Combinations and methods for enhancing checkpoint inhibitor therapy in cancer treatment

Abstract

A combination, composition, and method of use of (i) a conjugate comprising a targeted immune modulator and (ii) one or more immune checkpoint inhibitors for reprogramming M2-like macrophages into M1-like macrophages to reverse the pro-inflammatory response observed in cancer into an anti-inflammatory response. In at least one embodiment of a method of treating a subject having cancer, the subject is experiencing or at risk of experiencing cancer or cancer recurrence, and the step of administering a first treatment further comprises administering or applying to the subject a therapeutically effective amount of at least one compound or a pharmaceutically acceptable salt or hydrate thereof.

Description

[Technical Field]

[0001] Priority This patent application claims priority to U.S. Provisional Patent Application No. 63 / 454,306, filed on 23 March 2023. The contents of the aforementioned application are incorporated in their entirety by reference.

[0002] Technical field This disclosure relates to a method for reprogramming M2 macrophages into M1 macrophages using one or more compounds comprising a targeting moiety to enhance the efficacy of one or more checkpoint inhibitors (compared to the baseline efficacy of such checkpoint inhibitors). [Background technology]

[0003] background In a normal, healthy system, immune checkpoints are surface proteins that check the immune response and block any excessive stimulation. Their role is to prevent the immune response from becoming too strong and destroying healthy cells in the body. In cancer, when a checkpoint protein on a T cell (e.g., programmed cell death protein 1 (PD-1)) binds to its binding protein on a tumor cell (programmed cell death ligand 1 (PD-L1)), it sends a "stop" signal to the T cell, suppressing the anti-tumor immune response. In this way, tumor killer cells within the immune system are unable to kill the tumor because their attack method is blocked by the tumor's own checkpoints.

[0004] Immune checkpoint inhibitors work by inhibiting checkpoint proteins from binding to ligands on tumor cells. PD-1, CTLA-4 (cytotoxic T lymphocyte-associated protein 4), LAG3 (lymphocyte activation protein 3), TIM3 (T cell immunoglobulin and mucin domain-containing protein 3), TIGIT (T cell immune receptor with Ig and ITIM domains), and VISTA (V domain Ig suppressor of T cell activation) are some examples of checkpoint proteins. Checkpoint inhibitor therapy is approved by the U.S. Food and Drug Administration for several cancer types. Some of the approved checkpoint inhibitors include anti-PD1 antibodies, including pembrolizumab (Keytruda®) and nivolumab (Opdivo®), and the anti-CTLA4 antibody called ipilimumab (Yervoy®).

[0005] There remains unmet demand in the field of cancer immunotherapy. For example, approximately 50% of patients with PD-L1-positive tumors develop resistance to PD-1 / PD-L1 checkpoint therapy or experience subsequent relapse (Herbst et al., Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients, Nature 515(7528): 563-567 (2014)). One of the crucial factors behind resistance to checkpoint blockade may be the function of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) (Bai et al., Regulation of PD-1 / PD-L1 pathway and resistance to PD-1 / PD-L1 blockade, Oncotarget 8(66): 110693-110707 (2017)). MDSCs in the tumor microenvironment are known to contribute to the impairment of checkpoint blockade effectiveness (Meyer et al., Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab, Cancer Immunology Immunotherapy 63: 247-57 (2014)). Similarly, TAM reprogramming has been shown to increase the effectiveness of checkpoint blockade (Zhu et al., CSF1 / CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models, Cancer Research 74: 5057-69 (2014)). Despite the clear need for cancer prevention and treatment, these conditions remain a significant cause of death worldwide, due to the current lack of an effective therapeutic cure. Furthermore, when drugs or other treatments are available, such treatments typically involve highly potent drugs that pose a risk of systemic toxicity to the underlying target due to their low selectivity for the cancer cells of interest. What is needed is a treatment that is not only effective in disrupting the growth factor cycle initiated by activated M2-type (or activated) macrophages, but can also do so with very high specificity to the cells in question. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Herbst et al., Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients, Nature 515(7528): 563-567 (2014) [Non-Patent Document 2] Bai et al., Regulation of PD-1 / PD-L1 pathway and resistance to PD-1 / PD-L1 blockade, Oncotarget 8(66): 110693-110707 (2017) [Non-Patent Document 3] Meyer et al., Frequencies of circulating MDSC correlate with clinical outcome of melanoma patients treated with ipilimumab, Cancer Immunology Immunotherapy 63: 247-57 (2014) [Non-Patent Document 4] Zhu et al., CSF1 / CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models, Cancer Research 74: 5057-69 (2014) [Overview of the project] [Means for solving the problem]

[0007] overview In some cases, activated M2 phenotypic macrophages play a role in cancer by secreting anti-inflammatory cytokines that activate fibroblasts to synthesize collagen and other extracellular matrix proteins. In certain cases, these macrophages also trigger the release of problematic growth factors in cancer-experiencing subjects. In some cases, activated macrophages derived from tissue-resident macrophages or peripheral blood monocytes induce fibroblast activation via the secretion of chemokine (CC motif) ligand 18 (CCL18), transformed growth factor-β1 (TGFβ1), and / or platelet-derived growth factor (PDGF). This activation, in some cases, promotes collagen secretion by fibroblasts, which can lead to the associated cancer progression. In many late-stage cancers, activated macrophages and myofibroblasts can cross-stimulate each other, potentially promoting tumor growth (e.g., due to growth factors secreted by activated macrophages) and / or collagen formation in cancerous tumors (e.g., through downstream fibrous collagen production, which can result in cancerous tumors that are more difficult to treat by blocking drug penetration).

[0008] In some embodiments, compounds represented by the formula QLT are provided herein. In some embodiments, Q is a radical of a folate receptor-binding ligand. In some embodiments, L is a linker. In some embodiments, T is a radical of a Toll-like receptor (TLR) agonist. In some embodiments, QLT is a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the linker is a non-emission linker. In some embodiments, the non-emission linker is of formula: [ka] It is represented by [this].

[0010] In some embodiments, n is 1 to 30. In some embodiments, n is 1 to 24. In some embodiments, n is 1 to 12. In some embodiments, n is 1 to 3. In some embodiments, n is 12. In some embodiments, n is 3.

[0011] In some embodiments, w is between 0 and 5. In some embodiments, w is between 0 and 2. In some embodiments, w is 1.

[0012] Methods for treating subjects having cancer are also provided. In at least one embodiment of the method for treating subjects having cancer, the method comprises the steps of administering a first therapy to a subject, wherein the first therapy comprises at least one compound comprising a radical of an immunomodulator conjugated via a linker to a folate ligand or a functional fragment or analog thereof, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising at least one compound and one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or a combination thereof; and administering a second therapy to a subject, wherein the second therapy comprises one or more immune checkpoint inhibitors. The immunomodulator (e.g., a radical) or a pharmaceutically acceptable salt or hydrate thereof may target pattern recognition receptors or damage-associated molecular patterns (DAMPs). In certain embodiments, the step of administering a second therapy to a subject further comprises administering a therapeutically effective amount of one or more immune checkpoint inhibitors. In a particular embodiment, the step of administering to a target of a first treatment includes administering a therapeutically effective amount of at least one compound or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising at least one compound or a pharmaceutically acceptable salt or hydrate thereof.

[0013] In at least one embodiment, administration of at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof reprograms the target M2 type macrophages into the target M1 type macrophages and enhances the efficacy of one or more checkpoint inhibitors of the second treatment compared to the baseline efficacy of one or more checkpoint inhibitors.

[0014] In at least one embodiment of a method for treating a subject with cancer, the immunomodulator (i.e., radical) is or comprises a Toll-like receptor (TLR) 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR 9 agonist, or a TLR 7 / 8 agonist. In a particular embodiment of a method for treating a subject with cancer, the immunomodulator radical is or comprises a TLR7 agonist, and the linker is a non-releasing linker.

[0015] In at least one embodiment of a method for treating a subject having cancer, at least one compound is [ka] It is either the compound or a pharmaceutically acceptable salt or hydrate thereof.

[0016] In at least one embodiment of a method for treating a subject with cancer, at least one compound is of the following formula: [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0017] In at least one embodiment of a method for treating a subject with cancer, the immunomodulator (i.e., radical) is a TLR agonist of formula X or XX, or comprises a pharmaceutically acceptable salt or hydrate of formula X or XX. [ka] In equations X and XX, R1 is either -NH2 or -NH-R 1X And, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [Chemical formula] and is [Chemical formula] a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, In formula X, R3 is -OH, -SH, -NH2, or -NH-R 1X and is In formula XX, X is CH or N, R 1X R 2X and R 2Y each is independently selected from the group consisting of hydrogen (H), alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl.

[0018] In at least one embodiment of a method of treating a subject having cancer, the compound is [Chemical formula] comprises or is a pharmaceutically acceptable salt or hydrate thereof, and in formula (2-I), R 1 R 3 R 4 and R 5 are each independently H, alkyl, alkoxyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, halo, heteroaryl, -COR 2x and [Chemical formula] and is R 2 is H, -OH, -NH2, -NHR 2x N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [Chemical formula] and is Y is a binding site to the linker and / or targeting ligand of at least one compound, and is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] Includes, R 2x and R 2y Each of them is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, and each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q Independently selected from the group consisting of R 2q and R 2q’ Each of them is independently an alkyl group or H group. [ka] It is a non-aromatic monocyclic or bicyclic 3-10 member nitrogen-containing heterocycle, In equation 2-I, X 1 , X 2 , and X 3 Each of them independently, CR q or N, and each R q These are independently H, halogens, or optionally substituted alkyl groups. In equation 2-I, n is between 0 and 30, and m is between 0 and 4.

[0019] In at least one embodiment of a method for treating a subject having cancer, the subject is experiencing or at risk of experiencing cancer or a recurrence of cancer, and the step of administering a first treatment further comprises administering or applying to the subject a therapeutically effective amount of at least one compound or a pharmaceutically acceptable salt or hydrate thereof.

[0020] In at least one embodiment of a method for treating a subject with cancer, at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof is administered to the subject intravenously, orally, intramuscularly, intraperitoneally, topically, or by inhalation.

[0021] In at least one embodiment of a method for treating a subject with cancer, the M2 macrophages of the subject include bone marrow-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), or both MDSCs and TAMs.

[0022] In at least one embodiment of this method, the subject has a tumor that is positive for programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

[0023] In at least one embodiment of the method, at least one compound of the first treatment constitutes a composition comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or a combination thereof.

[0024] In at least one embodiment of a method for treating a subject with cancer, the method is performed to treat cancer recurrence or resistance to checkpoint blocker therapy in the subject. The subject may be a human, a mouse, or any other mammal.

[0025] In certain embodiments of the method for treating a subject with cancer, one or more immune checkpoint inhibitors of the second therapy each comprise a small molecule or other agent that destroys immune checkpoints on the target cells. In certain embodiments of the method, one or more immune checkpoint inhibitors of the second therapy include pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islelizumab, osiperlimab and islelizumab Each of the following combinations is independently selected from the group consisting of BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab. In at least one embodiment of a method for treating a subject with cancer, one or more checkpoint inhibitors of the second treatment include pembrolizumab, nivolumab, and one or more of ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, or small molecules or other drugs that disrupt immune checkpoints in the target cells.

[0026] In at least one embodiment of a method for treating a subject with cancer, the method further includes the steps of obtaining a biological sample from the subject and quantifying the expression level of one or more biomarkers in the biological sample. In at least one embodiment of a method for treating a subject with cancer, the biological sample is obtained from a certain amount of peripheral blood taken from the subject. In at least one embodiment of a method for treating a subject with cancer, quantification is performed using a process selected from the group consisting of qPCR, mass spectrometry, ELISA, and another modality capable of measuring or quantifying biomarker expression. One or more biomarkers may be selected from the group consisting of, for example, CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, TNFα, IFN-γ, CD206, CD163, IL-6, CXCL10, IFNα, and CD86.

[0027] In at least one embodiment of a method for treating a subject having cancer, the method further includes the step of comparing the expression level of one or more biomarkers with the expression level of such biomarkers in a control, wherein the control is a healthy individual or an individual that has not experienced cancer.

[0028] In at least one embodiment of a method for treating a subject with cancer, the radical of the immunomodulator is given by the following formula: [ka] A TLR agonist having, or containing, During the ceremony, R 1 It is an amine group, R 2 It is a single bond -NH-, R 3 is H, alkyl, hydroxyl, or any other substituted group thereof. X is CH2, NH, O, or S. Linker is R1 , R 2 , or R 3 Combine.

[0029] In at least one embodiment of a method for treating a subject with cancer, the linker of at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof is a release linker. In at least one embodiment of a method for treating a subject with cancer, the linker of at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof is a non-release linker. In at least one embodiment of a method for treating a subject with cancer, the linker of at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof comprises a polyethylene glycol (PEG) linker or a PEG derivative linker and is a non-release linker.

[0030] In at least one embodiment of a method for treating a subject with cancer, one or more immune checkpoint inhibitors of the second therapy include PD-L1, PD-1, CTLA-4, V-domain Ig suppressor of T cell activation (VISTA), lymphocyte activation 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immune receptor having Ig and ITIM domains (TIGIT), programmed death ligand 2 (PD-L2), and indoleamine 2,3-dioxygenase (indoleamine It inhibits cellular immune checkpoints selected from the group consisting of 2,3-deioxygenase (IDO), arginase-1 (AGR1), B7 family inhibitory ligand B7-H3 (B7-H3), B7 family inhibitory ligand B7-H4 (B7-H4), 2B4 (differentiation cluster 244), B and T lymphocyte attenuation factor (BTLA), adenosine A2A receptor (A2aR), and / or members of the killer cell immunoglobulin-like receptor (KIR) family, such as KIR and C-type lectin receptors, as well as signaling factors and transcriptional activators (STAT3).

[0031] In at least one embodiment of a method for treating a subject with cancer, the first and second treatments are administered simultaneously or sequentially (in either order). In a particular embodiment, the step of administering at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof activates antitumor cells or an anti-inflammatory signaling cascade in the subject. The antitumor cells may be, for example, T cells, macrophages, or both T cells and macrophages.

[0032] An additional method is provided for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a subject. In a particular embodiment, the method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a subject comprises the steps of: administering to the subject one or more compounds comprising an immune modulator radical conjugated via a linker to a folate ligand or a functional fragment or analog thereof, wherein the immune modulator radical targets a pattern recognition receptor or DAMP; and contacting the subject's targeted cells with one or more compounds or a pharmaceutically acceptable salt or hydrate thereof to reprogram the subject's M2 macrophages into M1 macrophages.

[0033] In some cases, activated M2 phenotypic macrophages play a role in fibrotic diseases by secreting anti-inflammatory cytokines that activate fibroblasts to synthesize collagen and other extracellular matrix proteins. In certain cases, these macrophages also trigger the release of problematic growth factors in cancer-experiencing subjects. For example, such growth factors can promote the growth of cancerous tumors. Furthermore, in some cases, macrophages release immunosuppressive cytokines (for example, simultaneously). Thus, macrophages may play a crucial role in promoting the construction and growth of fibrotic diseases and / or cancers.

[0034] In some cases, activated macrophages derived from tissue-resident macrophages or peripheral blood monocytes induce fibroblast activation via the secretion of chemokine (CC motif) ligand 18 (CCL18), transformed growth factor-β1 (TGFβ1), and / or platelet-derived growth factor (PDGF). This activation, in some cases, promotes collagen secretion by fibroblasts, which can lead to associated cancer progression. In many late stages of cancer, activated macrophages and myofibroblasts can cross-stimulate each other, leading to the growth of cancerous tumors (e.g., due to growth factors secreted by activated macrophages) and / or collagen formation in cancerous tumors (e.g., through downstream fibrous collagen production, which can result in cancerous tumors that are more difficult to treat by blocking their drug penetration).

[0035] In certain embodiments of a method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a subject, the immune modulator radical is or includes a TLR 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR 9 agonist, or a TLR 7 / 8 agonist. In certain embodiments of a method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a subject, the immune modulator radical is or includes a TLR 7 agonist, and the linker is a releasing linker. In certain embodiments of a method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a subject, the immune modulator radical is or includes a TLR 7 agonist, and the linker is a non-releasing linker.

[0036] In one particular embodiment of a method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a target, one or more compounds are: [ka] is a structure represented by or includes the same, or a pharmaceutically acceptable salt or hydrate thereof.

[0037] The linker can be a non-cleavable linker. The linker can be a cleavable linker. In certain embodiments, the cancerous disease state includes cancer recurrence.

[0038] In some embodiments, a compound represented by the formula Q-L-T, or a pharmaceutically acceptable salt or hydrate thereof, is provided herein. In some embodiments, Q is a radical of a folate receptor-binding ligand. In some embodiments, L is a linker. In some embodiments, T is a radical of a TLR agonist. In some embodiments, Q-L-T is a pharmaceutically acceptable salt thereof.

[0039] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker has the formula:

Chemical formula

[0040] In some embodiments, n is from 1 to 30. In some embodiments, n is from 1 to 24. In some embodiments, n is from 1 to 12. In some embodiments, n is from 1 to 3. In some embodiments, n is 12. In some embodiments, n is 3.

[0041] In some embodiments, w is from 0 to 5. In some embodiments, w is from 0 to 2. In some embodiments, w is 1.

[0042] In some embodiments, the TLR agonist is a TLR7 agonist. In some embodiments, the radical of the TLR agonist has the formula X:

Chemical formula

[0043] In some embodiments, R1 is -NH2 or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] In some embodiments, R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In some embodiments, [ka] R3 is a 3- to 10-membered nitrogen (N)-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, R3 is -OH, -SH, -NH2, or -NH-R 1X In some embodiments, R1 is -NH2 or -NH-R 1X R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] R3 is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle, where R3 is -OH, -SH, -NH2, or -NH-R1X is as follows.

[0044] In some embodiments, the radical of the TLR agonist has the structure represented by Formula XX:

Chemical formula

Chemical formula

[0045] In some embodiments, the radical of the TLR7 agonist is given by formula XXX: [ka] It has a structure represented by [this].

[0046] In some embodiments, the compound has a linker L between the targeting moiety and the immunomodulator or a pharmaceutically acceptable salt thereof. n It further includes Linker L n The linker L is configured to avoid the release of the free form of the TLR7 agonist, where n is an integer equal to or less than 50. In some embodiments, the linker L n The compound comprises PEG or a PEG derivative, n is an integer selected from the range of 1 to 32, and the radical of the folate receptor-binding ligand is a folate receptor β (FBβ)-binding ligand.

[0047] In some embodiments, the compound is [ka] It has the structure represented by, or a pharmaceutically acceptable salt or hydrate thereof.

[0048] In some embodiments, the compound is [ka] It has the structure represented by, or a pharmaceutically acceptable salt or hydrate thereof.

[0049] In some embodiments, the compound is [ka] It has the structure represented by, or a pharmaceutically acceptable salt or hydrate thereof.

[0050] In some embodiments, the compound is [ka] It has the structure represented by, or a pharmaceutically acceptable salt or hydrate thereof.

[0051] In some embodiments, pharmaceutical compositions comprising one or more compounds described herein or pharmaceutically acceptable salts or hydrates thereof, wherein the immunomodulator radical is or comprises a TLR7 agonist having a structure represented by formula XX.

[0052] In certain specific examples, methods for treating subjects suffering from fibrotic disease or cancer are provided herein, comprising the step of contacting cells of the subject with at least one compound comprising a compound described herein or a pharmaceutically acceptable salt or hydrate thereof, wherein the immunomodulator radical comprises a TLR 3, 7, 8, 9, or 7 / 8 agonist.

[0053] In some embodiments, a compound comprising a folate ligand or a functional fragment or analog thereof bound to a TLR agonist via a linker, wherein the TLR agonist has the following formula: [ka] Compounds having or pharmaceutically acceptable salts thereof are provided herein.

[0054] In some embodiments, R 1 is an amine group, R 2 It is a single bond -NH-, and R 3is H, alkyl, hydroxyl, or any other substituted group thereof, X is CH2, NH, oxygen (O), or sulfur (S), and the linker is R 1 , R 2 , or R 3 Combine.

[0055] In some embodiments, a pharmaceutical composition comprising any one compound of the formulas provided herein, wherein the linker comprises a PEG linker or a PEG derivative linker, 3 It is a non-emission linker that binds to R 1 , R 2 , or R 3 A pharmaceutical composition is provided herein, which is either a releasing linker that binds to a .

[0056] In some embodiments, pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, or fumarate.

[0057] Combinations for use utilizing any of the compounds / conjugates described herein are also provided. In certain embodiments, the combination is for use in the treatment of cancer in a subject, and the combination comprises a first pharmaceutically acceptable

[0058] In the combination, the radical of the immunomodulator of the first medicament may comprise a TLR 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR9 agonist, or a TLR 7 / 8 agonist (i.e., a TLR 3, 7, 8, 9, or 7 / 8 agonist). At least one compound of the first medicament of the combination has the following formula: [Chemical formula] or may be its pharmaceutically acceptable salt or hydrate. In certain embodiments, the radical of the immunomodulator comprises a TLR agonist of formula X or XX, or is a pharmaceutically acceptable salt or hydrate of formula X or XX, [Chemical formula] In formulas X and XX, R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , [Chemical formula] and [Chemical formula] is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, In formula X, R3 is -OH, -SH, -NH2, or -NH-R 1X and In formula XX, X is CH or N, R 1X 、R 2X 、and R 2Y each of which is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl.

[0059] In certain embodiments of the combination, at least one compound of the first medicament is [Chemical formula] comprises or is its pharmaceutically acceptable salt or hydrate, and in Formula 2-I, R 1 R 3 R 4 and R 5 are each independently H, alkyl, alkoxyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, halo, heteroaryl, -COR 2x [Chemical formula] and R 2 is H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [Chemical formula] [[ID=�9]] and [[ID=[]41]] Y is a linker of at least one compound and / or a point of attachment to a targeting ligand, and is H, -OH, -NH2, -NHR 2x , -O-R 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x ] , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [Chemical formula] and comprises R 2x and R 2y ​Each of them is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, and each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q Independently selected from the group consisting of R 2q and R 2q’ Each of them is independently an alkyl group or H group. [ka] It is a non-aromatic monocyclic or bicyclic 3-10 member nitrogen-containing heterocycle, In equation 2-I, X 1 , X 2 , and X 3 Each of them independently, CR q or N, and each R q These are independently H, halogens, or optionally substituted alkyl groups. In equation 2-I, n is between 0 and 30, and m is between 0 and 4.

[0060] In certain embodiments of the combination, the radical of the immunomodulator of the first pharmaceutical comprises a TLR agonist having the following formula or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, R 1 It is an amine group, R 2 It is a single bond -NH-, R 3 is H, alkyl, hydroxyl, or any other substituted group thereof. X is CH2, NH, O, or S. Linker is R 1 , R2 , or R 3 Combine.

[0061] In a particular embodiment of the combination, at least one compound of the first pharmaceutical is [ka] It is either the compound or a pharmaceutically acceptable salt or hydrate thereof.

[0062] The linker of the first pharmaceutical in the combination may be a release linker. The linker of the first pharmaceutical in the combination may be a non-release linker. The linker of the first pharmaceutical in the combination may be a PEG linker or a PEG derivative linker, or may contain one, and may be a non-release linker.

[0063] In certain embodiments of the combination, the subject has a PD-L1, PD-1, or CTLA-4 positive tumor.

[0064] In certain embodiments, one or more immune checkpoint inhibitors of the second pharmaceutical include small molecules or other agents that disrupt immune checkpoints on target cells. In certain embodiments of the combination, one or more checkpoint inhibitors of the second pharmaceutical include pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islerizumab, a combination of osiperlimab and islerizumab, BM Each of the following is independently selected from the group consisting of S-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab.

[0065] In certain embodiments of the combination, one or more immune checkpoint inhibitors of the second pharmaceutical are one or more agents that bind to or inhibit immune checkpoints in cells selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, LAG3, TIM3, TIGIT, PD-L2, IDO, AGR1, B7-H3, B7-H4, 2B4, BTLA, A2aR, and / or members of the KIR family, e.g., KIR and C-type lectin receptors, and STAT3.

[0066] Cancer can be, or may include, cold tumors, hot tumors, or immune desert tumors.

[0067] In certain embodiments, any of the combinations described herein may be used to enhance the efficacy of one or more immune checkpoint inhibitors administered to a subject with cancer and / or to treat cancer.

[0068] In some embodiments, a method for preventing or treating a cancerous disease state is provided herein, comprising the step of contacting cells with at least one compound (e.g., any compound provided by a formula provided herein) comprising an immunomodulator or a pharmaceutically acceptable salt thereof conjugated via a linker to a folate ligand or a functional fragment or analog thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor or DAMP. In some embodiments, the cells include cells of a subject experiencing or at risk of experiencing a fibrous disease state, and the step of contacting the cells with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject. In some embodiments, the subject is a patient experiencing IPF, and the at least one compound is administered to the subject intravenously, orally, intramuscularly, intraperitoneally, topically, or by inhalation. In some embodiments, the fibrous disease state includes IPF, or fibrous diseases of the liver, skin, bladder, heart, pancreas, prostate, or kidneys.

[0069] In some embodiments, the method includes the steps of obtaining or completing the acquisition of a sample from a subject, quantifying the expression level of one or more biomarkers in the sample, wherein each of the one or more biomarkers is selected from the group consisting of CCL18, Arg1, MMP9, TIMP3, IL-1β, hydroxyproline, collagen, PDGF, TGFβ, FRβ, TNFα, IFN-γ, CD206, CD163, CD86, IL-6, CXCL10, and IFNα, comparing the expression level of each of the one or more biomarkers in the sample with the expression level of such biomarkers in a control, and CCL18, Arg1, MMP9, TIMP 3. The procedure further includes administering or completing administration of a therapeutically effective dose of an unconjugated agonist or inhibitor to a subject if IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline, or collagen is upregulated compared to control levels, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα, or CD86 is downregulated or absent compared to control levels.

[0070] In some embodiments, the folate ligand or a functional fragment or analog thereof is specific to FRβ and binds to FRβ on cells. In some embodiments, a method is provided for treating a subject experiencing a cancerous disease state (e.g., cancer or cancer recurrence / relapse), comprising the step of administering one or more compounds (or pharmaceutically acceptable salts or hydrates thereof) comprising a targeting moiety (e.g., a folate ligand or a functional fragment or analog thereof) conjugated via a linker to an immunomodulator radical or a pharmaceutically acceptable salt thereof (e.g., any TLR agonist, including, without limitation, TLR 3, 7, 8, 9, or 7 / 8 agonists), wherein the targeting moiety targets a cellular pattern recognition receptor or DAMP. In certain embodiments, the M2 macrophages of the subject are reprogrammed into M1 macrophages by contacting the target cells of the subject with one or more compounds or a pharmaceutically acceptable salt or hydrate thereof. In at least one exemplary embodiment, the immunomodulator radical or a pharmaceutically acceptable salt thereof is a TLR7 agonist, and the linker is a releasing linker. In at least one additional embodiment, the linker is a non-releasing linker.

[0071] In certain embodiments, the step of administering at least one compound (e.g., a first therapeutic or first pharmaceutical) or a pharmaceutically acceptable salt or hydrate thereof activates antitumor cells or an anti-inflammatory signaling cascade in the subject. In at least one embodiment, such antitumor cells are T cells. Additionally, or alternatively, such antitumor cells may be macrophages.

[0072] The embodiments disclosed, as well as other features, advantages, and aspects included herein, and those associated therewith, will become apparent from the following detailed descriptions of various exemplary embodiments of this disclosure. Such detailed descriptions will be better understood in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0073] [Figure 1-1] Figure 1A shows the chemical structure of an exemplary compound having a targeting moiety (e.g., a folate receptor ligand) bound to an immunomodulator (e.g., a radical of a Toll-like receptor (TLR) 7 (TLR7) agonist) via a non-releasing linker (e.g., containing a polyethylene glycol (PEG) backbone).

[0074] [Figure 1-2] Figure 1B shows the chemical structure of an exemplary compound having a targeting moiety (e.g., a folate receptor ligand) bound to an immunomodulator (e.g., a TLR7 agonist radical) via a releasing linker (e.g., one containing a disulfide moiety in its skeleton), as well as an exemplary drug release mechanism.

[0075] [Figure 1-3] Figure 1C shows the chemical structure of an exemplary compound provided herein.

[0076] [Figure 1-4] Figure 1D illustrates how checkpoint proteins, such as programmed cell death ligand 1 (PD-L1) on tumor cells and programmed cell death protein 1 (PD-1) on T cells, help maintain the immune response in a checked state. PD-L1 binding to PD-1 prevents T cells from killing tumor cells in the body (left panel). Blocking PD-L1 binding to PD-1 with immune checkpoint inhibitors (anti-PD-L1 or anti-PD-1) allows T cells to kill tumor cells (right panel).

[0077] [Figure 2] Figure 2 shows a flowchart illustrating methods for treating subjects who have or are at risk of having fibrous disease or cancer.

[0078] [Figure 3-1] Figures 3A–3F show graphical data of various marker levels measured from human M2 macrophages when exposed to exemplary free (untargeted) TLR7 agonists or exemplary targeted (e.g., those with folate receptor-binding ligands) TLR7 agonists at various concentrations for each compound. Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; treated group vs. untreated M2 group, Dunnett's multiple comparison test. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above.

[0079] [Figure 4-1] Figures 4A–4E and 5A–5D show graphical data representing the levels of various markers measured from M2 macrophages incubated with exemplary free or targeted TLR7 agonists at various concentrations for 2 hours (Figures 4A–4E) or 46 hours (Figures 5A–5D). Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; in Figures 4A–5D, compound 1A and compound 1B treated groups versus untreated M2 groups, by Dunnett's multiple comparison test. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Same as above. [Figure 5-2] Same as above.

[0080] [Figure 6-1]Figures 6A–6D show graphical data representing the levels of various markers measured from M2 macrophages treated with (i) 48 hours with representative free and targeted TLR7 agonists at various concentrations (Figures 6A and 6B), or (ii) 2 hours with fresh medium followed by 46 hours of incubation (Figures 6C and 6D). Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; compound 1A and compound 1B treated group vs. M2 untreated group, Dunnett's multiple comparison test. [Figure 6-2] Same as above.

[0081] [Figure 6-3] Figure 6E shows flow cytometry data that supports the finding that macrophages induced by THP-1 (a human monocytic cell line derived from patients with acute monocytic leukemia) were folate receptor beta (FRβ) positive (FRβ+).

[0082] [Figure 6-4] Figure 6F shows that an exemplary targeted TLR7 agonist is stable.

[0083] [Figure 7-1] Figure 7A shows stained images of lungs taken from mice with BM-induced experimental fibrosis at 7, 14, and 21 days after BM-induced lung injury, stained with anti-mouse FRβ antibody and then stained with hematoxylin-eosin (H&E).

