Composition and method for regulating IL-2

Abstract

This disclosure relates to modulating IL-2 expression / activity to prevent or treat diseases (cancer, autoimmune diseases, inflammatory diseases, or infections). Compounds of formula (I), as well as compositions comprising their conjugates, complexes, and pharmaceutical compositions, as well as methods of using them to modulate (e.g., increase) IL-2 expression / activity to achieve desired results (e.g., reduction in cancer cell viability, growth, proliferation, or metastasis; reduction in tumor volume; increase in survival; increase or decrease in immune cell proliferation or activation; increase or decrease in cytokine production; decrease in autoimmunity; decrease in inflammation, etc.). Related pharmaceuticals, kits, and methods of delivering such compositions are described. Compounds of formula (I) and methods for developing or manufacturing their pharmaceutical compositions are provided. Diagnostic and test methods for detecting levels of IL-2 expression / activity, as well as compositions constituting diagnostic or test kits, are further described. JPEG2026513223000025.jpg3642

Description

[Technical Field]

[0001] The present invention relates to compositions, systems, and methods for modulating the expression and / or activity of IL-2 in cells, animals, or human subjects. Such compositions, systems, and methods are useful for treating, preventing, or improving diseases, including cancer, autoimmune diseases, inflammatory diseases, infections, and other diseases associated with IL-2.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 491,783 (filed on 23 March 2023), the disclosure of which is incorporated in its entirety by reference.

[0003] Sequence List This application includes a sequence listing electronically submitted in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 17 March 2023, named IL2_sequence_listing.xml, and has a size of 8,854 bytes. [Background technology]

[0004] The following discussion describes certain articles and processes for background and introductory purposes. Nothing included herein should be construed as an "acknowledgment" of prior art. The applicant expressly reserves the right, where applicable, to indicate that the articles and processes referenced herein do not constitute prior art under applicable legal provisions.

[0005] Interleukin-2 (IL-2 or IL2) is a cytokine produced by T cells, natural killer (NK) cells, and activated dendritic cells (DCs) that play a crucial role in immune tolerance and immune activation. IL-2 is also known as T cell growth factor, TCGF, ​​or lymphokine. In some embodiments, IL-2 refers to a polymorph, isoform, homolog, pseudogene, or variant of IL-2. As used herein, IL-2 may refer to a protein product obtained from the expression of a gene, RNA transcript, or nucleic acid encoding IL-2, unless otherwise specified or indicated.

[0006] IL-2 binds to different forms of IL-2 receptors (IL-2Rs) that can contain α(CD25), β(CD122), and / or γ(CD132) chains. IL-2 binds to monomeric IL-2Rs containing the α chain with low affinity (dissociation constant of 10 nM), which do not result in intracellular signaling. IL-2 is also found in memory T cells and resting CD8 + It binds to the dimer IL-2R, which contains β and γ chains and is expressed on T cells and NK cells, with moderate affinity (dissociation constant of 1 nM). IL-2 is used in regulatory T cells (T regs ) is constitutively expressed on and effector CD8 upon exposure to the antigen. +IL-2 binds with high affinity (dissociation constant of 10 pM) to the trimer IL-2R, which contains α, β, and γ chains and is induced in T cells and NK cells. IL-2 can also bind to the trimer IL-2R, which is formed in trans by the α chain on activated DCs, and by the β and γ chains on antigen-naive T cells and NK cells. Binding of IL-2 to dimers and trimer IL-2R leads to activation of intracellular signaling pathways, including the JAK / STAT5 pathway, PI3K / AKL / mTOR pathway, and MAP kinase (MAPK) pathway, and therefore to the activation of genes that increase immune cell proliferation, immune cell activation, induce differentiation, inhibit apoptosis, and promote survival. Activation of IL-2R also leads to NF-κB activation, which leads to the upregulation of genes involved in T cell development, maturation, and proliferation. The functions of IL-2 are described in Abbas et al. (Abbas et al., The American Journal of Pathology, 190(9):1776-1781 (2020)), Wuest et al. (Wuest et al., Nature Medicine, 17(5):604-609 (2011)), and Zhang et al. (Zhang et al., European Journal of Inflammation, 19:1-7 (2021)), and these disclosures, along with their references, are incorporated herein by reference in their entirety.

[0007] Low doses of IL-2 are used in conjunction with T regs This is favorable for binding to the high-affinity IL-2R mentioned above, leading to immune tolerance. Low-dose IL-2 ranges from 90,000 to 5.4 million units (IU) per dose, or a cumulative dose of 1.5 million to 5.25 million IU over the course of treatment. In some embodiments, low-dose IL-2 refers to a dose of 1 million IU or less per day, and T regsThis leads to the expansion and activation of IL-2, as well as a decrease in B cells. Low-dose IL-2 has been shown to be effective in treating graft-versus-host disease (GVHD), a major cause of death and other complications after allogeneic hematopoietic stem cell transplantation, which is mainly caused by an excessive immune response of B and T cells against autologous and allogeneic cells. Low-dose IL-2 leads to T regs Low-dose IL-2 has been shown to be effective in treating systemic lupus erythematosus (SLE), which can be caused by IL-2 deficiency resulting in a decrease in the number and function of IL-2 cells, as well as abnormal activation of other immune cells, leading to immune complex deposition that can result in autoantibody formation and multiple organ failure. regs Low-dose IL-2 has been shown to be effective in treating type 1 diabetes, which is caused by effector T cell-mediated killing of insulin-producing cells due to abnormally low function and quantity of IL-2. Low-dose IL-2 has also been shown to be effective in treating hepatitis C infection-associated mixed cryoglobulinemia and vasculitis. Low-dose IL-2 has also shown therapeutic potential for treating other autoimmune diseases, such as amyotrophic lateral sclerosis, alopecia areata, ankylosing spondylitis, autoimmune encephalitis, autoimmune hepatitis, Behçet's disease, corneal transplantation, Crohn's disease, dermatomyositis, granulomatosis with polyangiitis, hypomyosopathic dermatomyositis with refractory dermatitis, polymyositis, primary immune thrombocytopenia, primary Sjögren's syndrome, psoriasis, psoriatic arthritis, rheumatoid arthritis, sclerosing cholangitis, Takayasu's arteritis, ulcerative colitis, and Wiscott-Aldrich syndrome. In addition, low-dose IL-2 has shown therapeutic potential for treating T regsIt can suppress inflammation by multiple mechanisms, including consuming IL-2, reducing co-stimulation of effector T cells, and producing immunosuppressive cytokines such as IL-10, and thus can be useful for treating inflammatory diseases. Low-dose IL-2 has shown treatment potential for diseases such as rheumatoid arthritis where inflammation leads to joint destruction. Low-dose IL-2 has also shown treatment potential for atherosclerosis where inflammation leads to plaque formation. Low-dose IL-2 has also shown promise for treating other inflammatory conditions such as acute lung injury, beryllium-induced granulomatous inflammation, muscular dystrophy, and obesity. The advantages of low-dose IL-2 for the treatment of autoimmune and inflammatory diseases are described in Zhang et al. (Zhang et al., European Journal of Inflammation, 19:1-7 (2021)), Graβhoff et al. (Graβhoff et al., Front. Immunol., 12:648408 (2021)), Overwijk et al. (Overwijk et al., Annu. Rev. Med., 72:281-311 (2021)), Klatzmann et al. (Klatzmann et al., Nat. Rev. Immunol., 15(5):283-294 (2015)), Zhao et al. (Zhao et al., NEJM Evid., 1(1)(2022)), and Choi et al. (Choi et al., bioRxiv, 351841 (2018)), and these disclosures, together with those references, are incorporated herein in their entirety. Thus, increasing or decreasing the expression and / or activity of IL-2 to achieve levels of IL-2 equivalent to low-dose IL-2 is useful for the treatment of autoimmune and inflammatory diseases, and specific examples thereof are described herein.

[0008] High-dose IL-2 is IL-2 and CD8 +High-dose IL-2 induces binding to moderate-affinity IL-2R on T cells and NK cells, leading to immune activation. High-dose IL-2 can range from 600,000 to 720,000 IU / kg per dose in up to 15 consecutive doses every 8 hours over 5 days, or in cumulative doses exceeding 100 million IU. High-dose IL-2 is useful in treating various types of cancer. High-dose IL-2 has been clinically proven effective in treating patients with metastatic renal cell carcinoma, metastatic melanoma, and non-Hodgkin lymphoma. High-dose IL-2 is also effective in treating patients with neuroblastoma in combination with dinutuximab, GM-CSF, and 13-cis-retinoic acid. High-dose IL-2 has shown and / or is being tested as a promising treatment for a variety of cancers, including but not limited to acute myeloid leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer (e.g., triple-negative breast cancer), cervical cancer, cholangiocarcinoma, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, gastric cancer, glioblastoma, HPV-related cancers, lymphoma, malignant pleural effusion, mesothelioma, Merkel cell carcinoma, nasopharyngeal adenocarcinoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, recurrent myeloid disease, sarcoma, squamous cell carcinoma, thyroid cancer, urothelial carcinoma, and uveal melanoma. The benefits of high-dose IL-2 for cancer treatment are described in Overwijk et al. (Overwijk et al., Annu. Rev. Med., 72:281-311 (2021)), MacDonald et al. (MacDonald et al., Journal of Immunology Research, 2021:7855808 (2021)), Tang et al. (Tang et al., Cytokine X, 1(1):100001 (2018)), and Dutcher et al. (Dutcher et al., Journal for ImmunoTherapy of Cancer, 2:26 (2014)), and these disclosures, along with their references, are incorporated herein in their entirety. Furthermore, IL-2 has shown promise for treating infectious diseases such as HIV, severe respiratory syncytial virus, and pulmonary tuberculosis.The benefits of IL-2 for the treatment of infectious diseases are described in Zhou et al. (Zhou et al., PLoS Pathog., 17(10):e1009858 (2021)), Sananez et al. (Sananez et al., The Journal of Infectious Diseases, 218(1):75-83 (2018)), and Sheng et al. (Sheng et al., Journal of Tropical Medicine, 2022:5071816 (2022)), and these disclosures, along with their references, are incorporated herein by reference in their entirety. Thus, increasing the expression and / or activity of IL-2 is useful in treating diseases such as cancer or infectious diseases, and specific examples thereof are described herein.

[0009] Clinically approved or currently under development IL-2 drugs are based on recombinant peptides similar to wild-type IL-2, or modified versions thereof. Recombinant IL-2 (proleukin), also known as aldesleukin, is approved by the FDA for the treatment of metastatic melanoma and metastatic renal cell carcinoma. However, proleukin has significant drawbacks, such as rapid systemic clearance (it has a short half-life of approximately 15 minutes), which necessitates frequent IV administration in a hospital setting. In addition, proleukin has a narrow therapeutic range due to dose-limiting toxicity. Serious adverse effects of proleukin include vasoleak syndrome (also known as capillary leak syndrome), pulmonary edema, cardiac arrhythmias, liver and kidney damage, arthralgia, and fever. Proleukin is synthesized by heterologous expression in E. coli and lacks post-translational modifications such as glycosylation, which can reduce its stability and activity compared to naturally occurring IL-2 synthesized in the body. Furthermore, a significant number of patients receiving proleukin develop anti-drug antibodies due to the immunogenicity of heterologously expressed recombinant IL-2, which reduces activity and further shortens the drug's half-life due to increased clearance. Efforts are underway to develop modified forms of IL-2 that contain sequence mutations or are fused to other antibodies or proteins, which can preferentially bind to and activate the moderate-affinity dimer IL-2R rather than the high-affinity trimer IL-2R, thereby increasing therapeutic efficacy against cancer. regsIt has the goal of preferentially stimulating effector T cells and NK cells. However, modified IL-2 has shown limited efficacy in clinical practice, likely due to reduced stimulation of antigen-specific (activated) T cells expressing the trimer IL-2R, and has similar drawbacks to proleukins, such as serious side effects. Current IL-2 drugs and other peptide-based IL-2 drugs under development, as well as their limitations, are described in Overwijk et al. (Overwijk et al., Annu. Rev. Med., 72:281-311 (2021)), Pol et al. (Pol et al., J. Exp. Med., 217(1):e20191247 (2020)), Tang et al. (Tang et al., Cytokine X, 1(1):100001 (2018)), and MacDonald et al. (MacDonald et al., Journal of Immunology Research, 2021:7855808 (2021)), and these disclosures, along with their references, are incorporated herein by reference in their entirety.

[0010] In contrast to current peptide-based IL-2 therapies, small molecule-based therapies that can modulate IL-2 expression and / or activity include, for example, CD4 localized to specific tissues, including tumors. +Local production of IL-2 by T cells can offer clear advantages such as increased efficacy and reduced toxicity, ease of delivery and administration (most can be administered orally), modifiable properties via medicinal chemistry (e.g., increased metabolic stability, reduced toxicity), modifiableness allowing systemic distribution with or without central nervous system (CNS) distribution, scalable manufacturing routes, lower development and manufacturing costs in most cases, ease of dose adjustment to reduce or avoid serious adverse effects, better penetration into tumors with poor angiogenesis, cellular permeability to reach intracellular targets (e.g., nucleic acids), and a rapid, established regulatory pathway for approval. In addition, small molecule-based therapies that increase the expression and / or activity of endogenous IL-2 expressed by human immune cells can circumvent problems associated with recombinant IL-2 (such as lack of post-translational modification, as well as the formation of anti-drug antibodies which can lead to increased clearance, decreased stability, and lower efficacy), thus resulting in longer-lasting, higher therapeutic efficacy.

[0011] Therefore, in the art, there is a need for small molecule therapies to modulate the expression and / or activity of IL-2, which are useful in treating, preventing, or improving diseases such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. This disclosure addresses this need and other unmet needs in the art. A range of small molecule compounds for modulating the expression and / or activity of IL-2 in cells, animals, or human subjects, as well as their conjugates, complexes, and pharmaceutical compositions, as well as methods of use thereof, are disclosed. Such compositions, systems, and methods are useful in treating, preventing, or improving diseases, particularly cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. [Overview of the Initiative]

[0012] The present invention relates to a compound of formula (I), [ka] In the formula, A1 , R 1 , and R 4 However, this specification relates to compounds and pharmaceutically acceptable salts thereof, as described herein.

[0013] This specification describes compounds of formula (I) disclosed herein, as well as compositions comprising the same conjugates, complexes, pharmaceutical compositions, or medicinal products, which are useful for treating, preventing, or improving diseases such as cancer, autoimmune diseases, inflammatory diseases, or infections. Methods of using such compositions for treating, preventing, or improving diseases such as cancer, autoimmune diseases, inflammatory diseases, or infections are also described. Compositions comprising conjugates and complexes of compounds of formula (I) that are useful in the methods described herein are also described.

[0014] Methods are described that involve the use of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, for increasing IL-2 expression or activity. Methods are also described that involve the use of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, for decreasing IL-2 expression or activity. Furthermore, methods are described that involve the use of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, for achieving one or more phenotypic outcomes, such as a decrease in cancer cell growth or proliferation, a decrease in cancer cell viability, a decrease in tumor volume (i.e., tumor regression), a decrease in cancer metastasis, an increase in survival of an animal or human subject, an increase or decrease in immune cell proliferation, an increase or decrease in immune cell activation, an increase or decrease in cytokine production, a decrease in autoimmunity, a decrease in inflammation, a decrease in vascular leakage, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). Related pharmaceuticals, kits, and methods for delivering such compositions are also described.

[0015] Methods for the development, manufacture, and / or synthesis of compounds of formula (I) disclosed herein, or their conjugates, complexes, and pharmaceutical compositions are also described herein. Furthermore, methods for diagnostic and testing, including detecting IL-2 expression or activity levels, and compositions constituting kits for diagnostic and testing are also described herein.

[0016] Other features and advantages of the present invention will become apparent from the following detailed description and examples. [Modes for carrying out the invention]

[0017] The following description is provided to enable those skilled in the art to create and use the present invention and is provided in the context of a patent application and its requirements. Exemplary embodiments described herein, as well as various modifications to the genetic principles and features, will readily become apparent. Exemplary embodiments are described primarily with respect to specific processes and systems provided in a particular implementation. However, the processes and systems will also operate effectively in other implementations. Terms such as “exemplary embodiment,” “one embodiment,” and “another embodiment” may refer to the same or different embodiments.

[0018] Exemplary embodiments are described with respect to methods and configurations having specific components. However, methods and configurations may include more or fewer components than those shown, and the arrangement and type of components can be changed without departing from the scope of the invention.

[0019] Exemplary embodiments are also described in the context of methods having specific steps. However, the present method and configuration operate effectively with additional steps and steps in a different order that are not inconsistent with the exemplary embodiments. Therefore, the present invention is not intended to be limited to the embodiments shown, but should be given the broadest scope consistent with the principles and features described herein and limited only by the appended claims.

[0020] Unless expressly stated otherwise, terms used herein are intended to have plain and ordinary meanings that will be understood by those skilled in the art. The following definitions are intended to help the reader understand the invention, but are not intended to alter or otherwise limit the meaning of such terms unless specifically indicated. All publications referenced herein are incorporated by reference for the purpose of describing and disclosing formulations and processes described in those publications and that may be used in connection with the inventions currently described.

[0021] Those skilled in the art can recognize or confirm, by routine experimentation alone, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the claims.

[0022] For simplicity, this specification describes specific embodiments with respect to the use of specific methods. A person skilled in the art who has read this disclosure will see that the invention is not intended to be limited to specific uses and can be used in a wide range of implementations.

[0023] To clarify the disclosure rather than to limit it, the detailed description of the present invention is divided into subsections that describe or illustrate specific features, embodiments, or uses of the invention.

[0024] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom the embodiments relate.

[0025] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference.

[0026] The terms "optional" or "optional" indicate that the event or situation described thereafter may occur, but does not have to occur, and that the description includes both cases in which the event or situation may or may not occur.

[0027] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “and,” and “the” refer to multiple subjects unless the context clearly indicates otherwise. For example, a reference to the effect of “a compound” may refer to the effect of one or more compounds, and a reference to “a method” may include references to equivalent steps and processes known to those skilled in the art.

[0028] If a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range, and any other stated or intermediate values ​​within that stated range, are included within the present invention. If the stated range includes an upper and lower limit, the range excluding either of those limits is also included within the present invention.

[0029] The terms "nucleic acid base" or "base" are used interchangeably and refer to nitrogen-containing compounds that form nucleosides, which are then components of nucleotides. The five main or naturally occurring nucleic acid bases are adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). Other nucleic acid bases, such as synthetic or modified nucleic acid bases, are included herein and are described in detail below.

[0030] A "nucleotide" refers to a compound containing a nucleoside and a linking group, usually a phosphate linking group. Nucleotides include both natural and modified nucleotides.

[0031] A "motif" refers to a region or subsequence within an oligonucleotide or polypeptide sequence that has specific functional or biological significance. Examples of motifs include nucleic acid base sequences within oligonucleotides such as DNA or RNA, which are recognized by DNA or RNA-binding proteins or by functional RNA (e.g., miRNA). Other examples of motifs include nucleic acid base sequences within RNA that perform a specific function, or amino acid sequences within a polypeptide that perform a specific function. A motif may also refer to a target site of a modulator on a DNA, RNA, or polypeptide target.

[0032] The terms "nucleic acid sequence," "nucleic acid base sequence," "nucleotide sequence," or simply "sequence" are used interchangeably and refer to the sequence of nucleic acid bases on a nucleic acid molecule or oligonucleotide. A nucleic acid molecule can refer to a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.

[0033] As used herein, the term “gene” refers to a DNA sequence that is transcribed into mRNA and subsequently translated into polypeptides, and / or a DNA sequence that is transcribed into functional RNA that is not translated into polypeptides.

[0034] As used herein, the term “RNA” refers to a ribonucleic acid molecule. The transcription process first results in the formation of precursor mRNA (premRNA). In the case of protein-coding genes, premRNA is then processed into mature mRNA by splicing, which removes introns and adds a 5' cap and poly(A) tail. Mature mRNA is used as a template by ribosomes for translation into polypeptides. As used herein, the term “RNA” includes premRNA (sometimes also called heteronuclear RNA), mature mRNA, and RNA at any stage of processing. As used herein, the term “RNA” includes coding RNA that is translated into polypeptides, and non-coding RNA (e.g., miRNA, tRNA, rRNA, etc.).

[0035] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably and refer to polymers of two or more amino acids.

[0036] An "oligonucleotide" refers to a polymer containing two or more nucleotides.

[0037] An "allele," also called a "mutant" or "polymorphism," refers to one of at least two different nucleotide sequence variations at a given location (locus) in the genome. Therefore, a specific allele of a polymorphic site refers to a particular version of the sequence relating to that site. A "mutant" or "polymorphism" can also refer to a specific allele of a polymorphic site that differs from that of a reference genome.

