the present invention and optionally other medicaments includes powders, sprays, ointments, pastes, creams, ointments, lotions, gels, solutions and patches. Active ingredient can be mixed with a pharmaceutically acceptable carrier and any preservative, buffer or propellant that may be needed under aseptic conditions. In addition to therapeutic agents, ointments, pastes, creams, lotions, solutions, foams and gels can also contain excipients such as animal and plant fats, oils, waxes, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, organosilicon, bentonite, silicic acid, talc and zinc oxide or their mixtures. Injectable formulations comprising the compounds of the present invention can also be achieved by using purified oils (soybean oil, safflower oil, triolein and castor oil, fractionated coconut oil, miglyol 810, 812, Neobee MS, Captex 300) and FDA-approved dermal fillers (hydroxyapatite, hyaluronic acid, poly-L-lactic acid, collagen, hydrogels) as components of the injectable formulation. Topical, intracapsular, and intratumoral formulations comprising the compounds of the present invention can be designed, which are optimized by selecting the specific ingredients of the formulation (including enhancers, vehicles, and matrices) (Karande 2004 Nature Biotechnology, Karande 2005 PNAS, and related references in and citing these works). Specific formulations can have a profound effect on the efficacy and target indications of the drug. The physicochemical properties of the active compound can be derived from its structure and chemical composition. The chemical descriptor of the penetration enhancer can be estimated, which allows balancing its efficacy and safety. Based on this, a chemical penetration enhancer combination can be determined containing the chemicals in the following list at a total concentration range of 0-2% wt / vol, in a weight fraction range of 0 to 100%. The formulation can contain 2 or 3 individual chemicals in a solvent to prepare the formulation.

[0348] The compounds of the present invention may be combined in a formulation containing one or more of the following additives: sodium octyl sulfate, sodium decyl sulfate, sodium lauryl sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate, sodium eicosyl sulfate, sodium laureth sulfate, nicotine sulfate, sodium taurocholic acid sulfate, dimethyl sulfoxide, sodium tridecyl phosphate, ChemBetaine CAS, ChemBetaine Oleyl, ChemBetaine C, hexadecyldimethylammonium propane sulfonate, decyldimethylammonium propane sulfonate, dodecyldimethylammonium propane sulfonate, myristyldimethylammonium propane sulfonate, benzylpyridinium chloride, dodecylpyridinium chloride, hexadecylpyridinium 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, Triton X-100, caproic acid, caprylic acid, capric acid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid, palmitic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, cholic acid, methyl caproate, 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 sarcosinate, sodium caprylate, sodium caprate, palm Sodium palmitate, sodium oleate, octylamine, decylamine, dodecylamine, tetradecylamine, oleylamine, urea, methylpyrrolidone, cyclohexylpyrrolidone, octylpyrrolidone, decylpyrrolidone, decylmethylpyrrolidone, methylpiperazine, phenylpiperazine, octylamide, hexadecylamide, caprolactam, carveol, pinene oxide, limonene, menthol, pulegone, carvacrol, pinene, menthone, terpineol, eucalyptol, fenchone, glyceryl triacetate, trimethoxypropylenemethylbenzene, linalool, geraniol, and octyldodecanol. Others include sulfoxides, alcohols, polyols, alkanes, fatty acids, esters, amines and amides, terpenes, surfactants, cyclodextrins, C2 or C3 alcohols and higher alcohols, C3 or C4 diols or higher diols, DMSO, DMF, DMA and related solvents, 1-n-dodecyl-cycloazacycloheptan-2-one, N-methyl-pyrrolidone and N-(2-hydroxyethyl)pyrrolidone, and the broader class of azones and mixtures (binary, ternary or higher).

[0349] The compounds of the present 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.

[0350] (Single or double) unsaturation in the hydrocarbon chain can also contribute to the fluidization of the skin lipid bilayer and the penetration of the compounds of the present invention. Oleic acid, linoleic acid, linolenic acid, palmitic acid, myristic acid, etc. are examples of fatty acids. Esters of fatty acids with small alkyl groups such as methyl, ethyl, propyl or butyl, such as palmitate, myristate, oleate, linoleate, etc., are examples of fatty esters. Salts of fatty esters such as sodium lauryl sulfate, sodium oleate, etc. are examples of surfactants. Short-chain alcohols including ethanol, isopropanol, butanol, hexanol, etc. are examples of solvents. If the compounds of the present invention are formulated into topical formulations, the combination of chemicals and solvents can significantly enhance the permeability of the compounds of the present invention across the skin. Combinations of such examples are viable formulations with different efficacy and safety, as well as different PK / PD properties for the API being the compounds of the present invention.

[0351] Examples of formulations comprising the above chemicals and combinations thereof are as follows: a compound of the present invention at a concentration of 0.01, 0.02, 0.03, 0.04 to 1 wt / vol%, wherein the compound is preferably combined with one or more and preferably all of the components listed in i, ii, iii, iv, v, vi, vii, viii, ix, x, xi, xii or xiii below:

[0352] i. Oleic acid (50%) and isopropyl myristate (50%) at a total concentration of 1% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0353] ii. Isopropyl myristate (50%) and sodium lauryl sulfate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0354] iii. Oleic acid (50%) and sodium lauryl sulfate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0355] iv. Oleic acid (33%), isopropyl myristate (33%), and sodium lauryl sulfate (33%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0356] v. Isopropyl palmitate (50%) and sodium oleate (50%) at a total concentration of 2% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0357] vi. Sodium lauryl sulfate (25%) and linoleic acid (75%) at a total concentration of 1.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0358] vii. Palmitic acid (50%) and isopropyl laurate (50%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0359] viii. Oleic acid (50%) and linoleic acid (50%) at a total concentration of 1.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0360] ix. Linoleic acid (25%), oleic acid (25%), and isopropyl linoleate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0361] x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0362] xi. A solution containing 50 ml of phosphate-buffered saline and 50 ml of ethanol.

[0363] xii. Oleic acid (10%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0364] xiii. Oleic acid (2%) and sodium lauryl sulfate (5%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol.

[0365] Pharmaceutical and other uses of the compounds and compositions of the present invention

[0366] The present invention as described comprises providing a composition according to the invention for use as a medicament.

[0367] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient will depend on many factors, including the patient's size, weight, body surface area, age, the specific compound to be administered, the activity of the compound used, the time and route of administration, general health, and combination with other therapies or treatments. Proteinaceous pharmaceutical active substances can be present in an amount of 1 g to 100 mg / kg body weight per dose; however, doses below or above this exemplary range are also envisioned. If the regimen is continuous infusion, it can be in the range of 1 pg to 100 mg per kilogram of body weight per minute.

[0368] The specific amount or dosage to be administered can be determined by a clinician, also based on the above factors. The useful dosage 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; for example, referring to US 4,938,949.

[0369] Generally, the clinician will be able to determine the appropriate dosage depending on the particular disease, disorder or condition to be treated, the efficacy of the particular compound of the invention, the particular route of administration, and the particular pharmaceutical formulation or composition used.

[0370] The desired dosage can be conveniently provided as a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more divided doses per day. The divided dose itself can be further divided into, for example, multiple discrete, loosely spaced administrations. Preferably, the dosage is administered once a week or even less frequently, such as once every two weeks, once every three weeks, once a month or even once every two months.

[0371] The administration regimen may include long-term treatment. "Long-term" refers to a duration of at least two weeks, preferably several weeks, months, or years. Necessary adjustments to the dosage range can be determined by one of ordinary skill in the art using only routine experimentation as taught herein. See Remington's Pharmaceutical Sciences (Martin, EW, ed. 4), Mack Publishing Co., Easton, PA. The individual physician may also adjust the dosage if any complications arise.

[0372] Typically, in the above methods, a compound of the present invention will be used. However, it is 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 other drugs.

[0373] Likewise, the clinician will be able to select such other compounds or drugs and appropriate combination therapy regimens based on the above factors and his or her professional judgment.

[0374] When two or more substances or ingredients are to be used as part of a combination therapy regimen, they can be administered via the same route of administration or via different routes of administration at substantially the same time or at different times (e.g., substantially simultaneously, sequentially, or according to an alternating regimen). When these substances or ingredients are to be administered simultaneously via the same route of administration, they can be administered as different pharmaceutical formulations or compositions, or can be administered as part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

[0375] In addition, when two or more active substances or ingredients are to be used as part of a combined treatment regimen, each substance or ingredient can be used in the same amount and according to the same regimen as when the compound or ingredient is used alone, and such combined use may or may not produce a synergistic effect. However, when the combined use of two or more active substances or ingredients produces a synergistic effect, it is also possible to reduce the amount of one, more or all of the substances or ingredients to be administered while still achieving the desired therapeutic effect. This can, for example, be useful in avoiding, limiting or reducing any undesirable side effects associated with the use of one or more substances or ingredients when they are used in their usual amounts while still achieving the desired drug effect or therapeutic effect.

[0376] The effectiveness of the treatment regimen used in accordance with the present disclosure can be determined and / or tracked in any manner known per se for the disease, disorder, or condition involved, as will be apparent to the clinician. The clinician will also be able to change or modify a particular treatment regimen as appropriate and on a case-by-case basis to achieve the desired therapeutic effect, avoid, limit, or reduce undesirable side effects, and / or strike an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting, or reducing undesirable side effects on the other.

[0377] Typically, the treatment regimen will be followed until the desired therapeutic effect is achieved and / or as long as necessary to maintain the desired therapeutic effect. Again, this can be determined by the clinician.

[0378] Therefore, in yet another aspect, the present invention relates to a pharmaceutical composition comprising at least one compound according to the present invention and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use) and optionally one or more other active substances. In a particular aspect, 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 shown in the claims, more preferably selected from Examples 1 to 6 of the list shown in the claims) and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use) and optionally one or more other active substances.

[0379] The subject to be treated may be any warm-blooded animal, but in particular a mammal, more particularly a human. In veterinary applications, the subject to be treated includes any animal that is raised for commercial purposes or kept as a pet. As will be clear to the skilled person, the subject to be treated is in particular a human suffering from or at risk of developing the diseases, disorders and conditions mentioned 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 they may also be used for veterinary purposes.

[0380] Likewise, in such pharmaceutical compositions, one or more compounds of the invention may also be suitably employed in combination with one or more other active ingredients, such as those mentioned herein.

