Compounds and methods for treating and preventing fibrotic disease states and cancer

Abstract

Compounds, pharmaceutical compositions and methods are provided for reprogramming M2-like macrophages to M1-like macrophages that reverse the anti-fibrosis to fibrosis-promoting transition observed during fibrotic diseases and certain cancers. The compounds comprise immunomodulators that target pattern recognition receptors of cells and are specific for cells of interest by incorporating targeting moieties (e.g., folates or functional fragments or analogs thereof). Releasable and / or non-releasable linkers may be included and engineered to facilitate optimal delivery of immunomodulators. The compounds and compositions may be used in one or more methods of treating fibrotic diseases and / or cancer.

Description

[0001] This application is a divisional application of the patent application with the filing date of July 8, 2020, application number 202080058464.1, and title "Compounds and Methods for Treating and Preventing Fibrotic Disease States and Cancer".

[0002] Priority

[0003] This application claims priority to the following: a) U.S. Provisional Patent Application No. 62 / 871,686, filed on July 8, 2019; and b) U.S. Provisional Patent Application No. 62 / 872,146, filed on July 9, 2019. The contents of the above applications are hereby incorporated by reference in their entireties into this disclosure. Technical Field

[0004] This disclosure relates to compounds, pharmaceutical compositions, and methods for treating and preventing fibrotic disease states and / or cancer using one or more compounds that comprise a targeting moiety and reprogram M2 macrophages to M1 macrophages. Background Art

[0005] Although many fibrotic diseases such as idiopathic pulmonary fibrosis (IPF) are severe, there are few options for successful treatment, and almost all of the conventionally available options are designed to relieve symptoms and delay progression rather than cure the underlying disease. For example, for IPF, oxygen therapy can improve comfort and lifestyle but has little effect on disease progression. Similarly, while two FDA-approved drugs (i.e., pirfenidone and nintedanib) may slow the development of the disease, neither can reverse existing fibrosis or stop the production of further fibrosis. Given the high probability of mortality associated with many fibrotic diseases such as IPF, there is an urgent need to identify new strategies to slow or even stop disease progression.

[0006] In addition, cancer is treated with chemotherapy using highly potent drugs such as mitomycin, paclitaxel, and camptothecin. In many cases, these chemotherapeutic agents exhibit a dose-response effect, and tumor suppression is proportional to the drug dose. Therefore, aggressive dosing regimens are used to treat tumors; however, high-dose chemotherapy is hindered by poor selectivity for cancer cells and toxicity to normal cells. Lack of tumor specificity is one of many obstacles that conventional chemotherapy needs to overcome.

[0007] Although there is clearly a need to prevent and treat both fibrotic diseases and cancer, these conditions remain a significant cause of death and / or suffering worldwide because there are currently no effective treatment options that can cure the conditions. Moreover, where drugs or other therapies are available, such treatments typically employ highly potent drugs that carry a risk of systemic toxicity to potential subjects because of their poor selectivity for the fibrotic and / or cancerous cells of interest. What is needed is a treatment that can not only effectively disrupt the profibrotic and / or pro-growth factor cycle initiated by activated M2 (alternatively activated) macrophages, but also do so with extremely high specificity for the relevant cells, whether cancerous or other cells undergoing fibrotic disease. SUMMARY OF THE INVENTION

[0008] In some instances, activated M2-phenotype macrophages play a role in fibrotic diseases, for example, by secreting profibrotic cytokines that activate fibroblasts to synthesize collagen and other extracellular matrix proteins. In certain instances, these macrophages similarly cause the release of problematic growth factors in subjects undergoing cancer. For example, such growth factors can promote the growth of cancerous tumors. Additionally, in some instances, macrophages release immunosuppressive cytokines (e.g., simultaneously). Thus, macrophages can play an important role in promoting the establishment and growth of fibrotic diseases and / or cancer.

[0009] Idiopathic pulmonary fibrosis (IPF) is one such fibrotic disease, for example, an interstitial lung disease caused by excessive deposition of collagen. In some instances, this type of fibrosis leads to progressive stiffening of the lung, and in some instances, to an inevitable loss of the lung's ability to mediate gas exchange. Because of this progressive decrease in vital capacity, the median survival time after an IPF diagnosis is estimated to be only 2.5 - 5 years. In some instances, severe associated morbidities (e.g., chronic hypoxia, fatigue, weight loss, muscle and joint pain, persistent cough, and loss of mobility, etc.) continue to increase in the later stages of the pathology. In the United States, approximately 40,000 new cases of IPF are diagnosed each year, and the majority end in death.

[0010] In some cases, activated macrophages derived from tissue-resident macrophages or peripheral blood monocytes induce the activation of fibroblasts by secreting chemokine (C-C motif) ligand 18 (CCL18), transforming growth factor β1 (TGFβ1), and / or platelet-derived growth factor (PDGF). In some cases, this activation promotes the secretion of collagen by fibroblasts, which can cause fibrotic diseases and the development of associated cancers. In the later stages of many fibrotic diseases, activated macrophages and myofibroblasts can cross-stimulate each other, leading to a vicious cycle that ensures the spread of fibrosis throughout the lungs or other relevant parts of the body.

[0011] Similar pathologies have also been observed in other fibrotic diseases. Cancer can also involve similar immune responses, such as promoting the growth of cancerous tumors (e.g., attributed to growth factors secreted by activated macrophages) and / or promoting collagen formation in cancerous tumors (e.g., through downstream fibrotic collagen production, which can lead to more difficult-to-treat cancerous tumors by blocking their drug permeability).

[0012] In some embodiments, the present disclosure provides a compound represented by the formula Q-L-T. In some embodiments, Q is a group of a folate receptor-binding ligand. In some embodiments, L is a linker. In some embodiments, T is a group of a toll-like receptor (TLR) agonist. In some embodiments, Q-L-T is a pharmaceutically acceptable salt thereof.

[0013] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is represented by the following formula:

[0014]

[0015] 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.

[0016] In some embodiments, w is from 0 to 5. In some embodiments, w is from 0 to 2. In some embodiments, w is 1.

[0017] In some embodiments, the TLR agonist is a toll-like receptor 7 (TLR7) agonist. In some embodiments, the group of the TLR agonist has a structure represented by the formula X:

[0018]

[0019] In some embodiments, R1 is -NH2 or -NH-R 1X. In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , In some embodiments, R 1X , R 2X and R 2Y each independently is selected from the group consisting of: hydrogen (H), alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl. In some embodiments, is a 3- to 10-membered nitrogen (N)-containing non-aromatic monocyclic or bicyclic heterocyclic ring. 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, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X , R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocyclic ring; and R3 is -OH, -SH, -NH2, or -NH-R 1X .

[0020] In some embodiments, the group of the TLR agonist has a structure represented by formula XX:

[0021]

[0022] In some embodiments, R1 is -NH2 or -NH-R 1X . In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , In some embodiments, R 1X , R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl. In some embodiments, is a 3- to 10-membered nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, X is CH, CR2, or N. In some embodiments, R1 is -NH2 or -NH-R 1X ; R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X , R 2X and R 2Y each independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl, is a 3- to 10-membered nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle; and X is CH, CR2, or N.

[0023] In some embodiments, the group of the TLR7 agonist has a structure represented by formula XXX:

[0024]

[0025] In some embodiments, the compound further comprises a linker L between the targeting moiety and the immunomodulator or a pharmaceutically acceptable salt thereof n , wherein the linker L n is configured to avoid release of the free form of the TLR7 agonist, and n is an integer equal to or less than 50. In some embodiments, the linker L n comprises polyethylene glycol (PEG) or a PEG derivative, n is an integer selected from the range 1-32, and the group of the folate receptor-binding ligand is a folate receptor beta (FBβ) binding ligand.

[0026] In some embodiments, the compound has a structure represented by:

[0027]

[0028] In some embodiments, the compound has a structure represented by:

[0029]

[0030] In some embodiments, the compound has a structure represented by:

[0031]

[0032] In some embodiments, the compound has a structure represented by:

[0033]

[0034] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more compounds of the present disclosure, wherein the TLR7 agonist has a structure represented by Formula XX.

[0035] In certain instances, the present disclosure provides methods of treating a subject having a fibrotic disease state or cancer, the method comprising contacting cells of the subject with at least one compound comprising a compound described herein, wherein the immunomodulator comprises an agonist of TLR 7, 8, or 9.

[0036] In some embodiments, the present disclosure provides compounds comprising a folate ligand or a functional fragment or analogue thereof attached via a linker to a TLR agonist having the following formula or a pharmaceutically acceptable salt thereof:

[0037]

[0038] In some embodiments, R 1 is an amino group, R 2 is a single bond -NH-, and R 3 is H, alkyl, a hydroxy group, or any other substituent group, X is CH2, NH, oxygen (O), or sulfur (S), and the linker is attached at R 1 、R 2 or R 3 .

[0039] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a compound of any of the formulas provided herein, wherein the linker comprises a PEG linker or a PEG derivative linker and is a non-cleavable linker attached at R 3 or a cleavable linker attached at R 1 、R 2 or R 3 .

[0040] In some embodiments, the pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate, or fumarate.

[0041] In some embodiments, provided herein are methods of preventing or treating a fibrotic disease state, the methods comprising contacting a cell with at least one compound (e.g., any compound provided by a formula provided herein), the at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt thereof attached via a linker to a folate ligand or a functional fragment or analogue thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor. In some embodiments, the cell comprises a cell of a subject who has experienced or is at risk of experiencing a fibrotic disease state, and contacting the cell with at least one compound further comprises administering or applying to the subject a therapeutically effective amount of the at least one compound. In some embodiments, the subject is a patient experiencing IPF, and the at least one compound is administered to the subject by intravenous, intramuscular, intraperitoneal, topical, or inhalation routes. In some embodiments, the fibrotic disease state includes IPF or a fibrotic disease of the liver, skin, bladder, heart, pancreas, prostate, or kidney.

[0042] In some embodiments, the method further comprises obtaining or having obtained a sample from the subject; quantifying the expression level of one or more biomarkers in the sample, each of the one or more biomarkers selected from the group consisting of CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), tissue inhibitor of metalloproteinase 3 (TIMP3), interleukin 1β (IL-1β), hydroxyproline, collagen, PDGF, TGFβ, folate receptor β (FRβ), tumor necrosis factor-α (TNFα), interferon γ (IFN-γ), mannose receptor (CD206), cluster of differentiation 163 (CD163), cluster of differentiation 86 (CD86), interleukin 6 (IL-6), chemokine (C-X-C motif) ligand 10 (CXCL10), and immune interferon (IFNα); comparing the expression level of each of the one or more biomarkers in the sample to the expression level of such biomarkers in a control; and if the expression level of CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline, or collagen is upregulated relative to the control or the expression level of TNFα, IFN-γ, IL-6, CXCL10, IFNα, or CD86 is downregulated or not expressed relative to the control, administering or having administered to the subject a therapeutically effective amount of an unconjugated agonist or inhibitor.

[0043] In some embodiments, the folate ligand or a functional fragment or analogue thereof is specific for FRβ and binds to FRβ on the cell.

[0044] In some embodiments, provided herein are compounds comprising a targeting moiety attached to an immunomodulator or a pharmaceutically acceptable salt thereof that targets a pattern recognition receptor of a cell, the targeting moiety comprising a folate ligand or a functional fragment or analogue thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In view of the following detailed description of various exemplary embodiments of the present disclosure, the disclosed embodiments and other features, advantages, and aspects contained herein, as well as the things that implement them, will become apparent. Such detailed description will be better understood when taken in conjunction with the drawings, in which:

[0046] Figure 1 A shows the chemical structure of an exemplary compound having a targeting moiety (folate receptor ligand) attached to an immunomodulator (toll-like receptor 7 (TLR7) agonist group) via a non-cleavable linker (e.g., comprising a polyethylene glycol (PEG) backbone moiety).

[0047] Figure 1 B shows the chemical structure of an exemplary compound having a targeting moiety (folate receptor ligand) attached to an immunomodulator (TLR7 agonist group) via a cleavable linker (e.g., comprising a disulfide moiety in its backbone), as well as an exemplary drug release mechanism.

[0048] Figure 2 Shows a flowchart representing a method for treating a subject experiencing a fibrotic disease or cancer or at risk of experiencing a fibrotic disease or cancer.

[0049] Figure 3 A-3F shows graphical data of various marker levels measured from human M2 macrophages when contacted with an exemplary free (non-targeted) TLR7 agonist or an exemplary targeted (e.g., having a folate receptor-binding ligand) TLR7 agonist at various concentrations of each compound. In Figure 3 the data shown in A-3C support that the administration of a non-targeted TLR7 agonist or a targeted TLR7 agonist successfully reprograms M2 macrophages to M1 macrophages (i.e., downregulates M2 pro-fibrotic macrophages), and Figure 3 the data shown in D-3F support that the administration of the tested compounds upregulates M1 macrophages; each value represents the mean ± S.D. of each group; #P < 0.05, ##P < 0.01, P < 0.005, #P < 0.0001; by Dunnett's multiple comparison test, the treatment groups relative to the M2 untreated group.

[0050] Figure 4 A-4E and Figure 5A-5D shows graphical data representing levels of various markers measured from M2 macrophages incubated for 2 hours ( Figure 4 A-4E) or 46 hours ( Figure 5 A-5D) with exemplary free or targeted TLR7 agonists at various concentrations. Figure 4 A-4E and Figure 5 A-5D support that the M2 pro-fibrotic phenotype is downregulated following administration of free and targeted TLR7 agonists. Each value represents the mean ± S.D. for each group; #P<0.05, ##P<0.01, P<0.005, #P<0.0001; by Dunnett's multiple comparison test, Figure 4 for the compound 1A and compound 1B treatment groups in A-5D relative to the M2 untreated group.

[0051] Figure 6 A-6D shows graphical data representing levels of various markers measured from M2 macrophages treated with exemplary free and targeted TLR7 agonists at various concentrations for (i) 48 hours ( Figure 6 A and 6B) or (ii) 2 hours then replaced with fresh medium and cultured for the remaining 46 hours ( Figure 6 C and 6D). Each value represents the mean ± S.D. for each group; #P<0.05, ##P<0.01, P<0.005, #P<0.0001; by Dunnett's multiple comparison test, for the compound 1A and compound 1B treatment groups relative to the M2 untreated group.

[0052] Figure 6 E shows flow cytometry data supporting that THP-1 (a human monocyte cell line derived from a patient with acute monocytic leukemia)-induced macrophages are positive for folate receptor β (FRβ) (FRβ+).

[0053] Figure 6 F shows that the exemplary targeted TLR7 agonist is stable.

[0054] Figure 7 A shows stained images of lungs obtained from mice with bleomycin (BM)-induced experimental fibrosis and stained with anti-mouse FRβ antibody, where hematoxylin and eosin (H&E) staining was performed on days 7, 14, and 21 after BM-induced lung injury.

[0055] Figure 7 B shows Figure 7 quantification of FRβ staining in the inset of A.

[0056] Figure 7 C and 7D show human idiopathic pulmonary fibrosis (IPF) lung tissue ( Figure 7C) and healthy human lung tissue ( Figure 7 D) FRβ immunohistochemistry (IHC) staining.

[0057] Figure 7 E shows images of mouse tissues / organs obtained from mice induced with experimental fibrosis with or without (phosphate buffered saline (PBS) control) BM and imaged with a folate receptor-targeted fluorescent dye.

[0058] Figure 7 F shows fluorescence-activated cell sorter (FACS) analysis of mice with BM-induced experimental fibrosis.

[0059] Figure 8 A shows the treatment regimens of free and targeted TLR7 agonists in the BM model.

[0060] Figure 8 B-8G shows measuring the levels of profibrotic markers ( Figure 8 B-8D) and antifibrotic marker levels ( Figure 8 E-8G) from mice treated with the BM model of Figure 8 A. Figure 8 H shows the cell counts in the bronchoalveolar lavage fluid (BALF) from mice treated with the BM model of Figure 8 A.

[0061] Figure 9 A and 9B show the survival curves ( Figure 9 A) and body weight changes ( Figure 9 B) of mice with pulmonary fibrosis treated with non-targeted and targeted TLR7 drugs.

[0062] Figure 10 A shows the hydroxyproline content (μg / lung) of lung tissue as a measure of fibrosis.

[0063] Figure 10 B and 10C show lung tissue with H&E staining ( Figure 9 B) and Masson's trichrome (collagen) staining ( Figure 10 C) in Figure 10 A.

[0064] Figure 11 A and 11B show the survival curves ( Figure 11 A) and body weight changes ( Figure 11 B) of mice with pulmonary fibrosis treated with an exemplary targeted TLR7 agonist, with each value representing the mean ± S.D. of each group.

[0065] Figure 12Shows the dose-dependent effect of an exemplary TLR7 agonist of the present disclosure on the inhibition of fibrosis in BM-induced mice. Figure 12 A shows graphical data related to the body weight of BM-induced mice over time. Figure 12 B shows the measurement of hydroxyproline content in lung tissues treated with various doses of the exemplary conjugates provided herein (e.g., Compound 1B). Figure 12 C shows images of histological analysis of lung tissues with various stainings. Each value represents the mean ± S.D. of each group; *P < 0.05, **P < 0.005, *** < 0.0005; by Student's t-test, saline vs. vehicle group, treatment groups vs. vehicle group.

[0066] Figure 13 A-13D shows the levels of various markers measured from M2 macrophages reprogrammed with various concentrations of an exemplary TLR7 agonist according to the method of the present disclosure for 48 hours, and each value represents the mean ± S.D. of each group.

[0067] Figure 14 A-14C shows the levels of various markers measured from M2 macrophages reprogrammed with various concentrations of exemplary free and targeted TLR7 agonists according to the method of the present disclosure. Figure 14 Each value shown in A-14C represents the mean ± S.D. of each group; #P < 0.05, ##P < 0.005, P < 0.0005, #P < 0.0001; by Dunnett's multiple comparison test, Compound 3A, Compound 3B, and Compound 3C treatment groups vs. M2 untreated group.

[0068] Figure 15 Shows after treatment with exemplary free and targeted TLR7 agonists in Figure 14 The protein levels of chemokine (C-C motif) ligand 18 (CCL18) secreted in each group of cells in A-14C.

[0069] Figure 16 Demonstrates the methodology for the BM mouse model.

[0070] Figure 17 A and 17B show the purity of the exemplary TLR7 agonists provided herein.

[0071] Figure 18 A-18F shows data from Figure 16 the in vivo research methodology, including survival curves ( Figure 18 A), body weight changes ( Figure 18 B and 18D), concentration of cells present in BALF ( Figure 18C), in live mice ( Figure 18 E) and in all mice (i.e., including live mice and mice that died before day 21)( Figure 18 F) hydroxyproline concentration (μg HP / lobe of lung).

[0072] Figure 19 Shows that both targeted and untargeted TLR7 agonists reprogram human monocyte-derived profibrotic macrophages into an anti-fibrotic phenotype( Figure 19 A-19F). Mean ± SD. Unpaired two-tailed t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) were used to determine statistical significance between groups.

