Compounds and methods for the treatment and prevention of fibrous disease conditions and cancer

Abstract

Compounds, pharmaceutical compositions, and methods are provided for reprogramming M2-like macrophages into M1-like macrophages and reversing the anti-fibrotic to pro-fibrotic shift observed during the course of fibrotic diseases and certain cancers. The compounds include immunomodulators that target cellular pattern recognition receptors and are specific to the target cells through the incorporation of a targeting moiety (e.g., folic acid or a functional fragment or analog thereof). Releasable and / or non-releasable linkers can be included and engineered to facilitate optimal delivery of the immunomodulator. The compounds and compositions can be employed in one or more treatment methods for fibrotic diseases and / or cancer.

Description

[Technical Field]

[0001] Priority This application relates to (a) U.S. Provisional Patent Application No. 62 / 871,686 filed on 8 July 2019, and (b) U.S. Provisional Patent Application No. 62 / 872,146 filed on 9 July 2019, and claims the benefit of their priority. The contents of the aforementioned applications are incorporated herein by reference in their entirety.

[0002] This disclosure relates to compounds, pharmaceutical compositions, and methods for treating and preventing fibrous disease conditions and / or cancer using one or more compounds comprising a targeting moiety that reprograms M2 macrophages into M1 macrophages. [Background technology]

[0003] Despite the severity of many fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), there are few treatment options for successful management, and almost everything currently available is designed to alleviate symptoms and slow progression rather than cure the underlying condition. For example, in IPF, oxygen therapy can improve comfort and lifestyle, but has little effect on disease progression. Similarly, two FDA-approved drugs, pirfenidone and nintedanib, may slow disease progression, but they cannot restore existing fibrosis or stop the production of further fibrosis. Given the high mortality rate associated with many fibrotic diseases, including IPF, there is a great need to identify new strategies to slow, and possibly halt, disease progression.

[0004] Furthermore, cancer is treated with chemotherapy using very potent drugs such as mitomycin, paclitaxel, and camptothecin. Often, these chemotherapy agents exhibit a dose-response effect, with tumor inhibition proportional to the drug dose. Therefore, aggressive dosing regimens are used to treat neoplasms, but high-dose chemotherapy is hampered by its low selectivity for cancer cells and toxicity to normal cells. The lack of tumor specificity is one of many hurdles that conventional chemotherapy must overcome.

[0005] Despite the clear need for the prevention and treatment of both fibrous disease and cancer, these conditions remain a significant cause of death and / or suffering worldwide, as there are currently no effective treatment options available to cure the condition. Furthermore, when drugs or other therapies are available, such treatments employ very potent agents that typically carry a risk of systemic toxicity in the underlying target due to their poor selectivity for the fibrous and / or cancer cells being targeted. What is needed is a treatment that not only disrupts the pro-fibrotic and / or pro-growth factor cycle initiated by activated M2-type (or alternatively activated) macrophages, but can also do so with very high specificity to the cells in question (whether cancer cells or other cells experiencing fibrous disease). [Overview of the project] [Problems that the invention aims to solve]

[0006] In some cases, activated M2 phenotypic macrophages play a role in fibrotic diseases, for example, by secreting pro-fibrotic cytokines that activate fibroblasts to synthesize collagen and other extracellular matrix proteins. In certain cases, these macrophages also trigger the release of growth factors that are problematic in cancer-experiencing subjects. For example, such growth factors can promote the growth of cancerous tumors. Furthermore, in some cases, macrophages release immunosuppressive cytokines (e.g., concurrently). Thus, macrophages may play a significant role in promoting the establishment and growth of fibrotic diseases and / or cancer.

[0007] Idiopathic pulmonary fibrosis (IPF) is one such fibrotic disease, an interstitial lung disease resulting from excessive collagen deposition. In some cases, this type of fibrosis leads to progressive rigidification of the lungs and, in some cases, loss of the lungs' ability to mediate gas exchange. Due to this progressive decline in vital capacity, the median survival time after diagnosis of IPF is estimated to be only 2.5–5 years. In some cases, serious associated pathological conditions (e.g., chronic hypoxia, fatigue, weight loss, muscle and joint pain, persistent cough, and loss of mobility) continuously increase during the later stages of the pathology. In the United States, approximately 40,000 new cases of IPF are diagnosed annually, most of which are fatal.

[0008] In some cases, activated macrophages, either from within the tissue or derived from peripheral blood monocytes, induce fibroblast activation via the secretion of chemokine (CC motif) ligand 18 (CCL18), transforming growth factor-β1 (TGFβ1), and / or platelet-derived growth factor (PDGF). This activation, in some cases, promotes collagen secretion by fibroblasts, which can drive the progression of fibrotic diseases and associated cancers. In the later stages of many fibrotic diseases, activated macrophages and myofibroblasts can cross-stimulate each other, creating a vicious cycle that ensures the spread of fibrosis throughout the lungs or other relevant parts of the body.

[0009] Similar pathological conditions are observed in other fibrous diseases. Cancer may also be accompanied by similar immune responses that promote the growth of cancerous tumors (e.g., by growth factors secreted by activated macrophages) and / or promote collagen formation in cancerous tumors (e.g., through downstream fibrous collagen production, which can result in cancerous tumors that are more difficult to treat by blocking their drug permeability). [Means for solving the problem]

[0010] In some embodiments, compounds represented by the formula QLT are provided herein. In some embodiments, Q is a radical of a folate receptor-binding ligand. In some embodiments, L is a linker. In some embodiments, T is a radical of a Toll-like receptor (TLR) agonist. In some embodiments, QLT is a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the linker is a non-discharge linker. In some embodiments, the non-discharge linker is of the formula, [ka] It is represented as follows.

[0012] In some embodiments, n is 1 to 30. In some embodiments, n is 1 to 24. In some embodiments, n is 1 to 12. In some embodiments, n is 1 to 3. In some embodiments, n is 12. In some embodiments, n is 3.

[0013] In some embodiments, w is between 0 and 5. In some embodiments, w is between 0 and 2. In some embodiments, w is 1.

[0014] In some embodiments, the TLR agonist is a toll-like receptor 7 (TLR7) agonist. In some embodiments, the radical of the TLR agonist has a structure represented by the following formula X. [Chemical formula]

[0015] In some embodiments, R1 is -NH2 or -NH-R 1X where. In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , [Chemical formula] where. In some embodiments, each of R 1X , R 2X , and R 2Y is independently selected from the group consisting of hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In some embodiments, [Chemical formula] is a 3- to 10-member nitrogen (N)-containing non-aromatic monocyclic or bicyclic heterocyclic ring. In some embodiments, R3 is -OH, -SH, -NH2, or -NH-R 1X where. In some embodiments, R1 is -NH2 or -NH-R 1X where, and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -O-R 2X , -S-R 2X , [Chemical formula] And R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] R3 is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle, where R3 is -OH, -SH, -NH2, or -NH-R 1X That is the case.

[0016] In some embodiments, the radical of the TLR agonist has a structure represented by formula XX. [ka]

[0017] In some embodiments, R1 is -NH2 or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] In some embodiments, R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In some embodiments, [ka] X is a 3- to 10-membered N-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, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And R 1X , R 2X , and R 2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] X is a 3- to 10-membered N-containing non-aromatic monocyclic or bicyclic heterocycle, where X is CH, CR2, or N.

[0018] In some embodiments, the TLR7 agonist radical has a structure represented by formula XXX. [ka]

[0019] In some embodiments, a linker L is placed between the targeting moiety and the immunomodulator or its pharmaceutically acceptable salt. n It further includes Linker L n The linker L is configured to avoid the release of the free form of the TLR7 agonist, where n is an integer less than or equal to 50. In some embodiments, the linker L nThe compound comprises polyethylene glycol (PEG) or a PEG derivative, n is an integer selected from the range of 1 to 32, and the radical of the folate receptor binding ligand is a folate receptor β (FBβ) binding ligand.

[0020] In some embodiments, the compound has the structure shown below. [ka]

[0021] In some embodiments, the compound has the structure shown below. [ka]

[0022] In some embodiments, the compound has the structure shown below. [ka]

[0023] In some embodiments, the compound has the structure shown below. [ka]

[0024] In some embodiments, pharmaceutical compositions comprising one or more of the compounds of the Disclosure are provided herein, wherein the TLR7 agonist has a structure represented by formula XX.

[0025] In certain specific examples, a method for treating a subject suffering from a fibrotic disease condition or cancer is provided herein, the method comprising contacting the cells of the subject with at least one compound comprising a compound described herein, wherein the immunomodulator comprises a TLR7, 8, or 9 agonist.

[0026] In some embodiments, compounds comprising a folic acid ligand or a functional fragment or analog thereof attached to a TLR agonist via a linker are provided herein, wherein the TLR agonist has the following formula and is a pharmaceutically acceptable salt thereof. [ka]

[0027] In some embodiments, R 1 is an amine group, R 2 It is a single bond -NH-, and R 3 is H, alkyl, hydroxyl group, or any other substituent thereof, X is CH2, NH, oxygen (O), or sulfur (S), and the linker is R 1 、 R 2 , or R 3 It adheres to the surface.

[0028] In some embodiments, a pharmaceutical composition is provided comprising a compound of any one of the formulas provided herein, wherein the linker described above comprises a PEG linker or a PEG derivative linker, R 3 It is a non-release linker attached in or R 1 、 R 2 , or R 3 It is a releaseable linker attached thereto.

[0029] In some embodiments, pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbic acid, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, or fumarate.

[0030] In some embodiments of this specification, a method is provided for preventing or treating a fibrous disease state, comprising contacting cells with at least one compound (e.g., any compound provided by a formula provided herein) comprising an immunomodulator or a pharmaceutically acceptable salt thereof attached to a folate ligand or a functional fragment or analog thereof via a linker, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets pattern recognition receptors. In some embodiments, the cells include cells of a subject who is experiencing or at risk of experiencing a fibrous disease state, and contacting the cells with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject. In some embodiments, the subject is a patient experiencing IPF, and the at least one compound is administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation. In some embodiments, the fibrous disease state includes IPF or fibrous diseases of the liver, skin, bladder, heart, pancreas, prostate, or kidneys.

[0031] In some embodiments, the method involves obtaining a sample from a subject, or obtaining a sample and quantifying the expression level of one or more biomarkers in the sample, each of which is CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), metalloproteinase 3 (TIMP3), interleukin 1β (IL-1β), hydroxyproline, collagen, PDGF, TGFβ, folate receptor β (FRβ), tumor necrosis α-α (TNFα), interferon gamma (IFN-γ), mannose receptor (CD206), and differentiation antigens (cluster of The method further includes quantifying a biomarker selected from the group consisting of differentiation antigen group 163 (CD163), differentiation antigen group 86 (CD86), interleukin-6 (IL-6), chemokine-10 (CXCL10), and immune interferon (IFNα), comparing the expression level of each of one or more biomarkers in the sample to the expression level of such biomarkers in the control, and administering, or having administered, a therapeutically effective dose of an unconjugated agonist or inhibitor to the subject if CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline, or collagen is upregulated compared to the control's expression level, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα, or CD86 is downregulated or not expressed compared to the control's expression level.

[0032] In some embodiments, the folate ligand or its functional fragment or analogue is specific to FRβ and binds to FRβ on the cell.

[0033] In some embodiments of this specification, compounds are provided that include a targeting moiety attached to an immunomodulator or a pharmaceutically acceptable salt thereof that targets a cellular pattern recognition receptor, wherein the targeting moiety includes a folic acid ligand or a functional fragment or analog thereof. [Brief explanation of the drawing]

[0034] The embodiments and other features, advantages, and aspects disclosed herein, as well as the means by which they are achieved, will become apparent in light of the following detailed descriptions of various exemplary embodiments of this disclosure. Such detailed descriptions will be better understood in conjunction with the accompanying drawings: [Figure 1A] The chemical structures of exemplary compounds having a targeted moiety (folate receptor ligand) attached to an immunomodulator (Toll-like receptor 7 (TLR7) agonist radical) via a non-release linker (e.g., containing a polyethylene glycol (PEG) backbone) are shown. [Figure 1B] The chemical structure of an exemplary compound having a targeting moiety (folate receptor ligand) attached to an immunomodulator (TLR7 agonist radical) via a releaseable linker (e.g., containing a disulfide moiety in its skeleton), as well as an exemplary drug release mechanism, are shown. [Figure 2] This flowchart shows typical treatment methods for individuals who have or are at risk of having fibrous disease or cancer. [Figure 3A-3F] Graphs of various marker levels measured from human M2-type macrophages when exposed to exemplary free (untargeted) TLR7 agonists or exemplary targeted (e.g., those with folate receptor-binding ligands) TLR7 agonists at various concentrations for each compound are shown. The data shown in Figures 3A-3C support that administration of either an untargeted or targeted TLR7 agonist successfully reprogrammed M2-type macrophages to M1-type macrophages (i.e., downregulated M2-type pro-fibrotic macrophages), while the data shown in Figures 3D-3F support that administration of the tested compounds upregulated M1-type macrophages. Each value represents the mean ± SD for each group: #P<0.05, ##P<0.01, ##P<0.005, ####P<0.0001, and Dunnett's multiple comparison test for treated vs. untreated M2-type macrophages. [Figure 4A-4E]Representative graphical data of various marker levels measured from M2 macrophages incubated for 2 hours (Figures 4A–4E) or 46 hours (Figures 5A–5D) with exemplary free or targeted TLR7 agonists at various concentrations are shown. Figures 4A–4E and 5A–5D support the downregulation of the M2 type pro-fibrillation phenotype after administration of free and targeted TLR7 agonists. Each value represents the mean ± SD for each group: #P<0.05, ##P<0.01, ##P<0.005, ####P<0.0001, Dunnett's multiple comparison test for compound 1A and compound 1B treated groups versus M2 untreated groups in Figures 4A–5D. [Figures 5A-5D] Representative graphical data of various marker levels measured from M2 macrophages incubated for 2 hours (Figures 4A–4E) or 46 hours (Figures 5A–5D) with exemplary free or targeted TLR7 agonists at various concentrations are shown. Figures 4A–4E and 5A–5D support the downregulation of the M2 type pro-fibrillation phenotype after administration of free and targeted TLR7 agonists. Each value represents the mean ± SD for each group: #P<0.05, ##P<0.01, ##P<0.005, ####P<0.0001, Dunnett's multiple comparison test for compound 1A and compound 1B treated groups versus M2 untreated groups in Figures 4A–5D. [Figures 6A-6D] Graph data representing various marker levels measured from treated M2 macrophages with exemplary free and targeted TLR7 agonists at various concentrations are shown. These agonists were (i) incubated for 48 hours (Figures 6A and 6B), or (ii) incubated for 2 hours, then in fresh medium, and incubated for the remaining 46 hours (Figures 6C and 6D). Each value represents the mean ± SD for each group: #P<0.05, ##P<0.01, ##P<0.005, ####P<0.0001, compound 1A and compound 1B treated groups versus untreated M2 groups by Dunnett's multiple comparison test. [Figure 6E] Flow cytometry data supporting the finding that THP-1 (a human monocytic cell line derived from patients with acute monocytic leukemia)-induced macrophages were folate receptor beta (FRβ) positive (FRβ+) are shown. [Figure 6F] This demonstrates the stability of an exemplary targeted TLR7 agonist. [Figure 7A] The images show stained lungs taken from mice with bleomycin (BM)-induced experimental fibrosis and stained using anti-mouse FRβ antibody, with hematoxylin-eosin (H&E) staining performed on days 7, 14, and 21 after BM-induced lung injury. [Figure 7B] The quantification of FRβ staining in the panel shown in Figure 7A is presented. [Figure 7C-7D] Figure 7C shows FRβ immunohistochemistry (IHC) staining of lung tissue from a human with idiopathic pulmonary fibrosis (IPF) and healthy human lung tissue (Figure 7D). [Figure 7E] Images of mouse tissues / organs, taken from mice with or without BM-induced experimental fibrosis (phosphate-buffered saline (PBS) control), and imaged with folate receptor-targeted fluorescent dyes, are shown. [Figure 7F] This shows the fluorescence-activated cell sorting (FACS) analysis of mice with BM-induced experimental fibrosis. [Figure 8A] This shows the treatment plans for free and targeted TLR7 agonists in the BM model. [Figures 8B-8G] Figure 8A shows the levels of fibrosis-promoting markers (Figures 8B-8D) and anti-fibrosis markers (Figures 8E-8G) measured from mice treated with the BM model. [Figure 8H] Figure 8A shows the number of cells in bronchoalveolar lavage fluid (BALF) from mice treated with the BM model. [Figure 9A-9B] The survival curves (Figure 9A) and weight changes (Figure 9B) of pulmonary fibrosis mice treated with untargeted and targeted TLR7 drugs are shown. [Figure 10A] This shows the hydroxyproline content (μg / lung) in lung tissue as a measure of fibrosis. [Figure 10B-10C] Figure 9A shows lung tissue stained with H&E (Figure 10B) and Masson's trichrome (collagen) stain (Figure 10C). [Figures 11A-11B]The survival curves (Figure 11A) and weight changes (Figure 11B) of mice with pulmonary fibrosis treated with exemplary targeted TLR7 agonists are shown, with each value representing the mean ± SD for each group. [Figure 12] This disclosure demonstrates the dose-dependent effect of an exemplary targeted TLR7 agonist on the suppression of fibrosis in BM-induced mice. [Figure 12A] This graph shows data relating to the body weight of BM-induced mice over time. [Figure 12B] This specification shows the measurement of hydroxyproline content in lung tissue treated with various exemplary doses of conjugates (e.g., compound 1B). [Figure 12C] Images for histological analysis of lung tissue using various stains are shown. Each value represents the mean ± SD for each group, with *P<0.05, **P<0.005, and ***<0.0005, and Student's t-tests for saline vs. vehicle group and treatment group vs. vehicle group. [Figures 13A-13D] The following shows various marker levels measured from M2-like macrophages reprogrammed over 48 hours with exemplary targeted TLR7 agonists at various concentrations according to the method of this disclosure, and each value represents the mean ± SD for each group. [Figure 14A-14C] The following shows various marker levels measured from M2 macrophages reprogrammed according to the method of this disclosure using exemplary free and targeted TLR7 agonists at various concentrations. The values ​​shown in Figures 14A–14C represent the mean ± SD for each group; #P<0.05, ##P<0.005, ###P<0.0005, ####P<0.0001; Dunnett's multiple comparison test shows compound 3A, compound 3B treated, and compound 3C treated groups versus the untreated M2 group. [Figure 15] The levels of secreted chemokine (CC motif) ligand 18 (CCL18) protein in each group of 14A-14C cells after treatment with exemplary free TLR7 agonists are shown. [Figure 16] This paper presents the methodology of the BM mouse model. [Figures 17A-17B]The purity of exemplary targeted TLR7 agonists provided herein is shown. [Figures 18A-18F] Figure 16 shows data from the in vivo study methodology, including survival curves (Figure 18A), body weight changes (Figures 18B and 18D), cell concentration by BALF (Figure 18C), and hydroxyproline concentration (μgHP / leaf) in surviving mice (Figure 18E) and all mice (i.e., including both surviving mice and mice that died before day 21) (Figure 18F). [Figure 19] Both targeted and untargeted TLR7 agonists reprogram human monocyte-derived profibrosis macrophages to an antifibrosis phenotype (Figures 19A-19F). Mean ± SD. Statistical significance between groups was determined using unpaired two-sided t-tests (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 20] Figures 20A–20F show a comparison of plasma cytokine levels in healthy mice after treatment with compound 1A and compound 1B. Figure 20G shows the change in body weight after treatment of mice with the exemplary compounds provided herein, with body weight change as a measure of systemic toxicity during every-other-day administration (n=2), mean ± SD. Statistical significance between groups was compared using unpaired two-sided t-tests (*P<0.05, **P<0.01, ***P<0.001). [Figure 21] Figure 6 shows healthy and fibrous lungs stained with 4',6-diamido-2-phenylindole (DAPI) (nucleus, blue), anti-F4 / 80 (macrophage, red), and anti-mannose receptor (CD206).