[0381] The present invention also relates to a pharmaceutical composition for use in vitro (e.g., in an in vitro or cellular assay) or in vivo (e.g., in a unicellular or multicellular organism, particularly in a mammal, more particularly in a human, such as a human at risk of developing or suffering from a disease, disorder or condition of the present invention).

[0382] It will be understood that references to treatment include treatment of established conditions as well as prophylactic treatment unless expressly stated otherwise.

[0383] Typically, for pharmaceutical use, the compound of the present invention can be formulated as a pharmaceutical preparation or composition, which includes at least one compound used in the present invention and at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more pharmaceutically active polypeptides and / or compounds. As a non-limiting example, such a preparation can be suitable for oral administration, parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration (such as intra-articular administration), by inhalation, by skin patch, by implant, by suppository administration, etc., wherein intra-articular administration is preferred. Such suitable administration forms - depending on the mode of administration, which may be solid, semi-solid or liquid - and the methods and carriers used in their preparation will be clear to the skilled person and are further described herein. Such pharmaceutical preparations or compositions are generally referred to herein as "pharmaceutical compositions".

[0384] As exemplary excipients, disintegrants, binding agents, fillers and lubricants can be mentioned. Examples of disintegrants include agar, alginate, calcium carbonate, cellulose, colloidal silicon dioxide, gum, magnesium aluminum silicate, methylcellulose and starch. Examples of binding agents include microcrystalline cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and polyvinyl pyrrolidone. Examples of fillers include calcium carbonate, tricalcium phosphate, tribasic calcium sulfate, carboxymethyl cellulose calcium, 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, glycerol, glyceryl palmitostearate, hydrogenated vegetable oils, magnesium oxide, stearates, mannitol, poloxamers, glycols, sodium benzoate, sodium lauryl sulfate, sodium stearyl, sorbitol, and talc. Conventional stabilizers, preservatives, wetting and emulsifying agents, consistency improvers, flavor improvers, salts for varying the osmotic pressure, buffer substances, solubilizers, diluents, emollients, colorants and masking agents, and antioxidants can all be considered as pharmaceutical adjuvants.

[0385] 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, polyols, glycerin, vegetable oils, and the like.

[0386] In general, the compounds of the present invention can be formulated and administered in any suitable manner known per se. For example, reference is made to the general background art cited above (particularly with reference to WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079) and reference standard handbooks such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, USA (1990), Remington; the Science and Practice of Pharmacy, 21st edition, Lippincott Williams and Wilkins (2005); or Handbook of Therapeutic Antibodies (ed. S. Dubel), Wiley, Weinheim, 2007 (see, for example, pages 252-255).

[0387] In a particular aspect, the invention relates to a pharmaceutical composition comprising a compound according to the invention and further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant and optionally one or more other pharmaceutically active compounds.

[0388] Suitable formulations and methods for their preparation will be clear to the skilled person, and include, for example, preferably formulations suitable for parenteral administration (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial, intrathecal, intranasal or intrabronchial administration) and for topical (e.g., intraarticular, transdermal or intradermal) administration.

[0389] Preparations for topical or parenteral administration may, for example, be sterile solutions, suspensions, dispersions or emulsions suitable for infusion or injection. Suitable carriers or diluents for such preparations include, for example, those mentioned in WO 08 / 020079, page 143. Typically, aqueous solutions or suspensions will be preferred.

[0390] In yet another aspect, the present invention also provides a kit comprising at least one compound according to the present invention. It is envisaged that the kit may be provided in different forms.

[0391] The invention will now be further described with the aid of the following non-limiting preferred aspects, examples and figures.

[0392] Example

[0393] The following examples illustrate the methods of the present disclosure and the products of the present invention.

[0394] A series of novel pharmaceutical TLR7 agonists were designed, chemically prepared and biologically evaluated, as detailed below. Unexpectedly, compared to prior art compounds such as R484 and some recently reported TLR7 / 8 agonists (see Figure 2 ), several new TLR7 agonists not only showed low nanomolar activity in cellular TLR7 assays, but also exhibited high selectivity between TLR7 and TLR8. Even more surprisingly, these new highly selective TLR7 agonists could elicit high cytokine and chemokine (IL-6, IL1-b, and TNF-a) production in cellular monocyte assays and induce strong upregulation of CD40 and CD86.

[0395] Chemical synthesis of TLR7 agonists

[0396] Chemical synthesis of Example 1

[0397]

[0398] Step 1

[0399] To a stirred solution of R848 (750.00 mg; 2.36 mmol; 1.00 eq.) in acetonitrile (15.00 ml; 20.00 V) was added triethylamine (0.93 ml; 7.09 mmol; 3.00 eq.) and then chloro-(diphenyl)methyl]benzene (1.33 g; 4.72 mmol; 2.00 eq.) under a nitrogen atmosphere. The resulting suspension was irradiated in a microwave reactor at 100° C. for 1.15 hours. The reaction was monitored by TLC and the solvent was then removed in vacuo to obtain a crude product. The crude product was purified by flash column chromatography on 100-200 silica gel using ethyl acetate and hexane. The desired product was eluted in 25-30% ethyl acetate and hexane, and the eluent was concentrated to give compound 1 (1.30 g; 2.31 mmol; 97.8%; white solid; purified product).

[0400] Yield: 1.30 g (2.31 mmol), yield %: 97.8.

[0401] Analyze the data:

[0402] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0403] RT (min): 2.13; purity: 98.94%; M+H: 557.10.

[0404] Step 2:

[0405] 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, and then 1,2,3-oxathiazolidine-3-carboxylic acid tert-butyl ester 2,2-dioxide (441.12 mg; 1.98 mmol; 1.10 eq.) was added. The reaction mixture was stirred at room temperature for 12 hours, and the reaction progress was monitored by LCMS. After completion, the reaction mixture was quenched with cold water and extracted with ethyl acetate (2 x 100 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give a crude product. The crude product was purified by silica gel column chromatography. The desired product was eluted in 60% ethyl acetate / 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).

[0406] Yield: 550.00 mg (0.75 mmol), yield %: 41.9.

[0407] Analyze the data:

[0408] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0409] RT (min): 2.60; purity: 95.70%; M+H: 700.00.

[0410] Step 3:

[0411] 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 room temperature for 3 hours. The progress of the reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure. The crude product was washed with diethyl ether (2 x 25 ml) and dried under reduced pressure to give compound 3 (360.00 mg; 0.73 mmol; 97.4%; pale white solid; purified product) as a trifluoroacetate salt.

[0412] Yield: 360.00 mg (0.73 mmol), yield %: 97.4.

[0413] Analyze the data:

[0414] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0415] RT (min): 0.94; purity: 95.90%; M+H: 358.00.

[0416] Step 4:

[0417] 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 DMF (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 progress of the reaction 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% NH4OAc / water and acetonitrile as eluents. The desired fractions were lyophilized to give Example 1 (4.50 mg; 0.01 mmol; 2.4%; brown gummy solid; purified product).

[0418] Yield: 4.50 mg (0.01 mmol), yield %: 2.4.

[0419] Analyze the data:

[0420] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0421] RT (min): 1.76; purity: 95.81%; M+H: 441.30.

[0422] HPLC: column: XBridge C8, 3.5 μm, 4.6 x 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.

[0423] RT (min): 1.72; purity: 96.98% (maximum); purity: 88.09% (220 nm).

[0424] 1H NMR (400MHz, DMSO-d6): 9.12 (s, 2H), 8.55 (d, J = 8.00Hz, 1H), 7.80 (d, J = 8.00Hz, 1H), 7.71 (t, J = 8.00Hz, 1H), 7.54 (t, J = 7 .20Hz,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=6.80Hz,3H).

[0425] Chemical synthesis of Example 2

[0426]

[0427] Step 1:

[0428] 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) at 0°C under a nitrogen atmosphere was added dropwise compound 1 (900.00 mg; 1.53 mmol; 1.00 eq.) in DMSO (5 ml) and the reaction mixture was stirred at room temperature for 30 minutes. (3-Bromopropoxy)(tert-butyl)dimethylsilane (0.78 ml; 4.77 mmol; 3.12 eq.) was then added at 0°C and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. 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 / hexane to give compound 4 (215.00 mg; 0.28 mmol; 18.4%; gum; purified product).

[0429] Yield: 215.00 mg (0.28 mmol), yield %: 18.4.

[0430] Analyze the data:

[0431] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0432] RT (min): 3.59; purity: 95.53%; M+H: 729.30.

[0433] Step 2:

[0434] 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.) at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 3 hours and the completion of the reaction was confirmed by TLC. 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).

[0435] Yield: 150.00 mg (0.24 mmol), yield %: 100.

[0436] Analyze the data:

[0437] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0438] RT (min): 2.22; purity: 99.51%; M+H: 615.00.

[0439] Step 3:

[0440] To a stirred solution of compound 5 (160.00 mg; 0.26 mmol; 1.00 eq.) in DCM (3.30 ml; 20.63 V) was added 1,1,1-tris(acetoxy)-1,1-dihydro-1,2-benzoiodine oxalanine-3-(1H)-one (0.34 g; 0.77 mmol; 3.00 eq.) at 0°C and the reaction mixture was stirred at room temperature for 30 minutes. Completion of the reaction was confirmed by TLC. The reaction mixture was washed with 10% NaHCO3 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).

[0441] Yield: 170.00 mg (0.21 mmol), yield %: 81.8.

[0442] Analyze the data:

[0443] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0444] RT (min): 2.36; purity: 75.96%; M+H: 613.00.

[0445] Step 4:

[0446] 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 room temperature. The reaction mixture was stirred at room temperature 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 room temperature 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 yield compound 7 (140.00 mg; 0.15 mmol; 71.7%; gum; crude product).

[0447] Yield: 140.00 mg (0.15 mmol), yield %: 71.7.

[0448] Analyze the data:

[0449] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0450] RT (min): 1.75; purity: 75.26%; M+H: 697.00.