[0038] A “polymorphic marker,” also called a “polymorphic site” or simply a “marker,” refers to a genomic site that has at least two sequence variants or at least two alleles. Therefore, a genetic association with a polymorphic marker refers to an association with at least one specific allele of that polymorphic marker. A “marker” can also refer to a specific allele of a polymorphic marker. A polymorphic marker can refer to any type of sequence variant found in the genome, including, but is not limited to, single nucleotide polymorphisms (SNPs), curated SNPs (cSNPs), insertions, deletions, copy number variations (CNVs), codon extensions, methylation states, translocations, duplications, repeat extensions, rearrangements, polynucleotide polymorphisms, splice variants, microsatellite polymorphisms, etc. A “marker” can also refer to a “biomarker.”

[0039] A single nucleotide polymorphism (SNP) is a type of DNA variation in which a single nucleotide at a specific location in the genome differs between two or more individuals or populations. Most SNPs have two alleles, in which case an individual is either homozygous for one allele at the polymorphic site or heterozygous for both alleles.

[0040] An "insertion" or "deletion" is a variant that has additional or fewer nucleotides compared to a reference DNA sequence.

[0041] A "microsatellite" is a type of polymorphic marker that consists of multiple small repeats of a base, each having a length of 2 to 8 nucleotides.

[0042] The term “associated with” can mean “in relation to,” or “correlated with,” or “in linkage disequilibrium with,” or “functionally related with,” or any combination of these terms, and can be used interchangeably. “Linkage disequilibrium” refers to the non-random association of alleles at different loci in a given population.

[0043] "Susceptibility" refers to an individual's tendency, trait, or risk of developing a particular phenotype (e.g., trait or disease), or their ability to be more or less resistant to developing a particular phenotype. This term encompasses reduced susceptibility to disease, reduced risk of disease, or protection against disease. It also encompasses increased susceptibility to disease, or increased risk of developing disease.

[0044] The term "and / or" indicates "one or the other or both." In other words, it means that both or either of the items are involved.

[0045] The term "biomarker" refers to biomolecules such as proteins, polypeptides, small molecules, metabolites, or nucleic acid sequences that are associated with a phenotype, such as a disease, and whose measurement can be used to determine susceptibility to a disease, the prognosis of a disease, the diagnosis of a disease, or the response to treatment for a disease.

[0046] The term "lookup table" refers to a table that links one form of data to another, or one or more forms of data to a predicted outcome (e.g., trait, disease, or other phenotype). A lookup table may include information about the expression or activity levels of one or more targets or one or more polymorphic markers, and information about the correlation between the expression or activity levels of one or more targets or alleles of polymorphic markers and a particular phenotype (e.g., trait or disease).

[0047] A "computer-readable medium" is a medium for storing information that is accessible through a custom-made or commercially available computer interface. Some examples of computer-readable mediums include, but are not limited to, optical storage media, magnetic storage media, memory, punch cards, or other commercially available media.

[0048] A "nucleic acid sample" refers to a DNA or RNA sample obtained from an individual. Nucleic acid samples can be obtained from any source containing DNA or RNA, such as blood, saliva, tissue samples, cerebrospinal fluid, or amniotic fluid.

[0049] The term "sample" generally refers to any sample, such as a biological sample obtained from an individual.

[0050] "Subject" may be interchangeable with "patient" or "individual," and unless otherwise indicated, refers to a living multicellular vertebrate, including both humans and non-human mammals.

[0051] "Subjects requiring treatment" may include subjects having a disease, disorder, or condition that would respond to treatment with the compounds disclosed herein. For example, "Subjects requiring treatment" as described herein may include subjects having diseases such as cancer, autoimmune diseases, inflammatory diseases, or infections.

[0052] The term "therapeutic agent" refers to a drug that can be used to prevent, treat, or improve symptoms associated with a disease.

[0053] The terms “response to treatment,” “response to treatment,” or “response to modulator administration” refer to the outcome of any type of treatment on an individual, including beneficial, neutral, and adverse effects.

[0054] The term "therapeutic dose" refers to the amount of a therapeutic agent, administered alone or in combination with one or more additional therapeutic agents, that elicits a desired response, such as a reduction in disease-related signs and symptoms. Often, a therapeutic dose provides the desired response without causing serious side effects in the recipient. The effective dose of a drug administered to a particular subject in a particular case is not always effective in treating the conditions / diseases described herein, even if such a dose is considered therapeutically effective by those skilled in the art.

[0055] The term “modulator” refers to a compound that affects (also called “regulates”) the signaling, activity, or expression of a polypeptide or nucleic acid sequence, and includes both activators and inhibitors. A modulator that increases or upregulates the signaling, activity, or expression of a polypeptide or nucleic acid sequence is called an “activator.” A modulator that inhibits, reduces, decreases, or downregulates the signaling, activity, or expression of a polypeptide or nucleic acid sequence is called an “inhibitor.” “Regulation” refers to the act of regulating as defined above, which can be performed using a modulator. Unless otherwise specified, “regulate” or “modulate” refers to the act of regulating as defined above, and includes both increasing or upregulating the signaling, activity, or expression of a polypeptide or nucleic acid sequence, as well as inhibiting, reducing, decreasing, or downregulating the signaling, activity, or expression of a polypeptide or nucleic acid sequence.

[0056] The term "antisense modulator" refers to a modulator that affects the signaling, activity, or expression of at least one nucleic acid sequence through some form of complementary binding or hybridization to a nucleic acid molecule. Common forms of antisense modulators include, but are not limited to, antisense oligonucleotides (ASOs) and nucleic acids used in RNAi mechanisms for gene regulation, such as miRNAs, siRNAs, and small hairpin RNAs (shRNAs).

[0057] The terms "amplification" or "to amplify" refer to increasing the copy number of a nucleotide sequence. One example of amplification is a polymerase chain reaction, in which a sample containing a nucleotide sequence is brought into contact with a pair of oligonucleotide primers. The primers hybridize with the nucleotide sequence, extend under favorable conditions, and then dissociate from the nucleotide sequence. This process is repeated to increase the copy number of the nucleotide sequence. Other methods can be used for amplification, and these are known to those skilled in the art.

[0058] The term "isolated" refers to a purified, enriched, or concentrated collection of molecules. "Isolated" also refers to the act of enriching or concentrating a particular molecule, compound, or complex to increase its purity.

[0059] The term "tissue" refers to a collection of cells in an organism that perform a specific physiological function.

[0060] When used in the context of drugs, pharmaceuticals, or pharmaceutical compositions, the term “delivery” refers to the administration of a drug, pharmaceutical, or pharmaceutical composition to an assay mixture, cultured cells, animals, or human subjects or patients.

[0061] A "carrier," also known as a "vehicle" or "excipient," is one or more molecules used to assist in the delivery of one or more other molecules when used in the context of drugs, pharmaceuticals, or pharmaceutical compositions. Examples of carriers include, but are not limited to, gelatin, cellulose, cellulose derivatives, polyvinylpyrrolidone, starch, sucrose, and polyethylene glycol.

[0062] "Improvement" is a reduction in the severity of a disease, measured by at least one indicator of the disease. The indicator may be a symptom of the disease or a marker associated with the disease, and may be evaluated objectively or subjectively. In certain embodiments, "improvement" may mean slowing, stopping, or reversing the progression of the disease.

[0063] "Dose" refers to a specific unit of a pharmaceutical composition provided for administration. In some embodiments, a dose can refer to a specific amount of a pharmaceutical composition administered over a period of time. A dose can refer to the total amount of pharmaceutical composition administered, or the amount of pharmaceutical composition administered per unit time.

[0064] Cancer refers to the abnormal growth of cells that proliferate in an uncontrolled manner and, in some cases, have a tendency to metastasize. The term "cancer" or "cancer(s)" includes, but is not limited to, astrocytoma, breast cancer, low-grade brain glioma, Burkitt lymphoma, cervical cancer, colorectal cancer (e.g., colon adenocarcinoma, rectal adenocarcinoma, etc.), esophageal cancer, stomach cancer (e.g., gastric adenocarcinoma, etc.), glioblastoma (e.g., glioblastoma multiforme, etc.), head and neck squamous cell carcinoma, leukemia (e.g., myeloid leukemia, etc.), liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, etc.), non-Burkitt lymphoma, medullary thyroid carcinoma, medulloblastoma, melanoma (e.g., cutaneous melanoma, uveal melanoma, etc.), mesothelioma, multiple myeloma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, paraganglioma, pheochromocytoma, prostate cancer, kidney cancer (e.g., clear cell renal carcinoma, etc.), retinoblastoma, rhabdomyosarcoma, and testicular cancer.

[0065] An "autoimmune disease" refers to a condition in which the body's immune system mistakenly identifies its own healthy tissues as foreign substances, attacks them, and causes inflammation that can affect various parts of the body. While autoimmune diseases are traditionally defined as being driven by components of adaptive immunity such as B lymphocytes and T lymphocytes, innate immunity can also play a significant role. Examples of autoimmune diseases include type 1 diabetes, graft-versus-host disease, hepatitis C virus-associated vasculitis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), juvenile idiopathic arthritis, primary Sjögren's disease, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, vasculitis, amyotrophic lateral sclerosis, alopecia areata, ankylosing spondylitis, autoimmune encephalitis, autoimmune hepatitis, Behçet's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, corneal transplantation, and dermatomyositis. Examples include, but are not limited to, Duchenne muscular dystrophy, Graves' disease, granulomatosis with polyangiitis, Guillain-Barré syndrome, Hashimoto's thyroiditis, hypomyositis with refractory dermatitis, inflammation of the intestines and enterotoxosis, multiple sclerosis, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, polymyositis, primary immune thrombocytopenia, scleroderma, sclerosing cholangitis, Sjögren's syndrome, Takayasu's arteritis, uveitis, and Wiscott-Aldrich syndrome. Autoimmune diseases are described in Zhang et al. (Zhang et al., European Journal of Inflammation, 19:1-7 (2021)), Graβhoff et al. (Graβhoff et al., Front.Immunol., 12:648408 (2021)), Klatzmann et al. (Klatzmann et al., Nat.Rev.Immunol., 15(5):283-294 (2015)), and Hartemann et al. (Hartemann et al., Lancet Diabetes Endocrinol., 1:295-305 (2013)), and these disclosures, along with the references cited therein, are incorporated herein by reference in their entirety.

[0066] "Inflammatory diseases," also known as "inflammatory disorders," "chronic inflammation," or "autoinflammatory diseases," refer to conditions in which the body's immune system attacks its own healthy tissues, resulting in recurrent episodes of frequent inflammation associated with fever. Inflammatory diseases are traditionally defined as being driven by components of innate immunity, such as cytokines, and other pro-inflammatory chemicals, in the absence of high titers of autoantibodies. However, autoimmune diseases and inflammatory diseases are part of a spectrum of diseases driven by the immune system, with purely autoimmune diseases and purely autoinflammatory diseases at either end of the spectrum, as well as a variety of diseases in between that are driven by both innate and adaptive immune components. Therefore, in some embodiments, the terms "autoimmune disease" and "inflammatory disease" can be used interchangeably. Examples of inflammatory diseases include, but are not limited to, acne, acute lung injury, adult-onset Still's disease, adenitis syndrome, aphthous stomatitis, atherosclerosis, Behçet's disease, beryllium-induced granulomatous inflammation, chronic relapsing multifocal osteomyelitis syndrome, Crohn's disease, osteoporosis, muscular dystrophy, obesity, osteitis syndrome, periodic fever, pharyngitis, pustulosis, rheumatoid arthritis, Still's disease, Sweet's syndrome, and synovitis. Inflammatory diseases can also include neuroinflammatory diseases. Inflammatory diseases are described in El-Shebiny et al. (El-Shebiny et al., The Egyptian Journal of Internal Medicine, 33:11 (2021)), Zhang et al. (Zhang et al., European Journal of Inflammation, 19:1-7 (2021)), and Klatzmann et al. (Klatzmann et al., Nat. Rev. Immunol., 15(5):283-294 (2015)), and these disclosures, along with the references cited therein, are incorporated herein by reference in their entirety.

[0067] An "infectious disease" refers to an illness caused by a virus, bacteria, fungus, or parasite. Examples of infectious diseases include, but are not limited to, chickenpox, the common cold, diphtheria, giardiasis, HIV, infectious mononucleosis, influenza, Lyme disease, malaria, measles, meningitis, mumps, poliomyelitis, pneumonia, Rocky Mountain spotted fever, rubella, salmonella infection, severe acute respiratory syndrome, sexually transmitted infections, herpes zoster, tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, and pertussis. st This information is found in Edition, Nov 16 (2019), Zhou et al. (Zhou et al., PLoS Pathog., 17(10):e1009858 (2021)), Sananez et al. (Sananez et al., The Journal of Infectious Diseases, 218(1):75-83 (2018)), and Sheng et al. (Sheng et al., Journal of Tropical Medicine, 2022:5071816 (2022)), and these disclosures, along with the references cited therein, are incorporated herein by reference in their entirety.

[0068] "Neurological inflammatory diseases" refer to diseases characterized by neurological damage caused by an immune response, and include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

[0069] Obesity is defined as an abnormal or excessive accumulation of fat that poses a health risk. A body mass index (BMI) of 25 or higher is considered overweight, and a BMI of 30 or higher is considered obese.

[0070] "Immune cells" refers to one or more specific types of immune cells, including basophils, dendritic cells, eosinophils, mast cells, monocytes, macrophages, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, B lymphocytes (B cells), T lymphocytes (T cells), cytotoxic T cells, effector T cells, memory T cells, CD4+ T cells, CD8+ T cells, and regulatory T cells (T). regs Examples include, but are not limited to, ), or tumor-infiltrating lymphocytes (TILs).

[0071] The term "cytokine" refers to one or more specific cytokines, including but not limited to chemokines (e.g., CCL1, CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17, CCL22, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), interleukins (e.g., IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-10, IL-13, IL-15, IL-19, IL-33, IL-35, etc.), lymphokines (e.g., IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, etc.), tumor necrosis factor (e.g., TNF-α, etc.), and TGF-β. The term "cytokine" includes both pro-inflammatory and anti-inflammatory cytokines.

[0072] Chemical substance definition This specification discloses novel chemical substances and their uses. Chemical substances may be described using terms known in the art, and are further discussed below.

[0073] The nomenclature used herein is based on the IUPAC systematic nomenclature or other nomenclature commonly used and understood by those skilled in the art.

[0074] Any open valence appearing on carbon, oxygen, sulfur, or nitrogen atoms in the structures described herein indicates the presence of hydrogen, unless otherwise indicated.

[0075] The term "substituent" refers to an atom or group of atoms that substitutes for a hydrogen atom on the parent molecule.

[0076] The term "substituted" indicates that the specified group has one or more substituents. Any group can have multiple substituents, and if a variety of possible substituents are provided, the substituents are independently selected and may or may not be the same. The term "unsubstituted" means that the specified group has no substituents. The term "optionally substituted" means that the specified group is either unsubstituted or substituted with one or more substituents independently selected from the group of possible substituents.

[0077] When indicating the number of substituents, the term "one or more" refers to the range from one substituent to the maximum possible number of substitutions, i.e., from the substitution of one hydrogen by a substituent to the substitution of all hydrogens.

[0078] The term "part" refers to an atom or group of chemically bonded atoms that are bonded to another atom or molecule by one or more chemical bonds, thereby forming a part of a molecule. For example, the variable A in equation (I). 1 , R 1 , and R 4 This refers to the part that is connected to the core structure of equation (I) by one or more covalent bonds.

[0079] As used herein, an asterisk "*", a plus sign "+", or a dotted line may be used to specify the bonding point of any radical group, moiety, and / or substituent.

[0080] The term "alkyl" refers to all isomerized forms of linear or branched saturated hydrocarbon groups. In certain embodiments, alkyl refers to linear or branched groups of 1 to 12, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 4, or 1 to 2 carbon atoms, respectively as used herein. 1-12 -alkyl, C 1-10 -alkyl, C 1-8 -alkyl, C 1-7 -alkyl, C1-6 -alkyl, C 1-4 -alkyl, or C 1-2 -Also called alkyl. Some specific examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl.

[0081] The term "alkylene" refers to the diradical of an alkyl group (e.g., -(CH2) n -, where n is an integer, for example, an integer between 1 and 20). An exemplary alkylene group is -CH2CH2-. Other examples of alkylene groups include methylene, ethylene, propylene, 2-methylpropylene, butylene, 2-ethylbutylene, pentylene, and hexylene.

[0082] The terms “halo,” “halogen,” and “halide” are used interchangeably herein and refer to fluoro, chloro, bromo, or iodine. One specific example of a halogen is fluoro.

[0083] The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom of the alkyl group is replaced by the same or a different halogen. Examples of haloalkyls include monofluoromethyl (-CH2F), difluoromethyl (-CHF2), trifluoromethyl (-CF3), and 2,2,2-trifluoroethyl (-CH2CF3). "Perhaloalkyl" refers to an alkyl group in which all hydrogen atoms of the alkyl group are replaced by the same or a different halogen.

[0084] As used herein, the term “heteroalkyl” refers to an “alkyl” group in which at least one carbon atom is replaced by a heteroatom (e.g., an O, N, or S atom). An example of a heteroalkyl group is an “alkoxy” group.

[0085] As used herein, the term "alkenyl" refers to an unsaturated linear or branched hydrocarbon having at least one carbon-carbon double bond, for example, a linear or branched group of 2 to 12, 2 to 10, 2 to 8, 2 to 7, 2 to 6, or 2 to 4 carbon atoms, where C is used as the term. 2-12 -Alkenil, C 2-10 -Alkenil, C 2-7 -Alkenil, C 2-6 -Alkenyl, or C 2-4 - Also known as alkenil

[0086] As used herein, the term "alkynyl" refers to an unsaturated linear or branched hydrocarbon having at least one carbon-carbon triple bond, for example, a linear or branched group of 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 carbon atoms, where C is used as the term. 2-12 -Alkinyl, C 2-10 -Alkinyl, C 2-8 -Alkinyl, C 2-6 -Alkinyl, or C 2-4 - Also known as alkinyl.

[0087] The terms "amine" and "amino" are derived from formula -NR a R c It refers to the base of, and in the formula, R a and R c R is independently a hydrogen, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, heterocyclyl, or another group. a and R c These can combine with the nitrogen to which they are bound to form heterocycloalkyl groups. The term "primary amino" refers to R a and R c This indicates a group in which both sides are hydrogen. The term "secondary amino" is R a is hydrogen, R c This indicates a group that is not hydrogen. The term "tertiary amino" is R a and R cThis indicates a group in which both atoms are non-hydrogen groups. Examples of secondary and tertiary amino groups include methylamino, ethylamino, propylamino, isopropylamino, phenylamino, benzylamino, dimethylamino, diethylamino, dipropylamino, and diisopropylamino.

[0088] The terms "alkoxy," "alkoxyl," or "-O-alkyl" refer to the alkyl groups defined above, which are bonded to the rest of the molecule by an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, and tert-butoxy. For example, if other parts, such as alkenyl, alkynyl, or heteroalkyl groups, are bonded to the rest of the molecule by an oxygen atom, they may be represented by -O-alkenyl, -O-alkynyl, and -O-heteroalkyl groups, respectively.

[0089] The term "ether" refers to two hydrocarbons covalently bonded together by an oxygen atom.

[0090] As used herein, the term "carbonyl" refers to the radical -C(O)-.

[0091] The term "oxo" refers to an oxygen atom that is double-bonded to carbon or another element.

[0092] As used herein, the term "carboxamide" refers to radical-C(O)NRR f This refers to R and R f These can be the same or different substituents. R and R f These may, for example, be independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.

[0093] As used herein, the terms "carboxy" or "carboxyl" refer to the radical -COOH or its corresponding salt, such as -COONa.

[0094] As used herein, the terms “amide,” “amido,” or “amidyl” are in morphological order. g C(O)N(R d )-,-R g C(O)N(R d )R e -, -C(O)NR d R e , or refers to the radical of -C(O)NH2, where R g , R d , and R e These include, for example, alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro, each independently.

[0095] The term "bicyclic ring system" can refer to two rings fused together via a common single or double bond (fused bicyclic ring system), two rings fused together via an arrangement of three or more common atoms (bridging bicyclic ring system), or two rings fused together via a common single atom (spironicyclic ring system). Bicyclic ring systems can be saturated, partially unsaturated, unsaturated, or aromatic. A bicyclic ring system may include rings in which one or more carbon atoms are substituted by identical or different heteroatoms selected from N, O, and S.