[0084] [Figure 7-2] Figure 7B shows the quantification of FRβ staining in the panel shown in Figure 7A.

[0085] [Figure 7-3] Figures 7C and 7D show FRβ immunohistochemistry (IHC) staining of lung tissue from a human with idiopathic pulmonary fibrosis (IPF) (Figure 7C) and lung tissue from a healthy human (Figure 7D).

[0086] [Figure 7-4] Figure 7E shows images of mouse tissues / organs taken from mice with or without BM-induced experimental fibrosis (phosphate-buffered saline (PBS) control) and imaged with folate receptor-targeted fluorescent dyes.

[0087] [Figure 7-5] Figure 7F shows fluorescence-activated cell sorting (FACS) analysis of mice with BM-induced experimental fibrosis. [Figure 7-6] Same as above. [Figure 7-7] Same as above.

[0088] [Figure 8-1] Figure 8A illustrates the treatment plans for free and targeted TLR7 agonists in the BM model.

[0089] [Figure 8-2] Figures 8B-8G show the levels of pro-fibrosis markers (Figures 8B-8D) and pro-inflammatory markers (Figures 8E-8G) measured from mice treated with the BM model shown in Figure 8A. [Figure 8-3] Same as above. [Figure 8-4] Same as above.

[0090] [Figure 8-5] Figure 8H shows the cell count in bronchoalveolar lavage fluid (BALF) obtained from mice treated with the BM model shown in Figure 8A.

[0091] [Figure 9-1] Figures 9A and 9B show the survival curves (Figure 9A) and body weight changes (Figure 9B) of mice with pulmonary fibrosis treated with untargeted and targeted TLR7 drugs. [Figure 9-2] Same as above.

[0092] [Figure 10-1]Figure 10A shows the hydroxyproline content (μg / lung) in lung tissue as a measure of fibrosis.

[0093] [Figure 10-2] Figures 10B and 10C show lung tissue from Figure 9A after H&E staining (Figure 10B) and Masson's trichrome (collagen) staining (Figure 10C). [Figure 10-3] Same as above.

[0094] [Figure 11-1] Figures 11A and 11B show the survival curves (Figure 11A) and weight change (Figure 11B) of mice with pulmonary fibrosis treated with exemplary targeted TLR7 agonists, where the values ​​represent the mean ± standard deviation for each group. [Figure 11-2] Same as above.

[0095] [Figure 12-1] Figure 12 shows the dose-dependent effect of exemplary targeted TLR7 agonists on the suppression of fibrosis in BM-induced mice. Figure 12A shows graph data related to body weight over time in BM-induced mice. Figure 12B shows the measurement of hydroxyproline content in lung tissue treated with various doses of exemplary compounds provided herein (e.g., compound 1B). Figure 12C shows images of histological analysis of lung tissue by various stains. Each value represents the mean ± standard deviation for each group; *P<0.05, **P<0.005, ***<0.0005; saline vs. vehicle group, treatment group vs. vehicle group, by Student's t-test. [Figure 12-2] Same as above. [Figure 12-3] Same as above.

[0096] [Figure 13-1] Figures 13A–13D show the levels of various markers measured from M2 macrophages reprogrammed by the method herein for 48 hours using exemplary targeted TLR7 agonists at various concentrations, with each value representing the mean ± standard deviation for each group. [Figure 13-2] Same as above.

[0097] [Figure 14-1] Figures 14A–14C show the levels of various markers measured from M2 macrophages reprogrammed by the method herein using exemplary free and targeted TLR7 agonists at various concentrations. Figure 14A shows CCL18, Figure 14B shows CD206, and Figure 14C shows IL-1β. The values ​​shown in Figures 14A–14C represent the mean ± standard deviation for each group; #P<0.05, ##P<0.005, ###P<0.0005, ####P<0.0001; Compound 3A, Compound 3B treatment, and Compound 3C treatment group vs. M2 untreated group, by Dunnett's multiple comparison test. [Figure 14-2] Same as above. [Figure 14-3] Same as above.

[0098] [Figure 15] Figure 15 shows the levels of secreted chemokine (CC motif) ligand 18 (CCL18) protein in each group of cells in Figures 14A–14C after treatment with exemplary free and targeted TLR7 agonists.

[0099] [Figure 16] Figure 16 shows the methodology for the BM mouse model.

[0100] [Figure 17-1] Figures 17A and 17B show the purity of exemplary targeted TLR7 agonists. [Figure 17-2] Same as above.

[0101] [Figure 18-1]Figures 18A–18F show data obtained from the in vivo study in Figure 16, including survival curves (Figure 18A), body weight changes (Figures 18B and 18D), concentration of cells containing BALF (Figure 18C), hydroxyproline concentration (μg HP / lung lobe) in live mice (Figure 18E), and hydroxyproline concentration (μg HP / lung lobe) in all mice (i.e., including both live mice and those that died before day 21) (Figure 18F). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above.

[0102] [Figure 19-1] Figures 19A–19F show that both targeted and untargeted TLR7 agonists reprogram human monocyte-derived anti-inflammatory macrophages into an anti-fibrotic phenotype. Figure 19A shows Arg1, Figure 19B shows CD206, Figure 19C shows CD163, Figure 19D shows CCL18, Figure 19E shows CXCL10, and Figure 19F shows IL-6. Mean ± standard deviation. Statistical significance between groups was determined using unpaired two-sided t-tests (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 19-2] Same as above. [Figure 19-3] Same as above.

[0103] [Figure 20-1] Figure 20 shows plasma cytokine levels in healthy mice treated with compound 1A, compared to those of compound 1B (Figures 20A-20F). Figures 20A and 20D show IL-6, Figures 20B and 20E show IFNα, and Figures 20C and 20F show tumor necrosis factor α (TNF-α). Figure 20G shows the change in body weight after treatment of mice with exemplary compounds, with the change in body weight being a measure of systemic toxicity during every-other-day administration (n=2); mean ± standard deviation. Statistical significance between groups was compared using unpaired two-sided t-tests (*P<0.05, **P<0.01, ***P<0.001). [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above.

[0104] [Figure 21] Figure 21 shows healthy lung and fibrous lung as shown in Figure 6, stained with 4',6-diamidino-2-phenylindole (DAPI) (nucleus, blue), anti-F4 / 80 (macrophage, red), and anti-mannose receptor (CD206).

[0105] [Figure 22] Figure 22 shows the effects of various exemplary compounds on interleukin-6 (IL-6) expression in peripheral blood mononuclear cells.

[0106] [Figure 23-1] Figures 23A and 23B show the in vitro effects of various exemplary compounds on IL-6 and CXC motif chemokine 10 (CXCL-10) induction in monocyte-derived M2 macrophages over 48 hours. [Figure 23-2] Same as above.

[0107] [Figure 23-3] Figures 23C and 23D show the in vivo effects of various exemplary compounds on IL-6 and tumor necrosis factor α (TNF-α) production. [Figure 23-4] Same as above.

[0108] [Figure 24-1]Figures 24A-24F show data obtained from in vivo combined studies of FA-TLR7-1A non-releasing conjugate (compound 1000) (3 nmol / mouse) and anti-CTLA-4 (10 mg / kg, twice weekly) in a 4T1 orthotopic solid tumor model. Figure 24A shows tumor volume measured every other day after treatment, Figure 24B shows a graph of tumor weight measured at the end of the study, Figure 24C shows graphs of tumor volume of individual mice in different treatment groups, Figure 24D shows graphs of tumor volume after treatment with combinations of FA-TLR7-1A, PD-1, and FA-TLR7-1A+PD-1, Figure 24E shows a plot of tumor volume of a single mouse after treatment with either PD-1 or the FA-TLR7-1A+PD-1 combination, and Figure 24F shows a graph of tumor volume against the number of days after the start of treatment. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 24-5] Same as above.

[0109] [Figure 25] Figure 25 shows a graph of immunocytological analysis results obtained from digestive tumor tissue after in vivo combined treatment with FA-TLR7-1A slow-release conjugate (3 nmol / mouse) and anti-CTLA-4 (10 mg / kg, twice weekly) in a 4T1 orthotopic solid tumor model.

[0110] [Figure 26] Figure 26 shows IHC images of 4T1 tumor tissue after treatment with FA-TLR7-1A (compound 1000), stained with DAPI (white, solid circles), antibody against Arg-1 (white arrows, top row), antibody against iNOS (white, dashed circles), and antibody against CD8 (white arrows, bottom row).

[0111] [Figure 27]Figure 27 shows an overview of in vivo combination therapy approaches, including the use of FA-TLR7-1A in combination with anti-PD-1 / anti-PD-L1 antibodies in a 4T1 tumor model.

[0112] [Figure 28] Figure 28 shows a graph of tumor volume obtained from the in vivo combination study shown in Figure 27, measured after treatment with FA-TLR7-1A conjugate (compound 1000) (3 nmol / mouse) in combination with anti-PD-1 (5 mg / kg, twice weekly) in the MC38 solid tumor model.

[0113] [Figure 29-1] Figure 29A shows the immunocytological analysis data of the digestive MC38 tumor sample after the treatment shown in Figure 27.

[0114] [Figure 29-2] Figure 29B shows the tumor volume measured after re-challenging cured mice and healthy control mice from the combined study outlined in Figure 28 with 500,000 MC38 cells.

[0115] [Figure 30] Figure 30 shows an overview of in vivo combination therapy techniques, including the use of FA-TLR7-1A non-releasing conjugate (compound 1000) in combination with an anti-CTLA-4 antibody in an LL / 2 Lewis lung cancer tumor model.

[0116] [Figure 31] Figure 31 shows a graph of tumor volume obtained from the in vivo combination study shown in Figure 30, measured every other day after treatment with FA-TLR7-1A conjugate (compound 1000) (3 nmol / mouse) in combination with anti-CTLA-4 (10 mg / kg, twice weekly) in an LL / 2 Lewis lung cancer tumor model.

[0117] [Figure 32-1] Figure 32A shows a graph of the weight change of mice during combination therapy in the 4T1 tumor model of Example 16.

[0118] [Figure 32-2] Figure 32B shows a graph of the weight change of mice during the combination therapy treatment of Example 17.

[0119] [Figure 32-3] Figure 32C shows a graph of mouse body weight change as a measure of systemic toxicity after chronic administration of 60 nanomoles / mouse / day of TLR7-1A (compound 1) or FA-TLR7-1A (compound 1000) to healthy mice.

[0120] This disclosure can take various modifications and alternative forms, but exemplary embodiments are shown in the drawings as an example and are described in detail herein. [Modes for carrying out the invention]

[0121] Detailed explanation The compounds, combinations, compositions, and methods of this disclosure generally target the innate immune system of interest and reprogram macrophage polarization from M2 to M1, thereby making the pro-inflammatory properties of the M1 phenotype dominant. For example, in at least one exemplary embodiment, such compounds and compositions include a targeting moiety that targets folate receptor β (FRβ), such as a folate receptor-binding ligand, or its analogue, functional fragment, derivative, or radical (e.g., a pteroyl amino acid), conjugated to or linked to an immunomodulator or a pharmaceutically acceptable salt thereof. The term "ligand" refers to a molecule, ion, or atom that binds to a central atom or ion (e.g., a drug) of a compound.

[0122] As will be described in detail below, such embodiments utilize limited FRβ expression to directly localize systemically administered compounds to FRβ-expressing cells (e.g., those of cancerous tissue), thereby enabling the immune modulator component to then convert, for example, reprogram activated myeloid cells (e.g., M2-like macrophages) to pro-inflammatory M1 polarization. This targeted design is beneficial because it can prevent systemic activation of the immune system (i.e., systemic exposure to such compounds is reduced), and therefore toxicity is avoided.

[0123] Further exemplary embodiments may include a linker positioned between the targeting moiety and the immunomodulator. Such a linker may be release-oriented or non-release-oriented. Compounds / compositions containing a release-oriented linker may, upon administration, result in the release of the targeting moiety and the immunomodulator from each other at or approximately the time the immunomodulator becomes activated. Additionally, or alternatively, in embodiments in which the compound contains a non-release-oriented linker, upon administration, the targeting moiety and the immunomodulator are not rapidly released under physiological conditions. In this form, the components of the compound remain together after uptake by the targeted cells and / or activation of the immunomodulator.

[0124] Various embodiments and supporting examples related to them will now be described.

[0125] compound

[0126] The compound may comprise a drug (e.g., its radical) (e.g., an immunomodulator), or a pharmaceutically acceptable salt or hydrate thereof, conjugated to a targeting moiety (e.g., its radical). The immunomodulator (e.g., its radical) may be conjugated directly to the targeting moiety (e.g., its radical) or via a linker (e.g., including a spacer as appropriate).

[0127] Figure 1A shows at least one embodiment of compound 100, where compound 100 comprises, for example, an immunomodulator 102 (or a drug or its radical) having formula I, where R 3 is a hydroxyl group. The immunomodulator 102 (e.g., its radical) may be conjugated to the targeting moiety 104 (e.g., its radical) via the linker 106, where the targeting moiety 104 (e.g., its radical) is a folate, and the (e.g., non-releasing) linker 106 is a polyethylene glycol (PEG) linker repeated n times, where n is an integer from 1 to 32.

[0128] In a particular embodiment, compound 100 is of formula: QLT It can be represented by the formula, where Q is the radical of the folate receptor-binding ligand / targeting moiety 104, L is the linker 106, and T is the radical of the TLR agonist / immunomodulator 102. The linker L may include any of the linker formulas presented herein.

[0129] Similarly, Figure 1B shows at least one embodiment of compound 150. Compound 150 has an immunomodulator / drug 152 (e.g., its radical) which is a Toll-like receptor 7 agonist (e.g., its radical) having formula III, for example, conjugated to a targeting moiety 154 (e.g., its radical) via a (e.g., releasing) linker 156.

[0130] Immunomodulator

[0131] "Immune modulator" means any drug, warhead, or other composition or compound that stimulates or otherwise affects the immune system of interest by inducing the activation of or increasing the activity of one or more components of the immune system. For example, without limitation, an immune modulator may include a compound or composition that targets one or more pattern recognition receptors or damage-associated molecular patterns (DAMPs) in addition to, or instead of, targeting signaling pathways in immune cells. "Pattern recognition receptor" means, including, any immune receptor expressed on the membrane of leukocytes, e.g., at least macrophages, that can bind to a specific ligand that activates the receptor and ultimately result in an innate immune response (in certain cases, ultimately resulting in the development of antigen-specific adaptive immunity). An immune modularizer (e.g., a radical of an immune modularizer) may include its pharmaceutically acceptable salts or hydrates.

[0132] Exemplary examples of immune modulators include, but are not limited to, TLR agonists, interferon gene stimulators (STINGs), nucleotide-binding oligomerized domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), absent-in-melanoma 2 (AIM2)-like receptors (ALRs), advanced glycation end product receptors (RAGEs), or any other pattern recognition receptors located in the cell's endosomes or cytoplasm. Immune modulators may also include, as an addition or alternative, activated B cell nuclear factor kappa light chain enhancer (NFκβ) activators or Iκβ kinase inhibitors, which function further downstream in the pathway. Table 1 provides examples of such NFκβ activators or Iκβ kinase inhibitors that can be used as immune modulators.

[0133] [Table 1-1] [Table 1-2] [Table 1-3]

[0134] "TLRs" are a class of proteins that play a role in the innate immune system and are examples of pattern recognition receptors. TLRs can be single transmembrane receptors that recognize structurally conserved molecules derived from microorganisms. TLRs can be expressed on the membranes of leukocytes, including dendritic cells, macrophages, natural killer cells, adaptive immune cells (e.g., T and B lymphocytes), as well as non-immune cells (epithelial and endothelial cells and fibroblasts). Non-limiting examples of TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. In some embodiments, the TLR agonists provided herein bind to one or more TLRs. In some embodiments, the TLR agonists provided herein bind to TLR7, TLR8, or TLR9. In some embodiments, the TLR agonists provided herein bind to TLR7. In some embodiments, the TLR agonists provided herein bind to TLR7 and TLR8 (TLR7 / 8 agonists). In some embodiments, the agonist is a ligand that binds to a receptor and activates it.

[0135] In some cases, such as when a compound is a (potent) TLR-7 / 8 agonist, the immunomodulator (or its radical) can be highly toxic if delivered systemically in an unconjugated form. In some cases, it is desirable to reduce and / or eliminate the systemic toxicity associated with such compounds. In some cases, the radical of a compound has reduced toxicity compared to the free form of such a compound (e.g., reduced by 10%–15%, 15%–20%, 20%–30%, 30%–40%, 40%–50%, 50%–60%, 60%–70%, 70%–75%, 75%, 75%–80%, 80%–9%, ​​90%, or 90%–99% (all enumerated ranges include the endpoint mentioned and all 1% increments are encompassed therein)). Furthermore, in some cases, the conjugated form of the compound is effective at equivalent or lower concentrations (e.g., 120% or less, 100% or less, 80% or less, 60% or less, or 40% or less of the free form's effective dose (ED)) compared to the free form. 50 It is effective (having a median concentration).

[0136] The immunomodulator or its pharmaceutically acceptable salts or hydrates may include any therapeutic agent (e.g., a drug) suitable for reprogramming activated macrophages (M2-like phenotype) to an M1-like phenotype. In certain embodiments, the immunomodulator operates in the endosomes and / or cytoplasm of cells (e.g., depending on its structure). In at least one embodiment, the immunomodulator positively modulates pattern recognition receptors and / or their downstream signaling pathways (in each case, a part of the innate immune system), e.g., TLR, NLR, RLR, ALR, RAGE, and / or STING agonists, and / or kinases of the Pelle / interleukin-1 receptor-associated kinase (IRAK) family (e.g., IRAK-M inhibitors). In other embodiments, the compound includes a phosphoinositide 3-kinase (PI3K) inhibitor or other inhibitors negatively modulating the adaptive immune system (e.g., this can be used alone or in combination with an immunomodulator targeting a pattern recognition receptor or DAMP). In some embodiments, particularly when used in the treatment of cancer, the compound (or a pharmaceutically acceptable salt or hydrate thereof) includes (a) an immunomodulator radical that targets a pattern recognition receptor or DAMP and / or is an agonist of a downstream signaling pathway of the target innate immune system, and (b) a combination with a mammalian target (mTOR) inhibitor of rapamycin (ATP competitive or otherwise), e.g., rapamycin or CZ415.

[0137] In some embodiments, the immunomodulator of a compound (or a pharmaceutically acceptable salt or hydrate thereof) includes TLR agonists (e.g., its radical), for example, but not limited to, TLR3 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR8 agonists, or TLR9 agonists (all of which bind to TLRs present in cellular endosomes). For example, in at least one exemplary embodiment, but not limited to, the immunomodulator of a compound (or a pharmaceutically acceptable salt or hydrate thereof) may be selected from the compounds (e.g., including its radicals) listed in Table 2 below.

[0138] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0139] In some embodiments, the immunomodulator comprises a TLR7 agonist or its radical. In some embodiments, the immunomodulator (or radical) (e.g., TLR7 agonist) is of formula I: [ka] It has the structure of or a pharmaceutically acceptable salt or hydrate thereof. In some embodiments, R 1 is an amine. In some embodiments, R 2is (for example, a single) bond, or an amine (for example, -NH-). In certain embodiments, R 3 X is H, alkyl, hydroxyl, or any other suitable substituent (e.g., as described herein). In some embodiments, X is CH2, NH, O, or S. In some embodiments, if the compound contains a radical of formula I, the targeted moiety is in this compound at any suitable position, for example, R 1 , R 2 , and / or R 3 They are then conjugated or connected through them (for example, through the linker and / or directly).

[0140] In at least one exemplary embodiment, the compound is of formula Ia: [ka] An immunomodulator (or its radical) (e.g., a TLR7 agonist) or containing thereof, or a pharmaceutically acceptable salt or hydrate thereof. In some embodiments, X is CH or N. In some embodiments, R1 is -NH2 or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] In a particular embodiment, R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In some embodiments, [ka] The compound is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, if the compound contains a radical of formula Ia, the targeting moiety can be placed at any preferred position, for example, R 1 , R 2 , and / or R 3 They are conjugated or connected to, or through, them (for example, through the linker and / or directly).

[0141] In some embodiments, the compound is of formula II: [ka] The immunomodulator comprises having or containing the structure of, or a pharmaceutically acceptable salt or hydrate thereof. In some embodiments, R 1 is an amine. In some embodiments, R 2 is (for example) a single bond or -NH-. In some embodiments, R 3 X is H, alkyl, hydroxyl, or any other substituent, for example, as described herein. In some embodiments, X is CH2, NH, O, or S. In some embodiments, if the compound contains a radical of formula II, the targeted moiety is at any preferred position, for example, R 1 , R 2 , and / or R 3 They are conjugated or connected to, or through, them (for example, through the linker and / or directly).

[0142] In other embodiments, the immunomodulator of the compound may include or may include a drug comprising a TLR agonist of formula III (e.g., or its radical) or a pharmaceutically acceptable salt or hydrate thereof. [ka] In the formula, R 1 is an amine group, R 3This is a hydroxyl group. Furthermore, if desired, the targeting moiety (e.g., or its radical) or other ligand can be added to the agonist of formula III. 1 or R 3 It can be conjugated (either through a linker or directly). The TLR agonist of Equation III (e.g., or its radical) is a TLR7 agonist and is at least 10 times (10 ×) more potent than conventionally available TLR7 agonists.

[0143] In a particular embodiment, the immunomodulator of the compound is formula IV: [ka] This may be a TLR7 agonist (e.g., or its radical) or a pharmaceutically acceptable salt or hydrate thereof, or may contain the same. In the formula, R 1 is an amine group, R 2 It is a single bond -NH-.

[0144] In certain embodiments, the immunomodulator comprises a TLR agonist of formula X or XX, or a pharmaceutically acceptable salt of formula X or XX. [ka] [ka] In equations X and XX, R1 is -NH2 or -NH-R 1X R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And, [ka] R3 is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle, where R3 is -OH, -SH, -NH2, or -NH-R 1X In formula XX, X is CH or N, and R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0145] In certain embodiments, the immunomodulator (e.g., TLR7 agonist) group of the compound is of formula XX, and more specifically, formula XX': [ka] A radical having the structure, During the ceremony, R 1B -NH2 or -NH-R 1X And, R 2B Hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And, R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. X is either CH or nitrogen (N).

[0146] Alkyl, alkoxy, etc., represent a straight chain (i.e., an unbranched chain), a branched chain, or a combination thereof, and these may be fully saturated, monounsaturated, or polyunsaturated, and have a specified number of carbon atoms (i.e., C1-C1). 10 This can include divalent and polyvalent radicals having 1 to 10 carbon atoms. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl-2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-penadienyl(3-(1,4-penadienyl))), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher-order homologs and isomers. Alkoxy is an alkyl group bonded to the remainder of the molecule via an oxygen linker (-O-). In some embodiments, alkoxy refers to a radical bonded through the oxygen atom of the formula -O-alkyl.

[0147] Generally, the terms "acyl" or "acyl substituent" refer to groups derived from oxoacids, including inorganic acids, by the removal of one or more hydroxyl groups, and include double-bonded oxygen atoms and alkyl groups. Furthermore, references to individual radicals, such as "propyl," encompass only linear radicals, while branched isomers, such as "isopropyl," are specifically mentioned.

[0148] In a particular embodiment, the TLR7 agonist has formula X, and the radical of the TLR7 agonist is R 1A , R 1B , R 3A , or R 3BIf the TLR7 agonist is conjugated to the targeting portion via a linker in any one of the following ways, and the TLR7 agonist has formula XX', then the TLR7 agonist is R 1A , R 1B , R 3A , or R 3B It is one of these components and is conjugated to the target portion via the linker.

[0149] In a particular embodiment, a compound comprising a targeting moiety containing a folate ligand or a functional fragment or analog thereof, conjugated via a linker to an immunomodulator containing a TLR agonist, wherein the TLR agonist has the following structure represented by formula XXX: [ka] It has, In the formula, R 1 is an amine group, R 2 It is a single bond -NH-, and R 3 is H, alkyl, hydroxyl, or any other substituted group thereof, X is CH2, NH, O, or S, and the linker is R 1 , R 2 , or R 3 A compound that binds to is provided. Additionally, or alternatively, R1 may be -NH2 or -NH-R 1X It could be, and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] It could be, R 1X , R 2X , and R 2Y Each of these can be independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] X may be a 3-10 member N-containing non-aromatic monocyclic or bicyclic heterocycle, and / or X may be CH, CR2, or N.

[0150] In some embodiments, a pharmaceutical composition comprising any formula or compound provided, wherein the linker comprises PEG or a PEG derivative, and in some examples, R 3 It is a non-emission linker that binds to R 1 , R 2 , or R 3 A pharmaceutical composition is provided which is either a release linker that binds to a certain object.

[0151] In some embodiments, the immunomodulator group of the compound (e.g., TLR7 or TLR7 / 8 agonist) is of formula XXX, and more specifically, formula XXX': [ka] A radical having the structure, During the ceremony, R 1C -NH2 or -NH-R 1X And, R 2C This is combined, NH, -NR 1X , or CH2, If applicable, [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. X A This is CH2, NH2, or -NH-R 1X And, Each R 1X This is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. The TLR7 agonist is R 1C , R 2C , or R 3BIt is one of these components and is conjugated to the target portion via the linker.

[0152] One embodiment is given by formula (2-II): [ka] We provide a compound represented by the structure of, or a pharmaceutically acceptable salt or hydrate thereof, in which, R 1 , R 3 , R 4 , R 5 These are, independently, H, alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, and -COR. 2x , [ka] And, R 2 H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] And, Z is represented by the formulas GL-, GO-, GLO-, GLO-alkyl-, GLS-, G-SO2-NH-, and GL-NR a R b -, GLS(O) x -alkyl-, GL-CO-, GL-aryl-, GL-NH-CO-NH-, GL-NH-O-, GL-NH-NH-, GL-NH-CS-NH, GLC(O)-alkyl-, GL-SO2-, [ka] It is the basis of, L is the linker, and G is the folate receptor-binding ligand. R a and R bEach of these is independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c And R c x is alkyl, aryl, oxy, or alkoxy, and x is 0 to 3, R 2x and R 2y Each of these is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. Each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q Independently selected from the group consisting of, Each R 2q and R 2q’ These are independently alkyl or H, [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. In Equation 2-II, X 1 , X 2 , and X 3 Each of them operates independently, CR q or N, and each R q These are independently hydrogen, halogen, or optionally substituted alkyl groups. In equation 2-II, n is between 0 and 30, and m is between 0 and 4.

[0153] At least one embodiment is given by formula (2-IIA): [ka] We provide an immunomodulator (or its radical) represented by the structure of or a pharmaceutically acceptable salt or hydrate thereof, During the ceremony, R 1 This is an optionally substituted alkyl (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each substituent independently being a halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3, R 2x and R 2y These are, independently, hydrogen and -N(R). z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (for example, substituted with one or more substituents where each substituent is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and each R z These are independently hydrogen, halogen, or optionally substituted alkyl, or R 2x and R 2y These combine to form a heterocycloalkyl group as needed (for example, the heterocycloalkyl group as needed may be monocyclic or bicyclic heterocycloalkyl groups, and / or the heterocycloalkyl group as needed may be a 3- to 10-membered heterocycloalkyl group), Each R 3 These are independently halogens, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, where alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl may be substituted as needed. R 4 and R 5Each of these is independently an alkyl, alkoxy, halogen, or cycloalkyl, and the alkyl, alkoxy, and cycloalkyl are substituted as needed. X 1 , X 2 , and X 3 Each of them independently, CR q or N, and each R q These are independently hydrogen, halogen, or optionally substituted alkyl groups. Z is LG, L is the linker, and G is the folate receptor-binding ligand. n is between 1 and 6, and m is between 0 and 4.

[0154] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1.

[0155] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 1 R is an alkyl group that is substituted as needed. In one embodiment, R 1 is a C3-C6 alkyl group, which is substituted as needed. In another embodiment, R 1 R is an acyclic C3-C6 alkyl which is substituted as needed. In at least one embodiment, R 1 It is butyl.

[0156] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 2 -NR2x R 2y In at least one embodiment, R 2 It is NH2.

[0157] One embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 3 H is H.

[0158] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 is alkyl. In at least one embodiment, R 4 It is methyl.

[0159] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 5 is alkyl. In at least one embodiment, R 5 It is methyl.

[0160] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 and R 5 Each of these is an alkyl group. In at least one embodiment, R 4 and R 5 These are methyl compounds, respectively.

[0161] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, where m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0162] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein X 1 , X 2 , and X 3 Each of these is N. In at least one embodiment, X 1 In another embodiment, X 2 is N. In at least another embodiment, X 3 It is N.

[0163] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-II) or (2-IIA), where the compound has the structure: [ka] [ka] It is represented by, or is a pharmaceutically acceptable salt or hydrate of any of the aforementioned structures.

[0164] At least one embodiment is given by formula (2-III): [ka] We provide an immunomodulator (or its radical) represented by the structure of the formula, or a pharmaceutically acceptable salt or hydrate thereof, in which, R 1 , R 3 , R 4 , and R5 These are, independently, H, alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, and -COR. 2x , [ka] And, R 2x and R 2y Each of them is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, and each R 2z -NH2, -NR 2x R 2y , -OR 2x , -SO-R 2x , and -COR 2x Independently selected from the group consisting of, Z is a group of the formula GL-, GL-CO-, GLC(O)-alkyl-, L is a linker, and G is a folate receptor-binding ligand. X 1 , X 2 , and X 3 Each of them independently, CR q or N, Each R q These are independently hydrogen, halogen, or optionally substituted alkyl groups. In equation 2-III, n is between 0 and 30, and m is between 0 and 4.

[0165] At least one embodiment is given by formula (2-IIIA): [ka] We provide an immunomodulator (or its radical) represented by the structure of or a pharmaceutically acceptable salt or hydrate thereof, During the ceremony, R 1This is an optionally substituted alkyl (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each substituent independently being a halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), Y is the binding site to the linker or targeting moiety of the compound, H, -OR z , -NR 2x R 2y , -SR z -SOR z , -SO3R z -N3, -COR z ,-COOR z ,-CONR z 2. -COSR z , -SO2N(R z )2, or -CON(R z )2 included, R 2x and R 2y These are H and -N(R) independently of each other. z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (for example, optionally substituted with one or more substituents, each substituent independently being oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), Each R z These are independently H, halogens, or optionally substituted alkyls, or R 2x and R 2y These combine to form a heterocycloalkyl group as needed (for example, the heterocycloalkyl group as needed may be monocyclic or bicyclic heterocycloalkyl groups, and / or the heterocycloalkyl group as needed may be a 3- to 10-membered heterocycloalkyl group), Each R 3These are independently halogens, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, where alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl may be substituted as needed. R 4 and R 5 Each of these is independently an alkyl, alkoxy, halogen, or cycloalkyl, and the alkyl, alkoxy, or cycloalkyl may be substituted as needed. Each X 1 , X 2 , and X 3 CR is independent. q or N, and each R q These are independently H, halogens, or optionally substituted alkyl groups. Z is LG, L is the linker, and G is the folate receptor-binding ligand. n is between 1 and 6, and m is between 0 and 4.