[0073] Figure 20 Shows a comparison of plasma cytokine levels in healthy mice after treatment with compound 1A and compound 1B( Figure 20 A-20F). Figure 20 G shows the change in body weight after treating mice with the exemplary compounds provided herein, where the change in body weight is a measure of systemic toxicity during alternate-day dosing (n = 2); mean ± SD. Unpaired two-tailed t-tests (*P < 0.05, **P < 0.01, ***P < 0.001) were used to compare statistical significance between groups.

[0074] Figure 21 Shows healthy and fibrotic lungs stained with 4',6-diamidino-2-phenylindole (DAPI) (cell nuclei; blue), anti-F4 / 80 (macrophages; red), and anti-mannose receptor (CD206) in Figure 6 as described in

[0075] While the present disclosure is susceptible to various modifications and alternative forms, its exemplary embodiments are shown by way of example in the drawings and are described in detail herein. Detailed Description

[0076] To facilitate understanding of the principles of the present disclosure, reference will now be made to the embodiments shown in the drawings, and the same embodiments will be described using specific language. However, it should be understood that the description of these embodiments is not intended to limit the scope. On the contrary, the present disclosure is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the present application as defined by the appended claims. As previously mentioned, while this technology may be shown and described in one or more preferred embodiments, its compositions, compounds, and methods may include many different configurations, forms, materials, and attachments.

[0077] In the following description, numerous specific details are set forth to provide a thorough understanding of the present disclosure. Certain examples may be practiced without some or all of these specific details, and it should be understood that the present disclosure is not limited to a particular biological system, a particular fibrotic disease or cancer, or a particular organ or tissue, which may of course vary, but remains applicable in view of the data provided herein.

[0078] The various techniques and mechanisms of the present disclosure will sometimes describe a connection (connection or link) between two components. Words such as attach, link, couple, connect, and similar terms and their non-inflected forms may be used interchangeably, unless a difference is indicated from the context or otherwise becomes clear. These words and expressions do not necessarily denote a direct connection, but include connections through intervening components. It should be noted that a connection between two components does not necessarily imply a direct unobstructed connection, as multiple other components may reside between the two components being noted. Thus, unless otherwise stated, a connection does not necessarily imply a direct unobstructed connection.

[0079] In addition, where feasible and convenient, the same reference numerals are used in the drawings and the description to refer to the same or similar parts or steps. The drawings are in simplified form and are not drawn to precise scale. It should be understood that the present disclosure is presented in this manner for purposes of explanation only, and the principles and embodiments described herein may be applied to compounds and / or composition components having configurations other than those specifically described herein. In fact, it is expressly contemplated that the components and compounds of the compositions of the present disclosure may be customized to facilitate their desired applications.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of chemistry and biology. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the subject matter of this application, the preferred methods and materials are described herein. Additionally, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a" and "the" include plural referents. Thus, for example, where a compound / composition is "a" alkyl or aryl substituted, the compound / composition is optionally substituted with at least one alkyl and / or at least one aryl. Further, unless otherwise specifically stated, the term "about" refers to a range of ±10% of the percentage value and ±1.0 unit of the unit value; for example, "about 1.0" refers to a range of values from 0.9 to 1.1.

[0081] In certain embodiments, the compounds, compositions, and methods of the present disclosure can be used for the prevention and / or treatment of fibrotic diseases. In certain embodiments, the provided compounds and / or compositions can also be used for the prevention and / or treatment of cancer. In some embodiments, the compounds, compositions, and methods provided herein utilize strategies to (e.g., selectively) target the innate immune system and reprogram macrophage polarization from M2 to M1 and, for example, utilize their anti-fibrotic properties.

[0082] Generally and without being bound by any particular theory, the novel compounds, compositions, and methods of the present disclosure target the innate immune system of a subject and reprogram macrophage polarization from M2 to M1 to favor the anti-fibrotic properties of the M1 phenotype. For example, in at least one exemplary embodiment, such compounds and compositions comprise a targeting moiety (such as a folate receptor-binding ligand or a group thereof) that targets folate receptor beta (FRβ) conjugated to an immunomodulator or a pharmaceutically acceptable salt thereof. As described in detail below, such embodiments utilize the restricted expression of FRβ to direct systemically administered compounds directly to cells that express FRβ (e.g., those of fibrotic and / or cancerous tissues) such that the immunomodulator component can then convert (e.g., reprogram) activated myeloid cells (e.g., M2-like macrophages) to an anti-fibrotic M1 polarization. This targeted design advantageously prevents systemic activation of the immune system and thus avoids toxicity.

[0083] Further exemplary embodiments can include a linker disposed between the targeting moiety and the immunomodulator. Such linkers can be cleavable or non-cleavable. As described herein, compounds / compositions of the present disclosure that comprise a cleavable linker will upon administration result in the targeting moiety and the immunomodulator releasing from each other at or about the time the immunomodulator becomes active. Additionally or alternatively, in embodiments where the compounds / compositions of the present disclosure comprise a non-cleavable linker, there is no rapid release under physiological conditions when the targeting moiety and the immunomodulator are administered. In this way, the components remain together after uptake by the targeted cell and / or activation of the immunomodulator.

[0084] Various embodiments of the present disclosure will now be described, along with data related to examples that support the present disclosure. First, there are two major immune strategies in vertebrates: the innate immune system and the adaptive immune system. The innate or non-specific immune response is the first line of defense against non-self pathogens and consists of physical, chemical, and cellular defenses. On the other hand, the adaptive immune system is called upon to act against pathogens that evade or overcome the primary innate immune defenses.

[0085] The inflammatory response plays a key role in immunity. When tissue damage or a pathogen is detected, for example, an inflammatory response is triggered and the immune system is mobilized. Immune cells of the innate immune system (i.e., neutrophils and eosinophils) are the first to be recruited via the blood vessels and lymphatic system to the site of tissue damage or injury or the location of the pathogen, followed by macrophages.

[0086] 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. As used herein, "pattern recognition receptor" means and includes any immune receptor expressed on the membrane of a leukocyte (e.g., at least a macrophage) and that can bind to specific ligands that activate the receptor and ultimately lead to an innate immune response (and in some cases, ultimately the development of antigen-specific acquired immunity).

[0087] Examples of two classes of molecules that can bind to pattern recognition receptors include pathogen-associated molecular patterns associated with microbial pathogens and damage-associated molecular patterns associated with components of host cells released during cell damage or death. Recognition of these protein sequences by pattern recognition receptors can initiate signal transduction pathways that trigger the expression of certain genes, the products of which control the innate immune response (e.g., in some cases, direct the development of antigen-specific acquired immunity). Thus, pattern recognition receptors mediate these signal transduction pathways and, in some cases, can be used to positively or negatively control innate and even adaptive immune responses.

[0088] Macrophages are a diverse group of leukocytes known to eliminate pathogens by phagocytosis and are widely classified as having an M1 phenotype or an M2 phenotype depending on the 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 (such as 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, strong microbicidal properties, the production of high levels of reactive nitrogen and oxygen intermediates, and / or a T helper type 1 (Th1) response. In some cases, M1 polarization is associated with the "attack and kill" phase of the innate immune response. In certain cases, M1 polarization functions to inhibit or prevent the initial establishment of infection and / or to remove damaged tissue.

[0089] In some cases, after the innate immune system executes this "attack and kill" phase, macrophages can reprogram themselves to become the healing system (i.e., M2 type) and, for example, release growth factors to promote healing. Such growth factors can include (but are not limited to) certain cytokines such as interleukin 4 (IL-4), interleukin 10 (IL-10), platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGFβ), chemokine (C-C motif) ligand 18 (CCL18), and / or interleukin 13 (IL-13). In some cases, exposure to such cytokines / growth factors can alternatively activate the M2 macrophage phenotype.

[0090] Compared with M1, M2 macrophages may be associated with wound healing and tissue repair. In some cases, M2 macrophages are characterized by their involvement in tissue remodeling, immune regulation / suppression, and / or tumor promotion. In specific examples, M2 macrophages produce polyamines to induce cell proliferation and / or produce proline to induce collagen production. Although this healing response is beneficial in healthy subjects, the presence of M2 macrophages may have significant adverse effects by immunosuppressing and / or promoting tumor growth and fibrosis in those subjects with fibrotic diseases or cancer.

[0091] For example, fibrotic pathologies can begin with an unknown trauma or injury to the epithelium. In response to the resulting tissue damage, chemokines and other factors can be released to promote infiltration of immune cells (e.g., innate immune response) into the damaged tissue, such immune cells including, for example, monocytes and macrophages that adopt an M2-like phenotype and, for example, release profibrotic cytokines. Then, the chronic secretion of these cytokines can activate tissue-resident and infiltrating fibroblasts / fibrocytes to become myofibroblasts, which in turn secrete collagen and other extracellular matrix proteins that can harden the surrounding tissue. In some cases, these M2 macrophages worsen the disease by promoting fibrosis. For example, in subjects with idiopathic pulmonary fibrosis (IPF), for example, M2 macrophages can infiltrate the lungs and promote fibrosis therein, which further reduces their functionality. In some cases, growth factors and other cytokines produced by the M2 phenotype drive cancerous tumor growth through similar pathways.

[0092] Reprogramming of M2-like macrophages to M1-like macrophages

[0093] In certain cancers and fibrotic diseases, macrophages can be disproportionately biased towards an anti-inflammatory (M2-like) phenotype. In certain instances, immunomodulators can convert (e.g., reprogram) activated myeloid cells (e.g., M2-like macrophages) to an anti-fibrotic M1 polarization (e.g., in which case they produce little or no growth factors and / or associated cytokines and, e.g., slow or even eliminate the progression of the disease state). In certain instances, the compositions and methods provided herein reverse the anti-fibrotic to pro-fibrotic transition observed during the development of fibrotic diseases (e.g., IPF and certain cancers). In some embodiments, the compositions and methods provided herein reduce the amount / expression of fibrotic biomarkers (e.g., those associated with pro-fibrotic activity (e.g., CCL18, hydroxyproline, and collagen)) in an individual or in a sample obtained from a subject. As used herein, "individual", "subject", or "patient" is a mammal (preferably human), but can also be an animal.

[0094] A "marker" or "biomarker" as the term is used herein can be described as differential expression when the expression level of a subject experiencing an active disease state is significantly different from the expression level of a subject or a sample collected from a healthy subject or a subject not experiencing the disease state. A differentially expressed marker can be overexpressed or underexpressed compared to the expression level of a normal or control sample or the baseline of the subject (in the embodiments mentioned in the immediately preceding paragraph, the biomarker is reduced or underexpressed). An increase or decrease or quantification of a marker 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. The marker level can be determined as an absolute value, or relative to a baseline value, and the subject marker level compared to a cut-off index. Alternatively, the relative abundance of one or more markers can be determined relative to a control, which can be a clinically normal subject. Additionally, as used herein, the terms "gene overexpression" and "overexpression" (when used in connection with a gene) and their formative words have the meaning given to them by one of ordinary skill in the relevant art, which includes (but is not limited to) overexpression or misexpression of a wild-type gene product that can cause a mutant phenotype and / or result in the expression of a large amount of the target protein.

[0095] In some embodiments, the compositions and methods provided herein increase anti-fibrotic biomarkers (e.g., TNFα and IFN-γ). In some embodiments, compositions are provided that reverse the M2-like phenotype transition (e.g., provide effective treatment for fibrotic diseases, their conditions, or symptoms).

[0096] In at least one embodiment, a drug comprising an immunomodulator is used to prepare a compound for use in the methods described herein. As used herein, "immunomodulator" means any drug, warhead, or other composition or compound that stimulates or otherwise affects the immune system of a subject by inducing activation or increased activity of one or more components of the immune system. For example, but not limited to, immunomodulators can include compounds or compositions that target one or more pattern recognition receptors, in addition to or alternatively targeting signaling pathways in immune cells.

[0097] Exemplary examples of the immunomodulators of the present disclosure include, but are not limited to, toll-like receptor (TLR) agonists, stimulator of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), receptor for advanced glycation end products (RAGE), or any other pattern recognition receptor located in the endosomes or cytoplasm of cells. Additionally or alternatively, the immunomodulators of the present disclosure can include activators of B cells that act more downstream in the pathway activators or nuclear factor kappa-light-chain enhancer of kinase inhibitors. Table 1 provides examples of such activators or kinase inhibitors that can be used as immunomodulators of the present disclosure.

[0098] Table 1. Activator / Inducer

[0099]

[0100]

[0101] As used herein, "TLR" is a class of proteins that function in the innate immune system and are examples of pattern recognition receptors. A TLR can be a single transmembrane receptor that recognizes structurally conserved molecules derived from microorganisms. TLRs can be expressed on the membranes of leukocytes, including, for example, dendritic cells, macrophages, natural killer cells, adaptive immune cells (e.g., T and B lymphocytes), and 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. In some embodiments, the agonist is a ligand that binds to and activates the receptor.

[0102] Any therapeutic agent (e.g., drug) suitable for reprogramming activated macrophages (M2-like phenotype) to an M1-like phenotype can be used, and the drug (or warhead) can operate in the endosomes and / or cytoplasm of the cell (e.g., depending on its structure). In at least one embodiment, the therapeutic agent comprises an immunomodulator (e.g., an immunomodulator of a positive control pattern recognition receptor and / or its downstream signaling pathway (in each case, a part of the innate immune system), e.g., a TLR, NLR, RLR, ALR, RAGE, and / or STING agonist and / or an inhibitor of the Pelle / interleukin 1 receptor-associated kinase (IRAK) family such as IRAK-M inhibitor). In other embodiments, the compounds provided herein comprise a phosphoinositide 3-kinase (PI3K) kinase inhibitor or other inhibitor that negatively controls the adaptive immune system (e.g., which can be used alone or in combination with an immunomodulator targeting a pattern recognition receptor). In some embodiments, particularly when used to treat IPF or other fibrotic conditions, the composition or compound (e.g., drug) comprises a combination of: (a) an immunomodulator that targets a pattern recognition receptor and / or is an agonist of its downstream signaling pathway in the innate immune system, and (b) a mammalian target of rapamycin (mTOR) inhibitor (ATP-competitive or otherwise), such as rapamycin or CZ415.

[0103] In certain embodiments, provided herein are compounds comprising a targeting moiety (or a group thereof) attached to an immunomodulator (or a group thereof) that targets a pattern recognition receptor of a cell, wherein the targeting moiety comprises a folate ligand or a functional fragment or analogue thereof. "Folate" means a folate receptor-binding molecule (including, for example, folic acid and analogues and derivatives of folic acid such as, but not limited to, leucovorin, pteroylpolyglutamate, pteroyl-D-glutamate) and folate receptor-binding pteridines such as tetrahydrobiopterin, dihydrofolic acid, tetrahydrofolic acid, and their aza and diaza analogues.

[0104] The terms "aza" and "diaza" analogues refer to analogues recognized in the art having a carbon atom substituted for one or two nitrogen atoms in the structure of a naturally occurring folate or an analogue or derivative thereof. For example, aza analogues can include 1-aza, 3-aza, 5-aza, 8-aza, and 10-aza analogues of folate, leucovorin, pteroylpolyglutamate, and folate receptor-binding pteridines such as tetrahydrobiopterin, dihydrofolate, and tetrahydrofolate. Diaza analogues include, for example, 1,5-diaza, 5,10-diaza, 8,10-diaza, and 5,8-diaza analogues of folate. Other folates useful as complex-forming ligands in the context of the present disclosure are the folate receptor-binding analogues pemetrexed, pyrimethamine, trimethoprim, pralatrexate, raltitrexed, aminopterin, methotrexate (also known as amethopterin), N 10 -methylfolate, 2-deamino-dihydrofolate, aza analogues such as l-deaza-methotrexate or 3-deaza-methotrexate, and 3′,5′-dichloro-4-amino-4-deoxy-N 10 -methylpteroylglutamate (dichloromethotrexate).

[0105] Folic acid and the foregoing analogues and / or derivatives are also referred to as "a folate / the folate / folates", reflecting their ability to bind to folate receptors. As described herein, such molecules can effectively enhance transmembrane transport when conjugated to an exogenous molecule, such as via folate-mediated endocytosis. The foregoing substances can be used as folate receptor-binding ligands as described herein. As used herein, the term "ligand" is a molecule, ion, or atom attached to a central atom or ion (e.g., a drug) of a compound.

[0106] Certain embodiments of the novel compounds of the present disclosure will now be provided. Those skilled in the art will appreciate that the compounds of the present disclosure may exhibit polymorphism. In fact, the compounds of the present disclosure may include any racemic, optically active, polymorphic, or stereoisomeric form of the compounds described herein that exhibit the described useful properties, or mixtures thereof. It is well known in the art how to prepare optically active forms (e.g., resolving a racemic form by recrystallization techniques, synthesizing from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine anti-tumor activity using the standard tests described herein or other similar tests well known in the art. Additionally, unless otherwise explicitly stated, the structures depicted herein are also intended to include all stereochemical forms of the structure, i.e., the right-handed (R) and left-handed (S) configurations at each asymmetric center. Thus, single stereoisomers as well as enantiomeric and diastereomeric mixtures of the compositions of the present disclosure are within the scope of the present disclosure.

[0107] Unless otherwise specified, the specific values listed herein for groups, substituents, and ranges are for illustrative purposes only; such examples do not exclude other defined values or other values within the defined ranges for the groups and substituents. For example, (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C1-C3)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C1-C3)alkoxy can be methoxy, ethoxy, or propoxy; and (C2-C6)alkanoyloxy can be acetoxy, propionyloxy, butyryloxy, isobutyryloxy, pentyryloxy, or hexanoyloxy.

[0108] In addition, when a moiety is substituted by an R substituent or a substituent group, the moiety may be referred to as "R-substituted". When a moiety is R-substituted or otherwise described as generally comprising a substituted group, the moiety is substituted by at least one R substituent and each substituent is optionally different. It should be understood that the substituted group (or R substituent) may include any molecule or combination of molecules provided that it comprises a group that does not substantially affect the overall structure and shape of the compound and does not alter any hydrogen bonds necessary for the potential compound to achieve its intended purpose (e.g., binding to a targeted pattern recognition receptor).

[0109] When substituent groups are specified by a conventional chemical formula written from left to right, they equally encompass chemically identical substituents resulting from writing the structure from right to left, e.g., —CH2O— is equivalent to —OCH2—.