[0096] The term "cycloalkyl" refers to a saturated, bicyclic, or bridged (e.g., adamantyl) hydrocarbon group having 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 8, or 4 to 6 carbon atoms per ring, respectively, as used herein. 3-12 -Cycloalkyl, C 3-10 -Cycloalkyl, C 3-8 -Cycloalkyl, C 3-6 -Cycloalkyl, C 4-8 -Cycloalkyl, or C 4-6-Also called cycloalkyl. Unless otherwise specified, a cycloalkyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0097] The term “partially unsaturated carbocyclyl” refers to a cyclic hydrocarbon containing at least one double bond between ring atoms, where at least one ring of the carbocyclyl is non-aromatic. Partially unsaturated carbocyclyls can be characterized according to the number of ring carbon atoms. For example, a partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-10, 5-8, or 5-6 ring carbon atoms, and is therefore called a 5-14 member, 5-12 member, 5-10 member, 5-8 member, or 5-6 member partially unsaturated carbocyclyl, respectively. Partially unsaturated carbocyclyls may be in the form of monocyclic, bicyclic, tricyclic, bridging, spirocyclic, or other carbocyclic ring systems. Examples of partially unsaturated carbocyclyl groups also include cycloalkenyl groups and partially unsaturated bicyclic carbocyclyl groups. Unless otherwise specified, the partially unsaturated carbocyclyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is unsubstituted, i.e., it is unsubstituted.

[0098] The terms "cycloheteroalkyl" or "heterocycloalkyl" refer to a ring system that is saturated or partially unsaturated, non-aromatic, monocyclic or polycyclic (e.g., having two, three, or four fused rings), or a bridged ring system containing 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 8, or 4 to 6 carbon atoms per ring, where one or more of the ring-forming atoms are heteroatoms, such as N, O, or S. Cycloheteroalkyl or heterocycloalkyl groups may include spiro rings. Examples of monocyclic saturated cycloheteralkyl groups or heterocycloalkyl groups include azilidinyl, oxyranil, azetidinil, oxetanil, pyrrolidinyl, tetrahydrofuranil, tetrahydrothienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, tetrahydropyranil, tetrahydrothiopyranil, piperazinyl, morpholinil, thiomorpholinil, 1,1-dioxo-thiomorpholin-4-yl, azepanil, diazepanil, homopiperazinyl, and oxazepanil. Examples of bicyclic saturated cycloheteralkyl groups or heterocycloalkyl groups include 8-aza-bicyclo[3.2.1]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, and 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated cycloheteralkyl groups or heterocycloalkyl groups include dihydrofuryl, imidazolinyl, dihydro-oxazolyl, tetrahydropyridinyl, and dihydropyranyl. Furthermore, the definition of cycloheteralkyl or heterocycloalkyl includes moieties having one or more aromatic rings condensed (i.e., sharing a common bond) to a non-aromatic heterocycle, such as phthalimidyl, naphthalimidyl, and heterocyclic benzo derivatives, such as 2,3-dihydrobenzofuryl, 1,3-benzodioxole, and benzo-1,4-dioxane. Cycloheteralkyl or heterocycloalkyl groups having one or more condensed aromatic rings may be linked via either an aromatic or non-aromatic moiety.The definition of a cycloheteralkyl or heterocycloalkyl group also includes a moiety in which one or more ring-forming atoms are substituted with one or two oxo or sulfide groups. In some embodiments, a cycloheteralkyl or heterocycloalkyl group has 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 heteroatoms (e.g., N, O, or S). In some embodiments, a cycloheteralkyl or heterocycloalkyl group contains 0, 1, 2, 3, 4, or 5 double bonds. In some embodiments, a cycloheteralkyl or heterocycloalkyl group contains 0, 1, or 2 triple bonds.

[0099] The term "N-heterocycloalkyl" refers to a heterocycloalkyl group containing at least one nitrogen ring atom, where the bond site to the rest of the heterocycloalkyl radical molecule is via the nitrogen ring atom. Examples of N-heterocycloalkyls include 1,4-diazepanyl, hexahydropyrrolo[1,2-a]pyrazinyl, piperidinyl, piperazinyl, and pyrrolidinyl, where the bond site to the rest of the heterocycloalkyl radical molecule is via the nitrogen ring atom.

[0100] The term "cycloalkylene" refers to a cycloalkyl group that is unsaturated in one or more ring bonds.

[0101] The term "aryl" refers to a carbocyclic aromatic group. Typical aryl groups include phenyl, naphthyl, and anthracenyl. The term "aryl" also includes polycyclic ring systems (rings are "fused rings") having two or more carbocyclic rings in which two or more carbons are common to two adjacent rings, where at least one of the rings is aromatic, and the other rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocycloalkyl, and / or aryl. Unless otherwise specified, the aromatic ring may be substituted at one or more ring positions with, for example, halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos, nitros, sulfhydryls, iminos, amides, carboxylic acids, -C(O)alkyls, -CO2alkyls, carbonyls, carboxyls, alkylthios, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aryl or heteroaryl moieties, -CF3, -CN, etc. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogens, alkyls, hydroxyls, or alkoxyls. In certain other embodiments, the aromatic ring is unsubstituted, i.e., unsubstituted. In certain embodiments, the aryl group has a 6- to 10-membered ring structure.

[0102] The terms “heterocyclyl” and “heterocyclic group” refer to saturated, partially unsaturated, or aromatic cyclic systems, or combinations thereof, containing 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 8, or 4 to 6 carbon atoms per ring, where one or more of the ring-forming atoms are heteroatoms, such as N, O, or S. As used herein, “heterocyclyl” includes an “aryl” group in which at least one ring-forming atom is a heteroatom, such as N, O, or S. In some embodiments, the heterocyclyl group has 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 heteroatoms (e.g., N, O, or S). The number of ring atoms in a heterocyclyl group can be specified using Cx-y nomenclature, where x and y are integers specifying the number of ring atoms. For example, C 3-6 A heterocyclyl group refers to a saturated or partially unsaturated 3- to 6-membered ring structure in which one or more ring atoms are heteroatoms (e.g., N, O, or S). 3-6 " indicates that the heterocycle contains a total of 3 to 6 ring atoms, including any heteroatoms that are ring atoms. "Heterocyclyl" includes "N-heterocycloalkyl", "heterocycloalkyl", and a moiety containing one or more rings in which at least one ring-forming atom of at least one ring is a heteroatom such as N, O, or S.

[0103] The terms used herein are intended to be added to form chemically related combinations, such as “carboxyheterocycloalkyl,” “arylalkylheteroaryl,” or “aminoalkylheterocyclyl.” The definitions provided herein apply whether the terms in question appear alone or in combination. The last member of a combination is a radical bonded to the rest of the molecule. The other members of a combination are bonded to the bonded radical in reverse order with respect to their literal arrangement. For example, the combination “aminoalkylheterocyclyl” refers to a heterocyclyl radical substituted with an alkyl group substituted with an amino group.

[0104] The terms “compounds of this invention,” “compounds of this disclosure,” “compounds of this disclosure,” and “compounds of the present invention” refer to the compounds disclosed herein, as well as their stereoisomers, tautomers, solvates, and salts (e.g., pharmaceutically acceptable salts).

[0105] If the compounds of the present invention are solids, it will be understood by those skilled in the art that these compounds, as well as their solvates and salts, may exist in different solid forms, particularly different crystalline forms, all of which are intended to be within the scope of the present invention and specific formulas.

[0106] The term "chiral center" refers to a carbon atom bonded to four non-identical substituents. The term "chiral" refers to embodiments that cannot be superimposed on their mirror images, while the term "achiral" refers to embodiments that can be superimposed on their mirror images. Chiral molecules are optically active; that is, they have the ability to rotate the plane of plane-polarized light.

[0107] The definitions and conventions of stereochemistry used herein generally follow those of SP. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994. When describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule around its chiral center (or multiple center). Substituents attached to the chiral center under consideration are ranked according to the Sequence Rule of Cahn, Ingold, and Prelog. (Cahn et al., Angew. Chem. Inter. Edit., 5:385; errata 511 (1966)). The prefixes D and L or (+) and (-) are used to indicate the sign of the rotation of plane-polarized light by the compound, with (-) or L indicating that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory.

[0108] The compounds of the present invention may have one or more chiral centers and / or double bonds, and therefore may exist as stereoisomers such as geometric isomers, enantiomers, or diastereomers. Whenever a chiral center and / or double bond is present in the chemical structure, all stereoisomers associated with that chiral center and / or double bond are intended to be included in the present invention. The term “stereoisomer,” as used herein, includes all geometric isomers, enantiomers, or diastereomers, and mixtures thereof.

[0109] The compounds of the present invention may exist in the form of optically pure enantiomers or mixtures of enantiomers, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, racemates of diastereoisomers, or mixtures of racemates of diastereoisomers. Compositions comprising, essentially consisting of, or comprising enantiopure compounds are also contemplated herein, and such compositions may comprise, essentially consisting of, or may consist of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of formula (I) disclosed herein.

[0110] It is understood that a schematic representation of a chemical structure, such as a general chemical structure, encompasses all stereoisomeric forms of a given compound unless otherwise shown. Those skilled in the art will recognize that a structure may implicitly indicate a chiral center. Thus, throughout this specification, the compounds of this disclosure include all enantiomers, stereoisomers, racemic mixtures, and optically pure isomeric forms.

[0111] The term "pharmaceutically acceptable salt" refers to a salt that is not biologically or otherwise undesirable. Both acid addition salts and base addition salts are considered pharmaceutically acceptable salts.

[0112] The term "pharmaceutically acceptable acid addition salt" refers to pharmaceutically acceptable salts formed from inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid) as well as organic acids of the classes aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic acid, and sulfonic acid (e.g., formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid).

[0113] The term "pharmaceutically acceptable base addition salt" refers to a pharmaceutically acceptable salt formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperizine, piperidine, N-ethylpiperidine, and polyamine resins).

[0114] compound The present invention relates to a compound of formula (I), [ka] During the ceremony, A 1 However, hydrogen, halo, C 1-7 -alkyl, C2-7 -alkenyl, C 3-8 -cycloalkyl, aryl, or heterocyclyl, and R 1 and R 4 are each independently hydrogen, halo, C 1-7 -alkyl, C 2-7 -alkenyl, C 3-8 -cycloalkyl, aryl, or heterocyclyl, and C 1-7 -alkyl, C 2-7 -alkenyl, C 3-8 -cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted, a compound and its pharmaceutically acceptable salts, tautomers, N-oxides, and solvates.

[0115] Particular embodiments of the invention are compounds of formula (I), and their pharmaceutically acceptable salts.

[0116] All embodiments disclosed herein with respect to a particular A 1 , R 1 , R 2 , R 3 , and R 4 are understood to be combinable with any other embodiments disclosed herein with respect to another A 1 , R 1 , R 2 , R 3 , and R 4 .

[0117] Particular embodiments of the invention are compounds of formula (I) wherein A 1 is each independently hydrogen, halo, C 1-7 -alkyl, C 2-7 -alkenyl, C 3-8 -cycloalkyl, aryl, or heterocyclyl, and R 1 and R 4 are each independently hydrogen, halo, C 1-7 -alkyl, C2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 - Compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted. The present invention relates to pharmaceutically acceptable salts, tautomers, N-oxides, and solvates thereof.

[0118] A particular embodiment of the present invention is a compound of formula (I), wherein R 1 However, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, in particular hydrogen, methyl, or C 1-7 - Regarding alkyl compounds.

[0119] A particular embodiment of the present invention is a compound of formula (I), wherein R 4 However, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, in particular hydrogen, methyl, or C 1-7 - Regarding alkyl compounds.

[0120] A particular embodiment of the present invention relates to a compound of formula (I) in which one or more hydrogens are substituted with deuterium, thereby conferring useful properties to the compound, such as, for example, extending the residence time of the active drug species in plasma to achieve better efficacy and / or avoiding harmful side effects.

[0121] A particular embodiment of the present invention is a compound of formula (I), wherein A 1 However, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8-Cycloalkyl, aryl, or heterocyclyl, in particular, C 3-8 - Relating to compounds that are cycloalkyl, aryl, or heterocyclyl.

[0122] A particular embodiment of the present invention is a compound of formula (I), wherein A 1 but, [ka] Here, X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 -Alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C 3-8 -Cycloalkyl (e.g., cyclopropyl, cyclobutyl, etc.), halo (e.g., bromo, chloro, fluoro, or iodine), haloalkyl (e.g., monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, etc.), cyano, thiocyano, cyanato, thiocyanato, methoxy, hydroxy, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino. 0, 1, or more X substituents replace hydrogen with A 1 It is independently covalently bonded to one or more ring atoms, provided that the valence of X does not exceed the maximum valence of the ring atom (or number of ring atoms) to which it is bonded. R 2 However, independently, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -The invention relates to compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted.

[0123] A particular embodiment of the present invention is a compound of formula (I), wherein A 1 but, [ka] Selected from the group consisting of, X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 -Alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C 3-8 -Cycloalkyl (e.g., cyclopropyl, cyclobutyl, etc.), halo (e.g., bromo, chloro, fluoro, or iodine), haloalkyl (e.g., monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, etc.), cyano, thiocyano, cyanato, thiocyanato, methoxy, hydroxy, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino. 0, 1, or more X substituents replace hydrogen with A 1 It is independently covalently bonded to one or more ring atoms, provided that the valence of X does not exceed the maximum valence of the ring atom (or number of ring atoms) to which it is bonded. R 2 However, independently, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -The invention relates to compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted.

[0124] A particular embodiment of the present invention is a compound of formula (I), wherein A 1 but, [ka] Selected from the group consisting of R 2 However, independently, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -The invention relates to compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted.

[0125] A particular embodiment of the present invention is a compound of formula (I), wherein R 2 but, [ka] A group consisting of is selected, where X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 -Alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C 3-8 -Cycloalkyl (e.g., cyclopropyl, cyclobutyl, etc.), halo (e.g., bromo, chloro, fluoro, or iodine), haloalkyl (e.g., monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, etc.), cyano, thiocyano, cyanato, thiocyanato, methoxy, hydroxy, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino. 0, 1, or more X substituents replace hydrogen with R 2 This relates to a compound in which X is independently covalently bonded to one or more ring atoms, but the valence of X does not exceed that of the ring atom (or number of ring atoms) to which it is bonded.

[0126] A particular embodiment of the present invention is a compound of formula (I), wherein R 2However, this relates to compounds selected from the following group. [ka]

[0127] A particular embodiment of the present invention is a compound of formula (I), wherein A 1 but, [ka] And, X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 -Alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C 3-8 -Cycloalkyl (e.g., cyclopropyl, cyclobutyl, etc.), halo (e.g., bromo, chloro, fluoro, or iodine), haloalkyl (e.g., monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, etc.), cyano, thiocyano, cyanato, thiocyanato, methoxy, hydroxy, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino. 0, 1, or more X substituents replace hydrogen with A 1 It is independently covalently bonded to one or more ring atoms, provided that the valence of X does not exceed the maximum valence of the ring atom (or number of ring atoms) to which it is bonded. R 3 However, independently, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -The invention relates to compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted.

[0128] A particular embodiment of the present invention is a compound of formula (I), wherein R 3 but, [ka] A group consisting of is selected, where X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 -Alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.), C 3-8 -Cycloalkyl (e.g., cyclopropyl, cyclobutyl, etc.), halo (e.g., bromo, chloro, fluoro, or iodine), haloalkyl (e.g., monofluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, etc.), cyano, thiocyano, cyanato, thiocyanato, methoxy, hydroxy, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino. 0, 1, or more X substituents replace hydrogen with R 3 This relates to a compound in which X is independently covalently bonded to one or more ring atoms, but the valence of X does not exceed that of the ring atom (or number of ring atoms) to which it is bonded.

[0129] A particular embodiment of the present invention is a compound of formula (I), wherein R 2 However, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8 -Cycloalkyl, aryl, or heterocyclyl, in particular, C 3-8 - Relating to compounds that are cycloalkyl, aryl, or heterocyclyl.

[0130] A particular embodiment of the present invention is a compound of formula (I), wherein R 3 However, hydrogen, halo, C 1-7 -alkyl, C 2-7 -Alkenil, C 3-8-Cycloalkyl, aryl, or heterocyclyl, in particular, C 3-8 - Relating to compounds that are cycloalkyl, aryl, or heterocyclyl.