[0035] This disclosure is susceptible to various modifications and alternative forms, but exemplary embodiments are shown as examples in the drawings and described in detail herein. [Modes for carrying out the invention]

[0036] For the purpose of facilitating an understanding of the principles of this disclosure, embodiments shown in the drawings are referred to and described herein using specific language. Nevertheless, it will be understood that the description of these embodiments is not intended to limit the scope. Rather, this disclosure is intended to cover all substitutes, modifications, and equivalents that may fall within the spirit and scope of this application as defined by the appended claims. As stated above, the art may be illustrated and described in one or more preferred embodiments, but the compositions, compounds, and methods herein may include many different configurations, forms, materials, and accessories.

[0037] The following description includes numerous specific details to provide a complete understanding of the disclosure. Certain embodiments may be carried out without some or all of these specific details, and the disclosure is not limited to specific biological systems, specific fibrous diseases or cancers, or specific organs or tissues, and is naturally subject to change, but should be understood as continuing to be applicable in light of the data provided herein.

[0038] Various technologies and mechanisms in this disclosure may describe connections or linkages between two components. Terms such as attached, linked, joined, connected, and similar terms with their derived morphemes are used interchangeably unless the difference is noted or otherwise evident from the context. These words and expressions do not necessarily mean direct connections, but include connections via intermediate components. It should be noted that connections between two components do not necessarily mean direct and unimpeded connections, as they may exist between two components that various other components focus on. Therefore, unless specifically stated, connections do not necessarily mean direct and unimpeded connections.

[0039] Furthermore, wherever it is feasible and convenient, similar reference numerals are used in the figures and descriptions to refer to identical or similar parts or processes. The drawings are simplified forms and not to exact scale. Naturally, this disclosure is presented in this manner solely for illustrative purposes, and it is understood that the principles and embodiments described herein may apply to compounds and / or compositional components having configurations other than those specifically described herein. In fact, it is expressly intended that the compositions and compound components of this disclosure may be modified to facilitate their desired application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology. 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, but preferred methods and materials are described herein. Furthermore, where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the content otherwise explicitly indicates. Thus, for example, when a compound / composition is substituted with “an” alkyl or aryl, the compound / composition is optionally substituted with at least one alkyl and / or at least one aryl. Furthermore, unless otherwise stated, the term “about” means a range of plus or minus 10% of a value for percentages, and plus or minus 1.0 unit for unit values, for example, “about 1.0” means a range of values ​​from 0.9 to 1.1.

[0041] In certain embodiments, the compounds, compositions, and methods of this disclosure are useful for the prevention and / or treatment of fibrotic diseases. In certain embodiments, the compounds and / or compositions provided are also useful for the prevention and / or treatment of cancer. In some embodiments, the compounds, compositions, and methods provided herein utilize strategies that (e.g., selectively) target the innate immune system to reprogram macrophage polarization from M2 to M1 and, for example, utilize its anti-fibrotic properties.

[0042] In general, and without any intended limitation, the novel compounds, compositions, and methods of this disclosure target the innate immune system of interest 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 include a targeting moiety that targets folate receptor β (FRβ), such as a folate receptor-binding ligand or its radical, conjugated to an immunomodulator or a pharmaceutically acceptable salt thereof. As described in detail below, such embodiments utilize the limited expression of FRβ to directly localize systemically administered compounds to FRβ-expressing cells (e.g., cells in fibrous and / or cancerous tissues), thereby enabling the immunomodulatory component to convert activated bone marrow cells (e.g., M2-like macrophages) to anti-fibrotic M1 polarization. This targeting design favorably prevents systemic activation of the immune system and thus avoids toxicity.

[0043] Further exemplary embodiments may include a linker positioned between the targeting moiety and the immunomodulator. Such linkers may be releaseable or non-releaseable. As described herein, in compounds / compositions of the Disclosure including a releaseable linker, when administered, the targeting moiety and the immunomodulator are released from each other at or around the time the immunomodulator becomes active. Additionally or alternatively, in embodiments of the Compound / Composition of the Disclosure including a non-releaseable linker, the targeting moiety and the immunomodulator are not released immediately under physiological conditions when administered. Thus, after uptake by the target cell and / or activation of the immunomodulator, the components remain together.

[0044] Various embodiments of this disclosure, as well as supporting data relating to examples, will now be described. There are primarily two main immune strategies found in vertebrates: the innate immune system and the adaptive immune system. The innate immune response, or nonspecific immune response, is the first line of defense against non-self pathogens and consists of physical, chemical, and cellular defenses. The adaptive immune system, on the other hand, is called to act against pathogens that evade or overcome the primary innate immune defense.

[0045] Inflammatory responses play a crucial role in immunity. When tissue is damaged or pathogens are detected, for example, an inflammatory response is initiated and the immune system is activated. Immune cells of the innate immune system (i.e., neutrophils and eosinophils) are initially recruited to the site of tissue damage or the location of the damage or pathogen via the vascular and lymphatic systems, followed by the recruitment of macrophages.

[0046] Cells of the innate immune system can express special pattern recognition receptors that sense and bind to specific protein sequences present on microbial pathogens or other non-self molecules. As used herein, “pattern recognition receptor” means and includes any immune receptor expressed on the membrane of leukocytes—for example, at least macrophages—that can be activated and to which specific ligands can be bound, ultimately leading to an innate immune response (and, in specific cases, ultimately to the development of antigen-specific adaptive immunity).

[0047] Examples of two classes of molecules that can bind to pattern recognition receptors include pathogen-associated molecular patterns related to microbial pathogens, and injury-associated molecular patterns related to host cell components released during cell injury or death. Recognition of these protein sequences by pattern recognition receptors can initiate signaling pathways in which the product triggers the expression of specific genes that control the innate immune response (e.g., in some cases directing the development of antigen-specific adaptive immunity). Thus, pattern recognition receptors can be used to mediate these signaling pathways and, in certain cases, to positively or negatively regulate the innate immune response, and even the adaptive immune response.

[0048] Macrophages are a diverse group of leukocytes known to eliminate pathogens through phagocytosis and are broadly classified as either having an M1 or M2 phenotype, depending on the specific differentiation that occurs in response to the local tissue environment. In some cases, macrophages are polarized toward the M1 phenotype by exposure to interferon-gamma (IFN-γ), lipopolysaccharides (LPS), and / or granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain cases, the M1 phenotype is characterized by the production of high levels of pro-inflammatory cytokines (e.g., interleukin-1β (IL-1β), tumor necrosis factor (TNF), interleukin-12 (IL-12), interleukin-18 (IL-18), and / or interleukin-23 (IL-23)), the ability to mediate resistance to pathogens, potent bactericidal properties, high production of reactive nitrogen and oxygen intermediates, and / or enhancement of the 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 instances, M1 polarization functions to inhibit or prevent the initial establishment of infection and / or to remove damaged tissue.

[0049] In certain cases, after the innate immune system has performed this “attack and kill” phase, macrophages may reprogram themselves to become a healing system (i.e., M2 type), and for example, release growth factors to promote healing. Such growth factors may 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 (CC motif) ligand 18 (CCL18), and / or interleukin-13 (IL-13). In certain cases, exposure to such cytokines / growth factors alternatively activates the M2 macrophage phenotype.

[0050] In contrast to M1 macrophages, M2 macrophages may be associated with wound healing and tissue repair. In some cases, M2 macrophages are characterized by involvement in tissue remodeling, immunomodulation / suppression, and / or tumor promotion. In certain cases, M2 macrophages produce polyamines that induce cell proliferation and / or proline that induces collagen production. While this healing response is beneficial in healthy subjects, the presence of M2 macrophages can have significantly detrimental effects in subjects suffering from fibrotic disease or cancer through immunosuppression and / or promotion of tumor growth and fibrosis.

[0051] For example, fibrotic conditions may begin with an unknown trauma or damage to the epithelium. In response to the resulting tissue damage, immune cells may release chemokines and other factors to promote the infiltration of immune cells into the damaged tissue (e.g., innate immune response), which may include monocytes and macrophages that release pro-fibrotic cytokines, such as the M2-like phenotype. Chronic secretion of these cytokines may then activate endogenous and invasive fibroblasts / fibrous cells to become myofibroblasts, which then secrete collagen and other extracellular matrix proteins to harden the surrounding tissue. In some cases, these M2 macrophages exacerbate the disease by promoting fibrosis. For example, in subjects with idiopathic pulmonary fibrosis (IPF), M2 macrophages may infiltrate the lungs, promoting fibrosis within them and further impairing their function. In some cases, growth factors and other cytokines produced by the M2 phenotype promote the growth of cancerous tumors through a similar pathway.

[0052] Reprogramming of M2-like macrophages into M1-like macrophages In certain cancers and fibrotic diseases, macrophages may be disproportionately biased toward an anti-inflammatory (M2-like) phenotype. In certain cases, immunomodulators can convert—e.g., reprogram—activated myeloid cells (e.g., M2-like macrophages) to anti-fibrotic M1 polarization (e.g., they produce little to no growth factors and / or related cytokines, e.g., slowing or eliminating the progression of the disease state). In certain cases, compositions and methods provided herein reverse the anti-fibrotic to pro-fibrotic shift observed during the course of development of fibrotic diseases (e.g., IPF and certain cancers). In some embodiments, 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 samples taken from an individual or subject. Where used herein, “individual,” “subject,” or “patient” is a mammal, preferably a human, but may be an animal.

[0053] The terms “marker” or “biomarker” as used herein may be described as differentially expressed when the level of expression in a subject experiencing an active disease state is significantly different from the level of expression in a healthy subject or a subject or sample not experiencing a disease state. Differentially expressed markers may be overexpressed or underexpressed (in the embodiments described in the preceding paragraph, the biomarker is reduced or underexpressed) compared to the baseline level of a normal or control sample or the subject. Increases or decreases in or quantification of markers in a biological sample may be determined by any of several methods known in the Art for measuring the presence and / or relative abundance of a gene product or transcript. Marker levels may be determined as absolute values, compared to baseline values, or as the level of the marker in a subject compared to a cutoff index. Alternatively, the relative abundance of one or more markers may be determined compared to a control that may be a clinically normal subject. Furthermore, as used herein, the terms “gene overexpression” and “overexpression” (when used in relation to a gene) and their constituent elements have meanings to those skilled in the art, including (but not limited to) the overexpression or misexpression of a wild-type gene product that may result in a mutant phenotype and / or abundant expression of a target protein.

[0054] In some embodiments, compositions and methods provided herein increase anti-fibrotic biomarkers (e.g., TNFα and IFN-γ). In some embodiments, compositions are provided that reverse an M2-like phenotypic shift (e.g., providing an effective treatment for fibrotic diseases, disorders, or conditions).

[0055] In at least one embodiment, a compound used in the method described herein is prepared using a drug containing an immunomodulator. As used herein, “immunomodulator” means any drug, warhead, or other composition or compound that stimulates or otherwise affects an immune system of interest by inducing the activation or increased activity of one or more components of the immune system. For example, but not limited to, an immunomodulator may include a compound or composition that targets one or more pattern recognition receptors in addition to, or instead of, a signaling pathway in immune cells.

[0056] Examples of immunomodulators of this disclosure include, but are not limited to, agonists of Toll-like receptors (TLRs), interferon gene stimulants (STINGs), nucleotide-binding oligomerized domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), absent-in-melanoma 2 (AIM2)-like receptors (ALRs), advanced glycation end product (RAGE) receptors, or any other pattern recognition receptors located in the endosomes or cytoplasm of cells. The immunomodulators of this disclosure may additionally or alternatively include activated β-cell nuclear factor kappa-light chain enhancer (NFκβ) activators or Iκβ kinase inhibitors that function further downstream in the pathway. Table 1 provides examples of such NFκβ activators or Iκβ kinase inhibitors that may be employed as immunomodulators of this disclosure. [Table 1] JPEG0007904596000019.jpg141170

[0057] As used herein, "TLR" refers to a class of proteins that play a role in the innate immune system and is an example of a pattern recognition receptor. 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 lymphocytes and B lymphocytes), and non-immune cells (epithelial cells, 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.

[0058] Any therapeutic agent (e.g., a drug) suitable for reprogramming activated macrophages (M2-like phenotype) into an M1-like phenotype may be used, and the drug (or warhead) may act in the endosomes and / or cytoplasm of cells (e.g., depending on their structure). In at least one embodiment, the therapeutic agent comprises an immunomodulator (e.g., one that positively modulates pattern recognition receptors and / or their downstream signaling pathways (in each case, part of the innate immune system), e.g., TLR, NLR, RLR, ALR, RAGE, and / or STING agonists, as well as / or Pelle / interleukin-1 receptor-associated kinase (IRAK) family, e.g., IRAK-M inhibitors). In other embodiments, the compounds provided herein comprise phosphoinositide 3-kinase (PI3K) kinase inhibitors or other inhibitors that negatively modulate the adaptive immune system (e.g., one alone or in combination with an immunomodulator targeting a pattern recognition receptor). In some embodiments, particularly when used for the treatment of IPF or other fibrotic conditions, the composition or compound (e.g., a drug) comprises (a) an immunomodulator that targets pattern recognition receptors and / or an immunomodulator that is an agonist of a downstream signaling pathway of its innate immune system, and (b) a combination of mammalian target of rapamycin (mTOR) inhibitors (ATP competitive or otherwise), such as rapamycin or CZ415.

[0059] In certain embodiments, the Specified Provisions provide compounds comprising a targeting moiety (or radical) attached to an immunomodulator (or radical thereof) that targets a cellular pattern recognition receptor, wherein the targeting moiety comprises a folate ligand or a functional fragment or analog thereof. "Folic acid" includes, but is not limited to, folate and its analogs and derivatives, such as folate, pteroylpolyglutamic acid, pteroyl-D-glutamic acid, and folate receptor-binding pterdins, such as tetrahydropterin, dihydrofolate, tetrahydrofolate, and their deaza and dideza analogs.

[0060] The terms “deaza” and “dideaza” analogs refer to analogs, or derivatives thereof, recognized in the art that have carbon atoms instead of one or two nitrogen atoms in the natural folate structure. For example, deaza analogs may include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate, folic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterin, dihydrofolate, and tetrahydrofolate. Examples of dideza analogs include 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of folate. In the context of this disclosure, other folate useful as complex-forming ligands include the folate receptor binding analogs pemetrexed, proguanil, pyrimethamine, trimethoprim, pralatrexate, larcitrexed, aminopterin, ametopterin (also known as methotrexate), and N 10 -Methylfolate, 2-deaminodihydroxyfolate, deaza analogs, e.g., 1-deazametopterin or 3-deazametopterin, and 3',5'-dichloro-4-amino-4-deoxy-N 10 - Examples include methylpteroylglutamic acid (dichloromethotrexate).

[0061] Folic acid, as well as the aforementioned analogues and / or derivatives, are referred to as “folate,” “the folate,” or “folates” to reflect their ability to bind to folate receptors. As described herein, these molecules are effective in enhancing transmembrane transport, such as via folate-mediated endocytosis when conjugated with exogenous molecules. The aforementioned literature can be used in the folate receptor-binding ligands described herein. As used herein, the term “ligand” is a molecule, ion, or atom attached to the central atom or ion of a compound (e.g., a drug).

[0062] Specific embodiments of the novel compounds of the present disclosure are provided herein. Those skilled in the art will understand that the compounds of the present disclosure may be polymorphic. In fact, the compounds of the present disclosure may include any racemates, optically active compounds, polymorphs, or stereoisomers, or mixtures thereof, of the compounds described herein that exhibit useful properties, and methods for preparing optically active compounds (e.g., by recrystallization techniques, by synthesis from photoactive starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase), and methods for determining antitumor activity using the standard tests described herein or other similar tests well known in the art are well known in the art. Furthermore, unless otherwise expressly stated, the structures described herein also mean all stereochemical forms of the structure, i.e., right-handed (R) and left-handed (S) configurations of each asymmetric center configuration. Thus, single stereoisomers of the compositions, as well as enantiomers and diastrome mixtures, are within the scope of the present disclosure.