[0110] In some embodiments, the immunomodulatory agent (e.g., TLR7 agonist) moiety of the compounds provided herein is a moiety having the structure of Formula XX (and more specifically Formula XX'):

[0111]

[0112] wherein,

[0113] R 1B is -NH2 or -NH-R 1X ,

[0114] R 2B is hydrogen (H), alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X ,

[0115] R 1X R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl, and

[0116] is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and

[0117] X is CH or nitrogen (N);

[0118] As used herein, alkyl, alkoxy, etc. denote straight-chain (i.e., unbranched) or branched or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated and may include divalent and polyvalent groups having the specified number of carbon atoms (i.e., C1-C 10 means one to ten carbons). Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl; homologs and isomers such as n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Unsaturated alkyl groups are alkyl groups having one or more double or triple bonds. Examples of unsaturated alkyls include, but are not limited to, vinyl, 2-propenyl, butenyl-2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl group attached to the remainder of the molecule via an oxygen linking group (—O—). In some embodiments, alkoxy refers to a group bonded through the oxygen atom of the formula –O-alkyl.

[0119] Generally, the term "acyl" or "acyl substituent" refers to a derivative produced by removing one or more hydroxy groups from an oxo acid (including inorganic acids) and containing a double-bonded oxygen atom and an alkyl group. In addition, reference to a single group such as "propyl" includes only the straight-chain group, and branched isomers such as "isopropyl" are specifically mentioned.

[0120] In some embodiments, the TLR7 agonist has formula X, and the TLR7 agonist is conjugated to a targeting moiety at any one of R 1A , R 1B , R 3A or R 3B via a linker; and in the case where the TLR7 agonist has formula XX', the TLR7 agonist is conjugated to a targeting moiety at one of R 1A , R 1B , R 3A or R 3B via a linker.

[0121] As used herein, the term "linker" includes a chain of atoms that is biologically functional to form chemical bonds with A, B, or S and that links two or more functional moieties of a molecule to form the compounds of the present disclosure. Illustratively, the chain of atoms can 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 link different functional capabilities of compounds such as folates and drugs. The linker can comprise a variety of linkages, such as in the range of about 2 to about 100 atoms in a continuous backbone, and can comprise releasable or non-releasable linkers. In some embodiments, the immunomodulatory agent (e.g., TLR7 agonist) moiety of the compounds provided herein is a moiety having the structure of formula XXX (and more specifically formula XXX'):

[0122]

[0123] wherein,

[0124] R 1C is -NH2 or -NH-R 1X ,

[0125] R 2C is a bond, NH, -NR 1X or CH2,

[0126] and

[0127] if applicable, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle;

[0128] X A is CH2, NH2, or -NH-R1X ; and

[0129] each R 1X is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl,

[0130] wherein the TLR7 agonist is conjugated to the targeting moiety at one of R 1C , R 2C or R 3B .

[0131] In some embodiments, the compound further comprises a linker ("L" or "L n ") that connects the targeting moiety to the immunomodulator or otherwise links the targeting moiety to the immunomodulator. In some embodiments, the linker L n is configured to avoid release of the immunomodulator, and n is an integer equal to or less than 50. In some embodiments, the linker L n comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, n is an integer selected from the range of 1 - 32, and the targeting moiety is specific for folate receptor β. In some embodiments, n is 1 - 50, 1 - 10, 2 - 8, or 2 - 4.

[0132] In some embodiments, L is a hydrolysable linker. In some embodiments, L is a non - hydrolysable linker. In some embodiments, L is an optionally substituted heteroalkyl.

[0133] Unless otherwise specified, the term "alkylene" by itself or as part of another substituent means a divalent group derived from an alkyl group, such as exemplified but not limited to —CH2CH2CH2CH2—. Generally, an alkyl (or alkylene) group has 1 to 24 carbon atoms. "Lower alkyl" or "lower alkylene" is a shorter - chain alkyl or alkylene group, typically having eight or fewer carbon atoms.

[0134] Unless otherwise specified, the term "heteroalkyl" by itself or in combination with another term means a stable straight, branched, 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, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, P, S, and Si may be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —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, such as —CH2—NH—OCH3.

[0135] Similarly, the term "heteroalkylene" by itself or as part of another substituent means (unless otherwise specified) a divalent group derived from heteroalkyl, as exemplified but not limited to —CH2—CH2—S—CH2—CH2 and —CH2—S—CH2—CH2—NH—CH2. For heteroalkylene groups, the heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkylamino, alkylenediamino, etc.). Further, for alkylene and heteroalkylene linking groups, the direction of the linking group does not imply the direction of writing the formula of the linking group. For example, the formula —C(O)2R′— represents both —C(O)2R′— and —R′C(O)2—. As described above, heteroalkyl groups as used herein include those groups attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and / or —SO2R′. In instances where "heteroalkyl" is recited followed by a recitation of a specific heteroalkyl group such as —NR′R″, etc., it should be understood that the terms heteroalkyl and —NR′R″ are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited to add clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups such as —NR′R″, etc.

[0136] In some embodiments, L is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxy, oxo, PEG, carboxylate, and halo. Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent means a fluorine, chlorine, bromine, or iodine atom.

[0137] In some embodiments, L comprises a spacer (e.g., as described elsewhere herein). In some embodiments, the spacer comprises peptidoglycan or sugar.

[0138] In some embodiments, L is a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, L is a peptide having at least one disulfide bond in its backbone.

[0139] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues, a polypeptide, or a fragment of a polypeptide, peptide or fusion polypeptide. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, and to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0140] In some embodiments, L comprises -CONH-CH(COOH)-CH2-S-S-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-S-S-CH2-CR a R b -O-CO-, where R a and R b are independently H, alkyl or heteroalkyl (e.g., PEG).

[0141] In some embodiments, L comprises the following structure:

[0142]

[0143] where n and m are each independently 0 to 10.

[0144] In some embodiments, L comprises the following structure:

[0145]

[0146] where n is 1 to 32. In at least one exemplary embodiment, n is 1 to 30, and w is 0 to 5.

[0147] In some embodiments, L comprises the following structure:

[0148]

[0149] where n is from 1 to 30, and w is from 0 to 5.

[0150] In some embodiments, the compound has a structure represented by the following formula:

[0151]

[0152] In some embodiments, the compound has a structure represented by the following formula:

[0153]

[0154] In some embodiments, the compound has a structure represented by the following formula:

[0155] In some embodiments, the compound has a structure represented by the following formula:

[0156]

[0157] In certain embodiments, provided herein are compounds comprising a targeting moiety that comprises a folate ligand or a functional fragment or analogue thereof attached via a linker to an immunomodulator comprising a TLR agonist, the TLR agonist having a structure represented by the following formula XXX:

[0158]

[0159] where R 1 is an amino group, R 2 is a single bond -NH-, and R 3 is H, alkyl, a hydroxy group or any other substituent group, X is CH2, NH, O or S, and the linker is attached at R 1 , R 2 or R 3 . Additionally or alternatively, R1 can be -NH2 or -NH-R 1X ; R2 can be H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X , R 2X and R 2Y each independently can be selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl and heteroaryl; It can be a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle; and / or X can be CH, CR2 or N.

[0160] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the formulas or compounds provided herein, wherein the linker comprises PEG or a PEG derivative, and in some cases, is an unreleasable linker attached at R 3 or a releasable linker attached at R 1 , R 2 or R 3 .

[0161] In some embodiments, the pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate or fumarate.

[0162] In some embodiments, the compound comprises a TLR agonist (e.g., its moiety), such as but not limited to a TLR3 agonist, a TLR7 agonist, a TLR7 / 8 agonist, a TLR8 agonist or a TLR9 agonist (e.g., all of them bind to toll-like receptors present in endosomes). For example but not limited to, in at least one exemplary embodiment, the immunomodulator of the drug / compound can be selected from the compounds listed in Table 2 below.

[0163] Table 2. TLR agonists.

[0164]

[0165]

[0166]

[0167]

[0168] In some embodiments, the compound provided herein is or comprises a compound (or moiety) of Formula I (e.g., a TLR7 agonist):

[0169]

[0170] Or a pharmaceutically acceptable salt thereof. In some embodiments, R 1 is an amine. In some embodiments, R 2 is (e.g., a single) bond or an amine (e.g., -NH-). In certain embodiments, R 3is H, an alkyl group, a hydroxy group or any other suitable substituent (e.g., as described herein). In some embodiments, X is CH2, NH, O or S. In some embodiments in which the compounds provided herein comprise a group of formula I, the targeting moiety is conjugated or linked (e.g., via a linker and / or directly) thereto at any suitable position (such as at and / or through R 1 、R 2 and / or R 3 ).

[0171] In at least one exemplary embodiment, the compounds described herein are or comprise a compound (or group) of formula Ia (e.g., a TLR7 agonist):

[0172]

[0173] or a pharmaceutically acceptable salt 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, an alkyl group, an alkenyl group, an alkynyl group, an alicyclic group, an aryl group, a biaryl group, a heteroaryl group, -NH-R 2X , -O-R 2X , -S-R 2X , In a specific embodiment, each of R 1X , R 2X and R 2Y is independently selected from the group consisting of H, an alkyl group, an alkenyl group, an alkynyl group, an alicyclic group, an aryl group, a biaryl group and a heteroaryl group. In some embodiments, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments in which the compounds provided herein comprise a group of formula Ia, the targeting moiety is conjugated or linked (e.g., via a linker and / or directly) thereto at any suitable position (such as at and / or through R 1 , R 2 and / or R 3 ).

[0174] In some embodiments, the compounds provided herein are or comprise a compound (or group) of formula II (e.g., a TLR7 agonist):

[0175]

[0176] or a pharmaceutically acceptable salt thereof. In some embodiments, R 1 is an amine. In some embodiments, R 2 is (e.g., a single) bond or -NH-. In some embodiments, R 3is H, an alkyl group, a hydroxy group, or any other substituent as described herein. In some embodiments, X is CH2, NH, O, or S. In some embodiments in which the compounds provided herein comprise a group of Formula II, the targeting moiety is conjugated or linked (e.g., via a linker and / or directly) thereto at any suitable position (such as at R 1 、R 2 and / or R 3 or through them).

[0177] In other embodiments, the compounds of the present disclosure may comprise a medicament comprising a TLR agonist of Formula III (e.g., or a group thereof) or a pharmaceutically acceptable salt thereof:

[0178]

[0179] wherein R 1 is an amino group, and R 3 is a hydroxy group. Additionally, if desired, the targeting moiety (e.g., or a group thereof) or other ligand may be conjugated (via a linker or directly) to the agonist of Formula III at R 1 or R 3 . The TLR agonist of Formula III (e.g., or a group thereof) is a TLR7 agonist and has at least ten-fold (10x) potency compared to a conventionally available TLR7 agonist.

[0180] In some embodiments, provided herein is a TLR7 agonist of Formula IV (e.g., or a group thereof) or a pharmaceutically acceptable salt thereof:

[0181]

[0182] wherein R 1 is an amino group, and R 2 is a single bond -NH-.

[0183] In some embodiments, administration of the compounds provided herein (e.g., to an individual) converts macrophages in fibrotic tissue from an M2-like phenotype to an M1-like phenotype. In some embodiments, a decrease in cytokines that stimulate collagen synthesis (i.e., CCL18, PDGF, and IL-1β) occurs following administration of the compounds provided herein, and cytokines that inhibit collagen production (e.g., IFN-γ) increase concomitantly. Notably, in at least one embodiment, following administration of the compounds provided herein, the cytokine profile is consistent with reprogramming from an M2-like phenotype to an M1-like phenotype. As used herein, "profile" or "assay" is a set of one or more markers and their presence, absence, and / or relative levels or abundances (relative to one or more controls). For example, a cytokine profile is a dataset of the presence, absence, relative levels, or abundances of cytokines present within a sample. A genomic or nucleic acid profile is a dataset of the presence, absence, relative levels, or abundances of expressed nucleic acids (e.g., transcripts, mRNAs, etc.). Alternatively, a profile may be referred to as an expression profile.

[0184] In some embodiments, the net consequence of reprogramming is an increase in alveolar air sacs, a decrease in extracellular matrix deposition, and a decrease in hydroxyproline / collagen biosynthesis; an effective reversal of the disease (e.g., see Example 4).

[0185] It should be understood that although specific drugs and formulas are described herein, any compound (e.g., drug) that can be used to reprogram activated myeloid cells to an anti-fibrotic M1-like phenotype can be used in the novel compounds and methods herein (e.g., any compound (e.g., drug) capable of binding to a pattern recognition receptor and inhibiting at least a portion of the downstream innate immune system response). In some embodiments, analogs and / or derivatives of the compounds described herein can be used in the targeted compounds provided herein.

[0186] In addition, more than one compound can be administered, and in some cases, the compounds can comprise different drugs. For example, the different drugs can be selected from TLR7 agonists and TLR9 agonists. In yet another embodiment, one or more compounds can be administered in a composition together with one or more conjugated and / or unconjugated drugs (e.g., the conjugated embodiments described below). In some embodiments, any of the compounds and drugs described herein can be used according to the methods described herein, and in some cases, depending on the desired application, can be combined with other drugs and / or any other anti-fibrotic and / or anti-cancer drugs and therapies that deplete or inhibit myeloid-derived suppressor cells (e.g., relevant to cancer treatment), down-regulate the production of growth factors (e.g., pirfenidone relevant to IPF treatment), directly modify fibroblasts via inhibition of the mammalian target of rapamycin complex 1 (mTORC1) signaling (e.g., CZ415 relevant to the treatment of IPF or other fibrotic disease states). As used herein, "downregulation" and words formed by its morphological elements (e.g., "down-regulation" or "down-regulated", for example) can be used interchangeably and refer to a decrease in the level of a biomarker such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. Similarly, "upregulation" and words formed by its morphological elements (e.g., "up-regulation" or "up-regulated") can also be used interchangeably and refer to an increase in the level of a biomarker such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. Moreover, a pathway such as a signal transduction or metabolic pathway can be upregulated or downregulated.

[0187] Targeting moiety

[0188] In some cases, the toxicity associated with the systemic administration of at least the conventional drugs identified herein has precluded their practical use in treating fibrotic diseases, cancer, or any other disease state. For example, TLR agonists may not be tolerated by an individual and in some cases can cause death of the subject (e.g., if administered systemically via a conventional mode). In some embodiments, the compounds provided herein (e.g., compounds having Formula I and / or II) are significantly more potent than the conventional drugs that can be used in conjunction with the compounds of the present disclosure, and in some cases, the mechanism for circumventing systemic toxicity is preferred.

[0189] In certain embodiments, provided herein are therapeutic agents conjugated to targeting moieties (e.g., drugs as previously described). In some embodiments, the targeting moiety comprises a ligand or other atom or molecule that targets a specific region or tissue of an individual (e.g., with high specificity), and in certain instances can comprise, for example, a hormone, an antibody, and / or a vitamin. As described in further detail below, in at least one embodiment, the targeting moiety comprises a molecule that has (e.g., high) affinity for FRβ. In some instances, the targeting moiety has specific affinity for any receptor that is unique to the cells or tissue of a fibrotic disease or cancer, as the case may be.

[0190] In some instances, FRβ is significantly upregulated in activated myeloid cells (e.g., predominantly activated monocytes and M2-like macrophages), e.g., all recorded data to date support induction of FRβ only in bone marrow-derived cells following exposure to anti-inflammatory or pro-inflammatory stimuli. Folate receptor can be upregulated in (e.g., more than 90%) non-mucinous ovarian cancers. In certain instances, folate receptor is present in renal, brain, lung, and breast cancers. For example, although there are many cancers that do not express a sufficient amount of folate receptor on their own to provide the required specificity, cancerous tumors do express myeloid-derived suppressor cells (MDSC) (e.g., which do express FRβ), and can, for example, be targeted by the targeting moieties provided herein. In some embodiments, folate receptor is substantially absent (e.g., present only at very low levels) in healthy (non-myeloid) tissues (e.g., regardless of lung, liver, spleen, heart, brain, muscle, intestine, pancreas, bladder, etc.). In some instances, even quiescent tissue-resident macrophages that are abundant throughout the body are predominantly FRβ-negative. In some instances, there is uptake of folate-targeted imaging agents in inflamed tissue, malignant lesions, and kidney. In certain instances, subjects without cancer retain folate-targeted drugs only in the kidney and sites of inflammation. In some instances, the differential expression of folate receptor provides a mechanism for selectively targeting fibrotic cancer cells.

[0191] In some embodiments, the compounds and methods provided herein utilize the limited expression of FRβ to target / deliver a potent compound (e.g., a conjugate or a drug) administered systemically to fibrotic and / or cancerous tissue. In some instances, the compounds provided herein are directly delivered to cells expressing FRβ, which advantageously prevents systemic activation of the immune system and can, for example, avoid (e.g., at least in part) the toxicity that has thus far precluded the systemic use of non-targeted compounds (e.g., drugs) described herein. In some embodiments, the methods described herein are used to treat fibrotic diseases and / or cancer, e.g., regardless of whether the cancer expresses the folate receptor. In some embodiments, folic acid and other folate receptor-binding ligands (or moieties thereof) (e.g., folates) are used as targeting moieties because, for example, they have an affinity for FRβ.

[0192] Folic acid is a member of the vitamin B family and can play a key 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 immunomodulatory drugs by targeting activated myeloid cells and the specificity of conjugated anti-cancer drugs by targeting folate receptor-positive cancer cells. In some instances, compounds are provided herein that comprise a folate ligand (or moiety thereof) or a functional fragment or analogue thereof as a targeting moiety and an immunomodulator (e.g., a TLR7, TLR8, TLR 7 / 8, TLR9, or TLR3 agonist). In some instances, TLR7, TLR8, TLR 7 / 8, TLR9, and TLR3 are present in endosomes. In some embodiments, the compound or moiety thereof binds to a TLR. In some embodiments, the TLR is TLR7.

[0193] A pyrido[2,3-d]pyrimidine analogue ligand (e.g., or moiety thereof), a functional fragment or analogue thereof, or any other molecule, fragment, or atom having an affinity (e.g., but not limited to, high specificity) for FRβ can alternatively be used as the targeting moiety (or moiety thereof). For example, such folate analogue molecules can have a relative affinity for binding FRβ of about 0.01 or greater compared to folic acid at about 20 °C / 25 °C / 30 °C / physiological temperature. Similarly, galectin 3 ligands, translocator protein (TSPO) ligands, and any other ligand or targeting moiety having a highly specific affinity for fibrotic and / or cancerous cells or tissue can be employed.

[0194] Specific examples of suitable targeting moieties (or moieties thereof) will now be provided; however, it should be understood that the targeting moieties (or moieties thereof) of the present disclosure can comprise any ligand (or moiety thereof) that can be used to target FRβ and are not limited to the structures specified herein. The ligand (or moiety thereof) can bind to FRβ.

[0195] In at least one embodiment, the compounds provided herein include a targeting moiety (or a group thereof) having the structure of Formula V or a functional fragment or analogue thereof:

[0196]

[0197] wherein

[0198] X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S;

[0199] Y is C, CH, CH2, N, NH, O, or S;

[0200] Z is glutamate, valine, or a substrate;

[0201] R1 and R2 are each independently NH2, OH, SH, CH3, or H;

[0202] R3 is hours or an alkyl group;

[0203] m and n are each independently 0, 1, or between 0 and 1; and

[0204] represents a single or double bond C-C.

