Drug conjugates of imidazo quinoline amine derivatives, compositions and methods thereof

Abstract

The present invention provides novel drug-linker conjugates and antibody-drug conjugates of imidazo quinoline amine derivatives, which have agonistic activity against Toll-like receptors (TLRs), in particular TLR7 and / or TLR8, also provided are pharmaceutical compositions and methods of treatment against certain diseases or conditions mediated by or associated with TLR7 and / or TLR8 (e.g., graft rejection, autoimmunity, inflammation, allergy, asthma, infection, sepsis, cancer, and immunodeficiency).

Description

[0001] Priority claims and related applications This application claims priority to PCT International Application No. PCT / CN2023 / 108272, filed on July 20, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention generally relates to a novel compound and its therapeutic applications. More specifically, the invention provides novel drug-linker conjugates and antibody-drug conjugates of imidazoquinolineamine derivatives that have agonistic activity against Toll-like receptors (TLRs), particularly TLR7 and / or TLR8, and pharmaceutical compositions and treatment methods for treating certain diseases or conditions mediated or associated with TLR7 and / or TLR8 (e.g., graft rejection, autoimmunity, inflammation, allergy, asthma, infection, sepsis, cancer, and immunodeficiency). Background Technology

[0003] Toll-like receptors (TLRs) are an important component of the mammalian immune system, functioning by recognizing pathogen-associated molecular patterns (PAMPs), thus bridging the gap between innate and adaptive immunity (Iwasaki, et al. "Control of adaptive immunity by the innate immune system" Nat Immunol 2015, 16:343-353; O'Neill, et al. "The history of Toll-like receptors - redefininginnate immunity" Nat Rev Immunol 2013, 13:453-460.). TLRs play a central role in regulating innate immunity, and over the past decade, research has begun to reveal their significance as important therapeutic targets for infectious diseases, cancer, rheumatoid arthritis, and allergies. Currently, many TLR agonists are in clinical trials or have been approved as immunostimulants (Kaczanowska, 2013, 13:453-460). et al . "TLR agonists: our best frenemy in cancerimmunotherapy" J Leukoc Biol2013, 93:847-863.). Innate immunity begins with antigen-presenting cells (APCs), primarily dendritic cells (DCs) in peripheral tissues, which recognize PAMPs via TLRs (Iwasaki, 2013, 93:847-863). et al . "Toll-like receptor control of the adaptive immune responses" Nat Immunol 2004, 5:987-995.). The binding of TLR ligands to dendritic cells initiates a downstream signaling cascade, thereby inducing dendritic cell (DC) maturation. This process is characterized by the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-12), upregulation of co-stimulatory molecules (CD40, CD80, and CD86), enhanced antigen-presenting capacity, and the migration of DCs from peripheral tissues to draining lymph nodes. There, antigen-presenting DCs stimulate naive T cells, thereby initiating an adaptive immune response (Guo, 2004, 5:987-995.). et al . "TheNovel Toll-Like Receptor 2 Agonist SUP3 Enhances Antigen Presentation and TCell Activation by Dendritic Cells" Front Immunol 2017, 8:158; Silva-Cardoso, et al . "CXCL4 Exposure Potentiates TLR-Driven Polarization of Human Monocyte-Derived Dendritic Cells and Increases Stimulation of T Cells" J Immunol 2017, 199:253-262.). B cells also express TLRs and present antigens upon activation, thereby producing specific antibodies against the encountered antigens (Pone, et al . "B cell TLRs and induction of immunoglobulin class-switchDNA recombination" Front Biosci (Landmark Ed) 2012, 17:2594-2615.). Since the maturation of T cells and B cells is influenced by TLRs, co-stimulation, and cytokine signaling, TLR agonists play a crucial role in establishing the types of cellular and humoral immune responses produced (Bessa, 2012, 17:2594-2615). et al. "T cell-dependent and -independent IgA responses: role of TLR signaling" Immunol Invest 2010, 39:407-428; Booth, et al . "Modulation of B cell responses by Toll-like receptors" Cell Tissue Res (2011, 343:131-140.). Utilizing the potent immunostimulatory properties of TLR agonists, they hold great potential in the development of active immunotherapies for cancer.

[0004] Antibody-drug conjugates (ADCs) are a rapidly developing class of drugs designed to provide targeted therapy with long plasma half-lives, representing a promising treatment modality in cancer therapy. To date, 13 ADCs have received marketing approval from the U.S. Food and Drug Administration (FDA).

[0005] Currently available therapies and methods for managing diseases or conditions associated with TLR 7 and 8 are insufficient. There remains an urgent and ongoing need for novel and improved therapies to effectively treat these diseases and conditions. Summary of the Invention

[0006] This invention is partly based on novel drug-linker conjugates and antibody-drug conjugates (ADCs) of imidazoquinolineamine derivatives, which are agonists of Toll-like receptors (TLRs), particularly TLR7 and / or TLR8; the invention is also based on pharmaceutical compositions thereof, and methods for treating, alleviating or preventing certain diseases or conditions mediated by or associated with TLR7 and / or TLR8, such as graft rejection, autoimmunity, inflammation, allergy, asthma, infection, sepsis, cancer and immunodeficiency, and related diseases and conditions.

[0007] On the one hand, the present invention generally relates to a drug-linker conjugate having the structural formula (I): (I) in, L' is a group, including functional or reactive groups; D is the drug component, which has the following structure: in, R 1It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X is a single key or a connecting part; Or its pharmaceutically acceptable form or isotopic derivative.

[0008] On the other hand, the present invention generally relates to an antibody-drug conjugate comprising a pharmaceutical moiety having the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

[0009] On the other hand, the present invention generally relates to an antibody-drug conjugate having structural formula (II): (II) in, Ab is the antigen-binding portion; L stands for connector; D represents the drug component, which has the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 For H or P(=O)(CH3)2; n It is an integer selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

[0010] On the other hand, the present invention generally relates to a pharmaceutical composition comprising the antibody-drug conjugates disclosed herein.

[0011] On the other hand, the present invention generally relates to a unit dosage form comprising the pharmaceutical composition of the present invention.

[0012] On the other hand, the present invention generally relates to a method for treating, alleviating or preventing a disease or condition, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such treatment.

[0013] On the other hand, the present invention generally relates to a method for modulating an immune response, which includes administering the antibody-drug conjugate of the present invention to a subject in need of doing so.

[0014] On the other hand, the present invention generally relates to a method for modulating TLR7 and / or TLR8-mediated signal transduction, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such action.

[0015] On the other hand, the present invention generally relates to a method for treating or alleviating a condition or disease that can be treated by modulating TLR7 and / or TLR8-mediated cell activity, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such treatment.

[0016] On the other hand, the present invention generally relates to the use of the antibody-drug conjugates of the present invention in the treatment or relief of diseases or conditions.

[0017] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. General principles of organic chemistry, as well as specific functional moieties and reactivity, are described in the following references: Thomas Sorrell, “Organic Chemistry”, University Science Books, Sausalito: 1999; and Smith, MB and March, J., “March's Advanced Organic Chemistry”, 5th edition, John Wiley & Sons, New York: 2001; the entire contents of which are incorporated herein by reference.

[0018] The term "at least" as used in this article should be understood to mean that value as well as all values ​​greater than that value.

[0019] When used to define compositions and methods, the term "comprising" is intended to indicate that the composition and method include the stated element, but does not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" means that the composition and method include the stated element and exclude other elements that have any substantial significance for the composition and method. For example, "consisting essentially of" means the application of a explicitly listed pharmacologically active agent and excludes pharmacologically active agents that are not explicitly listed. The term "consisting essentially of" does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" means excluding trace elements and substantial method steps that are other components. Examples defined by each of these transitional terms are within the scope of this invention.

[0020] Certain compounds of this invention may exist in specific geometric or stereoisomeric forms. This invention contemplates that all such compounds, including cis and trans isomers, R- and S-enantiomers, diastereomers, (d)-isomers, (l)-isomers, racemic mixtures thereof, and other mixtures thereof, fall within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included in this invention.

[0021] According to the present invention, mixtures of isomers containing any of a variety of isomer ratios can be used. For example, when only two isomers are combined, the present invention contemplates mixtures containing isomer ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. Those skilled in the art will understand that the above ratios are also applicable to more complex mixtures of isomers.

[0022] For example, if a specific enantiomer of the compound of the present invention is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is isolated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with a suitable optically active acid or base, and the resulting diastereomeric salt is then resolved by stepwise crystallization or chromatographic methods well known in the art, and the pure enantiomer is subsequently recovered.

[0023] This invention also covers solvates and polymorphs of the compounds of this invention. Solvates of the compounds of this invention include, for example, hydrates.

[0024] The definitions of specific functional groups and chemical terms are described in more detail below. When numerical ranges are listed, the aim is to cover every value and subrange within that range. For example, "C1-6 alkyl" is intended to cover C1 and C2, C3, C4, C5, C6, C6, C7, C8, C9 ... 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 C 5-6 alkyl.

[0025] As used herein, the term "alkyl" refers to a straight-chain, branched, or cyclic hydrocarbon group consisting only of carbon and hydrogen atoms, without unsaturated bonds, and having one to ten carbon atoms (e.g., C1-10 alkyl). Whenever it appears herein, numerical ranges such as "1 to 10" refer to each integer within a given range; for example, "1 to 10 carbon atoms" means that an alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although this definition also covers the occurrence of the term "alkyl" where no numerical range is specified. In some embodiments, "alkyl" can be a C1-6 alkyl. In some embodiments, an alkyl group has 1 to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms.

[0026] Representative saturated straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; while saturated branched alkyl groups include, but are not limited to, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, and 2,3-dimethylbutyl. The alkyl group is linked to the parent molecule via a single bond.

[0027] Unless otherwise specified in the specification, the alkyl group may optionally be substituted with one or more substituents, which independently include: acyl, alkyl, alkenyl, alkynyl, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amide, amidine, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxyl, cyano, halogen (F, Cl, Br, I), haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate ester, phosphonate, hypophosphonate, silyl, sulfinyl, sulfonyl, sulfonamide, sulfoxide, sulfonate, urea, -Si(R a 3. -OR a -SR a -OC(O)-R a -N(R) a )2、-C(O)R a -C(O)OR a -OC(O)N(R) a )2、-C(O)N(R a )2、-N(R a )C(O)OR a -N(R) a )C(O)R a -N(R) a )C(O)N(R a )2、-N(R a )C(NR a )N(R a )2、-N(R a S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a (R) a ) or -OP(=O)(OR a )2, where each R a Independently, it is hydrogen, alkyl, haloalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocyclic alkyl, heterocyclic alkylalkyl, heteroaryl, or heteroarylalkyl, and each of these moieties may be optionally substituted as defined herein. In non-limiting embodiments, the substituted alkyl group may be selected from fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, and phenethyl.

[0028] As used herein, the term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, or phosphorus (including any oxidized form of nitrogen, sulfur, or phosphorus; any quaternized form of basic nitrogen; or a substituted nitrogen of a heterocycle, such as N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR). + (e.g., in N-substituted pyrroleyl groups).

[0029] As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). As used herein, the terms "halide" or "halogenated" refer to fluorinated, chlorinated, brominated, or iodinated compounds. The terms "haloalkyl," "haloalkenyl," "haloalkynyl," and "haloalkoxy" include alkyl, alkenyl, alkynyl, and alkoxy structures substituted with one or more halogenated groups or combinations thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy" respectively include halogenated alkyl and halogenated alkoxy groups with fluorine as the halogen, such as, but not limited to, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc. Each alkyl, alkenyl, alkynyl, and alkoxy group is as defined herein and may optionally be further substituted as defined herein.

[0030] As used herein, the terms "aromatic" or "aryl" refer to a group having 6 to 14 ring atoms (e.g., C64 ... 6-14 Aromatic or C 6-14An aryl group having at least one carbon ring with a conjugated π-electron system (e.g., phenyl, fluorenyl, and naphthyl). In some embodiments, the aryl group is a C6-10 aryl group. For example, a divalent group formed from a substituted benzene derivative and having a free valence on a ring atom is named a substituted phenylene. In other embodiments, a divalent group derived from a monovalent polycyclic hydrocarbon group ending in "-yl", formed by removing a hydrogen atom from a carbon atom having a free valence, is named by adding "-idene" to the name of the corresponding monovalent group; for example, a naphthyl group with two connection points is called a naphthylidene. Whenever it appears herein, numerical ranges such as "6 to 14 aryl" refer to every integer within a given range; for example, "6 to 14 ring atoms" means that an aryl group can consist of 6 ring atoms, 7 ring atoms, etc., up to and including 14 ring atoms. This term includes monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups. Polycyclic aryl groups include bicyclic, tricyclic, and tetracyclic groups. In polycyclic groups, only one ring is required to be aromatic; therefore, groups such as indanyl are included in the definition of aryl. Non-limiting examples of aryl groups include phenyl, phenalenyl, naphthalenyl, tetrahydronaphthyl, phenanthryl, anthraceneyl, fluorenyl, indole, and indanyl. Unless otherwise specified in the specification, the aryl moiety may optionally be substituted with one or more substituents, which independently include: acyl, alkyl, alkenyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amide, amidine, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxyl, cyano, halogen, haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate ester, phosphonate, hypophosphonate, silyl, sulfinyl, sulfonyl, sulfonamide, sulfoxide, sulfonate, urea, -Si(R a 3. -OR a -SR a -OC(O)-R a -N(R) a )2、-C(O)R a -C(O)OR a -OC(O)N(R) a )2、-C(O)N(R a )2、-N(R a )C(O)OR a -N(R) a )C(O)R a -N(R) a )C(O)N(Ra )2、-N(R a )C(NR a )N(R a )2、-N(R a S(O) t N(R a )2 (where t is 1 or 2), -P(=O)(R a (R) a ), or -OP(=O)(OR a )2, where each R a Independently, each of these moieties is hydrogen, alkyl, haloalkyl, carbocyclic, carbocyclic alkyl, aryl, aralkyl, heterocyclic alkyl, heterocyclic alkylalkyl, heteroaryl, or heteroarylalkyl, and each of these moieties may be optionally substituted as defined herein.

[0031] As used herein, the terms "TLR7 and / or TLR8 ligand," "TLR7 and / or TLR8 ligand," and "TLR7 and / or signal transduction agonist" refer to molecules other than the compounds disclosed herein that interact directly or indirectly with TLR7 and / or TLR8 through TLR7 and / or TLR8 domains other than the TLR8 domain, and induce TLR7 and / or TLR8-mediated signal transduction. In some embodiments, the TLR7 and / or TLR8 ligand is a natural ligand, i.e., a TLR7 and / or TLR8 ligand found in nature. In some embodiments, the TLR7 and / or TLR8 ligand refers to molecules other than natural ligands of TLR7 and / or TLR8, such as molecules prepared through human activities.

[0032] As used herein, the term "modulator" is defined as a compound that binds to and / or activates or inhibits a target with measurable affinity, or a compound that directly or indirectly affects the normal regulation of receptor activity. In some embodiments, the modulator has an EC50 of less than about 50 µM, less than about 1 µM, less than about 500 nM, less than about 100 nM, or less than about 10 nM. 50 and / or binding constant.

[0033] As used herein, the term "agonist" refers to a compound that, when bound to a receptor (e.g., a TLR), produces a cellular response. An agonist can be a ligand that binds directly to the receptor. Alternatively, an agonist can bind indirectly to the receptor, for example by (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise causing modification of another compound, thereby enabling that other compound to bind directly to the receptor. An agonist may be referred to as an agonist of a specific TLR (e.g., a TLR7 and / or TLR8 agonist).

[0034] As used herein, the term "antagonist" refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of the agonist or inverse agonist on the receptor. However, antagonists have no effect on the constitutive activity of the receptor. More specifically, antagonists are compounds that inhibit the activity of TLR7 or TLR8 at the TLR7 or TLR8 receptor, respectively.

[0035] As used herein, the term “inhibit” refers to any measurable reduction in biological activity. Therefore, as used herein, “inhibit” or “inhibition” can be expressed as a percentage of the normal activity level.