[0063] The specific values ​​listed herein for radicals, substituents, and ranges are for illustrative purposes only unless otherwise specified, and such examples do not exclude other defined values ​​or other values ​​within the defined ranges for radicals and substituents. For example, (C1-C6) alkyls may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C1-C3) alkyls may be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C1-C3) alkoxys may be methoxy, ethoxy, or propoxy; and (C2-C6) alkanoyloxys may be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0064] Furthermore, if a portion is replaced by an R substituent or substituent, that group may be referred to as “R-substituted.” Where a portion is described as R-substituted, or otherwise as generally containing a substituent, the portion is replaced by at least one R substituent, each substituent being optionally distinct. The substituent (or R substituent) may contain any molecule or combination of molecules, provided that its inclusion does not substantially affect the overall structure and shape of the compound and does not alter any hydrogen bonds essential to the underlying compound in achieving its intended purpose (e.g., binding to a target pattern recognition receptor).

[0065] When substituents are specified by conventional chemical formulas written from left to right, they similarly encompass chemically identical substituents that arise from describing structures from right to left, such as -CH2O- being equivalent to -OCH2-.

[0066] In some embodiments, the immunomodulatory group (e.g., TLR7 agonist) of the compounds provided herein is a radical having the structure of formula XX, more specifically, the structure of formula XX'; [ka] During the ceremony, R 1B -NH2 or -NH-R 1X And, R 2B Hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And, R 1X , R 2X , and R2Y Each of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. X is either CH or nitrogen (N).

[0067] As used herein, alkyl, alkoxy, etc., refer to linear (i.e., unbranched) or branched chains, or combinations thereof, which may be fully saturated, monovalent or polyunsaturated, and may contain divalent and polyvalent radicals, having a specified number of carbon atoms (i.e., C1-C1). 10 (where n means 1 to 10 carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, and homologous and isomeric groups such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated alkyl groups are alkyl groups having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, clotyl-2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-penadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologous and isomeric groups. Alkoxy are alkyl groups attached to the rest of the molecule via an oxygen linker (-O-). In some embodiments, alkoxy refers to a radical bonded via the oxygen atom of the formula -O-alkyl.

[0068] Generally, the terms “acyl” or “acyl substituent” refer to those derived by removing one or more hydroxyl groups from oxoacids, including inorganic acids, and contain a double-bonded oxygen atom and an alkyl group. Furthermore, references to individual radicals such as “propyl” encompass only linear radicals, with branched isomers such as “isopropyl” specifically mentioned.

[0069] In some embodiments, the TLR7 agonist has formula X, and the TLR7 agonist is connected via the linker R 1A , R 1B , R 3A , or R 3B If the targeting portion is conjugated in any one of the following, and the TLR7 agonist has formula XX', then the TLR7 agonist is linked to R via the linker. 1A , R 1B , R 3A , or R 3B It is conjugated to the target portion in one of them.

[0070] As used herein, the term “linker” includes a chain of atoms that is biofunctionally adapted to form a chemical bond with A, B, or S, linking two or more functional parts of a molecule to form the compounds of this disclosure. Exemplary, the chain of atoms may be selected from carbon (C), N, oxygen (O), sulfur (S), silicon (Si), and phosphorus (P), or C, N, O, S, and P, C, N, O, and S. The chain of atoms may covalently bond different functional capabilities of a compound, such as folic acid and pharmaceuticals. The linker may include a wide variety of linkages, such as in the range of about 2 to about 100 atoms in a continuous skeleton, and may include releaseable or non-releaseable linkers. In some embodiments, the immunomodulatory (e.g., TLR7 agonist) group of the compounds provided herein is a radical having the structure of formula XXX, more specifically, the structure of formula XXX'. [ka] During the ceremony, R1C -NH2 or -NH-R 1X And, R 2C This is combined, NH, -NR 1X , or CH2 And, If applicable, [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. X A This is CH2, NH2, or -NH-R 1X And, Each R 1X These are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. The TLR7 agonist is R via the linker. 1C , R 2C , or R 3B In one of them, it is conjugated to the targeting portion.

[0071] In some embodiments, the compound is a linker between the targeted moiety and the immunomodulator, or otherwise a connecting linker ("L" or "L"). n This further includes ) in some embodiments, linker L n The linker L is configured to avoid the release of immunomodulators, where n is an integer less than or equal to 50. In some embodiments, the linker L n The linker comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, where n is an integer selected from the range of 1 to 32, and the targeting moiety is specific to the folate receptor β. In some embodiments, n is 1 to 50, 1 to 10, 2 to 8, or 2 to 4.

[0072] In some embodiments, L is a hydrolyzable linker. In some embodiments, L is a non-hydrolyzable linker. In some embodiments, L is an optionally substituted heteroalkyl.

[0073] The term "alkylene" refers to a divalent radical derived from an alkyl group, either alone or as part of another substituent, exemplified by -CH2CH2CH2CH2- unless otherwise specified, but not limited to this form. Typically, an alkyl (or alkylene) group has 1 to 24 carbon atoms. "Lower alkyl" or "lower alkylene" refers to a short-chain alkyl or alkylene group, generally having 8 or fewer carbon atoms.

[0074] The term "heteroalkyl," either alone or in combination with another term, means, unless otherwise specified, a stable linear or branched chain, or a combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quarternized. The heteroatoms O, N, P, S, and Si may be located at any internal position of the heteroalkyl group, or at a position where the alkyl group is attached to the rest of the molecule. Examples, though not limited to them, include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0075] Similarly, the term “heteroalkylene” means a divalent radical derived from a heteroalkyl group, either by itself or as part of another substituent (unless otherwise stated), exemplified by, but not limited to, -CH2-CH2-S-CH2-CH2 and -CH2-S-CH2-CH2-NH-CH2. For heteroalkylene groups, the heteroatom may also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2'. As stated above, heteroalkyl groups, as used herein, include groups attached to the rest of the molecule via heteroatoms, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO2R'. When “heteroalkyl” is enumerated, followed by an enumeration of specific heteroalkyl groups such as -NR'R'', it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are enumerated for clarity. Therefore, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups such as -NR'R''.

[0076] In some embodiments, L is a substituted heteroalkyl group comprising at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. Unless otherwise stated, “halo” or “halogen” means a fluorine, chlorine, bromine, or iodine atom, either by itself or as part of another substituent.

[0077] In some embodiments, L includes a spacer (as described elsewhere in this specification, for example). In some embodiments, the spacer includes a peptidoglycan or a sugar.

[0078] 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.

[0079] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers, polypeptides, or fragments of polypeptides, peptides, or fusion polypeptides of amino acid residues. The terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0080] In some embodiments, L is -CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO- is included in the formula, where R a and R b These are independently H, alkyl, or heteroalkyl (e.g., PEG).

[0081] In some embodiments, L includes the following structure: [ka] In the formula, n and m are independently between 0 and 10.

[0082] In some embodiments, L includes the following structure: [ka] In the formula, n is between 1 and 32. In at least one exemplary embodiment, n is between 1 and 30 and w is between 0 and 5.

[0083] In some embodiments, L includes the following structure [ka] In the formula, n is between 1 and 30, and w is between 0 and 5.

[0084] In some embodiments, the compound has a structure represented by the following formula. [ka]

[0085] In some embodiments, the compound has a structure represented by the following formula. [ka]

[0086] In some embodiments, the compound has a structure represented by the following formula. [ka]

[0087] In some embodiments, the compound has a structure represented by the following formula. [ka]

[0088] In certain embodiments, provided herein is a compound comprising a targeting moiety comprising a folic acid ligand or a functional fragment or analog thereof attached to an immunomodulatory substance comprising a TLR agonist via a linker, wherein the TLR agonist has the following structure represented by formula XXX,

Chemical formula

Chemical formula

Chemical formula

[0089] In some embodiments, provided herein are pharmaceutical compositions comprising any formula or compound provided herein, wherein the linker comprises PEG or a PEG derivative, and optionally R 3 It is a non-release linker attached in or R 1 , R 2 , or R 3 It is a releaseable linker attached thereto.

[0090] In some embodiments, pharmaceutically acceptable salts are selected from hydrobromide, citrate, trifluoroacetate, ascorbic acid, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, or fumarate.

[0091] In some embodiments, the compound includes a TLR agonist (e.g., its radical), for example, but not limited to, a TLR3 agonist, a TLR7 agonist, a TLR7 / 8 agonist, a TLR8 agonist, or a TLR9 agonist (all of which bind to Toll-like receptors present in cellular endosomes). For example, but not limited to, in at least one exemplary embodiment, the immunomodulator of the drug / compound may be selected from the compounds listed in Table 2 below. [Table 2] JPEG0007904596000036.jpg235170JPEG0007904596000037.jpg230170JPEG0007904596000038.jpg253170JPEG0007904596000039.jpg99170

[0092] In some embodiments, the compounds provided herein are compounds (or radicals) of formula I (e.g., TLR7 agonists), [ka] or a pharmaceutically acceptable salt thereof or containing thereof. In some embodiments, R 1 is an amine. In some embodiments, R 2 R is, for example, a single bond, or an amine (for example, -NH-). In certain embodiments, R 3 X is H, alkyl, hydroxyl, or any other suitable substituent (e.g., those described herein). In some embodiments, X is CH2, NH, O, or S. In some embodiments, the compounds provided herein comprise a radical of formula I, and the targeted moiety is at any suitable position, for example, R 1 , R 2 , and / or R 3 They are then conjugated or linked together, either through or via these (for example, via a linker and / or directly).

[0093] In at least one exemplary embodiment, the compounds described herein are compounds (or radicals) of formula Ia (e.g., TLR7 agonists). [ka] or a pharmaceutically acceptable salt thereof, or comprising thereof. In some embodiments, X is CH or N. In some embodiments, R1 is -NH2 or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] In a particular embodiment, R 1X , R 2X , and R 2YEach of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In some embodiments, [ka] The radical is a 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, the compounds provided herein comprise a radical of formula Ia, and the targeting moiety is at any suitable position, for example, R 1 , R 2 , and / or R 3 And, or through these (for example, via a linker and / or directly), they are conjugated or linked,

[0094] In some embodiments, the compounds provided herein are compounds (or radicals) of formula II (e.g., TLR7 agonists), [ka] or a pharmaceutically acceptable salt thereof, or comprising the same. In some embodiments, R 1 is an amine. In some embodiments, R 2 is (for example, a single) bond or -NH-. In certain embodiments, R 3 X is H, alkyl, hydroxyl, or any other suitable substituent, for example, as described herein. In some embodiments, X is CH2, NH, O, or S. In some embodiments, the compounds provided herein comprise the radical of formula II, and the targeted moiety is at any suitable position, for example, R 1 , R 2 , and / or R 3 They are then conjugated or joined together, either by means of or through these (for example, via a linker and / or directly).

[0095] In other embodiments, the compounds of the present disclosure may include agents comprising a TLR agonist of formula III (e.g., or its radical) or a pharmaceutically acceptable salt thereof. [ka] In the formula, R 1 R is an amine group, 3 R is a hydroxyl group. Furthermore, if desired, the targeting moiety (e.g., or its radical) or other ligand is R 1 or R 3 In this (either via a linker or directly), it may be conjugated to an agonist of formula III. The TLR agonist of formula III (e.g., or its radical) is a TLR7 agonist and is at least 10 times (10x) stronger than conventionally available TLR7 agonists.

[0096] In some embodiments of this specification, TLR7 agonists of formula IV (e.g., or radicals thereof) or pharmaceutically acceptable salts thereof are provided. [ka] In the formula, R 1 R is an amine group, 2 It is a single bond -NH-.

[0097] In some embodiments, administration of the compounds provided herein (e.g., to an organism) converts macrophages in fibrous tissue from an M2-like phenotype to an M1-like phenotype. In some embodiments, after administration of the compounds provided herein, a decrease in cytokines that stimulate collagen synthesis (i.e., CCL18, PDGF, and IL-1β) occurs, along with a simultaneous increase in cytokines that inhibit collagen production (e.g., IFN-γ). In particular, in at least one embodiment, after administration of the compounds provided herein, the cytokine profile coincides with the reprogramming of the M2-like phenotype to an M1-like phenotype. As used herein, “profile” or “assay” is a set of one or more markers, 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 in a sample. A genome or nucleic acid profile is a dataset of the presence, absence, relative levels, or abundances of expressed nucleic acids (e.g., transcripts, mRNA, etc.). Alternatively, a profile may also be called an expression profile.

[0098] In some embodiments, the net result of reprogramming is an increase in alveolar air sacs, a decrease in extracellular matrix deposition, and a decrease in hydroxyproline / collagen biosynthesis, leading to effective recovery from the disease (see, for example, Example 4).

[0099] While specific agents and formulations are described herein, it should be understood that any compound (e.g., an agent) useful for reprogramming activated myelocytes into an anti-fibrotic M1-like phenotype may be used in the novel compounds and methods herein (e.g., any compound (e.g., an agent) that can bind to pattern recognition receptors and inhibit at least a portion of their downstream innate immune response). In some embodiments, analogues and / or derivatives of the compounds described herein may be used in the targeted compounds provided herein.

[0100] Furthermore, two or more compounds can be administered, and in some cases, the compounds may include different agents. For example, the different agents may be selected from TLR7 agonists and TLR9 agonists. In yet another embodiment, one or more compounds may be administered in a composition together with one or more conjugated and / or unconjugated agents (e.g., the conjugated embodiments described below). In some embodiments, any of the compounds and agents described herein may be used according to the methods described herein and may be combined with other agents that directly modify fibroblasts by depleting or inhibiting myeloid suppressor cells (e.g., in relation to the treatment of cancer), downregulating the production of growth factors (e.g., pirfenidone, related to the treatment of IPF), inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling (e.g., CZ415, related to the treatment of IPF or other fibrotic conditions), and / or any other anti-fibrotic and / or anticancer agents and therapies, depending on the desired application. As used herein, “downregulation” and its constituents (e.g., “down-regulation” or “down-regulated”) may be used interchangeably and refer to a decrease in the level of a marker such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. As used herein, “upregulation” and its constituents (e.g., “p-regulation” or “up-regulated”) may be used interchangeably and refer to an increase in the level of a marker such as a gene, nucleic acid, metabolite, transcript, protein, or polypeptide. Pathways, such as signaling or metabolic pathways, may also be up- or down-regulated.

[0101] targeting part In some cases, the toxicity associated with the systemic administration of at least the conventional agents specified herein has hindered their practical application in the treatment of fibrotic diseases, cancer, or any other disease state. For example, TLR agonists may not be tolerable in individuals and may, in some cases, lead to death of the subject (e.g., when administered systemically via conventional means). In some embodiments, the compounds provided herein, for example, compounds having formulas I and / or II, are significantly more potent than conventional agents that can be used in combination with the compounds of this disclosure, and in some cases, a mechanism to avoid systemic toxicity is desirable.

[0102] In certain embodiments, therapeutic agents (e.g., drugs (as described above)) conjugated with a targeting moiety are provided herein. In some embodiments, the targeting moiety comprises a ligand (e.g., having high specificity) or other atom or molecule that targets a specific region or tissue of an individual, and in certain examples, may include, for example, hormones, antibodies, and / or vitamins. As will be described in more detail below, in at least one embodiment, the targeting moiety comprises a molecule having (e.g., high) affinity for FRβ. In some cases, the targeting moiety may have specific affinity for any receptor specific to cells or tissues of fibrous disease or cancer, as needed.

[0103] In some cases, FRβ is significantly upregulated in activated myeloid cells (e.g., primarily activated monocytes and M2-like macrophages), and all recorded data to date support the induction of FRβ only in cells of myeloid origin after exposure to anti-inflammatory or pro-inflammatory stimuli. Folate receptors can be upregulated in non-mucinous ovarian cancers (e.g., over 90%). In certain specific cases, folate receptors are present in kidney, brain, lung, and breast cancers. For example, there are numerous cancers that do not express folate receptors in sufficient numbers to provide the desired specificity, but the cancerous tumors express myeloid-derived suppressor cells (MDSCs) that express FRβ, for example, and can be targeted by the targeting moieties provided herein. In some embodiments, folate receptors are substantially absent (e.g., present only at very low levels) in healthy (non-myeloid) tissues (e.g., lung, liver, spleen, heart, brain, muscle, intestine, pancreas, bladder, etc.). In some cases, even quiescent macrophages, which are abundant throughout the body, are primarily FRβ-negative. In some cases, uptake of folate-targeted contrast agents occurs in, for example, inflammatory tissue, malignant lesions, and the kidney. In certain cases, non-cancerous subjects retain only folate-targeted agents in the kidney and inflammatory sites. In some cases, mismatches in folate receptor expression provide a mechanism for selectively targeting fibrous cancer cells.

[0104] In some embodiments, the compounds and methods provided herein utilize the limited expression of FRβ to target / localize systemically administered potent compounds (e.g., conjugates or drugs) to fibrous and / or cancerous tissue. In some cases, the compounds provided herein are delivered directly to FRβ-expressing cells, for example, to advantageously prevent systemic activation of the immune system, thereby avoiding the toxicity (e.g., at least some of it) that has so far hindered the systemic use of non-targeted compounds (e.g., drugs) described herein. In some embodiments, the methods described herein are used, for example, to treat fibrous diseases and / or cancer, regardless of whether the cancer expresses folate receptors. In some embodiments, folate and other folate receptor-binding ligands (or their radicals), such as folate, are used as targeting moieties because they have affinity for FRβ, for example.

[0105] Folic acid is a member of the vitamin B family and can play an important 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 agents by targeting activated myeloid cells and can enhance conjugated anticancer agents by targeting folic acid receptor-positive cancer cells. Compounds comprising a folic acid ligand (or its radical), or a functional fragment or analog thereof, are provided herein, in some cases as the targeting moiety and immunomodulator (e.g., TLR7, TLR8, TLR7 / 8, TLR9, or TLR3 agonist). In some cases, TLR7, TLR8, TLR7 / 8, TLR9, and TLR3 are present in endosomes. In some embodiments, the compound, or its radical, binds to a TLR. In some embodiments, the TLR is TLR7.