[0205] In a further aspect, as a non-limiting example, the targeting moiety (or a group thereof) of Formula V has the structure of VI (or a functional fragment or analogue thereof):

[0206]

[0207] wherein

[0208] X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S;

[0209] Y is C, CH, CH2, N, NH, O, or S;

[0210] Z is glutamate, valine, or a substrate;

[0211] R1 and R2 are each independently NH2, OH, SH, CH3, or H;

[0212] R3 is hours or an alkyl group;

[0213] m and n are each independently 0, 1, or between 0 and 1; and

[0214] represents a single or double bond C-C.

[0215] Another specific targeting moiety (or its group) of formula V (or its functional fragment or analogue) has the structure of formula VII:

[0216]

[0217] wherein

[0218] X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S;

[0219] Y is C, CH, CH2, N, NH, O or S;

[0220] Z is glutamate, valine or substrate;

[0221] R1 and R2 are each independently NH2, OH, SH, CH3 or H;

[0222] R3 is hour or alkyl;

[0223] m and n are each independently 0, 1 or between 0 and 1; and

[0224] represents a single or double bond C-C.

[0225] In some embodiments, the targeting moiety (or its group) of formula VI has the structure of formula VIII:

[0226]

[0227] wherein

[0228] X1, X2, X3, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S;

[0229] Y is C, CH, CH2, N, NH, O or S;

[0230] Z is glutamate, valine or substrate;

[0231] R1 and R2 are each independently NH2, OH, SH, CH3 or H;

[0232] R3 is hour or alkyl;

[0233] m is 0, 1 or between 0 and 1; and

[0234] represents a single or double bond C-C.

[0235] In some embodiments, the targeting moiety (or its group) of Formula VI has the structure of Formula IX:

[0236]

[0237] where

[0238] X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S;

[0239] Y is C, CH, CH2, N, NH, O, or S;

[0240] Z is glutamate, valine, or a substrate;

[0241] R1 and R2 are each independently NH2, OH, SH, CH3, or H;

[0242] R3 is hour or alkyl;

[0243] m is 0, 1, or between 0 and 1; and

[0244] represents a single bond or a double bond C-C.

[0245] In some embodiments, the targeting moiety (or its group) of Formula VII has the structure of Formula X or XI:

[0246] where

[0247] X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or Y is C, CH, CH2, N, NH, O, or S;

[0248] Z is glutamate, valine, or a substrate;

[0249] R1 and R2 are each independently NH2, OH, SH, CH3, or H;

[0250] R3 is hour or alkyl;

[0251] m is 0, 1, or between 0 and 1; and

[0252] represents a single bond or a double bond C-C; or

[0253]

[0254] where

[0255] X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or Y is C, CH, CH2, N, NH, O, or S;

[0256] Z is glutamic acid, valine, or a substrate;

[0257] R1 and R2 are each independently NH2, OH, SH, CH3, or H;

[0258] R3 is an hour or an alkyl group;

[0259] m is 0, 1, or between 0 and 1; and

[0260] represents a single bond or a double bond C-C.

[0261] The chemical structures and spectral data of some additional embodiments of the targeting moiety (e.g., or its group) of the present disclosure are provided in Tables 3, 4, 5, and 6 below.

[0262] Table 3 provides non-limiting examples of additional embodiments of the targeting moiety (e.g., or its group) having the structure of Formula VIII.

[0263] Table 3. Formula VIII

[0264]

[0265]

[0266]

[0267] Table 4 provides non-limiting examples of additional embodiments of the targeting moiety (e.g., or its group) having the structure of Formula IX.

[0268] Table 4. Formula IX

[0269]

[0270]

[0271] Table 5 provides non-limiting examples of additional embodiments of the targeting moiety having the structure of Formula X.

[0272] Table 5. Formula X

[0273]

[0274] As described above, instead of folate, the targeting moiety (e.g., its group) can be one or more non-classical antifolate analogs, such as pyrido[2,3-d]pyrimidine or similar analogs (or their groups) having the formulas set forth in Table 6 below (e.g., groups of the formulas):

[0275] Table 6. Non-classical antifolate analogs

[0276]

[0277]

[0278]

[0279]

[0280] In some instances, the compounds provided herein comprise a drug (e.g., its group) (e.g., an immunomodulator) conjugated to a targeting moiety (e.g., its group). The immunomodulator (e.g., its group) can be conjugated directly or through a linker (e.g., optionally comprising a spacer) to the targeting moiety (e.g., its group). Figure 1 A shows at least one embodiment of compound 100. Here, compound 100 comprises, for example, an immunomodulator (or its drug or group) 102 having formula I, where R 3 is a hydroxyl group. The immunomodulator (e.g., its group) 102 is conjugated to the targeting moiety (e.g., its group) 104 through a linker 106. Here, the targeting moiety (e.g., its group) 106 is folate, and the (e.g., non-cleavable) linker 106 is a PEG linker repeated n times, where n ranges from 1 to 32.

[0281] In at least one embodiment, but not limited to, compound 100 can be represented by the formula: Q-L-T, where Q is the group of the folate receptor-binding ligand / targeting moiety 104, L is the linker 106, and T is the group of the TLR agonist / immunomodulator 102. The linker L can comprise any of the linker formulas presented herein.

[0282] Similarly, Figure 1 B shows at least one embodiment of compound 150. Compound 150 has an immunomodulator / drug (e.g., its group) 152, which is a TLR7 agonist (e.g., its group), conjugated to the targeting moiety (e.g., its group) 154 through a (e.g., cleavable) linker 156.

[0283] The linker (L or L n) can be releasable or non-releasable. In some cases, for example, the target of a compound containing a non-releasable linker is an endosome (e.g., of the cell of interest), whereas the target of a releasable linker is, in some cases, an endosome, cytoplasm, or both (e.g., of the cell of interest).

[0284] In at least one exemplary embodiment, linker L n is disposed between a targeting moiety (e.g., its group) and an immunomodulator or a pharmaceutically acceptable salt thereof, wherein the linker L or L n is configured to avoid release of the free form of the TLR7 agonist, and n is an integer equal to or less than 50. Additionally or alternatively, the compound can comprise a linker L n that contains PEG or a PEG derivative, n can be an integer selected from the range of 1 to 32, and the targeting moiety (e.g., its group) can comprise a group containing a folate receptor binding ligand that contains an FRβ binding ligand.

[0285] In the context of a linker, the term "releasable" means a linker that includes at least one bond that can be cleaved under physiological conditions (e.g., by chemical or enzymatic hydrolysis) such as by a labile, pH-labile, acid-labile, base-labile, oxidation-labile, metabolically-labile, biochemically-labile, enzymatically-labile, or para-aminobenzyl-type multivalent cleavable bond. It should be understood that the physiological conditions that result in bond cleavage do not necessarily include biological or metabolic processes, but can include standard chemical reactions (e.g., hydrolysis reactions) at physiological pH or due to compartmentalization into an organelle (such as an endosome having a lower pH than cytoplasmic pH). The cleavable bond can connect two adjacent atoms within the releasable linker and / or, for example, connect other linker moieties or a targeting moiety and / or a drug as described herein at either or both ends of the releasable linker. In some cases, the releasable linker is cleaved into two or more fragments. In some cases, the releasable 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.

[0286] In contrast, in the context of a linking group, the term "non-cleavable" is meant to include a linking group comprising at least one bond that is not readily or rapidly cleaved under physiological conditions. In some embodiments, the non-cleavable linking group comprises a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (e.g., aqueous hydrolysis or enzymatic hydrolysis)). In some embodiments, the compositions provided herein that comprise a non-cleavable linking group do not release any components of the composition (e.g., a targeting ligand (e.g., a fully amorphous (FA)-ligand) or an immunomodulator (e.g., a TLR7 agonist). In some embodiments, the non-cleavable linking group lacks disulfide bonds (e.g., S-S) or esters in the backbone. In some embodiments, the composition comprises a targeting moiety and an immunomodulator linked by a backbone that is substantially stable throughout the duration of the composition's circulation (e.g., during endocytosis into the target cell endosome). In some embodiments, the composition comprising a non-cleavable linking group is particularly beneficial when the immunomodulator targets TLR, NOD-like receptors, and / or other pattern recognition receptors present in the endosome. Non-cleavable linking groups can comprise: amides, esters, ethers, amines, and / or thioethers (e.g., thio-maleimide). While specific examples are provided herein, it should be understood that any molecule(s) can be used in the non-cleavable linking group provided that at least one bond is formed that is not readily or rapidly cleaved under physiological conditions.

[0287] Perhaps more specifically, the non-cleavable linking group comprises a linking group that will hydrolyze in an aqueous solution (e.g., a buffered solution (e.g., phosphate buffered saline)) in a period of time (e.g., 24 hours) at a neutral pH, for example, less than ten 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%). In some embodiments, in the case of using a non-cleavable linking group, less than about ten percent (10%) and preferably less than five percent (5%) or zero of the conjugated compound administered releases free drug (e.g., in the systemic circulation prior to uptake by the targeted cell / tissue). In some embodiments, less than five percent (5%) of the free drug is released from the conjugate within one (1) hour of administration when the compound is in the systemic circulation.

[0288] In some embodiments, the targeting moiety does not cleave a therapeutically effective compound in vivo. In some embodiments, this is advantageous because it allows the use of targeted compositions that include a potent drug (e.g., a TLR7 agonist), for example because only a negligible amount (if any) of the drug (e.g., an immunomodulator, e.g., a TLR7 agonist) is released (e.g., systemically) prior to targeted delivery of the compound. In some embodiments, modulating the release profile of the active ingredient is a difficult aspect of preparing an effective pharmaceutical composition. In some embodiments, compositions that include the non-cleavable linkers provided herein avoid the difficulties of preparing an effective pharmaceutical composition (e.g., by removing the need to time the release). In some embodiments, the immunomodulator or warhead of the compounds provided herein is active when bound (e.g., conjugated to a targeting conjugate). In some embodiments, when the warhead / immunomodulator is active, the non-cleavable linker and the targeting moiety prevent (e.g., by the subject's body) the release of toxic cytokines (e.g., interleukin 6 (IL-6)) that activate the immune system (e.g., because the compound is specifically targeted (e.g., using folate or an analogue thereof)). In certain instances, e.g., even when the warhead / immunomodulator of the compound is active when linked to the non-cleavable linker, the immunomodulator cannot access the appropriate (e.g., targeted) receptor in the endosome until the compound binds to the targeted receptor (e.g., the folate receptor).

[0289] As a non-limiting example, Figure 1 Linker 106 of A is a non-cleavable PEG linker, whereas Figure 1 Linker 156 of B is a self-sacrificing cleavable linker (e.g., comprising a disulfide bond (e.g., S-S)). For example, Figure 1 The scheme shown for B demonstrates the self-sacrificing cascade of compound 150 after cleavage from the targeting moiety 154.

[0290] In some embodiments, the linker 156 is formed such that the drug is cleaved from the targeting moiety 154 only after sufficient time has elapsed for the compound to circulate in the systemic circulation of the subject following administration (e.g., cleared from non-target tissues and captured and internalized by the target cells and / or receptors). In some embodiments, the period of release will vary (e.g., between different subjects (e.g., based on a variety of factors)). In some embodiments, the cleavable linker can be engineered such that it is not cleaved / released until at least 24 hours after administration or even over a period of a week. In some embodiments, the compound can safely traverse the subject's body, and any amount not captured by the target cells (e.g., those expressing FRβ) can be excreted prior to release / activation, thereby preventing toxicity (e.g., because the immunomodulator is inactive when bound to the cleavable linker).

[0291] Both cleavable and non-cleavable linkers can be engineered to optimize (e.g., of the compound) biodistribution, bioavailability, and PK / PD and / or increase (e.g., of the compound) uptake in the target tissue according to methods known in the art or developed below such as by PEGylation and the like. In some embodiments, the linker is configured to avoid significant release in circulation of a pharmaceutically active amount of the drug prior to being captured by a cell (e.g., the cell of interest (e.g., macrophages in fibrotic or cancerous tissue to be treated)).

[0292] In some embodiments, a compound comprising the cleavable linker of the present disclosure can be designed to diffuse across the membrane of an endosome and, for example, into the cytoplasm of the target cell. In some embodiments, the cleavable linker can be designed such that the immunomodulator is not released until the compound reaches the cytoplasm.

[0293] In some embodiments, the conjugates provided herein can comprise a cleavable linker (e.g., to facilitate release of the immunomodulator in the cytoplasm, e.g., where the immunomodulator comprises a PI3K kinase, IRAK, or kinase activator (e.g., using Prostratin etc.) or nuclear factor kappa-light-chain-enhancer of activated B cells (see, for example, Table 1) or a myeloid differentiation primary response factor 88 (MyD88) agonist). In some embodiments, the cleavable linker prevents release of the immunomodulator, e.g., until after binding of the targeting moiety to the appropriate target (e.g., macrophage folate receptor), internalization into the endosome of the target cell, and / or diffusion into the cytoplasm (e.g., where the desired pattern recognition receptors are located). In some embodiments, the cleavable linker releases the immunomodulator in the endosome.

[0294] In some embodiments, the linkers provided herein can include one or more spacers (e.g., to facilitate a specific release time, promote increased uptake in a target tissue, and / or optimize the biodistribution, bioavailability, and / or PK / PD of the compounds provided herein). The spacer can include one or more of an alkyl chain, PEG, a peptide, a sugar, peptidoglycan, a clickable linker (e.g., a triazole), a rigid linker such as polyproline and poly(piperidine), and the like.

[0295] In some embodiments, a linker comprising PEG 12 significantly reduces (if not completely avoids) the non-specific uptake of the compounds provided herein (e.g., uptake into non-target organs (e.g., uptake into the liver and / or kidney of a subject after administration)). In some embodiments, the compound avoids delivery to the liver and kidney. In some embodiments, the targeting moiety (in its free form, its group, or its conjugate) does not bind to an uptake receptor on a non-target cell (e.g., as long as the organ is not the target site, and thus stimulation of immune complexes in those organs can be avoided, which is highly beneficial in a clinical setting).

[0296] In some embodiments, conjugates provided herein that include a non-cleavable linker reduce or eliminate the toxicity of components released from the conjugate in their free form (e.g., the free form of the compounds and / or ligands provided herein).

[0297] In at least one embodiment, the linker includes 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 to folate via a cleavable linker containing a first hydrophilic spacer):

[0298]

[0299] In some embodiments, the compound has the structure of Formula XIII (e.g., a substructure of a TLR7 agonist of Formula III conjugated to folate via a non-cleavable linker (covalent bond) containing a second hydrophilic spacer):

[0300]

[0301] Specific examples of exemplary conjugated compounds are provided herein.

[0302] In some embodiments, the compounds provided herein comprise a group of a targeting moiety conjugated to a group of an immunomodulator or a pharmaceutically acceptable salt thereof such that the immunomodulator (or a group thereof) or its pharmaceutically acceptable salt remains pharmaceutically active upon conjugation. The targeting moiety can comprise any of the targeting moieties described herein and, in at least one embodiment, comprises a folate ligand, any other folate receptor-binding molecule (e.g., or a functional fragment or analogue of any of the foregoing), or a pyrido[2,3-d]pyrimidine analogue. In some embodiments, the targeting moiety (or its conjugate or group) is specific for FRβ.

[0303] In some embodiments, the compounds provided herein comprise one or more linkers, wherein the group of the targeting moiety is conjugated to the group of the immunomodulator via the one or more linkers. For example, in the case where the immunomodulator or a pharmaceutically acceptable salt thereof has Formula I or II, the group of the immunomodulator can be conjugated to the group of the targeting moiety via a linker or directly at one of R 1 、R 2 or R 3 . Similarly, in the case where the immunomodulator or a pharmaceutically acceptable salt thereof has Formula III, the group of the immunomodulator can be conjugated to the group of the targeting moiety via a linker or directly at one of R 1 or R 3 . Alternatively, in the case where the immunomodulator or a pharmaceutically acceptable salt thereof has Formula IV, the group of the immunomodulator can be conjugated to the group of the targeting moiety via a linker or directly at one of R 1 or R 2 . As described herein, the linker can be releasable or non-releasable.

[0304] In some embodiments, one or more linkers of the compounds provided herein can comprise PEG, a PEG derivative, or any other linker known in the art or developed hereinafter that can achieve the purposes set forth herein. In some embodiments, the linker can be repeated n times, where n is a positive integer. For example, but not limited to, n can be any integer selected from the range 1-16, 1-32, 1-64, or 1-96. The number of repeats of the linker (i.e., n) can be selected to achieve the desired functionality, size, and / or potency of the compound and / or in view of the desired application. In some embodiments, one or more of the linkers comprise one or more spacers (e.g., which can also be used to specifically engineer the properties of the compound).

[0305] In some embodiments, the linking group is a hydrolysable linking group. In some embodiments, the linking group is a non-hydrolysable linking group. In some embodiments, the linking group is an optionally substituted heteroalkyl. In some embodiments, the linking group is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxy, oxo, PEG, carboxylate and halo. In some embodiments, the linking group comprises a spacer (e.g., as described elsewhere herein).

[0306] In some embodiments, the linking group is a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, the linking group is a peptide having at least one disulfide bond in its backbone.

[0307] In some embodiments, the linking group comprises -CONH-CH(COOH)-CH2-S-S-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 [[ID=?]]R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-S-S-CH2-CR a R b -O-CO-, wherein R a and R b are independently H, alkyl or heteroalkyl (e.g., PEG).

[0308] In some embodiments, the linking group comprises the following structure:

[0309]

[0310]

[0311] wherein n and m are each independently from 0 to 10.

[0312] In some embodiments, the linking group comprises the following structure:

[0313]

[0314] wherein n and m are each independently from 0 to 10.

[0315] In some embodiments, the linking group comprises the following structure:

[0316] It should be noted that there seems to be a typo in the original text where "R " appears multiple times without clear context. The above translation attempts to make sense of the overall text based on the provided rules. If there are any specific clarifications or corrections needed for the original text, it would be beneficial for a more accurate translation.

[0317] where n is from 1 to 32.

[0318] In some embodiments, the linker comprises the following structure:

[0319]

[0320]

[0321] where n is from 1 to 16.

[0322] It should be understood that any combination of groups of the compound (e.g., groups of the compounds in either Table 1 or Table 2), the linker (e.g., as provided herein), and the ligand (e.g., groups of the ligands in any of Tables 3 - 6) can be combined to form the conjugates provided herein. In some embodiments, the group of the compound or the ligand is a carbon atom or a heteroatom (e.g., O, S, N, etc.). In some embodiments, the group of the compound is C or O. In some embodiments, the group of the ligand is C or O. In some embodiments, the attachment points of the compound and the ligand are determined by the placement of the groups (e.g., via the linker). In some embodiments, the linker comprises a spacer (e.g., as described elsewhere herein). It should also be understood that any conjugate provided herein can be synthesized in a similar process as provided in the methods provided in the examples.