[0131] The specific compounds of formula (I) of the present invention are 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-2-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, and 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-Oxopyridazin-1-yl] [sopyridazine-1-yl]-2,2-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,6-difluoropiperidine-1-yl]-7,8- Dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3 -(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-3,5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-4-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-2,[5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,5-trifluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,6-trifluoropiperidine [1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridin-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,5,6-tetrafluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridin-3-carbonilicate, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(6-fluoroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazine-3-ol 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4,6-dimethylpyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5,6-dimethylpyrimidine-4-yl)piperidine-4-yl]pyridazin-3-one, 6-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]-3-methyl-1H-pyrimidine-2,4-dione, 2-[1 -(2,5-dimethylpyrazole-3-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methoxy-1-methylpyrazole-4-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methylsulfanylpyridine-3-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[6-(trifluoromethyl)pyridine-3-carbonyl]piperidine-4-yl]pyridazin-3-one, 2-[1-(5-bromofuran-2-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[3-(3-methylthiophen-2-yl)propanoyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2- [1-(6-propan-2-yloxypyridine-3-carbonyl)piperidine-4-yl]pyridazin-3-one, 2-[1-(3-chloro-5-fluorobenzoyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[1-[2-(2-chlorophenyl)acetyl]piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-fluoro-4-methoxybenzoyl)piperidine-3-one] [Zin-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-(2-methylphenoxy)acetyl]piperidine-4-yl]pyridazin-3-one, 2-[1-(5-chloro-2-methoxybenzoyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[1-[2-(2-chloro-6-fluorophenyl)acetyl]piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-di Methylpyrazole-1-yl)-2-[1-(4-propan-2-yloxybenzoyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-(4-fluorophenoxy)acetyl]piperidine-4-yl]pyridazin-3-one, tert-butyl4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-carboxylate, 2-[1-[2-(4-chlorophenoxy)acetyl]piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1,2,3,4-tetrahydronaphthalene-1-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1,3,5-trimethylpyrazole-4-yl)sulfonylpiperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methylimidazo[4,5-b]pyridine-2-yl) [Peridine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methylpyrazolo[1,5-a]pyrazine-4-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methyl-[1,2,4]triazolo[4,3-a]pyrazine-8-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(9-methylpurine-6-yl)piperidine-4-yl]pyridad Pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5-ethylthiophen-2-yl)sulfonylpiperidine-4-yl]pyridazin-3-one, 2-[1-(2,3-dihydro-1,4-benzodioxin-5-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5,6-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-7-yl)piperidine-4-yl]pyridazin Zin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-(1-pyrido[3,4-d]pyrimidine-4-ylpiperidine-4-yl)pyridazin-3-one, N-(3,4-dimethylphenyl)-4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-carboxamide, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-fluorophenyl)sulfonylpiperidine-4-yl]pyridazin-3-one, N-(2-chlorophenyl)-4-[3-(3,[5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-carboxamide, N-(2,6-difluorophenyl)-4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-carboxamide, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4-fluoro-2-methylphenyl)sulfonylpiperidine-4-yl]pyridazin-3-one, 4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl] -N-(2-methoxyphenyl)piperidine-1-carboxamide, 4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-N-[(4-fluorophenyl)methyl]piperidine-1-carboxamide, 2-[1-(2-chlorophenyl)sulfonylpiperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[1-(1,3-benzoxazole-2-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3 -one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1-methylbenzimidazole-2-yl)piperidine-4-yl]pyridazin-3-one, N-[(4-chlorophenyl)methyl]-4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-carboxamide, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-phenylethylsulfonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)- 2-(1-quinoline-2-ylpiperidine-4-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-(1-quinoline-4-ylpiperidine-4-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methylquinoxaline-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methoxyphenyl)sulfonylpiperidine-4-yl]pyridazin-3-one, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]quinoline-4-carbonitride, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4-fluoro-1,3-benzothiazole-2-yl)piperidine-4-yl]pyridazin-3-one, 2-[1-(1-benzofuran-2-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[1-(1,3-benzothiazole-6-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(isoquinoline-1-carbonyl)piperidine-4-yl]pyridadiz, Pyridazine-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5-pyridine-3-yl-1,2-oxazole-3-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-(1-methylindole-3-yl)acetyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(6-methoxy-1H-indole-2-carbonyl)piperidine-4-yl]pyridazin-3-one, 2-[1-[2-(1,2-benzoxazole-3-yl)acetyl]piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-pyrazole-1-ylbenzoyl)piperidine-4-yl]pyridazin-3-one, 3-[2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]-2-oxoethyl]-1,3-benzoxazole-2-one, 6-(3,5-dimethylpyrazole (Il-1-yl)-2-[1-(7-methoxy-1-benzofuran-2-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-phenyltriazole-4-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methoxyacetyl)piperidine-4-yl]pyridazin-3-one, 2-[1-(cyclohexa-3-en-1-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole- 1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(oxan-2-carbonyl)piperidine-4-yl]pyridazin-3-one, 2-[1-(4,4-difluorocyclohexanecarbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4,4,4-trifluorobutanoyl)piperidine-4-yl]pyridazin-3-one, 2-(1-cyclopropylsulfonylpiperidine-4-yl) -6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-N-(oxan-4-yl)piperidine-1-carboxamide, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1,3-thiazole-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5-fluoropyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]pyridine-4-carbonilicate, 6-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]pyridine-2-carbonilicate, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5-ethylpyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4-methoxy [Pyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-methyl-6-(trifluoromethyl)pyrimidine-4-yl]piperidine-4-yl]pyridazin-3-one, 2-[1-(5-bromopyrimidine-2-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5-methylthiophen-2-carbonyl)piperidine [-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-pyridine-3-ylacetyl)piperidine-4-yl]pyridazin-3-one, 2-[1-(2-cyclopropylpyrimidine-4-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[1-(5-cyclopropyl-1,3,4-thiadiazole-2-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3- On, 2-[1-(3-cyclopropyl-1,2,4-thiadiazole-5-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazine-3-one, 4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-N-(oxolan-2-ylmethyl)piperidine-1-carboxamide, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1,3,4-thiadiazole-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1-methyl-6-oxopyrimidine-4-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(pyridine-3-ylmethyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(1,2,4-oxadiazole-3-ylmethyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(pyridine-4-carbonyl) [Piperidin-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[[5-(trifluoromethyl)-1,3,4-oxadiazole-2-yl]methyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2,6-dimethylpyrimidine-4-yl)piperidine-4-yl]pyridazin-3-one, 2-[1-[(3,5-dimethyl-1,2-oxazol-4-yl)methyl]piperidine-4-yl]-6-(3,5-dimethylpyrazole-1 -yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[(3-propan-2-yl-1,2,4-oxadiazole-5-yl)methyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2,4-dimethyl-1,3-thiazole-5-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[(2-methyl-5,6-dihydro-4H-cyclopenta[c]pyrazole-3-yl )methyl]piperidine-4-yl]pyridazin-3-one, 2-[1-(6-cyclobutylpyrimidine-4-yl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-(trifluoromethyl)benzoyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[2-(2-fluorophenoxy)propanoyl]piperidine-4-yl]pyridazin-3-one, 2-[1-(3,4-dihydro-2H-chromen-2-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-(1-thieno[2,3-d]pyrimidine-4-ylpiperidine-4-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-(1-thieno[3,2-d]pyrimidine-4-ylpiperidine-4-yl)pyridazin-3-one, 2-[1-(3,4-dimethylphenyl)sulfonylpiper Lysine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]quinoline-3-carbonitride, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[(2-phenyl-1,3-thiazole-4-yl)methyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[(E)-3-phenylprop -2-enyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-[(2-methyl-1,3-oxazole-4-yl)methyl]piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-piperidine-4-ylpyridazin-3-one, 4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-N-(2-phenylethyl)piperidine-1-carboxamide, 2-[[1-(2, 3-Dihydro-1-benzofuran-5-ylsulfonyl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[5-(trifluoromethyl)pyridine-2-yl]azetidine-3-yl]methyl]pyridazin-3-one, 6-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]methyl]azetidine-1-yl]pyridine-3-carbonitrile, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[4-(trifluoromethyl)pyrimidine-2-yl]azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-7-yl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(1,6-dimethylpyrazolo[3,4-d]pyrimidine-4-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl) Pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[(1-imidazo[1,2-b]pyridazin-6-ylazetidine-3-yl)methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5,6,7,8-tetrahydroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5-fluoropyrimidine-4-yl)azetidine-3-yl]methyl]pyridazin-3-ol 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(2,5-dimethylpyrazolo[1,5-a]pyrimidine-7-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(7H-pyrrolo[2,3-d]pyrimidine-4-yl)azetidine -3-yl]methyl]pyridazin-3-one, 3-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]methyl]azetidine-1-yl]pyrazine-2-carbonitride, 2-[[1-(3-chloropyridine-4-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(7H-purine-6-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[3-(trifluoromethyl)-[1,2,4]triazolo[4,3-b]pyridazin-6-yl]azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5-methylpyrazolo[1,5-a]pyrimidine-7-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-, [[1-[(1-hydroxycyclohexyl)methyl]azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(1,2-dimethylimidazole-4-yl)sulfonylazetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[3-(trifluoromethyl)pyridine-2-yl]azetidine-3-yl]methyl]pyridazin-3-one, 2-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxop [ridazin-1-yl]methyl]azetidine-1-yl]pyridin-3-carbonitride, 2-[[1-(5-bromopyrimidine-2-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5-fluoropyrimidine-2-yl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(5-chloropyrimidine-2-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one Ridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[(1-thieno[2,3-d]pyrimidine-4-ylazetidine-3-yl)methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[6-(trifluoromethyl)pyrimidine-4-yl]azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[(1-thieno[3,2-d]pyrimidine-4-ylazetidine-3-yl)methyl]pyridazin-3-one, 2-[[1-( 6,7-dihydro-5H-cyclopenta[d]pyrimidine-4-yl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]methyl]azetidine-1-yl]-6,7-dihydro-5H-cyclopenta[b]pyridine-3-carbonitride, 2-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]methyl]azetidine-1-yl]-5,6,7,8-Tetrahydroquinoline-3-carbonitride, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[(1-hydroxycyclopentyl)methyl]azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(7-fluoroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(5-fluoro-1,3-benzoxazole-2-yl)azetidine-3-yl]methyl]py Ridazin-3-one, 2-[(1-benzylazetidine-3-yl)methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(1,2,5-thiadiazole-3-yl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[3-[[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]methyl]azetidine-1-yl]pyrido[1,2-a]pyrimidine-4-one, 6-(3,5-dimethylpyrazole-1 -yl)-2-[[1-(3-methylquinoxaline-2-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(quinoline-2-carbonyl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(isoquinoline-1-carbonyl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(quinoxaline-2-carbonyl)azetidine [3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(1-methylpyrazolo[3,4-d]pyrimidine-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(2-methylbenzoyl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(3-fluorobenzoyl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[2-(4-methylphenyl)acetyl]azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(3,4-dihydro-1H-isochromen-1-carbonyl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-[3-(trifluoromethyl)benzoyl]azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-[2-(4-chlorophenyl)acetyl]azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[[1-(2,4-dichlorobenzoyl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[[1-[2-(benzimidazole-1-yl)acetyl]azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3 ,5-dimethylpyrazole-1-yl)-2-[[1-(5-methyl-1-phenylpyrazole-4-carbonyl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(naphthalene-1-carbonyl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[(1-benzoylazetidine-3-yl)methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 2-[[1-(5-chloro-2-methoxy Selected from benzoyl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(4-phenylbenzoyl)azetidine-3-yl]methyl]pyridazin-3-one, 2-[[1-(2-chloro-6-fluorobenzoyl)azetidine-3-yl]methyl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, and pharmaceutically acceptable salts thereof.

[0132] The specific compounds of formula (I) of the present invention are 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-2-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, and 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-Oxopyridazin-1-yl] [sopyridazine-1-yl]-2,2-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,6-difluoropiperidine-1-yl]-7,8- Dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3 -(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-3,5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-4-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-2,5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,5-trifluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,6-trifluoropiperidine-1-yl]- 7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitride, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]-2,3,5,6-tetrafluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitride, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(6-fluoroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(6-fluoroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl) (Zol-1-yl)-2-[1-(4,6-dimethylpyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5,6-dimethylpyrimidine-4-yl)piperidine-4-yl]pyridazin-3-one, 6-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazin-1-yl]piperidine-1-yl]-3-methyl-1H-pyrimidine-2,4-dione, 2-[1-(2,5-dimethylpyrazole-3-carbony Selected from the group consisting of [(L)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methoxy-1-methylpyrazole-4-carbonyl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methylsulfanylpyridine-3-carbonyl)piperidine-4-yl]pyridazin-3-one, and pharmaceutically acceptable salts thereof.

[0133] The formulas of the compounds disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compound unless a specific stereoisomer, enantiomer, or epimer is indicated by the formula. The formulas of the compounds disclosed herein should be interpreted as encompassing their salts, esters, amides, or solvates.

[0134] In certain embodiments, compounds similar to the compound of formula (I) disclosed herein are provided herein. Similarities between low molecular weight compounds can be determined using methods well known in the art, such as deriving the Tanimoto index, Dice index, Cosine coefficient, or Soergel distance. Such methods are described in Bajusz et al. (Bajusz et al., Journal of Cheminformatics, 7: Article number 20 (2015)), which, along with the references cited therein, are incorporated herein in their entirety. In some embodiments, compounds having a Tanimoto index of at least 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00 compared to the compound of formula (I) disclosed herein are provided herein.

[0135] The compounds of formula (I) disclosed herein can be developed and produced by any number of methods known to those skilled in the art (see below for specific methods).

[0136] Target, target region, target segment, and nucleic acid sequence In certain embodiments, the compounds of formula (I) disclosed herein may target nucleic acids, the target nucleic acid being RNA or a portion thereof that encodes IL-2. RNA-targeted small molecules are useful because they combine the advantages of small molecule drugs (see background art) with the advantages of targeted RNA, enabling, for example, an extended targeting range that includes non-coding regulatory RNA beyond protein-coding genes, as well as the targeting of “drug-unavailable” proteins that lack deep binding pockets. In certain embodiments, the target nucleic acid may comprise a nucleic acid sequence encoding IL-2, including, but not limited to, RefSeq accession number NC_000004.12 (incorporated herein as SEQ ID NO: 1), RefSeq accession number NM_000586.4 (incorporated herein as SEQ ID NO: 2), or reverse complements thereof, cleaved from nucleotides 122451470 to 122456725, where the adenine base (A) is interchangeable with the uracil base (U). In some embodiments, the compounds of formula (I) disclosed herein may also target certain other RNA transcript variants encoding IL-2 or a portion thereof.

[0137] In certain embodiments, a compound of formula (I) disclosed herein may target at least one target region within a target nucleic acid. The target region is a structurally or functionally defined region of the target nucleic acid. Examples of target regions include, but are not limited to, exons, introns, exon-intron junctions, intron-exon junctions, exon-exon junctions, 3' untranslated regions (3'UTR), 5' untranslated regions (5'UTR), translation start regions, translation termination regions, 5' donor splice sites, 3' acceptor splice sites, start codons, upstream open reading frames (ORFs), repeat regions, hexanucleotide repeat extensions, splice enhancer regions, exon splicing enhancers (ESEs), splice suppressor regions, exon splicing silencers (ESSs), intron splicing enhancers (ISEs), intron splicing silencers (ISSs), RNA destabilization motifs, RNA stabilization motifs, miRNA binding sites, RNA-binding protein (RBP) sites, or other defined nucleic acid regions. Information regarding such structurally or functionally defined target regions in RNA, such as IL-2 RNA, may be obtained in part from accession numbers from databases such as NCBI, GENBANK, and ENSEMBL, as well as from the references therein, and such information in whole is incorporated herein.

[0138] The target region may include one or more overlapping or non-overlapping target segments. In certain embodiments, the target segments within the target region are less than 10,000, 5,000, 2,500, 1,000, 500, 250, 100, or 50 nucleotides in length. In some embodiments, the compounds of formula (I) disclosed herein may target one target segment within the target region. In some embodiments, the compounds of formula (I) disclosed herein may target two or more overlapping or non-overlapping target segments within the same target region. In other embodiments, the compounds of formula (I) disclosed herein may target two or more target segments originating from different target regions.

[0139] A suitable target segment may specifically include a structurally or functionally defined target region (e.g., a start codon or stop codon). Determining a suitable target segment may involve comparing the nucleic acid sequence of the target segment with other sequences across the entire transcriptome. For example, the BLAST algorithm can be used to identify similar regions between different sequences. This comparison can enable the selection of a unique target segment to develop compounds that specifically target the target nucleic acid while minimizing nonspecific targeting of other nucleic acids (i.e., off-target effects). In some embodiments, targeting involves determining at least one target segment within the target nucleic acid with which a compound can interact to produce a desired effect. The desired effect may be a decrease in the stability of the target nucleic acid, resulting in a decrease in RNA levels and corresponding decreases in encoded protein levels. The desired effect may be an increase in the stability of the target nucleic acid, resulting in an increase in RNA levels and corresponding increases in encoded protein levels. The desired effect may also be a change in the translation efficiency from the target nucleic acid, resulting in an increase or decrease in its encoded protein levels, but without a change in its RNA levels. The desired effect may also be a phenotypic change related to a change in the RNA level of the target nucleic acid or a change in the protein level encoded by the target nucleic acid.

[0140] In some embodiments, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is an increase in IL-2 RNA levels. In some embodiments, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is an increase in IL-2 protein levels. In yet other embodiments, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is a phenotypic change associated with an increase in IL-2 RNA or protein levels.

[0141] Accordingly, several embodiments provide compounds of formula (I) disclosed herein that can increase the expression or activity of IL-2. In one embodiment, a compound of formula (I) disclosed herein may target a nucleic acid encoding IL-2 and increase its transcription. In one embodiment, a compound of formula (I) disclosed herein may target a nucleic acid encoding IL-2 and stabilize it. In another embodiment, a compound of formula (I) disclosed herein may target a nucleic acid encoding IL-2 and increase its transcription. In yet another embodiment, a compound of formula (I) disclosed herein may target a nucleic acid encoding IL-2 and modulate its splicing, thereby increasing the expression or activity of IL-2.

[0142] In certain embodiments, an increase in IL-2 RNA or IL-2 protein can be achieved by targeting IL-2-related genes or pathways. Accordingly, in certain embodiments, compounds of formula (I) are provided herein that can increase IL-2 expression, activity, or signaling by targeting and increasing at least one gene or pathway that positively modulates or increases IL-2 expression, activity, or signaling. In other embodiments, compounds of formula (I) are provided herein that can increase IL-2 expression, activity, or signaling by targeting and decreasing the expression or activity of at least one gene or pathway that negatively modulates or inhibits IL-2 expression.

[0143] In certain embodiments, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is a reduction at the IL-2 RNA level. In other embodiments, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is a reduction at the IL-2 protein level. In yet another embodiment, the desired effect of using a compound of formula (I) disclosed herein to target at least one target segment within a target nucleic acid encoding IL-2 is a phenotypic change associated with a reduction in IL-2 RNA or protein levels.

[0144] Accordingly, several embodiments provide compounds of formula (I) disclosed herein, or conjugates and complexes thereof, which can inhibit or reduce the expression or activity of IL-2. In one embodiment, a compound of formula (I) disclosed herein, or a conjugate and complex thereof, may target a nucleic acid encoding IL-2 and inhibit its transcription. In one embodiment, a compound, or a conjugate and complex thereof, may target a nucleic acid encoding IL-2 and degrade or destabilize it. In one embodiment, a compound, or a conjugate and complex thereof, may target a nucleic acid encoding IL-2 and inhibit its translation. In yet another embodiment, a compound, or a conjugate and complex thereof, may target a nucleic acid encoding IL-2 and modulate its splicing, thereby reducing the expression or activity of IL-2.

[0145] In certain embodiments, inhibition or reduction of IL-2 RNA or IL-2 protein can be achieved by targeting IL-2-related genes or pathways. Accordingly, compounds of formula (I), or conjugates and complexes thereof, are provided herein that, in certain embodiments, can reduce or inhibit IL-2 expression, activity, or signaling by targeting and inhibiting at least one gene or pathway that positively modulates or increases IL-2 expression, activity, or signaling. In other embodiments, compounds of formula (I), or conjugates and complexes thereof, are provided herein that can reduce or inhibit IL-2 expression, activity, or signaling by targeting and increasing the expression or activity of at least one gene or pathway that negatively modulates or inhibits IL-2 expression.

[0146] Conjugates and complexes In certain embodiments, a compound of formula (I) disclosed herein may be conjugated to, bound to, or complexed with at least one other molecule, such as a peptide or polypeptide, antibody, lipid, sugar, nucleotide or oligonucleotide, other polymer, cleavage agent, transporter, insertor, molecular beacon, hybridization-induced crosslinking agent, lipophilic agent, or hydrophilic agent. These conjugates or complexes can provide the compound with many benefits, including, but not limited to, increased efficacy or activity, improved delivery to specific tissues or cells, increased oral bioavailability or absorption, enhanced cellular uptake, reduced toxicity, resistance to nuclease degradation, increased half-life or residence time, enhanced pharmacodynamic or pharmacokinetic properties, and / or improved selectivity for a specific target. Certain embodiments may include combinations of one or more of the conjugates or complexes described herein.

[0147] In some embodiments, the compounds of formula (I) disclosed herein are conjugated to or complexed with proteins or other polyamides, amines, or similar molecules. In other embodiments, the compounds of formula (I) disclosed herein are conjugated to or complexed with lipids, phospholipids, cholesterol or thiocholesterol, cholic acid, aliphatic chains, hexylamino-carbonyl-oxycholesterol, or other similar molecules. In yet another embodiment, the compounds of formula (I) disclosed herein are conjugated to or complexed with another organic molecule, such as an ether or thioether, a steroid, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, adamantane acetate, palmityl, fluorescein, rhodamine, coumarin, a dye or other marker molecule, or another polymer (e.g., polyethylene glycol). In other embodiments, the compound of formula (I) disclosed herein is conjugated to or complexed with another compound having special electromagnetic or optical properties, such as a photo-labile protecting group.

[0148] In some embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with other drugs or agents described herein that are used to treat, prevent or improve diseases such as cancer, autoimmune diseases, inflammatory diseases, or infections. In other embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with other drugs, for example, other drugs that relieve pain or other symptoms or improve uptake or delivery, for example, blood diluents (e.g., aspirin, warfarin), anti-inflammatory and analgesic agents (e.g., COX inhibitors, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, pranoprofen, carprofen, indomethacin, folinic acid, typrophenic acid, diclofenac, niflumic acid, diazepine or benzodiazepine, barbiturate), or antimicrobial agents, antiviral agents, antibiotics, or other drugs that promote at least one benefit in a therapeutic setting, including therapeutic efficacy, symptom relief, drug resistance, or side effect adjustment.

[0149] In some embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with antibodies or monobodies that recognize specific antigens characteristic of a particular cell type(s) (e.g., cancer cells, or immune cells such as T cells or NK cells) or tissue (i.e., to form an antibody-drug conjugate), thereby enabling the delivery of the compounds of formula (I) to a particular cell type(s) or tissue. In some embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with nanobodies or nanocarriers, thereby enabling targeted delivery of the compounds of formula (I) to a particular cell type(s) or tissue. In other embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with another agent that facilitates transport across the cell membrane or transport across the blood-brain barrier. In some embodiments, the compounds of formula (I) disclosed herein are conjugated or complexed with one or more GalNAc residues, which are recognized by asialoclycoprotein receptors, resulting in efficient uptake into cells. In some embodiments, the compound of formula (I) disclosed herein is conjugated or complexed with a protein, antibody, or nanobody that binds to a protein or receptor present on T cells, such as PD-1, the T cell receptor, IL-2R, CD3, CD4, CD8, CD25, CD122, or CD132. In other embodiments, the compound of formula (I) disclosed herein is conjugated or complexed with a protein, antibody, or nanobody that binds to a protein or receptor present on NK cells, such as CD4, CD16, CD56, CD107a, CD161, CD314, CD335, or NKG2D.

[0150] In some embodiments, the conjugate described herein is a prodrug, which, after administration, is converted (e.g., by enzymes in the body) into a pharmacologically active drug(s), such as a compound of formula (I) disclosed herein. Prodrugs are useful, for example, to improve stability, improve solubility, increase permeability, increase drug absorption, extend the duration of action, or achieve selective transport of a pharmacologically active drug(s). Currently, about 10% of drugs approved worldwide are administered as prodrugs. Prodrugs are described by Hajnal et al. (Hajnal et al., Acta Medica Marisiensis, 62(3):356-362 (2016)), Rautio et al. (Rautio et al., Nature Reviews Drug Discovery, 17:559-587 (2018)), and Rautio et al. (Rautio et al., Wiley-VCH Verlag GmbH&Co.KGaA, Prodrugs and Targeted Delivery: Towards Better ADME Properties, Volume 47 (2011)), and these, along with the references cited therein, are incorporated herein by reference in their entirety.

[0151] In some embodiments, the compounds of formula (I) disclosed herein can be conjugated to ribonucleases such as RNase L to induce degradation of target RNA. Such divalent compounds are known as RIBOTACs (ribonuclease-targeted chimeras) and are described by Dey et al. (Dey et al., Cell Chemical Biology, 26(8):1047-1049 (2019)), which, along with the references cited therein, are incorporated herein by reference. In other embodiments, the compounds of formula (I) disclosed herein can be conjugated to ligands of E3 ubiquitin ligases to induce ubiquitination and subsequent proteasome degradation of target proteins. Such divalent compounds are known as PROTACS (protein-degrading targeted chimeras) and have been described by Toure et al. (Toure et al., Angew. Chem. Int. Ed., 55:1966-1973 (2016)), which, along with the references cited therein, are incorporated herein by reference.