[0106] Alternatively, a pyrido[2,3-d]pyrimidine analog ligand (e.g., or a radical thereof), a functional fragment or analog thereof, or any other molecule, fragment or atom having an affinity for FRβ (e.g., but not limited to, high specificity) may be used as the targeting moiety (or a radical thereof). For example, such folic acid analog molecules may have a relative affinity for binding FRβ of about 0.01 or more compared to folic acid at about 20 °C / 25 °C / 30 °C / physiological temperature. Similarly, a Galectin-3 ligand, a translocator protein (TSPO) ligand, and any other ligand or targeting moiety having a highly specific affinity for fibrotic and / or cancerous cells or tissues may be employed.

[0107] Specific examples of suitable targeting moieties (or radicals thereof) are provided herein, but it will be understood that the targeting moieties (or radicals thereof) of the present disclosure may include any ligand (or radical thereof) useful for targeting FRβ and are not limited to the structures specified herein. The ligand (or radical thereof) may bind to FRβ.

[0108] In at least one embodiment, the compounds provided herein include a targeting moiety (or a radical thereof) having the structure of Formula V or a functional fragment or analog thereof,

Chemical formula

[0109] In a further aspect, by way of non-limiting example, the targeting moiety (or its radical) of formula V has the structure of VI (or its functional fragment or analog), [Chemical formula] wherein, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S, Y is C, CH, CH2, N, NH, O, or S, Z is glutamic acid, valine, or a substrate, R1 and R2 are each independently NH2, OH, SH, CH3, or H, R3 is time or an alkyl, m and n are each independently 0, 1, or between 0 and 1, [Chemical formula] represents either a single bond C-C or a double bond C-C.

[0110] [[ID=四十七]]Another specific targeting moiety (or its radical) of formula V (or its functional fragment or analog) has the structure of formula VII, [Chemical formula] wherein, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S, Y is C, CH, CH2, N, NH, O, or S, Z is glutamic acid, valine, or a substrate, R1 and R2 are each independently NH2, OH, SH, CH3, or H, R3 is time or alkyl, m and n are each independently 0, 1, or between 0 and 1,

Chemical formula

[0111] In some embodiments, the targeting moiety (or its radical) of formula VI has the structure of formula VIII,

Chemical formula

Chemical formula

[0112] In some embodiments, the targeted portion of formula VI (or its radical) has the structure of formula IX, [ka] During the ceremony, X1, X2, X3, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is time or alkyl, m is 0, 1, or between 0 and 1. [ka] This represents either a single bond or a double bond (CC).

[0113] In some embodiments, the targeted portion of formula VII (or its radical) has the structure of formula X or XI. [ka] During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is time or alkyl, m is 0, 1, or between 0 and 1. [ka] This represents either a single bond or a double bond CC, or [ka] During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently N, NH, CH, CH2, O, or S. Y is C, CH, CH2, N, NH, O, or S. Z is glutamate, valine, or a substrate. R1 and R2 are independently NH2, OH, SH, CH3, or H. R3 is time or alkyl, m is 0, 1, or between 0 and 1. [ka] This represents either a single bond or a double bond (CC).

[0114] Chemical structures and spectroscopic data of several additional embodiments of the targeted portion (e.g., or its radical) of the present disclosure are provided in Tables 3, 4, 5, and 6 below.

[0115] Table 3 provides non-limiting examples of additional embodiments of a targeted moiety (e.g., or its radical) having the structure of formula VIII. [Table 3] JPEG0007904596000062.jpg254170JPEG0007904596000063.jpg221170

[0116] Table 4 provides non-limiting examples of additional embodiments of a targeted moiety (e.g., or its radical) having the structure of formula IX. [Table 4] JPEG0007904596000065.jpg106170

[0117] Table 5 provides non-limiting examples of additional embodiments of the targeting portion having the structure of formula X. [Table 5]

[0118] As described above, instead of folic acid, the targeting moiety (e.g., its radical) may be one or more non-classical folic acid antimetabolite analogs (or their analogues or functional fragments), such as pyrido[2,3-d]pyrimidine or similar analogs (or their radicals) having the formulas listed in Table 6 below (e.g., the radicals of the formulas). [Table 6] JPEG0007904596000068.jpg219170JPEG0007904596000069.jpg219170JPEG0007904596000070.jpg254170JPEG0007904596000071.jpg87170

[0119] In some cases, the compounds provided herein include a targeted moiety (e.g., its radical) conjugated with a drug (e.g., its radical) (e.g., an immunomodulator). The immunomodulator (e.g., its radical) may be conjugated directly with the targeted moiety (e.g., its radical) or via a linker (e.g., optionally including a spacer). Figure 1A shows at least one embodiment of compound 100, where compound 100 includes, for example, an immunomodulator (or its drug or radical) 102 having formula I, where R 3 is a hydroxyl group. The immunomodulator (e.g., its radical) 102 is conjugated to the targeting moiety (e.g., its radical) 104 via the linker 106, where the targeting moiety (e.g., its radical) 106 is folic acid, and the (e.g., non-release) linker 106 is a PEG linker repeated n times, where n is between 1 and 32.

[0120] In at least one embodiment, and not limited thereto, compound 100 may be represented by the following formula, QLT, where Q is the radical of the folate receptor-binding ligand / targeting moiety 104, L is the linker 106, and T is the radical of the TLR agonist / immunomodulator 102. The linker L may include any of the linker formulas presented herein.

[0121] Similarly, Figure 1B shows at least one embodiment of compound 150. Compound 150 has an immunomodulator / drug (e.g., its radical) 152 which is a TLR7 agonist (e.g., its radical) having formula III, for example, conjugated to a targeting moiety (e.g., its radical) 154 via a linker 156 (e.g., releaseable).

[0122] Linker (L or L nThe linker may be releaseable or non-releaseable. In some cases, the target of the compound containing the non-releaseable linker is, for example, endosomes (e.g., of the target cell), while the target of the releaseable linker may, in some cases, be endosomes, cytoplasm, or both (e.g., of the target cell).

[0123] In at least one exemplary embodiment, linker L n It is positioned between the targeting moiety (e.g., its radical) and the immunomodulator or a pharmaceutically acceptable salt thereof, and linker L or L n The TLR7 agonist is configured to avoid the release of the free form, where n is an integer less than or equal to 50. Additionally or alternatively, the compound may contain a linker L containing PEG or a PEG derivative. n It may include n, where n is an integer selected from 1 to 32, and the targeting portion (e.g., its radical) may include a radical of a folate receptor-binding ligand containing an FRβ-binding ligand.

[0124] The term “releaseable” in relation to linkers means a linker containing at least one bond that can be broken under physiological conditions (e.g., by chemical or enzymatic hydrolysis), such as by reducing agent instability, pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability, enzymatic instability, or by a p-aminobenzyl-based multivalent releaseable bond. It is understood that the physiological conditions resulting in the breakdown of the bond do not necessarily involve biological or metabolic processes, but instead may include standard chemical reactions, such as hydrolysis reactions as a result of compartmentalization into organelles such as endosomes at physiological pH or at pH lower than cytoplasmic pH. A cleavable bond can, for example, connect two adjacent atoms within the releaseable linker and / or other linker or targeting moieties and / or drugs at either or both ends of the releaseable linker, as described herein. In some cases, the releaseable linker is split into two or more fragments. In some cases, the releaseable linker is separated from the targeting moiety. In some embodiments, the targeted moiety and the immunomodulator are released from each other, and the immunomodulator is activated.

[0125] In contrast, the term “non-released” in relation to linkers means a linker containing at least one bond that does not break easily or rapidly under physiological conditions. In some embodiments, the non-released linker contains a backbone that is stable under physiological conditions (e.g., the backbone is resistant to hydrolysis (e.g., aqueous hydrolysis or enzymatic hydrolysis)). In some embodiments, the compositions provided herein containing a non-released linker do not release any component of the composition (e.g., a targeted ligand (e.g., a fully amorphous (FA)-ligand) or an immunomodulator (e.g., a TLR7 agonist)). In some embodiments, the non-released linker lacks a disulfide bond (e.g., SS) or an ester in the backbone. In some embodiments, the composition contains a targeted moiety and an immunomodulator linked by a backbone that is substantially stable throughout the entire cycle of the composition (e.g., during endocytosis to target cell endosomes). In some embodiments, compositions containing a non-released linker are particularly beneficial when the immunomodulator targets TLRs, NOD-like receptors, and / or other pattern recognition receptors present in cellular endosomes. Non-release linkers may include amides, esters, ethers, amines, and / or thioethers (e.g., thio-maleimides). Specific examples are provided herein, but it will be understood that any molecule can be used as a non-release linker, provided that at least one bond is formed that does not break easily or rapidly under physiological conditions.

[0126] Perhaps more specifically, the non-release linker includes, for example, a linker that hydrolyzes in an aqueous solution (e.g., a buffered (e.g., phosphate buffer) solution) within a certain period (e.g., 24 hours) at a neutral pH, for example, less than 10 percent (10%) (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or 0.001%). In some embodiments, when a non-release linker is employed, less than 10 percent (10%), preferably less than 5 percent (5%), or none at all, of the administered conjugate compound releases free drug (e.g., in systemic circulation before uptake by target cells / tissues). In some embodiments, within one hour of administration, less than 5 percent (5%) of the free drug is released from the conjugate while the compound is in systemic circulation.

[0127] In some embodiments, the targeted portion is not cleaved from the drug / immunomodulator so that the compound is therapeutically effective in vivo. In some embodiments, this is advantageous because it allows the use of targeted compositions containing potent drugs (e.g., TLR7 agonists) because, for example, only a small amount of the drug (e.g., immunomodulator, e.g., TLR7 agonist) is released (e.g., systemically) before targeted delivery of the compound. In some embodiments, tuning the release characteristics of the active ingredient is a difficult aspect of preparing an effective pharmaceutical composition. In some embodiments, compositions containing a non-release linker provided herein avoid the difficulties of preparing an effective pharmaceutical composition (e.g., by eliminating the need for release timing). In some embodiments, the immunomodulator or warhead of the compound provided herein becomes active when conjugated (e.g., when conjugated to a targeted conjugate). In some embodiments, while the nuclear head / immunomodulator is active, the non-release linker and targeting moiety prevent the release of toxic cytokines (e.g., interleukin-6 (IL-6)) that activate the immune system (e.g., by the target body) (e.g., because the compound is specifically targeted (e.g., using folic acid or an analogue)). In certain examples, for example, even if the warhead / immunomodulator of the compound is active when it is attached to the non-release linker, the immunomodulator cannot access the appropriate receptor (e.g., the target) in the cell's endosome until the compound binds to the target receptor (e.g., the folic acid receptor).

[0128] As a non-limiting example, linker 106 in Figure 1A is a non-releaseable PEG linker, while linker 156 in Figure 1B is a self-immolative releaseable linker (e.g., containing a disulfide bond (e.g., SS)). For example, Figure 1B shows the self-destructive cascade of compound 150 upon cleavage from the targeted moiety 154.

[0129] In some embodiments, the linker 156 is configured such that the drug is cleaved from the targeting portion 154 only after sufficient time has elapsed since administration for the compound to circulate within the systemic circulation of the subject (e.g., disappear from non-target tissues and be captured and internalized by target cells and / or receptors). In some embodiments, the release period varies (e.g., per subject (e.g., based on various factors)). In some embodiments, the releaseable linker may be engineered not to be cleaved / released until at least 24 hours after administration, or even for a week. In some embodiments, toxicity can be prevented because the compound can safely pass through the system of the subject, and any amount not captured by target cells (e.g., those expressing FRβ) can be purged before release / activation (e.g., because the immunomodulator is not active when bound to the releaseable linker).

[0130] Both releaseable and non-releaseable linkers can be manipulated to optimize the in vivo distribution, bioavailability, and PK / PD of (e.g., compounds) and / or to increase uptake into target tissues (e.g., compounds) according to methodologies commonly known or to be developed in the art, such as PEGlaytion. In some embodiments, the linker is configured to avoid significant release of the pharmaceutically active amount of the circulating drug before capture by cells (e.g., target cells (e.g., macrophages in fibrous or cancerous tissue being treated)).

[0131] In some embodiments, compounds comprising the releaseable linker of the present disclosure may be designed to diffuse across the endosomal membrane, for example, into the cytoplasm of a target cell. In some embodiments, the releaseable linker may be designed so that the immunomodulator is not released until the compound reaches the cytoplasm.

[0132] In some embodiments, the conjugates provided herein include a releaseable linker (for example, to facilitate the release of an immunomodulator into the cytoplasm, in which case the immunomodulator includes an activator of PI3K kinase, IRAK, or 1-kappa-β (Iκβ) kinase (e.g., using prostratin, etc.) or the nuclear factor kappa-light chain enhancer (NF-κβ) of activated β cells (e.g., see Table 1), or a myeloid differentiation primary response 88 (MyD88) agonist). In some embodiments, the releaseable linker prevents the release of the immunomodulator until, for example, the targeting portion binds to a suitable target (e.g., macrophage folate receptor), is internalized into the endosome of the target cell, and / or diffuses into the cytoplasm (e.g., where the desired pattern recognition receptor is located). In some embodiments, the releaseable linker releases the immunomodulator into the endosome.

[0133] In some embodiments, the linkers provided herein may comprise one or more spacers (e.g., to facilitate a specific release time, to enhance uptake into target tissue, and / or to optimize the in vivo distribution, bioavailability, and / or PK / PD of the compounds provided herein). The spacers may comprise one or more of alkyl chains, PEGs, peptides, sugars, peptidoglycans, clickable linkers (e.g., triazoles), rigid linkers such as polyprolines and polypiperidines, and similar entities.

[0134] In some embodiments, PEG 12Linkers containing the above significantly reduce nonspecific uptake of the compounds provided herein (e.g., to non-targeted organs (e.g., the liver and / or kidneys of the target after administration)) unless they are completely avoided. In some embodiments, the compounds avoid delivery to the liver and kidneys. In some embodiments, the targeted moiety (its free form, its radical, or its conjugate) does not bind to uptake receptors on non-targeted cells (e.g., if the organ is not the target site, then stimulation of immune complexes in those organs can be avoided, which is highly beneficial in clinical situations).

[0135] In some embodiments, conjugates containing non-release linkers provided herein 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).

[0136] In at least one embodiment, the linker includes a hydrophilic spacer. In some embodiments, the compound has the structure of formula XII (for example, a substructure of the TLR7 agonist of formula III conjugated with folic acid via a releaseable linker containing a first hydrophilic spacer). [ka]

[0137] In some embodiments, the compound has the structure of formula XIII (for example, a substructure of the TLR7 agonist of formula III conjugated with folic acid via a non-release linker (covalent bond) containing a second hydrophilic spacer). [ka]

[0138] Specific examples of exemplary conjugate compounds are provided herein.

[0139] In some embodiments, the compounds provided herein include a radical of a targeted moiety conjugated with a radical of an immunomodulator or a pharmaceutically acceptable salt thereof, which remains pharmaceutically active when conjugated with an immunomodulator (or its radical) or a pharmaceutically acceptable salt thereof. The targeted moiety may include any targeted moiety described herein, and in at least one embodiment, includes a folate ligand, any other folate receptor binding molecule (e.g., or a functional fragment or analog of any of those described herein) or a pyrido[2,3-d]pyrimidine analog. In some embodiments, the targeted moiety (or its conjugate or radical) is specific to FRβ.

[0140] In some embodiments, the compounds provided herein comprise one or more linkers, and the radical of the targeted moiety is conjugated with the radical of the immunomodulator via one or more linkers. For example, if the immunomodulator or a pharmaceutically acceptable salt thereof has formula I or II, the radical of the immunomodulator is conjugated via or directly with the linker, R 1 , R 2 , or R 3 In one of these, the radical of the targeting portion may be conjugated. Similarly, if the immunomodulator or a pharmaceutically acceptable salt thereof has formula III, the radical of the immunomodulator may be conjugated via a linker or directly, R 1 or R 3 In one of these, the radical of the targeting portion may be conjugated. Alternatively, if the immunomodulator or a pharmaceutically acceptable salt thereof has formula IV, the radical of the immunomodulator may be conjugated via a linker or directly, R 1 or R 2 In one of these, the linker may be conjugated to the radical of the targeting portion. As described herein, the linker may be releaseable or non-releaseable.

[0141] In some embodiments, one or more linkers of the compounds provided herein may include PEG, PEG derivatives, or any other linkers known or hereafter developed in the art that can achieve the purposes specified herein. In some embodiments, the linker may be repeated n times, where n is a positive integer. For example, n may be any integer selected from the range of 1 to 16, 1 to 32, 1 to 64, or 1 to 96, but is not limited. The number of repetitions in the linker (i.e., n) may be selected to achieve the desired functionality, size, and / or potency of the compound, and / or to take into consideration the desired application. In some embodiments, one or more of the linkers may include one or more spacers (for example, these may also be used to specifically design the properties of the compound).

[0142] In some embodiments, the linker is a hydrolyzable linker. In some embodiments, the linker is a non-hydrolyzable linker. In some embodiments, the linker is an optionally substituted heteroalkyl. In some embodiments, the linker is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. In some embodiments, the linker comprises a spacer (e.g., as described elsewhere herein).

[0143] In some embodiments, the linker is a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, the linker is a peptide having at least one disulfide bond in its backbone.

[0144] In some embodiments, the linker is -CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CRa R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO- is included in the formula, where R a and R b These are independently H, alkyl, or heteroalkyl (e.g., PEG).

[0145] In some embodiments, the linker includes the following structure: [ka] In the formula, n and m are independently between 0 and 10.

[0146] In some embodiments, the linker includes the following structure: [ka] In the formula, n and m are independently between 0 and 10.

[0147] In some embodiments, the linker includes the following structure: [ka] In the formula, n is between 1 and 32.

[0148] In some embodiments, the linker includes the following structure: [ka] In the formula, n is between 1 and 16.

[0149] It is understood that any combination of a compound radical (e.g., a compound radical in either Table 1 or Table 2), a linker (e.g., one provided herein), and a ligand radical (e.g., a ligand radical in either Table 3-6) can be combined to form the conjugates provided herein. In some embodiments, the compound radical or ligand radical is a carbon atom or a heteroatom (e.g., O, S, N, etc.). In some embodiments, the compound radical is C or O. In some embodiments, the ligand radical is C or O. In some embodiments, the attachment site between the compound and ligand (e.g., via the linker) is determined by the arrangement of the radicals. In some embodiments, the linker includes a spacer (e.g., one described elsewhere herein). It is also understood that any conjugate provided herein can be synthesized by a process similar to that provided in the methods provided in the examples.