[0036] As used herein, the term "antigen" refers to any substance that elicits the production of antibodies or a specific cell-mediated immune response against it by the immune system. "Disease-associated antigen" refers to any substance associated with any disease that elicits the production of antibodies or a specific cell-mediated immune response against it by the immune system. Antigens are capable of being recognized by the immune system and / or inducing humoral and / or cellular immune responses, leading to the activation of B lymphocytes and / or T lymphocytes. Antigens may have one or more epitopes (B cell and / or T cell epitopes). Antigens preferably generally react with their corresponding antibodies or TCRs in a highly selective manner, rather than with a large number of other antibodies or TCRs that may be induced by other antigens. As used herein, antigens may also be a mixture of several individual antigens.

[0037] As used herein, the term "antibody" refers to a molecule capable of binding to an epitope or antigenic determinant. The term is intended to include whole antibodies and their antigen-binding fragments. It encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, Fab, Fv, single-chain antibodies, and monoimmunoglobulin variable chain or multiimmunoglobulin variable chain designs or CDR domain designs, as well as bispecific and multispecific antibodies. Antibodies can be derived from any animal. Preferably, antibodies are mammalian antibodies, such as those from humans, mice, rabbits, goats, guinea pigs, camels, horses, etc., or other suitable animals. Antibodies recognize polypeptide or polynucleotide antigens. The term includes active fragments, such as antigen-binding fragments comprising immunoglobulins, variable and / or constant regions of the heavy chain, variable and / or constant regions of the light chain, complementarity-determining regions (CDRs), and framework regions. These terms include polyclonal and monoclonal antibody formulations, as well as formulations including hybrid antibodies, altered antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (e.g., non-covalent heterodimers), dimer and trimer antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments derived from these molecules, wherein such fragments retain specific binding.

[0038] As used herein, the term “antigen-binding fragment” refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen epitope (e.g., through binding, steric hindrance, stabilization / instability, spatial distribution).

[0039] Examples of conjugated fragments include, but are not limited to: single-chain Fv (scFv), disulfide-bonded Fv (sdFv), Fab fragments, F(ab') fragments, and fragments composed of V... L V H C L and C H 1. A monovalent segment composed of structural domains; F(ab)2 segment, i.e., a divalent segment containing two Fab segments connected by disulfide bonds in the hinge region; composed of V H and C H Fd fragments composed of 1 structural domain; V-arms of the antibody L and V H Fv segments composed of structural domains; composed of V HdAb fragments composed of domains (Ward et al., 1989 Nature 341:544-546); and isolated complementarity-determining regions (CDRs), or epitope-binding fragments of other antibodies.

[0040] In addition, the two structural domains V of the Fv segment L and V H They can be linked by synthesizing linkers using recombinant methods, allowing them to be made into single protein chains, where V L and V H Regional pairing forms monovalent molecules (called single-chain Fvs (“scFvs”); see, for example, Bird et al., 1988). Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. (85:5879-5883). Such single-chain antibodies are also intended to be covered within the term "antigen-binding fragment". These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and their applicability is screened in the same manner as that of intact antibodies.

[0041] Antigen-binding fragments can also be incorporated into single-domain antibodies, macrobodies, minibody antibodies, nanobody antibodies, intrabody antibodies, diabody antibodies, triabody antibodies, tetrabody antibodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005). Nature Biotechnology 23:1126-1136). Antigen-binding fragments can be grafted into peptide-based scaffolds (such as type III fibronectin (Fn3)) (see, for example, U.S. Patent No. 6,703,199, which describes a fibronectin peptide monobody). The antigen-binding fragment can be incorporated into a single-chain molecule containing a pair of tandem Fv fragments (V...). H -C H 1-V H -C H 1) It forms a pair of antigen-binding regions together with complementary light chain polypeptides (Zapata et al., 1995 Protein Eng. 8:1057-1062; US Patent No. 5,641,870).

[0042] As used herein, the term "bispecific antibody" or "bispecific" refers to an antibody, typically a monoclonal antibody, that has binding specificity to at least two distinct antigenic epitopes. These epitopes may be derived from the same antigen or from two different antigens. Methods for preparing bispecific antibodies are known in the art. For example, bispecific antibodies can be generated recombinantly using the co-expression of two pairs of immunoglobulin heavy / light chain pairs. Alternatively, bispecific antibodies can be prepared using chemical linking. Bispecific antibodies comprise bispecific antibody fragments (see, for example, Milstein et al., 1983 Nature 305:537-39; Brennan et al., 1985 Science 229:81; Hollinger et al., 1994). Proc. Natl. Acad. Sci. USA 90:6444-48; Gruber et al., 1994 J. Immunol. 152:5368-74).

[0043] As used herein, the term "chimeric antibody" or "chimerism" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0044] As used herein, the term "fully human antibody" refers to an antibody with variable regions, where both the framework and CDR regions are derived from human sequences. Furthermore, if an antibody contains constant regions, those constant regions are also derived from such human sequences, such as human germline sequences, mutant versions of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis (e.g., Knappik et al., 2000). J. Mol. Biol. (As described in 296:57-86). Fully human antibodies may include amino acid residues encoded by non-human sequences, such as mutations introduced through random or site-directed mutagenesis in vitro, or through somatic mutations in vivo, or substitutions made to improve stability or facilitate production.

[0045] As used herein, the term "humanized antibody" refers to an antibody containing sequences derived from non-human (e.g., mouse) antibodies as well as human antibody sequences. Such antibodies are chimeric antibodies containing at least a minimum sequence derived from a non-human immunoglobulin. Typically, a humanized antibody comprises at least one, and usually substantially all, of two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of a non-human immunoglobulin, and all or substantially all frame regions (FR regions) are frame regions of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. (See, for example, Cabilly U.S. Patent No. 4,816,567; Queen et al., 1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; ANTIBODYENGINEERING: A PRACTICAL APPROACH, Oxford University Press 1996. ) As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically comprise multiple antibodies targeting (or specific to) different epitopes. "Monoclonal" indicates the property of the antibody being derived from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be prepared by various methods known in the art, including those described by Kohler et al., 1975. Nature The hybridoma method first described in 256:495, or the preparation method, can be used by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). For example, “monoclonal antibodies” can also be prepared using Clackson et al., 1991. Nature 352: 624-628 and Marks et al., 1991 J. Mol. Biol. The techniques described in 222: 581-597 are used to isolate monoclonal antibodies from phage antibody libraries. The binding dissociation constant (Kd) of these monoclonal antibodies is typically at least about 1 μM, more typically at least about 300 nM, typically at least about 30 nM, and preferably at least about 10 nM.

[0046] As used herein, the term "cleavable" linker refers to a linker or linker component that covalently connects two parts but breaks down under physiologically relevant conditions to sever the covalent connection between the parts. Typically, in vivo, cleavable linkers break down more rapidly in the intracellular environment than extracellularly, resulting in payload release preferentially occurring within the target cells. Cleavage can be enzymatic or non-enzymatic. The payload is typically released from the antibody without degrading the antibody. Cleavage may leave a portion of the linker or linker component still attached to the payload, or it may release the payload without any residual portion or component of the linker (i.e., traceless release).

[0047] As used herein, the term "non-cleavable" linker refers to a linker or linker component that is not easily degraded under physiological conditions; that is, its stability is at least comparable to that of the antibody or antigen-binding fragment of an immunoconjugate. Such linkers are sometimes referred to as "stable," meaning they possess sufficient degradation resistance to maintain the payload attached to the antigen-binding fragment until the antigen-binding fragment itself is at least partially degraded. In this case, in vivo, the degradation of the antibody precedes the cleavage of the linker. After partial degradation of the antibody in an immunoconjugate with a stable or non-cleavable linker, some or all of the linker, along with one or more amino acid groups from the antibody, may remain, allowing it to attach to the in vivo delivered payload or drug fragment.

[0048] As used herein, the terms “disease,” “condition,” or “disorder” are used interchangeably and refer to a pathological state, such as a state that can be identified by symptoms or other identifying factors as a deviation from a healthy or normal state. The term “disease” includes disorders, syndromes, symptoms, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0049] As used in this article, “in need of” refers to a subject who will benefit from the treatment in a biological, medical, or quality-of-life manner.

[0050] As used herein, the “effective amount” of an active agent means an amount sufficient to induce the desired biological response. Those skilled in the art will understand that the effective amount of the compounds of the present invention can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the route of administration, and the patient.

[0051] As used herein, the terms "treatment" or "treating" a disease or disorder refer to methods of reducing, delaying, or improving such a state before or after its occurrence. Treatment may target one or more effects or symptoms of the disease and / or its underlying pathology. Treatment can be any degree of relief and may include, but is not limited to, the complete elimination of the disease or its symptoms. Such relief or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an equivalent untreated control.

[0052] As used herein, the terms “prevention,” “preventive method,” or “for prevention” refer to a method of eliminating, delaying, avoiding, or stopping the onset, incidence, severity, or recurrence of a disease or condition. For example, a method is considered preventive if, compared to subjects who do not receive the method, the onset, incidence, severity, or recurrence of the disease or condition or one or more symptoms thereof is reduced or delayed. A disclosed method is also considered preventive if, after receiving the method, the onset, incidence, severity, or recurrence of osteoporosis or one or more symptoms of the disease or condition is reduced or delayed in subjects susceptible to the disease or condition compared to the progression of the disease before treatment. Therefore, a reduction or delay in the onset, incidence, severity, or recurrence of osteoporosis can be approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction between these values.

[0053] As used herein, the “pharmaceutically acceptable form” of the disclosed compounds includes, but is not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives thereof. In one embodiment, the “pharmaceutical acceptable form” includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives thereof. In some embodiments, the “pharmaceutical acceptable form” includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives thereof.

[0054] In some embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" means that, within the bounds of reliable medical judgment, it is suitable for contact with a subject's tissues without excessive toxicity, irritation, allergic reactions, etc., and has a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. in the *Journal of Pharmaceutical Science* (… J. Pharmaceutical SciencesPharmaceutically acceptable salts are described in detail in (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed by other methods commonly used in the art, such as ion exchange. Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptyl sulfate, glycerol phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodide, and 2-hydroxyethane. Sulfonates, lacturonates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, papoates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, p-valerates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. In some embodiments, organic acids from which salts can be derived include, for example: acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoroacetic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0055] The salts can be prepared in situ during the isolation and purification of the disclosed compounds, or they can be prepared separately, for example, by reacting the free base or free acid of the parent compound with a suitable base or acid, respectively. Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates). Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc., such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt may be selected from ammonium, potassium, sodium, calcium, and magnesium salts.

[0056] In some embodiments, the pharmaceutically acceptable form is a "solvent" (e.g., a hydrate). As used herein, the term "solvent" refers to a compound that also includes stoichiometric or non-stoichiometric amounts of solvent bound by non-covalent intermolecular forces. The solvate may be the disclosed compound or a pharmaceutically acceptable salt thereof. When the solvent is water, the solvate is a "hydrate". Pharmaceutically acceptable solvates and hydrates are, for example, complexes comprising, for example, 1 to about 100, 1 to about 10, 1 to about 2, about 3, or about 4 solvent or water molecules. It should be understood that the term "compound" as used herein encompasses the compound and its solvates, as well as mixtures thereof.

[0057] In some embodiments, the pharmaceutically acceptable form is a prodrug. As used herein, the term "prodrug" (or "pro-drug") refers to a compound that is converted in vivo to produce the disclosed compound or a pharmaceutically acceptable form of the compound. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound, for example, through hydrolysis (e.g., hydrolysis in the blood). In some cases, a prodrug has improved physical and / or delivery properties compared to the parent compound. A prodrug may increase the bioavailability of the compound when administered to a subject (e.g., by allowing enhanced absorption into the bloodstream after oral administration) or enhance delivery to a target biological compartment (e.g., the brain or lymphatic system) relative to the parent compound. Exemplary prodrugs include derivatives of the disclosed compound that have enhanced water solubility relative to the parent compound or active transport via the intestinal membrane.

[0058] Prodrug compounds often have advantages in solubility, tissue compatibility, or delayed release in mammalian organisms (see, for example, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). Discussions of prodrugs can be found in Higuchi, T. et al., "Pro-drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entirety. Exemplary advantages of prodrugs may include, but are not limited to, their physical properties, such as enhanced water solubility at physiological pH for parenteral administration compared to the parent compound, enhanced absorption in the digestive tract, or enhanced drug stability during long-term storage.

[0059] Commonly used prodrugs in this art include well-known acid derivatives, such as esters prepared by reacting a parent acid with a suitable alcohol; amides prepared by reacting a parent acid compound with an amine; and basic groups that react to form acylated base derivatives. Of course, other prodrug derivatives can be combined with other features disclosed herein to enhance bioavailability. Therefore, those skilled in the art will understand that certain compounds currently disclosed having free amino, amide, hydroxyl, or carboxyl groups can be converted into prodrugs. Prodrugs include compounds having amino acid residues, or polypeptide chains consisting of two or more (e.g., two, three, or four) amino acid residues covalently linked via peptide bonds to the free amino, hydroxyl, or carboxylic acid groups of the currently disclosed compounds. The amino acid residues include 20 naturally occurring amino acids, typically designated by three-letter symbols, and also include 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3-methylhistidine, norvaline, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. The prodrug also includes compounds having a carbonate, carbamate, amide, or alkyl ester moiety covalently bonded to any of the aforementioned substituents disclosed herein.

[0060] As used herein, the term "pharmaceutically acceptable" excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting a target drug from one organ or body site to another. Each carrier must be "acceptable" in the sense of compatibility with other components in the dosage form and harmlessness to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants, such as sodium dodecyl sulfate, magnesium stearate and polyoxyethylene-polyoxypropylene copolymer, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0061] As used herein, the terms “isolated” or “purified” refer to materials that are substantially or essentially free of the components that typically accompany them in their natural state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC).

[0062] As used herein, the term "subject" refers to any animal (e.g., a mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Generally, when referring to human subjects, the terms "subject" and "patient" are used interchangeably.

[0063] As used herein, the term "low dose" means at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) lower than the lowest standard recommended dose of a particular compound formulated for the treatment of any human disease or condition via a given route of administration. For example, a low dose formulated for inhalation administration will differ from a low dose formulated for oral administration of the same drug.

[0064] As used herein, the term “high dose” means at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dose of a particular compound for treating any human disease or condition.

[0065] Isotope-labeled compounds are also within the scope of this disclosure. As used herein, “isotope-labeled compound” or “isotope derivative” refers to the currently disclosed compounds described herein, including their pharmaceutical salts and prodrugs, wherein one or more atoms are replaced by atoms having an atomic mass or mass number different from those normally found in nature. Examples of isotopes that may be incorporated into the currently disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example… 2 H and 3 H, 13 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F, 36 Cl.

[0066] The compounds of this invention, labeled with isotopes, can be used for drug and / or substrate tissue distribution determination. (Tritium) 3 H) and carbon-14 ( 14 C) Labeled compounds are particularly preferred due to their ease of preparation and detectability. Furthermore, heavier isotopes (e.g., deuterium) are also preferred. 2 Substituting H) may offer certain therapeutic advantages due to its higher metabolic stability, such as prolonged in vivo half-life or reduced dosage requirements, and is therefore preferred in some cases. The isotope-labeled compounds disclosed in this invention, including their pharmaceutically acceptable salts, esters, and prodrugs, can be prepared by any method known in the art. Furthermore, those with high natural abundance... 12 C is replaced with 13 C also has beneficial effects. (See WO 2007 / 005643, WO 2007 / 005644, WO2007 / 016361 and WO 2007 / 016431) For example, deuterium ( 2H) is incorporated into the compounds disclosed in this invention to regulate the oxidative metabolism of the compounds through a first-order kinetic isotope effect. The first-order kinetic isotope effect refers to the change in chemical reaction rate due to isotopic nucleus substitution, which stems from the change in the ground-state energy required to form a covalent bond after isotopic substitution. Exchanging a heavier isotope typically leads to a decrease in the ground-state energy of the chemical bond, thereby reducing the rate-limiting bond breaking reaction rate. When this bond-breaking process occurs in or near a saddle point region in a multi-product reaction pathway, the product distribution ratio may change significantly. For example, if deuterium forms a non-exchangeable bond with a carbon atom, its reaction rate ratio (k...) is... M / k D The rate difference is typically between 2 and 7. If this rate difference is successfully applied to the easily oxidizable compounds disclosed in this invention, the characteristics of the compound in vivo can be significantly altered, and its pharmacokinetic properties can be improved.