[0152] Pharmaceutical composition and delivery method Some embodiments of the present invention provide pharmaceutical compositions or medicinal products comprising a compound of formula (I) disclosed herein, or its conjugate or complex, together with a pharmaceutically acceptable carrier, diluent, and / or other substance. Another embodiment relates to a method of using a compound of formula (I) disclosed herein, or its conjugate or complex, to prepare such a pharmaceutical composition or medicinal product. The carrier, diluent, and / or other substance of the pharmaceutical composition must be compatible with the other components of the composition and “acceptable” in the sense that it is not harmful to its recipient. In one embodiment, the carrier, diluent, and / or other substance is therapeutically inert. The terms “pharmaceutical composition” and “medicinal product” are used interchangeably herein.

[0153] Pharmaceutical compositions or medicinal products are formulated, administered, and given according to good medical practice. A list of common practices is provided by Wu & Chen (US2018 / 0112272A1), and the entire text is incorporated herein by reference therein. In some embodiments, pharmaceutical compositions or medicinal products may include formulations prepared by mixing at least one compound of formula (I) disclosed herein, or its conjugate or complex, with a carrier or excipient. Suitable carriers and excipients are described in detail by Ansel et al. (Ansel et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004), Lippincott, Williams & Wilkins, Philadelphia), Gennaro et al. (Gennaro et al., Remington: The Science and Practice of Pharmacy (2000), Lippincott, Williams & Wilkins, Philadelphia), and Rowe et al. (Rowe et al., Handbook of Pharmaceutical Excipients (2005), Pharmaceutical Press, Chicago), and these, along with the references cited therein, are incorporated herein by reference.

[0154] In some embodiments, a pharmaceutical composition or drug may contain at least one compound of formula (I) disclosed herein, or a conjugate or complex thereof, together with one or more substances common in pharmaceutical preparations, such as adjuvants, anesthetics, antioxidants, buffers, carriers, colorants, diluents, emulsifiers, flavorings, flow enhancers, lubricants, masking agents, opaquing agents, fragrances, pH adjusters, preservatives, processing aids, salts for altering osmotic pressure, solubilizers, stabilizers, surfactants, suspending agents, sweeteners, and wetting agents. The pharmaceutical composition or drug may also contain one or more substances to improve the appearance of the pharmaceutical product and / or to assist in its manufacture. The pharmaceutical composition or drug may also contain further therapeutically valuable substances. For example, the pharmaceutical composition or drug may contain a mixture of two or more compounds, each therapeutically beneficial, at least one of which is a compound of formula (I) disclosed herein, or a conjugate or complex thereof.

[0155] In some embodiments, the aforementioned pharmaceutical compositions or pharmaceuticals are prepared in unit dosage forms for ease of administration and uniformity of dosage. As used herein, unit dosage forms refer to physically distinct units suitable as unit doses, each unit containing a predetermined amount of the active ingredient (i.e., a compound of formula (I) disclosed herein, or its conjugate or complex) calculated to work in conjunction with a carrier and / or other substance in the pharmaceutical composition to produce a desired therapeutic effect. Examples of such unit dosage forms include tablets (including scored or coated tablets), capsules, pills, powder packets, cachets, suppositories, injectable solutions or suspensions, and multiple divisions thereof.

[0156] The dosage of the compound of formula (I) disclosed herein, or its conjugate or complex, can vary within a wide range and should be adjusted to the individual requirements of each case. For example, in some embodiments, the daily dose may range from about 0.01 mg to about 1000 mg per person for orally administered compounds, but may exceed the upper and lower limits as needed.

[0157] In certain embodiments, the pharmaceutical composition or drug may be administered by any preferred means, including intraintestinal, epidural, inhalation, intra-arterial, intracerebral, intraventricular, intradermal, intrafocal (if local treatment is desired), intramuscular, intranasal, intraocular, intraperitoneal, intrapulmonary, intrathecal, intravenous, intravitreous, oral, parenteral, transdermal, rectal, subcutaneous, topical (including buccal and sublingual), transdermal, transmucosal, and vaginal administration.

[0158] In some embodiments, the pharmaceutical composition or drug may be in any form convenient for administration, such as capsules, conjugates, creams, crystals, dispersions, elixirs, emulsions, nanoparticles, ointments, gels, patches, pills, powders, solutions, sprays, suppositories, suspensions, syrups, or tablets.

[0159] The following are examples of pharmaceutical compositions suitable for different routes of administration and methods for their preparation. In certain embodiments, pharmaceutical compositions for oral administration in liquid form, such as suspensions, syrups, elixirs, emulsions, and solutions, may include liquid carriers or diluents such as water, glycols, oils, and alcohols. In other embodiments, pharmaceutical compositions for oral administration in solid form, such as powders, pills, capsules, and tablets, may include solid carriers such as sugars (e.g., mannitol, lactose, sucrose, glucose, sodium saccharin, etc.), magnesium, carbonates, kaolin, diluents, lubricants (e.g., silicon dioxide, stearic acid, magnesium stearate, talcum, etc.), binders, and disintegrants that facilitate the dissolution of the solid (e.g., starch, clay, cellulose, aligning, gum, and polymers, etc.). Tablets and capsules are the most advantageous forms of oral administration because they are easy to administer, in which case solid pharmaceutical carriers are naturally used.

[0160] In some embodiments, a pharmaceutical composition administered parenterally, for example, in the form of an injectable solution or suspension, may contain a carrier such as sterile water, but may also contain other components, for example, to aid solubility. Injectable solutions can be prepared with a carrier containing physiological saline, glucose solution, or a mixture of physiological saline and glucose solution. Injectable solutions may be formulated in oil for sustained action. Suitable oils for this purpose include, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long-chain fatty acids, and mixtures of these and other oils. Injectable suspensions can be prepared using a suitable liquid carrier, suspending agent, etc. In certain embodiments, a pharmaceutical composition for parenteral administration may be a sterile solution, emulsion, or suspension prepared from a solid or lyophilized form before administration. In other embodiments, the pharmaceutical composition may contain certain adjuvants, anesthetics, buffers, or wetting agents to promote more effective distribution of the composition, facilitate administration of the composition, or improve the patient's response or health condition.

[0161] A particular embodiment provides a pharmaceutical composition for transdermal administration (e.g., transdermal or topical), wherein the carrier optionally comprises a penetration enhancer and / or a suitable humectant, and optionally, a suitable additive of any nature, which does not cause significant adverse effects on the skin, in small proportions. The additive can facilitate administration via the skin and / or can help prepare the desired composition. The pharmaceutical composition can be administered in various ways, such as a transdermal patch, spot-on, cream, gel, or ointment.

[0162] Some embodiments provide pharmaceutical compositions that can be administered transmucosally, such as in the form of a spray or suppository. Other embodiments provide pharmaceutical compositions that can be administered by nasal administration (e.g., inhalation) and / or via an aerosol delivery device such as an atomizer, nebulizer, or vaporizer. The aerosol delivery devices referred to herein, and other aerosol delivery devices, are well known to those skilled in the art and are included in the various embodiments herein.

[0163] Certain embodiments of the present invention provide methods for delivering compounds of formula (I) disclosed herein, or complexes, conjugates, or pharmaceutical compositions thereof, to cells, tissues, animals, or human subjects. In some embodiments, compounds of formula (I) disclosed herein, or complexes, conjugates, or pharmaceutical compositions thereof, are delivered using one of the above-described routes of administration or other routes of administration known to those skilled in the art. In some embodiments, compounds of formula (I) disclosed herein, or complexes, conjugates, or pharmaceutical compositions thereof, are delivered via targeted methods that directly introduce or direct the compounds, or complexes, conjugates, or pharmaceutical compositions thereof, to specific cells or tissues involved in disease, such as cancerous cells, tumors, or immune cells. Manish and Vimukta (Research Journal of Chemical Sciences, 1(2):135-138 (2011)) describe several methods for targeted drug delivery, which, together with the references cited therein, are incorporated herein by reference in their entirety.

[0164] In some embodiments, the delivery method of the compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, includes naked delivery to cells, animals, or human subjects. In some embodiments, the delivery method is electroporation or permeabilization. In other embodiments, the delivery method includes the use of liposome-based or amine-based transfection reagents. In other embodiments, the delivery method includes other forms of lipid-mediated transport. In yet another embodiment, the delivery method includes the use of membrane fusion. In yet another embodiment, the delivery method includes the use of a colloid or nanoparticle solution containing polymer particles. The nanoparticles may have specific properties (such as electromagnetic properties) that help target a particular region for delivery or otherwise facilitate delivery. In yet another embodiment, the delivery method includes the use of chemical-mediated transport, including the use of calcium phosphate. In yet another embodiment, the delivery method includes peptide-mediated transport, including the use of polylysine. In yet another embodiment, the delivery method includes the use of endocytosis. In yet another embodiment, the delivery method may include direct microinjection into cells. These delivery methods can be used with or without the aforementioned complexes or conjugates of the compound of formula (I) disclosed herein. Certain complexes or conjugates can improve the delivery rate or stability of compounds and are included in these embodiments. Other delivery methods are known to those skilled in the art and include standard practices incorporated into certain embodiments herein.

[0165] Analysis of the activity of compounds and pharmaceutical compositions Compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions, may have variable activity, as defined, for example, by a percentage increase in target RNA level, a percentage increase in target protein level, or a percentage increase in the activity of the target protein. In some embodiments, the target is IL-2. In certain embodiments, an increase in IL-2 RNA levels, including but not limited to RNA from the transcription of the IL-2 gene and RNA translated into IL-2 protein, indicates an increase in IL-2 expression. In certain embodiments, an increase in levels of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 indicates an increase in IL-2 expression. In some embodiments, an increase in levels of IL-2 protein indicates an increase in IL-2 expression. In certain embodiments, an increase in levels of one or more IL-2 proteins, which are the translation products of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 disclosed herein, indicates an increase in IL-2 expression. In some embodiments, an increase in IL-2 protein production by immune cells such as Jurcut cells, CD4+ T cells, CD8+ T cells, dendritic cells, or NK cells indicates an increase in IL-2 expression.

[0166] In other embodiments, an increase in the activity of an IL-2 protein, which is the translation product of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 herein, indicates an increase in IL-2 activity. IL-2 activity refers to one or more activities typically performed by the IL-2 transcripts or proteins described herein, such as binding to IL-2R or activating signaling pathways such as the JAK / STAT5, PI3K / AKL / mTOR, or MAPK pathways. In certain embodiments, a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, may selectively target one or more specific IL-2 transcript variants and the proteins encoded thereby, increasing their levels or activity, and such increase in the level or activity of one or more IL-2 transcript variants or proteins indicates an increase in IL-2 expression or activity.

[0167] In further embodiments, certain phenotypic changes resulting from the administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, to a cell, animal, or human subject may include, for example, increased IL-2 expression or activity such as reduced cancer cell growth or proliferation, reduced cancer cell viability, reduced cancer cell migration, reduced cancer cell invasion, reduced cancer cell metastasis, increased immune cell growth or proliferation, increased immune cell activation, increased immune cell sensitivity to antigens, increased immune cell infiltration into tissues, increased immune cell infiltration into tumors, increased cytokine production, reduced autoreactivity, or reduced inflammation.

[0168] In certain embodiments, changes in other genes, mRNAs, proteins, or pathways in cells resulting from an increase in IL-2 expression or activity, which are well known to those skilled in the art, are incorporated herein as indicating an increase in IL-2 expression or activity.

[0169] In other embodiments, compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, may have variable activity, defined, for example, by a percentage decrease in the target RNA level, a percentage decrease in the target protein level, or a percentage decrease in the activity of the target protein. In some embodiments, the target is IL-2. In certain embodiments, a decrease in IL-2 RNA levels, including but not limited to RNA from the transcription of the IL-2 gene and RNA translated into IL-2 protein, indicates a reduction or inhibition of IL-2 expression. In certain embodiments, a decrease in the levels of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 indicates a reduction or inhibition of IL-2 expression. In some embodiments, a decrease in the levels of IL-2 protein indicates a reduction or inhibition of IL-2 expression. In certain embodiments, a decrease in the levels of one or more IL-2 proteins, which are the translation products of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 disclosed herein, indicates a reduction or inhibition of IL-2 expression. In some embodiments, a decrease in IL-2 protein production by immune cells such as Jurcut cells, CD4+ T cells, CD8+ T cells, dendritic cells, or NK cells indicates a reduction or inhibition of IL-2 expression.

[0170] In other embodiments, a decrease in the activity of the IL-2 protein, which is the translation product of one or more IL-2 transcripts disclosed by SEQ ID NO: 1 or SEQ ID NO: 2 herein, indicates a reduction or inhibition of IL-2 activity. IL-2 activity refers to one or more activities typically performed by the IL-2 transcripts or proteins described herein, such as binding to IL-2R or activating signaling pathways such as the JAK / STAT5, PI3K / AKL / mTOR, or MAPK pathways. In certain embodiments, a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, may selectively target one or more specific IL-2 transcript variants and the proteins encoded thereby, reducing their levels or activity, and such a decrease in the level or activity of one or more IL-2 transcript variants or proteins indicates a reduction in IL-2 expression or activity.

[0171] In further embodiments, certain phenotypic changes resulting from the administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, to a cell, animal, or human subject may include, for example, reduced or inhibited IL-2 expression or activity, such as reduced immune cell growth or proliferation, reduced immune cell activation, reduced immune cell sensitivity to antigens, reduced immune cell infiltration into tissues, reduced cytokine production, reduced vascular leakage, reduced autoreactivity, or reduced inflammation.

[0172] In certain embodiments, changes in other intracellular genes, mRNAs, proteins, or pathways resulting from a reduction or inhibition of IL-2 expression or activity, which are well known to those skilled in the art, are incorporated herein as indicating a reduction or inhibition of IL-2 expression or activity.

[0173] RNA-level analysis In certain embodiments, the increase in IL-2 expression by the compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, can be evaluated by measuring the increase in the level of IL-2 RNA transcript. In certain embodiments, the reduction or inhibition of IL-2 expression by the compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, can be evaluated by measuring the decrease in the level of IL-2 RNA transcript. RNA analysis can be performed on poly(A)+ mRNA or whole cellular RNA. Methods for RNA isolation are well known in the art and include, for example, using TRIZOL reagents (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocol, or using RNA extraction kits (Qiagen). The target RNA level can be quantified using methods well known in the art, including, for example, Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR using an ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) after reverse transcription, as directed by the manufacturer.

[0174] Prior to quantitative real-time PCR, isolated RNA is first subjected to a reverse transcription reaction to produce complementary DNA (cDNA), which is then used as a substrate for the real-time PCR amplification reaction. Reagents for reverse transcription and real-time PCR are commercially available (e.g., Invitrogen, Carlsbad, CA). The reverse transcription and real-time PCR reactions can be performed sequentially in the same sample well or in different sample wells. The levels of target genes or RNA obtained by real-time PCR can be normalized using, for example, total RNA levels quantified by RIBOGREEN (Invitrogen, Carlsbad, CA), or using the expression levels of genes whose expression is relatively stable in cells, such as cyclophyllin A, beta-actin, or GAPDH. A method for RNA quantification using RIBOGREEN is described in Jones et al. (Jones et al., Analytical Biochemistry, 265:368-374 (1998)), which, along with the references cited therein, is incorporated herein by reference. RIBOGREEN fluorescence can be measured using the CYTOFLUOR4000 instrument (PE Applied Biosystems). The expression levels of cyclophyllin A, beta-actin, or GAPDH can be quantified by real-time PCR in the same well used to quantify the target RNA levels (i.e., by performing a multiplex reaction), or by performing it in a separate well. Probes and primers that hybridize to the target nucleic acid encoding IL-2 can be designed using methods well known in the art, including, for example, the use of software such as PRIMER EXPRESS software (Applied Biosystems, Foster City, CA).

[0175] Protein level analysis In certain embodiments, an increase in IL-2 expression by a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, can be evaluated by measuring an increase in the level of IL-2 protein. In other embodiments, a reduction or inhibition of IL-2 expression by a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, can be evaluated by measuring a decrease in the level of IL-2 protein. Several methods for quantifying or measuring IL-2 protein levels are well known in the Art, including Western blotting, homogeneous time-resolved fluorescence (HTRF) assays, enzyme-linked immunosorbent assays (ELISA), intracellular Western blotting, immunoprecipitation, immunocytochemistry, fluorescence-activated cell sorting (FACS), immunohistochemistry, protein activity assays, quantitative protein assays, and bicinchoninate assays (BCA assays), also known as Smith assays. Using such methods, the level of intracellular IL-2 protein and / or the level of IL-2 protein secreted from cells can be quantified or measured. Antibodies specific to target proteins such as IL-2 can be produced using conventional monoclonal or polyclonal antibody production methods well known in the art, or they can be identified from various sources such as the MSRS antibody catalog (Aerie Corporation, Birmingham, MI) and commercially available. Antibodies for the detection of mouse, rat, monkey, and human IL-2 are commercially available. Additional methods for measuring increases or decreases in IL-2 protein levels are described in Example 1 and are included in some embodiments herein.

[0176] In vitro testing of compounds The compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions, can be administered in vitro to cultured cells to evaluate their effects on the expression or activity of target genes (e.g., IL-2), or on one or more phenotypes such as cell growth or proliferation, cell viability, cell morphology, cell cycle arrest, cell migration, cell invasion, cell activation, cell sensitivity to antigens, cytokine production, and apoptosis. In some embodiments, administration of the compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions to cultured cells leads to an increase in IL-2 protein expression (see, for example, Example 1). In other embodiments, administration of the compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions to cultured cells leads to an increase in IL-2 RNA expression. In some embodiments, administration of a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, to cultured cells leads to one or more phenotypes, such as reduced cancer cell growth or proliferation, reduced cancer cell viability, reduced cancer cell migration, reduced cancer cell invasion, reduced cancer cell metastasis, increased immune cell growth or proliferation, increased immune cell activation, increased immune cell sensitivity to antigens, increased immune cell infiltration into tissues, increased immune cell infiltration into tumors, increased cytokine production, reduced autoreactivity, or reduced inflammation (see, for example, Example 2).

[0177] In other embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, to cultured cells leads to a decrease in the expression of IL-2 protein and / or IL-2 RNA. In some embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, to cultured cells leads to one or more phenotypes, such as reduced immune cell growth or proliferation, reduced immune cell activation, reduced immune cell sensitivity to antigens, reduced immune cell infiltration into tissues, reduced cytokine production, reduced vascular leakage, reduced autoreactivity, or reduced inflammation.

[0178] In certain embodiments, the cultured cells for in vitro testing of the compound may be, for example, breast cancer cells (e.g., MCF7, MDA-MB-231, MDA-MB-468, HS578T, BT-549, and T-47D), Burkitt lymphoma cells (e.g., BL-2, CA-46), colon cancer cells (e.g., COLO205, HCC-2998, HCT-116, HCT-15, HT29, KM12, and SW-620), CNS cells (e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, and U251), glioblastoma cells (e.g., U-87), immune cells (e.g., THP-1, Jarcut, etc.), leukemia cells (e.g., CCRF-CEM, HL-60, K-562, MOLT-4, RPMI-8226, and SR, etc.), liver cancer cells (e.g., HepG2, etc.), melanoma cancer cells (e.g., A375, LOX) Multiple myeloma cells (e.g., IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, and UACC-62), multiple myeloma cells (e.g., MM.1S), neuroblastoma cells (e.g., SH-SY5Y), non-small cell lung cancer cells (e.g., A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, and NCI-H522), ovarian cancer cells (e.g., IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI / ADR-RES, and SK-OV-3), pancreatic cancer cells (e.g., Mia These are animal or human cancer-derived cells such as PaCa-2, Panc-1, Capan-1, etc., prostate cancer cells (e.g., DU-145, LNCaP, and PC-3, etc.), renal cancer cells (e.g., 786-0, A498, ACHN, CAKI-1, RXF393, SN12C, TK-10, and UO-31, etc.), and small cell lung cancer cells (e.g., H2171, NCI-H82, SHP-77, etc.).Examples of cultured cells are listed in the catalogs of vendors such as Clonetics Corporation, Walkersville, MD; American Type Culture Collection, Manassas, VA; Zen-Bio, Inc., Research Triangle Park, NC, and are incorporated herein by reference. Such cells are cultured using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA) according to the supplier's instructions. Cells can be cultured and tested in multi-well plates, such as 6-well, 24-well, 48-well, 96-well, or 384-well plates.