[0323] Non - limiting examples of the conjugates provided herein are provided in Table 7.

[0324] Table 7. Examples of conjugates.

[0325]

[0326]

[0327]

[0328]

[0329] Non - limiting examples of the conjugates provided herein are provided in Table 8.

[0330] Table 8. Further examples of conjugates

[0331]

[0332]

[0333]

[0334]

[0335] In some instances, the conjugate compounds provided herein have the structure of Formula XIV (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula III conjugated to folate via a cleavable linker):

[0336]

[0337] In another embodiment, the conjugate compounds provided herein have the structure of Formula XV (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula II conjugated to folate via a cleavable linker (e.g., Compound 3B)):

[0338]

[0339] In yet another embodiment, the conjugate compounds provided herein have the structure of Formula XVI (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula II conjugated to folate via a non-cleavable linker comprising three PEGs (e.g., Compound 3D)):

[0340]

[0341] In still another embodiment, the conjugate compounds provided herein have the structure of Formula XVII (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula II conjugated to folate via a non-cleavable linker comprising twelve PEGs (e.g., Compound 3C)):

[0342]

[0343] Additional embodiments of the conjugate compounds provided herein have the structure of Formula XVIII (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula II conjugated to folate via a non-cleavable linker comprising sixteen PEGs (e.g., Compound 3D')):

[0344]

[0345] Additional embodiments of the conjugate compounds provided herein have the structure of Formula XIX (e.g., or a functional fragment or analogue thereof, including a TLR7 agonist of Formula III (Compound 1B) conjugated to folate):

[0346]

[0347] The compounds described herein can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. The general reaction sequences outlined below represent general methods that can be used to prepare the compounds of the present disclosure and are not intended to limit the scope or utility.

[0348] The description of the compounds of the present invention is limited by the principles of chemical bonding known to those skilled in the art. Thus, in instances where a group may be substituted by one or more of a number of substituents, such substitutions are chosen so as to conform to the principles of chemical bonding and to give compounds that are not inherently unstable and / or that are not known to be unstable to environmental conditions such as aqueous, neutral, and several known physiological conditions by those of ordinary skill in the art. For example, in accordance with the principles of chemical bonding known to those skilled in the art, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom so as to avoid inherently unstable compounds.

[0349] In the context of two or more polypeptide sequences, the term “identical” or “identity” percentage means that two or more sequences or subsequences are identical or that a specified percentage of the peptides are identical (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence in a comparison window or specified region such as a targeting end, folate end, linker, or warhead), as measured using sequence comparison algorithms known in the art or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” In other words, identity exists over one or more regions of the entire sequence, so long as the general shape and structure of the molecule and (where appropriate) hydrogen bonding are maintained such that it is substantially suitable for binding to the targeting site and acts as its agonist.

[0350] The compounds described herein may be administered in unit dosage forms and / or in compositions containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. As used herein, the term “administer” and words formed therefrom generally refer to any and all means of introducing the compounds described herein into a host subject, including but not limited to oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and similar routes of administration.

[0351] It may be appropriate to administer the compounds of the present disclosure as salts. Examples of acceptable salts include, but are not limited to, salts of alkali metals (e.g., sodium, potassium, or lithium) or alkaline earth metals (e.g., calcium); however, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable. Similarly, "pharmaceutically acceptable salts" refers to those salts having counterions that can be used in a drug. Such salts can include, but are not limited to: (1) acid addition salts, which can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as 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, or malonic acid; or (2) 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 metal ion, or an aluminum ion), or complexed with an organic base (such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc.). Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salts can be considered in connection with the embodiments described herein.

[0352] Acceptable salts can be obtained using standard procedures known in the art, which include (but are not limited to) reacting a sufficiently acidic compound with a suitable base that provides a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form non-toxic salts. Exemplary but non-limiting examples include salts of acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hydroxybenzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methanesulfate, naphthylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / monohydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, toluenesulfonate, and trifluoroacetate. Suitable base salts of the compounds described herein are formed from bases that form non-toxic salts. Exemplary but non-limiting examples include salts of arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc. Hemisalts of acids and bases can also be formed, e.g., hemisulfates and hemicalcium salts.

[0353] As used herein, the term "composition" generally refers to any article that contains more than one component, including the compounds described herein. It should be understood that the compositions described herein can be prepared from the isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It should be appreciated that certain functional groups (such as hydroxyl, amino, and like groups) can form complexes with water and / or various solvents in various physical forms of the compound. It should also be understood that the compositions can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other morphological forms of the compounds described herein, and the compositions can be prepared from various hydrates and / or solvates of the compounds described herein. Thus, the pharmaceutical compositions of the compounds described herein include each of the various morphological forms and / or solvate or hydrate forms of the compounds described herein, or any combination or individual form thereof.

[0354] The compounds of the present disclosure can be formulated into pharmaceutical compositions and administered to a mammalian host (such as a human patient) in various forms suitable for the selected route of administration. For example, the pharmaceutical compositions can be formulated for and administered orally or parenterally, intravenously, intraarterially, intraperitoneally, intrathecally, epidurally, intraventricularly, intraurethrally, intrasternal, intracranially, intratumorally, intramuscularly, topically, by inhalation, and / or subcutaneously. In fact, in at least one embodiment, the compounds and / or compositions as described herein can be administered directly into the bloodstream, muscle, or internal organ.

[0355] For example, in at least one embodiment, the compounds of the present invention can be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. The percentages of the compositions and formulations can vary and can be between about 1% and about 99% by weight of the active ingredient(s) and binder, excipient, disintegrant, lubricant, and / or sweetening agent (as known in the art). The amount of the active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0356] 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 compounds available for preparing parenteral compositions can be increased by using appropriate formulation techniques such as incorporating solubility enhancers.

[0357] As described above, the compounds / compositions of the present disclosure can also be administered via infusion or injection (e.g., using a needle (including a microneedle) syringe and / or a needleless syringe). The solution of the active composition can be aqueous, optionally mixed with a non-toxic surfactant and / or can contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at a pH from 3 to 9), but for some applications, they can be more suitably formulated as a sterile non-aqueous solution or in a dry form for use in combination with a suitable vehicle such as sterile, pyrogen-free water or phosphate buffered saline (PBS). For example, dispersions can be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof and in oils. Under conventional storage and use conditions, these formulations can also contain preservatives to prevent the growth of microorganisms.

[0358] Dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or insoluble solutions or dispersions, optionally encapsulated in liposomes. In all cases, the final dosage form should be a sterile liquid and stable under the conditions of production and storage. The liquid carrier or vehicle can be a solvent or a liquid dispersion medium, which includes, for example, but is not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glycerides, and / or suitable mixtures thereof. In at least one embodiment, the appropriate fluidity can be maintained by forming liposomes, in the case of a dispersion, by maintaining the desired particle size, or by using surfactants. The action of microorganisms can be prevented by adding various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it is necessary to include one or more isotonic agents such as sugars, buffers, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by incorporating agents formulated to delay absorption (such as aluminum monostearate and gelatin).

[0359] A sterile injectable solution can be prepared by incorporating the active compound and / or composition in an appropriate solvent in the required amounts with one or more of the other ingredients as set forth above, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional required ingredients present in its previously sterile-filtered solution.

[0360] For topical administration, it may be desirable to administer the compounds of the invention as a composition or formulation in combination with a dermatologically acceptable carrier, which can be solid or liquid. For example, in certain embodiments, solid carriers can include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Similarly, useful liquid carriers can comprise water, alcohols or diols, or water / alcohol / ethylene glycol mixtures in which the compounds of the invention are soluble or dispersible at effective levels, optionally with the aid of non-toxic surfactants. Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents can be added to optimize the properties for a given use. The resulting liquid compositions can be applied from absorbent layers, used to impregnate bandages and / or other dressings, sprayed onto the target area using a pump-type or aerosol sprayer, or simply applied directly to the desired area of the subject.

[0361] Thickeners (such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials) can also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, etc. for direct application to the skin of the subject.

[0362] As used herein, the terms "therapeutically effective," "therapeutically effective dose," "therapeutically effective amount," "preventively effective amount," or "preventively effective dose" mean (unless otherwise specifically stated) an amount of a compound that, when administered once or over the course of a treatment regimen, affects the health, well-being, or mortality of a subject (e.g., but not limited to delaying the onset and / or reducing the severity of one or more symptoms associated with a fibrotic disease or condition and / or cancer, as applicable). Useful doses of the compounds of the present disclosure can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective doses in mice and other animals are known in the art. In fact, the dose of a compound can vary significantly depending on the condition of the host subject, the cancer or fibrotic disease being treated, how the pathology progresses, the route of administration and tissue distribution of the compound, and the likelihood of co-use of other therapeutic treatments (such as additional drugs in radiation therapy or combination therapy). The amount of the composition required for treatment (e.g., a therapeutically or preventively effective amount or dose) will vary not only with the particular application but also with the salt selected (if applicable) and the characteristics of the subject (e.g., age, condition, gender, body surface area and / or mass of the subject, tolerance to the drug), and will ultimately be determined by the attending physician, clinician, or other factors. A therapeutically or preventively effective amount or dose can be in the range of, for example, from about 0.05 mg / kg of patient body weight to about 30.0 mg / kg of patient body weight, or from about 0.01 mg / kg of patient body weight to about 5.0 mg / kg of patient 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 per kg of patient body weight. The total therapeutically or preventively effective amount of the compound can be administered as a single dose or in divided doses and can fall outside the typical ranges given herein, depending on the decision of the practicing physician.

[0363] In another embodiment, the compound can be administered in a therapeutically or preventively effective amount of from about 0.5 g / m to about 500 mg / m 2 、from about 0.5 g / m 2 to about 300 mg / m 2 、or from about 100 g / m 2 to about 200 mg / m 2 . In other embodiments, the amount can be from about 0.5 mg / m 2to about 500 mg / m 2 from about 0.5 mg / m 2 to about 300 mg / m 2 from about 0.5 mg / m 2 to about 200 mg / m 2 from about 0.5 mg / m 2 to about 100 mg / m 2 from about 0.5 mg / m 2 to about 50 mg / m 2 from about 0.5 mg / m 2 to about 600 mg / m 2 from about 0.5 mg / m 2 to about 6.0 mg / m 2 from about 0.5 mg / m 2 to about 4.0 mg / m 2 or from about 0.5 mg / m 2 to about 2.0 mg / m 2 The total amount can be administered as a single dose or in divided doses and can fall outside the typical ranges given herein at the discretion of the physician. These amounts are based on m of body surface area.

[0364] In some embodiments, with regard to measuring the expression of certain biomarkers in a sample from a subject and / or the analysis of cytokine levels, the significance of the present disclosure is not the particular method for detecting a biomarker or set of biomarkers, but which biomarkers are used for detection. There are many methods available for detecting the expression, quantification, or profile of one or more biomarkers. Once the biomarker or set of biomarkers to be detected or quantified has been identified, any of several techniques (now known or developed hereinafter) can be used provided the appropriate reagents are available. When one or more biomarkers to be identified are provided, those skilled in the art will be able to select the appropriate assay for performing the methods disclosed herein (e.g., nucleic acid biomarker assays based on PCR or microarray detection, enzyme-linked immunosorbent assay (ELISA), protein or antibody microarrays, or similar immunoassays, etc.).

[0365] Chemical Examples

[0366] Example A: Synthesis of Compound 1A

[0367] Compound 1A was synthesized according to Scheme 1 below and as reported in the following literature: 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. February 9, 2012; 55(3):1106-1116.

[0368] Scheme 1

[0369]

[0370] Step 1: Synthesis of 1-amino-2-methylpropan-2-ol (Compound)

[0371] 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 h. The solvent was removed in vacuo and the residue was dissolved in methanol. Di-tert-butyl dicarbonate (0.75 g, 3.47 mmol) was added to the reaction mixture and stirred for 4 h. The mixture was purified by column chromatography (24% ethyl acetate (EtOAc) / hexane) to afford tert-butyl 2-hydroxy-2-methylpropylcarbamate. The pure tert-butyl 2-hydroxy-2-methylpropylcarbamate was dissolved in 5 mL of trifluoroacetic acid and stirred for 35 min. The solvent was removed under reduced pressure to afford 1-amino-2-methylpropan-2-ol as the trifluoroacetate 1'. 1H NMR 500 MHz (500 MHz, CDCl3, δ (in ppm)): δ 8.62 (s, 2H), 3.02 (d, 2H), 2.06 - 2.04 (m, 2H), 1.37 - 1.34 (s, 6H).

[0372] Step 2: Synthesis of 2-methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (Compound 2)

[0373] The trifluoroacetate of l-amino-2-methylpropan-2-ol (compound) (450 mg, 2.4 mmol) 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 half an hour until a solid began to precipitate. 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-l-(3-nitroquinolin-4-ylamino)propan-2-ol (compound 2). Liquid chromatography–mass spectrometry (LCMS) analysis: [M+H] + m / z = 261.

[0374] Step 3: Synthesis of l-(3-aminoquinolin-4-ylamino)-2-methylpropan-2-ol (compound 3)

[0375] 2-Methyl-l-(3-nitroquinolin-4-ylamino)propan-2-ol (compound 2) (450 mg, 1.72 mmol) was dissolved in methanol and hydrogenated with a hydrogen balloon for 4 hours using Pd / C as a catalyst. The solution was then filtered through diatomaceous earth, and the solvent was subsequently evaporated under reduced pressure to afford l-(3-aminoquinolin-4-ylamino)-2-ethylpropan-2-ol (compound 3). LCMS: [M+H] + m / z = 231. ΗNMR 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).

[0376] Step 4: Synthesis of l-(4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)-2-methylpropan-2-ol (compound 5, TLR7A)

[0377] To a solution of compound 3 (100 mg, 0.43 mmol) in anhydrous THF was added triethylamine (66 mg, 0.65 mmol) and valeryl chloride (62 mg, 0.52 mmol). The reaction mixture was then stirred for 6 - 8 h and then the solvent was removed in vacuo. The residue was dissolved in EtOAc, washed with water and brine, and then dried over Na2SO4 to obtain the intermediate amide compound. It was dissolved in methanol (MeOH), calcium oxide was then added, and it was microwave heated at ¹¹⁰ °C for 1 h. Then the solvent was removed and the residue was purified by 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) was added 3-chloroperoxybenzoic acid (84 mg, 0.49 mmol), and the solution was refluxed at 45 - 50 °C for 40 min. Then the solvent was removed and the residue was purified by column chromatography (20% MeOH / dichloromethane) to obtain the oxide derivative (55 mg). It was then dissolved in anhydrous dichloromethane, benzoyl isocyanate (39 mg, 0.26 mmol) was then added, and it was heated at 45 °C for 15 min. Then the solvent was removed in vacuo and the residue was dissolved in anhydrous MeOH, and then an excess of sodium methoxide was added. Then the reaction mixture was heated at 80 °C for one hour. The solvent was removed in vacuo and the residue was purified by column chromatography (11% MeOH / dichloromethane) to obtain compound 5. LCMS: [M+H] + m / z = 312. ¹H NMR 500 MHz (CDCl₃, 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).

[0378] Example B: Synthesis of Compound 1B

[0379] Compound 1A can then be used to synthesize compound 1B according to Scheme 2 below.

[0380] Scheme 2

[0381]

[0382] Compound 1A, folate, and the linker are commercially available or can be prepared by methods known to those skilled in the art.

[0383] At room temperature under a nitrogen atmosphere, the 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 dichloromethane, and the mixture was stirred at reflux temperature for 7 hours, at which time thin layer chromatography (TLC) analysis of the mixture indicated >80% conversion. The mixture was concentrated and purified by column chromatography using 10% acetonitrile in dichloromethane 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 3 portions at 20-minute intervals to a solution of the drug-linker intermediate compound 9 (1.0 equivalent - 1.5 equivalents) in DMSO with dimethylaminopyridine (1 equivalent). After stirring for 1 - 2 hours under argon at room temperature, LCMS analysis of the mixture indicated the formation of the desired folate-drug conjugate (compound 10) as the major product. The mixture was purified by preparative high performance liquid chromatography (HPLC). LCMS: [M+H] + m / z = 959. 1 H 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).

[0384] Example C: Synthesis of Compound 2A

[0385] Compound 2A can be synthesized according to Scheme 3 and Scheme 4.

[0386] Scheme 3

[0387]

[0388] Scheme 4

[0389]

[0390] The cysteine-loaded Wang resin (11) was initially deprotected using 20% piperidine in dimethylformamide (DMF). The free amine was treated with Fmoc-Glu(OtBu)-COOH in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), N,N-diisopropylethylamine (DIPEA), and DMF. The coupling product was deprotected using 20% piperidine in DMF and treated with pteroic acid in the presence of PyBop, DIPEA, and DMF to yield compound 12. The trifluoroacetyl group was deprotected using a 50% ammonia-DMF solution. Finally, the resin was cleaved using a solution of trifluoroacetic acid:triisopropylsilane:water:tris(2-carboxyethyl)phosphine mixture and purified using HPLC to afford folate-cysteine (13) as a yellow solid.

[0391] Compound 14 was initially treated with an isobifunctional linker reagent (15) to afford a folate-cysteine disulfide intermediate (16). This was then reacted with folate-cysteine (13) in DMSO and purified using HPLC to produce compound 17 (e.g., compound 2A). All compounds were characterized by LCMS using ammonium bicarbonate and acetonitrile as the buffer system. The LCMS observed mass for compound 2A was [M+H]+ = 1082.2.

[0392] In view of the state of the art in the relevant field, and in particular in accordance with Schemes 1, 2, and 3 set out above, the present disclosure provides sufficient detail such that a person of ordinary skill in the art can utilize the concepts set out herein to synthesize all other compounds of the present disclosure.

[0393] Therapeutic methods

[0394] In addition to the compounds described herein, methods for providing treatment and / or prophylaxis of fibrotic diseases or cancer are provided.

[0395] In some embodiments, provided herein are methods of treating a subject having a fibrotic disease state or cancer, the methods comprising contacting cells of the subject with any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutical) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, any compound provided herein such as a compound having a structure of any one of 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 or Formula XXX. In some embodiments, the immunomodulator comprises an agonist of TLR7, TLR8 or TLR9.

[0396] In some embodiments, provided herein are methods of treating a neoplastic, inflammatory, autoimmune or fibrotic disease or disorder in an individual in need thereof, the methods comprising administering to the individual any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutical) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, any compound provided herein such as a compound having a structure of any one of 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 or Formula XXX.

[0397] In some embodiments, provided herein are methods of treating a neoplastic disease or disorder in an individual in need thereof, the methods comprising administering to the individual any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutical) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, any compound provided herein such as a compound having a structure of any one of 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 or Formula XXX. In some embodiments, the neoplastic disease or disorder is cancer. In some embodiments, the cancer is selected from bladder cancer, brain cancer, liver cancer, kidney cancer, skin cancer, thymic cancer, gastrointestinal stromal tumor (GIST), esophageal cancer, pancreatic cancer, and breast cancer.