[0150] Non-limiting examples of conjugates provided herein are provided in Table 7. [Table 7] TIFF0007904596000079.tif244170TIFF0007904596000080.tif91170

[0151] Non-limiting examples of conjugates provided herein are provided in Table 8. [Table 8] JPEG0007904596000082.jpg254170JPEG0007904596000083.jpg230170TIFF0007904596000084.tif251170TIFF0007904596000085.tif207170

[0152] In some cases, the conjugate compounds provided herein have the structure of formula XIV (for example, or a functional fragment or analog thereof, including a TLR7 agonist of formula III conjugated with folic acid via a releaseable linker): [ka]

[0153] In another embodiment, the conjugate compounds provided herein have the structure of formula XV (for example, or a functional fragment or analog thereof, comprising a TLR7 agonist of formula II conjugated with folic acid via a releaseable linker (e.g., compound 3B)): [ka]

[0154] In yet another embodiment, the conjugate compound provided herein has the structure of formula XVI (for example, or a functional fragment or analog thereof, comprising a TLR7 agonist of formula II conjugated with folic acid via a non-release linker containing three PEGs (e.g., compound 3D)): [ka]

[0155] In yet another embodiment, the conjugate compound provided herein has the structure of formula XVII (for example, or a functional fragment or analog thereof, comprising a TLR7 agonist of formula II conjugated with folic acid via a non-release linker containing 12 PEGs (e.g., compound 3C)): [ka]

[0156] Further embodiments of the conjugate compounds provided herein have the structure of formula XVIII (e.g., or a functional fragment or analog thereof, comprising a TLR7 agonist of formula II conjugated with folic acid via a non-release linker containing 16 PEGs (e.g., compound 3D')): [ka]

[0157] Further embodiments of the conjugate compounds provided herein have the structure of formula XIX (for example, or a functional fragment or analog thereof, including a TLR7 agonist of formula III conjugated with folic acid (compound 1B)): [ka]

[0158] The compounds described herein can be prepared by conventional organic synthesis methods practiced by those skilled in the art. The general reaction sequences outlined below represent general methods useful for preparing the compounds of this disclosure and are not intended to limit their scope or usefulness.

[0159] The description of the compounds of the present invention is limited by the principles of chemical bonding known to those skilled in the art. Therefore, where a group can be substituted by one or more substituents, such substitutions are selected in accordance with the principles of chemical bonding to give a compound that is not inherently unstable and / or is known to those skilled in the art to be likely to be unstable under ambient conditions such as aqueous, neutral, and certain known physiological conditions. For example, heterocycloalkyl or heteroaryl groups, in accordance with the principles of chemical bonding known to those skilled in the art, attach to the rest of the molecule via ring heteroatoms, thereby avoiding an inherently unstable compound.

[0160] The term “identical” or “identical” in relation to two or more polypeptide sequences refers to two or more sequences or subsequences that, when measured using sequence comparison algorithms known in the art or by manual alignment and visual inspection, are identical or have a certain percentage of identical peptides (i.e., about 60% identity across a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared and aligned for maximum correspondence across a specified region such as a comparison window or targeting end, folate end, linker, or warhead). In such cases, such sequences are said to be “substantially identical.” In other words, identity exists over one or more regions of the whole sequence, insofar as the general shape and structure of the molecules, and, where appropriate, hydrogen bonds, are substantially fitted to the target binding site and maintained to function as agonists thereto.

[0161] The compounds described herein may be administered in unit dosage forms and / or compositions comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. As used herein, the term “administration” and its constituents generally refer to any means of introducing the compounds described herein into a host subject, including but not limited to oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, oral cavity, ocular, sublingual, vaginal, rectal, and similar routes of administration.

[0162] Administration of the compounds of this disclosure as salts may be appropriate. Examples of acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts; however, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable. Similarly, “pharmaceutically acceptable salt” refers to a salt having a counterion that can be used in a pharmaceutical product. Such salts may include, but are not limited to, (1) acid addition salts, which can be obtained by the reaction of a free base of the parent compound with an inorganic acid, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or by the reaction of an organic acid, 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 substituted with a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinated with an organic base, such as ethanolamine, diethanolamine, triethanolamine, trimamine, or N-methylglucamine. Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salt may be intended in connection with the embodiments described herein.

[0163] 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 to give a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form non-toxic salts. Exemplary and non-limiting examples include acetates, aspartates, benzoates, besilates, bicarbonates / carbonates, bisulfates / sulfates, borates, camsilates, citrates, edisylates, esylates, formates, fumarates, gluceptates, gluconates, glucuronates, hexafluorophosphates, hibenzates, hydrochlorides / chlorides, hydrobroms / bromids, hydroiodides / iodides, isethionates, lactates, malates, maleates, malons, mesilates, methylsulfates, naphthylates, 2-napsylates, nicotinates, nitrates, orotates, oxalates, palmitates, pamoates, phosphates / hydrogen phosphates / dihydrogen phosphates, sugarates, stearates, succinates, tartrates, tosylates, and trifluoroacetates. Suitable base salts of the compounds described herein are formed from bases that form non-toxic salts. Exemplary and non-limiting examples include, but are not limited to, salts of arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc. Hemi salts of acids and bases, such as hemisulfates and hemicalcium salts, may also be formed.

[0164] As used herein, the term “composition” generally refers to any product comprising two or more components, including the compounds described herein. It should be understood that the compositions described herein may 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 understood that certain functional groups, such as hydroxy, amino, and similar groups, may form complexes with water and / or various solvents in various physical forms of the compounds. Compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and / or other forms of the compounds described herein, and it should also be understood that compositions may be prepared from various hydrates and / or solvates of the compounds described herein. Thus, pharmaceutical compositions listing the compounds described herein may include each of the various forms and / or solvated or hydrated forms of the compounds described herein, or any combination thereof, or individual forms.

[0165] The compounds of this disclosure may be formulated as pharmaceutical compositions and administered to mammalian hosts such as human patients in various forms suitable for a selected route of administration. For example, pharmaceutical compositions may be formulated for and administered via oral or parenteral, intravenous, intra-arterial, intraperitoneal, subarachnoid, epidural, intraventricular, urethral, ​​intrasternal, intracranial, intratumoral, intramuscular, topical, inhalation, and / or subcutaneous routes. In fact, in at least one embodiment, the compounds and / or compositions described herein may be administered directly into the bloodstream, intramuscular, or visceral system.

[0166] For example, in at least one embodiment, the compound may be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilated food carrier. For oral therapeutic administration, the active compound may be used in combination with one or more excipients in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. The percentages of compositions and preparations may vary, ranging from about 1% by weight to about 99% by weight, and may be between the active ingredient and binders, excipients, disintegrants, lubricants, and / or sweeteners (known in the art). The amount of the active compound in such therapeutically useful compositions is such that an effective dose level is obtained.

[0167] For example, the preparation of parenteral compounds / compositions under sterile conditions by freeze-drying can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art. In at least one embodiment, the solubility of the compound used in the preparation of the parenteral composition can be increased by the use of appropriate formulation techniques, such as the incorporation of a solubility-enhancing agent.

[0168] As previously stated, the compounds / compositions of this disclosure may also be administered by injection or by infusion (e.g., using needle (including microneedle) syringes and / or needleless syringes). The solutions of the active compositions may be aqueous, optionally mixed with non-toxic surfactants, and / or contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), but for some applications they may be more appropriately formulated as sterile non-aqueous solutions or as dry forms used 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, as well as in oil. Under normal storage and use conditions, these preparations may further contain preservatives to prevent microbial growth.

[0169] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersants or sterile powders containing active ingredients adapted for immediate preparation of sterile injectable or injectable solutions or dispersants, optionally encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid, and stable under manufacturing and storage conditions. The liquid carrier or liquid vehicle may be a solvent or liquid dispersion medium, including, but not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, adequate fluidity can be maintained by liposome formation, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Microbial action can be prevented by the addition of various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In some cases, it is desirable to include one or more isotonic agents such as sugars, buffers, or sodium chloride. Long-term absorption of injectable compositions can be achieved by incorporating formulations that slow down absorption, such as aluminum monostearate and gelatin.

[0170] Sterile injectable solutions can be prepared by incorporating the active compound and / or composition, as needed, with one or more of the other components specified above, and the required amount of a suitable solvent, followed by filter sterilization. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which result in a powder of the active ingredient with any additional desired components present in the previously sterile filtered solution added.

[0171] For topical administration, it may be desirable to administer the compound to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be solid or liquid. For example, in certain embodiments, the solid carrier may include finely divided solids such as talc, clay, microcrystalline cellulose, silica, and alumina. Similarly, a useful liquid carrier may include water, alcohol or glycol, or a water-alcohol / glycol blend, and the compounds of the present invention can be optionally dissolved or dispersed at an effective level with the help of a non-toxic surfactant. Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents can be added to optimize the properties for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and / or other bandages, sprayed onto a target area using a pump or aerosol sprayer, or simply applied directly to the desired area of ​​the target.

[0172] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty acid alcohols, modified cellulose, or modified minerals can be used together with a liquid carrier to form pastes, gels, ointments, soaps, etc., that can be spread directly onto the target skin.

[0173] As used herein, the terms “therapeutically effective,” “therapeutic effective dose,” “therapeutic effective amount,” “preventive effective dose,” or “preventive effective dose” mean the amount of a compound that, when administered either once or in the course of a treatment cycle, would have an effect on the health, well-being, or mortality of the subject (e.g., delaying the onset and / or reducing the severity of one or more symptoms associated with fibrous disease or condition and / or cancer, where applicable, but not limited to). Useful doses of the compounds of this 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 to human subjects are known in the art. In practice, the dose of a compound can vary significantly depending on the condition of the host subject, the cancer or fibrous disease being treated, the stage of disease progression, the route and tissue distribution of the compound, and the possibility of concomitant use with other therapeutic treatments (such as additional agents in radiotherapy or combination therapy). The amount of composition required for use in treatment (e.g., a therapeutic or prophylactic effective dose or amount) varies not only with regard to the specific application but also with regard to the selected salt (if applicable) and the characteristics of the subject (e.g., age, condition, sex, body surface area and / or mass of the subject, drug tolerance, etc.), and is ultimately left to the discretion of the attending physician, clinician, or other person. The therapeutically effective or prophylactic effective dose or dosage may be, for example, in the range of approximately 0.05 mg / kg patient body weight to approximately 30.0 mg / kg patient body weight, or approximately 0.01 mg / kg patient body weight to approximately 5.0 mg / kg patient body weight, but is 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 in kg of patient body weight). The total therapeutic or prophylactic effective dose of the compound may be administered as a single dose or in divided doses, and may be outside the typical range given herein, at the discretion of the practitioner.

[0174] In another embodiment, the compound is present in concentrations of approximately 0.5 g / m to approximately 500 mg / m. 2 , about 0.5g / m 2 ~about 300mg / m 2 , or approximately 100g / m 2 ~about 200mg / m 2 It may be administered in a therapeutic or prophylactic effective dose. In other embodiments, the dose is about 0.5 mg / m². 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2 , about 0.5mg / m 2 ~about 200mg / m 2 , about 0.5mg / m 2 ~about 100mg / m 2 , about 0.5mg / m 2 ~about 50mg / m 2 , about 0.5mg / m 2 ~about 600mg / m 2 , about 0.5mg / m 2 ~about 6.0mg / m 2 , about 0.5mg / m 2 ~about 4.0mg / m 2 , or approximately 0.5 mg / m² 2 ~about 2.0mg / m 2 This may be the case. The total amount may be administered as a single dose or in divided doses, and may be outside the typical range given herein, at the discretion of the physician. These amounts are based on the body surface area in square meters.

[0175] In some embodiments, the significance of this disclosure lies in the use of methods for detecting markers, rather than in specific methods used for detecting a marker or set of markers, in relation to the measurement of the expression of a particular biomarker in a sample from a subject and / or the analysis of cytokine levels. There are many methods that can be used to detect the expression, quantification, or profile of one or more biomarkers. Once the marker or set of markers to be detected or quantified is identified, any of several techniques (currently known or to be developed in the future) may be used, along with the provision of appropriate reagents. Those skilled in the art will be able to select an appropriate assay (e.g., PCR-based or microassay-based assays for nucleic acid markers, enzyme-linked immunosorbent assays (ELISA), protein or antibody microarrays or similar immunoassays) for carrying out the methods disclosed herein, given that one or more biomarkers to be identified are provided.

[0176] Chemical examples Example A: Synthesis of Compound 1A: Compound 1A was synthesized according to Scheme 1 below, as reported in Nikunj M. Shukla, Cole A. Mutz, Subbalakshmi S. Malladi, Hemamli J. Warshakoon, Rajalakshmi Balakrishna, and Sunil A. David, “Regioisomerism-dependent TLR7 agonism and antagonism in an imidazoquinoline; Structure-Activity Relationships in Human Toll-Like Receptor 7-Active Imidazoquinoline Analogues,” J Med Chem. 2012 Feb 9;55(3):1106-1116. [ka]

[0177] Step 1: Synthesis of l-amino-2-methylpropan-2-ol (compound) 2,2-dimethyloxirane (0.1 g, 1.388 mmol) was added dropwise to 20 mL of ice-cold ammonium hydroxide solution. The reaction mixture was stirred at room temperature for 12 hours. The solvent was removed under vacuum, and the residue was dissolved in methanol. Di-tert-butyl dicarbonate (0.75 g, 3.47 mmol) was added to the reaction mixture, and the mixture was stirred for 4 hours. The mixture was purified by column chromatography (24% ethyl acetate (siRNA) / hexane) to obtain tert-butyl 2-hydroxy-2-methylpropyl carbamate. The pure tert-butyl 2-hydroxy-2-methylpropyl carbamate was dissolved in 5 mL of trifluoroacetic acid and stirred for 35 minutes. The solvent was removed under reduced pressure to obtain l-amino-2-methylpropan-2-ol as trifluoroacetate 1'. 1H NMR 500MHz (500MHz, CDC13, δ(ppm)): δ 8.62(s,2H),3.02(d,2H),2.06-2.04(m,2H),1.37-1.34(s,6H).

[0178] Step 2: Synthesis of 2-methyl-l-(3-nitroquinoline-4-ylamino)propan-2-ol (Compound 2) 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 30 minutes until the 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-nitroquinoline-4-ylamino)propan-2-ol (compound 2). Liquid chromatography-mass spectrometry (LCMS) analysis: [M+H] + m / z = 261.

[0179] Step 3: Synthesis of l-(3-aminoquinoline-4-ylamino)-2-methylpropane-2-ol (compound 3) 2-Methyl-l-(3-nitroquinoline-4-ylamino)propan-2-ol (compound 2) (450 mg, 1.72 mmol) was dissolved in methanol and hydrogenated for 4 hours on Pd / C as a catalyst using a hydrogen balloon. The solution was then filtered using Celite, and the solvent was evaporated under reduced pressure to obtain l-(3-aminoquinoline-4-ylamino)-2-ethylpropan-2-ol (compound 3). LCMS:[M+H] + m / z=231.Η NMR 500 MHz(CDC13, δ(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).

[0180] Step 4: Synthesis of l-(4-amino-2-butyl-lH-imidazo[4,5-c]quinoline-l-yl)-2-methylpropan-2-ol (Compound 5, TLR7A) To a solution of compound 3 (100 mg, 0.43 mmol) in anhydrous THF, triethylamine (66 mg, 0.65 mmol) and valeryl chloride (62 mg, 0.52 mmol) were added. The reaction mixture was then stirred for 6-8 hours, followed by removal of the solvent under vacuum. The residue was dissolved in RINKAN, washed with water and brine, and then dried over Na₂SiO₄ to obtain the intermediate amide compound. This was dissolved in methanol (MeOH), followed by the addition of calcium oxide, and heated in a microwave at 110°C for 1 hour. The solvent was then removed, and the residue was purified using column chromatography (9% MeOH / dichloromethane) to obtain compound 4 (58 mg). To a solution of compound 4 in a solvent mixture of MeOH:dichloromethane:chloroform (0.1:1:1), 3-chloroperoxybenzoic acid (84 mg, 0.49 mmol) was added, and the solution was refluxed at 45-50°C for 40 minutes. Next, the solvent was removed, and the residue was purified using column chromatography (20% MeOH / dichloromethane) to obtain the oxide derivative (55 mg). This was then dissolved in anhydrous dichloromethane, followed by the addition of benzoyl isocyanate (39 mg, 0.26 mmol), and the mixture was heated at 45°C for 15 minutes. Next, the solvent was removed under vacuum, the residue was dissolved in anhydrous MeOH, and then excess sodium methoxide was added. The reaction mixture was then heated at 80°C for 1 hour. The solvent was removed under vacuum, and the residue was purified using column chromatography (11% MeOH / dichloromethane) to obtain compound 5. LCMS:[M+H] + m / z=312. H NMR 500MHz(CDC13, δ(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).

[0181] Example B: Synthesis of Compound 1B: Subsequently, compound 1B can be synthesized using compound 1A according to scheme 2 below. [ka]

[0182] Compound 1A, folic acid, and linker are commercially available or can be prepared according to methods known to those skilled in the art.

[0183] Heterobifunctional linker 7 (88 mg, 0.213 mmol) was added to a solution of compound 5 (33 mg, 0.106 mmol) and dimethylaminopyridine (39 mg, 0.319 mmol) in 4 mL of methylene chloride at room temperature under a nitrogen atmosphere. The mixture was stirred at reflux temperature for 7 hours, during which thin-layer chromatography (TLC) analysis of the mixture showed >80% conversion. The mixture was concentrated and purified by column chromatography using 10% acetonitrile in methylene chloride as the eluent. The pure product compound 9 was obtained as a pale yellow solid. A solution of compound 8 (1 equivalent) in dimethyl sulfoxide (DMSO) was added in three portions at 20-minute intervals to a solution of the drug linker intermediate compound 9 (1.0 to 1.5 equivalents) in DMSO containing dimethylaminopyridine (1 equivalent). After stirring under argon at room temperature for 1-2 hours, LC-MS analysis of the mixture showed the formation of the desired folic acid-drug conjugate (compound 10) as the major product. The mixture was purified by preparative high-performance liquid chromatography (HPLC). LC-MS:[M+H] + m / z = 959. 1H 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).