[0067] In the discovery and development of therapeutic agents, those skilled in the art can optimize pharmacokinetic parameters while preserving favorable in vitro properties. It is reasonable to assume that many compounds with poor pharmacokinetic properties are susceptible to oxidative metabolism. Currently available in vitro liver microsomal assays provide important information about such oxidative metabolic processes, thereby enabling the rational design of tritium-substituted compounds of the present invention to enhance their stability by increasing resistance to oxidative metabolism. This can significantly improve the pharmacokinetic characteristics of the compounds of the present invention, which can be quantified by the following parameters: in vivo half-life (t / 2), maximum therapeutic concentration (C2). max Increased dose-response area (AUC) and bioavailability F, as well as reduced clearance, dosage and material costs.

[0068] The following aims to illustrate the above: A compound having multiple readily oxidative metabolic reaction sites, such as benzylic hydrogen atoms and hydrogen atoms bonded to nitrogen atoms, can be used to prepare a series of analogues in which these hydrogen atoms are replaced with deuterium atoms through different combinations, such that some, most, or all of the hydrogen atoms are replaced by deuterium atoms. Half-life determination allows for the advantageous and accurate measurement of the degree of improvement in antioxidant metabolic capacity. In this way, it can be determined that, through such deuterium-hydrogen exchange, the half-life of the parent compound can be extended by up to 100%.

[0069] The deuterium-hydrogen exchange in the compounds disclosed in this invention can also be used to advantageously modulate the metabolite profile of starting compounds to reduce or eliminate undesirable toxic metabolites. For example, if a toxic metabolite is formed through the oxidative breaking of carbon-hydrogen (CH) bonds, it can be reasonably assumed that even if this oxidation process is not a rate-determining step, the deuterated analogue can still significantly reduce or eliminate the formation of undesirable metabolites. Further information on deuterium-hydrogen exchange can be found in the following literature: Hanzlik et al., J. Org. Chem. 55, 3992-3997, 1990, Reider et al. , J. Org. Chem.52, 3326-3334, 1987, Foster, Adv. Drug Res.14, 1-40, 1985, Gillette et al, Biochemistry33(10) 2927-2937, 1994, and Jarman et al. Carcinogenesis16(4), 683-688, 1993.

[0070] After preparation, the compounds of the present invention are preferably separated and purified to obtain a component with a purity equal to or greater than 95% by weight (“substantially pure”), and then used or formulated as described herein. In some embodiments, the compounds of the present invention have a purity greater than 99%.

[0071] The combinations of substituents and variables contemplated in this invention are limited to those capable of forming stable compounds. "Stable" as used herein means having sufficient stability to allow its manufacture and to maintain the integrity of the compound for a sufficiently long period of time to be suitable for the applications described herein (e.g., therapeutic or prophylactic administration to a subject).

[0072] The enumeration of chemical groups for any variable in this specification includes defining the variable as a single listed group or any combination of listed groups. The description of embodiments of the variable also includes both single embodiments and combinations with any other embodiment or part thereof. Detailed Implementation

[0073] The present invention provides novel imidazoquinolineamine derivatives that are agonists of Toll-like receptors (TLRs), particularly TLR7 and / or TLR8; the present invention also provides pharmaceutical compositions thereof, and methods for treating, alleviating or preventing certain diseases or conditions mediated by or associated with TLR7 and / or TLR8 (e.g., graft rejection, autoimmunity, inflammation, allergy, asthma, infection, sepsis, cancer and immunodeficiency) or related diseases and conditions.

[0074] On the one hand, the present invention generally relates to a drug-linker conjugate having the structural formula (I): (I) in, L' is a group, including functional or reactive groups; D is the drug component, which has the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X is a single key or a connecting part; Or its pharmaceutically acceptable form or isotopic derivative.

[0075] In some embodiments, R 2 It's H.

[0076] In some embodiments, R 2 It is P(=O)(CH3)2.

[0077] In some embodiments, R 1 It is n-butyl and R 2 H is the drug-linker conjugate, which has the following structure: .

[0078] In some embodiments, R 1 It is n-butyl and R 2 It is P(=O)(CH3)2, and this drug-linker conjugate has the following structure: .

[0079] In some embodiments, n The value is 4.

[0080] In some embodiments, L' comprises para-aminobenzoic acid (PABA) and a dipeptide, and optionally, also comprises polyethylene glycol (PEG) units comprising m CH2-CH2O units, where m is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0081] In some embodiments, L' comprises Mal-Val-Cit-PABA (maleimide-valine-citrulline-p-aminobenzoic acid), Mal-Val-Ala-PABA (maleimide-valine-alanine-p-aminobenzoic acid), or Mal-Val-Cit-PABA, Mal-Val-Ala-PABA, Mal-Gly-Gly-Phe-Gly (maleimide-glycine-glycine-phenylalanine-glycine), M al-PEG2-Val-Cit-PABA, Mal-PEG8-Val-Ala-PABA, Mal-amido-PEG8-C2-acid, Py-MAA-Val-Cit-PAB (pyridyl-thioglycolic acid-valine-citrulline-p-aminobenzyl), CL2A, SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide), sulfonyl-SMCC Sodium (Sulfo-SMCC sodium, i.e., sodium 4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid sulfosuccinimide), SPDB (4-(2-pyridyldithio)butyric acid succinimide), SPDP (3-(2-pyridyldithio)propionic acid succinimide), DBCO (dibenzocyclooctylene), DBCO-NHCO-PEG4-amine (DBCO-NHCO-PEG4-amine), DBCO-PEG4-Gly-Gly-Phe-Gly, DBCO-PEG3-Val-Cit-PABA, DBCO-(PEG2-VC-PABA)2 or maleimide-DOTA (Maleimide-DOTA, i.e., 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-maleimide).

[0082] In some embodiments, L' is: .

[0083] In some embodiments, L' is selected from: .

[0084] In some embodiments, X is selected from: , , , , , , , .

[0085] Non-limiting examples of drug-linker conjugates include: On the other hand, the present invention generally relates to an antibody-drug conjugate comprising a pharmaceutical moiety having the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

[0086] On the other hand, the present invention generally relates to an antibody-drug conjugate having structural formula (II): (II) in, Ab is the antigen-binding portion; L stands for connector; D represents the drug component, which has the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 For H or P(=O)(CH3)2; n It is an integer selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

[0087] In some embodiments of antibody-drug conjugates, R 2 It's H.

[0088] In some embodiments of antibody-drug conjugates, R 2 It is P(=O)(CH3)2.

[0089] In some embodiments of antibody-drug conjugates, R 1 It is n-butyl and R 2 H is an antibody-drug conjugate having the following structure: .

[0090] In some embodiments of antibody-drug conjugates, R 1 It is n-butyl and R 2 It is P(=O)(CH3)2, and the antibody-drug conjugate has the following structure: .

[0091] In some embodiments of antibody-drug conjugates n The value is 4.

[0092] In some embodiments of antibody-drug conjugates, L represents a cleavable linker.

[0093] In some embodiments of antibody-drug conjugates, L is a linker sensitive to acid, lysosomal protease, β-glucuronide, or glutathione.

[0094] In some embodiments of antibody-drug conjugates, L represents a non-cleavable linker.

[0095] In some examples of antibody-drug conjugates, X is selected from: , , , , , , , .

[0096] In some embodiments of antibody-drug conjugates, Ab is an antibody.

[0097] In some examples of antibody-drug conjugates, Ab is a monoclonal antibody.

[0098] In some embodiments of antibody-drug conjugates, Ab is a chimeric antibody.

[0099] In some embodiments of antibody-drug conjugates, the Ab is a humanized antibody.

[0100] In some embodiments of antibody-drug conjugates, Ab is an antibody fragment.

[0101] In some examples of antibody-drug conjugates, the Ab is a polypeptide.

[0102] In some examples of antibody-drug conjugates, the Ab is selected from: trastuzumab, avelumab, mouse anti-human HER2 Ab, anti-mouse PD-L1 (B7-H1) Ab, atezolizumab, or other antibodies targeting the following targets: HER2, CD30, Netin-4, Trop3, CD79b, CD22, BCMA, TF, CD33, CD22, EGFR, TSHR, FSHR, LHR, CD19, FAP, and FR-alpha.

[0103] Non-limiting examples of antibody-drug conjugates include: On the other hand, the present invention generally relates to a pharmaceutical composition comprising the antibody-drug conjugates disclosed herein.

[0104] In some embodiments of antibody-drug conjugates, the pharmaceutical composition can effectively treat, prevent, or alleviate autoimmune diseases or related diseases or conditions.

[0105] In some embodiments of the antibody-drug conjugate, the pharmaceutical composition can effectively treat, prevent, or alleviate graft rejection or related diseases or conditions.

[0106] In some embodiments of antibody-drug conjugates, the pharmaceutical composition can effectively treat, prevent, or alleviate allergies or related diseases or conditions.

[0107] In some embodiments of antibody-drug conjugates, the pharmaceutical composition can effectively treat, prevent, or alleviate immunodeficiency or related diseases or conditions.

[0108] In some embodiments of the antibody-drug conjugate, the pharmaceutical composition can effectively treat, prevent, or alleviate infections, sepsis, or related diseases or conditions.

[0109] In some embodiments of the antibody-drug conjugate, the pharmaceutical composition can effectively treat, prevent, or alleviate cancer or related diseases or conditions.

[0110] On the other hand, the present invention generally relates to a unit dosage form comprising the pharmaceutical composition of the present invention.

[0111] On the other hand, the present invention generally relates to a method for treating, alleviating or preventing a disease or condition, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such treatment.

[0112] In some embodiments, the disease or condition is selected from: autoimmune diseases, graft rejection, allergies, immunodeficiency, infections, sepsis, cancer, and related diseases or conditions.

[0113] In some embodiments, the disease or condition is an autoimmune disease or a related disease or condition.

[0114] In some embodiments, the disease or condition is graft rejection or a related disease or condition.

[0115] In some embodiments, the disease or condition is an allergic or related disease or condition.

[0116] In some embodiments, the disease or condition is an immunodeficiency or related disease or condition.

[0117] In some embodiments, the disease or condition is an infection and / or sepsis, or a related disease or condition.

[0118] In some embodiments, the disease or condition is cancer or a related disease or condition.

[0119] On the other hand, the present invention generally relates to a method for modulating an immune response, which includes administering the antibody-drug conjugate of the present invention to a subject in need of doing so.

[0120] On the other hand, the present invention generally relates to a method for modulating TLR7 and / or TLR8-mediated signal transduction, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such action.

[0121] On the other hand, the present invention generally relates to a method for treating or alleviating a condition or disease that can be treated by modulating TLR7 and / or TLR8-mediated cell activity, comprising administering the antibody-drug conjugate of the present invention to a subject in need of such treatment.

[0122] On the other hand, the present invention generally relates to the use of the antibody-drug conjugates of the present invention in the treatment or relief of diseases or conditions.

[0123] On the other hand, the present invention generally relates to the use of the drug conjugates or antibody-drug conjugates of the present invention, as well as pharmaceutically acceptable excipients, carriers or diluents, in the preparation of medicaments for treating or alleviating diseases or conditions.

[0124] In some embodiments, the disease or symptom is associated with TLR7 and / or TLR8-mediated signal transduction.

[0125] In some embodiments, the disease or condition is selected from autoimmune diseases, graft rejection, allergies, immunodeficiency, infections, sepsis, cancer, or related diseases or conditions.

[0126] In some embodiments, the application is for treating or alleviating autoimmune diseases or related diseases or conditions.

[0127] In some embodiments, the application is for treating or alleviating graft rejection or related diseases or conditions.

[0128] In some embodiments, the application is used to treat or alleviate allergies or related diseases or conditions.

[0129] In some embodiments, the application is for treating or alleviating immune deficiencies or related diseases or conditions.

[0130] In some embodiments, the application is for the treatment or relief of infection and / or sepsis, or related diseases or conditions.

[0131] In some embodiments, the application is for treating or alleviating cancer or related diseases or conditions.

[0132] In some embodiments, the application is as a vaccine adjuvant.

[0133] In another aspect, the present invention relates to an antibody-drug conjugate consisting of an antibody (Ab), a linker (L), and a payload (D), as shown below: (DL) k -Ab (III) D, L, and Ab are as described above, and kThe value is an integer. The payload D can be coupled to different portions of the Ab, and can be linked, for example, via cysteine ​​or lysine residues. Typically, each Ab can link multiple payload D molecules. In some embodiments, k ranges from 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, k ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, k is 1, 2, 3, 4, 5, or 6. In some embodiments, k is 2, 3, or 4.

[0134] Drug-antibody ratio (DAR), or drug loading, can be characterized by conventional methods such as UV, mass spectrometry, ELISA assay, HIC, HPLC, or electrophoresis. In an exemplary embodiment, the DAR ranges from 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 2, 2 to 4, or about 1.

[0135] The DAR of immunoconjugates can be controlled through various methods, including limiting the molar excess of the payload-linker intermediate or linker reagent relative to the antibody; limiting the coupling reaction time or temperature; altering the reduction conditions for cysteine ​​thiol modification; and modifying the number and position of cysteine ​​residues and the linker-payload connection site. (See, for example, WO 2006 / 034488 A2.) To date, numerous unique antigens have been identified, and they may potentially be used as targets in antibody-based therapies. Several factors are typically considered when selecting an antigen. First, the target antigen should be highly expressed in tumors but not expressed or poorly expressed in healthy cells. An example is the HER2 receptor, which is expressed nearly 100 times more in tumor cells than in healthy cells. Second, the target antigen should be displayed on the surface of tumor cells so that circulating monoclonal antibodies can access it. Furthermore, the target antigen should possess endocytic properties, as this will facilitate ADC transport into cells, thereby enhancing the efficacy of the cytotoxic agent. Although some studies have demonstrated that non-endocytic ADC products targeting components of the tumor microenvironment can effectively dissociate their drugs in the extracellular space and mediate potent therapeutic activity in certain situations, ADCs often induce a strong "bystander effect." (Strohl2018) Protein & Cell 9(1):86-120; Damelin, et al 2015 Pharma. Res. 32(11):3494-507; Diamantis et al. 2016 British Journal of Cancer 114(4):362-7; Tipton et al. 2015 Blood125(12):1901-9; Donaghy et al.2016 mAbs 8(4):659-71; Casi et al.2015 Molecular Pharmaceutics 12(6):1880-4.) Typically, the antibody should preferably possess target specificity and target binding affinity, such as high binding affinity for tumor cell surface antigens. Furthermore, the antibody should preferably possess good retention, low immunogenicity, low cross-reactivity, and appropriate linker-binding properties. (Peters) et al 2015 Bioscience Reports 35(4);Hughes B2010 Nature Reviews Drug Discovery 9(9):665-7.).

[0136] In some embodiments, Ab is an antibody.

[0137] In some embodiments, Ab is a monoclonal antibody.

[0138] In some embodiments, Ab is a chimeric antibody.

[0139] In some embodiments, Ab is a humanized antibody.

[0140] In some embodiments, Ab is a bispecific antibody.

[0141] In some embodiments, Ab is an antibody fragment.

[0142] In some embodiments, Ab is a Fab fragment.

[0143] Antibodies and antibody fragments applicable to the immunoconjugates of the present invention include modified or engineered antibodies, for example, antibodies modified to introduce cysteine ​​residues or other reactive amino acids (including Pel, pyrrolidone, polypeptide tags and non-natural amino acids) to replace at least one amino acid in the natural sequence, thereby providing a reactive site on the antibody or antigen-binding fragment for conjugation with a cytotoxic agent.