[0451] Step 5:

[0452] To a stirred solution of compound 7 (100.00 mg; 0.11 mmol; 1.00 eq.) in DCM (5.00 ml; 50.00 V) at 0°C was added TFA (0.90 ml; 11.60 mmol; 9.00 V). The resulting solution was warmed to room temperature and stirred for 3 hours. The reaction was monitored by LCMS, and the solvent was then removed under reduced pressure below 35°C to give the crude product. The crude product was purified by preparative HPLC using the HCOOH method and the desired fractions were lyophilized to give Example 2 as a formate salt (32.91 mg; 0.07 mmol; 60.7%; off-white gum; purified product).

[0453] Yield: 32.91 mg (0.07 mmol), yield %: 60.7.

[0454] Analyze the data:

[0455] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0456] RT(min): 1.03; M+H: 455.20.

[0457] HPLC: column: XBridge C8, 3.5 μm, 4.6 x 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.

[0458] RT (min): 9.52; purity: 99.83% (220 nm).

[0459] 1 HNMR (400MHz, DMSO-d6): 8.36(dd,J=8.00,Hz,1H),8.15(s,1H),7.62(dd,J=0.80,8.20Hz,1H),7.45(t,J=7.20Hz,1H),7.25(t,J=6.80Hz,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,2 H),2.52-2.50(m,11H),2.10(m,2H),1.42-1.38(m,2H),1.24-1.12(m,9H).

[0460] Chemical synthesis of Example 3

[0461]

[0462] Step 1:

[0463] 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 room temperature under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 30 minutes. 1,4-Dibromobutane (4.83 ml; 39.46 mmol; 5.43 eq.) was added at room temperature, and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. 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 obtain a crude residue. The crude residue was purified by silica gel chromatography. The product was eluted with 15% ethyl acetate / hexane to give compound 8 (145.00 mg; 0.14 mmol; 2.0%; white gum; purified product); the unreacted starting material was eluted with 45% ethyl acetate / petroleum ether to give compound 1 (3.20 g; 5.12 mmol; 70.4%; white solid; purified product).

[0464] Yield: 145 mg (0.14 mmol), yield %: 2.0.

[0465] Analyze the data:

[0466] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0467] RT (min): 2.75; purity: 68.10%; M+H: 690.90.

[0468] Step 2:

[0469] To a stirred 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) was added potassium carbonate (189.73 mg; 1.35 mmol; 2.00 eq.) at room temperature. The suspension was heated to 50° C. for 16 hours and the reaction was monitored by TLC. After completion, the solvent was removed to obtain 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 obtain a crude material. The material was purified by silica gel column chromatography. The desired product was eluted in 7% MeOH / DCM to obtain compound 9 (400.00 mg; 0.55 mmol; 82.1%; off-white powder; purified product).

[0470] Yield: 400 mg (0.55 mmol), yield %: 82.1.

[0471] Analyze the data:

[0472] LCMS: column: Atlantis dC18 (50x4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0473] RT (min): 1.79; purity: 98.20%.

[0474] Step 3:

[0475] To a stirred 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 using the HCOOH method and the desired fractions were lyophilized to give Example 3 as a formate salt (44.00 mg; 0.08 mmol; 74.1%; off-white gum; purified product).

[0476] Yield: 44.00 mg (0.08 mmol), yield %: 74.1.

[0477] Analyze the data:

[0478] LCMS: column: Atlantis dC18 (50x4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0479] RT (min): 0.99; purity: 99.05%; M+H: 469.10.

[0480] 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.

[0481] RT (min): 7.96; purity: 99.22% (max); purity: 97.91% (220 nm).

[0482] 1HNMR(400MHz,DMSO-d6):8.33(d,J=8.40Hz,1H),7.59(d,J=8.40Hz,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=9.20Hz,4H),2.078-2.335(m,11H),1.13-1.26(m,14H),(s,H),(s,H),(s,H),(s,H).

[0483] Chemical synthesis of Example 4

[0484]

[0485] Step 1:

[0486] To a stirred solution of sodium hydride (104.78 mg; 2.62 mmol; 3.00 eq.) in DMSO (10.00 ml; 20.00 V) at 0 ° C under a nitrogen atmosphere was added a solution of compound 1 (500.00 mg; 0.87 mmol; 1.00 eq.) in DMSO (5 ml). The reaction mixture was stirred for 30 minutes. 1,5-dibromopentane (614.64 mg; 2.62 mmol; 3.00 eq.) was then added and the resulting solution was stirred at room temperature for 16 hours. The reaction was monitored by LCMS. After completion, the reaction mixture was quenched with cold water and extracted with ethyl acetate (2x100 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give a crude product. The crude product was purified by silica gel column chromatography. The desired product was eluted in 16% ethyl acetate / 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).

[0487] Yield: 150.00 mg (0.21 mmol), yield %: 23.7.

[0488] Analyze the data:

[0489] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0490] RT (min): 3.32; purity: 97.45%; M+H: 705.30.

[0491] Step 2:

[0492] To a stirred solution of compound 10 (150.00 mg; 0.21 mmol; 1.00 eq.) in acetonitrile (3.00 ml; 20.00 V) 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.) at room temperature. The suspension was heated to 50° C. and stirred for 16 hours. The reaction was monitored by LCMS. After completion, the reaction mixture was concentrated under reduced pressure to obtain a crude residue. The residue was dissolved in DCM (100 ml) and washed with brine (2 x 50 ml). The organic layer was concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography. The desired product was eluted in 7% MeOH / DCM. The combined pure fractions were concentrated under reduced pressure to obtain compound 11 (70.00 mg; 0.09 mmol; 41.1%; off-white gum; purified product)

[0493] Yield: 70.00 mg (0.09 mmol), yield %: 41.1.

[0494] Analyze the data:

[0495] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0496] RT (min): 1.72; purity: 88.13%; M+H: 725.20.

[0497] Step 3:

[0498] To a stirred 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 material. The material was purified by preparative HPLC using the HCOOH method. The desired fractions were lyophilized to give Example 4 (32.00 mg; 0.06 mmol; 72.6%; light yellow gum; purified product) as a formate salt.

[0499] Yield: 32.00 mg (0.06 mmol), yield %: 72.6.

[0500] Analyze the data:

[0501] LCMS: column: Atlantis dC18 (50x4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0502] RT (min): 1.09; purity: 99.24%; M+H: 483.10.

[0503] HPLC: column: Phenomenex Gemini C18 (150*4.6) mm, 3.0 μm; solvent A: 0.1% TFA / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.0 ml / min.

[0504] RT (min): 8.82; purity: 99.42% (maximum); purity: 98.26% (220 nm).

[0505] 1HNMR (400MHz, DMSO-d6): δ8.57(d,J=7.60Hz,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=4.40Hz,2H).

[0506] Chemical synthesis of Example 5

[0507]

[0508] Step 1:

[0509] To a stirred solution of sodium hydride (60%) (122.25 mg; 3.06 mmol; 2.50 eq.) in DMSO (7.00 ml; 10.00 V) at 0°C was added compound 1 (700.00 mg; 1.22 mmol; 1.00 eq.) in DMSO (5 ml) dropwise, and the reaction mixture was stirred at room temperature for 30 minutes. 1,6-Dibromohexane (0.58 ml; 3.67 mmol; 3.00 eq.) was then added to the reaction mixture at 0°C, and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. 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, and 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 / hexane to give compound 12 (200.00 mg; 0.21 mmol; 17.1%; off-white oil; purified product).

[0510] Yield: 200.00 mg (0.21 mmol), yield %: 17.1.

[0511] Analyze the data:

[0512] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0513] RT (min): 3.26; purity: 75.40%; M+H: 719.00.

[0514] Step 2:

[0515] 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) was added potassium carbonate (88.64 mg; 0.63 mmol; 3.00 eq.) at room temperature. The suspension was heated to 50° C. and stirred for 16 hours. The reaction was monitored by LCMS. After completion, the solvent was removed to obtain 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 obtain a crude residue. The residue was purified by silica gel chromatography. The desired product was eluted with 8% MeOH / DCM to obtain compound 13 (110.00 mg; 0.14 mmol; 67.6%; white solid; purified product).

[0516] Yield: 110.00 mg (0.14 mmol), yield %: 67.6.

[0517] Analyze the data:

[0518] LCMS: Column: Atlantis dC18 (50x4.6mm, 5μ), +ve mode; Mobile phase: A: 0.1% HCOOH / H2OB:ACN; Flow rate: 1.5ml / min

[0519] RT (min): 1.79; purity: 95.17%; M+H: 739.00.

[0520] Step 3:

[0521] 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 progress of 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 using the HCOOH method. The desired fractions were lyophilized to give Example 5 as a formate salt (60.00 mg; 0.11 mmol; 77.1%; white solid; purified product).

[0522] Yield: 60.00 mg (0.11 mmol), yield %: 77.1.

[0523] Analyze the data:

[0524] LCMS: column: Atlantis dC18 (50x4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0525] RT (min): 1.17; purity: 98.84%; M+H: 497.10.

[0526] HPLC: column: XBridge C8, 3.5 μm, 4.6 x 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.

[0527] RT (min): 5.39; purity: 99.49% (max); purity: 99.54% (220 nm).

[0528] 1 HNMR (400MHz, MeOD): δ8.60 (d, J = 8.40Hz, 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).

[0529] Chemical synthesis of Example 6

[0530] Step 1:

[0531] To a stirred solution of benzyl alcohol (1.00 g; 9.20 mmol; 1.00 eq.) in a 10% aqueous sodium hydroxide solution (0.92 g; 23.00 mmol; 2.50 eq.) dissolved in water (20.00 ml; 20.00 V) at 0°C was added tetrabutylammonium hydrogen sulfate (64.42 mg; 0.18 mmol; 0.02 eq.), followed by 1,4-dibromobutane (3.99 g; 18.4 mmol; 2 eq.). The resulting mixture was stirred at 70°C for 4 hours 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 yield the crude product. The product was purified by flash column chromatography on 100-200 silica gel using ethyl acetate and hexanes. The desired product was eluted in 1-5% ethyl acetate and hexane, and the eluate was concentrated to give compound 14 (1.00 g; 4.05 mmol; 44.0%; colorless oil; purified product)

[0532] Yield: 1 g (4.05 mmol), yield %: 44.

[0533] Analyze the data:

[0534] 1 HNMR (400MHz, DMSO-d6): δ7.40-7.29 (m, 5H), 4.53 (s, 2H), 3.53 (t, J = -12.40Hz, 2H), 3.46 (t, J = 6.80Hz, 2H), 2.04-1.79 (m, 4H).