[0184] Example C: Synthesis of Compound 2A: Compound 2A can be synthesized according to schemes 3 and 4. [ka] [ka]

[0185] Wang resin (11) loaded with cysteine ​​was first deprotected using 20% ​​piperidine in dimethylformamide (DMF). The free amine was treated with Fmoc-Glu(OtBu)-COOH in the presence of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), N,N-diisopropylethylamine (DIPEA), and DMF. The bound product was deprotected using 20% ​​piperidine in DMF and treated with pteroic acid in the presence of PyBop, DIPEA, and DMF to produce compound 12. The trifluoroacetyl group was deprotected with a 50% ammonia-DMF solution. Finally, the resin was cleaved using a trifluoroacetic acid:triisopropylsilane:water:tris(2-carboxyethyl)phosphine cocktail solution and purified by HPLC to obtain folate-cysteine ​​(13) as a yellow solid.

[0186] Compound 14 was first treated with a heterobifunctional linker reagent (15) to obtain a folic acid-cystine disulfide intermediate (16). This was then reacted with folic acid-cysteine ​​(13) in DMSO and purified using HPLC to produce compound 17 (e.g., compound 2A). All compounds were characterized using LC-MS with ammonium bicarbonate and acetonitrile as buffer systems. The mass observed by LC-MS of compound 2A was [M+H]+=1082.2.

[0187] In particular, taking into consideration schemes 1, 2, and 3 specified above, and in conjunction with the current state of the relevant technology, this disclosure provides sufficient detail so that those skilled in the art can synthesize all other compounds of this disclosure using the concepts specified herein.

[0188] Treatment method In addition to the compounds described herein, methods for the treatment and / or prevention of fibrotic diseases or cancer are also provided.

[0189] In some embodiments, provided herein are methods for treating a subject suffering from a fibrotic disease state or cancer, the method comprising contacting cells of the subject with any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutically) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, for example, the compound having any one structure of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XX, or XXX. In some embodiments, the immunomodulator comprises an agonist of TLR7, 8, or 9.

[0190] In some embodiments, provided herein are methods for treating a neoplastic, inflammatory, autoimmune, or fibrous disease or disorder in an individual requiring treatment, comprising administering to the individual any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutically) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, for example, the compound having any one structure of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XX, or XXX.

[0191] In some embodiments, provided herein are methods for treating a neoplastic disease or disorder in an individual requiring treatment, comprising administering to the individual any compound provided herein or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutically) composition comprising any compound provided herein or a pharmaceutically acceptable salt thereof, for example, the compound having any one structure from formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XX, or 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.

[0192] In some embodiments, provided herein are methods for treating a fibrous disease or disorder in an individual requiring treatment, comprising administering to the individual any compound provided herein, or a pharmaceutically acceptable salt thereof, or a (e.g., pharmaceutically) composition comprising any compound provided herein, or a pharmaceutically acceptable salt thereof, such, the compound having any one structure from formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XX, or XXX. In some embodiments, the fibrous disease or disorder is fibrosis. In some embodiments, fibrosis is selected from IPF, fatty liver disease, cirrhosis, colitis, chronic liver disease, cardiac fibrosis, and scleroderma.

[0193] In certain embodiments, provided herein is a method for preventing or treating a fibrotic disease state, comprising contacting cells via a linker with at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt thereof attached to a folate ligand or a functional fragment or analog thereof, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets pattern recognition receptors.

[0194] In some embodiments of the compounds, compositions, and / or methods provided herein, the immunomodulator comprises a TLR agonist, which has a structure represented by the following formula X or XX, or a pharmaceutically acceptable salt of formula X or XX. [ka] In the formulas, in formulas X and XX, R1 is either -NH2 or -NH-R 1X And, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X , [ka] And, [ka] These are 3-10 member nitrogen-containing non-aromatic monocyclic or bicyclic heterocyclic rings. In equation X, R3 is -OH, -SH, -NH2, or -NH-R 1X And, In formula XX, X is CH, CR2, or N. R 1X , R 2X , and R 2YEach of these is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0195] In some embodiments, the cells include cells of a subject experiencing or at risk of experiencing a fibrous disease state, and contacting the cells with at least one compound further includes administering or applying a therapeutically effective amount of at least one compound to the subject. In some embodiments, the subject is a patient experiencing IPF, and at least one compound is administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation. In some embodiments, the fibrous disease state includes IPF or fibrous diseases of the liver, skin, bladder, heart, pancreas, prostate, or kidneys.

[0196] In some embodiments, the method involves obtaining a sample from a subject, or obtaining a sample and quantifying the expression level of one or more biomarkers in the sample, wherein each of the one or more biomarkers is selected from the group consisting of CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), metalloproteinase 3 (TIMP3), IL-1β, hydroxyproline, collagen, PDGF, TGFβ, FRβ, TNFα, IFN-γ, anti-mannose receptor (CD206), differentiation antigen group 86 (CD86), differentiation antigen group 163 (CD163), IL-6, chemokine 10 (CXCL10), and immune interferon (IFNα), and the quantification is performed. The further includes comparing the expression level of one or more biomarkers in the sample with the expression level of such biomarkers in a control, and administering, or having administered, a therapeutically effective amount of an unconjugated agonist or inhibitor to the subject if CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline, or collagen is upregulated compared to the control's expression level, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα, or CD86 is downregulated or not expressed compared to the control's expression level. In some embodiments, the folate ligand or a functional fragment or analog thereof is specific to FRβ and binds to FRβ on the cell.

[0197] In at least one embodiment, a method is provided for treating and / or preventing a fibrotic disease (e.g., IPF). The method comprises administering to a subject a therapeutically effective dose of one or more compounds comprising a targeted moiety (e.g., a folate receptor-binding ligand) attached to a drug (by linker or other means) in order to reprogram M2-like macrophages in fibrous tissue or organs to an M1-like phenotype. For example, the drug may be a Toll-like receptor agonist (e.g., having formula I, III, or IV), or any other molecule or compound effective in conjugating macrophages with folate to reprogram them from an M2 phenotype to an M1 phenotype. In at least one embodiment, the drug may be selected from TLR3 agonists, TLR7 agonists, TLR7 / 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 pro-fibrotic cytokines and growth factors.

[0198] In at least one embodiment, a method is provided for treating a subject suffering from or at risk of experiencing a disease condition, wherein the disease condition includes a fibrotic disease condition or cancer, and the method comprises contacting the cells of the subject with at least one compound. The at least one compound may include any of the compounds of the Disclosure and, in at least one exemplary embodiment, includes a targeting moiety specific to FRβ. In some cases, contacting the cells may be achieved by administering the at least one compound to the subject intravenously, intramuscularly, intraperitoneally, topically, orally, or by inhalation, or by any other mode of administration described herein. Additionally or alternatively, the at least one compound may include 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 may be appropriately modified by the clinician, but the at least one compound is administered in a therapeutically effective or prophylactically effective amount, and in at least one embodiment, the dose is in the range of 1 nmol / kg subject body weight to 50 nmol / kg subject body weight.

[0199] Referring here to Figure 2, a representative flowchart of Method 1900 for preventing or treating a fibrous disease state or cancer using one or more of the compounds of this disclosure is shown. In at least one example, Method 1900 includes the step of contacting cells of interest with at least one compound comprising an immunomodulator (or a pharmaceutically acceptable salt thereof) attached to a folate ligand or a functional fragment or analog thereof via a linker (Step 1902). In at least one exemplary embodiment, the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor. The cells may include, for example, cells of interest that are experiencing or at risk of experiencing a fibrous disease state, and the at least one compound may include any of the compounds provided herein.

[0200] In at least one embodiment, step 1902, which involves contacting cells with at least one compound, further comprises administering or applying a therapeutically effective amount of at least one compound to the target. Additionally or alternatively, the at least one compound may comprise a composition containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, or a combination thereof.

[0201] In at least one embodiment, the disease condition includes IPF or fibrotic disease of the liver, skin, heart, or kidneys. Furthermore, the subjects may include mice, humans, or any other mammals.

[0202] Method 1900 may optionally include steps 1904-1910 in addition to step 1902. 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 may be obtained from a certain amount of peripheral blood collected from the subject.

[0203] The quantification step 1906 may be performed using any suitable method known in the Art, which may include, for example, qPCR, mass spectrometry, ELISA, and / or any other modality capable of measuring / quantifying biomarker expression. In at least one exemplary embodiment, one or more biomarkers are selected from the group consisting of CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, TNFα, IFN-γ, CD206, CD163, IL-6, CXCL10, IFNα, and CD86.

[0204] In step 1908, the expression level of each of one or more biomarkers in the sample is compared to the expression level of such biomarkers in a control. The control may be a healthy individual or simply an individual not experiencing the disease condition in question. In at least one embodiment, a clinical difference between the expression level of one or more biomarkers in the sample and the expression level of the relevant biomarkers in the control may indicate that the subject is suffering from the disease condition in question. For example, but not limited to, if comparison step 1908 shows that the expression of one or more of the biomarkers CCL18, Arg1, CD163, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, and / or CD206 (i.e., “pro-fibrosis biomarkers”) is upregulated compared to the control, this indicates that the subject is experiencing a pro-fibrosis immune response associated with the M2-like macrophage phenotype. Thus, in at least one embodiment, such results indicate the need to administer one or more compounds of the present disclosure to reprogram such M2-like macrophages to the M1 phenotype.

[0205] In contrast, if comparison step 1908 shows that the expression of the aforementioned biomarkers is downregulated compared to the control, or if the expression of one or more TNFα, IFN-γ, and / or CD86 ("anti-fibrotic biomarkers") is upregulated compared to the control, this indicates, in certain embodiments, that the subject either shows a positive response to a previously administered compound (if applicable) and / or that the subject is experiencing an anti-fibrotic immune response associated with the M1 phenotype.

[0206] Optionally, if in step 1910 the expression of one or more pro-fibrosis biomarkers in the sample is upregulated compared to their respective expression levels in the control, or if the expression of one or more anti-fibrosis biomarkers is downregulated in the sample compared to their respective expression levels in the control, an alternative therapy may be administered. In at least one embodiment, the alternative therapy may include administering a therapeutically effective dose of a derivative of at least one compound previously administered in step 1902, the derivative comprising at least one previously administered compound modified with respect to better optimizing the efficacy of the at least one compound by employing any of the following: a different targeting moiety, a different linker size, and / or a different immunomodulator. Additionally or alternatively, other therapies may be employed, including those conventionally known for the treatment of the fibrous disease in question. Steps 1904-1910 may be repeated as needed or as desired.

[0207] Additional embodiments may provide methods for treating and / or preventing cancer (whether folate receptor-positive or folate receptor-negative). For example, in certain examples, such methods include administering a therapeutically effective dose and / or prophylactically effective dose to a host subject of one or more compounds containing a targeted moiety attached (by linker or other means) to the agent in order to reprogram M2-like macrophages in cancerous and / or tumor cells to an M1-like phenotype. If the cancer is folate receptor-negative, such administration may further act to deplete or inhibit MDSCs present in such tissue / tumor. For example, but not limited to, agents may be selected from PI3k inhibitors, signaling and transcription activator 6 (STAT6) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, inducible nitric oxide synthase (iNOS) inhibitors, and anti-inflammatory agents (e.g., methotrexate). In at least one embodiment, the agent may inactivate MDSCs.

[0208] In describing representative embodiments, this disclosure may present methods and / or processes as a specific set of steps. Unless the method or process depends on a specific order of steps specified herein, the method or process should not be limited to a specific order of steps described. As those skilled in the art will understand, other orders of steps are possible. Therefore, a specific order of steps disclosed herein should not be construed as a limitation of the claims. Furthermore, claims directed to methods and / or processes should not be limited to the execution of those steps in the order described, and those skilled in the art will readily understand that the order may change and still remain in the spirit and scope of this disclosure. [Examples]

[0209] Human monocytic THP-1 cells were obtained from the American Type Culture Collection and cultured in folate-deficient RPMI1640 medium (Invitrogen, Carlsbad, CA) containing 10% thermoinactivated 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 significant amounts of pro-fibrotic cytokines when stimulated with IL-4, IL-6 plus IL-13.

[0210] IFN-γ, IL-4, interleukin-6 (IL-6), and interleukin-13 (IL-13) were obtained from Biolegend. Phorbol 12-myristot 13-acetate (PMA), lipopolysaccharide (LPS), and all other reagents and solvents were purchased from Sigma.

[0211] Example 1: In vitro differentiation and polarization of THP1 cells into M2-like macrophages THP-1 cells were seeded in 96-well plates at a density of 60,000 cells / well. The cells were differentiated into nonpolar macrophages by incubation with 200 nM PMA for 48 hours, followed by incubation 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, then reprogrammed with different concentrations of compound 1A and compound 1B for 48 hours, and collected for genetic analysis by quantitative polymerase chain reaction (qPCR). The cultures were maintained at 37°C in a humidified 5% CO2 incubator.

[0212] To evaluate whether potent TLR7 agonists (e.g., compound 1A of formula III) can reprogram profibrosis macrophages to a lower fibrosis phenotype, THP-1 cells stimulated with IL-4, IL-6 plus IL-13 were incubated with different concentrations of non-target compound 1A, and mRNA levels of several profibrosis markers, namely CCL18, CD206, IL-1β, and PDGFα and β, were examined.

[0213] As shown in Figures 3A-3C, 48-hour incubation with compound 1A induced a decrease in CCL18, CD206, and IL-1β expression, suggesting that the TLR7 agonist can indeed promote a shift in these fibrosis-polarized THP-1 cells to a lower fibrotic phenotype. Furthermore, an increase in TNFα expression, a marker of the anti-fibrotic phenotype, was observed (Figure 3D), confirming that THP-1 cells shifted from fibrosis-promoting characteristics to fibrosis-promoting characteristics.

[0214] Example 2: Evaluation of macrophage reprogramming To confirm that a folic acid-conjugated TLR7 agonist can induce the same THP-1 reprogramming as seen in Example 1, compound 1B was prepared, and a releaseable linker was constructed using a disulfide, self-destructing bond to connect folic acid to compound 1A, enabling the release of compound 1A following the internalization of compound 1B into the reducing environment of intracellular endosomes.

[0215] Either compound 1A or compound 1B at different concentrations was incubated with the polarized THP-1 macrophages described above for a specified time, and then the culture medium was collected for cell collection for secreted cytokine analysis and qPCR analysis.

[0216] Following the manufacturer's recommended protocol, use the Quick-RNA® MicroPrep kit (Zymo Research, Irvine, CA) to perform 1 × 10⁶ 5 ~2×10 5Total RNA was isolated from macrophages. The RNA samples were then reverse transcribed into cDNA using a high-volume cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, #4368814). qPCR analysis was performed using iTaq® Universal SYBR Green SuperMix (Bio-Rad Laboratories GmbH, Hercules, CA, #1725121), an iCycler thermocycler, and iCycler iQ 3.0 software (Bio-Rad Laboratories GmbH, Hercules, CA) to track the expression of markers characteristic of macrophage polarization. IL-6, CXCL10, IFNα, IFN-γ, and CD86 were used as markers for the M1 phenotype, and CCL18, CD206, CD163, and Arg1 were used as markers for the M2 phenotype. IL-1β, PDGFβ, MMP9, and TIMP 3 were measured as indicators of the pro-fibrillation phenotype. IRAK-4 was used as an indicator of TLR7 stimulation. Melting curve analysis was performed to control for the specificity of the amplified product. No amplification of nonspecific products was observed in any of the reactions. Each sample was analyzed independently in three ways for each marker.

[0217] Repeating the above studies (see Figures 3A-3F, gray bars) revealed the same qualitative changes, with only a slight decrease in the magnitude of the effect of compound 1B. This decrease in efficacy was expected because the untargeted TLR7 agonist enters cultured cells immediately, while its folate-targeted counterpart is designed to enter cells only after folate receptor binding and receptor-mediated endocytosis.

[0218] Figures 4A–4E and 5A–5D show graph data representing various marker levels measured from THP-1 cells induced into M2 macrophages, which were then incubated with different concentrations of compound 1B or compound 1A for 2 hours, washed with PBS, and incubated again for 46 hours for the data shown in Figures 5A–5D (for the data shown in Figures 4A–4E, cells were harvested immediately after the initial 2-hour incubation). Cells were harvested for genetic analysis by qPCR in both datasets. Figures 4A–4C show CCL18 mRNA levels (Figures 4A and 5A), CD206 mRNA levels (Figures 4B and 5B), IL-1β mRNA levels (Figures 4C and 5C), and PDGFβ mRNA levels (Figure 4E). The data support that the M2-like pro-fibrillation phenotype was downregulated after administration of the tested compounds. In particular, compound 1B downregulated macrophage fibrosis-promoting / M2 type markers more significantly than compound 1A. Furthermore, Figure 4D shows CD86 mRNA levels and Figure 5D shows TNFα levels; these data support the upregulation of the M1-like phenotype after administration of the tested compounds. Data on PDGFα are not shown because no significant post-treatment differences were observed during data collection.

[0219] Low molecular weight water-soluble drugs such as compounds 1A and 1B are often excreted from the body within 2 hours of injection. Therefore, a more physiologically appropriate in vitro model for in vivo drug exposure involves limiting the incubation of cells with the drug to only 2 hours and examining the drug's efficacy after a further 46 hours of incubation in the absence of the drug. As shown in Figures 4A-4E, after incubating THP-1 cells with a TLR7 agonist for 2 hours and then replacing the drug-containing medium with drug-free medium, compound 1B was observed to have superior potency compared to compound 1A, particularly dramatically improving folate-targeted conjugates in the case of TNFα induction. This is likely because the folate-targeted TLR7 agonist was captured by folate receptor-positive cells, whereas compound 1A was not retained by the same cells.