[0144] Antibodies and antibody fragments can be readily produced by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. (See, for example, Carvalho et al. 2016 “Production Processes for Monoclonal Antibodies”, DOI: 10.5772 / 64263(https: / / www.intechopen.com / chapters / 51512); Monoclonal Antibody Production, Committee on Methods of Producing Monoclonal Antibodies, Institute for Laboratory Animal Research, National Research Council, NATIONAL ACADEMY PRESSWashington, DC 1999; Jakobovits 1998) Adv. Drug Del. Rev 31:33-42; Marks, et al 1991 J. Mol. Biol 222:581; Cole, et al 1985 Monoclonal Antibodies And Cancer Therapy 77-96; Teng, et al. 1983 Proc. Natl. Acad. Sci. USA 80:7308-7312; Kozbor, et al 1983 Immunology Today 4:72-79; Olsson, et al 1982 Meth. Enzymol . 92:3-16; US Pat. No. 6,657,103 B2.).

[0145] The compounds according to the invention can be used as agonists of TLRs (especially TLR7 and TLR8).

[0146] The compounds according to the invention can provide a method for modulating TLR7 and / or TLR8-mediated signal transduction. The method of the invention is useful, for example, when it is necessary to alter TLR7 and / or TLR8-mediated signal transduction in response to suitable TLR7 and / or TLR8 ligands or TLR7 and / or TLR8 signal transduction agonists.

[0147] The compounds according to the invention can be used to treat or prevent diseases and conditions, including but not limited to: cancer, immune complex-related diseases, inflammatory diseases, immunodeficiency, graft rejection, graft-versus-host disease, allergies, asthma, infections, and sepsis. More specifically, they are applicable to methods for treating conditions involving autoimmunity, inflammation, allergies, asthma, and graft rejection. Alternatively, methods for treating conditions involving infections, cancer, and immunodeficiency will generally employ the compounds disclosed herein, which enhance TLR7 and / or TLR8-mediated signaling in response to suitable TLR7 and / or TLR8 ligands. In some cases, the compositions can be used to inhibit or promote TLR7 and / or TLR8-mediated signaling in response to TLR7 and / or TLR8 ligands or signaling agonists. In other cases, the compositions can be used to inhibit or promote TLR7 and / or TLR8-mediated immune stimulation in a subject.

[0148] The compounds according to the present invention can also be used to treat or prevent liver cancer, bile duct cancer and malignant mesothelioma, pancreatic cancer, head and neck cancer and hemangioma.

[0149] The compounds according to the invention can also be used in the treatment or prevention of obesity in patients. The invention provides a method for treating type II diabetes in a patient with this need, comprising administering to the patient an effective amount of the compound disclosed herein or a pharmaceutically acceptable salt thereof. Preferably, the patient is a human. The invention provides a method for treating non-alcoholic steatohepatitis in a patient with this need, comprising administering to the patient an effective amount of the compound disclosed herein or a pharmaceutically acceptable salt thereof.

[0150] This invention provides the use of the compounds disclosed herein or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating obesity. This invention also provides the use of the compounds disclosed herein or pharmaceutically acceptable salts thereof in the preparation of therapeutic medicaments for providing therapeutic weight loss.

[0151] The amount of compounds in the compositions of the present invention is such that they can effectively and measurably regulate TLRs, particularly TLR7 and / or TLR8 or mutants thereof, in biological samples or patients. In some embodiments, the amount of compounds in the compositions of the present invention is such that they can effectively and measurably regulate TLRs or mutants thereof in biological samples or patients. In some embodiments, the compositions of the present invention are formulated for administration to patients in need.

[0152] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the compounds described herein or their derivatives are mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (i) a filler or extender, such as starch, lactose, sucrose, glucose, mannitol, and silica, (ii) a binder, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic, (iii) a humectant, such as glycerin, (iv) a disintegrant, such as agar, calcium carbonate, potato or cassava starch, alginate, certain complex silicates, and sodium carbonate, (v) a solution barrier, such as paraffin, (vi) an absorption enhancer, such as a quaternary ammonium compound, (vii) a wetting agent, such as cetyl alcohol and glyceryl monostearate, (viii) an adsorbent, such as kaolin and bentonite, and (ix) a lubricant, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. For capsules, tablets, and pills, the dosage forms may also contain buffers. Similar types of solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules, using excipients such as lactose or toffee and high molecular weight polyethylene glycol. Solid dosage forms such as tablets, sugar-coated tablets, capsules, pills, and granules can be prepared with coatings and shells (such as enteric coatings and other coatings and shells known in the art).

[0153] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cosolvents, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, or mixtures of these substances. Besides such inert diluents, the composition may also include additional pharmaceutical agents such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, or fragrances.

[0154] The materials, compositions, and components disclosed herein can be applied to, combined with, or used to prepare for application to the disclosed methods and compositions, or as products of the disclosed methods and compositions. It should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, although specific references to every individual and collective combination and arrangement may not be explicitly disclosed, each combination and arrangement is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and many modifications that can be made to the many molecules included in the method are discussed, then every combination and arrangement of the method and its possible modifications is specifically contemplated unless the opposite is specifically stated. Similarly, any subset or combination of these materials is specifically contemplated and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in methods applying the disclosed compositions. Therefore, if various additional steps are available to be performed, it should be understood that each of these additional steps can be performed in conjunction with any particular method step or combination of method steps of the disclosed method, and each such combination or subset of combination is specifically contemplated and should be considered disclosed.

[0155] The compositions of the present invention can be administered orally, parenterally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. The term "parentereal" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrasheath, intrahepatic, intralesional, intratumoral, and intracranial injection or infusion techniques.

[0156] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form. Exemplary oral dosage forms are capsules, tablets, aqueous suspensions, or solutions. For tablets for oral administration, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are often added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension is required for oral administration, the active ingredient is combined with an emulsifier and a suspending agent. Optionally, sweeteners, flavoring agents, or coloring agents may also be added if desired.

[0157] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the pharmaceutical preparation with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0158] The pharmaceutically acceptable compositions of the present invention can also be administered topically, particularly when the therapeutic target includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower digestive tract. Suitable topical formulations can be readily prepared for each of these areas or organs.

[0159] Local application to the lower digestive tract can be achieved through rectal suppositories (see above) or suitable enemas. Transdermal patches can also be used.

[0160] For topical application, the provided pharmaceutically acceptable compositions are formulated in suitable ointments, wherein the active ingredient is suspended or dissolved in one or more carriers. Exemplary carriers for topical administration of the compounds of the present invention are mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the provided pharmaceutically acceptable compositions may be formulated in suitable lotions or creams, wherein the active ingredient is suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl wax, cetearyl alcohol, 2-octyldodecanool, benzyl alcohol, and water.

[0161] The pharmaceutically acceptable compositions of the present invention are optionally administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulation and are prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers for improving bioavailability, fluorocarbons and / or other conventional solubilizers or dispersants.

[0162] Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0163] The amount of the compounds of the invention, optionally combined with a carrier material to produce a single-dose composition, will vary depending on the host being treated and the specific route of administration. Preferably, the provided compositions should be formulated so that the compounds can be administered to patients receiving these compositions at doses between 0.01 and 100 mg / kg body weight / day.

[0164] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, sex, diet, timing of administration, excretion rate, drug combination, as well as the judgment of the treating physician and the severity of the specific disease being treated. The amount of the compounds of the present invention in the composition will also depend on the specific compounds in the composition.

[0165] The following examples are intended to illustrate the practice of the invention and not to limit it in any way.

[0166] Example As shown in the following examples, in some exemplary embodiments, the compounds are prepared according to the following general procedure. It should be understood that although the general method describes the synthesis of certain compounds of the present invention, the following general method, as well as other methods known to those skilled in the art, can be applied to all compounds as described herein and to each of the subclasses and species of these compounds.

[0167] The compound numbers used in the following examples correspond to the compound numbers described above.

[0168] 1 H and 13 C10 NMR was recorded at 400 MHz on a Varian Mercury 400 spectrometer. Proton chemical shifts are represented by residual proton resonances in internal standard reference CDCl3 (7.26 ppm). Carbon chemical shifts are represented by deuterated solvent signals in internal standard reference CDCl3 (77.20 ppm).

[0169] LC-MS spectra were recorded on a Shimadzu LC-MS2020 using an Agilent C18 column (Eclipse XDB-C18, 5 μm, 2.1 x 50 mm) at a flow rate of 1 mL / min. Mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid-acetonitrile solution. A general gradient method was used.

[0170] Analytical HPLC was performed on an Agilent 1200 HPLC system equipped with a Zorbax Eclipse XDB C18 column (2.1 x 150 mm) at a flow rate of 1 mL / min. Mobile phase A: 0.1% aqueous TFA solution; Mobile phase B: 0.1% TFA acetonitrile solution. The general method used the following gradient: Preparative HPLC was performed on a Varian ProStar using a Hamilton C18 PRP-1 column (15 x 250 mm) at a flow rate of 20 mL / min. Mobile phase A was 0.1% aqueous TFA solution; mobile phase B was 0.1% TFA acetonitrile solution. A typical gradient method was employed.

[0171] Example 1. Synthesis of 4-(4-acetylphenoxy)-N-(4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)butyramide (1) Synthesis Route 1 Step 1: In 0 o At C, ethyl 4-bromobutyrate (10.74 g, 55.06 mmol) was added to a mixture of compound 1-1 (5.00 g, 36.72 mmol) and potassium carbonate (10.15 g, 73.44 mmol) in acetone (75 mL). The mixture was slowly brought to room temperature and stirred for 5 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (100 mL) and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 10% ethyl acetate in petroleum ether) to give compound 1-2 (7.63 g, 83% yield) as a white solid.

[0172] Step 2: In 0 o At C, LiOH (383 mg, 15.99 mmol) was added to a solution of compounds 1-2 (2.00 g, 7.99 mmol) in tetrahydrofuran (20 mL) and water (10 mL). The mixture was slowly heated to room temperature and stirred for 2 hours. After the reaction was complete, the mixture was concentrated and the pH was adjusted to 3 with 1 N HCl. The aqueous solution was extracted with ethyl acetate (50 mL x 2). The combined organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Crude product 1-3 (1.49 g, 84% yield) was given as a white solid, which was directly used in the next step without further purification.

[0173] Step 3: Add EDCI (35 mg, 0.18 mmol) to a solution of compounds 1-4 (50 mg, 0.12 mmol), compounds 1-3 (35 mg, 0.16 mmol), DMAP (2 mg, 0.016 mmol), and DIPEA (31 mg, 0.24 mmol) in anhydrous DMF (3 mL). Stir the mixture at room temperature for 3 hours. After the reaction is complete, filter and concentrate the mixture. Purify the residue by preparative HPLC (C18 column, eluting with acetonitrile in water). Lyophilize the desired fraction to give compound 1 (40 mg, 54% yield) as a white solid.1 H NMR (400 MHz, DMSO- d 6) δ 8.28 –8.20 (m, 2H), 7.93 (ddd, J = 10.0, 8.2, 1.5 Hz, 1H), 7.89 – 7.80 (m, 2H), 7.03– 6.93 (m, 2H), 4.39 (t, J = 6.4 Hz, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.16 (t, J =6.8 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.45 (s, 3H), 2.27 (t, J = 7.3 Hz, 2H), 1.93 (dp, J = 21.6, 6.7 Hz, 4H), 1.83 (q, J = 7.6 Hz, 2H), 1.75 (d, J = 13.4 Hz, 6H), 1.66 (q, J = 7.6, 7.2 Hz, 2H), 1.49 – 1.35 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). LCMS: m / z C 32 H 42 Calculated N5O5P: 607.69; Measured: 608.64 [M+H] + .

[0174] Example 2.3 Synthesis of ((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-4-((2-aminoethyl)amino)cyclobut-3-ene-1,2-dione (2) Synthesis Route 2 Step 1: A solution of compound 2-2 (1.0 g, 6.24 mmol) in anhydrous methanol (10 mL) was added dropwise to a solution of compound 2-1 (1.06 g, 6.23 mmol) in anhydrous methanol (10 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with 30% ethyl acetate in petroleum ether) to give compound 2-3 (1.62 g, 91% yield).

[0175] Step 2: A solution of compound 2-4 (339 mg, 1.03 mmol) and DIPEA (333 mg, 2.57 mmol) in anhydrous methanol (4 mL) was slowly added to a solution of compound 2-3 (441 mg, 1.55 mmol) in anhydrous methanol (3 mL). The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with methanol in dichloromethane) to give compound 2-5 (445 mg, 76% yield) as a pale yellow solid. LCMS: m / z C 29 H 39 Calculated N7O5: 565.67; Measured: 566.72 [M+H] + . Step 3: Add trifluoroacetic acid (1 mL) to a solution of compounds 2-5 (445 mg, 0.78 mmol) in DCM (3 mL). Stir the mixture at room temperature for 1 hour. After the reaction is complete, concentrate the mixture. Purify the residue by preparative HPLC (C18 column, eluting with acetonitrile in water). Lyophilize the desired fraction to give compound 2 (295 mg, 81% yield) as a white solid. 1 H NMR (600 MHz, DMSO- d 6) δ 13.91 (s, 1H), 8.52 (s, 2H), 8.21(d, J = 8.2 Hz, 1H), 8.11 (s, 3H), 7.83 (d, J = 8.3 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.64 (t, J = 7.7 Hz, 1H), 4.45 (t, J= 6.5 Hz, 2H), 3.71 (s, 2H), 3.64 (s,2H), 3.04 – 2.97 (m, 4H), 2.03 (p, J = 6.7 Hz, 2H), 1.83 (dq, J = 20.6, 7.4 Hz, 4H), 1.44 (h, J = 7.4 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H). LCMS: m / z C 24 H 31 Calculated N7O3: 465.56; Measured: 466.49 [M+H] + .

[0176] Example 3. Synthesis of (4-amino-2-butyl-1-(4-(methylamino)butoxy)-1H-imidazo[4,5-c]quinoline-7-yl)dimethylphosphine oxide (3) Synthesis Route 3 Step 1: At room temperature, a mixture of compound 3-1 (2.0 g, 10.57 mmol), imidazole (1.8 g, 26.42 mmol), DMAP (129 mg, 1.06 mmol), and TBS-Cl (2.39 g, 15.85 mmol) in dichloromethane (20 mL) was stirred for 8 hours. After the reaction was complete, the mixture was diluted with dichloromethane (100 mL) and washed with water (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 5% ethyl acetate in petroleum ether) to give compound 3-2 (2.9 g, 90% yield) as a colorless oil.

[0177] Step 2: Under a nitrogen atmosphere, at 0 °C, NaH (60% of mineral oil, 554 mg, 13.84 mmol) was added to a solution of compound 3-2 (2.1 g, 6.92 mmol) in anhydrous DMF (8 mL). After stirring for 0.5 hours, iodomethane (1.47 g, 10.38 mmol) was added. The resulting mixture was brought to room temperature and stirred for 16 hours. After the reaction was complete, the mixture was partitioned between ice water (100 mL) and ethyl acetate (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 5% ethyl acetate in petroleum ether) to give compound 3-3 (1.8 g, 82% yield) as a colorless oil.

[0178] Step 3: At 0 °C, TBAF (1 M in THF, 8.5 mL, 8.51 mmol) was added to a solution of compound 3-3 (1.8 g, 5.67 mmol) in THF (5 mL). The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate (100 mL) and water (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 10% ethyl acetate in petroleum ether) to give compound 3-4 (1.05 g, 91% yield) as a colorless oil.

[0179] Step 4: Under a nitrogen atmosphere, at 0 °C, a solution of tetrabromomethane in THF (10 mL) was slowly added to a mixture of compounds 3-4 (1.05 g, 5.16 mmol) and PPh3 (2.03 g, 7.74 mmol) in THF (20 mL). After stirring at room temperature for 4 hours, the mixture was filtered and the filtrate was concentrated. The residue was purified by silica gel column chromatography (eluting with 5% ethyl acetate in petroleum ether) to give compound 3-5 (1.08 g, 78% yield) as a colorless oil.

[0180] Step 5: Under a nitrogen atmosphere, a mixture of compounds 3-6 (530 mg, 1.65 mmol), potassium carbonate (457 mg, 3.31 mmol), and compounds 3-5 (530 mg, 1.99 mmol) in DMF (5 mL) was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was partitioned between ethyl acetate (50 mL) and water (50 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 30% ethyl acetate in petroleum ether) to give compound 3-7 (530 mg, 63% yield) as a pale yellow solid. LCMS: m / z C 24 H 33 Calculated value of BrN4O3: 505.46; Measured value: 505.54 [M+H] + .