[0535] Step 2:

[0536] 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) at 0° C. under a nitrogen atmosphere was added dropwise compound 1 in DMSO (5 mL) and the reaction mixture was stirred at room temperature for 30 minutes. 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 room temperature under a nitrogen atmosphere for 16 hours. The reaction mixture was monitored by TLC. After 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 a crude material. The substance was purified by flash column chromatography on 100-200 silica gel using ethyl acetate and hexane. The desired product was eluted in 10-15% ethyl acetate and hexane, and the eluate was concentrated to give compound 15 (330.00 mg; 0.45 mmol; 24.8%; off-white gum; purified product)

[0537] Yield: 330 mg (0.45 mmol), yield %: 24.8.

[0538] Analyze the data:

[0539] LCMS: column: XBridge C8 (50x4.6 mm) 3.5 μm; solvent A: 0.1% TFA / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0540] RT (min): 3.35; purity: 97.21%; M+H: 719.3.

[0541] Step 3:

[0542] 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 room temperature. The resulting reaction mixture was stirred at room temperature in an autoclave under 5 kg hydrogen pressure. After completion, the reaction mixture was filtered through celite. The filtrate was concentrated under reduced pressure to give a crude material. The material was purified by preparative HPLC using 0.1% HCOOH / water and ACN as eluents. The combined pure fractions were lyophilized to give Example 6 (20.00 mg; 0.05 mmol; 37.9%; off-white solid; purified product) as a formate salt.

[0543] Yield: 20 mg (0.05 mmol), yield %: 37.9.

[0544] Analyze the data:

[0545] LCMS: column: Atlantis dC18 (50x4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H2O:ACN (95:5), solvent B: ACN; flow rate: 1.5 ml / min.

[0546] RT (min): 1.46; purity: 99.69%; M+H: 387.1

[0547] HPLC: Column: Atlantis dC18 (50x4.6mm) 3.5μm; Solvent A: 0.1% HCOOH / H2O:ACN (95:5), Solvent B: CAN

[0548] RT (min): 7.01; purity (maximum): 99.86%; purity (220nm): 99.32%.

[0549] 1 HNMR (400MHz, DMSO-d6): δ0.00(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 .20Hz, 2H), 3.35 (t, J = 6.00Hz, 2H), 3.21 (s, 1H), 1.32 (q, J = 2.00Hz, 4H), 1.26 (q, J = 10.80Hz, 4H), 1.14 (q, J = 7.20Hz, 4H).

[0550] Chemical synthesis of Example 7

[0551]

[0552] Step 1:

[0553] To a suspension of sodium hydride (60%) (0.21 g; 5.31 mmol; 2.30 eq.) in DMSO (13.00 ml; 10.00 V) at 0°C under a nitrogen atmosphere was added a solution of compound 1 (1.30 g; 2.31 mmol; 1.00 eq.) in DMSO (13.00 ml; 10.00 V) dropwise. The mixture was then stirred at room temperature for 30 minutes. 1,4-Dibromobutane (0.76 g; 3.46 mmol; 1.50 eq.) was added at 0°C, and the reaction mixture was stirred at room temperature for an additional hour. Upon completion, the reaction mixture was quenched with ice water, diluted with ethyl acetate, and 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 / hexane) to give compound 16 (120.00 mg; 0.17 mmol; 7.3%; white solid; purified product).

[0554] Yield: 120.00 mg (0.17 mmol), yield %: 7.3.

[0555] Analyze the data:

[0556] LCMS column: XBridge C8, 3.5 μm, 4.6×50 mm; mobile phase: A: 0.1% TFA / H 2 O:ACN (95:5); mobile phase B: 0.1% TFA / ACN; flow rate: 1.5 ml / min.

[0557] RT (min): 3.28; Purity (max): 97.18%; M+H: 691.20

[0558] Step 2:

[0559] 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) was added potassium carbonate (35.67 mg; 0.25 mmol; 3.00 eq.) at room temperature. The suspension was then heated to 70°C and stirred for 20 hours, and the progress of the reaction 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, dried over sodium sulfate, and then the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography (60% EtOAc / petroleum ether) to give compound 17 (60.00 mg; 0.08 mmol; 97.8%; gum; purified product).

[0560] Yield: 60.00 mg (0.08 mmol), yield %: 97.8.

[0561] Analyze the data:

[0562] LCMS-column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase A: 0.1% HCOOH / water; B: 0.1% HCOOH / ACN; flow rate: 0.8 ml / min.

[0563] RT (min): 1.88; Purity (max): 95.95%; M+H: 698.00

[0564] Step 3:

[0565] 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 room temperature and stirred for an additional 3 hours. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column chromatography (10% methanol / dichloromethane) to afford Example 7 (34.00 mg; 0.07 mmol; 89.5%; off-white gum; purified product).

[0566] Yield: 34.00 mg (0.07 mmol), yield %: 89.5.

[0567] Analyze the data:

[0568] LCMS-column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase A: 0.1% HCOOH / water; B: 0.1% HCOOH / ACN; flow rate: 0.8 ml / min.

[0569] RT (min): 1.15; Purity (max): 98.94%; M+H: 456.10.

[0570] HPLC-column: ATLANTIS dC18 (50x4.6 mm) 5 mm; mobile phase A: 0.1% TFA / MilliQ water; mobile phase B: acetonitrile; flow rate: 1.0 ml / min.

[0571] RT (min): 9.25; purity (max): 95.25%; M+H: 456.10; purity (220 nm): 96.41%.

[0572] Chemical synthesis of Example 8

[0573]

[0574] Step 1:

[0575] 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 room temperature. The suspension was heated to 70° C. and stirred for 20 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and the residue was subjected to flash chromatography (5% MeOH / dichloromethane) to give compound 18 (40.00 mg; 0.06 mmol, 66.9%; gum, purified product).

[0576] Yield: 40.00 mg (0.06 mmol), yield %: 66.9.

[0577] Analyze the data:

[0578] LCMS-column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase: A: 0.1% HCOOH / H2O:ACN (95:5), B: ACN flow rate: 1.5 ml / min.

[0579] RT (min): 2.01; Purity (max): 99.36%; M+H: 696.1

[0580] Step 2:

[0581] 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 allowed to warm to room temperature and stirred for an additional 3 hours. The progress of the reaction 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 / dichloromethane) to afford Example 8 (16.00 mg; 0.03 mmol; 61.0%; light brown gum; purified product).

[0582] Yield: 16.00 mg (0.03 mmol), yield %: 61.0.

[0583] Analyze the data:

[0584] LCMS-column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase: A: 0.1% HCOOH / H2O:ACN (95:5), B: ACN flow rate: 1.5 ml / min.

[0585] RT (min): 1.24; Purity (max): 98.78%; M+H: 454.10.

[0586] Chemical synthesis of Example 9

[0587]

[0588] Step 1:

[0589] 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 room temperature. The suspension was heated to 70° C. and stirred for 20 hours, and the reaction was monitored by TLC. After completion of the reaction, the solvent was removed, and the residue was dissolved in water and then 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 / petroleum ether) to give compound 19 (60.00 mg; 0.09 mmol; 99.8%; gum; purified product).

[0590] Yield: 60.00 mg (0.09 mmol), yield %: 99.8.

[0591] Analyze the data:

[0592] LCMS-column: Column: ATLANTIS dC18 (50x4.6mm) 5mm; Mobile phase: A: 0.1% HCOOH / H2O:ACN (95:5), B: ACN; Flow rate: 1.5ml / min.

[0593] RT (min): 1.99; Purity (max): 96.78%; M+H: 682.00.

[0594] Step 2:

[0595] 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 room temperature and stirred for 3 hours. The reaction was monitored by TLC. Upon completion, the reaction mixture was concentrated under reduced pressure and the crude product was purified by column chromatography (10% methanol / dichloromethane) to afford Example 9 (20.00 mg; 0.05 mmol; 54.3%; off-white gum; purified product).

[0596] Yield: 20.00 mg (0.05 mmol), yield %: 54.3.

[0597] Analyze the data:

[0598] LCMS-column: ATLANTIS dC18 (50×4.6 mm) 5 mm; mobile phase: A: 0.1% HCOOH / H 2 O:ACN (95:5), B: ACN; flow rate: 1.5 ml / min.

[0599] RT (min): 1.20; Purity (max): 99.56%; M+H: 440.10.

[0600] HPLC-column: ATLANTIS dC18 (50x4.6 mm) 5 mm; mobile phase A: 0.1% TFA / MilliQ water; mobile phase B: acetonitrile; flow rate: 1.0 ml / min.

[0601] RT (min): 5.76; purity (max): 99.55%; M+H: 456.10; purity (220 nm): 99.42%.

[0602] Chemical synthesis of Example 10

[0603]

[0604] Step 1:

[0605] To a stirred solution of compound 16 (50.0 mg; 0.07 mmol; 1.00 eq.) and tert-butyl piperazine-1-carboxylate (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 room temperature. The suspension was heated to 70° C. and stirred for 20 hours. The progress of the reaction 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 / petroleum ether) to give compound 20 (45 mg; 0.06 mmol; 78.5%; gum; purified product).

[0606] Yield: 45.00 mg (0.06 mmol), yield %: 78.5.

[0607] Analyze the data:

[0608] LCMS-column: Atlantis dC18 (50×4.6 mm, 5 μm), +ve mode; 0.1% HCOOH / H 2 O, B: ACN; flow rate: 1.5 ml / min.

[0609] RT (min): 2.07; Purity (max): 99.44%; M+H: 797.00

[0610] Step 2:

[0611] To a stirred 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 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, and the crude product was purified by preparative HPLC. The product fractions were lyophilized to give Example 10 (25.00 mg; 0.05 mmol; 88.1%; off-white gum; purified product) as a formate salt.

[0612] Yield: 25.00 mg (0.05 mmol), yield %: 88.1.

[0613] Analyze the data:

[0614] LCMS-column: Atlantis C18 (50×4.6 mm, 5 μm); solvent A: water+0.1% TFA; solvent B: ACN+0.1% TFA; flow rate: 2 ml / min.

[0615] RT (min): 9.46; Purity (max): 98.13%; M+H: 455.30.