[0179] In certain embodiments, the cultured cells for in vitro testing of a compound are a mixture of cancer-derived cells and immune cells, also known as a co-culture of cancer-derived cells and immune cells. Such a co-culture can be used to evaluate the efficacy of a compound that stimulates immune cell-mediated killing by cancer-derived cells. Cancer-derived cells can be commercially available cancer-derived cell lines or directly derived from tumor samples from patients. The immune cell types used for co-culture with cancer-derived cells can be peripheral blood mononuclear cells (PBMCs), T cells, or NK cells, etc. The immune cell types used for co-culture with cancer-derived cells can be isolated from healthy human donors or patients (e.g., those with cancer). Immune cells may be subjected to activation (e.g., using anti-CD3 antibodies, OKT3, anti-CD28 antibodies, anti-CD3 / CD28 beads, IL-2, phytohemagglutinin (PHA), ionomycin, phorbol 12-myristate 13-acetate (PMA), or irradiated feeder cells, etc.) or not to be activated before and / or during co-culture with cancer-derived cells. Immune cells can be co-cultured with cancer-derived cells grown in 2D or 3D culture (e.g., patient-derived organoids, explant cultures, spheroids, bioprints, organ chips, etc.). In some embodiments, immune cells and cancer-derived cells can be co-cultured in different compartments (e.g., using cell culture inserts, etc.), thus preventing direct contact between immune cells and cancer-derived cells but allowing exchange of media, cytokines, chemokines, or growth factors between compartments. In other embodiments, immune cells can be co-cultured with cancer-derived cells in the same compartment, allowing direct contact between immune cells and cancer-derived cells.In vitro co-culture methods for evaluating the activity of the compound of formula (I) described herein, or its complex, conjugate, or pharmaceutical composition are also described in Example 2, as well as in reference to Mackenzie et al. (Mackenzie et al., Clinical & Translational Immunology, e1400 (2022)), Pimentel et al. (Pimentel et al., Journal of Immunological Methods, 487:112899 (2020)), Liu et al. (Liu et al., Biosensors (Basel), 12(11):1045 (2022)), Mo et al. (Mo et al., Cell Chemical Biology, 26:331-339 (2019)), Yuki et al. (Yuki et al., Trends in Immunology, 41(8):652-664 (2020)), and Lanigan et al. (Lanigan Further details are provided in et al., Journal of Biological Methods, 7(2):e133(2020)), and these disclosures, along with their references, are incorporated herein by reference in their entirety.

[0180] One or more phenotypes resulting from the administration of the compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition, to cultured cells, such as a decrease or increase in cancer cell growth or proliferation, a decrease or increase in cancer cell viability, a decrease or increase in cancer cell migration, a decrease or increase in cancer cell invasion, a decrease or increase in cancer cell metastasis, an increase or decrease in immune cell growth or proliferation, an increase or decrease in immune cell activation, an increase or decrease in immune cell sensitivity to antigens, an increase or decrease in immune cell infiltration into tissues, an increase or decrease in immune cell infiltration into tumors, an increase or decrease in cytokine production, a decrease or increase in autoreactivity, a decrease or increase in inflammation, or a decrease or increase in apoptosis, can be evaluated using any method known to those skilled in the art. Examples of assays for evaluating cell growth and proliferation or cell viability include colorimetric assays utilizing dyes such as MTT, XTT, MTS, trypan blue, and CCK-8, fluorescence-based assays such as ApoTox-Glo, and luminescence-based assays such as CellTiter-Glo. Examples of methods for evaluating cell growth and proliferation in real time include the IncuCyte imaging system. Examples of assays for evaluating apoptosis include ApoTox-Glo, caspase-Glo (3 / 7, 8, and 9), and Annexin V. Examples of assays for evaluating cytokine production include RT-qPCR, ELISA, HTRF, AlphaScreen, AlphaLISA, and Lumit immunoassays. Examples of methods for evaluating immune cell growth and proliferation include flow cytometry.

[0181] In certain embodiments, the disclosed compound, its conjugate, complex, or pharmaceutical composition exhibits an IC50 of approximately 20 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.25 μM, 0.1 μM, 0.05 μM, less than 0.01 μM, or lower IC50 in a selected assay. 50or EC 50In other embodiments, the disclosed compounds, or their conjugates, complexes, or pharmaceutical compositions, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, less than 0.01 μM, or lower, reduce the growth or viability of cultured cancer cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a vehicle control. In other embodiments, the disclosed compounds, their conjugates, complexes, or pharmaceutical compositions increase apoptosis in cultured cancer cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or 1000% compared to a vehicle control, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 20 μM, 10 μM, 10 μM, 10 μM, 0.05 μM, 0.01 μM, or lower. In other embodiments, the disclosed compounds, their conjugates, complexes, or pharmaceutical compositions increase the growth or proliferation of cultured immune cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or 1000% compared to a vehicle control, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 20 μM, 10 μM, 10 μM, 10 μM, 0.05 μM, 0.01 μM, or lower. In other embodiments, the disclosed compounds, or their conjugates, complexes, or pharmaceutical compositions, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, less than 0.01 μM, or lower, reduce the growth or viability of cultured immune cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a vehicle control.In some embodiments, the disclosed compound, or its conjugate, complex, or pharmaceutical composition, increases cytokine production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or 1000% compared to a vehicle control, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 20 μM, 10 μM, 10 μM, 10.05 μM, 0.01 μM, or lower. In some embodiments, the disclosed compound, or its conjugate, complex, or pharmaceutical composition, increases cytokine production by at least 100, 500, 1000, 2000, 3000, 4000, 5000, or 10,000 pg / ml compared to a vehicle control, at concentrations of approximately 500 μM, 100 μM, 500 μM, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 pg / ml compared to a vehicle control. In some embodiments, the disclosed compound, or its conjugate, complex, or pharmaceutical composition, reduces cytokine production by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a vehicle control at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, or lower. In some embodiments, the cytokine is IL-2.

[0182] In vivo testing of compounds Compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions, can be administered in vivo to animals or human subjects to evaluate their properties, such as pharmacokinetic profiles, safety, maximum tolerated dose (MTD), ADME (absorption, distribution, metabolism, and excretion), pharmacodynamics, effects on the expression or activity of target genes (maybe multiple) (e.g., IL-2), or effects on one or more phenotypes, such as survival, cancer cell growth, cancer metastasis, behavior, body weight, metabolism, immune cell proliferation, immune cell activity, immune cell infiltration into cancer or tissue, and cytokine production. In some embodiments, administration of compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions to animals or human subjects leads to an increase in the expression and / or activity of IL-2 protein and / or RNA. In other embodiments, administration of compounds of formula (I) disclosed herein, or their complexes, conjugates, or pharmaceutical compositions to animals or human subjects leads to a decrease or inhibition of the expression and / or activity of IL-2 protein and / or RNA. In some embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition to an animal or human subject may result in one or more phenotypes, such as a reduction in tumor volume (i.e., tumor regression), a reduction in cancer cell growth or proliferation, a reduction in cancer metastasis, an increase or decrease in immune cell proliferation, an increase or decrease in immune cell activation, a reduction in autoimmunity, a reduction in inflammation, an increase or decrease in cytokine production, an increase in the survival of the animal or human subject, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). In some embodiments, the test may be conducted in a healthy animal or human subject. In other embodiments, the test may be conducted in a disease animal model (e.g., cell line-derived xenograft (CDX) model, patient-derived xenograft (PDX) model, syngeneic model, orthotopic model, humanized model, PBMC co-inoculation model, spontaneous or induced cancer model, etc.) or in a human subject diagnosed with cancer, autoimmune disease, inflammatory disease, or infection, etc.In vivo co-culture methods for evaluating the activity of the disclosed compounds, conjugates, complexes, or pharmaceutical compositions thereof are described by Chuprin et al. (Chuprin et al., Nat. Rev. Clin. Oncol., https: / / doi.org / 10.1038 / s41571-022-00721-2 (2023)), Ye et al. (Ye et al., Annu. Rev. Anim. Biosci., 10:395-417 (2022)), Morillon II et al. (Morillon II et al., Anticancer Res., 40(10):5329-5341 (2020)), and Cogels et al. (Cogels et al., A Frontiers in Oncology, 11:784947 (2021)), and these disclosures, along with their references, are incorporated herein by reference in their entirety.

[0183] In certain embodiments, the compound of formula (I) disclosed herein, or its complex or conjugate, is formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline, for administration to an animal. Administration includes any of the administration routes described herein, such as oral, subarachnoid, intraperitoneal, intravenous, and subcutaneous. Methods for calculating appropriate doses and administration frequencies of the compound, complex or conjugate disclosed herein are well known in the art and depend on factors such as animal body weight, administration route, ADME properties, and the potency of the compound. After administration of the compound, complex, conjugate, or pharmaceutical composition disclosed herein to an animal, the animal is monitored at defined time points for the expression levels of target genes, such as IL-2, and for effects on other phenotypes, such as survival, cancer cell growth, cancer metastasis, immune cell proliferation, immune cell activation, cytokine production, behavior, body weight, and metabolism. Levels of IL-2 RNA or IL-2 protein are known in the art and can be measured using the methods described herein in different tissues from animals, such as CSF, plasma, brain, spinal cord, lung, liver, kidney, and tumor tissue. Other modifications are known to those skilled in the art and are considered to be included in the embodiments described herein.

[0184] In certain embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in an increase in IL-2 RNA compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%, or any two of these values. In certain embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in an increase in IL-2 protein compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%, or any two of these values. In some embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in a reduction of IL-2 RNA by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values, compared to a vehicle control. In certain embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in a reduction of IL-2 protein by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values, compared to a vehicle control. In other embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in a reduction in tumor volume by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values, compared to a vehicle control. In certain embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in a reduction in tumor metastasis by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values, compared to a vehicle control.In certain embodiments, administration of a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, results in an increase in the survival of an animal or human subject by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000% compared to a vehicle control, or within a range defined by any two of these values.

[0185] In certain embodiments, administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition results in an increase in immune cell proliferation compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%, or any two of these values. In other embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in a decrease in immune cell proliferation compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values. In certain embodiments, administration of a compound of formula (I) disclosed herein, or its complex, conjugate, or pharmaceutical composition results in an increase in immune cell activation compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000%, or any two of these values. In some embodiments, administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition results in a decrease in immune cell activation compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values. In further embodiments, administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition results in an increase in cytokine production compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, 5000, or 10000%, or any two of these values.In some embodiments, administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition results in an increase in cytokine production compared to a vehicle control, within a range defined by at least 1, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 pg / ml, or any two of these values. In further embodiments, administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition results in a decrease in cytokine production compared to a vehicle control, within a range defined by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any two of these values. In some embodiments, the cytokine is IL-2.

[0186] Instructions for use and treatment methods This specification provides methods comprising administering a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition containing a compound of formula (I) disclosed herein, to cells, animals, or humans. Some embodiments provide methods for increasing the expression of IL-2 in cells or tissues, the methods comprising administering a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, to cells, animals, or humans such that the expression of IL-2 is increased. Other embodiments provide methods for increasing the activity of IL-2 in cells or tissues, the methods comprising administering a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, to cells, animals, or humans such that the activity of IL-2 is increased. Other embodiments provide methods for decreasing the expression of IL-2 in cells or tissues, the methods comprising administering a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, to cells, animals, or humans such that the expression of IL-2 is reduced. Other embodiments provide a method for reducing the activity of IL-2 in cells or tissues, the method comprising administering a compound of formula (I) disclosed herein, or a complex, conjugate, or pharmaceutical composition thereof, to cells, animals, or humans such that the activity of IL-2 is reduced.

[0187] Some embodiments provide methods for achieving one or more phenotypic outcomes, such as a decrease in cancer cell growth or proliferation, a decrease in cancer cell viability, a decrease in apoptosis, a decrease in tumor volume (i.e., tumor regression), a decrease in cancer metastasis, an increase in the survival of an animal or human subject, a decrease or increase in immune cell growth or proliferation, a decrease or increase in immune cell activation, a decrease or increase in immune cell sensitivity to antigens, increased immune cell infiltration into tumors, a decrease or increase in cytokine production, a decrease in inflammation, a decrease in autoimmunity, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.), the method comprising administering a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, to a cell, animal, or human. Further embodiments provide methods for treating diseases such as cancer, autoimmune diseases, inflammatory diseases, or infections, the method comprising administering an effective amount of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition, to an animal or human.

[0188] In certain embodiments, a method for treating a human subject diagnosed with cancer is provided herein, the method comprising administering to the human subject a compound of formula (I) disclosed herein, or a conjugate, complex thereof, or pharmaceutical composition thereof. In certain embodiments, a method for prophylactically increasing IL-2 expression or activity in a human subject to prevent a disease such as cancer, the human subject being at risk of developing cancer. In other embodiments, a compound of formula (I) disclosed herein, or a conjugate, complex thereof, or pharmaceutical composition thereof, is administered to a human subject to treat or improve the symptoms of cancer.

[0189] In certain embodiments, cancer refers to, for example, breast cancer, Burkitt lymphoma, cervical cancer, colorectal cancer (e.g., colonic adenocarcinoma, rectal adenocarcinoma, etc.), esophageal cancer, gastric cancer (e.g., gastric adenocarcinoma, etc.), glioblastoma (e.g., glioblastoma multiforme, etc.), head and neck squamous cell carcinoma, leukemia (e.g., myeloid leukemia, etc.), liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung squamous cell carcinoma, etc.), non-Burkitt lymphoma, medulloblastoma, melanoma (e.g., cutaneous melanoma, uveal melanoma, etc.), mesothelioma, multiple myeloma, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer (e.g., clear cell renal carcinoma), or rhabdomyosarcoma, etc. In certain embodiments, cancer refers to, for example, astrocytoma, low-grade brain glioma, breast cancer, glioblastoma, lung cancer (e.g., small cell lung cancer), medullary thyroid carcinoma, medulloblastoma, neuroblastoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., alveolar rhabdomyosarcoma), and testicular cancer.

[0190] Other embodiments provide the use of compounds of formula (I) disclosed herein, or its complexes, conjugates, or pharmaceutical compositions, to increase IL-2 expression or activity. Still other embodiments provide the use of compounds (I) disclosed herein, or its complexes, conjugates, or pharmaceutical compositions, to achieve one or more phenotypic outcomes, such as a decrease in cancer cell growth or proliferation, a decrease in cancer cell viability, a decrease in apoptosis, a decrease in tumor volume (i.e., tumor regression), a decrease in cancer metastasis, an increase in survival of an animal or human subject, an increase or decrease in immune cell growth or proliferation, an increase or decrease in immune cell activation, an increase or decrease in immune cell sensitivity to antigens, an increase in immune cell infiltration into tumors, an increase or decrease in cytokine production, a decrease in autoimmunity, a decrease in inflammation, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). Still other embodiments provide the use of compounds of formula (I) disclosed herein, or its conjugates, complexes, or pharmaceutical compositions, to treat, prevent or improve cancer, autoimmune diseases, inflammatory diseases, or infections.

[0191] Other embodiments provide the use of compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, to reduce the expression or activity of IL-2. Still other embodiments provide the use of compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, to achieve one or more phenotypic outcomes, such as increased survival in an animal or human subject, decreased growth or proliferation of immune cells, decreased immune cell activation, decreased immune cell sensitivity to antigens, decreased cytokine production, decreased autoimmunity, decreased inflammation, decreased vascular leakage, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). Still other embodiments provide the use of compounds of formula (I) disclosed herein, or their pharmaceutical compositions, to treat, prevent, or improve autoimmune or inflammatory diseases.

[0192] Certain embodiments provide compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions for use in increasing IL-2 expression or activity. Certain other embodiments provide compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions for use in treating, preventing or improving cancer, autoimmune diseases, inflammatory diseases, or infections.

[0193] Certain embodiments provide compounds of formula (I) disclosed herein, or conjugates, complexes, or pharmaceutical compositions thereof, for use in reducing the expression or activity of IL-2. Certain other embodiments provide compounds of formula (I) disclosed herein, or conjugates, conjugates, or pharmaceutical compositions thereof, for use in achieving one or more phenotypic outcomes, such as a decrease in immune cell growth or proliferation, a decrease in immune cell activation, a decrease in immune cell sensitivity to antigens, a decrease in cytokine production, an increase in survival of an animal or human subject, a decrease in autoimmunity, a decrease in inflammation, a decrease in vascular leakage, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). Certain other embodiments provide compounds of formula (I) disclosed herein, or conjugates, conjugates, or pharmaceutical compositions thereof, for use in treating, preventing, or improving autoimmune or inflammatory diseases.

[0194] Some embodiments provide the use of compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions in the manufacture of pharmaceuticals for increasing IL-2 expression or activity. In certain embodiments, compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions are used in the manufacture of pharmaceuticals to achieve one or more phenotypic outcomes, such as a decrease in cancer cell growth or proliferation, a decrease in cancer cell viability, a decrease in apoptosis, a decrease in tumor volume (i.e., tumor regression), a decrease in cancer metastasis, an increase or decrease in immune cell growth or proliferation, an increase or decrease in immune cell activation, an increase or decrease in immune cell sensitivity to antigens, increased immune cell infiltration into tumors, an increase or decrease in cytokine production, an increase in survival of an animal or human subject, a decrease in autoimmunity, a decrease in inflammation, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). In certain embodiments, compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions are used in the manufacture of pharmaceuticals to treat, prevent or improve cancer in patients diagnosed with or susceptible to cancer. In other embodiments, the compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, are used in the manufacture of pharmaceuticals to treat, prevent, or improve autoimmune or inflammatory diseases. In certain embodiments, the compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, are used in the manufacture of pharmaceuticals to treat, prevent, or improve infections in patients.

[0195] Some embodiments provide the use of compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions in the manufacture of pharmaceuticals for reducing IL-2 expression or activity. In certain embodiments, compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions are used in the manufacture of pharmaceuticals to achieve one or more phenotypic outcomes, such as a decrease in immune cell growth or proliferation, a decrease in immune cell activation, a decrease in immune cell sensitivity to antigens, a decrease in cytokine production, an increase in survival of an animal or human subject, a decrease in autoimmunity, a decrease in inflammation, a decrease in vascular leakage, or other desired outcomes relating to a particular phenotype (e.g., body weight, metabolism, etc.). In certain embodiments, compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions are used in the manufacture of pharmaceuticals to treat, prevent, or improve autoimmune or inflammatory diseases.

[0196] In other embodiments, methods for preventing, treating, or improving diseases other than cancer are provided herein, the methods comprising administering a compound of formula (I) disclosed herein, or a conjugate, complex, or pharmaceutical composition thereof, to cells, tissues, animals, or human subjects. In certain embodiments, such diseases are associated with excessively low or excessively high IL-2 expression or activity, and targeting IL-2 to increase or decrease its expression or activity, respectively, can provide therapeutic benefits. Examples of such diseases include autoimmune diseases or inflammatory diseases.

[0197] In certain embodiments, a method for treating a human subject in need of treatment is provided herein, by administering a therapeutically effective amount of a compound of formula (I) disclosed herein, or a conjugate, complex, or pharmaceutical composition thereof, which may target one or more IL-2 nucleic acids or portions thereof, to a human subject. In some embodiments, the administration of a therapeutically effective amount of a compound of formula (I) disclosed herein, or a conjugate, complex, or pharmaceutical composition thereof, is accompanied by monitoring of IL-2 RNA levels, protein levels, or activity in the subject to determine the subject's response to the administration of the compound, conjugate, complex, or pharmaceutical composition. The human subject's response to the administration of the compound may be used by a physician to determine the dose, schedule, and duration of the therapeutic intervention.

[0198] A therapeutic method involving the administration of a compound of formula (I) disclosed herein, or its conjugate, complex, pharmaceutical composition, or medicinal product, to a cell, tissue, animal, or human subject may vary with respect to the composition, the quantity of the dose, and the dose schedule. A unit dose is a predetermined therapeutically effective amount of the compound of formula (I) disclosed herein, or its conjugate, complex, pharmaceutical composition, or medicinal product, to be administered. The unit dose may vary depending on a variety of factors, including but not limited to body weight, age, sex, severity of symptoms, medical history, and the aggressiveness of the treatment. The schedule is the frequency of administration of the unit dose. The size of the unit dose and the administration schedule of the compound, conjugate, complex, pharmaceutical composition, and medicinal product can be determined by those skilled in the art and incorporated into certain embodiments herein.