[0181] Step 6: At 0 °C, hydrogen peroxide (30%, 0.5 ml) and m-CPBA (362 mg, 2.10 mmol) were added to a solution of compound 3-7 (530 mg, 1.05 mmol) in dichloromethane (10 mL). The mixture was slowly brought to room temperature and stirred for 10 hours. The resulting mixture was diluted with dichloromethane (50 mL) and washed with saturated sodium carbonate (50 mL x 2). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude intermediate was redissolved in dichloromethane (10 mL), and a solution of ammonium hydroxide (25%, 0.6 ml) and TsCl (500 mg, 2.62 mmol) in dichloromethane (10 mL) was added. After stirring at room temperature for 18 hours, the resulting mixture was diluted with dichloromethane (50 mL) and washed with saturated sodium bicarbonate. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in petroleum ether) to give compounds 3-8 (500 mg, 90% yield) as an off-white solid. LCMS: m / z C 24 H 34 Calculated value of BrN5O3: 520.47; Measured value: 520.55 [M+H] + .

[0182] Step 7: Under a nitrogen atmosphere, a mixture of compounds 3-8 (500 mg, 0.96 mmol), dimethylphosphine oxide (300 mg, 3.843 mmol), xantphos (167 mg, 0.288 mmol), palladium acetate (65 mg, 0.288 mmol), and potassium phosphate (510 mg, 2.40 mmol) in DMF (5 mL) was stirred at 120 °C for 16 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated. The residue was purified by reversed-phase rapid chromatography (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compounds 3-9 (360 mg, 72% yield) as a yellow solid. LCMS: m / z C 26 H 40 Calculated N₅O₄P: 517.61; Measured: 518.69 [M+H] + .

[0183] Step 8: Add HCl in ethyl acetate solution (4 N, 3 mL) to a solution of compounds 3-9 (360 mg, 0.695 mmol) in ethyl acetate and methanol (v:v = 3:1, 3 mL). After stirring at room temperature for 1 hour, concentrate the mixture. Purify the residue by preparative HPLC (C18 column, eluting with acetonitrile in water). Lyophilize the desired fraction to give compound 3 (250 mg, 86% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 8.28 (dd, J = 8.3, 2.5 Hz, 1H), 8.24 (d, J = 12.3 Hz, 1H), 7.95 (t, J = 9.2 Hz, 1H), 4.43 (t, J = 6.3Hz, 2H), 3.00 (dt, J = 14.4, 7.5 Hz, 4H), 2.57 (s, 3H), 2.00 (t, J = 7.7 Hz, 2H), 1.86 (p, J = 8.4, 7.5 Hz, 4H), 1.78 (s, 3H), 1.74 (s, 3H), 1.44 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H). LCMS: m / z C21 H 32 Calculated N₅O₂P: 417.49; Measured: 418.47 [M+H] + .

[0184] Example 4. Synthesis of 4-(4-acetylphenoxy)-N-(4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)-N-methylbutyramide (4) Synthesis Route 4 Step 1: EDCI (40 mg, 0.209 mmol) was added to a solution of compound 3 (56 mg, 0.134 mmol), compounds 1-3 (45 mg, 0.202 mmol), DMAP (2 mg, 0.016 mmol), and DIPEA (36 mg, 0.278 mmol) in anhydrous dichloromethane (5 mL). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 4 (62 mg, 74% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 8.31 – 8.19(m, 2H), 7.95 (d, J = 8.8 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.80 – 7.72 (m, 1H),7.01 (ddd, J = 8.6, 5.2, 2.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 1H), 4.41 (t, J = 7.3Hz, 2H), 4.07 (q, J = 6.2 Hz, 2H), 3.47 – 3.36 (m, 2H), 3.08 – 2.84 (m, 5H), 2.47 (d, J = 4.3 Hz, 3H), 2.40 (d, J = 9.9 Hz, 2H), 2.02 – 1.80 (m, 7H), 1.77 (s, 3H), 1.74 (s, 3H), 1.70 (d, J= 6.9 Hz, 1H), 1.48 – 1.36 (m, 2H), 0.92 (t, J =7.3 Hz, 3H). LCMS: m / z C 33 H 44 Calculated N5O5P: 621.72; Measured: 622.77 [M+H] + .

[0185] Example 5. Synthesis of 4-(4-acetylphenoxy)-N-(2-((2-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)butanamide (5) Synthesis Route 5 Step 1: EDCI (62 mg, 0.32 mmol) was added to a solution of compound 2 (100 mg, 0.21 mmol), compounds 1-3 (56 mg, 0.25 mmol), DMAP (3 mg, 0.02 mmol), and DIPEA (53 mg, 0.41 mmol) in anhydrous dichloromethane (8 mL). The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 5 (65 mg, 46% yield) as a white solid. 1 H NMR (600 MHz, DMSO- d 6) δ 13.68 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 8.12 (s, 1H), 8.04 (s, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.74 (t, J = 7.1 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 6.96(s, 2H), 4.42 (t, J = 6.5 Hz, 2H), 4.01 (t, J= 6.5 Hz, 2H), 3.62 (s, 2H), 3.23(s, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.48 (s, 3H), 2.25 (t, J = 7.3 Hz, 2H), 2.03 –1.97 (m, 2H), 1.94 (p, J = 6.5 Hz, 2H), 1.81 (tt, J = 14.3, 7.2 Hz, 4H), 1.43 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). LCMS: m / z C 36 H 43 Calculated N7O6: 669.78; Measured: 670.86 [M+H] + .

[0186] Example 6.3 Synthesis of ((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)amino)-4-((2-(methylamino)ethyl)amino)cyclobut-3-ene-1,2-dione (6) Synthesis Route 6 Step 1: Under a nitrogen atmosphere at 0 °C, a solution of compound 6-1 (1.1 g, 6.31 mmol) was added to a solution of compound 2-1 (1.1 g, 6.46 mmol) in MeOH (5 mL). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 10% ethyl acetate in petroleum ether) to give compound 6-2 (1.5 g, 79% yield) as a white solid.

[0187] Step 2: To a solution of compound 6-2 (620 mg, 2.08 mmol) in anhydrous MeOH (5 mL), add a solution of compound 2-4 (460 mg, 1.40 mmol) and DIPEA (365 mg, 2.80 mmol) in anhydrous MeOH (3 mL). Stir the mixture at room temperature for 4 hours. After the reaction is complete, concentrate the mixture. Purify the residue by silica gel column chromatography (eluting with 40% ethyl acetate in petroleum ether) to give compound 6-3 (700 mg, 86% yield) as a white solid.

[0188] Step 3: Trifluoroacetic acid (2 mL) was added to a solution of compound 6-3 (700 mg, 1.21 mmol) in dichloromethane (6 mL). After stirring at room temperature for 1 hour, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water) to give compound 6 (467 mg, 80% yield) as a white solid. 1 H NMR (600MHz, DMSO- d 6) δ 13.87 (s, 1H), 8.93 (s, 2H), 8.51 (s, 2H), 8.22 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 4.45 (t, J = 6.5 Hz, 2H), 3.78 (s, 2H), 3.65 (s, 2H), 3.14 – 3.06 (m, 2H), 3.01(t, J = 7.6 Hz, 2H), 2.58 (t, J = 5.4 Hz, 3H), 2.03 (p, J = 6.8 Hz, 2H), 1.83 (dp, J = 13.6, 7.5 Hz, 4H), 1.44 (dq, J = 14.8, 7.4 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H). LCMS: m / z C 25 H 33Calculated N7O3: 479.59; Measured: 480.42 [M+H] + .

[0189] Example 7.3 Synthesis of ((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-4-((2-aminoethyl)amino)cyclobut-3-ene-1,2-dione(7) Synthesis Route 7 Step 1: A solution of compounds 1-4 (270 mg, 0.67 mmol) and DIPEA (173 mg, 1.34 mmol) in anhydrous methanol (4 mL) was slowly added to a mixture of compounds 2-3 (285 mg, 1.00 mmol) in anhydrous methanol (3 mL). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 20% methanol in dichloromethane) to give compound 7-1 (209 mg, 48% yield) as a white solid. LCMS: m / z C 31 H 44 Calculated N7O6P: 641.71; Measured: 664.89 [M+Na] + .

[0190] Step 2: Trifluoroacetic acid (1 mL) was added to a solution of compound 7-1 (204 mg, 0.32 mmol) in DCM (4 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 7 (141 mg, 81% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 8.32 – 8.26 (m, 1H), 8.23 ​​(d, J =12.4 Hz, 1H), 7.96 (t, J = 9.3 Hz, 1H), 4.44 (t, J = 6.6 Hz, 2H), 3.74 – 3.68 (m,2H), 3.62 (s, 2H), 3.01 (t, J = 7.2 Hz, 4H), 2.00 (q,J = 7.3 Hz, 2H), 1.79 (t, J =15.0 Hz, 10H), 1.43 (p, J = 7.3 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H). LCMS: m / zC 26 H 36 Calculated N7O4P: 541.59; Measured: 564.87 [M+Na] + .

[0191] Example 8.3 Synthesis of ((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)amino)-4-((2-(methylamino)ethyl)amino)cyclobut-3-ene-1,2-dione (8) Synthesis Route 8 Step 1: A solution of compounds 1-4 (170 mg, 0.42 mmol) and DIPEA (110 mg, 0.85 mmol) in anhydrous methanol (3 mL) was slowly added to a mixture of compound 6-2 (188 mg, 0.63 mmol) in anhydrous methanol (3 mL). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 20% methanol in dichloromethane) to give compound 8-1 (160 mg, 58% yield) as a white solid. LCMS: m / z C 32 H 46 Calculated N7O6P: 655.74; Measured: 679.16 [M+Na] + .

[0192] Step 2: Trifluoroacetic acid (1 mL) was added to a solution of compound 8-1 (150 mg, 0.23 mmol) in DCM (4 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 8 (115 mg, 90% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 8.27 (d, J= 6.5 Hz, 1H), 8.19 (d, J = 12.4 Hz, 1H), 7.90 (t, J = 8.8 Hz, 1H), 4.44 (s, 2H), 3.76 (s, 2H), 3.69 (s,2H), 3.12 (s, 2H), 3.00 (s, 2H), 2.59 (s, 3H), 1.98 (s, 2H), 1.76 (d, J = 13.5Hz, 10H), 1.42 (d, J = 7.5 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). LCMS: m / z C 27 H 36 Calculated N7O4P: 555.62; Measured: 556.70 [M+H] + .

[0193] Example 9. Synthesis of (1-(4-aminobutoxy)-2-butyl-4-hydroxy-1H-imidazo[4,5-c]quinoline-7-yl)dimethylphosphine oxide (9) Synthesis Route 9 Step 1: At 0 °C, hydrogen peroxide (30%, 0.8 mL) and m-CPBA (552 mg, 3.20 mmol) were added to a solution of compound 9-1 (785 mg, 1.60 mmol) in dichloromethane (20 mL). The mixture was slowly brought to room temperature and stirred for 10 hours. The resulting mixture was diluted with dichloromethane (80 mL) and washed with saturated sodium carbonate (80 mL x 2). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude intermediate was redissolved in dichloromethane (10 mL), and a solution of ammonium hydroxide (25%, 0.2 mL) and TsCl (762 mg, 4.00 mmol) in dichloromethane (20 mL) was added. After stirring at room temperature for 18 hours, the resulting mixture was diluted with dichloromethane (50 mL) and washed with saturated sodium bicarbonate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 50% ethyl acetate in petroleum ether) to give compound 9-2 (495 mg, 61% yield) as a pale yellow solid.

[0194] Step 2: Under a nitrogen atmosphere, a mixture of compound 9-2 (470 mg, 0.93 mmol), dimethylphosphine oxide (216 mg, 2.77 mmol), xantphos (160 mg, 0.27 mmol), palladium acetate (30 mg, 0.13 mmol), and potassium phosphate (322 mg, 2.33 mmol) in DMF (5 mL) was stirred at 120 °C for 16 hours. The reaction mixture was cooled to room temperature, filtered, and concentrated. The residue was purified by reversed-phase rapid chromatography (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 9-3 (316 mg, 68% yield) as a yellow solid. 1 H NMR (400MHz, DMSO- d 6) δ 11.76 (s, 1H), 8.09 (d, J = 5.4 Hz, 1H), 7.90 (d, J = 12.5 Hz, 1H), 7.63 (t, J = 9.0 Hz, 1H), 7.01 – 6.89 (m, 1H), 4.33 (t, J = 6.3 Hz, 2H), 3.02 (d, J = 6.3 Hz, 2H), 2.94 – 2.87 (m, 2H), 1.87 (d, J = 7.5 Hz, 2H), 1.81 –1.76 (m, 2H), 1.71 (s, 3H), 1.67 (s, 3H), 1.63 (s, 2H), 1.45 – 1.41 (m, 2H),1.38 (s, 9H), 0.94 (t, J = 7.3 Hz, 3H). LCMS: m / z C 25 H 37 Calculated N4O5P: 504.57; Measured: 505.87 [M+H] + .

[0195] Step 3: To a solution of compound 9-3 (100 mg, 0.20 mmol) in ethyl acetate and methanol (v:v = 3:1, 3 mL), HCl in ethyl acetate solution (4 N, 3 mL) was added. After stirring at room temperature for 1 hour, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 9 (45 mg, 56% yield) as a white solid.1 H NMR (400 MHz, DMSO- d 6) δ 11.78 (s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 12.0 Hz, 3H), 7.70 – 7.58 (m, 1H), 4.36(t, J = 5.7 Hz, 2H), 2.91 (t, J = 7.4 Hz, 4H), 2.01 – 1.93 (m, 2H), 1.85 – 1.77(m, 4H), 1.70 (d, J = 13.3 Hz, 6H), 1.47 – 1.38 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). LCMS: m / z C 20 H 29 Calculated N4O3P value: 404.45; Measured value: 405.70 [M+H] + .

[0196] Example 10. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)carbamate (10) Synthesis Route 10 Step 1: 2-Chlorotriphenylmethyl chloride resin (0.71 mmol / g, 14.08 g, 10 mmol) was swollen under nitrogen atmosphere in dichloromethane (40 mL) for 30 minutes. After removing the dichloromethane, the first amino acid Fmoc-L-Cit-OH (4.76 g, 11.98 mmol), dichloromethane (80 mL), and DIPEA (6.18 g, 47.81 mmol) were added. The mixture was saturated for 2 hours, and unreacted Cl groups were blocked with methanol (7 mL) for 30 minutes. The resin was then washed with DMF and dichloromethane. Fmoc was then removed with 20% piperidine in DMF for 30 minutes. After removing the DMF solution containing Fmoc, the resin was washed again with DMF (80 mL x 4). The next amino acid was added under the following coupling conditions: Fmoc-AA-OH (30 mmol), HBTU (28.5 mmol), and DIPEA (60 mmol) were reacted in DMF for 30 min. The following amino acids were then coupled sequentially: Fmoc-L-Val-OH and 6-maleimide hexanoic acid. Finally, the desired linear peptide was cleaved from the resin using 20% ​​hexafluoroisopropanol in DCM for 30 min. The filtrate was concentrated under reduced pressure at 35 °C to give compound 10⁻¹ (1.42 g, 30% yield) as a white solid. The crude product was used directly in the next step without further purification.

[0197] Step 2: EEDQ (1.37 g, 5.56 mmol) and 4-aminobenzyl alcohol (514 mg, 4.17 mmol) were added to a solution of compound 10⁻¹ (1.3 g, 2.78 mmol) in DCM (20 mL) and MeOH (10 mL). The mixture was stirred at room temperature in the dark for 16 hours. The resulting mixture was filtered and concentrated. The residue was purified by silica gel column chromatography (eluting with 10% methanol in dichloromethane) to give compound 10⁻² (350 mg, 22% yield) as a white solid. LCMS: m / z C 28 H 40 Calculated N₆O₇: 572.66; Measured: 595.88 [M+Na] + .