[0616] HPLC-column: Atlantis dC18 (50x4.6mm, 5μm), +ve mode; 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, flow rate: 0.8mL / min.

[0617] RT (min): 8.47; Purity (max): 99.05%; Purity (220 nm): 98.46%.

[0618] 1H NMR(400MHz,MeOD):8.56(d,J=8.40Hz,1H),8.44(s,1H),7.78(d,J=0.52Hz,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).

[0619] Chemical synthesis of Example 11

[0620]

[0621] Step 1:

[0622] To a stirred solution of compound 16 (145.00 mg; 0.14 mmol; 1.00 eq.) and (2-piperazine-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 room temperature. The suspension was heated to 80° C. and stirred for 16 hours. 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 in 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).

[0623] Yield: 105.00 mg (0.12 mmol), yield %: 84.4

[0624] Analyze the data:

[0625] LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: A: 0.1% HCOOH / H2O B: ACN; Flow rate: 1.5ml / min

[0626] RT (min): 2.04; purity: 96.44%; M+H: 840.10

[0627] Step 2:

[0628] To a stirred 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 room temperature and stirred for 3 hours. 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 / H2O and ACN. The product fractions were lyophilized to give Example 11 (49.00 mg; 0.09 mmol; 77.9%; brown gum; purified product) as a formate salt.

[0629] Yield: 49 mg (0.09 mmol), yield %: 77.9

[0630] Analyze the data:

[0631] LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA / H 2 O, B: ACN;

[0632] Flow rate: 1.5ml / min.

[0633] RT (min): 0.1.88; purity: 99.42%; M+H: 498.30

[0634] HPLC: Column: 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; 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): 4.09; Purity (max): 99.14%; Purity (220 nm): 99.53%.

[0635] 1 HNMR (400MHz, DMSO-d6): δ8.44(d,J=8.40Hz,1H),8.16(s,1H),7.51-7.67(m,5H),7.34(t,J=7.60Hz,1H),4.75(s,4H),3.52(t,J=6.8 0Hz,3H),3.38(s,2H),3.21(s,2H),(s,3H),2.66(s,2H),2.51(d,J=2.00Hz,3H),2.46(d,J=36.00Hz,3H),1.13-1.20(m,13H),(s,H).

[0636] Chemical Synthesis of Example 12

[0637]

[0638] Step 1:

[0639] To a stirred solution of compound 16 (60.00 mg; 0.08 mmol; 1.00 eq.) and 2-(piperazin-1-yl)ethan-1-ol (20.30 mg; 0.15 mmol; 2.00 eq.) in acetonitrile (1.80 ml; 30.00 V) was added potassium carbonate (32.33 mg; 0.23 mmol; 3.00 eq.) at room temperature. The suspension was heated to 80° C. and stirred for 16 hours. The reaction 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 to give the crude product of compound 22 (47.00 mg; 0.06 mmol; 80.3%; off-white powder; crude product).

[0640] Yield: 47 mg (0.06 mmol), yield %: 80.3

[0641] Analyze the data:

[0642] LCMS: column: XBridge C8, 3.5 μm, 4.6 x 50 mm; mobile phase: A: 0.1% TFA / H 2 O, B: ACN; flow rate: 1.5 ml / min.

[0643] RT (min): 2.60; purity: 96.73%; M+H: 741.30

[0644] Step 2:

[0645] To a stirred 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 / HO and ACN. The product fractions were lyophilized to give Example 12 (19.00 mg; 0.03 mmol; 56.5%; off-white gum; purified product) as a formate salt.

[0646] Yield: 19 mg (0.03 mmol), yield %: 56.5

[0647] Analyze the data:

[0648] LCMS: Column: ATLANTIS dC18 (50x4.6mm) 5μm; Mobile phase: A: 0.1% HCOOH / H2O B: ACN; Flow rate: 1.5ml / min

[0649] RT (min): 0.98; purity: 97.32%; M+H: 499.10

[0650] HPLC: Column: 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; 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): 9.14; Purity (max): 95.04%; Purity (220 nm): 92.88%.

[0651] 1 HNMR (400MHz, DMSO-d6): δ8.54(d,J=0.80Hz,2H),7.76(d,J=0.80Hz,1H),7.65-7.74(m,1H),7.45-7.49(m,1H),3.77(t,J=11.20Hz ,2H),3.65(t,J=14.00Hz,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),

[0652] Chemical Synthesis of Example 13

[0653]

[0654] Step 1:

[0655] To a stirred solution of compound 16 (180.00 mg; 0.25 mmol; 1.00 eq.) was added sodium azide (20.00 mg; 0.30 mmol; 1.20 eq.) in DMF (3.60 ml; 20.00 V). The suspension was stirred at room temperature 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 the crude product of compound 23 (150.00 mg; 0.15 mmol; 57.7%; off-white powder; crude product).

[0656] Yield: 150 mg (0.15 mmol), yield %: 57.7

[0657] Analyze the data:

[0658] LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA / H2O; B: ACN;

[0659] Flow rate: 1.5ml / min

[0660] RT (min): 3.24; purity: 63.86%; M+H: 654.30.

[0661] Step 2:

[0662] 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) were added copper (I) iodide (7.90 mg; 0.04 mmol; 0.60 eq.) and ethyl-diisopropyl-amine (0.01 ml; 0.07 mmol; 1.00 eq.). The reaction mixture was stirred at room temperature 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 give a crude product. The crude product was purified by column chromatography using methanol / DCM as eluent, and the desired product was eluted in 8% methanol / DCM. The desired fraction was evaporated to give compound 24 (40.00 mg; 0.05 mmol; 70.6%; white solid; purified product).

[0663] Yield: 40 mg (0.05 mmol), yield %: 70.6

[0664] Analyze the data:

[0665] LCMS: column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase: A: 0.1% HCOOH / H2O B: ACN; flow rate: 1.5 ml / min.

[0666] RT (min): 2.23; purity: 85.71%; M+H: 710.00

[0667] Step 3:

[0668] 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. After completion, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography using methanol / DCM as eluent, and the desired product was eluted in 15% methanol / DCM. The desired fractions were evaporated to give Example 13 (18.00 mg; 0.04 mmol; 76.5%; light brown gum; purified product).

[0669] Yield: 18 mg (0.04 mmol), yield %: 76.5.

[0670] Analyze the data:

[0671] LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA / H 2 O, B: ACN;

[0672] Flow rate: 1.5ml / min

[0673] RT (min): 2.10; purity: 97.88%; M+H: 468.20

[0674] HPLC: column: Phenomenex Gemini C18 (150*4.6) mm, 3.0 μm; mobile phase A: 10 mM ammonium acetate / milli-q water; mobile phase B: acetonitrile; flow rate: 1.0 ml / min.

[0675] RT (min): 8.10; Purity (max): 95.26%; Purity (220nm): 90.72%

[0676] Chemical Synthesis of Example 14

[0677]

[0678] Step 1:

[0679] 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) was added copper (I) iodide (15.30 mg; 0.08 mmol; 0.60 eq.) and ethyldiisopropylamine (0.05 ml; 0.27 mmol; 2.00 eq.). The reaction mixture was stirred at room temperature 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 give the crude compound. The crude product was purified by column chromatography using methanol / DCM as eluent and the desired product was eluted in 8% methanol / DCM. The desired fractions were evaporated to give compound 25 (50.00 mg; 0.06 mmol; 45.2%; white solid; purified product).

[0680] Yield: 50 mg (0.06 mmol), yield %: 45.2.

[0681] Analyze the data:

[0682] LCMS: Column: ATLANTIS dC18 (50x4.6 mm) 5 mm; Mobile phase A: 0.1% HCOOH / water; B: 0.1% HCOOH / ACN; Flow rate: 0.8 ml / min.

[0683] RT (min): 2.51; purity: 96.95%; M+H: 809.00

[0684] Step 2:

[0685] 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 room temperature and stirred for 3 hours. The reaction was monitored by TLC. After completion, the reaction mixture was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography using methanol / DCM as eluent, and the desired product was eluted in 15% methanol / DCM. The desired fractions were evaporated to give Example 14 (15.00 mg; 0.03 mmol; 46.2%; off-white gum; purified product).

[0686] Yield: 15 mg (0.03 mmol), yield %: 46.2

[0687] Analyze the data:

[0688] LCMS: Column: XBridge C8, 3.5 μm, 4.6 x 50 mm; Mobile phase: A: 0.1% TFA / H 2 O:ACN (95:5); Mobile phase B: 0.1% TFA / ACN; Flow rate: 1.5 ml / min.

[0689] RT (min): 1.98; purity: 99.94%; M+H: 467.20

[0690] HPLC: column: ATLANTIS dC18 (50x4.6 mm) 5 mm; mobile phase A: 0.1% TFA / MilliQ water; mobile phase B: acetonitrile; flow rate: 1.0 ml / min.

[0691] RT (min): 5.42; purity (maximum): 99.86%; purity (220 nm): 99.30%.

[0692] Chemical Synthesis of Example 15

[0693]

[0694] Step 1:

[0695] 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) at 0°C was added sulfuric acid and chromium trioxide (0.10 ml). The resulting reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The crude product was washed with diethyl ether (10 mL) to afford compound 26 (45.00 mg; 0.10 mmol; 81.1%; blue gum; crude product). This crude product was used directly in the next step.

[0696] Yield: 45.00 mg (0.10 mmol), yield %: 81.1.

[0697] Analyze the data:

[0698] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 m; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 1.5 mL / min.

[0699] RT (min): 1.47; Purity (max): 92.18%; M+H: 401.10.

[0700] Step 2:

[0701] 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.). Ethyl-diisopropyl-amine (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° C. under a nitrogen atmosphere. The reaction mixture was then stirred at room temperature 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 afford Example 15 as a formate salt (13.00 mg; 0.02 mmol; 26.6%; brown gum; purified product).

[0702] Yield: 13.00 mg (0.02 mmol), yield %: 26.6.

[0703] Analyze the data:

[0704] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0705] RT (min): 8.48; Purity (max): 94.47%; M+H: 483.20.

[0706] HPLC-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% TFA / H 2 O:ACN (95:5), solvent B: ACN, flow rate: 0.8 mL / min.