[0166] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 1 and Y is OH. In another embodiment, n is 1 and Y is NH2.

[0167] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where Y is OH.

[0168] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where Y is NH2.

[0169] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 and Y is OH.

[0170] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 and Y is NH2.

[0171] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 0 and Y is NH2.

[0172] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 1 R is an alkyl group which is substituted as needed. In at least one embodiment, R 1 is a C3-C6 alkyl group, which is substituted as needed. In another embodiment, R 1 R is an acyclic C3-C6 alkyl which is substituted as needed. In at least one other embodiment, R 1 It is butyl.

[0173] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 3 H is H.

[0174] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 is alkyl. In at least one embodiment, R 4 It is methyl.

[0175] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 5 is alkyl. In at least one embodiment, R 5 It is methyl.

[0176] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 and R 5 Each of these is an alkyl group. In at least one embodiment, R 4 and R 5 These are methyl compounds, respectively.

[0177] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, where m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0178] At least one embodiment provides an immunomodulator (or its radical) having the structure of formula (2-III) or (2-IIIA) or a pharmaceutically acceptable salt or hydrate thereof, wherein X 1 , X 2 , and X 3Each of these is N. In at least one embodiment, X 1 In another embodiment, X 2 In another embodiment, X 3 It is N.

[0179] In some embodiments, the immunomodulator (or its radical) has the following structure: [ka] It is represented by one or more of the following, or a pharmaceutically acceptable salt or hydrate thereof.

[0180] In some embodiments, the immunomodulator (or its radical) has the following structure: [ka] It is represented by one or more of the above structures, or a pharmaceutically acceptable salt or hydrate of any of the above structures.

[0181] targeting part

[0182] As mentioned above, immunomodulators (or their radicals) containing TLR-7 / 8 compounds can be conjugated to the targeting moiety (e.g., via a linker).

[0183] The compound may further comprise a targeting moiety (or radical) bound to an immunomodulator (or radical thereof) that targets a cellular pattern recognition receptor or DAMP.

[0184] Due to the toxicity associated with systemic administration of at least some of the conventional immunomodulators identified herein, their practical application in the treatment of cancer has historically been excluded. For example, TLR agonists may not be tolerable by individuals and, in some cases, can lead to death of the subject (e.g., when administered systemically by conventional modalities). In some embodiments, the compounds present, for example, those having formulas I and / or II, are significantly more potent than conventional drugs that can be used with the compounds, and in some embodiments, mechanisms for avoiding systemic toxicity are preferred.

[0185] In some embodiments, the targeted portion includes a ligand that targets a specific region or tissue of an individual (e.g., with high specificity), and in certain examples, this may include, for example, hormones, antibodies, and / or vitamins.

[0186] In certain embodiments, the targeted portion comprises a folate ligand or a functional fragment or analog thereof. "Folate" means, for example, folic acid and analogs and derivatives of folate, for example, without limitation, folic acid, pteroyl polyglutamic acid, pteroyl-D-glutamic acid, and folate receptor-binding pteridines, for example, tetrahydropterin, dihydrofolate, tetrahydrofolate, and folate receptor-binding molecules including their deaza and dideza analogs.

[0187] The terms “deaza” and “dideaza” analogs refer to analogs recognized in the art that have a carbon atom in which one or two nitrogen atoms are replaced in a naturally occurring folate structure or its analogs or derivatives. For example, deaza analogs include folate, folic acid, pteropolyglutamic acid, and folate receptor-binding pteridines, such as tetrahydropterin, dihydrofolate, and 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of tetrahydrofolate. For example, dideaza analogs include 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of folate. Other folates useful as complex-forming ligands include folate receptor-binding analogs pemetrexed, proguanil, pyrimethamine, trimethoprim, pralatrexate, larcitrexed, aminopterin, ametopterin (also known as methotrexate), and N 10 -Methylfolate, 2-deamino-hydroxyfolate, deaza analogs, e.g., 1-deazametopterin or 3-deazametopterin, and 3',5'-dichloro-4-amino-4-deoxy-N 10 - It is methylpteroylglutamic acid (dichloromethotrexate).

[0188] Folic acid and the aforementioned analogs and / or derivatives are also referred to as “a folate,” “the folate,” or “folates,” reflecting their ability to bind to folate receptors. Folic acid is a member of the vitamin B family and can play an essential role in cell survival, for example, by participating in the biosynthesis of nucleic acids and amino acids. Folic acid can enhance the specificity of conjugated immunomodulator drugs by targeting activated myeloid cells, and can enhance the specificity of conjugated anticancer drugs by targeting folate receptor-positive cancer cells. Such molecules, when conjugated with exogenous molecules, may be effective in enhancing transmembrane transport via folate-mediated endocytosis, etc. The aforementioned can be used in the folate receptor-binding ligands described herein.

[0189] In at least one embodiment, the targeting moiety comprises a molecule having (e.g., high) affinity for folate receptor beta (FRβ). In some examples, the targeting moiety has a specific affinity for any receptor specific to cancer cells or tissues.

[0190] FRβ may be significantly upregulated in activated myeloid cells (e.g., primarily activated monocytes and M2-like macrophages), as documented data have shown that FRβ is induced only in myeloid cells after exposure to anti-inflammatory or pro-inflammatory stimuli. Folate receptors may be upregulated in non-mucinous ovarian cancers (e.g., in over 90% of them). In certain cases, folate receptors are present in renal, brain, lung, and breast carcinomas. For example, there are some cancers that do not express folate receptors in sufficient numbers to provide the desired specificity on their own, but the cancerous tumors express myeloid suppressor cells (MDSCs), which, for example, express FRβ, and which, for example, can be targeted by the targeting moieties provided herein. In some embodiments, folate receptors are substantially absent (e.g., present only at very low levels) in healthy (non-myeloid) tissues (e.g., lungs, liver, spleen, heart, brain, muscle, gastrointestinal tract, pancreas, bladder, etc.). In some cases, even dormant tissue-resident macrophages, which are abundant throughout the body, are primarily FRβ-negative. In some cases, uptake of folate-targeted imaging agents occurs, for example, in inflammatory tissue, malignant lesions, and the kidney. In certain specific cases, non-cancerous subjects retain folate-targeted drugs only in the kidney and inflammatory sites. In some cases, differences in folate receptor expression provide a mechanism for selectively targeting fibrous cancer cells.

[0191] This compound utilizes limited FRβ expression to target / localize a systemically administered potent immune modulator (e.g., the compound) to fibrous and / or cancerous tissue. The compound can be delivered directly to FRβ-expressing cells, which, for example, advantageously prevents systemic activation of the immune system and avoids the toxicity (e.g., at least part of it) that has previously hindered the systemic use of untargeted compounds (e.g., drugs).

[0192] In some examples, compounds are provided that include a folate ligand (or its radical) or a functional fragment or analog thereof as a targeting moiety, as well as an immunomodulator (e.g., a TLR7, TLR8, TLR7 / 8, TLR9, or TLR3 agonist). In some examples, TLR7, TLR8, TLR7 / 8, TLR9, and TLR3 are present in endosomes. In some embodiments, the compound or its radical binds to a TLR. In some embodiments, the TLR is TLR7.

[0193] Pyrido[2,3-d]pyrimidine analog ligands (e.g., or their radicals), their functional fragments or analogs, or any other molecule, fragment, or atom having affinity for FRβ (e.g., high specificity, without limitation) can be used as the targeting moiety (or its radical). For example, folate analog molecules may have a relative affinity for binding to FRβ of about 0.01 or more compared to folate at about 20°C / 25°C / 30°C / physiological temperatures. Similarly, galectin-3 ligands, translocator protein (TSPO) ligands, and any other ligands or targeting moieties having high specific affinity for cancer cells or tissues can be utilized.

[0194] Specific examples of suitable targeting moieties (or radicals) are provided below, however, it should be understood that the targeting moieties (or radicals) may include any ligand (or radical) useful for targeting FRβ and are not limited to the structures shown herein. The ligand (or radical) may bind to FRβ.

[0195] In a particular embodiment, the compound is of formula V: [ka] It may include a targeted portion (or its radical) having the structure or a functional fragment or analog thereof, During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m and n are independently either 0 or 1. [ka] This represents a CC that is either a single bond or a double bond.

[0196] In a further embodiment, as a non-limiting example, the targeting portion of formula V (or its radical) is the structure of VI (or its functional fragment or analog): [ka] It has, in the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m and n are independently either 0 or 1. [ka] This represents a CC that is either a single bond or a double bond.

[0197] Another specific targeting part (or radical) of formula V (or its functional fragment or analog) is formula VII: [ka] It has the structure, and in the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m and n are independently either 0 or 1. [ka] This represents a CC that is either a single bond or a double bond.

[0198] In some embodiments, the targeting portion of formula VI (or its radical) is formula VIII: [ka] It has the structure, and in the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m is either 0 or 1. [ka] This represents a CC that is either a single bond or a double bond.

[0199] In some embodiments, the targeted portion of formula VI (or its radical) is formula IX: [ka] It has the structure, and in the formula, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m is either 0 or 1. [ka] This represents a CC that is either a single bond or a double bond.

[0200] In some embodiments, the targeted portion of formula VII (or its radical) has the structure of formula X''' or XI: [ka] (In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m is either 0 or 1. [ka] (represents either a single bond or a double bond, or [ka] (In the formula, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamic acid, valine, or any other amino acid. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is either H or alkyl. m is either 0 or 1. [ka] (This represents either a single bond or a double bond.) It has.

[0201] Chemical structures and spectroscopic data for additional embodiments of a portion of the targeted portion (e.g., or its radical) are provided in Tables 3, 4, 5, and 6 below.

[0202] Table 3 provides non-limiting examples of additional embodiments of the targeting moiety (e.g., or its radical) having the structure of formula VIII.

[0203] [Table 3-1] [Table 3-2] [Table 3-3]

[0204] Table 4 provides non-limiting examples of additional embodiments of a targeted moiety (e.g., or its radical) having the structure of formula IX.

[0205] [Table 4-1] [Table 4-2]

[0206] Table 5 provides non-limiting examples of additional embodiments of the targeting moiety having the structure of formula X'''.

[0207] [Table 5-1] [Table 5-2]

[0208] As described above, instead of folate, the targeting moiety (e.g., its radical) may be one or more non-classical antifolate analogs, for example, pyrido[2,3-d]pyrimidine or similar analog (or its radical) having the formulas (e.g., the radical of the formula) (or its analog or functional fragment) listed in Table 6 below.

[0209] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0210] The present invention provides compounds having structural formulas (2-II), (2-IIA), (2-III), or (2-IIIA) (as described above), or pharmaceutically acceptable salts thereof, where G is a folate receptor-binding ligand. In one embodiment, G is folate, folic acid, or a functional fragment or derivative thereof, or derived therefrom. In one embodiment, G is folate or a folate derivative. In another embodiment, G is pteroic acid or a pteroyl derivative.

[0211] One embodiment provides a compound having structural embodiment formula (2-II), (2-IIA), (2-III), or (2-IIIA), or a pharmaceutically acceptable salt or hydrate thereof, where G is formula (2-IV): [ka] It is a base of, or contains a base of In the formula, each R is independent of the others. [ka] [ka] R is a naturally occurring or unnatural amino acid, or a derivative or fragment thereof.

[0212] One embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III), or (2-IIIA)), or a pharmaceutically acceptable salt or hydrate thereof, where G is formula (2-V): [ka] It is a base of, or contains a base of.

[0213] One embodiment provides a compound having structural formula (2-II), (2-IIA), (2-III), or (2-IIIA), or a pharmaceutically acceptable salt or hydrate thereof, where G is formula (2-VI): [ka] It is a base of, or contains a base of.

[0214] Linker

[0215] The compound may contain one or more linkers, and the radical of the targeted moiety is conjugated to the radical of the immunomodulator through one or more linkers. The term “linker” includes a chain of atoms biofunctionally adapted to form chemical bonds with A, B, or S, connecting two or more functional moieties of a molecule to form a compound. Exemplarily, the chain of atoms may be selected from carbon (C), N, oxygen (O), sulfur (S), silicon (Si), and phosphorus (P), or C, N, O, S, and P, C, N, O, and S. The chain of atoms can covalently connect different functional capability moieties of the compound, such as folate and drug (i.e., immunomodulator).

[0216] The linker may include a wide range of links, such as approximately 2 to 100 atoms in a continuous skeleton, and may include release or non-release linkers.

[0217] In some embodiments, the linker includes PEG, a PEG derivative, or any other linker known or to be developed in the art that can achieve the purposes described herein. In some embodiments, the compound has a linker ("L" or "L") connecting the targeting moiety and the immunomodulator, or otherwise connecting them. n This further includes ) in some embodiments, linker L n The linker L is configured to avoid the release of the immune modulator, where n is an integer equal to or less than 50. In some embodiments, the linker L nn comprises PEG or a PEG derivative, n is an integer selected from the range of 1 to 32, and the targeted moiety (e.g., its radical) comprises a radical of a folate receptor-binding ligand containing an FRβ-binding ligand. In some embodiments, n is 1 to 50, 1 to 10, 2 to 8, or 2 to 4.

[0218] In some embodiments, the linker is repeated n times ("L n ), where n is a positive integer. For example, n can be any integer selected from the range of 1 to 16, 1 to 32, 1 to 64, or 1 to 96, for instance. The number of iterations in the linker may be selected in order to achieve the desired functionality, size, and / or potency of the compound, and / or in accordance with the desired application.

[0219] The linker is either release-type (i.e., "rapidly release") or non-release-type (i.e., "slowly release"). In some examples, for instance, the target of a compound containing a non-release linker is endosomes (e.g., of the target cell), while the target of a release-type linker is, in some examples, endosomes, cytoplasm, or both (e.g., of the target cell). In some embodiments, L is a rapidly release hydrolyzable linker. In some embodiments, L is a slowly release non-hydrolyzable linker. In some embodiments, L is a heteroalkyl group as needed.

[0220] In the context of linkers, the terms “releasing” and “rapidly releasing” (these terms are used interchangeably herein) mean a linker that comprises at least one bond that can be cleaved to varying degrees under certain conditions (e.g., chemically or enzymatically hydrolyzed), and in particular can initiate a cascade of fragmentation or bond cleavage (which may result in the release of one or more of the parts connected through one or more parts of the linker (e.g., the targeting part and the immunomodulator)) and can be fragmented or cleaved within less than one week under certain metabolic, physiological, or cellular conditions, or when exposed to them. Rapidly releasing linkers may include, for example, reducing agent instability, pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability, enzymatic instability, or multivalent releasing bonds of the p-aminobenzyl system.

[0221] The cleavage of the linkage can occur, for example, by a standard chemical hydrolysis reaction occurring at physiological pH, or as a result of partitioning into cellular organelles such as endosomes, which have a pH lower than cytosolic pH. The cleavage of the linkage can also occur by acid-catalyzed elimination. Alternatively, fragmentation may be initiated by a nucleophilic attack on the disulfide group of the rapid-release linker, causing cleavage and, for example, the formation of a thiolate. In any of these cases, the rapid-release property of such a linker may be realized by some mechanism that may be related to the chemical, metabolic, physiological, or biological conditions present. In certain embodiments, the rapid-release linker comprises one or more sulfide bridges. In some examples, the releasing linker is fragmented into two or more fragments. In some examples, the releasing linker is separated from the targeting moiety. In some embodiments, the targeting moiety and the immunomodulator are released from each other, and the immunomodulator becomes active.

[0222] In contrast, the terms “non-releasing” and “slow-releasing” in the context of linkers (these terms are used interchangeably herein) mean a linker comprising at least one bond that is not easily or rapidly destroyed (i.e., the bond is not cleaved), and which, under certain conditions, is potentially cleavable or fragmentable to varying degrees, but which, when subjected to certain metabolic, physiological, or cellular conditions that can initiate a cascade of fragmentation (e.g., after administration to a subject), does not cleave, fragment, or otherwise release one or more of the parts connected through one or more parts of the linker (e.g., the targeting part and the immunomodulator) for more than about one week (e.g., one week), more than about one month (e.g., one month), more than about four months (e.g., four months), more than about six months (e.g., six months), or more than about one year (e.g., one year). In certain embodiments, a slow-releasing linker comprises one or more amide bonds.

[0223] In some embodiments, the compound includes a non-releasing linker that does not release any of the compound's components (e.g., a targeting ligand (e.g., a fully amorphous (FA) ligand or pteroyl amino acid) or an immunomodulator (e.g., a TLR7 agonist)). In some embodiments, the non-releasing linker lacks disulfide bonds (e.g., SS) or esters in its backbone. In some embodiments, the compound includes a targeting moiety and an immunomodulator linked by a backbone that is substantially stable for the entire duration of the compound's circulation in the target (e.g., during endocytosis of the target cell to endosomes). In some embodiments, compounds containing a non-releasing linker are particularly beneficial when the immunomodulator targets TLRs, NOD-like receptors, and / or other pattern recognition receptors present in the cell's endosomes. The non-releasing linker may include amides, esters, ethers, amines, and / or thioethers (e.g., thio-maleimides). While specific examples are provided herein, it will be understood that any molecule can be used in a non-releasing linker, as long as at least one bond is formed that is neither easily nor rapidly broken under physiological conditions.

[0224] Perhaps more specifically, in some embodiments, the non-releasing linker includes a linker in which, at neutral pH, less than 10 percent (10%) (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001%) of the administered compound will be hydrolyzed in an aqueous solution (e.g., buffered (e.g., phosphate-buffered) solution) over a period of time (e.g., 24 hours). In some embodiments, when a non-releasing linker is used, less than about 10 percent (10%), preferably less than 5 percent (5%), or none at all, of the administered compound will release free drug (e.g., into systemic circulation before uptake by targeted cells / tissues). In some embodiments, when the compound includes a non-releasing linker, less than 5 percent (5%) of the free drug will be released from the compound while the compound is in systemic circulation within a one-hour administration.

[0225] In some embodiments, the targeted portion is not cleaved from the drug / immunomodulator for the compound to be therapeutically effective in vivo. In some embodiments, this is beneficial because it allows the use of a targeted composition containing, for example, a potent drug (e.g., a TLR7 agonist), since the amount of the drug (e.g., an immunomodulator, e.g., a TLR7 agonist) released (e.g., systemically) before the targeted delivery of the compound is negligible (if any).

[0226] Controlling the release characteristics of the active component of a compound can be a challenging aspect of preparing effective pharmaceutical compositions. Compounds containing a non-releasing linker can circumvent this challenge in preparing effective pharmaceutical compositions (e.g., by eliminating the necessity of timing release). In some embodiments, the immunomodulator or warhead of a compound is active when conjugated (e.g., conjugated to a targeted compound). In some embodiments, while the warhead / immunomodulator is active, the non-releasing linker and targeting moiety prevent the release (e.g., by the target body) of toxic cytokines that activate the immune system (e.g., interleukin-6 (IL-6) as an example) (e.g., because the compound is specifically targeted (e.g., using folate or its analogues)). In certain specific examples, the immunomodulator cannot access the appropriate (e.g., targeted) receptor in the cell's endosome until the compound binds to the target receptor (e.g., a folate receptor), even if the warhead / immunomodulator of the compound is active when conjugated to a non-releasing linker.

[0227] As a non-limiting example, linker 106 in Figure 1A is a non-releasing PEG linker, while linker 156 in Figure 1B is a self-destructing releasing linker (e.g., containing a disulfide bond (e.g., SS)). For example, the scheme shown in Figure 1B illustrates the self-destructing cascade of compound 150 upon cleavage from the targeted portion 154.

[0228] In some embodiments, the linker 156 is configured such that the drug (e.g., an immunomodulator) is cleaved from the targeting portion 154 only after sufficient time has elapsed since administration for the compound to circulate within the systemic circulation of the subject (e.g., cleared from non-targeting tissues and captured and internalized by targeted cells and / or receptors). In some embodiments, the release period will vary (e.g., per subject (e.g., based on various factors)). In some embodiments, the releasing linker is engineered not to be cleaved / released for at least 24 hours or even up to a week after administration. In some embodiments, the compound can safely pass through the system of the subject, and any amount not captured by targeted cells (e.g., those expressing FRβ as an example) is purged before release / activation, thus preventing toxicity (e.g., since the immunomodulator is not active when bound to the releasing linker).

[0229] Both releasing and non-releasing linkers can be manipulated, through methods commonly known or to be developed in the art, such as PEGylation, to optimize biodistribution, bioavailability, and PK / PD (e.g., of a compound) and / or to increase uptake (e.g., of a compound) into targeted tissue. In some embodiments, the linker is configured to avoid significant release of a pharmaceutically active amount of drug into circulation before capture by cells (e.g., macrophages in cancer tissue to be treated).

[0230] In some embodiments, the compound containing the release linker is designed to diffuse across the endosomal membrane, for example, into the cytoplasm of a targeted cell. In some embodiments, the release linker is designed so that the immunomodulator is not released until the compound reaches the cytoplasm.

[0231] In some embodiments, the compound includes a release linker (for example, to facilitate the release of the immune modulator in the cytoplasm, if the immune modulator includes an activator of PI3K kinase, IRAK, or 1-kappa-β(Iκβ) kinase (e.g., using prostratin, etc.), or the nuclear factor kappa light chain enhancer (NF-κβ) of activated B cells (e.g., see Table 1), or a myeloid differentiation primary response 88 (MyD88) agonist). In some embodiments, the release linker prevents the release of the immune modulator until, for example, the targeting moiety binds to an appropriate target (e.g., macrophage folate receptor) and translocates to the endosome of the targeted cell and / or diffuses into the cytoplasm (e.g., where the desired pattern recognition receptor is located). In some embodiments, the release linker releases the immune modulator within the endosome.

[0232] In some embodiments, the linker may include one or more spacers (for example, these may be used to specifically design the compound's characteristics, such as, for example, facilitating a particular release time, facilitating increased uptake into the target tissue, and / or optimizing the compound's biodistribution, bioavailability, and / or PK / PD). The spacers may include one or more alkyl chains, PEGs, peptides, sugars, peptidoglycans, clickable linkers (e.g., triazoles), rigid linkers (e.g., polyprolines and polypiperidines), and the like.

[0233] In some embodiments, PEG 12Linkers containing the above significantly reduce, if not all, the nonspecific uptake of the compounds provided herein (e.g., to non-targeted organs (e.g., to the liver and / or kidneys of the target after administration)). In some embodiments, the compounds avoid delivery to the liver and kidneys. In some embodiments, the targeted moieties (in their free forms, their radicals, or the compounds themselves) do not bind to uptake receptors on non-targeted cells (e.g., provided that the organs are not targeting sites, thus avoiding stimulation of immune complexes in those organs, which is highly beneficial in a clinical setting).

[0234] Compounds containing a non-releasing linker can reduce or eliminate the toxicity of immunomodulators released from the compound in their free form (e.g., the free form of the compound and / or ligand provided herein). In certain embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof has or comprises the structure of formula (2-II), (2-IIA), (2-III), or (2-IIIA) described below, where L is a rapidly releasing linker (e.g., an amide, ester, ether, or sulfonamide).

[0235] In another embodiment, L is a heteroalkyl group which may be optionally substituted. The term "heteroalkyl group", by itself or in combination with another term, means, unless otherwise indicated, a stable linear or branched chain or combination thereof consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, S, and Si may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is bonded to the remainder of the molecule. Examples, without limitation, include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, in -CH2-NH-OCH3.

[0236] In some embodiments, the heteroalkyl is unsubstituted. In other embodiments, the heteroaryl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, oxo, PEG, carboxylate, and halo. Unless otherwise indicated, “halo” or “halogen” means a fluorine, chlorine, bromine, or iodine atom, either by itself or as part of another substituent.

[0237] In another embodiment, L is a substituted heteroalkyl having at least one disulfide bond in its skeleton.

[0238] In another embodiment, L is a peptide or peptidoglycan having at least one disulfide bond in its backbone. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers, polypeptides, or fragments of polypeptides, peptides, or fusion polypeptides of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0239] In some embodiments, L is -CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO- Includes, R a and R b These are independently H, alkyl, or heteroalkyl (e.g., PEG).

[0240] In another embodiment, L is a cleavable linker that can be cleaved by an enzymatic reaction, reactive oxygen species (ROS), or reducing conditions.

[0241] In some embodiments, L is given by the formula: -NH-CH2-CR 6 R 7 It has -SS-CH2-CH2-O-CO-, in the formula, R 6 and R 7 Each of these is independently H, alkyl, or heteroalkyl.

[0242] The linker may further include a spacer. In certain embodiments, the spacer is a hydrophilic spacer. In some embodiments, the compound has the structure of formula XII (e.g., a substructure of a TLR7 agonist of formula III conjugated with folate via a releasing linker containing a first hydrophilic spacer): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0243] In some embodiments, the compound has the structure of formula XIII (for example, a substructure of a TLR7 agonist of formula III conjugated with folate via a non-releasing linker (covalent bond) containing a second hydrophilic spacer): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0244] In some embodiments, L is expressed by the formula: [ka] It is a base of, or contains a base of During the ceremony, each [ka] is a junction, p is between 0 and 30, and d is between 1 and 40. R 8 and R 9 Each of these is independently H, alkyl, cyclic, aryl, or heteroalkyl.

[0245] One embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III), or (2-IIIA), or a pharmaceutically acceptable salt thereof, where L is a non-cleavable linker.

[0246] One embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III), or (2-IIIA), or a pharmaceutically acceptable salt or hydrate thereof, where L is a non-hydrolyzable linker.

[0247] In some embodiments, L is selected from the group consisting of alkylenes, heteroalkylenes, -O-alkylylenes, alkenylenes, acyls, aryls, heteroaryls, amides, oximes, ethers, esters, triazoles, PEGs, and carboxylates.

[0248] In one embodiment, L is an alkyl ether. In another embodiment, L is an amide. In another embodiment, L is a peptide or peptidoglycan. In another embodiment, L is an amino acid. In another embodiment, L is PEG (e.g., -OCH2-CH2-O-). In another embodiment, L is a polysaccharide.

[0249] In some embodiments, L is [ka] It includes the structure, During the ceremony, each [ka] is a junction, and n and m are independently between 0 and 10.

[0250] In some embodiments, the linker is [ka] It includes the structure, During the ceremony, each [ka] is a junction, and n and m are independently between 0 and 10.

[0251] In some embodiments, L is [ka] [ka] It includes the structure, During the ceremony, each [ka] is a junction point, and n is between 1 and 32. In at least one exemplary embodiment, n is between 1 and 30, and w is between 0 and 5.

[0252] In some embodiments, the linker is [ka] It includes the structure, In the formula, n is 1 to 16, and each [ka] This is a connection point.

[0253] In some embodiments, L is [ka] It includes the structure, During the ceremony, each [ka] is a junction, n is between 1 and 30, and w is between 0 and 5.

[0254] In one embodiment, L is the following list: [ka] [ka] Selected from, In the formula, n ranges from 0 to 30.

[0255] Specific examples of exemplary conjugated compounds are provided herein. It will be understood by those skilled in the art that a compound may exhibit pleomorphism, and that a compound may include any racemic form, optically active form, pleomorphic form, or stereoisomeric form, or mixtures thereof, of a compound exhibiting the useful properties described herein. Methods for preparing optically active forms (e.g., by decomposition of racemic forms by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase) and methods for determining antitumor activity using the standard tests described herein or other similar tests well known in the art are well known in the art. Furthermore, unless otherwise expressly indicated, the structures shown herein also mean that they include all stereochemical forms of that structure, i.e., right-handed (R) and left-handed (S) configurations of each chiral center. Thus, single stereochemical isomers of the compound, as well as mixtures of enantiomers and diastereomers, are within the scope of this disclosure.

[0256] The specific values ​​listed herein with respect to radicals, substituents, and ranges are illustrative unless otherwise indicated, and such examples do not exclude other defined values ​​or other values ​​within the defined ranges for radicals and substituents. For example, (C1-C6) alkyls may be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C1-C3) alkyls may be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C1-C3) alkoxys may be methoxy, ethoxy, or propoxy; and (C2-C6) alkanoyloxys may be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0257] Furthermore, if a portion is substituted with an R substituent or a substituted group, the group may be referred to as “R-substituted.” Where a portion is described as R-substituted or generally containing a substituent, that portion is substituted with at least one R substituent, each substituent being different as may be. It is understood that a substituted group (or R substituent) may contain any molecule or combination of molecules, provided that its inclusion does not substantially affect the overall structure and shape of the compound and does not alter any hydrogen bonds that are essential for the underlying compound to achieve its intended purpose (e.g., binding to a targeted pattern recognition receptor).

[0258] When substituents are represented by conventional chemical formulas written from left to right, they equally encompass chemically identical substituents that would result from writing the structure from right to left; for example, -CH2O- is equivalent to -OCH2-.

[0259] In some embodiments, the compound includes a radical of a targeted moiety conjugated with the immunomodulator radical or a pharmaceutically acceptable salt or hydrate thereof, such that the immunomodulator (or its radical) or a pharmaceutically acceptable salt or hydrate thereof remains pharmaceutically active when conjugated. The targeted moiety may include any targeted moiety described herein, and in at least one embodiment, includes a folate ligand, any other folate receptor-binding molecule (e.g., or any functional fragment or analog of the foregoing), or a pyrido[2,3-d]pyrimidine analog. In some embodiments, the targeted moiety (or its compound or radical) is specific to FRβ.

[0260] In some embodiments, the compound comprises one or more linkers, and the radical of the targeted moiety is conjugated to the radical of the immunomodulator through one or more linkers. For example, if the immunomodulator or a pharmaceutically acceptable salt or hydrate thereof has formula I or II, the radical of the immunomodulator is R1 , R 2 , or R 3 One of these can be conjugated to the radical of the targeting moiety, either through a linker or directly. Similarly, if the immunomodulator or its pharmaceutically acceptable salt or hydrate has formula III, the radical of the immunomodulator is R 1 or R 3 One of these can be conjugated to the radical of the targeting moiety, either through a linker or directly. Alternatively, if the immunomodulator or a pharmaceutically acceptable salt or hydrate thereof has formula IV, the radical of the immunomodulator is R 1 or R 2 One of these may be conjugated to the radical of the targeting moiety, either through a linker or directly. As described herein, the linker may be release-enabled or non-release-enabled.