[0198] Step 3: Add DIPEA (316 mg, 2.44 mmol) and bis(4-nitrophenyl) carbonate (372 mg, 1.22 mmol) to a mixture of compound 10⁻² (350 mg, 0.61 mmol) in DMF (3 mL). Stir the mixture at room temperature for 5 hours. After the reaction is complete, dilute the mixture with DCM (30 mL) and wash with water (30 mL). Concentrate the organic layer, and purify the residue by reversed-phase chromatography. Lyophilize the desired fraction to give compound 10⁻³ (210 mg, 47% yield) as a white solid. LCMS: m / z C 35 H 43 N7O 11 Calculated value: 737.77; Measured value: 738.87 [M+H] + .

[0199] Step 4: Add DIPEA (26 mg, 0.201 mmol), compounds 1-4 (45 mg, 0.108 mmol), and HOBt (14 mg, 0.102 mmol) to a solution of compound 10-3 (50 mg, 0.068 mmol) in DMF (1 mL). After stirring at room temperature under a nitrogen atmosphere for 12 hours, filter and concentrate the mixture. Residue was redissolved in dichloromethane (20 mL) and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative TLC (eluting with MeOH in dichloromethane) to give compound 10 (20 mg, 29% yield) as a white solid. LCMS: m / z C 49 H 68 N 11 O 10 Calculated value of P: 1002.12; Measured value: 1003.12 [M+H] + . 1 H NMR (400 MHz, DMSO- d 6) δ 10.00 (s, 1H), 8.13 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.96 (d, J = 13.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.65 – 7.56 (m, 3H), 7.34(s, 1H), 7.28 (d, J= 8.3 Hz, 2H), 6.99 (s, 2H), 6.80 (s, 2H), 5.98 (s, 1H), 5.41 (s, 2H), 4.96 (s, 2H), 4.35 (q, J = 7.2 Hz, 3H), 4.19 (q, J = 7.6 Hz, 1H), 3.13 (s, 2H), 2.96 (s, 4H), 2.14 (s, 2H), 1.91 (s, 3H), 1.82 (s, 2H), 1.70(d, J = 13.3 Hz, 9H), 1.64 – 1.37 (m, 11H), 1.27 (s, 2H), 0.94 (s, 3H), 0.84 (s, 6H).

[0200] Example 11. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)(methyl)carbamate (11) Synthesis Route 11 Step 1: To a solution of compound 10-3 (50 mg, 0.068 mmol) in DMF (1 mL), DIPEA (26 mg, 0.201 mmol), compound 3 (45 mg, 0.108 mmol), and HOBt (14 mg, 0.102 mmol) were added. After stirring at room temperature under a nitrogen atmosphere for 12 hours, the mixture was filtered and concentrated. The residue was redissolved in dichloromethane (20 mL) and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative TLC (eluting with MeOH in dichloromethane) to give compound 11 (19 mg, 27% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 10.03 (s, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 8.08(d, J= 7.5 Hz, 1H), 8.03 – 7.98 (m, 1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.65 (t, J = 9.3 Hz, 1H), 7.59 (s, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.20 (s, 1H),7.00 (s, 1H), 5.99 (t, J = 5.8 Hz, 1H), 5.41 (s, 2H), 5.00 (s, 2H), 4.36 (d, J =8.2 Hz, 3H), 4.18 (t, J = 7.8 Hz, 1H), 2.97 (dq, J = 12.8, 6.7 Hz, 4H), 2.89 (s,3H), 2.89 (s, 2H), 2.15 (tq, J = 14.1, 7.3 Hz, 2H), 2.04 – 1.92 (m, 3H), 1.85(d, J = 22.9 Hz, 3H), 1.72 (d, J = 13.3 Hz, 9H), 1.63 – 1.35 (m, 11H), 1.20 –1.13 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H), 0.86 – 0.80 (m, 6H). LCMS: m / zC 50 H 70 N 11 O 10 Calculated value of P: 1016.15; Measured value: 1007.25 [M+H] + .

[0201] Example 12. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(2-((2-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)(methyl)carbamate (12) Synthesis Route 12 Step 1: To a solution of compound 10-3 (68 mg, 0.092 mmol) in DMF (1 mL), DIPEA (36 mg, 0.276 mmol), compound 6 (44 mg, 0.092 mmol), and HOBt (19 mg, 0.138 mmol) were added. After stirring at room temperature under a nitrogen atmosphere for 12 hours, the mixture was filtered and concentrated. The residue was redissolved in dichloromethane (20 mL) and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative TLC (eluting with MeOH in dichloromethane) to give compound 12 (7 mg, 7% yield) as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 9.99 (s, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 7.80 (s, 1H), 7.74 (s, 1H), 7.62 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.47 (s, 1H),7.25 (s, 2H), 6.99 (s, 2H), 5.99 (s, 1H), 5.41 (s, 2H), 4.96 (s, 2H), 4.40(s, 3H), 4.18 (s, 1H), 3.64 (s, 4H), 3.41 (s, 2H), 3.01 (dd, J = 13.3, 6.7 Hz,1H), 2.96 (s, 3H), 2.87 (s, 3H), 2.16 (s, 1H), 2.12 (s, 1H), 1.97 (s, 4H),1.80 (d, J = 7.6 Hz, 4H), 1.69 (s, 1H), 1.59 (s, 1H), 1.45 (d, J = 25.2 Hz, 10H), 1.18 (s, 2H), 0.93 (s, 3H), 0.83 (d, J = 15.2 Hz, 9H). LCMS: m / z C54H71N13O11 Calculated value: 1078.24; Measured value: 1079.13 [M+H] + .

[0202] Example 13. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(2-((2-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate (13) Synthesis Route 13 Step 1: To a solution of compound 10-3 (50 mg, 0.068 mmol) in DMF (1 mL), DIPEA (26 mg, 0.204 mmol), compound 2 (50 mg, 0.108 mmol), and HOBt (14 mg, 0.102 mmol) were added. After stirring at room temperature under a nitrogen atmosphere for 12 hours, the mixture was filtered and concentrated. The residue was redissolved in dichloromethane (20 mL) and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative TLC (eluting with MeOH in dichloromethane) to give compound 13 (16 mg, 22% yield) as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 9.96 (s, 1H), 8.76 (d, J = 46.7 Hz, 1H), 8.17 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.72 (t, J =7.9 Hz, 1H), 7.57 (d, J = 8.1 Hz, 3H), 7.46 (s, 1H), 7.35 (d, J = 5.6 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 7.00 (s, 2H), 5.97 (s, 1H), 5.41 (s, 2H), 4.94 (s,2H), 4.43 (t, J = 6.5 Hz, 2H), 4.38 (t, J= 7.1 Hz, 1H), 4.18 (dd, J = 8.6, 6.7 Hz, 1H), 3.59 (d, J = 43.6 Hz, 4H), 3.36 (s, 3H), 3.18 (d, J = 6.0 Hz, 2H), 3.01 (dt, J = 15.5, 7.2 Hz, 3H), 2.93 (dd, J = 13.1, 6.5 Hz, 1H), 2.14 (ddt, J = 28.5, 14.2,7.1 Hz, 2H), 2.03 – 1.92 (m, 3H), 1.83 (dt, J = 15.5, 7.8 Hz, 4H), 1.68 (s,1H), 1.58 (d, J = 9.6 Hz, 1H), 1.45 (dp, J = 22.2, 7.3 Hz, 8H), 1.37 (s, 1H), 1.18 (p, J = 7.9 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H), 0.84 (d, J = 6.7 Hz, 3H), 0.81(d, J = 6.7 Hz, 3H). LCMS: m / z C 53 H 69 N 13 O 11 Calculated value: 1064.22; Measured value: 1065.21 [M+H] + .

[0203] Example 14. Synthesis of (1r,4r)-N-(4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide (14) Synthetic route 14 Step 1: At 0 °C, compound 14-1 (210 mg, 0.63 mmol) was added to a solution of compound 1-4 (210 mg, 0.52 mmol) and DIPEA (100 mg, 0.77 mmol) in DMF (3 mL). The mixture was slowly brought to room temperature and stirred for 3 hours. After the reaction was complete, the mixture was filtered and purified by preparative HPLC (eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 14 (180 mg, 55% yield) as a white solid. 1 H NMR (400MHz, DMSO- d 6) δ 8.13 (dd, J = 8.2, 3.0 Hz, 1H), 7.97 (d, J = 11.9 Hz, 1H), 7.79(t, J = 5.6 Hz, 1H), 7.61 (t, J = 8.5 Hz, 1H), 7.01 (s, 2H), 6.92 (s, 2H), 4.34(t, J = 6.5 Hz, 2H), 3.24 (d, J = 7.1 Hz, 2H), 3.13 (q, J = 6.4 Hz, 2H), 2.95 (t, J =7.7 Hz, 2H), 2.03 (d, J = 23.9 Hz, 1H), 1.93 – 1.85 (m, 2H), 1.82 (t, J = 7.6 Hz,2H), 1.72 (s, 3H), 1.68 (s, 4H), 1.67 – 1.59 (m, 4H), 1.56 – 1.49 (m, 1H),1.43 (q, J = 7.5 Hz, 2H), 1.35 – 1.23 (m, 3H), 0.95 (d, J = 7.4 Hz, 3H), 0.88(dd, J = 15.0, 11.7 Hz, 2H). LCMS: m / z C 32 H 43 Calculated N6O5P: 622.71; Measured: 623.80 [M+H] + .

[0204] Example 15. Synthesis of (1r,4r)-N-(2-((2-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide (15) Synthetic Route 15 Step 1: At 0 °C, compound 14-1 (255 mg, 0.76 mmol) was added to a solution of compound 2 (300 mg, 0.64 mmol) and DIPEA (125 mg, 0.97 mmol) in DMF (4 mL). The mixture was slowly brought to room temperature and stirred for 3 hours. After the reaction was complete, the mixture was filtered and purified by preparative HPLC (eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 15 (250 mg, 57% yield) as a white solid. 1 H NMR (400MHz, DMSO- d 6) δ 8.22 – 8.17 (m, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.75 (t, J = 7.8Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 6.97 (s, 2H), 4.44 (t, J = 6.4 Hz, 2H), 3.62(s, 4H), 3.19 (d, J = 7.1 Hz, 4H), 3.00 (t, J = 7.6 Hz, 2H), 2.04 – 1.93 (m, 3H), 1.83 (dt, J = 15.2, 7.6 Hz, 4H), 1.68 (d, J = 10.8 Hz, 2H), 1.57 (d, J = 11.5 Hz, 2H), 1.42 (dd, J = 14.7, 7.3 Hz, 3H), 1.24 (q, J = 12.9 Hz, 2H), 0.97 – 0.91 (m,3H), 0.85 (q, J= 12.5, 11.3 Hz, 2H). LCMS: m / z C 36 H 44 Calculated N8O6: 684.80; Measured: 685.75 [M+H] + .

[0205] Example 16. Synthesis of 2,5-dioxopyrrolidine-1-yl 2-((1-(((1r,4r)-4-((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidine-3-yl)thio)acetate (16) Synthesis Route 16 Step 1: Thiothioacetic acid (38 mg, 0.41 mmol) was added to a solution of compound 14 (170 mg, 0.27 mmol) in anhydrous acetonitrile (3 mL). The mixture was stirred at room temperature for 19 hours. After the reaction was complete, the mixture was concentrated and purified by HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 16-1 (101 mg, 51% yield) as a white solid. LCMS: m / z C 34 H 47 Calculated N6O7PS: 714.82; Measured: 716.01 [M+H] + .

[0206] Step 2: Under a nitrogen atmosphere, N-hydroxysuccinimide (24 mg, 0.212 mmol) and DCC (43 mg, 0.212 mmol) were added to a solution of compound 16-1 (39 mg, 0.054 mmol) in anhydrous DCM (1 mL) and anhydrous DMF (0.2 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, insoluble impurities were removed by filtration, and the filtrate was concentrated to give compound 16 (crude product, 42 mg, 95% yield) as a yellow solid. The crude product was used directly in the next step without further purification. LCMS: m / z C 38 H 50 Calculated N7O9PS: 811.89; Measured: 813.08 [M+H] + .

[0207] Example 17. Synthesis of 2,5-dioxopyrrolidine-1-yl 2-((1-(((1s,4s)-4-((2-((2-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamoyl)cyclohexyl)methyl)-2,5-dioxopyrrolidine-3-yl)thio)acetate (17) Synthesis Route 17 Step 1: Thiothioacetic acid (41.7 mg, 0.45 mmol) was added to a solution of compound 15 (124 mg, 0.18 mmol) in anhydrous acetonitrile (3 mL). The mixture was stirred at room temperature for 19 hours. After the reaction was complete, the mixture was concentrated and purified by preparative HPLC (C18 column, eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 17-1 (83 mg, 51% yield) as a white solid. LCMS: m / z C 38 H 48 Calculated N8O8S: 776.91; Measured: 778.02 [M+H] + .

[0208] Step 2: Under a nitrogen atmosphere, N-hydroxysuccinimide (24 mg, 0.212 mmol) and DCC (43 mg, 0.212 mmol) were added to a solution of compound 17-1 (55 mg, 0.07 mmol) in anhydrous DCM (5 mL) and anhydrous DMF (0.2 mL). The mixture was then stirred at room temperature for 2.5 hours. After the reaction was complete, insoluble impurities were removed by filtration, and the filtrate was concentrated to give compound 17 (crude product, 59 mg, 96% yield) as a yellow solid. LCMS: m / z C 42 H 51 N9O 10 Calculated value of S: 873.98; Measured value: 875.17 [M+H] + .

[0209] Example 18. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(2-((2-((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate (18) Synthesis Route 18 Step 1: To a solution of compound 10-3 (67 mg, 0.091 mmol) in DMF (2 mL), DIPEA (24 mg, 0.186 mmol), compound 7 (50 mg, 0.092 mmol), and HOBt (12 mg, 0.089 mmol) were added. After stirring at room temperature under a nitrogen atmosphere for 16 hours, the mixture was filtered and concentrated. The residue was purified by preparative HPLC (eluting with acetonitrile in water) to give compound 18 (36 mg, 34% yield) as a white solid. 1 H NMR (400MHz, DMSO- d 6) δ 8.22 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 7.5 Hz, 1H), 7.82 (d, J =8.6 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.3 Hz, 2H), 6.98 (s, 2H), 4.92 (s, 2H), 4.42 (t, J = 6.4 Hz, 2H), 4.37 (d, J = 5.6 Hz, 1H), 4.16 (dd, J =7.7, 5.0 Hz, 1H), 3.62 (s, 2H), 3.52 (s, 2H), 3.19 – 3.15 (m, 2H), 3.01 –2.93 (m, 4H), 2.90 (dd, J = 14.0, 6.9 Hz, 1H), 2.18 (dd, J= 14.2, 7.5 Hz, 1H), 2.11 (dd, J = 14.2, 7.0 Hz, 1H), 1.96 (dd, J = 14.1, 7.5 Hz, 4H), 1.81 (q, J = 7.6Hz, 4H), 1.73 (d, J = 13.4 Hz, 8H), 1.64 – 1.51 (m, 2H), 1.50 – 1.39 (m, 8H), 0.93 (t, J = 7.4 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.6 Hz, 3H). LCMS: m / z C 55 H 74 N 13 O 12 Calculated value of P: 1140.25; Measured value: 1141.63 [M+H] + .

[0210] Example 19. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(2-((2-((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)(methyl)carbamate (19) Synthetic route 19 Step 1: To a solution of compound 10-3 (70 mg, 0.095 mmol) in DMF (2 mL), DIPEA (24 mg, 0.186 mmol), compound 8 (54 mg, 0.092 mmol), and HOBt (12 mg, 0.089 mmol) were added. After stirring at room temperature under a nitrogen atmosphere for 16 hours, the mixture was filtered and concentrated. The residue was purified by preparative HPLC (eluting with acetonitrile in water) to give compound 19 (26 mg, 24% yield) as a white solid. 1 H NMR (600MHz, DMSO- d 6) δ 8.17 (d,J = 8.2 Hz, 1H), 8.08 (d, J = 7.1 Hz, 1H), 7.81 (d, J =8.6 Hz, 1H), 7.59 – 7.53 (m, 2H), 7.25 (d, J = 8.2 Hz, 2H), 6.98 (s, 2H), 4.95(d, J = 6.1 Hz, 2H), 4.37 (dt, J = 15.9, 7.0 Hz, 3H), 4.16 (t, J = 7.7 Hz, 1H), 3.64 (s, 4H), 3.35 (t, J = 7.0 Hz, 2H), 3.03 – 2.89 (m, 5H), 2.86 (d, J = 9.6 Hz, 3H), 2.17 (dt, J = 14.7, 7.5 Hz, 1H), 2.11 (q, J = 7.0 Hz, 1H), 1.96 (h, J = 7.8, 6.8 Hz, 4H), 1.80 (p, J = 7.6 Hz, 4H), 1.72 (d, J = 13.3 Hz, 8H), 1.49 (s, 2H), 1.48 – 1.35 (m, 8H), 0.92 (t, J = 7.4 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.7 Hz, 3H). LCMS: m / z C 56 H 76 N 13 O 12 Calculated value of P: 1154.28; Measured value: 1155.70 [M+H] + .