[0707] RT (min): 5.21; purity (maximum): 94.95%; purity (220nm): 92.87%.

[0708] 1H-NMR (400MHz, CD3OD-d6): δ8.83(d,J=8.00Hz,1H),7.72(d,J=8.40Hz,1H), 7.58(t,J=7.60Hz,1H),7.41(t,J=7.20Hz,1H),4.88(s,4H),3.65(t,J=6.80H z,2H),3.61(s,2H),3.50(d,J=3.20Hz,2H),3.15(d,J=4.00Hz,2H),2.34(s, 6H), 1.91 (t, J = 6.80Hz, 2H), 1.58 (q, J = 6.00Hz, 2H), 1.27 (q, J = 7.20Hz, 11H).

[0709] Chemical Synthesis of Example 16

[0710]

[0711] Step 1:

[0712] (4-Methyl-piperazin-1-yl)acetic acid (10,000 mg; 1,00 eq.) was dissolved in DMF (1,000 ml) and cooled to 0°C. 4-Methylmorpholine (17,374 μl; 2,50 eq.), [dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethylammonium hexafluorophosphate (26,439 mg; 1,10 eq.) were then added, and finally compound 3 (23,222 mg; 1,00 eq.) was added. The reaction mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC, eluting with water / acetonitrile (0.1% TFA). The fractions containing the product were combined and lyophilized overnight to give Example 16 (31,00 mg; 0,050 mmol; 79%) as a trifluoroacetate salt as a transparent solid.

[0713] Analyze the data:

[0714] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0715] RT (min): 0.98; Purity (max): 98.3%; M+H: 498.6.

[0716] Chemical Synthesis of Example 17

[0717]

[0718] Step 1:

[0719] 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, eluting with water / acetonitrile (0.1% TFA). The fractions containing the product were combined and lyophilized overnight to give Example 17 as a trifluoroacetate salt (8,5 mg; 22.5%, clear oil, purified product).

[0720] Analyze the data:

[0721] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0722] RT (min): 0.97; Purity (max): 94.0%; M+H = 455

[0723] Chemical Synthesis of Example 18

[0724]

[0725] Step 1:

[0726] To a solution of 4-chloro-3-nitroquinoline (4.50 g; 21.14 mmol; 1.00 eq.) in DCM (45.00 ml; 10.00 V) was added triethylamine (9.00 ml; 63.42 mmol; 3.00 eq.) at room temperature, 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 vacuum filtered to give compound 27 (5.30 g; 19.93 mmol; 94.3%; yellow solid; purified product).

[0727] Yield: 5.30 g (19.93 mmol), yield %: 94.3.

[0728] Analyze the data:

[0729] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0730] RT (min): 0.436; Purity (max): 98.27%; M+H: 260.20.

[0731] Step 2:

[0732] To a solution of compound 27 (5.30 g; 20.28 mmol; 1.00 eq.) in water (4.58 ml; 0.86 V) and methanol (50.00 ml; 9.43 V) was added nickel (II) chloride hexahydrate (0.19 g; 0.80 mmol; 0.04 eq.) at 0°C, followed by sodium borohydride (1.57 g; 40.67 mmol; 2.00 eq.). The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After completion, the black solution was filtered through celite and the filtrate was concentrated to give a crude product. The crude product was then purified by flash column chromatography (3% MeOH / DCM) to give compound 28 (4.20 g; 16.55 mmol; 81.6%; orange powder; purified product).

[0733] Yield: 4.20 g (16.55 mmol), yield %: 81.6.

[0734] Analyze the data:

[0735] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0736] RT (min): 0.37; Purity (max): 91.11%; M+H: 232.20.

[0737] Step 3:

[0738] To a stirred 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 room temperature for 3 hours. The reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo to obtain a residue. The residue was diluted with DCM and washed with water, and the organic layer was collected and concentrated in vacuo to give the 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 room temperature. The cyclization reaction was refluxed for 6 hours and the reaction was monitored by LCMS. After completion, the reaction mixture was concentrated in vacuo to give a crude product. The crude product was purified by silica gel chromatography using 7% MeOH / DCM to give compound 29 (4.90 g; 9.27 mmol; 51.0%; light brown solid; purified product).

[0739] Yield: 4.90 g (9.27 mmol), yield %: 51.0.

[0740] Analyze the data:

[0741] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0742] RT (min): 0.67; Purity (max): 82.73%; M+H: 399.20.

[0743] Step 4:

[0744] To a stirred solution of compound 29 (0.80 g; 1.92 mmol; 1.00 eq.) in acetonitrile (24.00 ml; 30.00 V) was added triethylamine (1.36 ml; 9.58 mmol; 5.00 eq.) under a nitrogen atmosphere, 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. After completion, the solvent was removed to give a crude product. The crude product was purified by flash column chromatography on 60-120 silica gel using ethyl acetate and petroleum ether. The product was eluted in 40-100% ethyl acetate and petroleum ether, and the eluent was concentrated to give compound 30 (800.00 mg; 1.22 mmol; 63.6%; white solid; purified product).

[0745] Yield: 800.00 mg (1.22 mmol), yield %: 63.6.

[0746] Analyze the data:

[0747] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0748] RT (min): 1.14; Purity (max): 93.25%; M+H: 656.20.

[0749] Step 5:

[0750] To a stirred solution of sodium hydride (60%) (0.05 g; 1.19 mmol; 1.50 eq.) in DMSO (5.60 ml; 10.00 V) at 0°C under a nitrogen atmosphere was added dropwise compound 30 (0.56 g; 0.80 mmol; 1.00 eq.) in DMSO (5.60 ml; 10.00 V) and the reaction mixture was stirred at room temperature for 30 minutes. 1,4-dibromobutane (0.53 g; 2.39 mmol; 3.00 eq.) was then added at 0°C and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. 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 in vacuo. The crude product was purified by silica gel chromatography and the product was eluted with 15% ethyl acetate / hexane. The desired fractions were evaporated to give compound 31 (23.00 mg; 0.03 mmol; 3.6%; white gum; purified product).

[0751] Yield: 23.00 mg (0.03 mmol), yield %: 3.6.

[0752] Analyze the data:

[0753] HPLC-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% TFA / H 2 O:ACN (95:5), solvent B: ACN, flow rate: 0.8 mL / min.

[0754] RT (min): 3.09; Purity (max): 96.95%; M+H: 790.30.

[0755] Step 6:

[0756] 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) was added potassium carbonate (23.91 mg; 0.17 mmol; 2.00 eq.) at room temperature. The suspension was heated to 50° C. and stirred for 16 hours. The reaction was monitored by LCMS. After completion, the solvent was removed to obtain a 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).

[0757] Yield: 55.00 mg (0.06 mmol), yield %: 67.4.

[0758] Analyze the data:

[0759] LCMS-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% HCOOH / H 2 O:ACN (95:5), solvent B: ACN; flow rate: 0.8 mL / min.

[0760] RT (min): 1.70; Purity (max): 84.18%; M+H: 810.40.

[0761] Step 7:

[0762] To a stirred 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 room temperature and stirred for 3 hours. The reaction was monitored by LCMS. After completion, the solvent was removed under reduced pressure below 35°C to give a crude product. The crude product was purified by preparative HPLC using a TFA method and the product fractions were lyophilized to give Example 18 (13.10 mg; 0.02 mmol; 51.6%; white gum; purified product) as a trifluoroacetate salt.

[0763] Yield: 13.10 mg (0.02 mmol), yield %: 51.6.

[0764] Analyze the data:

[0765] HPLC-column: Atlantis dC18 (50×4.6 mm) 3.5 μm; solvent A: 0.1% TFA / H 2 O:ACN (95:5), solvent B: ACN, flow rate: 0.8 mL / min.

[0766] RT (min): 8.32; Purity (max): 97.35%; M+H: 468.30.

[0767] 1 H-NMR (400MHz, DMSO-d6): δ9.23(s,br,2H),8.99(s,br,2H),8.61(d,J=8.00Hz,1H),7.83(d,J=8.00Hz,1H) ,7.75(t,J=15.20Hz,1H),7.55(t,J=15.20Hz,1H),4.68(s,br,6H),3.19-2.22(m,15H),1.20-1.31(m,13H).

[0768] Chemical Synthesis of Example 19

[0769]

[0770] Step 1:

[0771] 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 chloride (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. After completion, the mixture was cooled, then diluted with ethyl acetate and washed with 1N HCl. The organic phase was separated and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried over magnesium sulfate, filtered and concentrated in vacuo to give the crude product of compound 33 (25.20 mg, 0.070 mmol, white gum; crude product), which was used in the next reaction without further purification.

[0772] Analyze the data:

[0773] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0774] RT (min): 1.50; Purity (maximum): 30.81%; M+H = 445

[0775] Step 2:

[0776] Compound 33 obtained from step 1 (as crude material; 18.00 mg; 0.040 mmol; 1.0 eq.), (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 fractions containing the product were combined and lyophilized overnight to give compound 34 (16.70 mg, 0.02 mmol, 61.0%, white powder, purified product).

[0777] Yield: 16.70 mg (0.02 mmol), yield %: 61.0.

[0778] Analyze the data:

[0779] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0780] RT (min): 1.725; Purity (max): 93.5%; M+H = 631.7.

[0781] Step 3:

[0782] 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 a hydrazine solution (35%) in water (0.057 ml; 0.624 mmol; 23.6 eq.), the solution was stirred at room temperature for 4 hours and monitored by LCMS. After completion, the reaction mixture was directly injected onto a preparative HPLC, eluting with water / acetonitrile (0.1% TFA). The fractions containing the product were combined and lyophilized overnight to give Example 19 (3.70 mg; 0.01 mmol; 29.0%; white powder; purified product) as a trifluoroacetate salt.

[0783] Yield: 3.70 mg (0.01 mmol), yield %: 29.0.

[0784] Analyze the data:

[0785] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0786] RT (min): 0.902; Purity (max): 100%; M+H = 371.9.

[0787] Chemical Synthesis of Example 20

[0788]

[0789] Glycolic acid (6.482 mg; 1.00 eq.) was dissolved in DMF (2.00 ml), followed by the addition of 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.), 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 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]-dimethyl-ammonium 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 stirred again 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 solid).

[0790] Yield: 17.10 mg (0.041 mmol), yield %: 48.8.