[0199] In certain embodiments, the compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions or drugs, are administered co-administered with one or more other drugs. The compounds, conjugates, complexes, pharmaceutical compositions, or drugs described herein may be prepared together with one or more other drugs in a single formulation or separately. In some embodiments, one or more other drugs are designed to treat different diseases, disorders, symptoms, or conditions compared to the compounds, conjugates, complexes, pharmaceutical compositions, or drugs described herein. In other embodiments, one or more other drugs are designed to treat the same diseases, disorders, symptoms, or conditions as the compounds, conjugates, complexes, pharmaceutical compositions, or drugs described herein. In some embodiments, one or more other drugs are administered co-administered with the compounds, conjugates, complexes, pharmaceutical compositions, or drugs described herein to produce an additive effect. In certain embodiments, one or more other drugs are administered co-administered with the conjugates, complexes, compounds, pharmaceutical compositions, or drugs described herein to produce a synergistic or hyperadditive effect, where the co-administration of both results in an effect exceeding the sum of the effects of each administered individually. In other embodiments, one or more other agents are administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or medicinal products described herein to treat undesirable side effects of the compounds, conjugates, complexes, pharmaceutical compositions, or medicinal products described herein. In certain embodiments, the compounds, conjugates, complexes, pharmaceutical compositions, or medicinal products described herein are administered concurrently with one or more other agents to prevent or delay the onset of symptoms, delay disease progression, reduce side effects, improve therapeutic efficacy, or otherwise improve patient outcomes. Concurrent administration, as used herein, may mean that one or more other agents are administered simultaneously with the compounds, conjugates, complexes, pharmaceutical compositions, or medicinal products described herein, as well as that which may be administered before or after (e.g., several hours, several days, or several weeks) the compounds, conjugates, complexes, pharmaceutical compositions, or medicinal products described herein are administered.

[0200] In certain embodiments, one or more other agents administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein may include agents such as antisense oligonucleotides, other oligonucleotides (e.g., siRNA, ribozymes, deoxyribozymes, or aptamers), antibodies, peptides, small molecule compounds, nucleic acid vectors, cell therapies (e.g., CAR-T), or gene therapy agents.

[0201] In certain embodiments, one or more agents administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein are drugs typically administered to treat or improve symptoms in cancer, such as alkylating agents (e.g., altretamine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, melphalan, temozolomide, and trabectedin), antimetabolites (e.g., azacitidine, capecitabine, clopharabine, cytarabine, phloxuridine, fludarabine, 5-fluorouracil, gemcitabine, 6-mercaptopurine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, and tipiracil), and antitumor antibiotics (e.g., bleomycin, This includes daunorubicin, doxorubicin, doxorubicin liposomes, epirubicin, idarubicin, mitoxantrone, and barurubicin, etc.; biological response modifiers that enhance the body's immune system to fight cancer growth or disrupt processes necessary for cancer growth or spread (e.g., Avastin, Elbitux, Herceptin, and Rituxan); corticosteroid hormones (e.g., dexamethasone and prednisone, etc.); nitrosoureas (e.g., carmustine and lomustine, etc.); plant alkaloids and natural products (e.g., docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, and vinorelbine, etc.); and sex hormones (e.g., leuprolide and tamoxifen, etc.).

[0202] In certain embodiments, one or more agents administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein include immunotherapeutic agents, e.g., immune checkpoint (e.g., PD-1, PD-L1, CTLA-4, etc.) inhibitors (e.g., ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, etc.), adoptive cell immunotherapy (e.g., CAR-T cells, CAR-NK cells, etc.), cancer vaccines (e.g., DNA, peptides, dendritic cells, whole cells, etc.), cytokines (e.g., IL-2, IL-8, IL-10, IL-12, IL-15, IL-21, GM-CSF, IFN-α, TGF-β, TNF-α, etc.), monoclonal antibodies (e.g., bevacizumab, rituximab, trastuzumab, cetuximab, etc.), oncolytic immunotherapy, and the like. These and other examples of immunotherapies are described by Waldman et al. (Waldman et al., Nature Reviews Immunology, 20:651-668 (2020)), Dobosz et al. (Dobosz et al., Int.J.Mol.Sci., 23:2847 (2022)), Hoteit et al. (Hoteit et al., Oncology Letters, 22:655 (2021)), Yang et al. (Yang et al., Journal of Immunology Research, Article ID 8052212 (2022)), and Esfahani et al. (Esfahani et al., Curr.Oncol., 27(S2):87-97 (2020)), and these, along with the references cited therein, are incorporated herein in their entirety.

[0203] In certain embodiments, one or more agents administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein include drugs that relieve pain, inflammation, or other symptoms (e.g., COX inhibitors, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, pranoprofen, carprofen, indomethacin, folinic acid, tiaprofenic acid, diclofenac, niflumic acid, diazepines or benzodiazepines (e.g., diazepam), and barbiturates), drugs that improve uptake or delivery (e.g., blood thinners (e.g., aspirin and warfarin)), antibacterial agents, antiviral agents, antibiotics, or other drugs that provide at least one benefit in a therapeutic setting, including therapeutic efficacy, symptom relief, drug resistance, or side effect regulation.In certain embodiments, one or more agents administered concurrently with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein include drugs typically administered to treat or improve symptoms in autoimmune or inflammatory diseases, such as steroids (e.g., prednisone, methylprednisolone, dexamethasone), colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate, azathioprine, IL-1 targeting drugs (e.g., anakinra, canakinumab, lilonacept), TNF targeting drugs (e.g., infliximab, adalimumab, golimumab, etanercept, ceritolizumab), IL-6 targeting drugs (e.g., tocilizumab, sarilumab), complement targeting drugs (e.g., eculizumab), and CD20 targeting drugs. Drugs that target B cell growth factors (e.g., rituximab), drugs that target T cells (e.g., cyclosporine), drugs that target T cell activation (e.g., abatacept), drugs that target IL-4 / IL-13 (e.g., dupilumab), drugs that target IL-5 (e.g., mepolizumab, reslizumab, benralizumab), drugs that target IL-12 Examples include drugs (e.g., ustekinumab), drugs that target IL-17 (e.g., secukinumab, ixekizumab, brodalumab), drugs that target IL-23 (e.g., guselkumab), drugs that target IgE (e.g., omalizumab), drugs that target lymphocyte migration (e.g., vedolizumab), and JAK inhibitors (e.g., tofacitinib, upadacitinib, baricitinib).

[0204] In certain embodiments, one or more agents that can be co-administered with the compounds, conjugates, complexes, pharmaceutical compositions, or pharmaceuticals described herein include additional modulators that can increase or decrease the expression or activity of IL-2. In certain embodiments, the dose of the co-administered agent is lower than the dose that would be administered if the co-administered agent were administered alone.

[0205] Development methods In certain embodiments, methods for developing small molecule compounds that can target IL-2 or related genes or pathways, thereby increasing or decreasing IL-2 expression or activity, are provided herein. In certain embodiments, the compound development method is entirely computer-based. In some embodiments, the compound development method includes biochemical or cell-based methods such as screening and selection. In some embodiments, the compound development method includes a combination of computer-based and biochemical or cell-based methods. Such development methods include standard techniques known to those skilled in the art and incorporated into certain embodiments herein.

[0206] In some embodiments, the computer-based method may include artificial intelligence or machine learning software, rely on large molecular databases, utilize high-throughput analysis, or any combination thereof. Manigrasso et al.(Manigrasso et al.,Chem,7(11):2965-2988(2021)), Mendez-Lucio et al.(Mendez-Lucio et al.,Nature Communications,11,10(2020)), Dallakyan and Olson(Dallakyan and Olson,Hempel et al.(ed.)Chemical Biology:Methods and Protocols,Chapter 19,Methods in Molecular Biology pg 243-250(2015)), Zoete et al.(Zoete et al.,Journal of Chemical Information and Modeling,56:1399-1404(2016)), and Merk et al.(Merk et al.,Molecular The computer-aided methods described in Informatics, 37:1700153 (2018) are representative of some of the available computer-aided methods, and these, along with the references cited therein, are incorporated herein in their entirety. In some embodiments, the structure of a target RNA, such as IL-2, or a portion thereof, is predicted by computer. A computer library of small molecule compounds is then individually computer-docked to the target, and the binding energy of each small molecule compound to the target is determined using computer-aided methods. Small molecule compounds predicted to have a favorable binding energy to the target RNA are preferred for further analysis and development. In some embodiments, novel small molecule compounds are created for computer screening from fusions of existing molecules or atoms from one or more databases. Other computer-aided methods are well known to those skilled in the art and are included in various embodiments herein.

[0207] In certain embodiments, the method for developing small molecule compounds includes high-throughput biochemical or cell-based screening methods known to those skilled in the art. Physical libraries of small molecules are constructed or obtained from commercially available sources. These libraries are screened against selected target molecules, such as IL-2, which is done by introducing small molecules into the selected target molecules and then performing washing or separation methods to determine binding affinity and / or specificity. An example of such a method is affinity-selective mass spectrometry (ALIS), described by Rizvi et al. (Rizvi et al., Methods, 167:28-38 (2019)), which, along with the references cited therein, is incorporated herein in its entirety. In some embodiments, the binding of small molecule compounds to target molecules can be detected and measured using biophysical methods such as NMR, X-ray crystallography, small-angle X-ray scattering, microscale thermophoresis, surface plasmon resonance, fluorescence-based methods, isothermal titration calorimetry, and mass spectrometry. In other embodiments, a cell-based method is used to identify compounds that can modulate the expression or activity of targets such as IL-2, which includes treating cells with a library of small molecule compounds and subsequently detecting the level of target RNA (e.g., using RT-qPCR or RNA-seq) and / or protein (e.g., using Western blotting, ELISA, Alphascreen, or homogeneous time-resolved fluorescence (HTRF)). The structural and chemical properties of the small molecule or the molecular complex formed between the small molecule and the selected target molecule can be determined using a biochemical or cell-based assay or a series of biochemical or cell-based assays. A control or negative selection step can be used to screen for small molecules that have off-target binding activity to molecules other than the selected target molecule. Furthermore, screening of small molecules at different concentrations against the selected target molecule can be used to determine IC 50 , EC 50Determine other properties such as efficacy. Further descriptions of methods and procedures for developing low molecular weight compounds are described by Falese et al. (Falese et al., Chem. Soc. Rev., 50:2224 (2021)), and Cronk (Cronk, Drug Discovery and Development (Second Edition) Chapter 8, pp. 95-117 (2013)), which are incorporated by reference in their entirety together with the references cited therein. Other biochemical or cell-based screening methods are well known to those skilled in the art and are included in various embodiments of the present specification.

[0208] In certain embodiments, methods for developing small molecule modulators include fragment-based discovery techniques known to those skilled in the art. These methods include screening libraries of small molecule fragments that contain one or more binding epitopes for binding affinity and / or specificity to a target molecule such as IL-2. Typically, small molecule fragments have a molecular weight of about 120-250 daltons. In certain cases, these fragment-based discovery methods are combined with computer-based methods, some of which are described below. Examples of fragment-based discovery techniques include lead identification by fragment evolution, lead identification by fragment linking, lead identification by fragment self-assembly, and lead progression by fragment optimization, which are described herein. In many cases, evaluation of the target molecule binding site, development of fragment complexes, and subsequent determination of binding efficacy or specificity are informed by structural, morphological, and chemical data obtained from evaluation tools such as nuclear magnetic resonance spectroscopy, mass spectrometry, or X-ray crystallography.

[0209] In lead identification by fragment evolution, a library of fragments is applied to a selected target molecule, and the strength and specificity of binding are determined. Fragments with higher binding specificity are then reacted or evolved with various other fragments to form fragment complexes that are screened for even higher binding specificity.

[0210] In lead identification by fragment linking, a fragment library is screened through multiple binding sites of a target molecule selected for binding specificity. Two or more fragments with high binding specificity for two or more adjacent binding sites on the selected target molecule are chemically linked together.

[0211] In lead identification by fragment self-assembly, also known as combinatorial chemistry, a library of self-assembling fragments is introduced to a selected target molecule. The fragments can bind to the selected target molecule in a manner that produces a complex that decreases or increases the expression or activity of the selected target molecule. Various fragments can be assembled together while binding to the selected target molecule via complementary reactive groups. Once assembled, these fragment complexes can then be isolated and their chemical and structural properties can be evaluated.

[0212] In lead progression by fragment optimization, a library of fragments is used to modify the properties of an existing modulator or fragment complex. Typically, this method is used to address the optimization of specific properties such as selectivity, solubility, stability, or efficacy.

[0213] Examples of methods and procedures for the development of small molecule modulators via fragment-based discovery, and further considerations, are described by Rees et al. (Rees et al., Nature Reviews Drug Discovery, 3(8):660 (2004)), Erlanson et al. (Erlanson et al., Journal of Medicinal Chemistry, 47(14):3463-3482 (2004)), and Congress et al. (Congreve et al., Journal of Medicinal Chemistry, 51(13):3661-3680 (2008)), all of which are incorporated by reference in their entirety, along with the references cited therein. Other methods are well known to those skilled in the art and are included in the various embodiments herein.

[0214] Diagnostics and kits In some embodiments, measuring and detecting a decrease in IL-2 expression or activity, or a decrease in signaling via IL-2-related pathways, can be used to diagnose or determine an increased risk or increased susceptibility to cancer, autoimmune diseases, inflammatory diseases, or infections. In some embodiments, measuring and detecting an increase in IL-2 expression or activity, or an increase in signaling via IL-2-related pathways, can be used to determine a reduced risk to cancer, autoimmune diseases, inflammatory diseases, or infections. In other embodiments, measuring and detecting an increase in IL-2 expression or activity, or an increase in signaling via IL-2-related pathways, can be used to determine an increased risk or increased susceptibility to autoimmune diseases, inflammatory diseases, or leak-of-vasoconstriction syndromes. In some embodiments, measuring and detecting a decrease in IL-2 expression or activity, or a decrease in signaling via IL-2-related pathways, can be used to diagnose or determine a reduced risk or reduced susceptibility to autoimmune diseases, inflammatory diseases, or leak-of-vasoconstriction syndromes.

[0215] In some embodiments, a method for determining susceptibility of a subject to cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome includes obtaining a sample from the subject containing tissue, body fluid, or other biological specimens; detecting the expression level of at least one IL-2 RNA transcript associated with a sequence described by SEQ ID NO: 1 or SEQ ID NO: 2, wherein different expression levels of the transcript(s) are associated with different susceptibility to the disease; and determining susceptibility to the disease. In certain embodiments, a method for detecting IL-2 expression, IL-2 activity, or signaling via IL-2-related pathways includes the use of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition.

[0216] In certain embodiments, a method for determining risk or susceptibility to the aforementioned diseases, or a method for diagnosing a disease, can be applied to predict the prognosis of a human individual diagnosed with or experiencing symptoms associated with cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome. In other embodiments, a method for determining risk or susceptibility to the aforementioned diseases, or a method for diagnosing a disease, can be used to assess a human individual's likelihood of responding to therapeutic methods and / or modulators used to treat, prevent, or improve symptoms associated with cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome. In one embodiment, such a method can be used to select a therapeutic method or modulator to be used when treating a subject diagnosed with a disease.

[0217] Some embodiments also relate to kits and devices for determining a human individual's susceptibility to a disease, or for diagnosing a disease, or for predicting the prognosis of a human individual diagnosed with a disease or experiencing disease-related symptoms, or for evaluating a human individual's potential response to therapeutic methods and / or modulators used to treat, prevent, or improve disease-related symptoms. In some embodiments, the disease is cancer, autoimmune disease, inflammatory disease, infection, or vasodilatory leakage syndrome.

[0218] A kit useful for any of the methods described herein may include any components useful for any of the methods described herein, including, but are not limited to, probes (e.g., hybridization probes, allele-specific oligonucleotides), enzymes (e.g., for RFLP analysis, activity assays), reagents for nucleic acid amplification, reagents for direct analysis of at least one allele of at least one polymorphic marker in or related to IL-2, reagents for indirect analysis of at least one allele of at least one polymorphic marker in or related to IL-2, reagents for detecting IL-2 expression or activity, reagents for detecting signal transduction via IL-2-related pathways, and the like. In one embodiment, the kit may include necessary buffers. In another embodiment, the kit may further provide reagents for other disease-specific diagnostic methods known in the art that are carried out in conjunction with the methods described herein.

[0219] In certain embodiments, the reagents in the kit include at least one compound of formula (I) disclosed herein, or a complex or conjugate thereof, which can interact with IL-2 or otherwise detect IL-2 expression, IL-2 activity, or signaling via IL-2-related pathways. In other embodiments, the kit includes at least one labeled compound of formula (I) disclosed herein, and a reagent for detecting the label. Suitable labels include, but are not limited to, radioisotopes, fluorescent labels, enzyme labels, enzyme cofactor labels, magnetic labels, spin labels, or epitope labels.

[0220] In certain embodiments, the kit and apparatus include a lookup table containing correlation data between the expression level of at least one transcript associated with a sequence described by SEQ ID NO: 1 or SEQ ID NO: 2, which is selectively evaluated by the kit, and susceptibility to disease, or prognosis in the disease, or response to at least one therapy for the disease. Another set of embodiments relates to the use of compounds of formula (I) disclosed herein, or their conjugates or complexes, in the manufacture of reagents for diagnosing or evaluating susceptibility to disease, or prognosis in the disease, or response to therapy for the disease in a human individual. In one embodiment, the kit further includes a set of instructions for using the reagents comprising the kit. In another embodiment, the kit includes a set of instructions or guidelines for interpreting the results of tests using the reagents comprising the kit.

[0221] A further series of embodiments provides a kit (also known as a pharmaceutical pack, and used interchangeably) comprising a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug, and a series of instructions for the administration of the compound, conjugate, complex, or pharmaceutical composition and drug to humans. In some embodiments, an individual identified as a carrier of at least one allele of at least one polymorphic marker associated with IL-2-driven disease is instructed to take a prescription dose of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug. In other embodiments, an individual with increased risk or increased susceptibility to cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome is instructed to take a prescription dose of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug. In other embodiments, individuals diagnosed with cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome are instructed to take a prescribed dose of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug thereof.

[0222] Computer-readable media and devices The compositions, methods, and kits described herein may be implemented in whole or in part as computer executable instructions on a computer-readable medium. As will be understood by those skilled in the art, various steps of the compositions, methods, and kits described herein may be implemented as various blocks, operations, routines, tools, modules, and techniques, which may then be implemented in hardware, firmware, software, or any combination of hardware, firmware, and / or software. In certain embodiments, hardware implementations include, but are not limited to, custom integrated circuits (ICs), application-specific integrated circuits (ASICs), field-programmable logic arrays (FPGAs), programmable logic arrays (PLAs), and the like. In other embodiments, when implemented as software, the software may be stored on any computer-readable medium known in the art, including, but not limited to, solid-state disks, magnetic disks, optical disks, or other storage media, computer RAM or ROM or flash memory, processors, hard disk drives, thumb drives, optical disk drives, tape drives, and the like. In one embodiment, the software may be delivered to a user or computing system via any delivery method known in the art, including but not limited to communication channels such as the Internet, wireless connections, satellite connections, telephone lines, computer-readable disks, or other portable computer storage mechanisms.

[0223] A series of embodiments provide suitable computing system environments known in the art for implementing the compositions, methods, and kits described herein, including, but not limited to, distributed computing environments which include any of the above systems or devices, such as mobile phones, laptops, personal computers, server computers, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, cloud computing environments, and so on. In some embodiments, the steps of the compositions, methods, or kits described herein are implemented via computer executable instructions, such as program modules, which include, but not limited to, routines, programs, objects, components, data structures, etc., for performing a specific task or for implementing a specific abstract data type. In one embodiment, the methods and apparatus are implemented in a distributed computing environment in which tasks are performed by remote processing devices linked over a communication network. In one embodiment, the methods and apparatus are implemented in an integrated computing environment. In both integrated and distributed computing environments, program modules may reside on both local and / or remote computer storage media, including memory storage devices.

[0224] Accordingly, a series of embodiments provide a computer-readable medium having computer-executable instructions for determining the effects of administering a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug to a cell, animal, or human subject, comprising data indicating the level of at least one protein, RNA, biomarker, or other phenotype, and routines stored in the computer-readable medium and adapted to be executed by a processor to determine the effects of administering the compound, or its conjugate, complex, or composition from the data. In certain embodiments, the effects to be determined are changes in the level of IL-2 RNA, or changes in the level of IL-2 protein, or changes in one or more phenotypes, such as cell growth or proliferation, cell viability, cell morphology, cell cycle arrest, cell migration, cell invasion, apoptosis, survival, cancer cell growth or proliferation, cancer metastasis, immune cell proliferation, immune cell activation, cytokine production, autoimmunity, inflammation, vascular leakage, behavior, body weight, metabolism, etc. In one embodiment, a computer-readable medium is used to determine the progression of a disease such as cancer, autoimmune disease, inflammatory disease, infection, or vasoleap syndrome, as well as its response to administration of a compound of formula (I) disclosed herein, or its conjugate, complex, or pharmaceutical composition and drug to a human subject.

[0225] Another set of embodiments provides a computer-readable medium having computer-executable instructions for developing compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, using at least one computer method described herein, or other computer methods known to those skilled in the art and also included in the embodiments herein. The computer-readable medium may include data relating to specific nucleotide sequences, sequence numbers, or portions thereof disclosed herein, as well as any polypeptide sequences resulting from the transcription and translation of such nucleotide sequences. The computer-readable medium may also be adapted to be executed by a processor to develop compounds of formula (I) disclosed herein, or their conjugates, complexes, or pharmaceutical compositions, from such data.