[0211] Example 20. Synthesis of (1r,4r)-N-(2-((2-((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)cyclohexane-1-carboxamide (20) Synthesis Route 20 Step 1: At 0 °C, compound 14-1 (74 mg, 0.221 mmol) was added to a solution of compound 7 (100 mg, 0.184 mmol) and DIPEA (50 mg, 0.387 mmol) in DMF (4 mL). The mixture was then slowly brought to room temperature and stirred for 3 hours. After the reaction was complete, the mixture was filtered and purified by preparative HPLC (eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 20 (31 mg, 22% yield) as a white solid. 1 H NMR (400MHz, DMSO- d 6) δ 8.30 – 8.24 (m, 2H), 7.95 (t, J = 9.2 Hz, 1H), 6.96 (s, 2H), 4.46 (t, J = 6.5 Hz, 2H), 3.62 (s, 2H), 3.51 (s, 2H), 3.23 – 3.15 (m, 4H), 3.02(t, J = 7.6 Hz, 2H), 1.99 (td, J = 6.9, 3.5 Hz, 3H), 1.84 (p, J = 7.6 Hz, 4H), 1.78(s, 3H), 1.75 (s, 3H), 1.69 (d, J = 12.6 Hz, 2H), 1.60 – 1.53 (m, 2H), 1.49 –1.40 (m, 3H), 1.30 – 1.25 (m, 1H), 1.23 – 1.18 (m, 1H), 0.94 (t, J = 7.4 Hz, 3H), 0.90 – 0.79 (m, 2H). LCMS: m / z C 38 H 49Calculated N8O7P: 760.83; Measured: 762.04 [M+H] + .

[0212] Example 21. Synthesis of (1r,4r)-N-(2-((2-((4-((4-amino-2-butyl-7-(dimethylphosphoryl)-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)methyl)-N-methylcyclohexane-1-carboxamide (21) Synthesis Route 21 Step 1: At 0 °C, compound 14-1 (72 mg, 0.21 mmol) was added to a solution of compound 8 (100 mg, 0.18 mmol) and DIPEA (46 mg, 0.36 mmol) in DMF (4 mL). The mixture was slowly brought to room temperature and stirred for 3 hours. After the reaction was complete, the mixture was filtered and purified by preparative HPLC (eluting with acetonitrile in water). The desired fraction was lyophilized to give compound 21 (25 mg, 18% yield) as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 8.28 – 8.21 (m, 2H), 7.92 (q, J = 9.7 Hz, 1H), 6.89 (s, 2H), 4.44(d, J = 5.8 Hz, 2H), 3.62 (s, 4H), 3.20 – 3.09 (m, 3H), 3.04 – 2.95 (m, 4H), 2.79 (s, 2H), 1.98 (d, J = 20.5 Hz, 3H), 1.82 (dd, J = 7.5, 3.2 Hz, 2H), 1.76(dd, J = 13.5, 1.8 Hz, 8H), 1.59 (d, J = 23.6 Hz, 2H), 1.52 (d, J = 22.2 Hz, 2H), 1.42 (qd, J = 7.4, 2.7 Hz, 3H), 1.17 (d, J= 14.2 Hz, 2H), 0.93 (td, J = 7.3, 1.9Hz, 5H). LCMS: m / z C 39 H 51 Calculated N8O7P: 774.86; Measured: 775.90 [M+H] + .

[0213] Example 22. Synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl(3-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)(2-(heptadecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(methyl)carbamate (22) Synthesis Route 22 Step 1: Under a nitrogen atmosphere, at 0 °C, Et3N (2.37 g, 23.46 mmol) and 2-nitrobenzenesulfonyl chloride (22-2) (3.90 g, 17.60 mmol) were added to a solution of compound 22-1 (3.00 g, 15.93 mmol) in dichloromethane (30 mL). The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the mixture was diluted with dichloromethane (100 mL) and washed with water (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 30% EtOAc in petroleum ether) to give compound 22-3 (5.26 g, 88% yield) as a pale yellow oil. LCMS: m / z C 15 H 23 Calculated N3O6S: 373.42; Measured: 374.74 [M+H] + .

[0214] Step 2: 1-Bromo-4-chlorobutane (22-4) (4.59 g, 39.18 mmol) was added to a mixture of compound 22-3 (5.00 g, 13.39 mmol), potassium carbonate (5.55 g, 40.16 mmol), and potassium iodide (2.22 g, 13.37 mmol) in DMF (50 mL). The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. After the reaction was complete, the mixture was partitioned between ethyl acetate (300 mL) and water (200 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with 20% EtOAc in petroleum ether) to give compound 22-5 (5.90 g, 95% yield) as a pale yellow oil.

[0215] Step 3: Compound 22-5 (4.34 g, 9.35 mmol) was added to a mixture of compound 22-6 (2.00 g, 7.80 mmol) and potassium carbonate (2.16 g, 15.63 mmol) in DMF (20 mL). The mixture was stirred at room temperature under nitrogen atmosphere for 16 hours. The mixture was partitioned between ethyl acetate (200 mL) and water (150 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluting with 3% MeOH in DCM) to give compound 22-7 (2.15 g, 40% yield) as a yellow oil. LCMS: m / z C 33 H 45 Calculated N7O7S: 683.83; Measured: 685.13 [M+H] + .

[0216] Step 4: 4-Ethiophene (1.45 g, 10.49 mmol) was added to a mixture of compound 22-7 (2.00 g, 2.92 mmol) and potassium carbonate (2.02 g, 14.61 mmol) in acetonitrile (20 mL). The mixture was stirred at room temperature under a nitrogen atmosphere for 15 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 3% MeOH in DCM containing 0.1% Et3N) to give compound 22-8 (1.33 g, 91% yield) as a yellow oil. LCMS: m / z C 27 H 42 Calculated N6O3: 498.67; Measured: 500.24 [M+H] + .

[0217] Step 5: Add a solution of compound 22-8 (1.00 g, 2.01 mmol) and DIPEA (525 mg, 4.06 mmol) in anhydrous methanol (10 mL) to a solution of 3,4-dimethoxycyclobut-3-ene-1,2-dione (22-9) (313 mg, 2.20 mmol) in anhydrous methanol (5 mL). Stir the mixture at room temperature under a nitrogen atmosphere for 12 hours. After the reaction is complete, concentrate the mixture. Purify the residue by silica gel column chromatography (eluting with 5% MeOH in DCM) to give compound 22-10 (931 mg, 76% yield) as a yellow oil. LCMS: m / z C 32 H 44 Calculated N6O6: 608.74; Measured: 610.22 [M+H] + .

[0218] Step 6: Under a nitrogen atmosphere, at 0 °C, a solution of heptadecano-1-amine (22-11) (378 mg, 1.48 mmol) in anhydrous methanol (4 mL) was added dropwise to a solution of compound 22-10 (600 mg, 0.99 mmol) in anhydrous methanol (8 mL). The mixture was slowly brought to room temperature and stirred under a nitrogen atmosphere for 12 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 9% MeOH in DCM) to give compound 22-12 (784 mg, 95% yield) as a yellow foamy substance. LCMS: m / z C 48 H 77 Calculated N7O5: 832.19; Measured: 833.50 [M+H] + .

[0219] Step 7: Add 8 mL of EtOAc solution in 4 N HCl to a solution of compound 22-12 (710 mg, 0.85 mmol) in dichloromethane (8 mL). Stir the mixture at room temperature for 1 hour. After the reaction is complete, concentrate the mixture. The crude product is purified by reversed-phase rapid chromatography (C18 column, eluting with 73% acetonitrile aqueous solution containing 0.1% HCl). Lyophilize the desired fraction to give compound 22-13 (510 mg, 82% yield) as a pale yellow solid. 1 H NMR (500MHz, DMSO- d 6) δ 8.18 (d, J= 8.1 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.75 (t, J =7.3 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 4.44 (t, J = 6.4 Hz, 2H), 3.67 (s, 2H), 3.56 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.91 (s, 2H), 2.54 (s, 3H), 1.93 (d, J = 15.8 Hz, 4H), 1.81 (q, J = 7.5 Hz, 4H), 1.52 (t, J = 7.4 Hz, 2H), 1.46– 1.38 (m, 2H), 1.28 – 1.02 (m, 28H), 0.93 (t, J = 7.4 Hz, 3H), 0.83 (t, J = 7.0Hz, 3H). LCMS: m / z C 43 H 69 Calculated N7O3: 732.07; Measured: 733.36 [M+H] + .

[0220] Step 8: Under a nitrogen atmosphere, HOBt (23 mg, 0.17 mmol) and compound 10-3 (103 mg, 0.14 mmol) were added to a solution of compound 22-13 (100 mg, 0.14 mmol) and DIPEA (45 mg, 0.35 mmol) in anhydrous DMF (2.5 mL). The mixture was stirred at room temperature for 20 hours. After the reaction was complete, the mixture was filtered. The filtrate was purified directly by preparative HPLC (C18 column, eluted with 88% acetonitrile aqueous solution containing 0.1% HCl). The desired fraction was lyophilized to give compound 22 (121 mg, 65% yield) as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 8.17 (d, J = 8.0 Hz, 1H), 7.83 (dd, J= 8.4, 1.0 Hz, 1H), 7.74 (ddd, J = 8.4,7.1, 1.4 Hz, 1H), 7.57 (d, J = 8.1 Hz, 3H), 7.25 (d, J = 8.0 Hz, 2H), 6.97 (s,2H), 4.94 (s, 2H), 4.41 (s, 2H), 4.34 (s, 1H), 4.14 (s, 1H), 3.55 (s, 2H),3.35 (s, 2H), 3.25 (s, 2H), 2.96 (d, J = 21.0 Hz, 4H), 2.83 (s, 3H), 2.13 (d, J =19.9 Hz, 2H), 1.94 (s, 3H), 1.79 (d, J = 9.9 Hz, 6H), 1.67 (s, 1H), 1.58 (s,1H), 1.44 (dd, J = 32.6, 7.4 Hz, 11H), 1.18 (p, J = 14.6, 11.0 Hz, 33H), 0.91 (t, J = 7.4 Hz, 3H), 0.81 (q, J = 6.8 Hz, 9H). LCMS: m / z C 72 H 107 N 13 O 11 Calculated value: 1330.73; Measured value: 1332.15 [M+H] + .

[0221] Example 23. Synthesis of N-(3-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)(2-(heptadecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-methylhexanoamide (23) Synthesis Route 23 Step 1: 2,5-Dioxopyrrolidine-1-yl ester (23-1) (42 mg, 0.14 mmol) was added to a solution of compound 22-13 (100 mg, 0.14 mmol) and DIPEA (45 mg, 0.35 mmol) in anhydrous DMF (2.5 mL). The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was filtered. The filtrate was directly purified by preparative HPLC (C18 column, eluted with 87% acetonitrile aqueous solution containing 0.1% HCl). The desired fraction was lyophilized to give compound 23 (52 mg, 41% yield) as a pale yellow solid. 1 H NMR (500 MHz, DMSO- d 6) δ 8.18 (d, J = 8.1 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.8 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 6.97 (s, 2H), 4.44 (d, J = 6.3Hz, 2H), 3.56 (s, 2H), 3.34 (s, 2H), 3.29 (s, 2H), 2.99 (s, 2H), 2.92 (s,2H), 2.77 (s, 1H), 2.21 (s, 2H), 1.94 (s, 2H), 1.82 (s, 5H), 1.73 (s, 1H), 1.50 (s, 2H), 1.42 (d, J = 7.6 Hz, 7H), 1.22 (d, J = 23.8 Hz, 33H), 0.92 (t, J =7.4 Hz, 3H), 0.83 (t, J = 6.9 Hz, 3H). LCMS: m / z C 53 H 80 Calculated N8O6: 925.27; Measured: 926.62 [M+H] + .

[0222] Example 24. Synthesis of [4-[[(2S)-2-[[(2S)-2-[6-[3-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadecane-1(12),4(9),5,7,13,15-hexen-10-yn-2-yl)-4-oxo-butyryl]amino]ethylthio]-2,5-dioxo-pyrrolidine-1-yl]hexanoylamino]-3-methyl-butyrylamino]-5-ureo-pentanoylamino]phenyl]methyl N-[4-(4-amino-2-butyl-7-dimethylphosphoryl-imidazo[4,5-c]quinolin-1-yl)oxybutyl]carbamate (24) Synthesis Route 24 Step 1: At 0 °C, a solution of compound 24-1 (200 mg, 0.50 mmol) in dichloromethane (2 mL) was added to a solution of 2-aminoethane-1-thiol (24-2) (46 mg, 0.60 mmol) and DIPEA (96 mg, 0.74 mmol) in dichloromethane (4 mL). The mixture was stirred at 0 °C for 1 hour. After the reaction was complete, the mixture was concentrated. The residue was purified by reversed-phase rapid chromatography (C18 column, eluted with 75% acetonitrile aqueous solution containing 0.1% AcOH). The desired fraction was lyophilized to give compound 24-3 (62 mg, 34% yield) as a white solid. LCMS: m / z C 21 H 20 Calculated N₂O₂S: 364.12; Measured: 365.10 [M+H] + .

[0223] Step 2: Compound 24-3 (22 mg, 0.06 mmol) was added to a solution of compound 13 (50 mg, 0.05 mmol) and Et3N (10 mg, 0.10 mmol) in DMF (2 mL). The mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the mixture was filtered. The filtrate was purified directly by preparative HPLC (C18 column, eluted with 55% acetonitrile aqueous solution containing 0.1% AcOH). The desired fraction was lyophilized to give compound 24 (21 mg, 30% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 9.99 (s, 1H), 8.17 (s, 1H), 8.06 (d, J= 20.6 Hz,2H), 7.90 (s, 1H), 7.81 (s, 1H), 7.70 (s, 1H), 7.67 (s, 1H), 7.59 (t, J = 7.6Hz, 3H), 7.51 – 7.41 (m, 3H), 7.34 (s, 3H), 7.27 (d, J = 8.5 Hz, 3H), 5.96 (s,1H), 5.41 (s, 2H), 5.01 (d, J = 14.1 Hz, 1H), 4.95 (s, 2H), 4.36 (d, J = 5.8 Hz,3H), 4.18 (s, 1H), 3.94 (d, J = 8.2 Hz, 1H), 3.59 (d, J = 14.0 Hz, 1H), 3.14 (dt, J = 17.5, 6.5 Hz, 5H), 3.04 – 2.88 (m, J = 6.6 Hz, 4H), 2.75 (dq, J = 12.4, 6.4Hz, 1H), 2.58 (d, J = 16.4 Hz, 3H), 2.42 (d, J = 18.4 Hz, 1H), 2.27 – 2.05 (m,3H), 1.97 (d, J = 13.7 Hz, 2H), 1.91 (s, 2H), 1.80 (d, J = 7.7 Hz, 3H), 1.75 –1.62 (m, 10H), 1.57 (d, J = 9.2 Hz, 1H), 1.51 – 1.26 (m, 9H), 1.19 (d, J = 7.2Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H), 0.82 (dd, J = 12.5, 6.7 Hz, 6H). LCMS: m / zC 70 H 88 N 13 O 12 Calculated PS value: 1366.59; Measured value: 1367.30 [M+H] + .