[0398] In some embodiments, provided herein are methods of treating a fibrotic disease or disorder in an individual in need thereof, the method comprising administering to the individual any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutical) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, any compound provided herein such as a compound having a structure of any one of 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 or Formula XXX. In some embodiments, the fibrotic disease or disorder is fibrosis. In some embodiments, the fibrosis is selected from IPF, fatty liver disease, cirrhosis, colitis, chronic liver disease, cardiac fibrosis, and scleroderma.

[0399] In certain embodiments, provided herein are methods of preventing or treating a fibrotic disease state, the method comprising contacting a cell with at least one compound, the at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt thereof attached via a linker to a folate ligand or a functional fragment or analogue thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor.

[0400] In some embodiments of the compounds, compositions, and / or methods provided herein, the immunomodulator comprises a TLR agonist and has a structure represented by Formula X or XX, or is a pharmaceutically acceptable salt of Formula X or XX:

[0401]

[0402]

[0403] wherein in Formula X and XX:

[0404] R1 is -NH2 or -NH-R 1X ,

[0405] R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl,

[0406] -NH-R 2X , -O-R 2X , -S-R 2X , and

[0407] is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle;

[0408] wherein in Formula X, R3 is -OH, -SH, -NH2 or -NH-R 1X ;

[0409] wherein in formula XX, X is CH, CR2 or N; and

[0410] R 1X 、R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl and heteroaryl.

[0411] In some embodiments, the cells comprise cells of a subject who has experienced or is at risk of experiencing a fibrotic disease state, and contacting the cells with at least one compound further comprises administering or applying a therapeutically effective amount of the 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 by intravenous, intramuscular, intraperitoneal, topical or inhalation routes. In some embodiments, the fibrotic disease state includes IPF or fibrotic diseases of the liver, skin, bladder, heart, pancreas, prostate or kidney.

[0412] In some embodiments, the method further comprises obtaining or having obtained a sample from the subject; quantifying the expression level of one or more biomarkers in the sample, each of the one or more biomarkers being selected from the group consisting of: CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), tissue inhibitor of metalloproteinase 3 (TIMP 3), IL-1β, hydroxyproline, collagen, PDGF, TGFβ, FRβ, TNFα, IFN-γ, anti-mannose receptor (CD206), cluster of differentiation 86 (CD86), cluster of differentiation 163 (CD163), IL-6, chemokine 10 (CXCL10), 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 if the expression level of CCL18, Arg1, MMP9, TIMP 3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline or collagen is upregulated relative to the control or the expression level of TNFα, IFN-γ, IL-6, CXCL10, IFNα or CD86 is downregulated or not expressed relative to the control, administering or having administered a therapeutically effective amount of an unconjugated agonist or inhibitor to the subject. In some embodiments, the folate ligand or a functional fragment or analogue thereof is specific for FRβ and binds to FRβ on the cell.

[0413] In at least one embodiment, methods are provided for treating and / or preventing fibrotic diseases (e.g., IPF). The methods include administering to the subject a therapeutically effective amount of one or more compounds, the one or more compounds comprising a targeting moiety (such as a folate receptor-binding ligand) attached (via a linker or otherwise) to a drug for reprogramming M2-like macrophages in fibrotic tissue or organ to an M1-like phenotype. For example, the drug can be a toll-like receptor agonist (e.g., having Formula I, III, or IV) or any other molecule or compound conjugated to folate that is effective to reprogram macrophages from an M2 phenotype to an M1 phenotype. In at least one embodiment, the drug can be selected from TLR 3 agonists, TLR7 agonists, TLR 7 / 8 agonists, TLR8 agonists, and TLR9 agonists. In some embodiments, the drug can reprogram M2-like macrophages to an M1 phenotype, thereby reducing the production of profibrotic cytokines and growth factors.

[0414] In at least one embodiment, methods are provided for treating a subject having a disease state or at risk of experiencing a disease state, wherein the disease state includes a fibrotic disease state or cancer, and the method includes contacting cells of the subject with at least one compound. The at least one compound can comprise any compound of the present disclosure and, in at least one exemplary embodiment, comprises a targeting moiety specific for FRβ. In some instances, contacting the cells can be accomplished by administering the at least one compound to the subject intravenously, intramuscularly, intraperitoneally, topically, orally, or by inhalation or any other mode of administration described herein. Additionally or alternatively, the at least one compound can comprise a composition containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles or combinations thereof. The dose of the at least one compound administered can be modified at the discretion of the clinician; however, the at least one compound is preferably administered in a therapeutically effective or prophylactically effective amount and, in at least one embodiment, the dose ranges between 1 nmol / kg of subject body weight and 50 nmol / kg of subject body weight.

[0415] Now referring to Figure 2, the use of one or more of the compounds of the present disclosure is shown in a flowchart representing method 1900 for preventing or treating a fibrotic disease state or cancer. In at least one scenario, method 1900 includes the step of contacting a cell of a subject with at least one compound, the at least one compound comprising an immunomodulator (or a pharmaceutically acceptable salt thereof) attached via a linker to a folate ligand or a functional fragment or analogue thereof (step 1902). In at least one exemplary embodiment, the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor. The cell can include, for example, a cell of a subject experiencing a fibrotic disease state or at risk of experiencing a fibrotic disease state, and the at least one compound can include any of the compounds provided herein.

[0416] In at least one embodiment, step 1902 of contacting the cell with at least one compound further includes administering or applying to the subject a therapeutically effective amount of the at least one compound. Additionally or alternatively, the at least one compound can comprise a composition containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles or combinations thereof.

[0417] In at least one embodiment, the disease state includes IPF or a fibrotic disease of the liver, skin, heart, or kidney. Additionally, the subject can include a mouse, a human, or any other mammal.

[0418] In addition to step 1902, method 1900 can optionally include steps 1904 - 1910. In step 1904, a biological sample is obtained from the subject, and in step 1906, the expression level of one or more biomarkers in the sample is quantified. For example, the sample can be obtained from a quantity of peripheral blood drawn from the subject.

[0419] The quantification step 1906 can be performed using any suitable method known in the art and can include, for example, qPCR, mass spectrometry, ELISA, and / or any other modality capable of measuring / quantifying biomarker expression. In at least one exemplary embodiment, the one or more biomarkers are selected from the group consisting of: CCL18, Arg1, MMP9, TIMP 3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, TNFα, IFN-γ, CD206, CD163, IL-6, CXCL10, IFNα, and CD86.

[0420] In step 1908, the expression level of each of the one or more biomarkers in the sample is compared with the expression level of such biomarkers in the control. The control can be a healthy individual or an individual who has not experienced the disease state in question. In at least one embodiment, the clinical difference between the expression level of one or more biomarkers in the sample and the expression level of one or more related biomarkers in the control can indicate that the subject has the disease state in question. For example, but not limited to, if the comparison step 1908 indicates 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., "pro-fibrotic biomarkers") is upregulated compared to the control, it indicates that the subject experiences a pro-fibrotic immune response associated with the M2-like macrophage phenotype. Therefore, in at least one embodiment, such results indicate that it is necessary to administer one or more compounds of the present disclosure to reprogram such M2-like macrophages to the M1 phenotype.

[0421] Conversely, if the comparison step 1908 indicates that the expression of the aforementioned biomarkers is downregulated as compared to the control, or if the expression of one or more of TNFα, IFN-γ and / or CD86 ("anti-fibrotic biomarkers") is upregulated as compared to the control, then in certain embodiments this indicates that the subject exhibited a positive response to the previously administered compound (if applicable) and / or that the subject is experiencing an anti-fibrotic immune response associated with an M1 phenotype.

[0422] Optionally, in step 1910, if the expression of one or more pro-fibrotic biomarkers in the sample is upregulated compared to the corresponding expression levels in the control, or if the expression of one or more anti-fibrotic biomarkers is downregulated compared to the corresponding expression levels in the control, an alternative therapy can be administered. In at least one embodiment, an alternative therapy can include administering a therapeutically effective amount 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 different targeting moieties, different linker sizes, and / or different immunomodulators, in an attempt to better optimize the efficacy of the at least one compound on the subject. Additionally or alternatively, other treatments can be employed, including those generally known for treating the fibrotic disease in question. Steps 1904-1910 can be repeated as needed or as desired.

[0423] Additional embodiments can provide methods for treating and / or preventing cancer, whether folate receptor positive or folate receptor negative. For example, in certain instances, such methods include administering to a host subject a therapeutically effective amount and / or a prophylactically effective amount of one or more compounds, the one or more compounds comprising a targeting moiety attached (via a linker or otherwise) to a drug to reprogram M2-like macrophages in cancerous cells and / or tumor cells to an M1-like phenotype. In the case where the cancer is folate receptor negative, such administration can additionally serve to deplete or inhibit MDSCs present in such tissues / tumors. By way of example and not limitation, the drug can be selected from PI3k inhibitors, signal transducer and activator of transcription 6 (STAT6) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, inducible nitric oxide synthase (iNOS) inhibitors, and anti-inflammatory drugs (e.g., methotrexate). In at least one embodiment, the drug can inactivate MDSCs.

[0424] In describing representative embodiments, the present disclosure may have presented methods and / or processes in a particular sequence of steps. To the extent that the method or process is not dependent on the particular sequence of steps set forth herein, the method or process should not be limited to the described particular sequence of steps. As will be appreciated by one of ordinary skill in the art, other sequences of steps may be reasonable. Accordingly, the particular sequence of steps disclosed herein should not be construed as a limitation on the claims. Additionally, claims directed to a method and / or process should not be limited to performing their steps in the order written, and one of ordinary skill in the art can readily appreciate that the order may be varied and still remain within the spirit and scope of the present disclosure.

[0425] Examples

[0426] Human monocytic THP-1 cells were obtained from the American Type Culture Collection and cultured in folate-deficient RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). THP-1 cells were initially selected as a model system because this human monocytic cell line is known to acquire an M2-like phenotype and produce large amounts of profibrotic cytokines upon stimulation with IL-4, IL-6 plus IL-13.

[0427] 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.

[0428] Example 1: Differentiation and polarization of THP-1 cells into M2-like macrophages in vitro

[0429] THP-1 cells were seeded into 96-well plates at a density of 60,000 cells / well. The cells were differentiated into non-polarized macrophages by incubation with 200 nM PMA for 48 hours and then in fresh RPMI medium for 24 hours. The resulting macrophages were polarized to an M2-like phenotype by incubation with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / mL IL-6 for 3 days, and then reprogrammed with different concentrations of Compound 1A and Compound 1B for 48 hours and harvested for gene analysis by quantitative polymerase chain reaction (qPCR). The cultures were maintained at 37 °C in a humidified 5% CO2 incubator.

[0430] To evaluate whether a potent TLR7 agonist (e.g., Compound 1A; e.g., of Formula III) can reprogram pro-fibrotic macrophages to a less fibrotic phenotype, IL-4, IL-6 plus IL-13-stimulated THP-1 cells were incubated with different concentrations of non-targeting Compound 1A, and the mRNA levels of several pro-fibrotic markers (i.e., CCL18, CD206, IL-1β, and PDGFα and β) were examined.

[0431] As Figure 3 shown in A-3C, incubation with Compound 1A for 48 hours induced a decrease in the expression of CCL18, CD206, and IL-1β, indicating that the TLR7 agonist can indeed promote the transition of these pro-fibrotic polarized THP-1 cells to a less fibrotic phenotype. Additionally, when the expression of TNFα (a marker of the anti-fibrotic phenotype) was examined, an increase in its expression was observed ( Figure 3 D), confirming the transition of THP-1 from a pro-fibrotic to an anti-fibrotic nature.

[0432] Example 2: Evaluation of macrophage reprogramming

[0433] To confirm that the folate-conjugated TLR7 agonist can cause the same THP-1 reprogramming as seen in Example 1, Compound 1B was prepared, in which a releasable linker that connects folate to Compound 1A was constructed with a disulfide self-immolative bond to allow the release of Compound 1A upon reduction of the intracellular endosomal environment after the internalization of Compound 1B into the cell.

[0434] Different concentrations of Compound 1A or Compound 1B were incubated with the above-polarized THP-1 macrophages for a specified time, after which the culture medium was harvested for analysis of secreted cytokines and the cells were collected for qPCR analysis.

[0435] Total RNA was isolated from 1 x 10 5 – 2 x 10 5 macrophages using the Quick-RNATM MicroPrep kit (Zymo Research, Irvine, CA). The RNA samples were then reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA; #4368814). qPCR analysis was performed using the iTaqTM Universal SYBR Green SuperMix (Bio-Rad Laboratories GmbH, Hercules, CA; #1725121), an iCycler thermal cycler, and the iCycler iQ 3.0 software (Bio-Rad Laboratories GmbH, Hercules, CA) to track the expression of markers that characterize the macrophage polarization state. IL-6, CXCL10, IFNα, IFN-γ, and CD86 were used as markers of the M1 phenotype, while CCL18, CD206, CD163, and Arg1 were used as markers of the M2 phenotype. IL-1β, PDGFβ, MMP9, and TIMP 3, which are indicators of the pro-fibrotic phenotype, were measured. IRAK-4 was used as an indicator of TLR7 stimulation. To control for the specificity of the amplification products, melt curve analysis was performed. No amplification of non-specific products was observed in any of the reactions. Each sample was analyzed independently in triplicate for each marker.

[0436] After repeating the above study (see grey bars, Figure 3 A-3F), the same qualitative changes were observed, with only a slight decrease in the magnitude of the effect of compound 1B. This decrease in potency was expected since the non-targeted TLR7 agonist enters the cultured cells immediately, whereas its folate-targeted analogue was designed to enter the cells only after folate receptor binding and receptor-mediated endocytosis.

[0437] Figure 4 A-4E and Figure 5 A-5D show graphical data representing the levels of various markers measured in THP-1 cells induced to become M2 macrophages, which were then incubated with different concentrations of compound 1B or compound 1A for 2 hours, washed with PBS, and, in order to obtain the data shown in Figure 5 A-5D, incubated for a further 46 hours (for the data shown in Figure 4 A-4E, the cells were harvested immediately after the initial 2-hour incubation). In both datasets, the cells were harvested for gene analysis by qPCR. Figure 4A-4C shows the CCL18 mRNA levels ( Figure 4 A and Figure 5 A), the CD206 mRNA levels ( Figure 4 B and Figure 5 B), the IL-1β mRNA levels ( Figure 4 C and Figure 5 C) and the PDGFβ mRNA ( Figure 4 E). The data support that the M2 pro-fibrotic phenotype is down-regulated after administration of the test compound. In particular, compound 1B down-regulates macrophage pro-fibrotic / M2 type markers more than compound 1A. Additionally, Figure 4 D shows the CD86 mRNA levels and Figure 5 D shows the TNFα levels, and this data supports that the M1-like phenotype is up-regulated after administration of the test compound. At the time of collection, data for PDGFα was not shown because no significant difference was observed after treatment.

[0438] Since low molecular weight water-soluble drugs (such as compound 1A and compound 1B) are typically excreted from the body within 2 hours of injection, a more physiologically relevant in vitro model of in vivo drug exposure was used to limit the incubation of cells with the drug to only two hours, and then the drug efficacy was examined after incubating for an additional 46 hours in the absence of the drug. As Figure 4 shown in A-4E, when THP-1 cells were incubated with a TLR7 agonist for 2 hours before replacing the drug-containing medium with drug-free medium, compound 1B was observed to have superior potency to compound 1A, especially in the case of TNFα induction, where the folate-targeted conjugate was significantly improved. This is most likely because the folate-targeted TLR7 agonist is captured by folate receptor-positive cells, whereas compound 1A is not retained by the same cells.

[0439] These data support that compound 1B should be more effective in reprogramming pro-fibrotic macrophages in vivo, with the added advantage that the folate-conjugated drug (e.g., compound 1B) should also cause less systemic toxicity because it is concentrated in macrophages expressing FRβ and cannot enter folate receptor-negative cells that predominate throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0440] Figure 6 A-6D shows the representation from treating with different concentrations of the drug for 48 hours ( Figure 6 A and 6B) or treating for 2 hours, then replacing with fresh medium and culturing for the remaining 46 hours ( Figure 6Graphical data of the levels of various markers measured in IL-4, IL-6 plus IL-13-induced M2 THP-1 macrophages. In both cases, cell supernatants were collected and the secreted CCL18 protein and IL-1β were detected by ELISA. This data supports that the 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).

[0441] In addition, to ensure that the above mRNA analysis accurately reflects the levels of profibrotic cytokines produced by THP-1 cells stimulated with IL-4, IL-6 plus IL-13, the concentrations of CCL18 and IL-1β polypeptides in THP-1 supernatants were quantified by ELISA assay. As Figure 6 shown in FIGS. 6A and 6B, both Compound 1A and Compound 1B induced a decrease in CCL18 and IL-1β when continuously incubated with the agonist for 48 hours. However, when the drug exposure was limited to 2 hours, Compound 1B was again found to be superior (see Figure 6 FIGS. 6C and 6D).

[0442] Example 3: Characterization of FRβ expression by flow cytometry

[0443] To measure the expression of FRβ on THP-1-derived macrophages, fluorescence-activated cell sorter (FACS) analysis was performed. Cells were separated using Cell Dissociation Solution (Biolegend, San Diego, CA; #423201) and gently scraped off with a cell scraper. The cells were washed with PBS and non-specific binding was blocked by incubation with Fc Receptor Blocking Solution (Biolegend, San Diego, CA; #422301) for 10 minutes at room temperature. Then biotinylated anti-human FRβ monoclonal antibody (m909) was added and the cells were incubated on ice for an additional 30 minutes, followed by washing in staining buffer (PBS supplemented with 2% FBS). The cells were then incubated on ice for 20 minutes in fluorescein-labeled streptavidin (BD Biosciences, Franklin Lakes, NJ; #554060), washed twice in PBS, stained with 7AAD (vital staining) for 15 minutes and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ). Figure 6 FIG. 6E shows flow cytometry data that supports THP-1 macrophages and those that are FRβ+ and thus suitable for in vitro studies of Compound 1B and other studies described herein.

[0444] Figure 6F confirmed that Compound 1B remained stable during the incubation period in culture medium at 37°C. In fact, Compound 1B retained its original structure after 48 hours of incubation.

[0445] Example 4: Bleomycin-induced pulmonary fibrosis and in vivo reprogramming of profibrotic macrophages

[0446] Studies were also conducted to determine whether macrophages in fibrotic lungs could be specifically targeted in vivo with folate-conjugated drugs. After testing multiple protocols for inducing pulmonary fibrosis in mice, a protocol was selected in which 0.75 mg / kg bleomycin (BM) was instilled into the lungs of C57BL / 6 mice via an incision in the trachea, and the mice were allowed to progress through both the inflammatory and fibrotic stages of fibrosis before starting therapy. (The BM model is widely considered useful for enabling studies of the mechanisms associated with fibrogenesis in the in vivo environment.)