[0261] It is understood that any combination of a compound radical (e.g., a compound radical in either Table 1 or 2), a linker (e.g., provided herein), and a ligand radical (e.g., a ligand radical in any one of Tables 3-6) can be used to form the compounds provided herein. In some embodiments, the compound radical or ligand radical is a carbon atom or a heteroatom (e.g., O, S, N, etc.). In some embodiments, the compound radical is C or O. In some embodiments, the ligand radical is C or O. In some embodiments, the bond site between the compound and ligand (e.g., through a linker) is determined by the arrangement of the radicals. In some embodiments, the linker includes a spacer (e.g., described elsewhere herein). It is also understood that any compound provided herein can be synthesized by a process similar to that provided in the examples.

[0262] Non-limiting examples of compounds provided herein are given in Table 7.

[0263] [Table 7-1] [Table 7-2] [Table 7-3]

[0264] Non-limiting examples of compounds provided herein are provided in Table 8.

[0265] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0266] In some examples, the conjugated compounds provided herein have the structure of formula XIV (e.g., or a functional fragment or analog thereof, which includes a TLR7 agonist of formula III conjugated with folate via a releasing linker): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0267] In another embodiment, the conjugated compounds provided herein include the structure of formula XV (e.g., or a functional fragment or analog thereof (e.g., compound 3B), which comprises a TLR7 agonist of formula II conjugated with folate via a releasing linker): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0268] In yet another embodiment, the conjugated compound provided herein has the structure of formula XVI (e.g., or a functional fragment or analog thereof (e.g., compound 3D), which comprises a TLR7 agonist of formula II conjugated with folate via a non-releasing linker containing three PEGs): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0269] In yet another embodiment, the conjugated compound provided herein has the structure of formula XVII (e.g., or a functional fragment or analog thereof (e.g., compound 3C), which comprises a TLR7 agonist of formula II conjugated with folate via a non-releasing linker containing 12 PEGs): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0270] Further embodiments of the conjugated compounds provided herein include the structure of formula XVIII (e.g., or its functional fragment or analog (e.g., compound 3D'), which comprises a TLR7 agonist of formula II conjugated with folate via a non-releasing linker containing 16 PEGs): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0271] Further embodiments of the conjugated compounds provided herein include the structure of formula XIX (e.g., or its functional fragment or analog (compound 1B), which includes a TLR7 agonist of formula III conjugated with folate): [ka] It contains, or is a pharmaceutically acceptable salt or hydrate thereof.

[0272] In some embodiments, folate-conjugated TLR-7 / 8 agonists provide specificity for affected cell types. In one embodiment, a folate-TLR7 / 8 agonist compound can be delivered (e.g., specifically) to the endosomes of FRβ+ macrophages while limiting systemic exposure to the TLR-7 / 8 agonist, for example.

[0273] One embodiment is given by formula (2-I): [ka] We provide a compound represented by the structure, or a pharmaceutically acceptable salt or hydrate thereof. In equation (2-I), R 1 , R 3 , R 4 , and R 5 These are, independently, H, alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, and -COR. 2x , [ka] And, R 2 H, -OH, -NH2, -NHR 2x, N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] And, Y is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] And, R 2x and R 2y Each of them is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, and each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q Independently selected from the group consisting of, R 2q and R 2q’ Each of them is independently an alkyl group or H group. [ka] It is a non-aromatic monocyclic or bicyclic 3-10 member nitrogen-containing heterocycle, In equation (2-I), X 1 , X 2 , and X 3 Each of them independently, CR q or N, and each R qThese are independently H, halogens, or optionally substituted alkyl groups. In equation (2-I), n is between 0 and 30, and m is between 0 and 4.

[0274] Another embodiment is given by formula (2-IA): [ka] Compounds having the structure (In the formula, R 1 It is a C3-C8 alkyl group that is optionally substituted (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each substituent independently being a halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3, Y is H, -OR z , -NR 2x R 2y , -SR z -SOR z , -SO3R z -N3, -COR z ,-COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2, R 2x and R 2y These are, independently, hydrogen and -N(R). z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z)2, or optionally substituted alkyl (for example, substituted with one or more substituents where each substituent is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and each R z R is independently hydrogen, halogen, or optionally substituted alkyl, or R 2x and R 2y These combine to form a heterocycloalkyl group as needed (for example, the heterocycloalkyl group as needed may be monocyclic or bicyclic heterocycloalkyl groups, and / or the heterocycloalkyl group as needed may be a 3- to 10-membered heterocycloalkyl group), Each R 3 These are, independently, halogen, -N3, -CN, -NO2, and -COR z ,-COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, -CON(R z )2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, where alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl may be substituted as needed. R 4 and R 5 Each of these is independently an alkyl, alkoxy, halogen, or cycloalkyl, and the alkyl, alkoxy, and cycloalkyl are substituted as needed. n is between 1 and 6, and m is between 0 and 4. The present invention provides a pharmaceutically acceptable salt or hydrate thereof.

[0275] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 1 and Y is -OH. In another embodiment, n is 1 and Y is -NH2. In one embodiment, the compound is represented by the structure of Toll-like receptor (TLR) 7 (TLR7)-1 (Compound 1). In one embodiment, the compound is represented by the structure of TLR7-1 (Compound 2). In one embodiment, the compound is represented by the structure of TLR7-1 (Compound 3). The structures of such compounds are shown in Figure 1C.

[0276] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where Y is -OH, OCH3, -NH2, -NHNH2, -NHCONH2, -SH, -SO2NH2, -N3, -COOH, -COCH3, -COOCH3, or -CONH.

[0277] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt thereof, where Y is H, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] That is the case.

[0278] One embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where Y is OH.

[0279] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where Y is NH2.

[0280] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 and Y is OH.

[0281] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 1 and Y is NH2.

[0282] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where n is 0 and Y is NH2.

[0283] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 1 R is an alkyl group that is substituted as needed. In one embodiment, R 1 is a C3-C6 alkyl group, which is substituted as needed. In another embodiment, R 1 is an acyclic C3-C6 alkyl which is substituted as needed. In another embodiment, R 1 It is butyl.

[0284] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 2 -NR 2x R 2yIn one embodiment, R 2 It is NH2.

[0285] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 3 H is H.

[0286] One embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 is alkyl. In one embodiment, R 4 It is methyl.

[0287] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 5 is alkyl. In one embodiment, R 5 It is methyl.

[0288] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, wherein R 4 and R 5 Each of these is an alkyl group. In one embodiment, R 4 and R 5 These are methyl compounds, respectively.

[0289] At least one embodiment provides a compound having the structure of formula (2-I) or (2-IA) or a pharmaceutically acceptable salt or hydrate thereof, where m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0290] At least one embodiment provides a compound having the structure of formula (2-I) or a pharmaceutically acceptable salt or hydrate thereof, wherein X 1 , X 2, and X 3 These are N, respectively. In one embodiment, X 1 In another embodiment, X 2 In another embodiment, X 3 It is N.

[0291] At least one embodiment provides a compound having the structure of formula (2-I) or a pharmaceutically acceptable salt or hydrate thereof, except for compounds where n is 0.

[0292] At least one embodiment provides a compound having the structure of formula (2-I) or a pharmaceutically acceptable salt or hydrate thereof, except for compounds in which n is 0 and Y is OH.

[0293] At least one embodiment provides a compound having the structure of formula (2-I) or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 0, Y is OH, and R 1 is butyl, and R 2 NH2 is R 3 H is R 4 and R 5 Compounds in which each of the two components is methyl are excluded.

[0294] At least one embodiment provides a compound having the structure of formula (2-I) or a pharmaceutically acceptable salt or hydrate thereof, except for compound TLR7-1.

[0295] In some embodiments, the compound is given by formula: [ka] Represented by one or more of the above, or a pharmaceutically acceptable salt or hydrate of any of the above structures, in the formula, R 1It is a C3-C8 alkyl group that is optionally substituted (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each substituent independently being a halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y , or N3, Y is a binding site to a linker or targeting ligand of the compound (e.g., the first therapeutic or first pharmaceutical of this specification), and H, -OR z , -NR 2x R 2y , -SR z -SOR z , -SO3R z -N3, -COR z ,-COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, or -CON(R z )2 included, R 2x and R 2y These are, independently, hydrogen and -N(R). z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (for example, substituted with one or more substituents where each substituent is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and each R z R is independently hydrogen, halogen, or optionally substituted alkyl, or R 2x and R 2yThese combine to form a heterocycloalkyl group as needed (for example, the heterocycloalkyl group as needed may be monocyclic or bicyclic heterocycloalkyl groups, and / or the heterocycloalkyl group as needed may be a 3- to 10-membered heterocycloalkyl group), Each R 3 These are, independently, halogen, -N3, -CN, -NO2, and -COR z ,-COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, -CON(R z )2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, where alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl may be substituted as needed. R 4 and R 5 Each of these is independently an alkyl, alkoxy, halogen, or cycloalkyl, and the alkyl, alkoxy, and cycloalkyl are substituted as needed. n is between 1 and 6, and m is between 0 and 4.

[0296] In some embodiments, the compound is given by formula: [ka] It is represented by one or more of the following, or a pharmaceutically acceptable salt or hydrate thereof.

[0297] In some embodiments, the compound is given by formula: [ka] [ka] It is represented by one or more of the following, or a pharmaceutically acceptable salt or hydrate thereof.

[0298] As described above, this disclosure further relates to compounds (e.g., their radicals) (e.g., TLR 7 and / or 8 (TLR7 / 8) agonists) conjugated directly or via a linker to a targeting moiety (e.g., their radical) that targets a cellular pattern recognition receptor or DAMP. In some embodiments, the targeting ligand comprises a folate ligand or a functional fragment or analog thereof, e.g., a pteroyl amino acid. In some embodiments, the linker is non-releasing. In some embodiments, the compound has a non-releasing linker, thereby providing a targeting molecule (e.g., their radical) that reduces systemic exposure to a TLR7 / 8 agonist. In some embodiments, the compound has a non-releasing linker, thereby providing a targeting molecule (e.g., their radical) that reduces systemic adverse effects of a TLR7 / 8 agonist.

[0299] One embodiment has the following structure: [ka] [ka] [ka] [ka] [ka] The present invention provides compounds having the same property or pharmaceutically acceptable salts or hydrates thereof.

[0300] One embodiment has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] The present invention provides compounds having the same property or pharmaceutically acceptable salts or hydrates thereof.

[0301] The compounds can be prepared by conventional organic synthesis methods practiced by those skilled in the art. The general reaction flows outlined below represent general methods useful for preparing the compounds and are not intended to limit their scope or usefulness.

[0302] The description of a compound is limited by the principles of chemical bonding known to those skilled in the art. Therefore, if a group can be substituted by one or more of several substituents, such substitutions are selected, according to the principles of chemical bonding, such as not being inherently unstable and / or likely to be unstable under ambient conditions, e.g., aqueous, neutral, and certain known physiological conditions, as is known to those skilled in the art. For example, heterocycloalkyl or heteroaryl groups are bonded to the remainder of the molecule via ring heteroatoms, according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0303] The term “identical” or “identity” percentage refers to two or more polypeptide sequences or subsequences that, when measured using sequence comparison algorithms known in the art or by manual alignment and visual inspection, have peptides that are identical or identical by a specified percentage (i.e., approximately 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity across a specified region, e.g., when compared and aligned for maximum match across a comparison window or specified region, e.g., a targeting end, a folate end, a linker, or a warhead). Such sequences are therefore referred to as “substantially identical.” In other words, identity exists across one or more regions of the entire sequence, insofar as the general shape and structure of the molecule and, where appropriate, hydrogen bonds are maintained to substantially fit the target binding site and function as an agonist to it.

[0304] The compounds may be administered in unit dosage forms and / or compositions comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, and combinations thereof. The term “administer” and its derivatives generally refer to any means by which the compounds described herein are introduced into a host subject, including, but not limited to, routes of administration such as oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, intrabuccal, intraocular, sublingual, vaginal, and rectal administration.

[0305] Salts and hydrates

[0306] A compound (e.g., its conjugate or part) may be presented as a pharmaceutically acceptable salt. A “pharmaceutically acceptable salt” of a compound means a salt whose counterion can be used pharmacopoeia. Such salts include (i) acid addition salts that can be obtained by the reaction of a free base of the parent compound with an inorganic acid, e.g., hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, etc., or with an organic acid, e.g., acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, malonic acid, etc., and (ii) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinated with an organic base, e.g., ethanolamine, diethanolamine, triethanolamine, trimamine, N-methylglucamine, etc. Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salt is intended herein.

[0307] In various embodiments, suitable basic salts are formed from bases that form non-toxic salts. Exemplary examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemi salts of acids and bases, such as hemisulfates and hemicalcium salts, can also be formed.

[0308] In some embodiments, pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, or fumarate.

[0309] Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acidic or basic forms of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two, generally in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is incorporated herein by reference.

[0310] Compounds or pharmaceutically acceptable salts thereof may exist in insoluble forms as well as solvated forms, including hydrated forms. Solvated forms may be equivalent to non-solvated forms. In each embodiment herein, it will be understood that a formula includes and represents not only all pharmaceutically acceptable salts of a compound, but also all hydrates and / or solvates of the compound formula or its salts. The term "solvate" means a compound or its salt further comprising a stoichiometric or non-stoichiometric amount of solvent bonded by non-covalent intermolecular forces. If the solvent is water, the solvate is a hydrate.

[0311] Certain functional groups, such as hydroxyl and amino, can form complexes and / or coordination conjugates with water and / or various solvents. Therefore, the formula is understood to include and represent their various hydrates and / or solvates. The nonhydrates and / or nonsolvates of the dual specificity adapters are also included.

[0312] Pharmaceutical composition

[0313] Based on the foregoing, compositions for the treatment of cancer (e.g., pharmaceutical compositions) comprising at least one of the compounds of this specification and a pharmaceutically acceptable carrier or additive are also provided. The term “composition” generally refers to any product comprising one or more components, including the compounds described herein. “pharmaceutically acceptable carriers” include, but are not limited to, any standard pharmaceutically acceptable carriers, such as buffering agents, preservatives, anesthetics, solubilizers, isotonic agents, wetting agents, and stabilizers. The term also encompasses any drugs that are approved by a regulatory authority, e.g., the U.S. Food and Drug Administration, or listed in the United States Pharmacopeia, for use in animals (e.g., mammals, e.g., humans). The carrier may be phosphate-buffered saline, water, or an emulsion, e.g., oil / water or water / oil emulsion.

[0314] It is understood that compositions may be prepared from isolated compounds described herein, or from salts, solutions, hydrates, solvates, and other forms of the compounds. It is understood that certain functional groups, such as hydroxyl and amino groups, may form complexes with water and / or various solvents in various physical forms of the compounds. It is also understood that compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the compounds described herein, and that compositions may be prepared from various hydrates and / or solvates of the compounds described herein. Therefore, pharmaceutical compositions containing the compounds may include each of the various morphological forms and / or solvates or hydrates of the compounds described herein, or any combination thereof, or individual forms.

[0315] The compounds can be formulated as pharmaceutical compositions and administered to mammalian hosts, such as human patients, in various forms adapted to a selected route of administration. For example, pharmaceutical compositions can be formulated for oral or parenteral, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, topical, inhalation, and / or subcutaneous routes, and administered thereby. In fact, in at least one embodiment, the compounds and / or compositions described herein can be administered directly into the bloodstream, muscle, or internal organs.

[0316] For example, in at least one embodiment, the compound can be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an absorbable food carrier. For oral therapeutic administration, the active compound can be combined with one or more additives and may be used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. The percentages of compositions and preparations may vary and may be about 1 to about 99% by weight of the active ingredient, as well as binders, additives, disintegrants, lubricants, and / or sweeteners (known in the art). The amount of the active compound in such therapeutically useful compositions is such that an effective dosage level is obtained.

[0317] The preparation of parenteral compounds / compositions under sterile conditions, for example by lyophilization, can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art. In at least one embodiment, the solubility of the compound used in the preparation of the parenteral composition can be increased by using appropriate formulation techniques, for example, by incorporating a solubility enhancer.

[0318] As previously stated, the compounds / compositions may also be administered by injection or injection (e.g., using needle (including microneedle) syringes and / or needle-free syringes). Solutions of the active composition may be aqueous and, if necessary, may be mixed with non-toxic surfactants and / or contain carriers or additives, such as salts, carbohydrates, and buffering agents (preferably pH 3-9), but for some applications they may be more preferably formulated as sterile non-aqueous solutions or as dry forms used in combination with a suitable vehicle, such as sterile pyrogen-free or phosphate-buffered saline (PBS). For example, dispersions may be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as in oils. Under normal storage and use conditions, these preparations may further contain preservatives to prevent microbial growth.

[0319] Suitable pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions or dispersions, or sterile powders adapted for the immediate preparation of sterile injection or infusion solutions or dispersions containing active ingredients encapsulated in liposomes as needed. In all cases, the final dosage form must be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, for example, without limitation, containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, adequate fluidity can be maintained by liposome formation, by maintaining the required particle size in the case of dispersants, or by the use of surfactants. Microbial action can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it may be desirable to include one or more isotonic agents, such as sugars, buffering agents, or sodium chloride. Long-term absorption of injectable compositions can be achieved by incorporating drugs formulated to delay absorption, such as aluminum monostearate and gelatin.

[0320] Sterile injectable solutions can be prepared by incorporating the active compound and / or composition, along with one or more of the other components mentioned above as needed, into the required amount of a suitable solvent, followed by sterilization by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder containing the active ingredient plus any additional desired components that were present in the previously sterile filtered solution.

[0321] For topical administration, it may be preferable to administer the compound to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be solid or liquid. For example, in certain embodiments, the solid carrier may be a finely ground solid, such as talc, clay, microcrystalline cellulose, silica, or alumina. Similarly, a useful liquid carrier may contain water, alcohol or glycol, or a water-alcohol / glycol blend, in which the compound can be dissolved or dispersed at an effective level, with the assistance of a non-toxic surfactant as needed. Additionally, or alternatively, adjuvants, such as fragrances and antimicrobial agents, may be added to optimize the properties for a given use. The resulting liquid composition can be applied from an absorbent pad, impregnated into a bandage and / or other dressing, sprayed onto the target area using a pump or aerosol spray, or simply applied directly to the desired area of ​​interest.

[0322] Thickening agents, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials, can also be used in conjunction with liquid carriers to form spreadable pastes, gels, ointments, soaps, etc., for direct application to the target skin.

[0323] The terms “therapeutically effective,” “therapeutic effective dose,” “therapeutic effective amount,” “preventive effective dose,” or “preventive effective dose” mean the amount of a compound that, when administered either once or over the course of a treatment cycle, affects the health, well-being, or mortality of a subject (e.g., delaying the onset of one or more cancer-associated symptoms and / or reducing their severity) unless otherwise specifically indicated. The effective dosage of a compound can be determined by comparing its in vitro activity with its in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to human subjects are known in the art. In practice, the dosage of a compound can vary significantly depending on the host subject’s condition, the type of cancer being treated, the stage of pathology, the route and tissue distribution of the compound, and the possibility of concurrent use of other therapeutic treatments (e.g., additional drugs in radiotherapy or combination therapy). The amount of a composition required for use in a treatment (e.g., a therapeutically or prophylactically effective amount or dose) will vary not only depending on the specific application but also on the salt selected (if applicable) and the characteristics of the subject (e.g., age, condition, sex, body surface area and / or weight of the subject, tolerance to the drug), and will ultimately be at the discretion of the attending physician, clinician, or other authority. Therapeutically or prophylactically effective doses or amounts may range, for example, from approximately 0.05 mg / kg to approximately 30.0 mg / kg based on the patient's body weight, or from approximately 0.01 mg / kg to approximately 5.0 mg / kg based on the patient's body weight, including, but not limited to, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg, all based on 1 kg of patient body weight. All ranges mentioned in this paragraph include the endpoints mentioned and all 0.05 g / m 2Each increment is included within its respective mentioned range. The total therapeutic or prophylactic effective dose of the compound may be administered as a single dose or in divided doses and, at the physician's discretion, may be outside the typical range provided herein.

[0324] In another embodiment, the compound is present in concentrations of approximately 0.5 g / m to approximately 500 mg / m. 2 , about 0.5g / m 2 ~about 300mg / m 2 , or approximately 100g / m 2 ~about 200mg / m 2 It may be administered in a therapeutic or prophylactic effective dose. In other embodiments, the dose is about 0.5 mg / m². 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2 , about 0.5mg / m 2 ~about 200mg / m 2 , about 0.5mg / m 2 ~about 100mg / m 2 , about 0.5mg / m 2 ~about 50mg / m 2 , about 0.5mg / m 2 ~about 600mg / m 2 , about 0.5mg / m 2 ~about 6.0mg / m 2 , about 0.5mg / m 2 ~about 4.0mg / m 2 , or approximately 0.5 mg / m² 2 ~about 2.0mg / m 2 It is possible. All ranges mentioned in this paragraph include the endpoints mentioned and all 0.5 g / m 2 Each increment falls within its respective mentioned range. The total amount may be administered as a single dose or in divided doses and, at the physician's discretion, may be outside the typical range provided herein. These amounts are based on body surface area in square meters.

[0325] In other embodiments, the amount of the compound (or its pharmaceutically acceptable salt or hydrate) to be administered to the subject is, for example, based on the subject's body weight, about 50 nmol / kg to about 3,000 nmol / kg, about 50 nmol / kg to about 2,000 nmol / kg, about 50 nmol / kg to about 1,000 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 800 nmol / kg, about 50 nmol / kg to about 700 nmol / kg, and about 50 nmol / kg to about 600 nmol / kg. It may be in the range of mol / kg, approximately 50 nmol / kg to approximately 500 nmol / kg, approximately 50 nmol / kg to approximately 400 nmol / kg, approximately 50 nmol / kg to approximately 300 nmol / kg, approximately 50 nmol / kg to approximately 200 nmol / kg, approximately 50 nmol / kg to approximately 100 nmol / kg, approximately 100 nmol / kg to approximately 300 nmol / kg, approximately 100 nmol / kg to approximately 500 nmol / kg, approximately 100 nmol / kg to approximately 1,000 nmol / kg, or approximately 100 nmol / kg to approximately 2,000 nmol / kg. In other embodiments, the dose may be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1,000 nmol / kg, about 2,000 nmol / kg, or about 3,000 nmol / kg based on the subject's body weight. In other embodiments, doses of about 20 μg / kg to about 3 mg / kg based on the subject's body weight may be administered. The amount may be about 0.2 mg / kg to about 0.4 mg / kg based on the subject's body weight, or about 50 μg / kg based on the subject's body weight. All ranges mentioned in this paragraph include the endpoints mentioned, and all 1 nmol / kg or 10 μg / kg increments are, as appropriate, included within their respective ranges.

[0326] In some embodiments, in connection with measuring the expression of a particular biomarker in a sample obtained from a subject and / or analyzing cytokine levels, the focus of this disclosure is not on the specific method used to detect the marker or set of markers, but rather on what is detected using the marker. Numerous methods exist that can be used to detect the expression, quantification, or profile of one or more biomarkers. Once the marker or set of markers to be detected or quantified is identified, one of several techniques (currently known or to be developed in the future) can be used, provided that appropriate reagents are provided. Those skilled in the art will be able to select an appropriate assay (e.g., a PCR-based or microassay-based nucleic acid marker assay, enzyme-linked immunosorbent assay (ELISA), protein or antibody microarray, or similar immunoassay) to perform the methods disclosed herein, given that one or more biomarkers to be identified are provided.

[0327] Combinations

[0328] Further provided are combinations of a first treatment comprising any of the compounds or pharmaceutical compositions of this specification and a second treatment comprising one or more checkpoint inhibitors.

[0329] A second treatment may include one or more immune checkpoint inhibitors that can bind to and / or antagonize immune checkpoint molecules. An "immune checkpoint inhibitor" or "antagonist" (as used in this context) is a molecule that inhibits, reduces, or blocks the activity of an immune checkpoint molecule, thereby inhibiting the suppressive effect that the immune checkpoint molecule has on the immune system. The inhibitor or antagonist may bind directly to the immune checkpoint molecule, or it may bind to a ligand of the immune checkpoint molecule that mediates the activity of the immune checkpoint molecule.

[0330] Drugs that selectively bind to immune checkpoint molecules may, without limitation, be antibodies or their antigen-binding fragments (including humanized antibodies or their antigen-binding fragments), proteins or peptides, small molecules, or nucleic acids. Immune checkpoint molecules that are nucleic acids may be, for example, antisense molecules, single-stranded or double-stranded DNA oligonucleotides, single-stranded or double-stranded RNA oligonucleotides, peptide nucleic acids (PNAs), single-stranded or double-stranded RNAi molecules, shRNA, or siRNA. Small molecules are organic compound drugs. Drugs that selectively bind to immune checkpoint molecules may bind to nucleic acids or amino acids in the immune checkpoint molecule sequence. Drugs that selectively bind to immune checkpoint molecules may bind to any region of the immune checkpoint molecule.

[0331] Checkpoint inhibitors target immune checkpoints in cells, such as programmed death 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), T cell activation V-domain Ig suppressor (VISTA), lymphocyte activation 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immune receptor with Ig and ITIM domains (TIGIT), programmed death ligand 2 (PD-L2), and indoleamine 2,3-dioxygen This disclosure may include small molecules or other agents that disrupt signaling factors and activators of transcription (STAT3), such as immune checkpoint inhibitors (IDO), arginase-1 (AGR1), B7 family inhibitory ligand B7-H3 (B7-H3), B7 family inhibitory ligand B7-H4 (B7-H4), 2B4 (differentiation cluster 244), B and T lymphocyte attenuation factor (BTLA), adenosine A2A receptor (A2aR), and / or members of the killer cell immunoglobulin-like receptor (KIR) family, such as KIR and C-type lectin receptors, as well as signaling factors and activators of transcription (STAT3). This disclosure is not limited to targeting the aforementioned immune checkpoint blockade receptors, and other inhibitory checkpoint molecules may be targeted by immune checkpoint inhibitors in combination therapy with the compounds and / or compositions herein. The activity of an immune checkpoint molecule refers to its inhibitory effect on immune checkpoints. Immune checkpoint inhibitors may reduce or block the activity of immune checkpoint molecules.

[0332] Immune checkpoints refer to inhibitory pathways integrated into the immune system that are crucial for maintaining self-tolerance and modulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize associated tissue damage. Immune checkpoint molecules may be stimulant or inhibitory to immune checkpoints. In this disclosure and claims, inhibitory molecules of immune checkpoints are referred to as “immune checkpoint molecules.” Preliminary clinical findings with agents blocking immune checkpoint molecules (e.g., PD-1, PD-L1, or CTLA-4) suggest an opportunity to enhance antitumor immunity that may result in an effective clinical response. Combining immune checkpoint blockade with immune checkpoint inhibitors with the administration of the compounds and / or compositions herein may enhance treatment efficacy in subjects with cancer or recurrent cancer.

[0333] Immune checkpoint inhibitors are a type of drug that blocks the signaling of immune checkpoint molecules carried out by certain types of immune system cells, such as T cells, and some cancer cells. Immune checkpoint inhibitors can therefore cause immune checkpoint blockade.

[0334] Immune checkpoint molecules (e.g., PD1) can help maintain the immune response in a checked state and can suppress the killing of cancer cells by T cells. Blocking these molecules releases a “brake” on the immune system (reduction or blockage of immune system inhibition), allowing T cells to more effectively kill cancer cells. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4. In some embodiments, the immune checkpoint molecule is a protein. In certain embodiments, the immune checkpoint molecule is a nucleic acid encoding a protein. In some embodiments, the immune checkpoint inhibitor binds to and / or antagonizes the immune checkpoint molecule. In some embodiments, the immune checkpoint inhibitor is used in combination with the compounds and / or compositions herein to treat subjects having cancer.

[0335] In certain embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islerizumab, a combination of osiperlimab and islerizumab, BMS-9365 The following may be selected from the group consisting of 59 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab.

[0336] In certain embodiments, the checkpoint inhibitor is a PD-1 antagonist or a PD-L1 antagonist. In certain embodiments, the checkpoint inhibitor is a CTLA-4 antagonist. In certain embodiments, the checkpoint inhibitor is a VISTA antagonist. In certain embodiments, the checkpoint inhibitor is a LAG3 antagonist. In certain embodiments, the checkpoint inhibitor is a TIM3 antagonist. In certain embodiments, the checkpoint inhibitor is a TIGIT antagonist.

[0337] A PD-1 antagonist may be, for example, a drug that binds to PD-1 and antagonizes it. In some embodiments, the drug that binds to and antagonizes PD-1 is a peptide that binds to PD-1. In some embodiments, the drug that binds to and antagonizes PD-1 is a humanized antibody that selectively binds to PD-1. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, pidilizumab, the monoclonal antibody MEDI-0680, the monoclonal antibody REGN2810, or the PD-1-targeting fusion protein AMP-224. In some embodiments, the humanized antibody that selectively binds to PD-1 is nivolumab, pembrolizumab, or pidilizumab. The PD-1 antagonist may be osiperlimab, islerizumab, or a combination of osiperlimab and islerizumab.

[0338] A PD-L1 antagonist may be, for example, a drug that binds to PD-L1 and antagonizes it. A drug that binds to PD-L1 and antagonizes it may be, for example, a peptide that binds to PD-L1. In certain embodiments, the drug that binds to PD-L1 and antagonizes it is a humanized antibody that selectively binds to PD-L1. A humanized antibody that selectively binds to PD-L1 may be BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab. A PD-L1 antagonist may be tiragolumab, atezolizumab, avelumab, durvalumab, or zimbererimab.

[0339] Examples of PD-L1-targeting drugs include, but are not limited to, atezolizumab, avelumab, and durvalumab. In certain embodiments, the checkpoint inhibitor is an antibody targeting CTLA-4, such as ipilimumab. Furthermore, checkpoint inhibitors may include those targeting transmembrane proteins such as T cell immunoglobulin and mucin domain-containing protein-3 (CD366 or TIM3). In alternative embodiments, checkpoint inhibitors include small molecules or other agents that disrupt immune checkpoints that cancer cells utilize to evade cell-mediated or other immune-mediated targeting.

[0340] A CTLA-4 antagonist may be, for example, a drug that binds to CTLA-4 and antagonizes it. In some embodiments, the drug that binds to CTLA-4 and antagonizes it is a peptide that binds to CTLA-4. In some embodiments, the drug that binds to CTLA-4 and antagonizes it is a humanized antibody that selectively binds to CTLA-4. In some embodiments, the humanized antibody that selectively binds to a CTLA-4 inhibitor is ipilimumab or tremelimumab. In some embodiments, a CTLA-4 antagonist is (i) an antisense molecule directed to CD80, CD86, and / or CTLA-4, (ii) adnectin directed to CD80, CD86, and / or CTLA-4, (iii) a single-stranded or double-stranded RNAi inhibitor of CD80, CD86, and / or CTLA-4, or (iv) a small molecule inhibitor of CD80, CD86, or CTLA-4.