[0791] Analyze the data:

[0792] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0793] RT (min): 1.10; Purity (max): 99.9%; M+H = 416.1.

[0794] Chemical Synthesis of Example 21

[0795]

[0796] 2-Hydroxyisobutyric acid (8.675 mg; 1.00 eq.) was dissolved in DMF (2.000 ml), followed by the addition of 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.) and stirring 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 fractions containing the product were combined and lyophilized overnight to give Example 21 (29.000 mg; 0.052 mmol; 63.7%; white solid; purified product) as a trifluoroacetate salt.

[0797] Yield: 29.000 mg (0.052 mmol), yield %: 63.7.

[0798] Analyze the data:

[0799] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0800] RT (min): 1.103; Purity (max): 100%; M+H = 444.0.

[0801] Chemical Synthesis of Example 22

[0802]

[0803] Monomethyl malonate (14.248 μl; 1.00 eq.) was dissolved in DMF (2,000 ml), followed by the addition of 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.) and stirring 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). The fractions containing the product were combined and lyophilized overnight to give Example 22 (52.300 mg; 0.084 mmol; 61.9%; clear glass solid; purified product) as a trifluoroacetate salt.

[0804] Yield: 52.300 mg (0.084 mmol), yield %: 61.9.

[0805] Analyze the data:

[0806] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0807] RT (min): 1.11; Purity (max): 92.0%; M+H = 458.0.

[0808] Chemical Synthesis of Example 23

[0809]

[0810] 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, followed by the addition of compound 3 (33.339 mg; 1.00 eq.). The reaction mixture was stirred at room temperature for 2 hours and monitored by LCMS. After completion, the reaction mixture was purified by preparative HPLC over a Sunfire column eluting with water / acetonitrile (0.1% TFA). The fractions containing the product were combined, concentrated, and lyophilized to give Example 23 (16.500 mg; 0.028 mmol; 30.3%; beige solid; purified product) as a trifluoroacetate salt.

[0811] Yield: 16.500 mg (0.028 mmol), yield %: 30.3.

[0812] Analyze the data:

[0813] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0814] RT (min): 1.177; Purity (max): 95.9%; M+H = 446.2.

[0815] Chemical Synthesis of Example 24

[0816]

[0817] 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 before the addition of compound 3 (26.000 mg; 0.073 mmol; 1.0 eq.). The reaction mixture was stirred overnight and monitored by LCMS. Upon completion, the reaction mixture was purified by preparative HPLC eluting with water / acetonitrile (0.1% TFA). The product containing fractions were collected and lyophilized overnight to give Example 24 as a trifluoroacetic acid salt (9.10 mg; 0.01 mmol; 20.5%; light beige solid; purified product).

[0818] Yield: 9.10 mg (0.01 mmol), yield %: 20.5.

[0819] Analyze the data:

[0820] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0821] RT (min): 1.14; Purity (max): 97.0%; M+H = 478.1.

[0822] Chemical Synthesis of Example 25

[0823]

[0824] 2-Sulfonic 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 before adding compound 3 (26.000 mg; 0.073 mmol; 1.0 eq.). The reaction mixture was stirred at room temperature overnight and monitored by LCMS. Upon completion, the reaction mixture was purified by preparative HPLC, eluting with water / acetonitrile (0.1% TFA). The fractions containing the product were collected and lyophilized overnight to give Example 25 (26.3 mg; 0.04 mmol; 60.3%; white powder; purified product) as a trifluoroacetic acid salt.

[0825] Yield: 26.3 mg (0.04 mmol), yield %: 60.3.

[0826] Analyze the data:

[0827] HPLC-MS: Column: Chromolith HR C18 5.0 μm; 50-4.6 mm; Method information (Chromolith): A: H2O + 0.05% HCOOH | B: MeCN + 0.04% HCOOH | T: 40°C | Flow rate: 3.3 ml / min | MS: 100-2000 amu positive mode | 1% -> 100% B: 0 -> 2.0 min | 100% B: 2.0 -> 2.5 min

[0828] RT (min): 1.108; Purity (max): 99%; M+H = 480.1.

[0829] Chemical Synthesis of Example 26

[0830]

[0831] Step 1:

[0832] 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) was added potassium carbonate (122.33 mg; 0.87 mmol; 3.00 eq.) 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).

[0833] Yield: 152.73 mg (0.19 mmol), yield %: 67.3.

[0834] Analyze the data:

[0835] HPLC: column: XBridge C8, 3.5 μm, 4.6 x 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.

[0836] RT (min): 2.59; Purity (max): 98.2%; M+H = 771.40.

[0837] Step 2:

[0838] To a solution of compound 35 (175.00 mg; 0.23 mmol; 1.00 eq.) in DCM (1.75 ml) was added trifluoroacetic acid (0.18 ml; 2.27 mmol; 10.00 eq.) dropwise and stirred at room temperature 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).

[0839] Yield: 21.4 mg (0.04 mmol), yield %: 17.5.

[0840] Analyze the data:

[0841] HPLC: column: XBridge C8, 3.5 μm, 4.6 x 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.

[0842] RT (min): 1.96; Purity (max): 97.4%; M+H = 529.20.

[0843] 1 H-NMR (400MHz, DMSO-d6): δ8.58(d,J=8.00Hz,1H),7.81(d,J=7.60Hz,1H),7.71(t,J=15.60Hz,1H),7.54(t,J=15.60Hz,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).

[0844] Chemical Synthesis of Example 27

[0845]

[0846] To a solution of Example 10 (100.00 mg; 0.22 mmol; 1.00 eq.) dissolved in acetonitrile (2.00 ml) was added 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.) 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 concentrated under reduced pressure to give a crude residue. The crude residue was purified by preparative HPLC using 0.1% TFA / water and ACN to give Example 27 (5.80 mg; 0.01 mmol; 4.7%; purified product).

[0847] Yield: 5.80 mg (0.01 mmol), yield %: 4.7.

[0848] Analyze the data:

[0849] LCMS-column: ATLANTIS dC18 (50x4.6 mm) 5 μm; mobile phase: A: 0.1% HCOOH / H2O:ACN (95:5), B: ACN flow rate: 1.5 ml / min.

[0850] RT (min): 2.00; Purity (max): 99.48%; M+H: 557.40.

[0851] Biological evaluation of TLR7 agonists

[0852] TLR7 / 8 reporter gene assay

[0853] Materials and general procedures

[0854] HEK-Blue TM TLR7 and TLR8 cell lines were obtained from InvivoGen (San Diego, CA, USA). HEK-Blue cells were cultured in high glucose DMEM medium (Gibco-10569010) supplemented with 10% HI-FBS (from Australia) (Gibco-10100-147) and 1% penicillin / streptomycin (Gibco-15140-122-100 ml) according to the manufacturer's recommendations. In addition, a specific antibiotic cocktail was added to the medium. For HEK-Blue TM hTLR7 cells, add 100 μg / ml Zeocin (11006-33-0), 10 μg / ml Blasticidin (ant-bl-05), 50 μg / ml Normocin (ANT-NR-1-500MG), for HEK-Blue TM hTLR8 cells were supplemented with 100 μg / ml Zeocin, 30 μg / ml Blasticidin, and 50 μg / ml Normocin (all from InvivoGen).

[0855] General procedure for in vitro agonist screening

[0856] All compounds were prepared in 100% DMSO (Sigma-D2650). Compounds were serially diluted (3-fold) in 100% DMSO, starting from 30 μM, up to a 10-point dose-response curve; the final concentration of DMSO in the well 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 minimum and maximum values, respectively, and activation percentage was calculated using the following formula:

[0857] Activation %: {100-(maximum absorbance-absorbance) / (maximum absorbance-minimum absorbance)}x100.

[0858] For stimulation with TLR7 / 8 agonists, cells were resuspended in complete medium and 40 μl of cell suspension (10,000 cells / well) were seeded in 384-well assay plates (Thermo Scientific TM Nunc–164688) were pre-dispensed with compounds using an Echo Liquid Handler. The plates were spun at 100 g for 1 minute to sediment the cells and incubated at 37°C, 5% CO2 for 24 hours. To measure SEAP production after 24 hours of incubation, 10 μl of Quanti-Blue HCl, prepared according to the manufacturer's instructions, was added to the assay plates in the dark. TM Solution (InvivoGen rep-qbs2) was added and incubated for 30 minutes at 37°C under 5% CO2. After incubation, the absorbance was measured at 620 nm in a Tecan spark (Spark control Magellan).

[0859] EC was determined using Graph Pad Prism statistical software (version 7.05). 50 , individual data points represent the absorbance values of replicates. Table 1 describes the compounds of the present disclosure, wherein EC 50 Values less than 0.01 μM are designated as "A" activity, values between 0.01 μM and 0.1 μM are designated as "B" activity, values between 0.1 μM and 1 μM are designated as "C" activity, values between 1 μM and 10 μM are designated as "D" activity, and values greater than 10 μM are designated as "E" activity.

[0860] Table 1: In vitro screening of TLR7 / 8 agonists in HEKBlue cells

[0861]

[0862]

[0863]

[0864] Overall, most of our examples showed much higher potency against TLR7 than the benchmark.

[0865] TNFα secretion from PBMCs

[0866] Materials and general procedures

[0867] Peripheral blood mononuclear cells (PBMC) are isolated from healthy volunteers and stored in liquid nitrogen. The bottle of freezing PBMC is thawed in 37 ℃ of water baths and is no more than 2 minutes. The content of pipe is aseptically transferred in preheated complete medium, this medium contains RPMI basal medium (Gibco-21875034) and is supplemented with 10% HI-FBS (originating from Australia) (Gibco-10100-147) and 1% penicillin / streptomycin (Gibco-15140-122-100ml), then centrifuged. Cells are resuspended in complete medium (3ml) and counted using a hemocytometer.

[0868] General procedure for in vitro agonist screening

[0869] All selected compounds (five benchmark molecules and 15 example molecules) were prepared in 100% DMSO (Sigma-D2650) (10mM stock solution). The compounds were serially diluted (4-fold) in 100% DMSO in a U-bottom 96-well plate (Cellstar-650180) until an 8-point DRC. 3 μl of this serially diluted DRC was mixed with 97 μl of complete culture medium (1:33.33) as an intermediate dilution of (300 μM) 3% DMSO. 11 μl of the compound from the intermediate dilution was added to the PBMC plate (1:10) so that the final concentration of DMSO in the culture plate was 0.3% and the starting concentration of the compound was 30 μM. Resiquimod (30 μM) and DMSO (0.3%) were used as positive and negative controls, respectively.