[0226] Many modifications and variations can be made to the compositions, methods, and kits described herein without departing from the spirit and scope of the invention. Therefore, it should be understood that the compositions, methods, and kits described herein are illustrative and do not limit the scope of the invention. [Examples]

[0227] The following embodiments are provided to those skilled in the art to provide a complete disclosure and explanation of methods of carrying out and using the invention, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent or imply that the following experiments are all or only the experiments in which the invention was performed. Those skilled in the art will understand that numerous variations and / or modifications can be made to the invention shown in particular embodiments without departing from the spirit or scope of the invention as broadly described. It should also be understood that the embodiments provide possible guidance on the use of combined features of the disclosure for applying such compositions, methods and systems to other uses. Accordingly, these embodiments should be considered in all respects to be illustrative and not restrictive.

[0228] In certain embodiments, these examples can be implemented by a computer or other processing device that incorporates and / or executes software, in which case the methods and functions, the software, and the processor utilize special methods to analyze data.

[0229] Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, strengths, temperatures, etc.), but some experimental errors and deviations should be taken into account.

[0230] Example 1: Increase in IL2 protein expression by the compound of formula (I) Using a homogeneous time-resolved fluorescence (HTRF) assay, according to the following protocol, the level of IL-2 protein expressed by a human cell line (e.g., Jurkat) is quantified after treatment with the compound of formula (I) described herein. Jurkat cells are seeded at 1×10 cells 6Cells are seeded at a density of cells / ml, with 100,000 cells per well in a 96-well plate. After 1 hour, cells are treated with the test compound at final concentrations of approximately 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, or 30 μM. After a 2-hour incubation period, cells are stimulated by the addition of 12.5 ng / ml phorbol 12-myristate 13-acetate (PMA) and 0.25 μg / ml phytohemagglutinin (PHA), or alternatively, left unstimulated. Cell condition medium (supernatant) is assayed using the HTRF kit (PerkinElmer, 62HIL02PEG) according to the kit manufacturer's protocol at 3 hours and 24 hours after PMA / PHA stimulation or PMA / PHA unstimulation. In short, 16 μL of supernatant is added to 4 μL of pre-mixed IL-2 antibody solution (anti-human IL-2-Eu cryptotate antibody and anti-human IL-2-d2 antibody) prepared in detection buffer in a 384-well white-walled plate. After incubation at room temperature for 3 hours, fluorescence readings are performed at the emission wavelengths of the donor and acceptor fluorophores (620 nm and 665 nm, respectively) using an HTRF-compatible plate reader. For each sample, the ratio of acceptor and donor emission signals is calculated by dividing the fluorescence signal at 665 nm by the signal at 620 nm. The ratio thus obtained is converted to the concentration of IL-2 (pg / ml) using a standard curve prepared according to the kit manufacturer's protocol. The concentration of IL-2 in each sample can also be presented as a percentage of vehicle controls, such as non-compound, solvent-only (DMSO) treated samples. A percentage of IL-2 protein in the sample exceeding 100% compared to the vehicle control indicates an increase in IL-2 protein expressed and secreted due to the test compound, and vice versa.

[0231] Table 1 lists the IL-2 protein concentrations in each sample, as determined using the HTRF assay protocol described above, after 3-hour treatment with different concentrations of a specific compound of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitride) in the presence (+) or absence (-) of PMA / PHA stimulation. Certain compounds of formula (I) disclosed herein increase the IL-2 concentration level in the supernatant by at least 10, 20, 30, 50, 100, 200, 300, 500, 100, 100, 1000, 2000, 300, 500, 1000, 2000, 300, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 pg / ml at concentrations of approximately 500 μM, 100 μM, 500 μM, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 pg / ml. Certain compounds of formula (I) disclosed herein increase IL-2 concentration levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or 1000% compared to a vehicle control, at concentrations of about 500 μM, 100 μM, 50 μM, 20 μM, 10 μM, 10 μM, 0.05 μM, less than 0.01 μM, or lower.

[0232] Example 2: Effect of compound (I) on the growth and proliferation of cancer cells co-cultured with human PBMCs The effect of the compound of formula (I) described herein on the growth and proliferation of cancer cells co-cultured with human peripheral blood mononuclear cells (PBMCs) is determined using the CCK-8 assay. Five cancer cell lines derived from different types of cancer are tested: A375 (melanoma), BT549 (triple-negative breast cancer), HT-29 (colorectal cancer), PC-3 (prostate cancer), and Mia PaCa-2 (pancreatic cancer). Cancer cells are grown in complete RPMI-1640 medium and seeded in 96-well plates at 5,000 cells per well. Pooled PBMCs from different human donors are added to each well to obtain a final PBMC-to-cancer cell ratio of 10:1 (50,000 PBMCs vs. 5,000 cancer cells per well). After adding PBMCs, the test compound is added to the wells to achieve final concentrations of approximately 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, or 30 μM, and incubated at 37°C for 120 hours in a 5% CO2 atmosphere. After the incubation period, the plate is removed from the incubator, and the CCK-8 assay is performed according to the kit manufacturer's protocol to evaluate cancer cell growth and proliferation. Absorbance readings are measured at 450 nm using a spectrophotometer (Tecan® Infinite 200Pro). Cell viability in wells treated with the test compound is presented as a percentage of the viability of vehicle control wells treated with 0.5% DMSO instead of the test compound. Lower cell viability compared to the vehicle indicates an increase in cancer cell proliferation inhibition attributable to the compound of formula (I) described herein, and vice versa.

[0233] Table 2 provides percentage survival rates of cancer cells after treatment with compounds of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile) in concentrations of approximately 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, or 30 μM. Certain compounds of formula (I) result in a decrease in cancer cell growth and proliferation, as evidenced by the dose-dependent decrease in percentage survival rates of cancer cells after treatment with the test compound (Table 2). Certain compounds of formula (I) reduce the growth or survival rate of cancer cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the vehicle at concentrations of approximately 500 μM, 100 μM, 50 μM, 20 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, less than 0.01 μM, or lower. Certain compounds of formula (I) reduce IC50 at approximately 10 μM, 1 μM, 0.1 μM, 0.05 μM, less than 0.01 μM, or lower IC50. 50 This reduces the growth or survival rate of cancer cells.

[0234] Example 3: Effect of compound (I) on cancer cell growth and proliferation in a mini-CDX humanized mouse model The effects of the compounds of formula (I) described herein on the growth and proliferation of cancer cells in vivo are determined using a humanized mini-CDX (hollow fiber) mouse model using human peripheral blood mononuclear cells (PBMCs). Three cancer cell lines derived from different types of cancer: A375 (melanoma), PC-3 (prostate cancer), and Mia PaCa-2 (pancreatic cancer) are tested. Cancer cells were grown in complete RPMI-1640 medium and co-inoculated with human PBMCs in hollow fibers of different colors (KrosoFlo® implant membranes, green, blue, and white fibers) at an E:T ratio of 10:1, with both ends sealed. Three hollow fibers, each containing a different cancer cell line, were surgically subcutaneously implanted into the left and right flanks of mice (a total of six hollow fibers per mouse). Drug treatment was initiated on day 0, three days after fiber implantation. Each treatment group consisted of two mice (a total of 3-4 hollow fibers per cancer type, and 2 hollow fibers for the PBMC-only control). Group 1: Vehicle control (0.25% carboxymethylcellulose (CMC)), Group 2: Pembrolizumab control administered intraperitoneally (ip) at 10 mg / kg on day 0, Group 3: Compound of formula (I) administered orally once daily at 50 mg / kg for 5 days. At the end of treatment on day 5, all animals were euthanized with an excess dose of CO2. Fibers were excised from the mice, and the viability of cancer cells within the hollow fibers was determined using the CCK-8 assay. The absorbance of hollow fiber samples containing cancer cell-PBMC co-cultures was first subtracted by the mean absorbance of the PBMC-only control in each group to determine the absorbance attributable to cancer cells. The survival rate of cancer cells in hollow fibers from mice treated with the test compound is shown as a percentage of the survival rate of mice treated with the vehicle control. A lower survival rate of cancer cells compared to the vehicle indicates an increase in cancer cell proliferation inhibition attributable to the compound of formula (I) described herein, and vice versa.

[0235] Table 3 provides the percentage survival rates of cancer cells in mice after oral treatment once daily with 50 mg / kg of a compound of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile). Certain compounds of formula (I) result in a reduction in cancer cell growth and proliferation, as evidenced by the decrease in the percentage survival rate of cancer cells after treatment with the test compound (Table 3). Certain compounds of formula (I) reduce the growth or survival rate of cancer cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a vehicle control, at daily doses of approximately 500 mg / kg, 100 mg / kg, 50 mg / kg, 20 mg / kg, 10 mg / kg, or lower.

[0236] Example 4: Dose-dependent increase in IL2 in vivo after oral administration of the compound of formula (I) in mice Animals were fasted for 2 hours and administered a single oral dose (oral gastric tube, dose volume of 10 ml / kg) of the compound of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile) at 0.05, 0.1, 0.5, 5, and 50 mg / kg. Food was given 1 hour after dose administration. Blood was collected from each animal via the saphenous vein or submandibular vein on days 3, 7, 10, 14, 21, and 28 post-administration according to the experimental design. Final blood sampling was performed by cardiac puncture. Blood was collected in a 1.5 mL Eppendorf tube containing 0.010 mL of 10% K2 EDTA, gently mixed, and placed on ice before centrifugation. The blood was centrifuged at 10,000 rpm for 2 minutes to collect plasma, which was stored at -80°C before use in ELISA. The collected plasma sample was diluted 1:2 with sample diluent PT1-df (ELISA kit), and 100 μl of the diluted sample was transferred to an ELISA plate. The level of IL-2 in the sample was estimated using a standard curve generated with a commercially available mouse IL-2 ELISA kit (Proteintech KE10004).

[0237] Table 4 provides the levels of IL-2 in mouse plasma 28 days after administration. A single oral dose of the compound of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile) induced a dose-dependent increase in plasma IL-2 in mice that persisted for more than 4 weeks. Certain compounds of formula (I) disclosed herein increase the IL-2 concentration level in mouse plasma by at least 1,5,10,20,30,40,50,100,100 pg / ml at doses of approximately 1,000 mg / kg, 500 mg / kg, 100 mg / kg, 5 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg, less than 0.005 mg / kg, or lower.

[0238] Example 5: Efficacy of the compound of formula (I) for treating autoimmune diseases determined using a mouse model of DSS-colitis The efficacy of the compounds of formula (I) described herein for the in vivo treatment of autoimmune diseases was determined using a dextran sulfate sodium (DSS)-colitis mouse model. Colitis was induced in mice by continuously providing a 5% (w / v) DSS solution in sterile drinking water for 8 days (days 0-7). On day 8, DSS treatment was discontinued, and mice were provided with sterile drinking water without DSS for a further 6 days. The body weight of individual animals was recorded daily, and the percentage change in body weight compared to the body weight on day 0 was calculated. The disease activity index (DAI) was determined daily by averaging individual scores for body weight loss (0=0%, 1=1%-5%, 2=6%-10%, 3=11%-15%, 4=>15%), stool consistency (0=normal, 1=soft and shaped, 2=loose, 4=diarrhea), and bloody stool (0=none, 2=occult blood in feces, 4=bleeding).

[0239] Table 5 provides DAI scores at day 14 post-treatment of DSS-colitis mice with a single oral dose of a compound of formula (I) described herein (e.g., 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile). Certain compounds of formula (I) result in a reduction in inflammation and / or autoimmunity, as evidenced by the decrease in DAI score. Certain compounds of formula (I) reduce the DAI score by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the vehicle control at daily doses of approximately 500 mg / kg, 100 mg / kg, 50 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, 0.01 mg / kg, 0.005 mg / kg, or lower.

[0240] table Table 1: Changes in IL-2 protein levels after treatment with specific compounds of formula (I) at different concentrations. [Table 1] Table 2: Percentage survival rates of cancer cells after treatment with specific compounds of formula (I) at different concentrations. [Table 2] Table 3: Percentage of cancer cell survival in mini-CDX humanized mice after treatment with specific compounds of formula (I). [Table 3] Table 4: Mouse plasma IL-2 levels after treatment with a specific compound of formula (I) in a single oral dose. [Table 4] Table 5: DAI scores after treatment of DSS-colitis mice with specific compounds of formula (I) in single oral doses. [Table 5]

Claims

1. A compound of formula (I), 【Chemistry 1】 During the ceremony, A 1 However, hydrogen, halo, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, R 1 and R 4 are, independently, hydrogen, halo, C 1-7 -alkyl, C 2-7 -alkenyl, C 3-8 -cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Compounds in which cycloalkyl, aryl, and heterocyclyl groups are independently substituted or unsubstituted. Furthermore, pharmaceutically acceptable salts, tautomers, N-oxides, and solvates thereof.

2. A 1 However, hydrogen, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, R 1 However, hydrogen, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, R 4 However, hydrogen, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - The compound according to claim 1, wherein the cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted. Furthermore, pharmaceutically acceptable salts, tautomers, N-oxides, and solvates thereof.

3. A 1 but, 【Chemistry 2】 And in the formula, X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 - Alkyl, C 3-8 -Cycloalkyl, halo, haloalkyl, cyano, thiocyano, cyanato, thiocyanato, methoxyl, hydroxyl, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino, 0, 1, or more X substituents replace hydrogen with A 1 It is independently covalently bonded to one or more ring atoms, provided that the valence of X does not exceed that of the ring atom (or number of ring atoms) to which X is bonded. R 2 However, independently, hydrogen, halo, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - The compound according to claim 1, wherein the cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted.

4. A 1 but, 【Transformation 3】 Selected from the group consisting of, in the formula, R 2 However, independently, hydrogen, halo, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - The compound according to claim 1, wherein the cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted.

5. R 2 but, 【Chemistry 4】 Selected from the group consisting of, where X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 - Alkyl, C 3-8 -Cycloalkyl, halo, haloalkyl, cyano, thiocyano, cyanato, thiocyanato, methoxyl, hydroxyl, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino, 0, 1, or more X substituents replace hydrogen with R 2 The compound according to claim 3, wherein X is independently covalently bonded to one or more ring atoms, provided that X does not exceed the maximum valence of the ring atom (or more) to which it is bonded.

6. R 2 The compound according to claim 4, selected from the group consisting of the following. 【Transformation 5】

7. R 1 and R 4 The compound according to claim 5, wherein the compound is methyl.

8. R 1 and R 4 The compound according to claim 6, wherein the compound is methyl.

9. A 1 but, 【Transformation 6】 In this formula, X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 - Alkyl, C 3-8 -Cycloalkyl, halo, haloalkyl, cyano, thiocyano, cyanato, thiocyanato, methoxyl, hydroxyl, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino, 0, 1, or more X substituents replace hydrogen with A 1 It is independently covalently bonded to one or more ring atoms, provided that the valence of X does not exceed that of the ring atom (or number of ring atoms) to which X is bonded. R 2 However, independently, hydrogen, halo, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - The compound according to claim 2, wherein the cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted.

10. A 1 but, 【Transformation 7】 Selected from the group consisting of, in the formula, R 2 However, independently, hydrogen, halo, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - Cycloalkyl, aryl, or heterocyclyl, C 1-7 - Alkyl, C 2-7 - Alkenil, C 3-8 - The compound according to claim 2, wherein the cycloalkyl, aryl, and heterocyclyl are each independently substituted or unsubstituted.

11. R 2 but, 【Transformation 8】 Selected from the group consisting of, where X represents a non-hydrogen substituent (multiple substituents are possible), and each X independently represents deuterium, C 1-7 - Alkyl, C 3-8 -Cycloalkyl, halo, haloalkyl, cyano, thiocyano, cyanato, thiocyanato, methoxyl, hydroxyl, formyl, acetyl, 2-hydroxyacetyl, 2-hydroxypropanal, formamidyl, thiol, S-methyl, sulfonyl, methylsulfonyl, ethylsulfonyl, 1-methylcarboxamide, N-ethylcarboxamide, or N,N-dimethylamino, 0, 1, or more X substituents replace hydrogen with R 2 The compound according to claim 9, wherein X is independently covalently bonded to one or more ring atoms, provided that X does not exceed the maximum valence of the ring atom (or more) to which it is bonded.

12. R 2 The compound according to claim 10, selected from the group consisting of the following. 【Chemistry 9】

13. 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,2-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,6-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3,3-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-3,5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonilicate, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-4-fluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,5-difluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,5-trifluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,6-trifluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 2-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]-2,3,5,6-tetrafluoropiperidine-1-yl]-7,8-dihydro-5H-pyrano[4,3-b]pyridine-3-carbonitrile, 6-(3,5-dimethylpyrazole-1-yl)-2-[[1-(6-fluoroquinazoline-4-yl)azetidine-3-yl]methyl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(4,6-dimethylpyrimidine-2-yl)piperidine-4-yl]pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(5,6-dimethylpyrimidine-4-yl)piperidine-4-yl]pyridazin-3-one, 6-[4-[3-(3,5-dimethylpyrazole-1-yl)-6-oxopyridazine-1-yl]piperidine-1-yl]-3-methyl-1H-pyrimidine-2,4-dione, 2-[1-(2,5-dimethylpyrazole-3-carbonyl)piperidine-4-yl]-6-(3,5-dimethylpyrazole-1-yl)pyridazin-3-one, 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(3-methoxy-1-methylpyrazole-4-carbonyl)piperidine-4-yl]pyridazin-3-one, and A compound according to claim 1, selected from the group consisting of 6-(3,5-dimethylpyrazole-1-yl)-2-[1-(2-methylsulfanylpyridine-3-carbonyl)piperidine-4-yl]pyridazin-3-one, or a pharmaceutically acceptable salt thereof.

14. The compound according to any one of claims 1 to 13, wherein one or more hydrogen atoms are replaced by deuterium.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, and at least one of a pharmaceutically acceptable carrier, excipient, or diluent.

16. The pharmaceutical composition according to claim 15, wherein one or more hydrogen atoms are replaced by deuterium.

17. A method comprising administering a compound according to any one of claims 1 to 13 to a cell, an animal, or a human.

18. A method comprising administering the pharmaceutical composition according to claim 15 to cells, animals, or humans.

19. The method according to claim 18, wherein one or more hydrogen atoms are replaced with deuterium.

20. A method for increasing the expression or activity of IL-2 in cells or tissues, comprising administering a compound according to any one of claims 1 to 13 to cells, animals, or humans such that the expression or activity of IL-2 is increased.

21. A method for increasing the expression or activity of IL-2 in cells or tissues, comprising administering the pharmaceutical composition according to claim 15 to cells, animals, or humans such that the expression or activity of IL-2 is increased.

22. The method according to claim 21, wherein one or more hydrogen atoms are replaced with deuterium.

23. A method for treating, preventing, or improving a disease in a subject, comprising administering to the subject a compound according to any one of claims 1 to 13 such that the expression or activity of IL-2 is increased.

24. The method according to claim 23, wherein the disease is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

25. A method for treating, preventing, or improving a disease in a subject, comprising administering the pharmaceutical composition according to claim 15 to the subject such that the expression or activity of IL-2 is increased.

26. The method according to claim 25, wherein the disease is cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

27. The method according to claim 26, wherein one or more hydrogen atoms are replaced with deuterium.

28. Use of a compound according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for increasing the expression or activity of IL-2 in cells or tissues.

29. Use of the compound according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for increasing the expression or activity of IL-2 in cells or tissues, wherein one or more hydrogen atoms are substituted with deuterium.

30. Use of a compound according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for treating, preventing or improving cancer, autoimmune disease, inflammatory disease, or infection.

31. Use of the compound according to any one of claims 1 to 13 in the manufacture of a pharmaceutical product for treating, preventing or improving cancer, autoimmune disease, inflammatory disease, or infection, wherein one or more hydrogen atoms are substituted with deuterium.

32. Use of the pharmaceutical composition according to claim 15 in the manufacture of a pharmaceutical product for increasing the expression or activity of IL-2 in cells or tissues.

33. Use of the pharmaceutical composition according to claim 15, in the manufacture of a pharmaceutical for increasing the expression or activity of IL-2 in cells or tissues, wherein one or more hydrogens are substituted with deuterium.

34. Use of the pharmaceutical composition according to claim 15 in the manufacture of a pharmaceutical product for treating, preventing, or improving cancer, autoimmune diseases, inflammatory diseases, or infections.

35. Use of the pharmaceutical composition according to claim 15, in the manufacture of a pharmaceutical for treating, preventing or improving cancer, autoimmune diseases, inflammatory diseases, or infections, wherein one or more hydrogen atoms are substituted with deuterium.

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