[0341] A VISTA antagonist can be, for example, a drug that binds to VISTA and antagonizes it. In some embodiments, the drug that binds to VISTA and antagonizes it is a peptide. In certain embodiments, the drug that binds to VISTA and antagonizes it is a VISTA-targeted inhibitory antibody. In some embodiments, the drug that binds to VISTA and antagonizes it is a humanized antibody. In some embodiments, the drug that binds to VISTA and antagonizes it is (i) a VISTA-targeted antisense molecule, (ii) a VISTA-targeted adnectin, (iii) a single-stranded or double-stranded RNAi inhibitor of VISTA, or (iv) a small molecule inhibitor of VISTA.

[0342] A LAG3 antagonist can be, for example, a drug that binds to LAG3 and antagonizes it. In some embodiments, the drug that binds to LAG3 and antagonizes it is a peptide that binds to LAG3. In some embodiments, the drug that binds to LAG3 and antagonizes it is a humanized antibody that selectively binds to LAG3. In some embodiments, the humanized antibody that selectively binds to LAG3 is relatrimab or the monoclonal antibody IMP321.

[0343] A TIM3 antagonist can be, for example, a drug that binds to TIM3 and antagonizes it. In some embodiments, the drug that binds to TIM3 and antagonizes it is a peptide that binds to TIM3. In some embodiments, the drug that binds to TIM3 and antagonizes it is a humanized antibody that selectively binds to TIM3. In some embodiments, the humanized antibody that selectively binds to TIM3 is INCAGN02390.

[0344] A TIGIT antagonist may be, for example, a drug that binds to and antagonizes TIM3. In some embodiments, the drug that binds to and antagonizes TIM3 is a peptide that binds to TIM3. In some embodiments, the drug that binds to and antagonizes TIM3 is a humanized antibody that selectively binds to TIM3. In some embodiments, the humanized antibody that selectively binds to TIM3 is etigirimab, domvanarimab, tilagolumab (RG6058), or vivostrimab.

[0345] As mentioned, immune checkpoint blockade can be used as a combination therapy with the compounds and compositions of this specification. In certain approaches, administering both the compounds / compositions and immune checkpoint inhibitor therapy results in inhibition of cancer growth that exceeds additive effects.

[0346] When multiple therapeutic agents and / or treatments are co-administered, the dosage may be adjusted accordingly, as is recognized in the art. "Co-administration" and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which a compound or composition is administered at least once during a course of treatment that includes administering an immune checkpoint inhibitor treatment to a subject. In some embodiments, one or more immune checkpoint inhibitors of a second treatment are administered on a different day from the first treatment which includes the compound / composition. In some embodiments, the second treatment is administered on the same day as the first treatment. In some embodiments, the second treatment (i.e., one or more immune checkpoint inhibitors) is administered on a different day from the first treatment (i.e., the compound / composition herein), but within 1 day, 5 days, 1 week, 8 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months from the administration of the first treatment to the subject.

[0347] In some embodiments, the second therapeutic immune checkpoint inhibitor is administered intravenously or subcutaneously.

[0348] In certain embodiments, the combination can be used to treat cancer recurrence or resistance to immune checkpoint blockade therapy in a subject. In certain embodiments, the combination can be used to treat cancer in a subject.

[0349] Usage and Method

[0350] In addition to the compounds described herein, methods for providing treatment for cancer and / or preventing cancer recurrence are also provided. In some embodiments, methods for treating an oncological disease or disorder in an individual in need thereof are provided herein, comprising the step of administering to the individual a composition (e.g., a pharmaceutical) containing any of the compounds described herein, for example, a compound having the structure of any one of the following: formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, formula XVII, formula XVIII, formula XX, formula XXX, formula 2-I, formula 2-II, formula 2-III, formula 2-IV, formula 2-V, or formula 2-VI, or a pharmaceutically acceptable salt or hydrate thereof, or any compound provided herein or a pharmaceutically acceptable salt thereof.

[0351] In some embodiments, a method is provided herein for treating an oncological disease or disorder in an individual in need thereof, comprising the step of administering to the individual any compound provided herein, for example, a compound having the structure of any one of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XX, XXX, 2-I, 2-II, 2-III, 2-IV, 2-V, or 2-VI, or a pharmaceutically acceptable salt or hydrate thereof, or a composition (e.g., a pharmaceutical) comprising any compound provided herein or a pharmaceutically acceptable salt or hydrate thereof. In some embodiments, the oncological disease or disorder is cancer. In some embodiments, the cancer is selected from bladder cancer, brain cancer, liver cancer, kidney cancer, skin cancer, thymic carcinoma, gastrointestinal stromal tumor (GIST), esophageal cancer, pancreatic cancer, and breast cancer.

[0352] In some embodiments, a method is provided herein for treating a cancer-related disease or disorder in an individual in need thereof, the method comprising the step of administering to the individual any compound provided herein, for example, a compound having the structure of any one of the following: formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, formula XVII, formula XVIII, formula XX, formula XXX, formula 2-I, formula 2-II, formula 2-III, formula 2-IV, formula 2-V, or formula 2-VI, or a pharmaceutically acceptable salt or hydrate thereof, or a composition (e.g., a pharmaceutical) comprising any compound provided herein or a pharmaceutically acceptable salt or hydrate thereof.

[0353] In at least one embodiment, a method is provided for treating a subject suffering from or at risk of experiencing a disease condition, wherein the disease condition includes cancer (or its recurrence), and the method comprises the step of contacting the cells of the subject with at least one compound. The at least one compound may include any of the compounds herein and, in at least one exemplary embodiment, includes a targeting moiety specific to FRβ. In some examples, the step of contacting the cells can be achieved by administering the at least one compound to the subject intravenously, orally, intramuscularly, intraperitoneally, topically, or by inhalation, or by any of the other administration modalities described herein. Additionally or alternatively, the at least one compound may constitute a composition comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or combinations thereof. The dosage of at least one compound administered may be modified as appropriate by the clinician, however, at least one compound is preferably administered in a therapeutically or prophylactically effective amount, and in at least one embodiment, the dosage is in the range of 1 nmol / kg to 50 nmol / kg based on the subject's body weight.

[0354] In certain embodiments, methods for treating or preventing the recurrence or recurrence of a cancerous disease state are provided herein, comprising the step of contacting cells with at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt or hydrate thereof conjugated via a linker to a folate ligand or a functional fragment or analog thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor or DAMP.

[0355] In certain embodiments, a method for treating a subject having cancer may include the steps of administering a first therapy comprising at least one of the compounds or compositions herein to the subject, and administering a second therapy comprising one or more checkpoint inhibitors (i.e., PD-1, PD-L1, CTLA-4, VISTA, LAG3, TIM3, TIGIT, PD-L2, IDO, AGR1, B7-H3, B7-H4, 2B4, BTLA, A2aR, and / or members of the KIR family, e.g., KIR and C-type lectin receptors, and one or more agents that bind to and / or antagonize STAT3). The disclosure is not limited to targeting the aforementioned immune checkpoint blockade receptors, and other inhibitory checkpoint molecules may be targeted by immune checkpoint inhibitors in combination therapy with the compounds and / or compositions herein.

[0356] At least one compound of the first therapeutic of the present method may include any of the compounds described, for example, a compound having the structure of any one of the following: formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII, formula XIII, formula XIV, formula XV, formula XVI, formula XVII, formula XVIII, formula XX, formula XXX, formula 2-I, formula 2-II, formula 2-III, formula 2-IV, formula 2-V, or formula 2-VI, or a pharmaceutically acceptable salt or hydrate thereof, or a composition (e.g., a pharmaceutical) comprising any compound provided herein or a pharmaceutically acceptable salt or hydrate thereof. In some embodiments, the immunomodulator includes an agonist of TLR 3, 7, 8, 9, or 7 / 8.

[0357] In certain embodiments, at least one compound comprises a radical of an immunomodulator or a pharmaceutically acceptable salt or hydrate thereof, conjugated via a linker to a targeted ligand, for example, a folate ligand or a functional fragment or analog thereof. The immunomodulator or a pharmaceutically acceptable salt or hydrate thereof may target a pattern recognition receptor. Similarly, a composition comprising at least one compound may comprise any of the pharmaceutical compositions described herein. In certain embodiments, a composition comprises at least one compound herein and one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or combinations thereof.

[0358] In certain embodiments of this method, the radical of the compound immunomodulator comprises a TLR 3, 7, 8, 9, or 7 / 8 agonist. In certain embodiments, at least one compound (e.g., of the first treatment) has the following formula: [ka] It has or is a pharmaceutically acceptable salt or hydrate thereof. In certain embodiments, the radical of the first therapeutic immunomodulator comprises a TLR agonist of formula X or XX, or A pharmaceutically acceptable salt or hydrate of formula X or XX, [ka] In equations X and XX, R1 is either -NH2 or -NH-R 1X And, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And, [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. In equation X, R3 is -OH, -SH, -NH2, or -NH-R 1X And, In formula XX, X is either CH or N. R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0359] At least one compound of the first treatment is [ka] It may contain, or may be a pharmaceutically acceptable salt or hydrate thereof, in formula 2-I, R 1 , R 3 , R 4 , and R 5 These are, independently, hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, and -COR. 2x , [ka] And, R 2 H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] And, Y is the linker and / or binding site to the targeting ligand of the conjugate, and is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2, [ka] Includes, R 2x and R 2y Each of them is independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, and each R 2z -NH2, -NR 2q R 2q’ , -OR 2q , -SO-R 2q , and -COR 2q Independently selected from the group consisting of R 2q and R 2q’ Each of them is independently an alkyl group or H group. [ka] It is a non-aromatic monocyclic or bicyclic 3-10 member nitrogen-containing heterocycle, In equation 2-I, X 1 , X 2 , and X 3 Each of them independently, CR q or N, and each R q These are independently H, halogens, or optionally substituted alkyl groups. In equation 2-I, n is between 0 and 30, and m is between 0 and 4.

[0360] In a particular embodiment, the radical of the first therapeutic immunomodulator is given by the following formula: [ka] Includes a TLR agonist having In the formula, R 1 is an amine group, R 2 It is a single bond -NH-, and R 3 is H, alkyl, hydroxyl, or any other substituted group thereof, X is CH2, NH, O, or S, and the linker is R 1 , R 2 , or R 3 Combine.

[0361] In certain embodiments of the methods described herein, at least one compound (e.g., of the first treatment) has the following formula: [ka] It is the structure of, or contains, or a pharmaceutically acceptable salt or hydrate thereof.

[0362] The linker of at least one compound of the first treatment (or a pharmaceutically acceptable salt or hydrate thereof) may be any linker described herein. In certain embodiments, the linker is a release linker. In certain embodiments, the linker is a non-release linker. In certain embodiments, the linker of at least one compound of the first treatment (or a pharmaceutically acceptable salt or hydrate thereof) includes a PEG linker or a PEG derivative linker and is a non-release linker.

[0363] The subjects may have experienced cancer or cancer recurrence, or may be at risk of experiencing it. In certain embodiments, the step of administering the first treatment further includes administering or applying to the subject a therapeutically effective amount of at least one compound or a pharmaceutically acceptable salt or hydrate thereof.

[0364] In certain embodiments, administration of a second treatment (i.e., one or more checkpoint inhibitors) destroys immune checkpoints in target cells. One or more checkpoint inhibitors of the second treatment may be or include small molecules or other agents that destroy immune checkpoints in target cells. Any of the checkpoint inhibitors described herein may be used. In certain embodiments, one or more of the checkpoint inhibitors of the second treatment may be pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islerizumab, osiperlimab and islerizumab The following combinations are independently selected from the group consisting of BMS-936559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), and vivostrimab. One or more immune checkpoint inhibitors of the second therapy may inhibit immune checkpoints in cells selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, LAG3, TIM3, TIGIT, PD-L2, IDO, AGR1, B7-H3, B7-H4, 2B4, BTLA, A2aR, and / or members of the KIR family, such as KIR and C-type lectin receptors, as well as STAT3.

[0365] Compounds (or pharmaceutically acceptable salts or hydrates thereof), compositions containing them, or combinations thereof, and one or more immune checkpoint inhibitors may be administered to a subject using any preferred method known in the art. Examples of preferred routes of administration include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal. Compounds, compositions, and / or combinations thereof, and one or more immune checkpoint inhibitors may be administered directly into the bloodstream, muscle, or internal organs. Preferred parenteral routes of administration include, but are not limited to, intravenous, intra-arterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous. Needle syringes, including microneedles, needle-free syringes, and injectors, may be used. Compounds, compositions, and / or combinations thereof, as well as one or more immune checkpoint inhibitors, may be administered either together in the same composition or as separate compositions in unit dosage forms and / or formulations comprising a conventional non-toxic, pharmaceutically acceptable carrier or excipient (or vehicle or adjuvant).

[0366] In this method, the compound or a pharmaceutically acceptable salt or hydrate thereof (or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or excipient), and one or more immune checkpoint inhibitors may be administered simultaneously or sequentially in any order, via the same or different routes. When administered simultaneously via the same route, the formulations may be the same or different. In various embodiments, the compound (or a pharmaceutically acceptable salt or hydrate thereof) may be administered to the subject after the immune checkpoint inhibitor. The timing between the administration of the immune checkpoint inhibitor and the administration of the compound may vary widely depending on factors including the type of immune checkpoint inhibitor used, the binding specificity of the compound (or a pharmaceutically acceptable salt or hydrate thereof), the identity of the targeting portion of the compound, the type of cancer, the location of the cancer in the subject, the means used to administer the immune checkpoint inhibitor and the compound to the subject, and the subject's health status, age, and weight.

[0367] The compound may be administered before or after an immune checkpoint inhibitor, for example, within approximately 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, or 24 hours, or within approximately 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or longer. The rate of administration of the compound and / or immune checkpoint inhibitor can be adjusted, for example (as a function of the dosing schedule, such as continuous, once daily, twice daily, three times daily, once weekly, twice weekly, or three times weekly). "Continuous" means a dosing regimen of at least 1 hour, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least 24 hours, or daily or weekly, e.g., once / day, twice / day, three times / day, every other day, once / week, twice / week, three times / week, or any other suitable regimen. In this method, immune checkpoint inhibitors (or pharmaceutical compositions comprising immune checkpoint inhibitors and pharmaceutically acceptable carriers or excipients) and compounds may be administered intravenously.

[0368] In certain approaches, administering both the first and second therapies targets results in inhibition of cancer growth that exceeds the additive effect.

[0369] The first treatment may be administered to the subject intravenously, orally, intramuscularly, intraperitoneally, topically, or by inhalation. The second treatment may be administered to the subject intravenously, orally, intramuscularly, intraperitoneally, topically, or by inhalation.

[0370] The term "subject" means an animal, such as a mammal, and in particular, a human. In veterinary applications, the subject may be a laboratory, agricultural, domesticated, or wild animal. Examples of such animals include, but are not limited to, rodents, rabbits, monkeys, chimpanzees, dogs, cats, cattle, horses, pigs, sheep, goats, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales.

[0371] In some embodiments, the method is used to treat cancer, for example, regardless of whether the cancer expresses folate receptors. In some embodiments, folate and other folate receptor-binding ligands (or their radicals), for example, folate, are used as the targeting moiety because, for example, they have affinity for FRβ.

[0372] The methods described herein may further include a step of imaging cancer in a subject. The step of imaging cancer may include, for example, imaging by optical imaging, positron emission tomography (PET), or single-photon emission computed tomography (SPECT).

[0373] In the methods described herein, cancer may be imaged additionally before administration to the subject of the first or second treatment. Cancer may also be imaged additionally or alternatively during or after administration of the first and / or second treatment, for example, to assess metastasis and the effectiveness of the treatment. For example, imaging may be performed by PET imaging, magnetic resonance imaging (MRI), or SPECT / computed tomography (CT) imaging. The imaging method may be any suitable imaging method known in the art.

[0374] Cancer can be any type of cancer. “Cancer,” as read herein, has its simple, ordinary meaning and may include, but is not limited to, a group of diseases involving abnormal cell growth that have the potential to invade or spread to other parts of the body (i.e., metastasize). Examples include, but are not limited to, cancers of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stroma, endometrium, breast, cervix, ovaries, colon, or prostate, leukemia, lymphoma, other blood-related cancers, and head and neck cancers. In certain embodiments, the cancer being treated is a tumor. In certain embodiments, cancer is malignant. In certain embodiments, cancer is PD-L1 + Includes tumors.

[0375] In certain embodiments, cancer includes hot tumors. In certain embodiments, cancer includes cold tumors. In certain embodiments, cancer includes immune desert tumors. Cold tumors are typically characterized by the absence of T cell infiltration (i.e., an immune desert) or by T cell accumulation only at the edges of the tumor site. These types of tumors exhibit immune neglect, low immunogenicity, and poor resistance to immune checkpoint blockade therapy. In contrast, hot tumors are typically characterized by high T cell infiltration or by the presence of pre-existing immunity, high immunogenicity, and generally exhibit a better response to immune checkpoint blockade therapy.

[0376] In some embodiments, the cancer is imaged before administering the first and second therapies to the subject. Imaging can be performed, for example, by PET, MRI, or SPECT / CT.

[0377] The administration of the first and second therapies may be performed to address cancer recurrence or resistance to checkpoint blocker therapy in the subject.

[0378] In certain embodiments, the use of (i) a compound, a pharmaceutically acceptable salt, hydrate, or solvate of the compound, or (ii) a composition comprising the compound, a pharmaceutically acceptable salt, hydrate, or solvate thereof, is provided in the manufacture of a pharmacopoeia for the treatment of cancer in a subject. The compound may be any compound described herein. The composition may be any composition described herein. The pharmacopoeia may be for use in combination with immune checkpoint inhibitor therapy to a subject, for example, the administration of one or more immune checkpoint inhibitors described herein.

[0379] Furthermore, a method for enhancing the efficacy of one or more immune checkpoint inhibitors is provided. This method may comprise the steps of administering one or more compounds of this specification (or a pharmaceutically acceptable salt or hydrate thereof), comprising an immunomodulator radical or a pharmaceutically acceptable salt or hydrate thereof, conjugated via a linker to a folate ligand or a functional fragment or analog thereof, to a subject; and contacting the target cells of the subject with one or more compounds to reprogram the subject's M2 macrophages into M1 macrophages. One or more compounds of such a method may comprise any of the compounds described herein. The immunomodulator or a pharmaceutically acceptable salt or hydrate thereof may target pattern recognition receptors. One or more compounds may be formulated into a composition of this specification by further comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or combinations thereof. The immunomodulator or a pharmaceutically acceptable salt or hydrate thereof may comprise TLR 3, 7, 8, 9, or 7 / 8 agonists. In certain embodiments, the step of administering at least one compound activates antitumor cells or an anti-inflammatory signaling cascade in the subject. The antitumor cells may be, for example, T cells, macrophages, or both.

[0380] In some embodiments, the method includes the steps of: obtaining or completing the acquisition of a sample from a subject; quantifying the level of expression of one or more biomarkers in the sample, wherein each of the one or more biomarkers is selected from the group consisting of CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), metalloproteinase 3 (TIMP3), IL-1β, hydroxyproline, collagen, PDGF, TGFβ, FRβ, TNFα, IFN-γ, anti-mannose receptor (CD206), differentiation cluster 86 (CD86), differentiation cluster 163 (CD163), IL-6, chemokine 10 (CXCL10), and immune interferon (IFNα); comparing the expression level of each of the one or more biomarkers in the sample with the expression level of such biomarkers in a control; and CCL18, Arg1, MMP9, TIMP 3. The procedure further includes administering or completing administration of a therapeutically effective dose of an unconjugated agonist or inhibitor to a subject if IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline, or collagen is upregulated compared to control expression levels, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα, or CD86 is downregulated or absent compared to control expression levels. In some embodiments, the folate ligand or its functional fragment or analog is specific to FRβ and binds to FRβ on cells.

[0381] Reprogramming of M2-type macrophages into M1-type macrophages

[0382] In at least one embodiment, a method for treating and / or preventing cancer recurrence is provided. The method includes administering to a subject a therapeutically effective amount of one or more compounds comprising a drug-conjugated targeting moiety (e.g., a folate receptor-binding ligand) (either via a linker or otherwise) for reprogramming M2-like macrophages in a tissue or organ to an M1-like phenotype. For example, the drug may be a TLR agonist (e.g., having formula I, III, or IV) conjugated to folate, or any other molecule or compound effective in reprogramming macrophages from an M2 phenotype to an M1 phenotype. In at least one embodiment, the drug may be selected from TLR 3 agonists, TLR 7 agonists, TLR 7 / 8 agonists, TLR 8 agonists, and TLR 9 agonists. In some embodiments, the drug may reprogram M2-like macrophages to an M1 phenotype, thereby reducing the production of anti-inflammatory cytokines and growth factors. For example, in at least one embodiment, reprogramming from an M2-like macrophage to an M1 phenotype results in the activation of antitumor cells and / or anti-inflammatory signaling cascades within the tumor microenvironment (TME).

[0383] Two major immune strategies are found in vertebrates: the innate immune system and the adaptive immune system. The innate immune response, or nonspecific immune response, is the first-line defense against non-self pathogens and consists of physical, chemical, and cellular defenses. The adaptive immune system, on the other hand, is the action that arises against pathogens that evade or overcome the primary innate immune defense.

[0384] Inflammatory responses play a crucial role in immunity. When tissue is damaged or pathogens are detected, for example, an inflammatory response is initiated, mobilizing the immune system. Immune cells of the innate immune system (i.e., neutrophils and eosinophils) are first mobilized through the vascular and lymphatic systems to the site of tissue injury or damage or the location of the pathogen, followed by the mobilization of macrophages.

[0385] Cells of the innate immune system can express special pattern recognition receptors that sense and bind to specific protein sequences present in microbial pathogens or other non-self molecules.

[0386] Two classes of molecules that can bind to pattern recognition receptors include pathogen-associated molecular patterns associated with microbial pathogens, and injury-associated molecular patterns associated with host cell components released during cell damage or death. Recognition of these protein sequences by pattern recognition receptors can initiate signaling pathways in which the product triggers the expression of certain genes whose products control the innate immune response (e.g., in some cases, lead to the development of antigen-specific adaptive immunity). Thus, pattern recognition receptors can be used to mediate these signaling pathways and, in certain cases, positively or negatively regulate the innate and even adaptive immune responses.

[0387] Macrophages are a diverse group of leukocytes known to eliminate pathogens through phagocytosis and are broadly classified as having either an M1 or M2 phenotype, depending on which specific differentiation they undergo in response to the local tissue environment. In some cases, macrophages are polarized to the M1 phenotype by exposure to interferon-gamma (IFN-γ), lipopolysaccharide (LPS), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain cases, the M1 phenotype is characterized by the production of high levels of pro-inflammatory cytokines (e.g., interleukin-1β (IL-1β), tumor necrosis factor (TNF), interleukin-12 (IL-12), interleukin-18 (IL-18), and / or interleukin-23 (IL-23)), the ability to mediate resistance to pathogens, potent microbiotacitative properties, high production of reactive nitrogen and oxygen intermediates, and / or enhancement of the type 1 helper T (Th1) cell response.

[0388] In other words, M1 polarization can activate antitumor cells, such as T cells (including, but not limited to, Th1 cells) and macrophages (including, but not limited to, immunoprotective M1 tumor-associated macrophages (TAMs) and "classically activated" / M1 myeloid-derived suppressor cells (MDSCs) in the TME). In some cases, M1 polarization is associated with the "attack and kill" phase of the innate immune response, which can induce lysis in various types of cancer cells. In addition, the release of pro-inflammatory antitumor cytokines (as well as similar chemokines, leukotrienes, prostaglandins, and complement releases from M1 macrophages) can lead to a global pro-inflammatory signaling cascade on reactive T cell function, influencing the regulation of multiple genes and their transcription factors, which may then result in the production of other cytokines and an increase in the number of surface receptors for other molecules, ultimately inducing the recruitment of inflammatory cells. Apart from local effects, these mediators can also have systemic effects, such as the production of acute inflammatory response proteins. In certain cases, M1 polarization may act to inhibit or prevent the initial establishment of infection and / or to remove damaged / cancerous tissue, and tumor growth and metastasis formation may be reduced by M2 TAM depletion, inhibition of M2 macrophage recruitment and pro-tumor function, and reprogramming M2 macrophages (e.g., TAMs and / or MDSCs) into a pro-inflammatory M1 phenotype.

[0389] In certain cases, after the innate immune system has carried out this “attack and kill” phase, macrophages can reprogram themselves to become a healing system (i.e., M2 type) and promote healing, for example, by releasing growth factors. Such growth factors could include (without limiting) certain cytokines, such as interleukin-4 (IL-4), interleukin-10 (IL-10), platelet-derived growth factor (PDGF), transformed growth factor-β1 (TGFβ), chemokine (CC motif) ligand 18 (CCL18), and / or interleukin-13 (IL-13). In certain cases, exposure to such cytokines / growth factors may activate the M2 macrophage phenotype.

[0390] In contrast to M1 macrophages, M2 macrophages may be associated with wound healing and tissue repair. In some cases, M2 macrophages are characterized by their involvement in tissue remodeling, immunomodulation / suppression, and / or tumor promotion. In specific cases, M2 macrophages produce polyamines that induce cell proliferation and / or proline that induce collagen production. While this healing response is beneficial in healthy subjects, the presence of M2 macrophages can have significantly detrimental effects in cancer subjects through immunosuppression and / or promotion of tumor growth and fibrosis. Chemokines and other factors may be released to promote the infiltration of immune cells into damaged tissue (e.g., innate immune response), including, for example, monocytes and macrophages that release anti-inflammatory cytokines, as is assumed in M2-like phenotypes. The chronic secretion of these cytokines can then activate tissue-resident and invasive fibroblasts / fibrous cells to become myofibroblasts, which secrete collagen and other extracellular matrix proteins that can harden surrounding tissue. In some cases, these M2 macrophages exacerbate the disease by promoting fibrosis. In some cases, growth factors and other cytokines produced by the M2 phenotype activate cancerous tumor growth through similar pathways.

[0391] In certain cancers, macrophages may be heterogeneously biased towards an anti-inflammatory (M2-like) phenotype. In certain cases, immune modulators can convert, for example, reprogram activated myeloid cells (e.g., M2-like macrophages) to a pro-inflammatory M1 polarized state (e.g., producing little to no growth factors and / or related cytokines, e.g., delaying or even eliminating the progression of a disease state (i.e., cancer)). In certain cases, combinations, compositions, and methods reverse the pro-inflammatory to anti-inflammatory shift observed during the development of certain cancers. In some embodiments, combinations, compositions, and methods reduce the amount / expression of fibrous biomarkers (e.g., those associated with anti-inflammatory activity (e.g., CCL18, hydroxyproline, and collagen)) in samples taken from an individual or subject, which indicates macrophage conversion to the M1 phenotype, and therefore activation of anti-tumor cells (e.g., T cells and / or macrophages) and initiation of an anti-inflammatory signaling cascade. "Individual," "subject," or "patient" may be a mammal, preferably a human, but may also be an animal.

[0392] A “marker” or “biomarker” may be described as differentially expressed if its expression level in a subject experiencing an active disease state is significantly different from that of a healthy subject or a subject not experiencing a disease state, or a sample taken from such a subject. Differentially expressed markers may be overexpressed or underexpressed compared to the baseline expression level of a normal or control sample or subject (in the embodiments referred to in the preceding paragraph, the biomarker may be reduced or underexpressed). Increases or decreases or quantifications of markers in a biological sample can be determined by any of several methods known in the art for measuring the presence and / or relative abundance of a gene product or transcript. Marker levels may be determined as absolute values ​​or compared to baseline values, and marker levels in a subject may be determined compared to a cutoff index. Alternatively, the relative abundance of one or more markers may be determined compared to a control that may be a clinically normal subject. Furthermore, the terms “gene overexpression” and “overexpression” (when used in relation to genes), and their derivatives, have meanings attributed to them by those skilled in the art in the relevant field, which include (without limitation) the overexpression or misexpression of wild-type gene products that may result in mutant phenotypes and / or abundant target protein expression.

[0393] In some embodiments, combinations, compositions, and methods increase antitumor biomarkers (e.g., TNFα and IFN-γ). In some embodiments, combinations and compositions that reverse an M2-like phenotypic shift are provided (e.g., providing an effective treatment for cancer or its condition).

[0394] By administering at least one compound (or a pharmaceutically acceptable salt or hydrate thereof) or composition of the first treatment, target M2 macrophages can be reprogrammed into M1 macrophages, and the efficacy of one or more checkpoint inhibitors of the second treatment can be enhanced compared to the baseline efficacy of one or more checkpoint inhibitors (for example, the baseline efficacy being that of the checkpoint inhibitors administered without combination with the first treatment).

[0395] In some embodiments, administration of a compound or composition (e.g., to a subject) can convert macrophages in the fibrous tissue of the subject from an M2-like phenotype to an M1-like phenotype. The M2-type macrophages of the subject may be MDSCs, TAMs, or both MDSCs and TAMs.

[0396] In some embodiments, a decrease in cytokines that stimulate collagen synthesis (i.e., CCL18, PDGF, and IL-1β) occurs after administration of the compounds or compositions herein, and similarly, a simultaneous increase in cytokines that inhibit collagen production (e.g., IFN-γ) occurs. Notably, in at least one embodiment, after administration of the compound or composition, the cytokine profile coincides with reprogramming from an M2-like phenotype to an M1-like phenotype. A “profile” or “assay” is a set of one or more markers, as well as their presence, absence, and / or relative levels or abundances (compared to one or more controls). For example, a cytokine profile is a dataset of the presence, absence, relative levels, or abundances of cytokines present in a sample. A genome or nucleic acid profile is a dataset of the presence, absence, relative levels, or abundances of expressed nucleic acids (e.g., transcripts, mRNA, etc.). A profile may alternatively be referred to as an expression profile.

[0397] In some embodiments, the net results of reprogramming are an increase in alveolar sacs, a decrease in extracellular matrix deposition, and a reduction in hydroxyproline / collagen biosynthesis, which is an effective reversal of the disease (see, for example, Example 4).

[0398] While specific drugs and formulas are described herein, it should be understood that any compound (e.g., a drug) useful for reprogramming activated myeloid cells into an anti-fibrotic M1-like phenotype (e.g., any compound (e.g., a drug) that can bind to pattern recognition receptors and inhibit at least a portion of the downstream innate immune response) may be used in the compounds, compositions, combinations, and methods herein. In some embodiments, analogs and / or derivatives of the compound or a pharmaceutically acceptable salt or hydrate thereof may be used in the targeted compounds, compositions, and combinations.