[0447] As Figure 7 shown in A-7D, mice treated with this protocol typically showed fibrosis on day 7 after BM treatment, and this nascent fibrosis developed into severe fibrosis on day 14. Then, the pathological progression continued for an additional 2 - 5 days before it began to spontaneously regress on day 21.

[0448] More specifically, eight-week-old C57BL6 male mice (average weight 22 g to 25 g) from Charles River were housed under pathogen-free conditions at room temperature (22°C) on a 12-hour light / dark cycle. Prior to BM or PBS instillation, the mice were fed a folate-deficient diet (Envigo Teklad Global rodent diet pellets) for 1 week. Fresh water and the folate-deficient diet were available ad libitum. All animal procedures were approved by the Purdue Animal Care and Use Committee in accordance with National Institute of Health guidelines.

[0449] Thereafter, the mice were anesthetized with ketamine / xylazine, and the necks of the mice were shaved with a 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 100 μL of sterile PBS or BM (Cayman Chemicals, Ann Arbor, MI; #13877) (0.75 mg / kg) dissolved in PBS was injected intratracheally using a 1 cc syringe with a 26G needle. Body weight was monitored every other day throughout the experiment.

[0450] To evaluate whether folate-conjugated drugs can specifically target pro-fibrotic lung macrophages in these mice, 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 with or without 200-fold excess of FA-glucosamine (a competitor of OTL38) 10 days after instillation, and dye uptake in major organs was evaluated.

[0451] After 2 hours, the mice were sacrificed by CO2 asphyxiation, and an incision was immediately made in the skin from the abdomen to the neck to expose the lungs and trachea. Then a small incision was introduced into the upper trachea for inserting a blunted 22-gauge needle, and a nylon thread was tied around the trachea to seal the trachea around the needle. Then the trachea (with the inserted needle), lungs, and heart were removed together by carefully cutting the connective tissue under the lungs, and the bronchus of the left lung was cut with a Dieffenbach vascular clamp. PBS was injected into the right lung and aspirated 3 times with a 1 ml syringe, and the recovered lavage fluid was kept on ice.

[0452] Then bronchoalveolar lavage fluid (BALF) was analyzed to determine how the targeted TLR7 agonist works. The BALF samples were centrifuged at 1500 rpm for 5 minutes at 4 °C, and the supernatant was aliquoted and stored at -80 °C for cytokine / chemokine analysis. The cell pellet was resuspended and cultured in pre-warmed RPMI 1640 medium for 2 hours, and then washed 3 times with pre-warmed PBS before harvesting for qPCR assay. Then the right lung was tied with a nylon thread and used for subsequent analysis of hydroxyproline content. The left lung was inflated with 1 ml PBS using the inserted syringe and transferred to 10% formalin solution for subsequent histological analysis.

[0453] The lung lobe of the right lung collected above was weighed, placed in a pressure-tight vial (Supelco Inc., Bellefonte, PA; #27003), and hydrolyzed with 6N HCl (10 ml / g, v / w) in a sand bath at 120 °C for 3.5 hours. The hydrolyzed solution was cooled at 4 °C for 15 minutes and transferred to a 1.5 ml Eppendorf tube, and then centrifuged at 12,000 rcf for 15 minutes at 4 °C. The supernatant was carefully collected, aliquoted, and used for hydroxyproline (HYP) analysis.

[0454] 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. Then isopropanol (40 μl) was added to each well, followed by 20 μl of oxidation buffer, and the mixture was incubated for 5 minutes at room temperature on an oscillator. The assay reagent (260 μl) was added, and the plate was incubated for 30 seconds at room temperature on an oscillator and then immediately incubated for 25 minutes at 60 °C. The absorbance (A 560 ) was measured at 560 nm within 15 minutes. All reagents were prepared according to the previously reported protocol.

[0455] For histological analysis of lung sections, the 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). The tissue sections were examined by a licensed pathologist in a blinded manner. Using Aperio-Image Scope (Leica Biosystems, Wetzlar, DE), more than 90x 10 6 cells were quantified per section.

[0456] CCL18 and IL-1β were quantified in the induced THP-1 cell supernatants using the Human DuoSet ELISA Development System (R&D Systems Europe, Abingdon, UK; #DY394-05) and the IL-1β Human ELISA Kit (Thermo Fisher Scientific, Waltham, MA; #BMS224-2) as described by the manufacturer. Mouse IFN-γ in BALF samples was analyzed using ELISA MAX TM Deluxe (Biolegend, San Diego, CA; #430804).

[0457] Finally, for in vivo folate imaging studies, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were excised and imaged using an AMI in vivo imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, lungs from mice were harvested immediately after euthanasia and digested as described using a gentleMACS Octo Dissociator with Heater (Miltenyi Biotec, Bergisch Gladbach, DE; #130-096-427) with a Lung Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, DE; #130-098-427) either manually or through a 70 μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, DE; #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 for 30 minutes on ice with antibodies against the desired macrophage markers (FITC-CD11b, Biolegend, San Diego, CA, #101205; PE-F4 / 80, Biolegend, San Diego, CA, #123109). The labeled macrophages were then washed twice in PBS, stained with 7AAD (vital stain) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, San Jose, CA).

[0458] As Figure 7 shown in

[0459] Panel Figure 7 A (upper panel), untreated lungs (PBS control column) and BM-treated lungs at day 7 showed similarly high densities of alveoli interconnected by minimal extracellular matrix. In contrast, at day 14 post-BM instillation, the size and frequency of air sacs were significantly reduced and the density of extracellular matrix was markedly increased, suggesting the development of significant fibrosis in treated mice. By day 21, the pathology in this model had begun to spontaneously regress and many mice eventually recovered from BM-induced injury by day 35. Figure 7 Evidence of inflammation development by day 7 was seen in the infiltration of macrophages expressing FRβ (see Figure 7A). The expression of FRβ is restricted to the inflamed lung (in IPF patients or BM-induced PF, but not in healthy lungs). Additionally, macrophages expressing FRβ were observed in the mouse lung on day 7 after BM administration, with maximal expression on day 14. Figure 7 B). These results corroborate the previously reported expression of FRβ on activated macrophages in the inflamed lung.

[0460] Then, it was demonstrated that these FRβ-expressing macrophages could be targeted with folate-conjugated molecules by the accumulation of OTL38 (i.e., folate-targeted fluorescent dye) in the lungs of BM-treated mice but not in healthy mice after tail vein injection. As Figure 7 shown in B, OTL38 fluorescence was observed only in the kidney (i.e., its major excretion site) of healthy mice, with little or no uptake in other tissues.

[0461] Figure 7 C and 7D show FRβ IHC staining of human IPF lung tissue Figure 7 C) and healthy human lung tissue Figure 7 D). Folate-deficient diet was fed to 8-week-old C57BL / 6 male mice for 1 week before BM or PBS infusion and for 10 days after infusion. 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 200-fold excess FA-glucosamine. After 2 hours, the mice were sacrificed prior to analysis. For in vivo folate imaging studies, major organs (heart, lung, spleen, liver, small intestine, large intestine, and kidney) were excised and imaged using an AMI in vivo imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, the lungs of mice were harvested immediately after euthanasia, digested and then labeled with antibodies against the desired macrophage markers (FITC-CD11b, PE-F4 / 80) and 7AAD (live / dead staining), and analyzed by flow cytometry.

[0462] Figure 7Panel E shows images of various mouse tissues / organs obtained from mice with (BM) or without (PBS control) BM-induced experimental fibrosis and imaged with the folate receptor-targeted fluorescent dye OTL38, where 10 nmol OTL38 was injected into the tail vein of healthy mice (column a) or BM-treated mice (columns b and c) on day 10 after fibrosis induction in the absence (b) or presence (c) of 200-fold excess folate-targeted glucosamine (a competitive reagent for FRβ that blocks the binding of OTL38), and the mice were euthanized 2 hours later for tissue excision and fluorescence imaging, supporting that the inventive FA-targeted conjugates of the present disclosure exhibit FRβ-specific binding and no uptake in other healthy tissues.

[0463] Intravenous injection of OTL38 into the tail vein of BM-treated mice produced not only the aforementioned fluorescence in the kidney but also significant accumulation in the fibrotic lung (see Figure 7 Panel E). When BM-treated mice were injected simultaneously with 200-fold excess folate-glucosamine (i.e., a competitive inhibitor of FRβ binding (see Figure 7 Panel E)), this lung uptake was shown to be mediated primarily by the folate receptor by almost quantitative blockade of lung accumulation. These data demonstrate that folate-targeted molecules selectively bind to cells expressing folate receptors in fibrotic tissue and do not accumulate to any significant extent in other tissues of the body. In other words, macrophages expressing FRβ can in fact be targeted with folate-linked molecules and are almost exclusively localized to fibrotic tissue in clinical applications. Thus, when the targeting moiety is used in the compounds of the present disclosure, any TLR7 agonist not captured by the targeted fibrotic (or cancerous) tissue will be minimal.

[0464] Next, to determine which cell types capture the folate dye conjugate in the lungs of BM-treated mice, the lungs from the above animals were digested with collagenase and cell-specific dye uptake was examined by flow cytometry. Figure 7 Panel F shows data from FACS analysis generated by in vivo labeling of such mice with BM-induced experimental fibrosis that received an intravenous injection of PBS (row 1) or 100 nmol OTL38 in the absence (row 2) or presence (row 3) of 200-fold excess folate-targeted [glucosamine]. As Figure 7As shown in F, when isolated from mice treated with BM without injection of OTL38, no macrophage-like cells showed any fluorescence (see line 1). In contrast, approximately 22% of macrophage-like cells from fibrotic mice injected with OLT38 showed significant folate-targeted dye retention (line 2), which supports that OTL38 targets FRβ-positive macrophages in the inflamed lung. Indeed, dye uptake is mediated by specific folate receptors, as demonstrated by the observation that intravenous injection of a 200-fold excess of folate-glucosamine substantially blocked all folate-dye retention, demonstrating that unoccupied folate receptors are required for dye accumulation. Importantly, this conclusion is also supported by the following data, which show that FRβ expression is substantially undetectable in untreated lungs (see Figure 7 A), but increases significantly during the development of fibrosis in BM-treated lungs (see Figure 7 A-7D). FRβ is also significantly expressed in the lungs of human IPF patients.

[0465] Example 5

[0466] In the ability to target attached drugs to established FRβ-expressing fibrotic macrophages, it was then investigated whether a folate-targeted TLR7 agonist could inhibit the signs and symptoms of fibrosis in BM-treated mice. For this, BM-treated mice were intravenously injected with vehicle (PBS containing 3% DMSO) or compound 1B every other day starting on day 10 (see Figure 8 A). Because the TLR7-54 agonist caused rapid weight loss followed by death (see Figure 9 A and 9B), compound 1A could not be similarly evaluated in vivo. In BM-induced experimental fibrosis in mice, inflammation is known to persist for approximately 9-10 days after BM infusion. Since the transition from inflammation to fibrosis occurs in this model around day 9 to day 14, and profibrotic markers start around day 10, dosing began on day 10 ( Figure 8 A).

[0467] Two doses were given every other day until day 21. The single dose for one day was separated by 6 hours to prevent any "tolerance" to the TLR agonist. Then the mice were sacrificed on day 21, and bronchoalveolar lavage was immediately performed, followed by resection of the lungs for immunohistochemistry and quantification of collagen and hydroxyproline.

[0468] Figure 8 B-8G shows the representation from using Figure 8Graphical data of the levels of various markers measured in mice treated with the BM model of A, where BALF was collected at day 21 and centrifuged at 4°C, the resulting pellet was resuspended in medium and seeded into 96-well plates, cultured for 2 hours, washed 3 times with pre-warmed PBS, and the cells were harvested for qPCR; the data showed Arg1( Figure 8 B), MMP9( Figure 8 C), TIMP 3( Figure 8 D) (e.g., profibrotic markers) were all downregulated. CD86( Figure 8 E) and IFN-γ( Figure 8 F) (e.g., antifibrotic markers) were both upregulated. In addition, the negative regulator of TLR7 signaling IRAK-4 was upregulated( Figure 8 G), as well as the number of BALF cells present( Figure 8 H). Indeed, after treatment with different doses of compound 1B, the total number of BALF cells in mice decreased in a dose-dependent manner. Figure 8 Each value shown in B-8G represents the mean ± S.D. of each group; *P < 0.05, **P < 0.005, *** < 0.0005; for saline vs. vehicle group, compound 1A and compound 1B treatment groups vs. vehicle group, calculated by Student's t-test, except for BALF cell count and protein concentration measurement (where compound 1B treatment group and vehicle group were calculated by Dunnett's multiple comparison test); and vehicle = PBS containing 3% DMSO.

[0469] As Figure 8 shown in B-8D, qPCR analysis of profibrotic markers in the macrophage subset of bronchoalveolar lavage cells revealed that the tissue inhibitors of Arg1, MMP9, and TIMP 3 were all elevated in BM-induced mice relative to control mice. More importantly, parallel studies demonstrated that when BM-induced mice were treated with compound 1B, the same profibrotic markers were all inhibited, resulting in fibrotic marker levels similar to those seen in healthy mice. Consistent with these data, quantification of antifibrotic markers revealed that the transcript of CD86 (qPCR) and the concentration of IFN-γ (ELISA of lavage fluid) were both elevated after treatment with compound 1B (see Figure 7 E and 7F). Along with the observed upregulation of IRAK-4 (i.e., a marker of TLR activation; Figure 7 results shown in G) and the total number of BALF cells present decreased in a dose-dependent manner 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 the lungs of mice treated with bleomycin in vivo from a pro-fibrotic M2-like phenotype to an anti-fibrotic M1-like phenotype.

[0470] Example 6

[0471] Additional studies were conducted to determine whether the above-described reprogramming of fibrotic lung macrophages results in an actual improvement in fibrotic disease conditions in fibrotic mice. Lung tissues from the above mice were embedded in paraffin and sectioned and stained with H&E and Masson's trichrome to evaluate tissue density and extracellular collagen deposition, respectively.

[0472] Figure 9 Figures 9A and 9B show the survival curves ( Figure 9 Figure 9A) and body weight changes ( Figure 9 Figure 9B) of mice with experimental pulmonary fibrosis treated with non-targeted and targeted TLR7 agonists. The data support that administration of the compounds of the present disclosure (here, for example, compound 1B) increases the survival of bleomycin-treated mice without causing significant weight loss. Each value represents the mean ± S.D. of each group.

[0473] Figure 10 Figure 10A shows the hydroxyproline content (μg / lung) of lung tissues to use collagen deposition as a measure of fibrosis. Shown are tissues at day 21 in each of the following: healthy control (saline) (●), disease control (vehicle) (■), treated with free drug TLR7 agonist (compound 1A) and treated with folate-targeted TLR7 agonist (compound 1B) (▲). Compared to the vehicle control (■), bleomycin-induced mice treated with either 10 nmol of compound 1B (▲) and compound 1A showed a significant reduction in the total hydroxyproline content per lung. Figure 9 Each value shown in Figure 10A represents the mean ± S.D. of each group; *P<0.05, **P<0.005, ***<0.0005; by Student's t-test, saline versus vehicle group, compound 1A and compound 1B treatment groups versus vehicle group.

[0474] Figure 10 Figures 10B and 10C show the stained images of lung tissues with H&E staining ( Figure 10 Figure 10B) and Masson's trichrome (collagen) staining ( Figure 10 Figure 10C) as represented in Figure 10 Figure 10A.

[0475] As Figure 10As shown by H&E staining of the small sections of B, healthy lungs contain numerous air sacs surrounded by a thin reticular membrane. In contrast, BM-induced lungs show significantly fewer alveoli with obvious deposition of extracellular matrix where air sacs used to be. Most importantly, mice infused with BM and treated with compound 1B starting from day 10 showed a lung structure similar to that of healthy mice ( Figure 10 B), suggesting that targeting fibrotic lung macrophages with compound 1A can effectively inhibit the main hallmarks of pulmonary fibrosis. This prevention of fibrosis was actually recorded by Masson's trichrome staining of parallel lung sections ( Figure 10 C), in which collagen staining was strongly inhibited in mice injected with compound 1B via the tail vein ( Figure 10 B). Thus, the data support that treatment of IPF mice with at least compound 1B (▲) demonstrated inhibition of IPF pathology (e.g., fibrosis).

[0476] Finally, to confirm that compound 1B actually affects collagen production in vivo, hydroxyproline (the main component of collagen) was quantified in the total hydrolysate of the affected lungs. More specifically, the lung tissues from the above mice were perfused with PBS, acid-hydrolyzed, and the hydroxyproline content was analyzed. As Figure 10 shown in A, induction of fibrosis induced a significant increase in hydroxyproline content and this increase was inhibited upon treatment with compound 1B. Thus, the data support that treatment with the TLR7 agonist compounds disclosed herein reduces (and even counteracts) collagen deposition in vivo and thus fibrosis.

[0477] In summary, the overall survival rate of mice injected with the optimized BM dose (0.75 mg / kg) was significantly improved by treatment with compound 1B, whereas in the case of compound 1A there was no benefit to survival except for showing a significant weight loss (>25%, Figure 7 ). Although the free drug performed better in reducing hydroxyproline content, the poor survival rate seen can be attributed to overall toxicity (i.e., weight loss, see Figure 7 B). This is not surprising as systemic administration of TLR7 agonists is known to cause toxicity.

[0478] Example 7

[0479] Since the use of non-targeted TLR7 agonists for the treatment of IPF (or other fibrotic diseases) has been precluded by the systemic activation of the immune system and the resulting toxicity, any apparent toxicity that might have accompanied the systemic administration of compound 1B in mice was evaluated. To this end, BM-induced mice were treated every other day starting on day 10 with 0, 1, 3 or 10 nmol of compound 1B, and body weight, lung hydroxyproline content and histological analysis were performed on day 21. Different from conventional systemic administration, the targeted drug not only improved survival, but also reduced weight loss, highlighting the significance of the targeted approach( Figure 11 A and 11B).

[0480] Figure 12 Data showing the dose-dependent effect of folate-targeted TLR7 agonists on fibrosis inhibition in BM-induced mice using collagen deposition as a measure of fibrosis are shown. The data are represented as: healthy control (PBS, ●), BM-induced mice treated with vehicle (■), 1 nmol compound 1B (○), 3 nmol compound 1B (□) or 10 nmol compound 1B (▲), where sub-section A shows graphical data related to the body weight of BM-induced mice over time, sub-section B shows the measured hydroxyproline content (μg / lung) of lung tissues treated with different doses (10 nmol, 3 nmol or 1 nmol of compound 1B), and sub-section C shows images of histological analysis of right lung tissues stained with H&E and trichrome.