[0220] These data support the idea that compound 1B should be more effective in reprogramming pro-fibrotic macrophages in vivo, and that folate-conjugated drugs (e.g., compound 1B) have the added advantage of causing less systemic toxicity because they concentrate in FRβ-expressing macrophages and cannot enter the predominantly folate receptor-negative cells throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0221] Figures 6A–6D show graphical data representing various marker levels measured from M2-induced THP-1 macrophages treated with different concentrations of the drug for 48 hours (Figures 6A and 6B) or 2 hours, then replaced with fresh medium and cultured for the remaining 46 hours (Figures 6C and 6D). In both cases, the cell supernatant was collected and secreted CCL18 protein and IL-1β were detected by ELISA. The data support that administration of TLR7 compounds or folic acid-targeted TLR7 compounds downregulates the secretion of CCL18 and IL-1β in the low concentration range (0.1–10 nM).

[0222] Furthermore, to stimulate THP-1 cells so that the above mRNA analysis accurately reflects the levels of pro-fibrotic cytokines produced by 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 shown in Figures 6A and 6B, both compound 1A and compound 1B induced a decrease in CCL18 and IL-1β when incubated continuously with the agonist for 48 hours, but compound 1B was again found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D).

[0223] Example 3: Characterization of FRβ expression by flow cytometry To measure FRβ expression in THP-1-derived macrophages, fluorescence-activated cell sorting (FACS) analysis was performed. Cells were sorted using Accutase ( Cells were detached using a registered trademark cell detachment solution (Biolegend, San Diego, CA, #423201) and gently lifted with a cell scraper. Cells were washed with PBS and nonspecific binding was blocked by incubation at room temperature for 10 minutes using Fc receptor blocking solution (Biolegend, San Diego, CA, #422301). Biotinylated anti-human FRβ monoclonal antibody (m909) was then added, and the cells were incubated on ice for a further 30 minutes, followed by washing in staining buffer (PBS supplemented with 2% FBS). Cells were then incubated on ice for 20 minutes in fluorescein-labeled streptavidin (BD Biosciences, Franklin Lakes, NJ, #554060), washed twice with PBS, stained with 7AAD (viability stain) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ). Figure 6E shows flow cytometry data, which confirms that THP-1 macrophages are FRβ+ and therefore suitable for in vitro studies of compound 1B and other studies described herein.

[0224] Figure 6F confirms that compound 1B remained stable during the incubation period at 37°C in the culture medium. In fact, compound 1B retained its original structure after 48 hours of incubation.

[0225] Example 4: In vivo reprogramming of bleomycin-induced pulmonary fibrosis and pro-fibrosis macrophages. Studies were also conducted to determine whether macrophages in pulmonary fibrosis can be specifically targeted with folic acid-coupled agents in vivo. After testing multiple protocols for inducing pulmonary fibrosis in mice, a protocol was selected in which 0.75 mg / kg of bleomycin (BM) was injected into the lungs of C57BL / 6 mice via tracheotomy, allowing the mice to progress through both the inflammatory and fibrotic phases of fibrosis before the initiation of treatment. (The BM model is widely considered useful because it allows for mechanistic investigations related to fibrosis in an in vivo setting.)

[0226] As shown in Figures 7A–7D, mice treated using this protocol typically exhibit fibrosis by day 7 after BM treatment, and this neofibrosis progresses to severe fibrosis by day 14. The progression of the disease then continues for 2–5 days and begins to resolve spontaneously by day 21.

[0227] More specifically, eight-week-old C57BL6 male mice (average body weight 22g–25g) from Charles River were housed in a pathogen-free environment at room temperature (22°C) under a 12-hour light-dark cycle. The mice were fed a folate-deficient diet (Envigo Teklad Global Rat Food Pellets) for one week prior to BM or PBS infusion. Fresh water and the folate-deficient diet were freely available. All animal procedures were approved by the Purdue Animal Care and Use Committee in accordance with National Institutes of Health guidelines.

[0228] Subsequently, the mice were anesthetized with ketamine / xylazine, their necks were shaved using depilatory lotion, and then sterilized with 70% alcohol. A small incision was made in the neck to visualize the trachea. The mice were positioned at a 75-degree angle, and 100 μL of sterile PBS or BM (Cayman Chemicals, Ann Arbor, MI, #13877) (0.75 mg / kg) dissolved in PBS was injected into the trachea using a 1 cc syringe with a 26G needle. Body weight was monitored every other day throughout the experiment.

[0229] To evaluate whether pro-fibrotic lung macrophages in these mice could be specifically targeted by folate-conjugated agents, folate-conjugated near-infrared fluorescent dye (OTL38) containing or not containing 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) of 200-fold excess FA-glucosamine (a competitor of OTL38) was injected into the tail vein of BM-treated mice 10 days after injection, and the uptake of the dye into major organs was evaluated.

[0230] Two hours later, the mouse was sacrificed using CO2 asphyxiation, and the skin from the abdomen to the neck was immediately incised to expose the lungs and trachea. A small incision was then made in the upper part of the trachea to insert a 22-gauge blunt needle, and a nylon string was tied around the trachea to seal the area around the needle. The trachea (including the insertion needle), lungs, and heart were then removed by carefully cutting the connective tissue below the lungs and clipping the left lung bronchus with a Diefenbach vascular clip. PBS was injected into the right lung, aspirated three times using a 1 ml syringe, and the collected lavage solution was stored on ice.

[0231] Next, bronchoalveolar lavage fluid (BALF) was analyzed to determine how the targeted TLR7 agonist acted. BALF samples were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was aliquoted and stored at -80°C for cytokine / chemokine analysis. The cell pellet was resuspended and cultured for 2 hours in pre-warmed RPMI1640 medium, then washed three times with pre-warmed PBS before collection for qPCR assay. The right lung was then ligated with nylon string and used for subsequent analysis of hydroxyproline content. The left lung was inflated with 1 ml of PBS using an inserted syringe and transferred to 10% formalin solution for subsequent histological analysis.

[0232] The right lung lobes collected above were weighed and placed in a pressure-resistant 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, transferred to a 1.5 ml Eppendorf tube, and centrifuged at 12,000 rcf at 4°C for 15 minutes. The supernatant was carefully collected, separated, and used for hydroxyproline (HYP) analysis.

[0233] In the subsequent HYP analysis, 10 μl of the sample was transferred to a 96-well plate and neutralized with 10 μl of 5.3 M sodium hydroxide solution. Isopropanol (40 μl) was then added to each well, followed by 20 μl of oxidative buffer. The mixture was incubated on a shaker at room temperature for 5 minutes. Analytical reagent (260 μl) was added, and the plate was incubated on a shaker at room temperature for 30 seconds, then immediately incubated at 60°C for 25 minutes. The absorbance was measured within 15 minutes at 560 nm (A). 560 Measurements were taken using [specific method / tool]. All reagents were prepared according to previously reported protocols.

[0234] For histological analysis of lung sections, fixed lungs (see above) were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E), Masson's trichrome, or F3 (anti-mouse FRβ antibody). Histological sections were examined in a blinded manner by a licensed pathologist. Each section was scanned using an Aperio-Image Scope (Leica Biosystems, Wetzlar, DE) at 90 × 10⁶ degrees. 6 We quantified cells exceeding a certain number.

[0235] The Human DuoSet ELISA Development System (R&D Systems Europe, Abingdon, UK, #DY394-05) and the IL-1 Beta Human ELISA Kit (Thermo Fisher Scientific, Waltham, MA; #BMS224-2) were used as described by the manufacturers to quantify CCL18 and IL-1β in the induced THP-1 cell supernatant. BALF samples were analyzed for mouse IFN-γ using ELISA MAX® Deluxe (Biolegend, San Diego, CA; #430804).

[0236] Finally, for the in vivo folate imaging study, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were resected and imaged using an AMI live imager (Spectral Instruments Imaging, Tucson, AZ). For the in vivo folate receptor labeling test, the lungs of mice were collected immediately after euthanasia and digested using a lung dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE, #130-098-427) according to the instructions for the gentleMACS Octo Dissociator with Heathers (Miltenyi Biotec, Bergisch Gladbach, DE, #130-096-427) as described in the manual, and filtered 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 with antibodies against desired macrophage markers for 30 minutes on ice (FITC-CD11b, Biolegend, San Diego, CA, #101205; FE-F4 / 80, Biolegend, San Diego, CA, #123109). The labeled macrophages were then washed twice in PBS, stained with 7AAD (viability stain) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, San Jose, CA).

[0237] As shown in Figure 7A (top panel), the untreated lung (PBS control column) and the BM-treated lung at day 7 show similar high-density alveoli interconnected by minimal extracellular matrix. In contrast, at day 14 after BM infusion, the size and frequency of air sacs were significantly reduced, and the density of extracellular matrix was clearly increased, suggesting the development of prominent fibrosis in the treated mice. By day 21, the disease in this model had already begun to resolve spontaneously, and many mice eventually recovered from BM-induced trauma by day 35.

[0238] Evidence of inflammation up to day 7 was found in the infiltration of FRβ-expressing macrophages (see lower panel of Figure 7A and quantification in Figure 7B), which are almost completely absent in healthy lungs but continue to accumulate in BM-exposed lungs until day 14. Furthermore, staining with F3 showed marked expression of FRβ in IPF lungs (primarily interstitial space), as previously reported in the literature (Figure 7A). FRβ expression was limited to inflamed lungs (either IPF patients or BM-induced PF, but not in healthy lungs). In addition, FRβ-expressing macrophages were observed in mouse lungs on day 7 after BM administration, with peak expression on day 14 (Figure 7B). These results confirm the previously reported FRβ expression in activated macrophages in inflamed lungs.

[0239] Next, we demonstrated that these FRβ-expressing macrophages can be targeted by folate-binding molecules by the accumulation of the folate-targeting fluorescent dye OTL38 in the lungs of BM-treated but unhealthy mice after tail vein injection. As shown in Figure 7B, OTL38 fluorescence was observed only in the kidneys of healthy mice (i.e., its primary excretion site), with little or no uptake in other tissues.

[0240] Figures 7C and 7D show FRβ IHC staining of human IPF lung tissue (Figure 7C) and healthy human lung tissue (Figure 7D). Eight-week-old C57BL / 6 male mice were fed a folate-deficient diet for one week prior to BM or PBS infusion. Ten days after infusion, the mice were injected via the tail vein with OTL38 containing or without 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) of FA-glucosamine in a 200-fold excess. Two hours later, the mice were sacrificed before analysis. For in vivo folate imaging studies, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were resected and imaged using an AMI live imager (Spectral Instruments Imaging, Tucson, AZ). For the in vivo folate receptor labeling test, the lungs of mice were collected 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.

[0241] Figure 7E shows images of various mouse tissues / organs taken from mice with or without BM-induced experimental fibrosis (BM) (PBS control) and imaged with the folate receptor-targeted fluorescent dye, OTL38. Healthy mice (column a) or BM-treated mice (columns b and c) were injected into the tail vein with 10 nmol of OTL38 10 days after fibrosis induction, either in the absence (b) or presence (c) of folate-targeted glucosamine (a competing reagent for FRβ that blocks OTL38 binding), and euthanized 2 hours later for tissue excision and fluorescence imaging. This supports the idea that the FA-targeted conjugate of the present invention exhibits FRβ-specific binding without being taken up by other healthy tissues.

[0242] Tail vein injection of OTL38 into BM-treated mice resulted in not only the aforementioned fluorescence in the kidneys but also significant accumulation in the fibrous lungs (see Figure 7E). This pulmonary uptake is primarily mediated by folate receptors, as demonstrated by the near-quantitative blockade of pulmonary accumulation when BM-treated mice were simultaneously injected with 200-fold excess folate-glucosamine (i.e., a competitive inhibitor of FRβ binding (see Figure 7E)). These data indicate that folate-targeting molecules selectively bind to folate receptor-expressing cells in fibrous tissues without significant accumulation in other tissues of the body. In other words, FRβ-expressing macrophages can, in practice, be targeted with folate-binding molecules, which in clinical applications localize almost exclusively to fibrous tissues. Therefore, when using the targeting moiety in the compounds of this disclosure, any TLR7 agonists that are not captured by the target fibrous (or cancerous) tissue are minimal.

[0243] Next, to determine which cell types capture the folate-pigment conjugate in the lungs of BM-treated mice, the lungs of the above animals were digested with collagenase, and cell-specific pigment uptake was examined by flow cytometry. Figure 7F shows data from FACS analysis obtained from in vivo labeling of mice experiencing BM-induced experimental fibrosis, which were injected into the tail vein with PBS (column 1) or 100 nmol OTL38 in the absence (column 2) or presence (column 3) of a 200-fold excess of folate-targeted glucosamine. As shown in Figure 7F, macrophage-like cells isolated from BM-treated mice not injected with OTL38 did not exhibit fluorescence (see column 1). In contrast, approximately 22% of macrophage-like cells from fibrous mice injected with OTL38 showed significant retention of folate-targeted pigment (column 2), supporting the idea that OTL38 targets FRβ-positive macrophages in the inflamed lung. Indeed, the observation that simultaneous tail vein injection of 200-fold excess folate-glucosamine essentially blocked all folate-pigment retention demonstrates that pigment uptake is particularly folate receptor-mediated and that pigment accumulation requires unoccupied folate receptors. Importantly, this conclusion is further supported by data showing that FRβ expression is essentially undetectable in untreated lungs (see Figure 7A) but dramatically increases during the development of fibrosis in BM-treated lungs (see Figures 7A-7D). FRβ expression is also prominently expressed in the lungs of human IPF patients.

[0244] Example 5 Next, using the established ability to target drugs attached to FRβ-expressing fibrotic macrophages, it was investigated whether folate-targeted TLR7 agonists could suppress the signs and symptoms of fibrosis in BM-treated mice. For this purpose, BM-treated mice were intravenously injected every other day starting on day 10 with either a vehicle (3% DMSO in PBS) or compound 1B (see Figure 8A). Compound 1A could not be similarly evaluated in vivo because the TLR7-54 agonist caused rapid weight loss and subsequent death (see Figures 9A and 9B). In BM-induced experimental pulmonary fibrosis in mice, inflammation is known to persist for approximately 9–10 days after BM induction. Therefore, the transition from inflammation to fibrosis occurred approximately 9–14 days in this model, fibrosis-promoting markers began to appear around day 10, and drug administration was initiated on day 10 (Figure 8A).

[0245] The drug was administered twice every other day until day 21. To prevent any "resistance" to the TLR agonist, the individual daily doses were divided every 6 hours. Next, on day 21, mice were sacrificed and immediately subjected to bronchoalveolar lavage, followed by lung resection for immunohistochemistry and quantification of collagen and hydroxyproline.

[0246] Figures 8B–8G show graph data representing various marker levels measured from mice treated with the BM model in Figure 8A. BALF cells were collected on day 21, centrifuged at 4°C, the resulting pellet was resuspended in culture medium, seeded in a 96-well plate, cultured for 2 hours, washed three times with pre-warmed PBS, and cells were harvested for qPCR. This data shows that Arg1 (Figure 8B), MMP9 (Figure 8C), and TIMP 3 (Figure 8D) (e.g., pro-fibrosis markers) were all downregulated. CD86 (Figure 8E) and IFN-γ (Figure 8F) (e.g., anti-fibrosis markers) were both upregulated. Furthermore, negative regulators of TLR7 signaling IRAK-4 were upregulated (Figure 8G), as were the number of BALF cells present (Figure 8H). Indeed, the total number of mouse BALF cells decreased in a dose-dependent manner after treatment with different doses of compound 1B. The values ​​shown in Figures 8B-8G represent the mean ± SD for each group, with *P<0.05, **P<0.005, and ***<0.0005; calculations for saline versus vehicle group and compound 1A and compound 1B treatment groups versus vehicle group were performed using Student's t-test, except for BALF cell count and protein concentration measurements calculated by Dunnett's multiple comparison test for the compound 1B treatment group and the vehicle group. Vehicle = 3% DMSO in PBS.

[0247] As shown in Figures 8B–8D, qPCR analysis of pro-fibrosis markers in macrophage subgroups of bronchoalveolar lavage cells revealed elevated tissue inhibitors Arg1, MMP9, and TIMP3 in BM-induced mice compared to control mice. More importantly, a parallel study demonstrated that treating BM-induced mice with compound 1B suppressed all of the same pro-fibrosis markers, resulting in levels similar to those seen in healthy mice. Consistent with these data, quantification of pro-fibrosis markers revealed that both CD86 transcript (qPCR) and IFN-γ concentration (ELISA of lavage solution) increased after treatment with compound 1B (see Figures 7E and 7F). The observed upregulation of IRAK-4 (i.e., a marker of TLR activation, results shown in Figure 7G) and the dose-dependent decrease in the total number of BALF cells present after treatment with different doses of compound 1B (Figure 7H) together, these data suggest that administration of a folate-targeted TLR7 agonist can reprogram macrophages in vivo from a pro-fibrotic M2-like phenotype to a pro-fibrotic M1-like phenotype in the lungs of BM-treated mice.

[0248] Example 6 Additional studies were conducted to determine whether the reprogramming of fibrous lung macrophages described above resulted in an actual improvement in the fibrous condition in fibrous mice. Lung tissue from the aforementioned mice was embedded in paraffin, sectioned, and stained with H&E and Masson's trichrome to assess tissue density and extracellular collagen deposition, respectively.

[0249] Figures 9A and 9B show survival curves (Figure 9A) and body weight changes (Figure 9B) of mice with experimental pulmonary fibrosis treated with untargeted and targeted TLR7 agonists. The data support that administration of the compounds of this disclosure (here, for example, compound 1B) increases the survival of BM-treated mice without causing significant body weight loss. Each value represents the mean ± SD for each group.