[0870] The plates were incubated at 37°C, 5% CO2 for 18 hours. Following incubation, the plates were spun at 2000 rpm for 5 minutes, and the supernatant was gently aspirated and collected in a protein low binding V-bottom 96-well plate (Thermo Fisher-249944). The supernatant plate was sealed, appropriately labeled, and stored at -80°C until cytokine analysis (<1 month).

[0871] TNF-α was measured using the MILLIPLEX MAP Human Cytokine / Chemokine Magnetic Bead Assay Kit (HCYTOMAG-60K) according to the kit's recommendations.

[0872] EC was determined using Graph Pad Prism statistical software (version 7.05). 50 , individual data points represent the absorbance values of replicates. Table 2 describes the compounds of the present disclosure, wherein EC 50 Values less than 0.1 μM are designated as "A" activity, values between 0.1 μM and 1 μM are designated as "B" activity, values between 1 μM and 10 μM are designated as "C" activity, and values greater than 10 μM are designated as "D" activity.

[0873] Table 2: In vitro screening of selected TLR7 / 8 agonists in PBMCs

[0874]

[0875]

[0876] Expression of activation marker CD86 in PBMCs

[0877] Materials and general procedures

[0878] PBMCs were purchased commercially from HemaCare Corporation and cultured in RPMI (Gibco-11875093) containing 10% HI-FBS. For stimulation, 50 μl of a cell suspension containing 2 × 10^5 cells was seeded in a 96-well flat-bottom, white-lid assay plate (Greiner Bio one–655180). Freshly cultured PBMCs were used to screen agonists. Flow cytometry staining was performed using 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.

[0879] General Procedure for In Vitro Testing of Agonists

[0880] All compounds were prepared at a 2-fold higher concentration to generate an intermediate stock solution (2 μM) in culture medium. Compounds were serially diluted in culture medium using 10-fold dilutions for a total of 4 DRCs (2-0.002 μM). The intermediate stock solution (50 μl) and the serially diluted stock solution 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.

[0881] The next day, cells were harvested using ice-cold FACS buffer (DPBS without cations and containing 2% HI-FBS) and transferred to a separate 96-well V-bottom plate (Greiner, 650201). Cells were washed once using FACS buffer by spinning at 1400 rpm for 2 minutes at 4 ° C. Subsequently, the cells were washed in PBS and then stained with zombie yellow stain (Biolegend, 423103; 1: 100) diluted in PBS according to the manufacturer's operating procedures. The cells were washed twice in FACS buffer and stained for 1 hour at 4 ° C in the dark using anti-CD14 FITC, anti-CD11c BV421, anti-CD123 PE and anti-CD86 APC antibodies (2 μl / test for each antibody). After staining, cells were washed twice at 1400 rpm at 4 ° C for 2 minutes using FACS buffer. Cells suspended in FACS buffer (100 μl / well) were analyzed using an ACEA NovoCyte flow cytometer after setting up a compensation matrix with compensation beads, except for live-dead staining, for which PBMCs stained with zombie yellow dye were used.

[0882] PBMCs were gated using FSC-H and SSC-H, and then single cells were further gated using FSC-H and FSC-A. Live-dead staining gated single cells (singlet) captured in the Pacific Orange channel was used to detect viability. Single cells that can survive were gated for CD14+ (monocyte) colonies, CD14-CD11c+ (cDC colonies), and CD14-CD11c-CD123+ (pDC) colonies. CD86+ expression of each colony in the corresponding channel was further assessed. Negative and positive gates were specified using NovoExpress software with corresponding FMO controls. Concentrations were plotted against cDC colonies ( Figure 4A ), pDC population ( Figure 4B ) and monocytes ( Figure 4C ) to generate an XY scatter plot for each stimulus.

Claims

1. A compound according to formula I or a pharmaceutically acceptable salt thereof, wherein X is an oxygen atom, a C1-C5-alkyl group (preferably CH2) or NH; where R 2 and R 3 Each is independently selected from hydrogen, C1-C5-alkyl, C4-C7-cycloalkyl, C4-C7-heterocycloalkyl, aryl and heteroaryl; preferably R 2 and R 3 are each hydrogen; And where L 1 、L 2 、L 3 and R 1 is as defined below under (a) or (b) or (c): (a)L 1 is a C2-C6 alkyl group; L 2 is a 5- or 6-membered heterocyclic ring or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and R 1 Absent or selected from hydrogen, NH2, OH and SCH3; (b)L 1 is a C2-C6 alkyl group; L 2 selected from 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 Absent or methyl or hydrogen; (c)L 1 is C(O)CH2; L 2 is a 6-membered heterocyclic ring or NH2; L 3 does not exist; and R 1 is methyl or hydrogen or is absent; And if L 3 If it does not exist, then L 2 Through covalent bond with R 1 Direct combination.

2. The compound according to claim 1, wherein R 2 and R 3 For hydrogen. The compound according to claim 1 or 2, wherein X is an oxygen atom.

4. The compound of claim 3, wherein the compound has a structure according to Formula II Among them L 1 、L 2 、L 3 and R 1 as defined below under (d), (e) or (f) (d)L 1 is a C2-C6 alkyl group; L 2 is a 5- or 6-membered heterocycle (preferably, triazole or piperazine) or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and R 1 Absent or selected from hydrogen, NH2, OH and SCH3; (e)L 1 is a C2-C3 alkyl group; L 2 selected from C(O), NHC(O)CH2 and NHC(O)C(CH3)2; L 3 is selected from a 6-membered heterocycle (preferably piperazine), OH, C(O)O, S, SO2 and SO3H; and R 1 Absent or methyl or hydrogen; (f)L 1 is C(O)CH2; L 2 is a 6-membered heterocycle (preferably piperazine) or NH2; L 3 does not exist; and R 1 is methyl or hydrogen or is absent.

5. The compound according to claim 4, wherein L 1 is a C2-C6 alkyl group; L 2 is a 5- or 6-membered heterocyclic ring or OH; L 3 is absent or selected from hydrogen, C1-C2 alkyl and CH2C(CH3)2; and R 1 Absent or selected from NH2, OH and SCH3.

6. A compound according to any one of the preceding claims, wherein the compound has a structure selected from the structures listed below designated as Examples 1 to 27:

7. The compound according to claim 1, wherein X is NH; L 1 is a C2-C6 alkyl group; L 2 is a 5- or 6-membered heterocyclic ring; L 3 does not exist; and R 1 is methyl or hydrogen; and R 2 and R 3 Each is hydrogen.

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

9. The compound according to any one of the preceding claims, wherein if the compound is contacted with peripheral blood mononuclear cells (PBMCs), the compound induces the secretion of TNFα from said cells.

10. The compound according to any one of the preceding claims, wherein the compound has an EC50 for TLR7 of less than 10 μM and preferably less than 0.01 μM when tested 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. The compound according to any one of claims 1 to 10 for use in the treatment of a disease, wherein the compound is used in said treatment in combination with the additional compounds imiquimod and / or resiquimod (R848).

14. A composition comprising a compound according to any one of claims 1 to 10 for use in treating a condition selected from the group consisting of cancer, viral infection, noncancerous skin lesions, precancerous skin lesions, cancerous skin lesions, bladder cancer, virally mediated skin diseases, the composition optionally being formulated to enhance penetration upon topical application, the composition preferably eliciting a local specific inflammatory cytokine response while limiting undesirable erythema and other inflammatory responses.

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

16. The composition according to claim 14 or 15, for use in treating bladder cancer, wherein the composition is formulated for intravesical administration.

17. The composition according to any one of claims 11, 14, 15 or 16, further comprising an additional component of one of the following groups i to xiii: i. a total concentration of 1% wt / vol oleic acid (50%) and isopropyl myristate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or ii. a total concentration of 0.5% wt / vol isopropyl myristate (50%) and sodium lauryl sulfate (50%) in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or iii. oleic acid (50%) and sodium lauryl sulfate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or iv. a total concentration of 0.5% wt / vol 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, or v. isopropyl palmitate (50%) and sodium oleate (50%) at a total concentration of 2% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or vi. sodium lauryl sulfate (25%) and linoleic acid (75%) at a total concentration of 1.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or vii. Palmitic acid (50%) and isopropyl laurate (50%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or viii. oleic acid (50%) and linoleic acid (50%) at a total concentration of 1.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or ix. linoleic acid (25%), oleic acid (25%), and isopropyl linoleate (50%) at a total concentration of 0.5% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, or x. Sodium oleate (33%), oleic acid (33%), and methyl palmitate (33%) at a total concentration of 2.0% wt / vol in a solution containing 50 ml of phosphate buffered saline and 50 ml of ethanol, 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. 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. A method for treating skin tumor lesions and viral-induced skin diseases, said method comprising topically administering a composition according to any one of claims 11 and 14-17, said composition preferably being effective in reducing or eliminating said lesions or diseases while limiting adverse skin reactions selected from erythema and inflammation, said composition preferably reducing the penetration of tumor lesions into surrounding tissues and metastasis to lymph nodes.

20. A method for treating bladder tumors, the method comprising intravesicularly administering a composition according to any one of claims 11 and 14-17, the composition preferably enhancing penetration and delivery of the composition to the bladder epithelium while limiting irritation, the composition preferably reducing tumor infiltration into surrounding muscle tissue and metastasis to lymph nodes.

21. The method of any one of claims 18-20, further comprising systemic administration of at least one immunomodulator selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD137 antibody, an agonist CD40 antibody, a CD134 (anti-OX40) agonist, and PLX3397.

22. The method of any one of claims 18-20, further comprising systemically administering interferon gamma.

23. The method of any one of claims 18-20, further comprising administering localized radiation with or without systemic anti-PDl antibodies.

24. The method of any one of claims 18-20, further comprising administering photodynamic therapy.

25. A method of activating TLR 7 and / or 8 in a biological sample, the method comprising contacting the biological sample with a compound according to any one of claims 1-10.

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

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