[0481] As Figure 11 B and Figure 12 As seen in sub-section A, no difference in weight loss was observed among mice treated with 0, 1, 3 or 10 nmol of compound 1B, indicating that repeated doses of the compound did not cause visible toxicity. However, these treatments were still seen to have the expected effect on pulmonary fibrosis when comparing the hydroxyproline content of various lung hydrolysates, with the order of efficacy being 10 nmol / mouse > 3 nmol / mouse > 1 nmol / mouse > 0 nmol / mouse( Figure 12 sub-sections B and C). More importantly, a detailed analysis of lung histology demonstrated an improvement in lung histology with increasing doses of compound 1B, indicating that the tissues with the highest concentration of TLR7 agonist were actually the most morphologically normal microscopically. Collectively, these data support that targeting FRβ+ macrophages in fibrotic tissues with TLR7 agonists can effectively prevent fibrosis without systemic activation of the immune system, which would otherwise limit the use of TLR7 agonists in humans.

[0482] Finally, to determine whether this anti-fibrotic effect could be achieved with lower doses, a treatment study with two lower doses (3 nmol / kg and 1 nmol / kg) was conducted ( Figure 9 and Figure 10 ). Interestingly, while the lower doses showed a significant decrease in hydroxyproline content and collagen deposition levels, the 10 nmol dose provided the best survival rate.

[0483] Example 8

[0484] To support that embodiments of the compounds of the present disclosure other than Compound 1A and Compound 1B perform similarly in applications, other representative embodiments of the compounds herein were examined in in vitro studies.

[0485] Figure 13 A-13D shows graphical data representing the levels of various markers measured from human THP-1 cells that were induced to M2 macrophages with 20 ng / mL IL-4, 20 ng / mL IL-13, and 5 ng / mL IL-6. The cells were then reprogrammed with different nM concentrations of a TLR7 agonist of Formula IV (e.g., Compound 2A) for 48 hours and harvested for gene analysis by qPCR. The following marker mRNA levels of expression relative to the M2-like macrophage control were obtained: CCL18 mRNA level ( Figure 12 A), IL-1β mRNA level ( Figure 13 B), and TNFα level ( Figure 13 C), and Figure 13 D shows the results of protein analysis after collecting the cell supernatants. The secreted CCL18 protein was detected by ELISA.

[0486] In Figure 13 A-13D, an agonist compound of Formula IV of the present disclosure (e.g., Compound 2A) was evaluated with respect to its ability to reprogram M2-like macrophages to M1-like macrophages.

[0487] First, human monocyte (THP-1) cells were induced to an M2-like phenotype using the methods and materials described previously. Specifically, THP-1 cells were seeded into 96-well plates at a density of 60,000 cells / well. The cells were differentiated into non-polarized macrophages by incubating with 200 nM PMA for 48 hours and then in fresh RPMI medium for 24 hours. The resulting macrophages were polarized to an M2-like phenotype by incubating with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / mL IL-6 for 48 hours. The cultures were maintained at 37 °C in a humidified 5% CO2 incubator.

[0488] To evaluate whether compound 2A can reprogram pro-fibrotic macrophages into a less fibrotic phenotype, THP-1 cells stimulated with IL-4, IL-6 plus IL-13 were incubated with different concentrations of compound 2A, and the mRNA levels of several pro-fibrotic markers (i.e., CCL18, IL-1β, and TNFα) were examined using qPCR and ELISA.

[0489] As shown in Figure 13 Figures A and 13B, incubation with compound 2A (free drug) for 48 hours induced a decrease in CCL18 and IL-1β expression, indicating that the TLR7 agonist can indeed promote the transformation of these pro-fibrotic polarized THP-1 cells into a less fibrotic phenotype. (Note: Figure 13 Figure B shows a bell-shaped curve indicating that compound 2A has an inhibitory response at lower concentrations and a stimulatory response at higher concentrations, which is a common response curve for some drugs.) Additionally, when examining the expression of TNFα (an anti-fibrotic phenotype marker), an increase in its expression was observed ( Figure 13 Figure C), confirming the transformation of THP-1 from a pro-fibrotic to an anti-fibrotic property.

[0490] In addition to the unconjugated TLR7 agonist, the conjugated compounds of the present disclosure were similarly evaluated. Human THP-1 cells were induced to macrophages with an M2-like phenotype according to the methods described herein (e.g., using 20 ng / mL IL-4, 20 ng / mL IL-13, 5 ng / mL IL-6), and then reprogrammed for 2 hours with various compounds of the present disclosure at different nM concentrations: i.e., unconjugated (free drug) TLR7 agonist compounds of formula I and / or II (data shown together as compound 3A), folate-conjugated TLR7 agonist compounds of formula XV (with a cleavable linker) (e.g., compound 3B), folate-conjugated TLR7 agonist compounds of formula XVII (with a non-cleavable linker) (e.g., compound 3C), and folate-conjugated TLR7 agonist compounds of formula XVI (with a non-cleavable linker) (e.g., compound 3D). Subsequently, the cells were harvested for gene analysis by qPCR and the relative expression of CCL18 ( Figure 14 Figure A), CD206 ( Figure 14 Figure B), and IL-1β ( Figure 14 Figure C) was analyzed.

[0491] The expression of various pro-fibrotic (M2 phenotype) markers CCL18, IL-1β, and CD206 markers was quantified. As shown in Figure 14As shown in A-14C, the expression of each of these profibrotic markers was reduced following administration of each of Compound 3B, Compound 3D, and Compound 3C, with Compound 3D and Compound 3C compounds (both of which have a non-releasable linker) being the most effective relative to the other compounds.

[0492] Figure 15 Shown are the results after treatment with Compound 3A, Compound 3B, Compound 3C, or Compound 3D. Figure 14 The secreted CCL18 protein levels in each of the THP-1 cell groups A-14C. Compound 3A and folate-targeted TLR7 compounds (eg, Compound 3B, Compound 3C, and Compound 3D) downregulated the secretion of CCL18 at low concentrations (0.1-10 nM).

[0493] In addition, cell supernatants were collected and secreted CCL18 protein was detected by ELISA. Figure 15 We demonstrated that compound 3A (free drug) and folate-targeted compounds (compounds 3B, 3C, and 3D) all downregulated CCL18 secretion at a low concentration range (0.1-10 nM), further supporting that similar to the example described for the combination of compound 1A and compound 1B, these compounds can similarly reprogram M2-like pro-fibrotic macrophages to M1-like anti-fibrotic macrophages through a similar mechanism.

[0494] Example 9

[0495] After repeating the above study (see grey bar, Figure 3 A-3F), the same qualitative changes were observed, with only a slight decrease in the magnitude of the effect of compound 1B. This decrease in efficacy is expected because non-targeted TLR7 agonists enter cultured cells immediately, whereas their folate-targeted analogs are designed to enter cells only after folate receptor binding and receptor-mediated endocytosis. Since low molecular weight water-soluble drugs (such as compound 1A and compound 1B) are typically excreted from the body within 2 hours of injection, a more physiologically relevant in vitro model of in vivo drug exposure is used to limit the cells to incubation with the drug for only two hours, and then to examine drug efficacy after incubation for another 46 hours in the absence of the drug. Figure 4 As shown in A-4E, when THP-1 cells were incubated with TLR7 agonists for 2 hours before the drug-containing medium was replaced with drug-free medium, compound 1B was observed to have superior potency to compound 1A, particularly under TNFα induction, where the folate-targeted conjugate was significantly improved. This is most likely because the folate-targeted TLR7 agonist was captured by folate receptor-positive cells, whereas compound 1A was not retained by the same cells.

[0496] These data support that compound 1B should be more effective in reprogramming pro-fibrotic macrophages in vivo, with the added advantage that folate-conjugated drugs (e.g., compound 1B) should also cause less systemic toxicity because it is concentrated in macrophages expressing FRβ and cannot enter folate receptor-negative cells that predominate throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0497] In addition, to ensure that the above mRNA analysis accurately reflects the levels of pro-fibrotic cytokines produced by THP-1 cells stimulated with IL-4, IL-6 plus IL-13, the concentrations of CCL18 and IL-1β polypeptides in the THP-1 supernatant were quantified by ELISA assay. As Figure 6 shown in FIGS. 6A and 6B, both compound 1A and compound 1B induced a decrease in CCL18 and IL-1β when continuously incubated with the agonist for 48 hours; however, when the drug exposure was limited to 2 hours, compound 1B was again found to be superior (see Figure 6 FIGS. 6C and 6D).

[0498] Example 10

[0499] Figure 16 An in vivo study method of at least one embodiment of the compounds of the present disclosure in a BM mouse model is presented, the compounds having formula XVII (e.g., compound 3C). Figure 17 FIGS. 17A and 17B are LC-MS spectra of compound 3C and support the high purity of the conjugate, and no free drug was detected.

[0500] Figure 18 FIGS. 18A-18F show the results of the subject mice from the Figure 16 in vivo study method, including survival curves ( Figure 18 FIG. 18A), body weight changes ( Figure 18 FIGS. 18B and 18D), concentration of cells with BALF ( Figure 17 FIG. 18C), hydroxyproline concentration (μg HP / lobe of lung) in live mice ( Figure 18 FIG. 18E) and in all mice (e.g., including live mice and mice that died before day 21) ( Figure 18 FIG. 18F). A 10 nmol concentration dose of the compound having formula XVII (e.g., compound 3C) increased the survival rate of the subject mice while reducing the number of HP and BALF cells. Moreover, a 3 nmol concentration dose did not show any measurable benefit to the subject mice.

[0501] Example 11

[0502] Human monocyte-derived macrophages induced by M2 were continuously treated with 100 nM Compound 1A or Compound 1B for 48 hours, or initially for 2 hours in the presence or absence of FA-glucosamine (competition), followed by 46 hours in the absence of the drug (2 + 46 h). As Figure 19 shown, the mRNA levels of profibrotic markers Arg1 ( Figure 19 A), CD206 ( Figure 19 B), and CD163 ( Figure 19 C), as well as the secreted profibrotic CCL18 ( Figure 19 D) and the anti-fibrotic cytokine CXCL10 ( Figure 19 E) and IL-6 ( Figure 19 F) protein levels (n = 3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blocking unoccupied folate receptors with excess FA-glucosamine (2 + 46 h, competition). This data supports the binding of Compound 1B to the folate receptor, as the downregulation of biomarkers was blocked by excess FA-glucosamine (competitor).

[0503] Example 12

[0504] Healthy mice were injected via the tail vein with 10 nmol of Compound 1A (circles) or Compound 1B (squares), and peripheral blood was collected at the indicated time points after drug injection. ( Figure 20 A-C) Plasma IL-6 ( Figure 20 A), IFNα ( Figure 20 B), and TNFα ( Figure 20 C) measurements (n = 3). ( Figure 20 D-F). The effects of drug concentration on the plasma levels of IL-6 ( Figure 20 D), IFNα ( Figure 20 E), and TNFα ( Figure 20 F) were determined at 1.5 hours, 1 hour, or 1 hour after treatment, respectively (n = 2) ( Figure 20 G). Compound 1A stimulated the release of systemic cytokines in healthy mice, while Compound 1B did not. Additionally, the release of inflammatory cytokines stimulated by Compound 1B was lower than half the dose of Compound 1A. These data suggest that if TLR7 agonists are targeted to lung macrophages with folate receptor-targeting ligands, TLR7 agonists can be safely employed to reprogram fibrotic lung macrophages to an anti-fibrotic state.

[0505] Example 13

[0506] From Figure 6Sections of the same healthy and fibrotic lungs described in were stained with DAPI (nuclei; blue), anti-F4 / 80 (macrophages; red), and anti-CD206 (M2 macrophage marker; green), and images were acquired using a Leica Versa 8 whole-slide scanner as described in the methods (n = 2). Scale bar, 100 μm. Differences between treatment groups indicate that Compound 1B produces a robust anti-fibrotic response in vivo.

[0507] Although various embodiments of compounds, compositions, and methods have been described herein in considerable detail, these embodiments are provided by way of non-limiting examples only. Given this disclosure, many variations and modifications of the embodiments described herein will be apparent to those of ordinary skill in the art. Accordingly, those skilled in the art will understand that various changes and modifications can be made without departing from the scope of this disclosure and that elements thereof can be replaced with equivalents. Indeed, this disclosure is not intended to be exhaustive or overly limiting. The scope of this disclosure will be defined by the appended claims and their equivalents.

[0508] In addition, although many of the examples provided herein use a mouse model, those of ordinary skill in the art will appreciate that gene expression patterns in the mouse model show a remarkably significant correlation with gene expression patterns of human pathologies, and that many pathways are commonly regulated by multiple pathologies in both humans and mice. Thus, the gene expression patterns and disease progression in the mouse model closely recapitulate those of human pathologies (particularly with respect to inflammatory diseases and cancer), and thus support the relevance of the working examples set forth herein to human data, specific pathologies, and applications.

[0509] Accordingly, it is expected that this specification and the appended claims will cover all modifications and changes that are obvious to those of ordinary skill in the art based on this disclosure.

Claims

1. A compound represented by the following formula: Q-L-T Wherein, Q is a group of folate receptor-binding ligands; L is a linker; and T is a group of toll-like receptor (TLR) agonists or pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, wherein the linker is a non-cleavable linker.

3. The compound according to claim 2, wherein the non-cleavable linker is represented by the following formula: Wherein: n is 1 - 30; and w is 0 - 5.

4. The compound according to any one of the preceding claims, wherein the TLR agonist is a toll-like receptor 7 (TLR7) agonist.

5. The compound according to any one of the preceding claims, wherein the group of the TLR agonist has a structure represented by formula X: Wherein, R1 is -NH2 or -NH-R 1X , R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X 、R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and R3 is -OH, -SH, -NH2 or -NH-R 1X .

6. The compound according to any one of claims 1 - 4, wherein the group of the TLR agonist has a structure represented by formula XX: Wherein, R1 is -NH2 or -NH-R 1X , R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X 、R 2X and R 2Y Each of is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and X is CH, CR2 or N.

7. The compound according to claim 6, wherein the group of the TLR is a TLR7 agonist having a structure represented by formula XXX:

8. A compound as claimed in any one of the preceding claims, wherein the compound further comprises a linker L between the targeting moiety and the immunomodulator or a pharmaceutically acceptable salt thereof n , wherein the linker L n is configured to avoid release of the free form of the TLR7 agonist, and n is an integer equal to or less than 50.

9. A compound according to any one of the preceding claims, said compound comprising a linker L n , wherein said linker L n comprises polyethylene glycol (PEG) or a PEG derivative, n is an integer selected from the range of 1-32, and the folate receptor binding ligand moiety is a folate receptor β binding ligand.

10. The compound according to claim 1, wherein the compound has a structure represented by the following:

11. The compound according to claim 1, wherein the compound has a structure represented by the following:

12. The compound according to claim 1, wherein the compound has a structure represented by the following:

13. The compound according to claim 1, wherein the compound has a structure represented by the following:

14. A pharmaceutical composition comprising the compound according to claim 4, wherein the TLR7 agonist has a structure represented by formula XX: Wherein, R1 is -NH2 or -NH-R 1X , R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , R 1X 、R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl and heteroaryl, is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and X is CH, CR2 or N.

15. A method of treating a subject having a fibrotic disease state or cancer, the method comprising contacting the cells of the subject with at least one compound including the compound according to any one of claims 1 - 13 or the pharmaceutical composition according to claim 14, wherein the group of the TLR agonist or its pharmaceutically acceptable salt comprises an agonist of TLR 7, 8 or 9.

16. A compound comprising a folate ligand or a functional fragment or analogue thereof attached via a linker to a toll-like receptor (TLR) agonist, the TLR agonist having the following formula or a pharmaceutically acceptable salt thereof: Wherein: R 1 is an amino group, R 2 is a single bond -NH- R 3 is H, an alkyl group, a hydroxyl group or any other substituent group, X is CH2, NH, O or S, and The linking group is attached at R 1 、 R 2 or R 3 at the position.

17. A pharmaceutical composition, the pharmaceutical composition comprising the compound according to any one of claims 1-13 or the composition according to claim 16, wherein the linker comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, and is an unreleasable linker attached at R 3 or a releasable linker attached at R 1 、 R 2 or R 3 .

18. The compound according to any one of claims 1 - 13 or claim 16 or the pharmaceutical composition according to claim 14, wherein the pharmaceutically acceptable salt is selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate or fumarate.

19. A method of preventing or treating a fibrotic disease state, the method comprising: Contacting a cell with at least one compound, the at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt thereof attached via a linker to a folate ligand or a functional fragment or analogue thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor.

20. The method according to claim 19, wherein the immunomodulator comprises a toll-like receptor (TLR) agonist of formula X or XX, or is a pharmaceutically acceptable salt of formula X or XX: Wherein in formulae X and XX: R1 is -NH2 or -NH-R 1X , R2 is H, alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , and is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle; Wherein in formula X, R3 is -OH, -SH, -NH2 or -NH-R 1X ; Wherein in formula XX, X is CH or N; and R 1X 、R 2X and R 2Y each independently is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl and heteroaryl.

21. The method according to claim 19, wherein the cell comprises a cell of a subject who has experienced or is at risk of experiencing a fibrotic disease state, and contacting the cell with at least one compound further comprises administering or applying a therapeutically effective amount of the at least one compound to the subject.

22. The method according to claim 21, wherein the subject is a patient suffering from idiopathic pulmonary fibrosis, and the at least one compound is administered to the subject by intravenous, intramuscular, intraperitoneal, topical or inhalation routes.

23. The method according to claim 21, wherein the fibrotic disease state includes idiopathic pulmonary fibrosis or fibrotic diseases of the liver, skin, bladder, heart, pancreas, prostate or kidney.

24. The method according to claim 21, further comprising: Obtaining a sample from or having obtained a sample from the subject; Quantifying the expression levels of one or more biomarkers in the sample, each of the one or more biomarkers being selected from the group consisting of chemokine (C-C motif) ligand 18 (CCL18), arginase 1 (Arg1), matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 3 (TIMP3), interleukin 1β (IL-1β), hydroxyproline, collagen, platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), folate receptor β (FRβ), tumor necrosis factor α (TNFα), interferon γ (IFN-γ), mannose receptor (CD206), cluster of differentiation 163 (CD163), cluster of differentiation 86 (CD86), interleukin 6 (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 If the expression levels of CCL18, Arg1, MMP9, TIMP 3, IL-1β, PDGF, TGFβ, CD206, CD163, FRβ, hydroxyproline or collagen are upregulated relative to the control or the expression levels of TNFα, IFN-γ, IL-6, CXCL10, IFNα and CD86 are downregulated or not expressed relative to the control, administering a therapeutically effective amount of an unconjugated agonist or inhibitor to the subject or having administered a therapeutically effective amount of an unconjugated agonist or inhibitor to the subject.

25. The method according to claim 21, wherein the folate ligand or a functional fragment or analogue thereof is specific for folate receptor β and binds to folate receptor β on the cell.

26. A compound comprising a targeting moiety attached to an immunomodulator or a pharmaceutically acceptable salt thereof that targets a pattern recognition receptor of a cell, the targeting moiety comprising a folate ligand or a functional fragment or analogue thereof.

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