[0399] Furthermore, more than one compound may be administered, and in some examples, the compounds may include different drugs. For example, the different drugs may be selected from TLR7 agonists and TLR9 agonists. In yet another embodiment, one or more compounds may be administered in a composition together with one or more conjugated drugs and / or unconjugated drugs. In some embodiments, any of the compounds and drugs may be used according to the methods described herein, and in some examples, depending on the desired application, they may be combined with other drugs that deplete or inhibit bone marrow-derived suppressor cells (e.g., in conjunction with cancer treatment), downregulate the production of growth factors (e.g., pirfenidone), directly modify fibroblasts through inhibition of rapamycin complex target 1 (mTORC1) signaling, and / or any other pro-inflammatory and / or anticancer drugs and therapies. "Downregulation" and its derivatives (e.g., "down-regulation" or "downregulated") can be used interchangeably and refer to a decrease in the level of a marker, such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. Similarly, "upregulation" and its derivatives (e.g., "p-regulation" or "upregulated") can also be used interchangeably and refer to an increase in the level of a marker, such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. Furthermore, pathways, such as signaling or metabolic pathways, can also be upregulated or downregulated.

[0400] In certain embodiments, the method may include the steps of providing a compound of the Specified, a pharmaceutical composition containing the same, or a combination of the Specified (e.g., a therapeutically effective amount of the compound or composition of the Specified and one or more immune checkpoint inhibitors), and exposing a target M2 macrophage thereto, the M2 macrophage being converted to an M1 phenotype that promotes activation of the target's own immune response simultaneously with the administration of the compound, composition, and / or combination of the Specified.

[0401] Figure 2, a flowchart of Method 1900 for using one or more compounds (or pharmaceutically acceptable salts or hydrates thereof), is referenced herein. In at least one example, Method 1900 includes the step of contacting (administering) cells of interest with at least one compound, which includes, for example, a TLR7 agonist, conjugated via a linker to an immunomodulator (or a pharmaceutically acceptable salt or hydrate thereof), such as a TLR7 agonist. In at least one exemplary embodiment, the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor or DAMP (i.e., utilizing its targeting ligand). The cells may include, for example, cells of interest that have experienced or are at risk of experiencing cancer recurrence or resistance, and the at least one compound may include any of the compounds provided herein.

[0402] In at least one embodiment, step 1902, which involves contacting cells with at least one compound (or a pharmaceutically acceptable salt or hydrate thereof), further comprises administering or applying a therapeutically effective amount of at least one compound (or a pharmaceutically acceptable salt or hydrate thereof) to the subject. Additionally, or alternatively, at least one compound (or a pharmaceutically acceptable salt or hydrate thereof) may be formulated into a composition further comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or combinations thereof.

[0403] In at least one embodiment, the disease state includes cancer that is resistant to or subsequently relapses to immune checkpoint therapy. Since checkpoint inhibitors target acquired immune cells, their combination with drugs that reprogram the innate immune system may be beneficial. Furthermore, since it has been established that MDSCs and TAMs can be reprogrammed to disrupt the growth-promoting cycle by FA-TLR7 and TLR7 / 8 agonists, systemic administration of these FA-TLR7 and / or FA-TLR7 / 8 agonists in combination with immune checkpoint inhibitors may target cancers that relapse, are resistant, or are treatment-ineffective with checkpoint blockade alone.

[0404] In addition to step 1902, method 1900 may optionally include steps 1904-1910. In step 1904, a biological sample is obtained from a subject, and in step 1906, the expression level of one or more biomarkers in the sample is quantified. For example, the sample may be obtained from a certain amount of peripheral blood taken from a subject.

[0405] The quantification step 1906 can be carried out using any suitable method known in the art, including, for example, qPCR, mass spectrometry, ELISA, and / or any other modality capable of measuring / quantifying biomarker expression. In at least one exemplary embodiment, one or more biomarkers are selected from the group consisting of CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, TNFα, IFN-γ, CD206, CD163, IL-6, CXCL10, IFNα, and CD86.

[0406] In step 1908, the expression level of one or more biomarkers in the sample is compared to the expression level of such biomarkers in a control. The control may be a healthy individual or simply an individual not experiencing the disease condition in question. In at least one embodiment, a clinical difference between the expression level of one or more biomarkers in the sample and the expression level of the relevant biomarkers in the control may indicate that the subject is suffering from the disease condition in question. For example, without limitation, if the comparison step 1908 shows that the expression of one or more of the biomarkers CCL18, Arg1, CD163, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, and / or CD206 (i.e., “anti-inflammatory biomarkers”) is upregulated compared to the control, this indicates that the subject is experiencing an anti-inflammatory immune response, which is associated with an M2-like macrophage phenotype. Thus, in at least one embodiment, such a result indicates the need to administer one or more compounds of the present disclosure to reprogram such M2-like macrophages into an M1 phenotype.

[0407] In contrast, if comparison step 1908 shows that the expression of the aforementioned biomarkers is downregulated compared to the control, or if the expression of one or more of TNFα, IFN-γ, and / or CD86 ("pro-inflammatory biomarkers") is upregulated compared to the control, this indicates, in certain embodiments, that the subject is showing a positive response to the previously administered compound (if applicable) and / or is experiencing a pro-inflammatory immune response, which is associated with the M1 phenotype and the activation of one or more antitumor cells and / or anti-inflammatory signaling cascades.

[0408] If necessary, in step 1910, if the expression of one or more anti-inflammatory biomarkers in the sample is upregulated compared to their respective expression levels in the control, or if the expression of one or more pro-inflammatory biomarkers in the sample is downregulated compared to their respective expression levels in the control, an alternative treatment may be administered. In at least one embodiment, the alternative treatment may include administering a therapeutically effective dose of a derivative of at least one compound previously administered in step 1902, wherein the derivative comprises at least one compound previously administered, modified with respect to utilizing any of the following: a different targeting moiety, a different linker size, and / or a different immunomodulator, in an attempt to better optimize the efficacy of at least one compound for the target. Additionally, or alternatively, other treatments may be utilized, including conventionally known treatments for the cancerous disease in question (e.g., imaging, surgery, chemotherapy, radiation, etc.). Steps 1904–1910 may be included and / or repeated as necessary or desired to meet established criteria and / or to ensure that the active ingredients are effective in alleviating the symptoms of cancerous disease conditions.

[0409] As described above, this method can be used to treat and / or prevent cancer or its recurrence (whether folate receptor-positive or folate receptor-negative). For example, in certain cases, such a method may include administering to a subject a therapeutically effective and / or prophylactically effective dose of a drug for reprogramming M2-like macrophages in cancerous and / or tumor cells to an M1-like phenotype, e.g., one or more compounds (or pharmaceutically acceptable salts or hydrates thereof) containing a targeted moiety bound to a targeted TLR-7 agonist, for example. If the cancer is folate receptor-negative, such administration may further act to deplete or inhibit MDSCs present in such tissue / tumor. Additional drugs, e.g., PI3k inhibitors, signaling and transcriptional activator 6 (STAT6) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, inducible nitric oxide synthase (iNOS) inhibitors, and anti-inflammatory drugs (e.g., methotrexate), may also be administered in connection with such a method. In at least one embodiment, the drug can inactivate MDSCs.

[0410] The compounds and compositions may be used alone or in combination with checkpoint inhibitor immunotherapy to inhibit cancer growth in the target. For example, in addition to step 1902, method 1900 may further include the step of administering checkpoint inhibitor immunotherapy (as described above).

[0411] Such combination therapies are particularly beneficial for treating cancers that are prone to recurrence or are resistant to monotherapy, leading to immune recognition of the cancer by producing a systemic immune response, and thus resulting in effective attack or even elimination (e.g., remission) of any remaining living cancer cells that have the recognized antigen. As a significant consequence, any remaining cancer can be eliminated or mitigated. Therefore, in certain particular approaches, the methods of the present disclosure include the step of treating a subject having cancer that is resistant to treatment with checkpoint inhibitors.

[0412] Such combination therapy methods can be carried out using any suitable checkpoint inhibitor (as described above), and may include using more than one of these types of agents. The immune checkpoint inhibitor used in combination with the compound or composition of the present invention may be any immune checkpoint inhibitor. As is known in the art, examples of immune checkpoint inhibitors include (without limiting) transmembrane programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand PD-L1 (also known as CD274). In normal non-malignant physiology, PD-L1 on the surface of cells binds to PD1 on the surface of immune cells, which inhibits the activity of immune cells. Upregulation of PD-L1 on the surface of cancer cells is thought to facilitate evasion of the host immune system, at least in part, by inhibiting T cells that would normally target tumor cells. In certain embodiments, one or more checkpoint inhibitors include pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islerizumab, a combination of osiperlimab and islerizumab, and BMS-936. Each patient is independently selected from the group consisting of 559 / MDX-1105, MPDL3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab.

[0413] In certain approaches, administering both the compounds described herein (or their pharmaceutically acceptable salts or hydrates) and checkpoint inhibitor therapy results in inhibition of cancer growth that exceeds additive effects.

[0414] When multiple therapeutic drugs are administered together, the dosage may be adjusted accordingly, as is recognized in the art.

[0415] If a combination therapy regimen involves administering more than one treatment to a target, it should be understood that the order, timing, frequency, concentration, and volume of administration are limited only by the medical requirements and limitations of the treatment (i.e., two treatments may be administered to the target, for example, simultaneously or sequentially (in either order), or according to any other regimen).

[0416] When describing typical embodiments, this disclosure may present methods and / or processes as a specific sequence of steps. Unless the method or process depends on a specific order of steps described herein, the method or process is not limited to that specific sequence of steps. Those skilled in the art will understand that other sequences of steps are possible. Therefore, a specific sequence of steps disclosed herein should not be construed as a limitation on the claims. In addition, claims relating to methods and / or processes are not limited to the function of those steps in the sequence described herein, and those skilled in the art will readily understand that the order may vary and still remain within the spirit and scope of this disclosure.

[0417] Various embodiments of compounds, compositions, and methods are described in great detail herein, but these embodiments are provided only as non-limiting examples. Numerous variations and modifications of the embodiments described herein will be apparent to those skilled in the art in light of this disclosure. Therefore, it will be understood that various changes and modifications may be made without departing from the scope of this disclosure, and that equivalents may be substituted for their elements. Indeed, this disclosure is not intended to be either thorough or limiting. The scope of this disclosure shall be defined by the appended claims and their equivalents.

[0418] In addition, while many of the examples provided herein utilize mouse models, those skilled in the art will understand that gene expression patterns in mouse models exhibit highly significant correlations with those under human conditions, and that numerous pathways are commonly regulated by multiple conditions in both humans and mice. Therefore, gene expression patterns and disease progression in mouse models closely replicate those under human conditions, particularly with respect to inflammatory diseases and cancer, and thus the working examples described herein are supported to correlate with human data, specified conditions, and applications.

[0419] Accordingly, this specification and the appended claims are intended to encompass all modifications and changes that would be obvious to those skilled in the art based on this disclosure.

[0420] overview

[0421] For the purpose of facilitating the understanding of the principles of this disclosure, references are made herein to embodiments illustrated in the drawings, and specific terms are used to describe them. In any case, it should be understood that the description of these embodiments is not intended to limit the scope. In contrast, this disclosure is intended to include alternative forms, modifications, and equivalents that may fall within the spirit and scope of this application as defined by the appended claims. As stated above, the art may be illustrated and described in one or more preferred embodiments, but the compositions, compounds, and methods herein may include a number of different configurations, forms, materials, and accessories.

[0422] All patents, patent application publications, articles, textbooks, and other publications referenced herein represent the skill level of a person skilled in the art relating to this disclosure. All such publications are incorporated herein by reference to the same extent that each individual publication is indicated as being incorporated by reference specifically and individually.

[0423] Numerous specific details are provided in the following description to provide a complete understanding of the disclosure. Certain embodiments may be implemented without some or all of these specific details, and it should be understood that the disclosure is not limited to any particular biological system, any particular cancer, or any particular organ or tissue, and is, of course, variable, but still applicable based on the data provided herein.

[0424] Various techniques and mechanisms in this disclosure may describe connections or couplings between two components. Terms such as attached, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably unless otherwise indicated or clearly indicated by context. These terms and expressions do not necessarily imply a direct connection and may include connections through intervening components. Note that a connection between two components does not necessarily imply a direct, uninterrupted connection, and various other components may exist between the two components described. Consequently, a connection does not necessarily imply a direct, uninterrupted connection unless otherwise indicated.

[0425] Furthermore, where preferable and convenient, similar reference numerals are used in the drawings and description to refer to the same or similar parts or steps. The drawings are in a simplified form and are not to exact scale. This disclosure is presented in this form solely for illustrative purposes, and it is understood that the principles and embodiments described herein may apply to components of compounds and / or compositions having configurations other than those specifically described herein. In fact, it is expressly intended that the components of compositions and compounds of this disclosure may be adapted to facilitate their desired application.

[0426] In certain embodiments, the compounds, compositions, and methods herein are useful for the prevention and / or treatment of cancer. In certain embodiments, the compounds and / or compositions provided herein are also useful for the treatment of cancer. In some embodiments, the compounds, compositions, and methods provided herein utilize strategies that target the innate immune system (e.g., selectively) and reprogram macrophage polarization from M2 to M1, for example, by leveraging its checkpoint blocking properties. In some embodiments, the compounds include Toll-like receptor TLR7 and / or 8 agonists. In certain embodiments, the compounds provided herein are provided or used alone, with targeted agents, and / or in combination therapy with other interventions.

[0427] A certain definition

[0428] The following terms and phrases have the meanings set forth below. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art.

[0429] The terms “about” or “approximately” mean within an acceptable range for a particular value, as determined by those skilled in the art, which will depend in part on how that value is measured or determined, for example, on the limitations of the measuring system. For example, “about” could mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. As further examples, “about” or “approximately” could mean within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or above the value mentioned or the range limit mentioned. Alternatively, particularly with respect to biological systems or processes, the term could mean within one order of magnitude of a value, preferably up to five times, and more preferably up to two times. Unless otherwise indicated, the term “about” means within an acceptable margin of error for a particular value, for example, ±1 to 20%, preferably ±1 to 10%, and more preferably ±1 to 5%.

[0430] When a range of values ​​is provided, it should be understood that this includes the respective intermediate values ​​between the upper and lower limits of that range, as well as any other mentioned or intermediate values ​​within that range. The upper and lower limits of these smaller ranges may independently be included within or encompassed within those smaller ranges, and any limit values ​​within that range may be specifically excluded. If the range mentioned includes one or both of the limit values, the range excluding one or both of those limit values ​​is also included.

[0431] The phrase "at least one of the items" in the list of items refers to any combination of these items, including a single member. For example, "at least one of a, b, or c" is intended to include a, b, c, ab, ac, bc, and abc.

[0432] The terms “a,” “an,” or “the” are used to imply one or more than one unless otherwise indicated by the context. The term “or” is used to refer to a non-exclusive “or” unless otherwise indicated. In addition, it should be understood that the words or terms used herein are for illustrative purposes only and not restrictive unless otherwise defined.

[0433] The term "or" is used to mean a non-exclusive "or" unless otherwise specified. Furthermore, it should be understood that any words or terms used herein are for illustrative purposes only, and not restrictive, unless otherwise defined.

[0434] The terms and expressions used are intended to be descriptive, not restrictive. If a particular term is defined and described or discussed elsewhere in the "Modes for Carrying Out the Invention," all such definitions, descriptions, and discussions are intended to belong to that term. Furthermore, the use of such terms and expressions is not intended to exclude any equivalent of the characteristics or parts thereof that are shown or described. In addition, subheadings may be used in the "Modes for Carrying Out the Invention," but such use is simply for ease of reference and is not intended to restrict any disclosure made in one section to that section only; rather, any disclosure made under one subheading is intended to constitute a disclosure under each of the other subheadings.

[0435] It is understood that various modifications are possible within the scope of the claimed invention. Therefore, although the present invention is specifically disclosed in the context of preferred embodiments and, as necessary, characteristics, those skilled in the art will be able to arrive at modified and varied forms of the concepts disclosed herein. Such modified and varied forms are considered to be within the scope of the claimed invention as described herein. [Examples]

[0436] The following embodiments serve to illustrate the present disclosure. The embodiments are not intended in any way to limit the scope of the claimed invention. Chemical Examples (Example A) Synthesis of compound 1A

[0437] Compound 1A was synthesized according to Scheme 1 below, as reported by Nikunj M. Shukla, Cole A. Mutz, Subbalakshmi S. Malladi, Hemamli J. Warshakoon, Rajalakshmi Balakrishna, and Sunil A. David, "Regioisomerism-dependent TLR7 agonism and antagonism in an imidazoquinoline; Structure-Activity Relationships in Human Toll-Like Receptor 7-Active Imidazoquinoline Analogues," J Med Chem. 2012 Feb 9; 55(3): 1106-1116. [ka] Step 1: Synthesis of 1-amino-2-methylpropan-2-ol (compound)

[0438] 2,2-dimethyloxirane (0.1 g, 1.388 mmol) was added dropwise to 20 mL of ice-cold ammonium hydroxide solution. The reaction mixture was stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was dissolved in methanol. Di-tert-butyl dicarbonate (0.75 g, 3.47 mmol) was added to the reaction mixture, and the mixture was stirred for 4 hours. The mixture was purified by column chromatography (24% ethyl acetate (siRNA) / hexane) to obtain tert-butyl 2-hydroxy-2-methylpropyl carbamate. The pure tert-butyl 2-hydroxy-2-methylpropyl carbamate was dissolved in 5 mL of trifluoroacetic acid and stirred for 35 minutes. The solvent was removed under reduced pressure to obtain 1-amino-2-methylpropan-2-ol as trifluoroacetate 1'. 1H NMR 500 MHz (500 MHz, CDC13, δ at ppm): δ 8.62 (s, 2H), 3.02 (d, 2H), 2.06-2.04 (m, 2H), 1.37-1.34 (s, 6H).

[0439] Step 2: Synthesis of 2-methyl-1-(3-nitroquinoline-4-ylamino)propan-2-ol (Compound 2)

[0440] 450 mg, 2.4 mmol of trifluoroacetate of 1-amino-2-methylpropan-2-ol (compound) was added to a solution of 4-chloro-3-nitroquinoline (compound 1) (250 mg, 1.2 mmol) and Et3N (0.5 ml, 3 mmol) in a 4:1 mixture of toluene and 2-propanol. The mixture was heated to 70°C for 30 minutes until solid sedimentation began. The reaction mixture was then cooled, filtered, and washed with toluene / 2-propanol (7:3), ether, and cold water. The residue was dried at 80°C to obtain 2-methyl-1-(3-nitroquinoline-4-ylamino)propan-2-ol (compound 2). Liquid chromatography-mass spectrometry (LCMS) analysis: [M+H] + m / z = 261.

[0441] Step 3: Synthesis of l-(3-aminoquinoline-4-ylamino)-2-methylpropan-2-ol (Compound 3)

[0442] 2-Methyl-1-(3-nitroquinoline-4-ylamino)propan-2-ol (compound 2) (450 mg, 1.72 mmol) was dissolved in methanol and hydrogenated using a hydrogen balloon with Pd / C as a catalyst for 4 hours. The solution was then filtered using Celite, and the solvent was evaporated under reduced pressure to obtain l-(3-aminoquinoline-4-ylamino)-2-ethylpropan-2-ol (compound 3). LCMS:[M+H] + m / z=231. Η NMR 500 MHz (CDC13, δ in ppm): δ 8.12 (s, 1H), 7.61-7.58 (m, 1H), 7.48-7.40 (m, 2H), 4.90 (s, 2H), 3.47 (2H), 1.35-1.21 (s, 6H).

[0443] Step 4: Synthesis of 1-(4-amino-2-butyl-1H-imidazo[4,5-c]quinoline]-1-yl)-2-methylpropan-2-ol (Compound 5, TLR7A)

[0444] To a solution of compound 3 (100 mg, 0.43 mmol) in anhydrous THF, triethylamine (66 mg, 0.65 mmol) and valeryl chloride (62 mg, 0.52 mmol) were added. The reaction mixture was then stirred for 6-8 hours, after which the solvent was removed under vacuum. The residue was dissolved in RINKAN, washed with water and brine, and dried over Na2SO4 to obtain the intermediate amide compound. This was dissolved in methanol (MeOH), calcium oxide was added, and the mixture was heated in a microwave apparatus at 110°C for 1 hour. The solvent was then removed, and the residue was purified using column chromatography (9% MeOH / dichloromethane) to obtain compound 4 (58 mg). To a solution of compound 4 in a solvent mixture of MeOH:dichloromethane:chloroform (0.1:1:1), 3-chloroperbenzoic acid (84 mg, 0.49 mmol) was added, and the solution was refluxed at 45-50°C for 40 minutes. Next, the solvent was removed, and the residue was purified using column chromatography (20% MeOH / dichloromethane) to obtain the oxide derivative (55 mg). This was then dissolved in anhydrous dichloromethane, and benzoyl isocyanate (39 mg, 0.26 mmol) was added, followed by heating at 45°C for 15 minutes. Next, the solvent was removed under vacuum, and the residue was dissolved in anhydrous MeOH, followed by the addition of an excess amount of sodium methoxide. The reaction mixture was then heated at 80°C for 1 hour. The solvent was removed under vacuum, and the residue was purified using column chromatography (11% MeOH / dichloromethane) to obtain compound 5. LCMS:[M+H] +m / z=312. Н NMR 500 MHz (CDC13, δ in ppm): δ 8.16-8.15 (d, 1H), 7.77-7.46 (d, 1H), 7.46-7.43 (m, 1H), 7.33-7.26 (m, 1H), 3.00-2.97 (m, 2H), 1.84-1.78 (m, 2H), 1.47-1.41 (m, 2H), 1.36 (s, 6H), 0.98-0.95 (m, 3H). (Example B) Synthesis of compound 1B

[0445] Subsequently, compound 1B can be synthesized using compound 1A according to scheme 2 below. [ka]

[0446] Compound 1A, folate, and linker are commercially available or can be prepared according to methods known to those skilled in the art.

[0447] Heterobifunctional linker 7 (88 mg, 0.213 mmol) was added to a solution of compound 5 (33 mg, 0.106 mmol) and dimethylaminopyridine (39 mg, 0.319 mmol) in 4 mL of methylene chloride under a nitrogen atmosphere at room temperature. The mixture was stirred at reflux temperature for 7 hours, and thin-layer chromatography (TLC) analysis of the mixture at that time showed a conversion of more than 80%. The mixture was concentrated and purified by column chromatography using 10% acetonitrile in methylene chloride as the eluent. The pure product, compound 9, was obtained as a pale yellow solid. A solution of compound 8 (1 equivalent) in dimethyl sulfoxide (DMSO) was added in three portions at 20-minute intervals to a solution of drug-linker intermediate compound 9 (1.0 to 1.5 equivalents) in DMSO containing dimethylaminopyridine (1 equivalent). After stirring at room temperature under argon for 1-2 hours, LC-MS analysis of the mixture revealed the formation of the desired folate-drug compound (compound 10) as the main product. The mixture was purified by preparative high-performance liquid chromatography (HPLC). LC-MS:[M+H] + m / z = 959. 11H NMR (500 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.49 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.83 - 7.74 (m, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.06 (s, 1H), 6.81 (d, J = 6.2 Hz, 1H), 6.61 (d, J = 8.3 Hz, 2H), 6.27 (s, 1H), 4.43 (d, J = 5.9 Hz, 2H), 4.28 (t, J = 6.6 Hz, 2H), 4.00 (d, J = 25.7 Hz, 3H), 3.03 (t, J = 7.5 Hz, 2H), 2.97 (dd, J = 13.0, 6.5 Hz, 1H), 2.09 (s, 2H), 1.81 (s, 7H), 1.40 (q, J = 7.4 Hz, 2H), 1.22 (s, 2H), 1.13 (s, 2H), 0.91 (t, J = 7.4 Hz, 3H). (Example C) Synthesis of Compound 2A

[0448] Compound 2A can be synthesized according to Scheme 3 and Scheme 4. [Chemical formula] [Chemical formula]

[0449] First, the cysteine-loaded Wang resin (11) was deprotected using 20% ​​piperidine in dimethylformamide (DMF). The free amine was treated with Fmoc-Glu(OtBu)-COOH in the presence of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), N,N-diisopropylethylamine (DIPEA), and DMF. The bound product was deprotected using 20% ​​piperidine in DMF and treated with pteroic acid in the presence of PyBop, DIPEA, and DMF to obtain compound 12. The trifluoroacetyl group was deprotected with a 50% ammonia-DMF solution. Finally, the resin was cleaved using a trifluoroacetic acid:triisopropylsilane:water:tris(2-carboxyethyl)phosphine cocktail solution and purified by HPLC to obtain folate-cysteine ​​(13) as a yellow solid.

[0450] Compound 14 was first treated with a heterobifunctional linker reagent (15) to obtain a folate-cystine disulfide intermediate (16). This was then reacted with folate-cysteine ​​(13) in DMSO and purified using HPLC to obtain compound 17 (e.g., compound 2A). All compounds were characterized by LC-MS using ammonium bicarbonate and acetonitrile as buffer systems. The mass observed by LC-MS for compound 2A was [M+H]+=1082.2. (Example D) Synthesis of the TLR7 agonist TLR7-1A

[0451] Solvents, reagents, and starting materials were purchased from commercial suppliers and used as received, unless otherwise indicated. All reactions were carried out at room temperature, unless otherwise indicated. Starting materials were purchased from commercial suppliers or synthesized according to the methods described herein or using procedures from the literature.

[0452] The synthesis of the TLR7 agonist TLR7-1A is described in Scheme 5. [ka] [ka] (Example E) Synthesis of the TLR7 agonist TLR7-1B

[0453] The synthesis of the TLR7 agonist TLR7-1B is described in Scheme 6. [ka] (Example F) Synthesis of the TLR7 agonist TLR7-1C

[0454] The synthesis of the TLR7 agonist TLR7-1C is described in Scheme 7. [ka] (Example G) Synthesis of release-type TLR7-folate compounds

[0455] The synthesis of the releaseable TLR7-folate compound is described in Scheme 8. [ka] [ka] (Example H) Synthesis of non-release TLR7-folate compounds

[0456] The synthesis of a non-release TLR7-folate compound is described in Scheme 9. [ka] (Example I) Synthesis of non-release TLR7-folate compounds

[0457] The synthesis of a non-releasing TLR7-folate compound (compound 1000) is described in Scheme 10. [ka] (Example J) Synthesis of non-release TLR7-folate compounds

[0458] The synthesis of non-releasing TLR7-folate compounds is described in schemes 11 and 12. [ka] [ka]

[0459] In conjunction with the state of the art in the relevant technical fields, and in particular in light of the scheme described above, this disclosure provides sufficient detail so that a person skilled in the art can synthesize all other compounds of this disclosure using the concepts described herein. Examples Methods and materials

[0460] Human monocyte THP-1 cells were obtained from the American Type Culture Collection and cultured in folate-deficient RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% thermo-inactivated fetal bovine serum and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). THP-1 cells were selected as a model system because this human monocyte cell line is known to acquire an M2-like phenotype and produce a considerable amount of anti-inflammatory cytokines upon stimulation with IL-4, IL-6, and IL-13.

[0461] All reagents were purchased from commercial suppliers and used without further purification. All other cell culture reagents, syringes, and disposable items were purchased from VWR (Chicago, IL).

[0462] Anti-CTLA-4 (catalog number BE0131, clone: ​​9H10), anti-PD-1 (catalog number BE0273, clone: ​​29F.1A12), and anti-PDL1 (catalog number BE0101, clone: ​​10F.9G2) antibodies were purchased from Bio X Cell (Lebanon, NH).

[0463] Flow cytometry antibodies, Zombie Violet® Fixable Viability Kit, fixation / permeabilization buffer, and RBC lysis buffer were obtained from BioLegend (San Diego, CA).

[0464] The mouse tumor dissociation kit (catalog number 130-096-730) was purchased from Miltenyi Biotech. All flow cytometry sample analysis was performed using an Attune NXT analyzer.

[0465] IFN-γ, IL-4, interleukin-6 (IL-6), and interleukin-13 (IL-13) were obtained from Biolegend. Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and all other reagents and solvents were purchased from Sigma. (Example 1) Differentiation and polarization of THP-1 cells into M2-like macrophages in vitro

[0466] THP-1 cells were seeded in 96-well plates at a density of 60,000 cells / well. The cells were differentiated into non-polarized macrophages by 48-hour incubation with 200 nM PMA, followed by 24-hour incubation in fresh RPMI medium. The resulting macrophages were polarized to an M2-like phenotype by 3-day incubation with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / mL IL-6, and then reprogrammed for 48 hours with different concentrations of compound 1A and compound 1B. They were then collected for genetic analysis by quantitative polymerase chain reaction (qPCR). The cultures were maintained at 37°C in a humidified 5% CO2 incubator.

[0467] To evaluate whether potent TLR7 agonists (e.g., compound 1A, e.g., formula III) can reprogram anti-inflammatory macrophages into a pro-inflammatory phenotype, THP-1 cells stimulated with IL-4, IL-6, and IL-13 were incubated with different concentrations of untargeted compound 1A, and mRNA levels of several anti-inflammatory markers, namely CCL18, CD206, IL-1β, and PDGFα and β, were tested.

[0468] As shown in Figures 3A-3C, incubation with compound 1A for 48 hours induced a decrease in the expression of CCL18, CD206, and IL-1β, suggesting that the TLR7 agonist can indeed promote the shift of these anti-inflammatory polarized THP-1 cells to a low-fibrility / high-inflammatory phenotype. Furthermore, when the expression of TNFα, a marker of the pro-inflammatory phenotype, was tested, an increase in its expression was observed (Figure 3D), confirming that a shift from anti-inflammatory to pro-inflammatory characteristics occurred in THP-1 cells. (Example 2) Evaluation of macrophage reprogramming

[0469] To confirm that a folate-conjugated TLR7 agonist could induce the same THP-1 reprogramming observed in Example 1, compound 1B was prepared in which a release linker connecting folate to compound 1A was constructed with a self-destructing disulfide bond, allowing for the release of compound 1A after the internal transfer of compound 1B into the reducing environment of intracellular endosomes.

[0470] Either compound 1A or compound 1B at different concentrations was incubated with the polarized THP-1 macrophages described above for the indicated period, after which the culture medium was collected for cell harvesting for analysis of secreted cytokines and qPCR analysis.