[0250] Figure 10A shows the hydroxyproline content (μg / lung) in lung tissue, utilizing collagen deposition as a measure of fibrosis. Tissue samples from each of the following groups at day 21 are shown: healthy control (saline) (●), disease control (vehicle) (■), treatment with a free drug TLR7 agonist (compound 1A) (▼), and treatment with a folic acid-targeted TLR7 agonist (compound 1B) (▲). BM-induced mice treated with either 10 nmol of compound 1B (▲) or compound 1A (▼) showed a significant decrease in total hydroxyproline content per lung compared to the vehicle control (■). The values ​​shown in Figure 9A represent the mean ± SD for each group, with *P<0.05, **P<0.005, ***<0.0005, and Student's t-tests for saline vs. vehicle group, and compound 1A and compound 1B treatment groups vs. vehicle group.

[0251] Figures 10B and 10C show stained images of the lung tissue shown in Figure 10A, using Figure H&E staining (Figure 10B) and Masson's trichrome (collagen) staining (Figure 10C).

[0252] As shown in the H&E staining of the panel in Figure 10B, healthy lungs are rich in air sacs surrounded by a thin reticular membrane. In contrast, BM-induced lungs show far fewer alveoli with marked deposition of extracellular matrix where air sacs once existed. Most importantly, BM-injected mice treated with compound 1B from day 10 exhibited lung structures similar to those of healthy mice (Figure 10B), suggesting that targeting of compound 1A to fibrotic lung macrophages is effective in suppressing the major and prominent features of pulmonary fibrosis. This prevention of fibrosis is indeed accompanied by blockade of collagen deposition, as documented by Masson's trichrome staining of parallel lunch sections (Figure 10C), and collagen staining is strongly suppressed in mice injected with compound 1B into the tail vein (Figure 10B). Thus, the data support that IPF mice treated with at least compound 1B(▲) exhibit suppression of IPF pathological conditions (e.g., fibrosis).

[0253] Finally, to confirm that compound 1B actually affected collagen production in vivo, hydroxyproline (a major component of collagen) in the total hydrolysate of the affected lung was quantified. More specifically, lung tissue from the mice described above was perfused with PBS, hydrolyzed with acid, and analyzed for hydroxyproline content. In Figure 10A, induction of fibrosis induced a significant increase in hydroxyproline content, and this increase was suppressed by treatment with compound 1B. Thus, these data support that treatment with the targeted TLR7 agonist compounds of this disclosure reduces (and even counteracts) collagen deposition and, consequently, fibrosis in vivo.

[0254] 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, while compound 1A offered no survival benefit except for significant weight loss (>25%, Figure 7). The free agent functioned well in reducing hydroxyproline content, but the observed poor survival rate may be due to overall toxicity (i.e., weight loss, see Figure 7B). This was not surprising, as systemic administration of TLR7 agonists is known to cause toxicity.

[0255] Example 7 Because the use of untargeted TLR7 agonists to treat IPF (or other fibrotic diseases) is discouraged by systemic activation of the immune system and resulting toxicity, we evaluated whether there is any apparent toxicity associated with systemic administration of compound 1B in mice. For this purpose, BM-induced mice were treated with 0, 1, 3, or 10 nmol of compound 1B every other day starting from day 10, and body weight, pulmonary hydroxyproline content, and histological analysis were performed on day 21. Unlike conventional systemic administration, the targeted agent not only improved survival but also reduced body weight loss, highlighting the significance of the targeted approach (Figures 11A and 11B).

[0256] Figure 12 shows data on the dose-dependent effect of folate-targeted TLR7 agonists on suppressing fibrosis in BM-induced mice, using collagen deposition as a measure of fibrosis. The data are represented as follows: healthy control (PBS, ●), vehicle (■), BM-induced mice treated with 1 nmol compound 1B (○), 3 nmol compound 1B (□), or 10 nmol compound 1B (▲); subpart A shows graph data on body weight of BM-induced mice over time; subpart B shows measurements of hydroxyproline content in lung tissue treated with different doses (10 nmol, 3 nmol, or 1 nmol of compound 1B); and subpart C shows images for histological analysis of right lung tissue using H&E staining and trichrome staining.

[0257] Figures 11B and 12 subpart A show no difference in weight loss among mice treated with 0, 1, 3, or 10 nmol of compound 1B, suggesting that repeated administration of the compound did not induce significant toxicity. The expected effect of these treatments on pulmonary fibrosis can still be seen from a comparison of the hydroxyproline content of various lung hydrolysates, with the order of effectiveness being 10 nmol / mouse > 3 nmol / mouse > 1 nmol / mouse > 0 nmol / mouse (see subparts B and C of Figure 12). More importantly, detailed analysis of lung tissue morphology showed improvement as the dose of compound 1B increased, suggesting that the tissue most strongly enriched with the TLR7 agonist was indeed the tissue with the most normal microscopic morphology. In summary, these data support the idea that targeting TLR7 agonist FRβ+ macrophages in fibrous tissue can effectively prevent fibrosis without the systemic activation of the immune system that limits the use of TLR7 agonists in humans.

[0258] Finally, to determine whether this anti-fibrotic effect could be achieved at low doses, we conducted therapeutic studies using two low doses (3 nmol / kg and 1 nmol / kg) (Figures 9 and 10). Interestingly, while the low doses showed a significant decrease in hydroxyproline content and collagen deposition levels, the 10 nmol dose resulted in the best survival rate.

[0259] Example 8 To support the idea that embodiments of the compounds of this disclosure other than Compound 1A and Compound 1B function similarly in applications, other representative embodiments of the compounds of this specification were examined in in vitro studies.

[0260] Figures 13A–13D show graph data of various marker levels measured from human THP-1 cells induced into M2 macrophages with 20 ng / mL IL-4, 20 ng / mL IL-13, and 5 ng / mL IL-6. The cells were then reprogrammed for 48 hours with different nM concentrations of a TLR7 agonist having formula IV (e.g., compound 2A) and collected for genetic analysis by qPCR. The mRNA levels of the following markers relative to the expression of M2-like macrophage controls are shown: CCL18 mRNA level (Figure 12A), IL-1β mRNA level (Figure 13B), and TNFα level (Figure 13C). Figure 13D shows the results of protein analysis after collection of the cell supernatant. The secreted CCL18 protein was detected by ELISA.

[0261] Figures 13A–13D show the evaluation of the agonist compounds of this disclosure having formula IV (e.g., compound 2A) in terms of their ability to reprogram M2-like macrophages into M1-like macrophages.

[0262] Primarily, human monocytic (THP-1) cells were induced into the M2-like phenotype using the methods and materials described above. Specifically, THP-1 cells were seeded at a density of 60,000 cells / well in 96-well plates. The cells were differentiated into nonpolar macrophages by incubation with 200 nM PMA for 48 hours, followed by incubation in fresh RPMI medium for 24 hours. The resulting macrophages were polarized into the M2-like phenotype by incubation 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.

[0263] To evaluate whether compound 2A can reprogram profibrosis-promoting macrophages to a less fibrillatory phenotype, THP-1 cells stimulated with IL-4, IL-6 plus IL-13 were incubated with different concentrations of compound 2A, and mRNA levels of several profibrosis-promoting markers, namely CCL18, IL-1β, and TNFα, were examined using qPCR and ELISA.

[0264] As shown in Figures 13A and 13B, 48-hour incubation with compound 2A (free agent) induced a decrease in the expression of CCL18 and IL-1β, suggesting that the TLR7 agonist can indeed promote a shift in these pro-fibrotic polarized THP-1 cells to a lower fibrotic phenotype. (Note: Figure 13B shows a bell-shaped curve indicating compound 2A, which has an inhibitory response at low concentrations and a stimulative response at high concentrations; this is a response curve common to specific agents.) Furthermore, an increase in the expression of TNFα (anti-fibrotic phenotype marker) was observed (Figure 13C), confirming that THP-1 shifted from pro-fibrotic to anti-fibrotic properties.

[0265] In addition to non-conjugate TLR7 agonism, the conjugate compounds of the present disclosure were also evaluated. Human THP-1 cells were induced into macrophages having an M2-like phenotype according to the method described herein (e.g., using 20 ng / mL of IL-4, 20 ng / mL of IL-13, and 5 ng / mL of IL-6), and then reprogrammed for 2 hours at different nM concentrations of the following various compounds of the present disclosure: non-conjugate (free agent) TLR7 agonist compounds having formula I and / or II (data are collectively shown as compound 3A), folate-conjugate TLR7 agonist compounds having formula XV (with a releaseable linker) (e.g., compound 3B), folate-conjugate TLR7 agonist compounds having formula XVII (with a non-releaseable linker) (e.g., compound 3C), and folate-conjugate TLR7 agonist compounds having formula XVI (with a non-releaseable linker) (e.g., compound 3D). Next, cells were collected for gene analysis by qPCR, and the relative expression of CCL18 (Figure 14A), CD206 (Figure 14B), and IL-1β (Figure 14C) was analyzed.

[0266] The expression of various pro-fibrosis (M2 phenotype) markers, including CCL18, IL-1β, and CD206, was quantified. As shown in Figures 14A-14C, the expression of each of these pro-fibrosis markers was reduced after administration of compound 3B, compound 3D, and compound 3C, respectively, with compounds 3D and 3C (both containing non-release linkers) being the most effective compared to the other compounds.

[0267] Figure 15 shows the secreted CCL18 protein levels in each group of THP-1 cells in Figures 14A–14C after treatment with compound 3A, compound 3B, compound 3C, or compound 3D. Compound 3A and folate-targeted TLR7 compounds (e.g., compounds 3B, 3C, and 3D) downregulate CCL18 secretion in the low concentration range (0.1–10 nM).

[0268] Furthermore, the cell supernatant was collected and the secreted CCL18 protein was detected by ELISA. Figure 15 confirms that compound 3A (free agent) and the folate-targeting compounds (compounds 3B, 3C, and 3D) all downregulated CCL18 secretion in the low concentration range (0.1–10 nM), and further supports the idea that these compounds can similarly reprogram M2-like pro-fibrillation macrophages into M1-like anti-fibrillation macrophages via a similar mechanism, similar to the examples described in relation to compounds 1A and 1B.

[0269] Example 9 Repeating the above studies (Figures 3A-3F) revealed the same qualitative changes, with only the magnitude of the effect of compound 1B being slightly reduced. This reduction in potency was expected because the untargeted TLR7 agonist enters cultured cells immediately, whereas its folate-targeted counterpart is designed to enter cells only after folate receptor binding and receptor-mediated endocytosis. Since low molecular weight, water-soluble drugs like compounds 1A and 1B are often excreted from the body within two hours of injection, a more physiologically appropriate in vitro model of in vivo drug exposure would limit cell incubation with the drug to only two hours, and then examine the drug's efficacy after a further 46 hours of incubation in the absence of the drug. As shown in Figures 4A-4E, after incubating THP-1 cells with a TLR7 agonist for 2 hours and then replacing the drug-containing medium with a drug-free medium, compound 1B was observed to have superior efficacy compared to compound 1A, and in particular, folate-targeted conjugates were dramatically improved in the case of TNFα induction. This is thought to be because the folate-targeted TLR7 agonist was captured by folate receptor-positive cells, whereas compound 1A was not retained by the same cells.

[0270] These data support the idea that compound 1B should be more effective in reprogramming pro-fibrotic macrophages in vivo, and that folate-conjugated drugs (e.g., compound 1B) have the added advantage of causing less systemic toxicity because they concentrate in FRβ-expressing macrophages and cannot enter the predominantly folate receptor-negative cells throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0271] Furthermore, to stimulate THP-1 cells so that the above mRNA analysis accurately reflects the levels of pro-fibrotic cytokines produced by 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 shown in Figures 6A and 6B, both compound 1A and compound 1B induced a decrease in CCL18 and IL-1β when incubated continuously with the agonist for 48 hours, but compound 1B was again found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D).

[0272] Example 10 Figure 16 shows an in vivo study methodology for at least one embodiment of the compound of this disclosure in a BM mouse model, where the compound has formula XVII (e.g., compound 3C). Figures 17A and 17B are LC-MS spectra of compound 3C, supporting high purity of the conjugate and no free agent detected.

[0273] Figures 18A–18F show the results from the in vivo study methodology of the control mice in Figure 16, and include survival curves (Figure 18A), body weight changes (Figures 18B and 18D), cell concentration by BALF (Figure 17C), hydroxyproline concentration (μgHP / leaf) in surviving mice (Figure 18E), and hydroxyproline concentration (μgHP / leaf) in all mice (e.g., including both surviving mice and mice that died before day 21) (Figure 18F). A 10 nmol concentration dose of the compound having formula XVII (e.g., compound 3C) increased the survival rate of the control mice while simultaneously decreasing the number of HP and BALF cells. A 3 nmol concentration dose did not show any measurable benefit to the control mice.

[0274] Example 11 M2-induced human monocyte-derived macrophages were treated with either 100 nM compound 1A or compound 1B for 48 consecutive hours, or initially for 2 hours in or without FA-glucosamine (a competitor), followed by 46 hours in the absence of the agent (2+46 hours). mRNA levels of the pro-fibrosis markers Arg1 (Figure 19A), CD206 (Figure 19B), and CD163 (Figure 19C), as well as protein levels of secreted pro-fibrosis CCL18 (Figure 19D) and anti-fibrotic cytokines CXCL10 (Figure 19E) and IL-6 (Figure 19F) (n=3, technically replicated), were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46 hours, competitive). This data supports the idea that compound 1B binds to the folate receptor because the downregulation of the biomarker was blocked by excessive FA-glucosamine (a competitor).

[0275] Example 12 Healthy mice were injected via tail vein with 10 nmol of compound 1A (round) or compound 1B (square), and peripheral blood was collected at specified time points after drug injection (Figures 20A-C). Plasma IL-6 (Figure 20A), IFNα (Figure 20B), and TNFα (Figure 20C) levels were measured (n=3) (Figures 20D-F). The effect of drug concentration on plasma levels of IL-6 (Figure 20D), IFNα (Figure 20E), and TNFα (Figure 20F) was determined at 1.5 hours, 1 hour, or 1 hour post-treatment, respectively (n=2) (Figure 20G). Compound 1A stimulated systemic cytokine release in healthy mice, while compound 1B did not. Furthermore, compound 1B stimulated inflammatory cytokine release at a dose lower than half that of compound 1A. These data suggest that TLR7 agonists, when targeting lung macrophages with folate receptor-targeting ligands, can safely be used to reprogram fibrotic lung macrophages into a pro-fibrotic state.

[0276] Example 13 Sections of the same healthy and fibrous lung tissues shown in Figure 6 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-hole slide scanner as described in the Methods (n=2). Scale bar, 100 μm. Differences between treatment groups indicate that compound 1B elicits a potent pro-fibrotic response in vivo.

[0277] Various embodiments of compounds, compositions, and methods are described in considerable detail herein, but these embodiments are provided merely as non-limiting examples. Many variations and modifications of the embodiments described herein will be obvious to those skilled in the art in light of this disclosure. Thus, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the scope of this disclosure, and that equivalents may be substituted for their elements. In fact, this disclosure is not intended to be exhaustive or overly limiting. The scope of this disclosure is defined by the appended claims and their equivalents.

[0278] Furthermore, while many of the examples provided herein utilize mouse models, it will be understood by those skilled in the art that gene expression patterns in mouse models show a very significant correlation with gene expression patterns in the human state, and that many pathways are generally controlled by multiple conditions in humans and mice. Therefore, gene expression patterns and disease progression in mouse models closely replicate those in human pathologies, particularly with respect to inflammatory diseases and cancer, thus supporting the correlation of the examples described herein with human data, specific pathologies, and applications.

[0279] Accordingly, this description and the attached claims are intended to include all modifications and changes that would be apparent to those skilled in the art based on this disclosure.

Claims

1. A compound represented by the following formula, Q-L-T During the ceremony, Q is a radical of the folate receptor binding ligand, L is a non-release linker, T is a radical of a Toll-like receptor (TLR) agonist or a pharmaceutically acceptable salt thereof. The aforementioned non-dischargeable linker is as follows: 【Chemistry 1】 Represented by, During the ceremony, n is between 1 and 30. w is between 0 and 5. The radical of the TLR agonist has a structure represented by formula X; 【Chemistry 2】 During the ceremony, R １ Ha-NH ２ And, R ２ is H or alkyl, R ３ -OH, -SH, -NH ２ , or -NH-R １Ｘ And, R １Ｘ is a compound selected from the group consisting of H, alkyl, alkenyl, and alkynyl.

2. The compound according to claim 1, wherein the TLR agonist is a Toll-like receptor 7 (TLR7) agonist.

3. The compound according to claim 1, wherein Q is a radical of the compound of the following formula: 【Chemistry 20】

4. The compound according to any one of claims 1 to 3, wherein the radical of the TLR agonist is a radical having the following structure. 【Transformation 8】

5. The compound according to claim 1, wherein the compound has the structure represented below. 【Chemistry 9】

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use in the manufacture of a pharmaceutical for the treatment of a fibrotic disease state or cancer, wherein the compound according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6, wherein the radical of the TLR agonist or a pharmaceutically acceptable salt thereof comprises a TLR7, 8, or 9 agonist.

8. The compound comprises the compound described in any one of claims 1 to 5 or the composition described in claim 6, The linker comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, and the linker is R ３ A pharmaceutical composition conjugated with T.

9. The compound according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable salt is selected from hydrobromide, citrate, trifluoroacetate, ascorbic acid, hydrochloride, tartrate, triflate, maleate, mesylate, formate, acetate, or fumarate.

10. Use of a compound according to any one of claims 1 to 5 and 9 or a pharmaceutical composition according to claim 6 or 8 in the manufacture of a pharmaceutical for preventing or treating a fibrous disease state.

11. The use according to claim 10, wherein the fibrous disease state is idiopathic pulmonary fibrosis, and the pharmaceutical agent is prescribed to be administered intravenously, intramuscularly, intraperitoneally, topically, or by inhalation to the subject.

12. The use according to claim 10, wherein the fibrotic disease state includes idiopathic pulmonary fibrosis, or fibrotic diseases of the liver, skin, bladder, heart, pancreas, prostate, or kidneys.

13. The use according to claim 10, wherein the folate receptor binding ligand is specific to folate receptor β and binds to folate receptor β on a cell.

14. Q is, Folic acid; Pyrido[2,3-d]pyrimidine; or, Structures shown in Tables 3-6; The compound according to claim 1, which is a radical of [the compound]. Table 3 Table 4 Table 5 Table 6

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