[0471] Total RNA was collected using the Quick-RNA® MicroPrep kit (Zymo Research, Irvine, CA) according to the manufacturer's recommended protocol, in a 1x10⁶ fraction. 5 ~2×10 5RNA samples were isolated from macrophages. The RNA samples were then reverse transcribed to cDNA using a high-performance cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, No. 4368814). qPCR analysis was performed using iTaq® Universal SYBR Green SuperMix (Bio-Rad Laboratories GmbH, Hercules, CA, No. 1725121), an iCycler thermal cycler, and iCycler iQ 3.0 software (Bio-Rad Laboratories GmbH, Hercules, CA) to track the expression of markers characteristic of macrophage polarization. IL-6, CXCL10, IFNα, IFN-γ, and CD86 were used as markers for the M1 phenotype, while CCL18, CD206, CD163, and Arg1 were used as markers for the M2 phenotype. IL-1β, PDGFβ, MMP9, and TIMP 3 were measured as indicators of the anti-inflammatory phenotype. IRAK-4 was used as an indicator of TLR7 stimulation. Melting curve analysis was performed to control for the specificity of the amplified product. Amplification of nonspecific products was not observed in any of the reactions. Each sample was analyzed independently in triplicate for each marker.

[0472] Repeating the above studies (see Figures 3A–3F, gray bars), the same qualitative changes were observed, except that the magnitude of the effect of compound 1B was somewhat reduced. This reduction in potency was predicted to be because the non-targeted TLR7 agonist enters cultured cells immediately, while its folate-targeted counterpart is designed to enter cells only after folate receptor binding and receptor-mediated endocytosis.

[0473] The data shown in Figures 3A-3C confirm that administration of either untargeted or targeted TLR7 agonists successfully reprogrammed M2 macrophages into M1 macrophages (i.e., downmodulated M2 anti-inflammatory macrophages), while the data shown in Figures 3D-3F confirm that administration of the tested compounds upmodulated M1 macrophages.

[0474] Figures 4A–4E and 5A–5D show graph data representing the levels of various markers measured from THP-1 cells that were induced into M2 macrophages, incubated with different concentrations of compound 1B or compound 1A for 2 hours, washed with PBS, and, for the data shown in Figures 5A–5D, incubated again for 46 hours (for the data shown in Figures 4A–4E, cells were collected immediately after the initial 2-hour incubation). In all datasets, cells were collected for genetic analysis by qPCR. Figures 4A–4C show CCL18 mRNA levels (Figures 4A and 5A), CD206 mRNA levels (Figures 4B and 5B), IL-1β mRNA levels (Figures 4C and 5C), and PDGFβ mRNA levels (Figure 4E). The data support the downregulation of the M2 type anti-inflammatory phenotype after administration of the test compounds. In particular, compound 1B downregulated macrophage anti-inflammatory / M2 type markers more than compound 1A. Furthermore, Figure 4D shows CD86 mRNA levels, and Figure 5D shows TNFα levels. This data supports the upmodulation of the M1-like phenotype after administration of the tested compound. Figures 4A-4E and 5A-5D support the downmodulation of the M2-type anti-inflammatory phenotype after administration of free and targeted TLR7 agonists. Although collected, data for PDGFα are not shown because no significant difference was observed after treatment.

[0475] Because low molecular weight water-soluble drugs such as compound 1A and compound 1B are often eliminated from the body within two hours of injection, in a more physiologically relevant in vitro model of drug exposure in vivo, the incubation of cells and drugs is limited to only two hours, followed by further incubation for 46 hours in the absence of the drug before testing the efficacy of the drug. As shown in Figures 4A-4E, when THP-1 cells were incubated with a TLR7 agonist for two hours, and then the drug-containing medium was replaced with a drug-free medium, compound 1B was observed to have superior potency compared to compound 1A, and was dramatically improved, especially in the case of TNFα induction, by the folate-targeted compound. This is most likely because the folate-targeted TLR7 agonist was captured by folate receptor-positive cells, while compound 1A was not retained by the same cells.

[0476] These data support the idea that compound 1B is more effective in reprogramming anti-inflammatory macrophages in vivo, and that folate-conjugated drugs (e.g., compound 1B) also have the added advantage of being enriched in FRβ-expressing macrophages and therefore unable to enter folate receptor-negative cells, which are dominant throughout the body, thus resulting in lower systemic toxicity (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0477] Figures 6A–6D show graph data representing the levels of various markers measured from THP-1 macrophages induced to M2, treated with different drug concentrations for 48 hours (Figures 6A and 6B) or 2 hours, then cultured for the remaining 46 hours in fresh medium (Figures 6C and 6D). In all cases, cell supernatant was collected and secreted CCL18 protein and IL-1β were detected by ELISA. The data support that administration of TLR7 compounds or folate-targeted TLR7 compounds downregulates the secretion of CCL18 and IL-1β in the low concentration range (0.1–10 nM).

[0478] Furthermore, to ensure that the above mRNA analysis accurately reflected the levels of anti-inflammatory cytokines produced by THP-1 cells stimulated with IL-4, IL-6, and IL-13, the concentrations of CCL18 and IL-1β polypeptides in the THP-1 supernatant were quantified by ELISA assay. As shown in Figures 6A and 6B, both compound 1A and compound 1B induced reductions in CCL18 and IL-1β when incubated continuously with the agonist for 48 hours; however, compound 1B was found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D). (Example 3) Characterization of FRβ expression by flow cytometry

[0479] Fluorescence-activated cell separation (FACS) analysis was performed to measure FRβ expression in THP-1-derived macrophages. Cells were detached using Accutase® cell detachment solution (Biolegend, San Diego, CA, No. 423201) and gently removed with a cell scraper. Cells were washed with PBS, and nonspecific binding was blocked by incubation with Fc receptor blocking solution (Biolegend, San Diego, CA, No. 422301) at room temperature for 10 minutes. Biotinylated anti-human FRβ monoclonal antibody (m909) was then added, and the cells were incubated on ice for a further 30 minutes before being washed with staining buffer (PBS supplemented with 2% FBS). Next, the cells were incubated on ice for 20 minutes in streptavidin labeled with fluorescein (BD Biosciences, Franklin Lakes, NJ, No. 554060), washed twice in PBS, stained with 7AAD (viability dye) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ). Figure 6E shows flow cytometry data supporting that THP-1 macrophages were FRβ+ and therefore suitable for in vitro studies of compound 1B and other studies described herein.

[0480] Figure 6F confirms that compound 1B remained stable in the culture medium during the incubation period at 37°C. In fact, compound 1B retained its original structure after 48 hours of incubation. (Example 4) In vivo bleomycin-induced pulmonary fibrosis and reprogramming of anti-inflammatory macrophages.

[0481] Furthermore, we conducted studies to determine whether macrophages in pulmonary fibrotic lungs could be specifically targeted in vivo with folate-linked drugs. After testing several protocols for inducing pulmonary fibrosis in mice, we selected a protocol in which 0.75 mg / kg of bleomycin (BM) was injected into the lungs of C57BL / 6 mice through a tracheal incision, thereby progressing the mice to both the inflammatory and fibrotic stages of fibrosis before the initiation of treatment. (The BM model is widely recognized as useful in enabling mechanistic investigations related to fibrogenesis in an in vivo setting.)

[0482] As shown in Figures 7A–7D, mice treated with this protocol typically show fibrosis by day 7 after BM treatment, and this developmental fibrosis progresses to severe fibrosis by day 14. The progression of the pathology then continues for another 2–5 days before spontaneously resolving by day 21.

[0483] More specifically, eight-week-old male C57BL6 mice (average body weight 22g–25g) obtained from Charles River were housed at room temperature (22°C) under a 12-hour light-dark cycle in pathogen-free conditions. The mice were placed on a folate-deficient diet (Envigo Teklad Global Rat Food Pellets) for one week, after which they were given BM or PBS infusions. Fresh water and the folate-deficient diet were provided freely. All animal procedures were approved by the Purdue Animal Experimentation Committee in accordance with the guidelines of the National Institutes of Health.

[0484] Next, the mice were anesthetized with ketamine / xylazine, their necks were shaved using depilatory lotion, and then sterilized with 70% alcohol. A small incision was made in the neck to visualize the trachea. The mice were positioned at a 75-degree angle and intratracheally injected with 100 μL of sterile PBS, or BM (Cayman Chemicals, Ann Arbor, MI, No. 13877) (0.75 mg / kg) dissolved in PBS, using a 1 cc syringe with a 26 G needle. Body weight was monitored every other day during the experiment.

[0485] To evaluate whether anti-inflammatory lung macrophages in these mice could be specifically targeted with folate-conjugated drugs, 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) of folate-conjugated near-infrared fluorescent dye (OTL38) was injected into the tail vein of BM-treated mice 10 days post-infusion, with or without a 200-fold excess of FA-glucosamine (a competitor of OTL38), and dye uptake in major organs was evaluated.

[0486] Two hours later, the mice were euthanized using CO2 asphyxiation, and an immediate incision was made in the skin from the abdomen to the neck to expose the lungs and trachea. A small incision was then introduced into the upper trachea for insertion of a 22-gauge blunt needle, and nylon sutures were tied around the trachea to seal it around the needle. The trachea (including the inserted needle), lungs, and heart were then removed together by carefully cutting the connective tissue below the lungs, and the bronchus of the left lung was secured with a Diefenbach duct clip. PBS was injected into the right lung, and the lavage solution was aspirated three times using a 1 ml syringe. The collected lavage solution was stored on ice.

[0487] Bronchoalveolar lavage fluid (BALF) was then analyzed to determine how targeted TLR7 agonists function. BALF samples were centrifuged at 1500 rpm for 5 minutes at 4°C, and aliquots of the supernatant were taken and stored at -80°C for cytokine / chemokine analysis. Cell pellets were resuspended in pre-warmed RPMI 1640 medium, cultured therefor for 2 hours, then washed three times with pre-warmed PBS, and collected for qPCR assay. The right lung was then tied with nylon thread and used for subsequent analysis of hydroxyproline content. The left lung was inflated with 1 ml of PBS using an inserted syringe and transferred to 10% formalin solution for subsequent histological analysis.

[0488] The right lung lobe collected as described above was weighed and placed in an airtight vial (Supelco Inc., Bellefonte, PA, No. 27003). Hydrolysis was performed in a sand bath at 120°C for 3.5 hours with 6N HCl (10 ml / g, v / w). The hydrolyzed solution was cooled at 4°C for 15 minutes, transferred to a 1.5 ml Eppendorf tube, and centrifuged at 12,000 rcf for 15 minutes at 4°C. The supernatant was carefully collected, aliquots were taken, and used for hydroxyproline (HYP) analysis.

[0489] For subsequent HYP analysis, 10 μl of the sample was transferred to a 96-well plate and neutralized with 10 μl of 5.3 M sodium hydroxide solution. Isopropanol (40 μl) was then added to each well, followed by 20 μl of oxidative buffer, and the mixture was incubated in a shaker at room temperature for 5 minutes. Analytical reagent (260 μl) was added, and the plate was incubated in a shaker at room temperature for 30 seconds, and immediately incubated at 60°C for 25 minutes. The absorbance was measured within 15 minutes at 560 nm (A). 560 Measurements were taken using [specific method / tool]. All reagents were prepared according to previously reported protocols.

[0490] For histological analysis of lung sections, fixed lungs (see above) were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E), Masson's trichrome, or F3 (anti-mouse FRβ antibody). Tissue sections were examined in a blinded manner by a qualified pathologist. 90 × 10 6 Cells exceeding 100 were quantified in each section using an Aperio-Image Scope (Leica Biosystems, Wetzlar, DE).

[0491] CCL18 and IL-1β were quantified in the supernatant of induced THP-1 cells using the Human DuoSet ELISA Development System (R&D Systems Europe, Abingdon, UK, No. DY394-05) and the IL-1 Beta Human ELISA Kit (Thermo Fisher Scientific, Waltham, MA, No. BMS224-2) as described by the manufacturers. BALF samples were analyzed for mouse IFN-γ using ELISA MAX® Deluxe (Biolegend, San Diego, CA, No. 430804).

[0492] Finally, for in vivo folate imaging studies, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were resected and imaged using an AMI live imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, mouse lungs were collected immediately after euthanasia and digested using a lung dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE, no. 130-098-427) as described in the gentleMACS Octo Dissociator with Heathers (Miltenyi Biotec, Bergisch Gladbach, DE, no. 130-096-427), and filtered through a 70 μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, DE, no. 130-098-462). Cells collected in the filtrate were depleted of red blood cells by ammonium sulfate lysis, washed twice in cold PBS, and labeled with antibodies against desired macrophage markers (FITC-CD11b, Biolegend, San Diego, CA, no. 101205; FE-F4 / 80, Biolegend, San Diego, CA, no. 123109) on ice for 30 minutes. The labeled macrophages were then washed twice in PBS, stained with 7AAD (a viability dye) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, San Jose, CA).

[0493] As shown in Figure 7A (upper panel), untreated lungs (PBS control column) and BM-treated lungs exhibit similar high-density alveoli interconnected by minimal extracellular matrix on day 7. In contrast, on day 14 after BM infusion, the size and frequency of alveolar sacs were significantly reduced, and the density of extracellular matrix was visibly increased, suggesting significant fibrosis development in treated mice. By day 21, the pathology in this model had already begun to resolve spontaneously, with a large number of mice eventually recovering from BM-induced trauma by day 35.

[0494] The basis for inflammation development up to day 7 was confirmed by the infiltration of FRβ-expressing macrophages (see the lower panel in Figure 7A and the quantification in Figure 7B), which are almost completely absent in healthy lungs but continue to accumulate until day 14 in BM-exposed lungs. Furthermore, staining with F3 showed significant expression of FRβ (primarily in the interstitial space) in IPF lungs, as previously reported in the literature (Figure 7A). FRβ expression was restricted to inflamed lungs (either IPF patients or BM-induced PF, not healthy lungs). In addition, FRβ-expressing macrophages were observed in mouse lungs on day 7 after BM administration, with maximum expression on day 14 (Figure 7B). These results support the previously reported FRβ expression on activated macrophages in inflamed lungs.

[0495] The ability to target these FRβ-expressing macrophages with folate-linked molecules was then demonstrated by the accumulation of the folate-targeting fluorescent dye OTL38 in the lungs of BM-treated mice after tail vein injection, but not in healthy mice. As shown in Figure 7B, OTL38 fluorescence was observed only in the kidneys of healthy mice (i.e., its primary excretion site), with little to no uptake into other tissues.

[0496] Figures 7C and 7D show FRβ IHC staining of human IPF lung tissue (Figure 7C) and healthy human lung tissue (Figure 7D). Eight-week-old C57BL / 6 male mice were fed a folate-deficient diet for one week, followed by BM or PBS infusion. Ten days after infusion, the mice were injected via the tail vein with 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) of OTL38, with or without a 200-fold excess of FA-glucosamine. After two hours, the mice were euthanized and analyzed. For in vivo folate imaging studies, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were resected and imaged using an AMI Live Imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, mouse lungs were collected immediately after euthanasia, digested, and then labeled with antibodies against desired macrophage markers (FITC-CD11b, PE-F4 / 80) and 7AAD (live / dead staining), and analyzed by flow cytometry.

[0497] Figure 7E shows images of various mouse tissues / organs, imaged with the folate receptor-targeted fluorescent dye OTL38, collected from mice with or without experimental fibrosis induced by BM (BM) or without (PBS control). These tissues were injected into the tail vein of healthy (column a) or BM-treated (columns b and c) mice 10 days after induction of fibrosis with 10 nmol of OTL38 in the absence (b) or presence (c) of a 200-fold excess of folate-targeted glucosamine (a competing reagent for FRβ and blocking OTL38 binding). The mice were euthanized 2 hours later for tissue excision and fluorescence imaging, and imaged with the folate receptor-targeted fluorescent dye OTL38. This demonstrates that the FA-targeted compounds of the present invention exhibit FRβ-specific binding without being taken up by other healthy tissues.

[0498] Tail vein injection of OTL38 into BM-treated mice resulted in significant accumulation not only in the kidneys (as described above) but also in the fibrous lungs (see Figure 7E). This pulmonary uptake was largely mediated by folate receptors, which can be demonstrated by the near-quantitative blockade of pulmonary accumulation when BM-treated mice were simultaneously injected with a 200-fold excess of folate-glucosamine (i.e., a competitive inhibitor of FRβ binding (see Figure 7E)). These data indicate that folate-targeting molecules selectively bind to cells expressing folate receptors in fibrous tissue without significant accumulation in other tissues of the body. In other words, FRβ-expressing macrophages can indeed be targeted with folate-linked molecules and, in clinical applications, can localize almost exclusively to fibrous tissue. Therefore, when using the targeting portion in the compounds of this disclosure, any TLR7 agonists that are not captured by the targeted fibrous (or cancerous) tissue will be minimal.

[0499] Next, to determine which cell types capture folate-pigment compounds in the lungs of BM-treated mice, lungs obtained from the above animals were digested with collagenase and tested by flow cytometry for cell-specific pigment uptake. Figure 7F shows data from FACS analysis obtained by in vivo labeling of such mice experiencing BM-induced experimental fibrosis, injected via tail vein with PBS (column 1) or 100 nmol of OTL38 in the absence (column 2) or in the presence (column 3) of folate-targeted glucosamine. As shown in Figure 7F, no macrophage-like cells showed any fluorescence when isolated from BM-treated mice that had not been injected with OTL38 (see column 1). In contrast, approximately 22% of macrophage-like cells obtained from fibrous mice injected with OLT38 showed significant retention of folate-targeted dye (Column 2), supporting the idea that OLT38 targets FRβ-positive macrophages in the inflammatory lung. Indeed, dye uptake is specifically mediated by folate receptors, as evidenced by the observation that simultaneous tail vein injection of a 200-fold excess of folate-glucosamine essentially blocked all folate-dye retention, indicating that dye accumulation required unoccupied folate receptors. Importantly, this conclusion is further supported by data showing that FRβ expression was essentially undetectable in untreated lungs (see Figure 7A) but dramatically increased during the development of fibrosis in BM-treated lungs (see Figures 7A-7D). FRβ expression was also markedly expressed in the lungs of human IPF patients. (Example 5)

[0500] Having established the ability to target bound drugs to fibrous macrophages expressing FRβ, we then investigated whether folate-targeted TLR7 agonists could suppress the signs and symptoms of fibrosis in BM-treated mice. To this end, BM-treated mice were intravenously injected with either a vehicle (3% DMSO in PBS) or compound 1B every other day, starting on day 10 (see Figure 8A). Compound 1A could not be similarly evaluated in vivo because the TLR7-54 agonist caused rapid weight loss followed by death (see Figures 9A and 9B). In BM-induced experimental pulmonary fibrosis in mice, inflammation is known to persist for approximately 9–10 days after BM infusion. Since the switch from inflammation to fibrosis occurs approximately 9–14 days in this model, and anti-inflammatory markers begin to appear around day 10, drug administration was started on day 10 (Figure 8A).

[0501] Two doses were administered every other day until day 21. Each dose within a day was spaced 6 hours apart to prevent any "tolerance" to the TLR agonist. The mice were then euthanized on day 21, immediately subjected to bronchoalveolar lavage, and subsequently resected for immunohistochemistry and quantification of collagen and hydroxyproline.

[0502] Figures 8B–8G show graph data representing the levels of various markers measured from mice treated with the BM model in Figure 8A. BALF cells were collected on day 21, centrifuged at 4°C, the resulting pellet was resuspended in culture medium, seeded in a 96-well plate, cultured for 2 hours, washed three times with pre-warmed PBS, and the cells were collected for qPCR. The data show that Arg1 (Figure 8B), MMP9 (Figure 8C), and TIMP 3 (Figure 8D) (e.g., anti-inflammatory markers) were all downregulated. CD86 (Figure 8E) and IFN-γ (Figure 8F) (e.g., pro-inflammatory markers) were both upregulated. Furthermore, IRAK-4, a negative regulator of TLR7 signaling, was upregulated (Figure 8G), and the number of BALF cells present was similarly upregulated (Figure 8H). In fact, the total number of mouse BALF cells decreased in a dose-dependent manner after treatment with different doses of compound 1B. The values ​​shown in Figures 8B to 8G represent the mean ± standard deviation for each group; * P<0.05, ** P<0.005, *** <0.0005; calculated by Student's t-test, with respect to physiological saline versus vehicle group, and treatment groups of compound 1A and compound 1B versus vehicle group, excluding BALF cell counts and protein concentration measurements calculated by Dunnett's multiple comparison test for the compound 1B treatment group and vehicle group. Vehicle = 3% DMSO in PBS.

[0503] As shown in Figures 8B–8D, qPCR analysis of anti-inflammatory markers in a macrophage subpopulation of bronchoalveolar lavage fluid cells revealed that tissue inhibitors of Arg1, MMP9, and TIMP3 were all elevated in BM-inducible mice compared to control mice. More importantly, parallel studies showed that the same anti-inflammatory markers were all suppressed when BM-inducible mice were treated with compound 1B, resulting in anti-inflammatory marker levels similar to those observed in healthy mice. Consistent with these data, quantitative analysis of pro-inflammatory markers revealed that the concentrations of CD86 transcript (qPCR) and IFN-γ (ELISA of lavage fluid) were both elevated after treatment with compound 1B (see Figures 7E and 7F). Together with the observation of upregulation of IRAK-4 (i.e., a marker of TLR activation, results shown in Figure 7G) and the dose-dependent decrease in the total number of BALF cells present after treatment with different doses of compound 1B (Figure 7H), these data demonstrate that administration of a folate-targeted TLR7 agonist can reprogram macrophages in vivo from an anti-inflammatory M2-like phenotype to an anti-fibrotic M1-like phenotype in the lungs of BM-treated mice. (Example 6)

[0504] Further studies were conducted to determine whether the reprogramming of the fibrous lung macrophages described above resulted in an actual improvement in the fibrous / anti-inflammatory condition in fibrous mice. Lung tissue obtained from the mice described above was embedded in paraffin, sectioned, and stained with H&E and Masson's trichrome to assess tissue density and extracellular collagen deposition, respectively.

[0505] Figures 9A and 9B show survival curves (Figure 9A) and weight changes (Figure 9B) of mice with experimental pulmonary fibrosis treated with untargeted and targeted TLR7 agonists. The data support that administration of the compounds of this disclosure (here, for example, compound 1B) increases the survival of BM-treated mice without causing significant weight loss. Each value represents the mean ± standard deviation for each group.

[0506] Figure 10A shows the hydroxyproline content (μg / lung) in lung tissue for the use of collagen deposition as a measure of fibrosis. Tissue at day 21 is shown for each of the following: healthy control (saline) (●), disease control (vehicle) (■), treatment with free drug TLR7 agonist (compound 1A) (inverted triangle), and treatment with folate-targeted TLR7 agonist (compound 1B) (triangle). BM-inducible mice treated with either 10 nmol of compound 1B (triangle) or compound 1A (inverted triangle) showed a significant decrease in total hydroxyproline content per lung compared to the vehicle control (■). Each value shown in Figure 9A represents the mean ± standard deviation for each group; * P<0.05, ** P<0.005, *** <0.0005; Saline solution vs. vehicle group, compound 1A and compound 1B treatment groups vs. vehicle group, as determined by Student's t-test.

[0507] Figures 10B and 10C show stained images of the lung tissue shown in Figure 10A, obtained by H&E staining (Figure 10B) and Masson's trichrome (collagen) staining (Figure 10C).

[0508] As shown in the H&E staining of the panel in Figure 10B, healthy lungs contain abundant alveolar sacs surrounded by a thin reticular membrane. In contrast, lungs induced with BM show far fewer alveoli and marked deposition of extracellular matrix where alveolar sacs once existed. Most importantly, BM-injected mice, initiated with compound 1B treatment on day 10, exhibit a lung architecture similar to that of healthy mice (Figure 10B), suggesting that targeting of fibrotic lung macrophages with compound 1A is effective in suppressing the main features of pulmonary fibrosis. This prevention of fibrosis actually involves blocking collagen deposition, as shown by Masson's trichrome staining of parallel lung sections (Figure 10C), and collagen staining is strongly suppressed in mice injected with compound 1B via the tail vein (Figure 10B). Thus, the data support that IPF mice treated with at least compound 1B (triangle) exhibit suppression of IPF pathology (e.g., fibrosis).

[0509] Finally, to confirm that compound 1B actually affected collagen production in vivo, hydroxyproline (a major component of collagen) was quantified in the total hydrolysate of affected lung tissue. More specifically, lung tissue obtained from the mice described above was perfused with PBS, hydrolyzed with acid, and analyzed for hydroxyproline content. As shown in Figure 10A, induction of fibrosis led to a significant increase in hydroxyproline content, and this increase was suppressed by treatment with compound 1B. Thus, the data support that treatment with the targeted TLR7 agonist compounds of this disclosure reduces (and even counteracts) collagen deposition, and therefore fibrosis, in vivo.

[0510] In summary, overall survival in mice injected with the optimized BM dose (0.75 mg / kg) was significantly improved by treatment with compound 1B, while compound 1A showed no survival benefit but demonstrated significant weight loss (over 25%, Figure 7). The free drug performed better in terms of reducing hydroxyproline content, but the observed lower survivability may be due to overall toxicity (i.e., weight loss, see Figure 7B). This was not surprising, as systemic administration of TLR7 agonists is known to cause toxicity. (Example 7)

[0511] The use of untargeted TLR7 agonists to treat IPF (or other fibrotic diseases) has been contraindicated by the resulting systemic activation of the immune system and the toxicity it causes. Therefore, we evaluated whether any apparent toxicity could have been associated with systemic administration of compound 1B in mice. To this end, BM-inducible mice were treated with 0, 1, 3, or 10 nanomoles of compound 1B every other day, starting on day 10. Body weight, pulmonary hydroxyproline content, and histological analysis were performed on day 21. Unlike conventional systemic administration, targeted drugs not only improved survival but also reduced weight loss, highlighting the importance of a targeted approach (Figures 11A and 11B).

[0512] Figure 12 shows data on the dose-dependent effect of folate-targeted TLR7 agonists on the suppression of fibrosis in BM-inducible mice, using collagen deposition as a measure of fibrosis. The data are represented as follows: healthy control (PBS, ●), BM-inducible mouse, treated with vehicle (■), treated with 1 nmol of compound 1B (○), treated with 3 nmol of compound 1B (□), or treated with 10 nmol of compound 1B (triangle)); Part A shows graph data related to body weight over time in BM-inducible mice; Part B shows measurements of hydroxyproline content in lung tissue treated with different doses (10 nmol, 3 nmol, or 1 nmol of compound 1B) (μg / lung); and Part C shows images of histological analysis of right lung tissue after H&E staining and trichrome staining.

[0513] As shown in Figure 11B and Part A of Figure 12, no difference in weight loss was observed among mice treated with 0, 1, 3, or 10 nanomoles of compound 1B, suggesting that repeated administration of the compound did not induce significant toxicity. The fact that these treatments still possessed the expected effect against pulmonary fibrosis could be understood from the comparison of hydroxyproline content of various lung hydrolysates, with the order of effectiveness being 10 nmol / mouse > 3 nmol / mouse > 1 nmol / mouse > 0 nmol / mouse (Parts B and C of Figure 12). More importantly, detailed analysis of lung histology showed that lung histology improved as the dose of compound 1B increased, suggesting that the tissues that were most potently enriched with the TLR7 agonist were, in fact, the tissues with the most normal microscopic morphology. In summary, these data support the idea that targeting TLR7 agonist FRβ+ macrophages in fibrous / anti-inflammatory tissues can effectively prevent fibrosis (i.e., the M2 phenotype) without the systemic activation of the immune system that typically limits the use of TLR7 agonists in humans.

[0514] Finally, to determine whether this pro-inflammatory effect could be achieved at lower doses, therapeutic studies were conducted using two lower doses (3 nmol / kg and 1 nmol / kg) (Figures 9 and 10). Interestingly, while the lower doses showed a significant reduction in hydroxyproline content and collagen deposition levels, the best survival rate was achieved at a dose of 10 nmol. (Example 8)

[0515] Other representative embodiments of the compounds described herein were tested in in vitro studies.

[0516] Figures 13A–13D show graph data representing the levels of various markers measured from human THP-1 cells induced into M2 macrophages with 20 ng / mL IL-4, 20 ng / mL IL-13, and 5 ng / mL IL-6. The cells were then reprogrammed for 48 hours with TLR7 agonists (e.g., compound 2A) of different nM concentrations, and collected for gene analysis by qPCR. The mRNA levels of the following markers—CCL18 mRNA level (Figure 12A), IL-1β mRNA level (Figure 13B), and TNFα level (Figure 13C)—were compared to the expression levels of M2-like macrophage controls. Figure 13D shows the results of protein analysis after collection of the cell supernatant. The secreted CCL18 protein was detected by ELISA.

[0517] In Figures 13A to 13D, the agonist compounds of this disclosure having formula IV (e.g., compound 2A) were evaluated for their ability to reprogram M2-like macrophages into M1-like macrophages.

[0518] Primarily, human monocyte (THP-1) cells were induced into the M2-like phenotype using the methods and materials described above. Specifically, THP-1 cells were seeded in 96-well plates at a density of 60,000 cells / well. The cells were differentiated into non-polarized macrophages by 48-hour incubation with 200 nM PMA, followed by 24-hour incubation in fresh RPMI medium. The resulting macrophages were polarized into the M2-like phenotype by 48-hour incubation with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / mL IL-6. The cultures were maintained at 37°C in a humidified 5% CO2 incubator.

[0519] To evaluate whether compound 2A can reprogram anti-inflammatory macrophages into a low-fibrillation / pro-inflammatory phenotype, THP-1 cells stimulated with IL-4, IL-6, and IL-13 were incubated with different concentrations of compound 2A, and mRNA levels of several anti-inflammatory markers, namely CCL18, IL-1β, and TNFα, were tested using qPCR and ELISA.

[0520] As shown in Figures 13A and 13B, incubation with compound 2A (free drug) for 48 hours induced a decrease in the expression of CCL18 and IL-1β, suggesting that the TLR7 agonist can indeed promote the shift of these anti-inflammatory polarized THP-1 cells to a low-fibrinogenic / pro-inflammatory phenotype. (Note that Figure 13B shows a bell-shaped curve indicating that compound 2A has an inhibitory response at low concentrations and a stimulative response at high concentrations, which is a response curve common to certain drugs.) Furthermore, when the expression of TNFα (a marker for pro-inflammatory phenotype) was tested, an increase in its expression was observed (Figure 13C), confirming that a shift from anti-inflammatory to pro-inflammatory properties had occurred in THP-1.

[0521] In addition to the unconjugated TLR7 agonists, the conjugated compounds of this disclosure were similarly evaluated. Human THP-1 cells were induced into macrophages with an M2-like phenotype according to the method described herein (e.g., using 20 ng / mL of IL-4, 20 ng / mL of IL-13, and 5 ng / mL of IL-6), and then reprogrammed for 2 hours with various compounds of the present disclosure at different nM concentrations, namely, unconjugated (free drug) TLR7 agonist compounds having formula I and / or II (data are collectively shown as compound 3A), TLR7 agonist compounds conjugated to folate having formula XV (with a releasing linker) (e.g., compound 3B), TLR7 agonist compounds conjugated to folate having formula XVII (with a non-releasing linker) (e.g., compound 3C), and TLR7 agonist compounds conjugated to folate having formula XVI (with a non-releasing linker) (e.g., compound 3D). The cells were then collected for gene analysis by qPCR, and the relative expression of CCL18 (Figure 14A), CD206 (Figure 14B), and IL-1β (Figure 14C) was analyzed.

[0522] The expression of various anti-inflammatory (M2 phenotype) markers, CCL18, IL-1β, and CD206, was quantified. As shown in Figures 14A-14C, the expression of each of these anti-inflammatory markers was reduced after administration of compound 3B, compound 3D, and compound 3C, respectively, with compounds 3D and 3C (both containing non-releasing linkers) being the most effective compared to the other compounds.

[0523] Figure 15 shows the levels of secreted CCL18 protein in each of the THP-1 cell populations shown in Figures 14A–14C after treatment with compound 3A, compound 3B, compound 3C, or compound 3D. Compound 3A and folate-targeted TLR7 compounds (e.g., compounds 3B, 3C, and 3D) downregulate CCL18 secretion in the low concentration range (0.1–10 nM).

[0524] Additionally, cell supernatant was collected and the secreted CCL18 protein was detected by ELISA. Figure 15 confirms that compound 3A (free drug) and the folate-targeting compounds (compounds 3B, 3C, and 3D) all downregulated CCL18 secretion in the low concentration range (0.1–10 nM), further supporting the idea that these compounds can reprogram M2-like anti-inflammatory macrophages into M1-like pro-inflammatory macrophages through a similar mechanism, similar to the examples described in relation to compounds 1A and 1B. (Example 9)

[0525] Repeating the above studies (see Figures 3A–3F, gray bars), the same qualitative changes were observed, except that the magnitude of the effect of compound 1B was somewhat reduced. This reduction in potency was predicted to be because the untargeted TLR7 agonist enters cultured cells immediately, while its folate-targeted counterpart is designed to enter cells only after folate receptor binding and receptor-mediated endocytosis. Since low molecular weight water-soluble drugs such as compounds 1A and 1B are often eliminated from the body within two hours of injection, in a more physiologically relevant in vitro model of drug exposure in vivo, the incubation of cells and drugs is limited to only two hours, followed by further incubation for 46 hours in the absence of the drug before testing the efficacy of the drug. As shown in Figures 4A-4E, when THP-1 cells were incubated with a TLR7 agonist for 2 hours, and then the drug-containing medium was replaced with a drug-free medium, compound 1B was observed to have superior efficacy compared to compound 1A, and in particular, TNFα induction was dramatically improved by the folate-targeting compound. This is most likely because the folate-targeting TLR7 agonist was captured by folate receptor-positive cells, while compound 1A was not retained by the same cells.

[0526] These data support the idea that compound 1B is more effective in reprogramming anti-inflammatory macrophages in vivo, and that folate-conjugated drugs (e.g., compound 1B) also have the added advantage of being enriched in FRβ-expressing macrophages and therefore unable to enter folate receptor-negative cells, which are dominant throughout the body, thus resulting in lower systemic toxicity (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0527] Furthermore, to ensure that the above mRNA analysis accurately reflected the levels of anti-inflammatory cytokines produced by THP-1 cells stimulated with IL-4, IL-6, and IL-13, the concentrations of CCL18 and IL-1β polypeptides in the THP-1 supernatant were quantified by ELISA assay. As shown in Figures 6A and 6B, both compound 1A and compound 1B induced reductions in CCL18 and IL-1β when incubated continuously with the agonist for 48 hours; however, compound 1B was found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D). (Example 10)

[0528] Figure 16 illustrates an in vivo study technique for at least one embodiment of the compound of this disclosure, a compound having formula XVII (e.g., compound 3C), in a BM mouse model. Figures 17A and 17B are LC-MS spectra of compound 3C, confirming high purity of the compound and showing no detection of free drug.

[0529] Figures 18A–18F show the results obtained from the in vivo study mice described in Figure 16, including survival curves (Figure 18A), body weight changes (Figures 18B and 18D), concentration of cells with BALF (Figure 17C), hydroxyproline concentration (μgHP / lung lobe) in live mice (Figure 18E), and hydroxyproline concentration (μgHP / lung lobe) in all mice (e.g., including both live mice and those that died before day 21) (Figure 18F). A 10 nmol dose of the compound of formula XVII (e.g., compound 3C) increased the survival rate of the study mice, but simultaneously decreased the number of HP and BALF cells. Furthermore, a 3 nmol dose did not show any measurable benefit to the study mice. (Example 11)

[0530] M2-induced human monocyte-derived macrophages were treated with 100 nM of compound 1A or compound 1B for 48 consecutive hours, or for the first 2 hours in or without FA-glucosamine (competitive), followed by 46 hours in the absence of the drug (2+46 hours). mRNA levels of the anti-inflammatory markers Arg1 (Figure 19A), CD206 (Figure 19B), and CD163 (Figure 19C), as well as protein levels of the secreted anti-inflammatory CCL18 (Figure 19D) and pro-inflammatory cytokines CXCL10 (Figure 19E) and IL-6 (Figure 19F) (n=3, technical replicas), were then assessed, as shown in Figure 19. Changes in any of the cytokine sets were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46 hours, competitive). This data supports the idea that compound 1B binds to the folate receptor because the downregulation of the biomarker was blocked by an excess amount of FA-glucosamine (a competing substance). (Example 12)

[0531] Healthy mice were injected via tail vein with 10 nmol of compound 1A (circular) or compound 1B (square), and peripheral blood was collected at the indicated time points after drug injection (Figures 20A-C). Plasma IL-6 (Figure 20A), IFNα (Figure 20B), and TNFα (Figure 20C) were measured (n=3) (Figures 20D-F). The effect of drug concentrations on plasma levels of IL-6 (Figure 20D), IFNα (Figure 20E), and TNFα (Figure 20F) was assessed at 1.5 hours, 1 hour, or 1 hour after treatment, respectively (n=2) (Figure 20G). Compound 1A stimulated systemic cytokine release in healthy mice, while compound 1B did not. Furthermore, compound 1B stimulated less inflammatory cytokine release than half the dose of compound 1A. These data suggest that TLR7 agonists, when targeted to lung macrophages by folate receptor-targeting ligands, can be safely used to reprogram fibrotic lung macrophages into an anti-fibrotic / pro-inflammatory state. (Example 13)

[0532] Sections from the same healthy and fibrous lungs, shown in Figure 6, were stained with DAPI (nucleus, blue), anti-F4 / 80 (macrophage, red), and anti-CD206 (M2 macrophage marker, green), and images were obtained using a Leica Versa 8 full-slide scanner as described in the methods (n=2). Scale bar, 100 μm. Differences between treatment groups indicate that compound 1B produces potent anti-fibrotic and pro-inflammatory responses in vivo. (Example 14)

[0533] To evaluate the efficacy of TLR-7 agonists, compound 1 (TLR7-1A), compound 2 (TLR7-1B), and compound 3 (TLR7-1C) were treated with peripheral blood mononuclear cells (PBMCs) for 24 hours. TLR7-1 was used as a control. Cell culture supernatants were isolated and tested for IL-6 using enzyme-linked immunosorbent assay (ELISA) (Figure 22). As shown in Figure 22, TLR7 agonists resulted in increased IL-6 expression in PBMCs.

[0534] When these compounds were treated with human primary monocyte-derived M2 macrophages for 48 hours, they more efficiently induced IL-6 and CXCL-10 compared to the parent compound TLR7-1 (Figures 23A and 23B). Compounds 1, 2, and 3 polarize M2 macrophages into M1 macrophages, as indicated by the increase in M1 markers IL-6 (Figure 3A) and CXCL10 (Figure 23B). (Example 15)

[0535] Healthy mice were injected via tail vein with 10 nmol of compound A (TLR-1) or compound 1 (TLR-1A), and peripheral blood was collected at the indicated time points after drug injection (Figures 23C and 23D). The effects of the drugs on plasma levels of IL-6 (Figure 23C) and TNFα (Figure 23D) were assessed 1 or 1.5 hours after treatment. Both compounds stimulated systemic cytokine release in healthy mice. (Example 16) Combination therapy of folate-TLR7-1A (compound 1000) and checkpoint inhibitors in a 4T1 tumor model

[0536] Six-to-eight-week-old female BALB / c mice were purchased from Charles River Laboratories, housed in a sterile environment under a standard 12-hour light-dark cycle, and maintained on a folate-deficient diet. Upon arrival, the mice were transferred to the folate-deficient diet.

[0537] On the 14th day, the mice were given 5.0 × 10 4 Individual 4T1 cells were implanted orthotopically, and the tumor was reduced to approximately 50 mm. 3 The cells were grown until they reached a certain size. 4T1 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured as monolayers in 1640 RPMI supplemented with 10% thermoinactivated fetal bovine serum and 1% penicillin-streptomycin at 37°C under a 5% CO2 / 95% humidified air atmosphere. The study was performed on cells thawed from frozen stocks and passaged 10 times.

[0538] Mice were then randomized into groups based on their tumor volume and treated with folate-TLR7-1A (compound 1000), folate-TLR7-1A (compound 1000) + CKI, or CKI. In all experiments, folate-TLR7-1A (compound 1000) was administered by daily intravenous injection for 5 days per week (100 μL, 3 nanomoles / mouse), and checkpoint inhibitor (CKI) antibodies, anti-PD-1 and anti-CTLA-4, were administered intraperitoneally (ip) at a dose of 200 μg / mouse on days 3, 6, 10, 13, 17, and 20. The control group received PBS on the same schedule.

[0539] The tumor volume is calculated using the formula (a × b 2 The tumors were simultaneously measured with calipers using ) / 2 (where a is the largest diameter of the tumor and b is the smallest diameter), and subsequently digested using a mouse tumor dissociation kit (catalog no. 130-096-730, Miltenyi Biotech, Bergisch Gladbach, DE) according to the manufacturer's protocol. The digested tumor cells were resuspended in 1× RBC lysis buffer (catalog no. 420301, BioLegend, San Diego, CA) for 10 minutes, washed, filtered through a 70 mm filter, and resuspended in 2% FBS in PBS to obtain a single-cell suspension. The resulting single-cell suspension was stained for antibody, and the samples were then analyzed by flow cytometry. The results were plotted using GraphPad prism software, and immunohistochemistry was performed on post-treatment 4T1 tumor tissue (Figures 24A-24D-26). In addition, changes in mouse body weight during combination therapy were monitored (Figure 32A). (Example 17) Combination therapy of folate-TLR7-1A (compound 1000) and checkpoint inhibitors in an MC38 tumor model

[0540] Six-to-eight-week-old male C57BL / 6 mice (Jackson Laboratory, Bar Harbor, ME) were housed in a sterile environment under a standard 12-hour light-dark cycle and maintained on a folate-deficient diet. Upon arrival, the mice were transferred to a folate-deficient diet (TD.95247, Envigo Ltd., Indianapolis, IN).

[0541] MC38 cell lines were cultured at 37°C in a humidified environment of 5% CO2 in DMEM containing sodium pyruvate and L-glutamine, supplemented with 10% FBS, 1% non-essential amino acid (NEAA) cell culture adjuvant, 1% penicillin / streptomycin, gentamicin, and 1% 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The study was performed on cells that had been passaged 10 times from thawed frozen stocks.

[0542] On day 14, 500,000 MC38 cells were suspended in 100 μl of PBS and subcutaneously injected into the right flank of mice. The tumor volume was approximately 50 mm². 3 Upon reaching a certain tumor size, mice were randomly divided into groups based on their tumor volume and treated with folate-TLR7-1A (compound 1000), folate-TLR7-1A (compound 1000) + anti-PD-1, and anti-PD-1 antibody. The anti-PD-1 antibody was administered intraperitoneally (ip) at a dose of 100 μg / mouse on days 3, 5, 10, and 12. The control group received PBS on the same schedule. Figure 27 outlines the research methodology.

[0543] The tumor volume is calculated using the formula (a × b 2The tumors were simultaneously measured with calipers using ) / 2 (where a is the largest diameter of the tumor and b is the smallest diameter), and subsequently digested using a mouse tumor dissociation kit (catalog no. 130-096-730, Miltenyi Biotech, Bergisch Gladbach, DE) according to the manufacturer's protocol. The digested tumor cells were resuspended in 1× RBC lysis buffer (catalog no. 420301, BioLegend, San Diego, CA) for 10 minutes, washed, filtered through a 70 mm filter, and resuspended in 2% FBS in PBS to obtain a single-cell suspension. The resulting single-cell suspension was stained for antibody, and the samples were then analyzed by flow cytometry. The results were plotted using GraphPad prism software (Figures 28 and 29A).

[0544] Subsequently, 500,000 MC38 cells in 100 μl of PBS were subcutaneously injected into the left flank of both healthy and cured mice derived from the aforementioned experiment. Tumor volume was calculated using the formula (a × b 2 The diameters were simultaneously measured using a caliper with ) / 2 (where a is the largest diameter of the tumor and b is the smallest diameter) and plotted using GraphPad prism software (Figure 29B). In addition, changes in mouse body weight during combination therapy were monitored (Figure 32B). (Example 18) Combination therapy of folate-TLR7-1A and checkpoint inhibitors in an LL / 2 Lewis lung cancer tumor model

[0545] The LL / 2 (Lewis lung) cancer model was then evaluated. The 5-year survival rate for Lewis lung cancer is only about 20%, which is considerably lower than that of other cancers. If lung cancer is detected early, the 5-year survival rate rises to about 56%, but only about 16% of lung cancers are detected early enough to achieve this increase in survival rate. For detection at a later stage, the 5-year survival rate can be as low as 5%. Therefore, identifying effective and non-toxic treatments for Lewis lung cancer would be extremely beneficial.

[0546] Six-to-eight-week-old male C57BL / 6 mice (Jackson Laboratory, Bar Harbor, ME) were housed in a sterile environment under a standard 12-hour light-dark cycle and maintained on a folate-deficient diet. Upon arrival, the mice were transferred to a folate-deficient diet.

[0547] LL / 2(LLC1) Lewis lung cancer cells were cultured at 37°C in a humidified environment with 5% CO2 in DMEM containing sodium pyruvate and L-glutamine, supplemented with FBS, glucose, penicillin / streptomycin, and sodium bicarbonate. The cells were cultured in a 75 cm³ environment. 2 The cells were maintained in tissue culture flasks. The study was conducted using cells that had been passaged 10 times after being thawed from frozen stock.

[0548] On day 14, 500,000 LL / 2 cells were suspended in 100 μl of PBS and subcutaneously injected into the right flank of mice. The tumor volume was approximately 50 mm². 3 Upon reaching a certain tumor size, mice were randomly divided into groups based on their tumor volume and treated with folate-TLR7-1A (compound 1000), folate-TLR7-1A (compound 1000) + anti-CTLA-4, and anti-CTLA-4 antibody. Anti-CTLA-4 antibody was administered intraperitoneally (ip) at a dose of 200...

Claims

1. A method for treating a subject with cancer, A step of administering the first treatment, wherein the first treatment is At least one compound or a pharmaceutically acceptable salt or hydrate thereof comprising a radical of an immunomodulator conjugated via a linker to a folate ligand or a functional fragment or analog thereof, or A composition comprising at least one of the aforementioned compounds and one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles, or a combination thereof. Steps including, A step of administering a second treatment to the subject, wherein the second treatment comprises one or more immune checkpoint inhibitors. Methods that include...

2. The method according to claim 1, wherein the step of administering the second treatment to the subject further comprises administering a therapeutically effective amount of one or more immune checkpoint inhibitors.

3. The method according to claim 1, wherein by administering at least one compound of the first treatment, the target M2 macrophages are reprogrammed into M1 macrophages, and the efficacy of the one or more immune checkpoint inhibitors of the second treatment is enhanced compared to the baseline efficacy of the one or more immune checkpoint inhibitors.

4. The method according to claim 1, wherein the radical of the immunomodulator or a pharmaceutically acceptable salt or hydrate thereof comprises a Toll-like receptor (TLR) 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR 9 agonist, or a TLR 7 / 8 agonist.

5. The above at least one compound is of the formula: 【Chemistry 187】 Having or a pharmaceutically acceptable salt or hydrate thereof, The method according to claim 1.

6. The above at least one compound has the following formula: 【Chemical 188】 Having or a pharmaceutically acceptable salt or hydrate thereof, The method according to claim 1.

7. The radical of the immunomodulator is a TLR agonist of formula X or XX, or contains a TLR agonist of formula X or XX, or is a pharmaceutically acceptable salt or hydrate of formula X or XX. 【Chemical 189】 【Chemistry 190】 In equations X and XX, R １ is, -NH ２ or -NH-R １Ｘ And, R ２ H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R ２Ｘ , -O-R ２Ｘ , -S-R ２Ｘ , 【Chemistry 191】 And, 【Chemistry 192】 It is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle, In formula X, R ３ is -OH, -SH, -NH ２ or -NH-R １Ｘ and In equation XX, X is either CH or N. R １Ｘ , R ２Ｘ , and R ２Ｙ Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. The method according to any one of claims 1 to 4.

8. The at least one compound of the first treatment described above is 【Chemistry 193】 It contains, or a pharmaceutically acceptable salt or hydrate thereof, in formula 2-I, R １ , R ３ , R ４ , and R ５ These are, independently, hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, and -COR. ２ｘ , 【Chemistry 194】 And, R ２ は、H、-OH、-NH ２ 、-NHR ２ｘ N ３ 、-NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ 、-NH-CS-NH ２ 、 【Chemistry 195】 And, Y is a binding site of the at least one compound to the linker and / or targeting ligand, and is H, -OH, -NH ２ , - NHR ２ｘ , -O-R ２Ｘ , -SO-R ２ｘ -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ NH ２ Alkenyl, Alkinyl, Alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ NH ２ , -NH-CS-NH ２ , 【Chemistry 196】 Includes, R ２ｘ and R ２ｙ Each of these is H, -OH, and -CH. ２ -OH, -NH ２ ien-CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ Independently selected from the group consisting of alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, each R ２ｚ is, -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ , and -COR ２ｑ Independently selected from the group consisting of, R ２ｑ and R ２ｑ’ Each of them is independently an alkyl group or H group. 【Chemistry 197】 It is a non-aromatic monocyclic or bicyclic 3- to 10-membered nitrogen-containing heterocycle, In Equation 2-I, X １ , X ２ , and X ３ Each of these independently, CR ｑ or N, and each R ｑ These are independently H, halogens, or optionally substituted alkyl groups. In Equation 2-I, n is between 0 and 30, and m is between 0 and 4. The method according to claim 1.

9. The method according to any one of claims 1 to 6 and 8, wherein the subject is experiencing or at risk of experiencing cancer or a recurrence of cancer, and the step of administering the first treatment further comprises administering or applying to the subject a therapeutically effective amount of the at least one compound or a pharmaceutically acceptable salt or hydrate thereof.

10. The method according to any one of claims 1 to 6 and 8, wherein the first treatment is administered to the subject intravenously, orally, intramuscularly, intraperitoneally, locally, or by inhalation.

11. The method according to claim 3, wherein the target M2 type macrophage is a bone marrow-derived suppressor cell (MDSC), a tumor-associated macrophage (TAM), or both MDSC and TAM.

12. The method according to any one of claims 1 to 6, 8, and 11, wherein the subject has a tumor positive for programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

13. The method according to any one of claims 1 to 6, 8, and 11, which is performed to treat cancer recurrence or resistance to checkpoint blocker therapy in the subject.

14. The method according to any one of claims 1 to 6, 8, and 11, wherein the subject is a human, a mouse, or any other mammal.

15. The method according to claim 1, wherein the one or more immune checkpoint inhibitors of the second treatment each comprise a small molecule or other agent that destroys an immune checkpoint on the target cell.

16. The method according to any one of claims 1 to 6, 8, 11, and 15, wherein the one or more immune checkpoint inhibitors of the second treatment each comprise a small molecule or other agent that destroys an immune checkpoint on the target cell.

17. The one or more immune checkpoint inhibitors of the second treatment are pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, isrelizumab, a combination of osiperlimab and isrelizumab, BMS-936559 / MDX-1105, MPD The method according to any one of claims 1 to 6, 8, 11, and 15, wherein each is independently selected from the group consisting of L3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab.

18. The radical of the immunomodulator is given by the following formula: 【Chemistry 198】 Includes a TLR agonist having, During the ceremony, R １ It is an amine group, R ２ It is a single bond -NH-, R ３ is H, alkyl, hydroxyl, or any other substituted group thereof. X is CH ２ , NH, O, or S, The linker is R １ , R ２ , or R ３ Join, The method according to any one of claims 1 to 6, 8, 11, and 15.

19. The method according to any one of claims 1 to 6, 8, 11, and 15, wherein the linker is a non-emission linker.

20. The method according to any one of claims 1 to 6, 8, 11, and 15, wherein the linker is a release linker.

21. The method according to any one of claims 1 to 6, 8, 11, and 15, wherein the linker of the at least one compound or a pharmaceutically acceptable salt or hydrate thereof of the first treatment comprises a polyethylene glycol (PEG) linker or a PEG derivative linker and is a non-releasing linker.

22. The one or more immune checkpoint inhibitors of the second treatment are PD-1, PD-L1, CTLA-4, T cell activation V-domain Ig suppressor (VISTA), lymphocyte activation 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immune receptor having Ig and ITIM domains (TIGIT), programmed death ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase-1 (AGR1), B7 family inhibitory ligand B7-H3 ( The method according to any one of claims 1 to 6, 8, 11, and 15, which inhibits a cellular immune checkpoint selected from the group consisting of B7-H3), B7 family inhibitory ligand B7-H4 (B7-H4), 2B4 (differentiation cluster 244), B and T lymphocyte attenuation factor (BTLA), adenosine A2A receptor (A2aR), and / or members of the killer cell immunoglobulin-like receptor (KIR) family, e.g., KIR and C-type lectin receptors, and signaling factors and transcriptional activators (STAT3).

23. The method according to any one of claims 1 to 6, 8, 11, and 15, wherein the first and second treatments are administered simultaneously or sequentially in either order.

24. The method according to any one of claims 1 to 23, wherein the step of administering the at least one compound of the first treatment or a pharmaceutically acceptable salt or hydrate thereof activates antitumor cells or an anti-inflammatory signaling cascade in the subject.

25. A method for enhancing the efficacy of one or more immune checkpoint inhibitors administered to a target, The steps include administering to the subject one or more compounds containing a radical of an immunomodulator bound to a folate ligand or a functional fragment or analog thereof via a linker, or a pharmaceutically acceptable salt or hydrate thereof, The steps include: contacting the target cells of the subject with one or more compounds or their pharmaceutically acceptable salts or hydrates to reprogram the M2 macrophages of the subject into M1 macrophages; Methods that include...

26. The method according to claim 25, wherein the radical of the immunomodulator is a Toll-like receptor (TLR) 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR 9 agonist, or a TLR 7 / 8 agonist.

27. The method according to claim 25, wherein the radical of the immunomodulator is or comprises a TLR7 agonist, and the linker is a release linker.

28. The method according to claim 25, wherein the radical of the immunomodulator is or comprises a TLR7 agonist, and the linker is a non-releasing linker.

29. The one or more compounds mentioned above are of the formula: 【Chemistry 199】 It is either or contains it, or a pharmaceutically acceptable salt or hydrate thereof. The method according to claim 25.

30. The method according to claim 25 or claim 26, wherein the linker is a non-ejection linker.

31. The method according to any one of claims 25 to 27 and 29, wherein the cancerous disease state includes cancer recurrence.

32. The method according to claim 24, wherein the antitumor cells are T cells, macrophages, or both T cells and macrophages.

33. A combination for use in the treatment of cancer in the subject, The first type of medicine, At least one compound or a pharmaceutically acceptable salt or hydrate thereof comprising a radical of an immunomodulator conjugated via a linker to a folate ligand or a functional fragment or analog thereof, wherein the radical of the immunomodulator targets a pattern recognition receptor or a damage-associated molecular pattern (DAMP), or A composition comprising at least one of the compounds or a pharmaceutically acceptable salt or hydrate thereof, and one or more pharmaceutically acceptable carriers, adjuvants, diluents, additives, and / or vehicles. The first pharmaceutical product, including, A second pharmaceutical product comprising one or more immune checkpoint inhibitors and A combination that includes this.

34. The combination according to claim 33, wherein the radical of the immunomodulator comprises a Toll-like receptor (TLR) 3 agonist, a TLR 7 agonist, a TLR 8 agonist, a TLR 9 agonist, or a TLR 7 / 8 agonist.

35. At least one compound has the following formula: 【Chemistry 200】 Having or a pharmaceutically acceptable salt or hydrate thereof, The combination described in claim 33.

36. The radical of the immunomodulator comprises a TLR agonist of formula X or XX, or a pharmaceutically acceptable salt or hydrate of formula X or XX. 【Chemical Engineering 201】 In equations X and XX, R １ is, -NH ２ or -NH-R １Ｘ And, R ２ H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R ２Ｘ , -O-R ２Ｘ , -S-R ２Ｘ , 【Chemical Engineering 202】 And, 【Chemical 203】 It is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle, In equation X, R ３ -OH, -SH, -NH ２ , or -NH-R １Ｘ And, In equation XX, X is either CH or N. R １Ｘ , R ２Ｘ , and R ２Ｙ Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. The combination described in claim 33.

37. The at least one compound of the first pharmaceutical is 【Chemical 204】 It contains, or a pharmaceutically acceptable salt or hydrate thereof, In Equation 2-I, R １ 、R ３ 、R ４ 、and R ５ are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR ２ｘ 、 【Chemical 205】 And, R ２ は、H、-OH、-NH ２ 、-NHR ２ｘ N ３ 、-NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ 、-NH-CS-NH ２ 、 【Chemical 206】 And, Y is the linker and / or targeting ligand binding site of the at least one compound, and is H, -OH, -NH ２ , - NHR ２ｘ , -O-R ２Ｘ , -SO-R ２ｘ -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ NH ２ Alkenyl, Alkinyl, Alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ NH ２ , -NH-CS-NH ２ , 【Chemical 207】 Includes, R ２ｘ and R ２ｙ Each of these is H, -OH, and -CH. ２ -OH, -NH ２ ien-CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ Independently selected from the group consisting of alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, each R ２ｚ is, -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ , and -COR ２ｑ Independently selected from the group consisting of, R ２ｑ and R ２ｑ’ Each of them is independently an alkyl group or H group. 【Chemical 208】 is a non-aromatic monocyclic or bicyclic 3- to 10-membered N-containing heterocycle, and in formula 2-I, X １ , X ２ , and X ３ Each of these independently, CR ｑ or N, and each R ｑ These are independently H, halogens, or optionally substituted alkyl groups. In Equation 2-I, n is between 0 and 30, and m is between 0 and 4. The combination described in claim 33.

38. The radical of the immunomodulator of the first pharmaceutical product comprises a TLR agonist having the following formula or a pharmaceutically acceptable salt thereof, 【Chemical Engineering 209】 During the ceremony, R １ It is an amine group, R ２ It is a single bond -NH-, R ３ is H, alkyl, hydroxyl, or any other substituted group thereof. X is CH ２ , NH, O, or S, The linker is R １ , R ２ , or R ３ Join, The combination described in claim 33.

39. The at least one compound of the first pharmaceutical is of the formula: 【Chemical 210】 It is either or contains it, or a pharmaceutically acceptable salt or hydrate thereof. The combination described in claim 33.

40. The combination according to claim 33, wherein the linker of the at least one compound of the first pharmaceutical or a pharmaceutically acceptable salt or hydrate thereof is a release linker.

41. The combination according to claim 33, wherein the linker of the at least one compound of the first pharmaceutical or a pharmaceutically acceptable salt or hydrate thereof is a non-releasing linker.

42. The combination according to claim 33, wherein the linker of the at least one compound of the first pharmaceutical or a pharmaceutically acceptable salt or hydrate thereof comprises a polyethylene glycol (PEG) linker or a PEG derivative linker and is a non-release linker.

43. The combination according to any one of claims 33 to 42, wherein the subject has a tumor positive for programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

44. The combination according to claim 33, wherein the one or more immune checkpoint inhibitors of the second pharmaceutical product include a small molecule or other agent that destroys the immune checkpoint of the target cell.

45. The one or more checkpoint inhibitors of the second pharmaceutical product are pembrolizumab, nivolumab, ipilimumab, semiprimab, atezolizumab, avelumab, durvalumab, pidilizumab, monoclonal antibody MEDI-0680, monoclonal antibody REGN2810, or PD-1 targeting fusion protein AMP-224, osiperlimab, islerizumab, a combination of osiperlimab and islerizumab, BMS-936559 / MDX-1105, MPD A combination according to any one of claims 33 to 42 and 44, each independently selected from the group consisting of L3280A / RG7446 / atezolizumab, MSB0010718C / avelumab, or MEDI4736 / durvalumab, tilagolumab, zimbererimab, tremelimumab, relatrimab, monoclonal antibody IMP321, nivolumab, etigirimab, dombanarimab, tilagolumab (RG6058), vivostrimab, avelumab, and durvalumab.

46. The one or more immune checkpoint inhibitors of the second pharmaceutical product are PD-1, PD-L1, CTLA-4, T cell activation V-domain Ig suppressor (VISTA), lymphocyte activation 3 (LAG3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), T cell immune receptor having Ig and ITIM domains (TIGIT), programmed death ligand 2 (PD-L2), indoleamine 2,3-dioxygenase (IDO), arginase-1 (AGR1), B7 family inhibitory ligand B7-H3 (B7-H3), B7 family A combination according to any one of claims 33 to 42 and 44, comprising one or more agents selected from the group consisting of Millie inhibitory ligands B7-H4 (B7-H4), 2B4 (differentiation cluster 244), B and T lymphocyte attenuation factor (BTLA), adenosine A2A receptor (A2aR), and / or members of the killer cell immunoglobulin-like receptor (KIR) family, e.g., KIR and C-type lectin receptors, and signaling factors and transcriptional activators (STAT3), which bind to or inhibit an immune checkpoint on a cell.

47. The combination according to any one of claims 33 to 42 and 44, wherein the cancer includes a cold tumor, a hot tumor, or an immune desert tumor.

48. A combination according to any one of claims 33 to 47, used to enhance the efficacy of one or more immune checkpoint inhibitors administered to a subject with cancer and / or to treat cancer.

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