[0224] Example 25. Synthesis of N-(3-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)(2-(heptadecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-N-methyl-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (25) Synthesis Route 25 Step 1: EDCI (42 mg, 0.22 mmol) was added to a mixture of compounds 22-13 (80 mg, 0.11 mmol), 25-1 (86 mg, 0.16 mmol), HOBt (15 mg, 0.11 mmol), and DIPEA (43 mg, 0.33 mmol) in DMF (4 mL). The mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was filtered and purified directly by preparative HPLC (C18 column, eluted with 80% acetonitrile aqueous solution containing 0.1% TFA). The desired fraction was lyophilized to give compound 25 (67 mg, 49% yield) as a white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 8.18 (dd, J = 8.1, 1.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.75 (t, J = 7.8Hz, 1H), 7.65 (d, J = 22.9 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.02 (s, 2H), 4.44(t, J = 6.4 Hz, 2H), 3.63 – 3.53 (m, 8H), 3.49 (s, 20H), 3.46 (d, J = 7.1 Hz,9H), 3.30 (s, 3H), 3.00 (t, J = 7.6 Hz, 2H), 2.94 (s, 2H), 2.79 (s, 1H), 2.55(dd, J= 13.4, 6.8 Hz, 4H), 1.94 (s, 2H), 1.82 (d, J = 7.6 Hz, 6H), 1.51 (s, 2H), 1.43 (d, J = 7.6 Hz, 2H), 1.21 (s, 28H), 0.93 (t, J = 7.4 Hz, 3H), 0.84 (t, J = 6.9Hz, 3H). LCMS: m / z C 66 H 106 N8O 14 Calculated value: 1234.78; Measured value: 1235.60 [M+H] + .

[0225] Example 26. N-((34S,37S,40S)-40-(((S)-65-(1-(2-((3-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinoline-1-yl)oxy)butyl)(2-(heptadecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(methyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)-28,35,63-trioxo-2,5,8,11,14,17,20,23,26,39,42,45,48,51,54,57,60-heptadeca-29,36,64-triazahexapentadecane-34- Synthesis of (26) carbamoyl)-34-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide)-28,35,38-trioxo-37-(28-oxo-2,5,8,11,14,17,20,23,26-nonoxa-29-azatritriacontane-33-yl)-2,5,8,11,14,17,20,23,26-nonoxa-29,36,39-triazatetradecane-44-yl)-2,5,8,11,14,17,20,23,26-nonoxaheptadecane-28-amide Synthetic Route 26 Step 1: 2-Chlorotriphenylmethyl chloride resin (1.56 mmol / g, 3.22 g, 5.02 mmol) was swollen under nitrogen atmosphere in dichloromethane (40 mL) for 30 min. After removing the dichloromethane, DIPEA (2.34 g, 18.10 mmol), 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonoxa-4-azatrione-31-acid (26-1) (3.00 g, 4.52 mmol), and dichloromethane (40 mL) were added. The mixture was saturated with nitrogen atmosphere for 2 h, and unreacted Cl groups were blocked with methanol (3 mL) for 30 min. The resin was then washed with DMF and dichloromethane. Fmoc was then removed with 20% piperidine in DMF for 30 min. The following four Fmoc-Lys(Boc)-OH (26-2) compounds were coupled sequentially under the following conditions: Fmoc-Lys(Boc)-OH (6.35 g, 13.55 mmol), HBTU (4.89 g, 12.90 mmol), and DIPEA (3.48 g, 26.92 mmol) were reacted in DMF under nitrogen atmosphere for 30 minutes. The final Fmoc protective resin was washed with DMF and MeOH and dried under nitrogen atmosphere to obtain the desired resin.

[0226] The desired resin was added to a mixed solution of hexafluoroisopropanol (40 mL) and dichloromethane (160 mL). The reaction mixture was shaken at room temperature for 30 minutes and then filtered. The filter cake was washed with a 20% hexafluoroisopropanol solution in dichloromethane. The filtrate was concentrated to dryness to give compound 26-3 (6.42 g, 90% yield) as a pale yellow solid. LCMS: m / z C 78 H 129 N9O 24 Calculated value: 1575.92; Measured value: 1576.85 [M+H] + .

[0227] Step 2: EDCI (546 mg, 2.85 mmol) was added to a solution of compound 26-3 (3.00 g, 1.90 mmol) and propargylamine (210 mg, 3.81 mmol) in anhydrous dichloromethane (35 mL). The mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was diluted with DCM (100 mL) and washed with water (80 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with 9% MeOH in DCM) to give compound 26-4 (1.96 g, 64% yield). LCMS: m / z C 81 H 132 N 10 O 23 Calculated value: 1613.99; Measured value: 1614.79 [M+H] + .

[0228] Step 3: To a solution of compound 26-4 (1.50 g, 0.93 mmol) in dichloromethane (6 mL) and methanol (1.5 mL), a solution of EtOAc in 4 N HCl (7.5 mL) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated. The residue was ground with EtOAc and petroleum ether to give compound 26-5 (hydrochloride, 1.16 g, 92% yield). LCMS: m / z C 61 H 100 N 10 O 15 Calculated value: 1212.74; Measured value: 1213.87 [M+H] + .

[0229] Step 4: A solution of 2,5,8,11,14,17,20,23,26-nonoxaoctacosano-28-acid (26-6) (2.20 g, 4.97 mmol), DIPEA (1.06 g, 8.20 mmol), and HATU (2.50 g, 6.58 mmol) in anhydrous DMF (11 mL) was stirred at room temperature for 20 minutes. Compound 26-5 (1.40 g, 1.03 mmol) and DIPEA (1.06 g, 8.20 mmol) were added to this solution. The reaction mixture was stirred at room temperature for 5 hours. After the reaction was complete, the mixture was concentrated. The residue was extracted with DCM (100 mL x 2) and water (100 mL). The combined organic layers were washed with saturated NaHCO3 solution and 1 N HCl solution. The final organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluting with 15% MeOH in DCM) to give compound 26-7 (1.36 g, 45% yield) as a pale yellow paste. LCMS: m / z C 137 H 244 N 10 O 55 Calculated value: 2911.47; Measured value: 971.78 [M+3H] 3+ .

[0230] Step 5: The mixture of compound 26-7 (1.30 g, 0.45 mmol) and diethylamine (2.6 mL) in dichloromethane (10.4 mL) was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated. The residue was dispersed in isopropanol (20 mL), and the insoluble slurry was centrifuged and dried under vacuum. The crude product was purified by reversed-phase rapid chromatography (C18 column, eluted with 43% acetonitrile aqueous solution containing 0.1% HCl). The desired fraction was lyophilized to give compound 26-8 (847 mg, 70% yield) as a colorless oil. LCMS: m / z C 122 H 234 N 10 O 53 Calculated value: 2689.23; Measured value: 897.37 [M+3H] 3+ .

[0231] Step 6: Add EDCI (27 mg, 0.14 mmol) to a solution of compound 26-8 (210 mg, 0.08 mmol), HOBt (11 mg, 0.08 mmol), DIPEA (35 mg, 0.27 mmol), and 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-acid (26-9) (53 mg, 0.10 mmol) in anhydrous dichloromethane (5 mL). The reaction mixture was stirred at room temperature for 14 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by reversed-phase rapid chromatography (C18 column, eluted with 75% acetonitrile aqueous solution containing 0.1% TFA). The desired components were lyophilized to give compound 26-10 (105 mg, yield 42%) as a colorless oil. LCMS: m / z C 122 H 234 N 10 O 53 Calculated value: 3192.78; Measured value: 1065.27 [M+3H] 3+ .

[0232] Step 7: Add EDCI (79 mg, 0.42 mmol) to a mixture of compounds 22-13 (200 mg, 0.27 mmol), 2-azidoacetic acid (26-11) (42 mg, 0.42 mmol), HOBt (37 mg, 0.27 mmol), and DIPEA (106 mg, 0.82 mmol) in anhydrous DMF (4 mL). Stir the reaction mixture at room temperature for 16 hours. After the reaction is complete, filter the mixture. Purify the filtrate directly by reversed-phase rapid chromatography (C18 column, eluting with 86% acetonitrile aqueous solution containing 0.1% TFA). Lyophilize the desired fraction to give compound 26-12 (144 mg, 65% yield) as a colorless oil. LCMS: m / z C 45 H 70 N 10 Calculated O4 value: 814.56; Measured value: 815.64 [M+H] + .

[0233] Step 8: Under a nitrogen atmosphere, a mixture of compound 26-10 (50 mg, 0.016 mmol), compound 26-12 (16 mg, 0.020 mmol), CuI (6 mg, 0.031 mmol), and DIPEA (16 mg, 0.124 mmol) in THF (2.5 mL) was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by preparative HPLC (C18 column, eluted with 73% acetonitrile aqueous solution containing 0.1% HCl). The desired fraction was lyophilized to give compound 26 (35 mg, 55% yield) as an off-white solid. 1 H NMR (500 MHz, DMSO- d 6) δ 8.41 – 8.34(m, 1H), 8.15 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.89 (s, 2H), 7.86 – 7.77 (m,2H), 7.75 (d, J = 7.5 Hz, 2H), 7.72 – 7.63 (m, 6H), 7.02 (s, 2H), 5.39 (d, J =15.7 Hz, 2H), 4.42 (s, 2H), 4.29 (d, J = 5.5 Hz, 2H), 4.24 – 4.13 (m, 4H), 3.85(d, J = 1.9 Hz, 8H), 3.55 (s, 26H), 3.52 (s, 18H), 3.50 (q, J = 1.8 Hz, 138H), 3.47 (t, J = 2.2 Hz, 16H), 3.48 – 3.41 (m, 25H), 3.23 (s, 12H), 3.06 (d, J = 6.6Hz, 6H), 3.04 (s, 3H), 2.97 (t, J = 7.3 Hz, 2H), 2.40 (s, 1H), 2.44 – 2.32 (m,5H), 1.94 (s, 3H), 1.81 (d, J = 7.3 Hz, 5H), 1.62 (s, 4H), 1.49 (d, J= 8.2 Hz, 6H), 1.44 – 1.33 (m, 12H), 1.22 (d, J = 12.6 Hz, 36H), 0.93 (td, J = 7.4, 2.0 Hz,3H), 0.85 – 0.83 (m, 3H). LCMS: m / z C 190 H 341 N 21 O 68 Calculated value: 4007.90; Measured value: 1002.93 [M+4H] 4+ .

[0234] Example 27. Synthesis of 4-((47S,50S,53S)-47-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoylamino)-50-isopropyl-41,48,51-trioxo-53-(3-ureopropyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-tetraoxa-42,49,52-triazapentacosan-54-acylamino)benzyl(3-((4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)oxy)butyl)(2-(heptadecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(methyl)carbamate (27) Synthesis Route 27 Step 1: The mixture of compound 27-1 (5.00 g, 8.31 mmol) and diethylamine (12 mL) in DMF (48 mL) was stirred at room temperature for 2 hours. After the reaction was complete, MTBE (300 mL) was added to the mixture to form a precipitate. The crude product was ground with EtOAc, filtered, and dried under vacuum to give compound 27-2 (3.04 g, 95% yield) as a white solid. LCMS: m / z C 18 H 29 Calculated N₅O₄: 379.22; Measured: 380.26 [M+H] + .

[0235] Step 2: The mixture of 27-3 (1.00 g, 1.58 mmol), N-hydroxysuccinimide (364 mg, 3.16 mmol), and EDCI (606 mg, 3.16 mmol) in DCM (10 mL) was stirred at room temperature for 1 hour. After the preparation of the active NHS ester was complete, the mixture was diluted with DCM (100 mL) and washed with water (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the NHS ester intermediate. DMF (10 mL) and the NHS ester intermediate were added to another flask containing compound 27-4 (467 mg, 1.90 mmol) and DIPEA (815 mg, 6.31 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by silica gel column chromatography (eluting with 6% MeOH in DCM) to give compound 27-5 (1.00 g, 74% yield) as a pale yellow oil. LCMS: m / z C 39 H 76 N2O 18 Calculated value: 860.51; Measured value: 861.57 [M+H] + .

[0236] Step 3: EDCI (433 mg, 2.26 mmol) was added to a solution of compound 27-5 (970 mg, 1.13 mmol), compound 27-2 (641 mg, 1.69 mmol), HOBt (152 mg, 1.13 mmol), and DIPEA (730 mg, 5.64 mmol) in anhydrous DMF (10 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was concentrated. The residue was purified by reversed-phase rapid chromatography (C18 column, eluted with 50% acetonitrile aqueous solution containing 0.1% TFA). The desired fraction was lyophilized to give compound 27-6 (1.04 g, 75% yield) as a colorless oil. LCMS: m / z C 57 H 103 N7O 21 Calculated value: 1221.72; Measured value: 1222.71 [M+H] + .

[0237] Step 4: The mixture of compound 27-6 (1.16 g, 0.95 mmol) and TFA (6 mL) in DCM (24 mL) was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was concentrated. The residue was redissolved in a 5% K2CO3 solution in a mixed solvent of MeOH and H2O (v:v = 1:1, 15 mL) and stirred for another 2 hours. After complete product formation, the resulting mixture was concentrated. The crude product was purified directly by reversed-phase rapid chromatography (C18 column, eluted with 40% acetonitrile aqueous solution containing 0.1% TFA). The desired fraction was lyophilized to give compound 27-7 (832 mg, 78% yield) as a colorless oil. LCMS: m / z C 52 H 95 N7O 19 Calculated value: 1121.67; Measured value: 1122.75 [M+H] + .

[0238] Step 5: To a solution of compound 27-7 (832 mg, 0.74 mmol) and DIPEA (370 mg, 2.86 mmol) in DMF (10 mL), 2,5-dioxopyrrolidine-1-yl ester of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (23-1) (251 mg, 0.81 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was filtered. The filtrate was purified directly by reversed-phase rapid chromatography (C18 column, eluted with 50% acetonitrile aqueous solution containing 0.1% AcOH). The desired fraction was lyophilized to give compound 27-8 (623 mg, 64% yield) as a colorless oil. LCMS: m / z C 62 H 106 N8O 22 Calculated value: 1314.74; Measured value: 1315.87 [M+H] + .

[0239] Step 6: The mixture of compound 27-8 (500 mg, 0.38 mmol), DIPEA (200 mg, 1.55 mmol), and bis(4-nitrophenyl) carbonate (232 mg, 0.76 mmol) in DMF (5 mL) was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was filtered. The filtrate was purified directly by reversed-phase rapid chromatography (C18 column, eluted with 65% acetonitrile aqueous solution containing 0.1% TFA). The desired fraction was lyophilized to give compound 27-9 (422 mg, 75% yield) as a white solid. LCMS: m / z C 69 H 109 N9O 26 Calculated value: 1479.75; Measured value: 1480.83 [M+H] + .

[0240] Step 7: Under a nitrogen atmosphere, a mixture of compounds 22-13 (42 mg, 0.057 mmol), DIPEA (22 mg, 0.17 mmol), HOBt (8 mg, 0.059 mmol), and 27-9 (100 mg, 0.068 mmol) in DMF (3 mL) was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was filtered. The filtrate was purified directly by preparative HPLC (C18 column, eluted with 65% acetonitrile aqueous solution containing 0.1% TFA). The desired fraction was lyophilized to give compound 27 (25 mg, 21% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 9.99 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.09 (d, J = 7.1 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.86 –7.77 (m, 2H), 7.72 (t, J = 7.8 Hz, 1H), 7.65 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 8.2Hz, 3H), 7.25 (d, J = 8.1 Hz, 2H), 6.97 (s, 2H), 4.95 (s, 2H), 4.39 (d, J= 22.5Hz, 3H), 4.26 – 4.13 (m, 2H), 3.55 (s, 4H), 3.51 – 3.47 (m, 46H), 3.46 (d, J =1.8 Hz, 4H), 3.34 (d, J = 7.1 Hz, 2H), 3.25 (s, 2H), 3.22 (s, 3H), 3.03 – 2.89(m, 6H), 2.83 (s, 3H), 2.28 (t, J = 6.5 Hz, 2H), 2.09 (dd, J = 8.9, 6.1 Hz, 2H),1.99 – 1.89 (m, 3H), 1.81 (p, J = 7.7 Hz, 6H), 1.61 (d, J = 23.1 Hz, 2H), 1.45(dt, J = 14.2, 7.2 Hz, 10H), 1.39 – 1.31 (m, 4H), 1.21 (d, J = 17.2 Hz, 34H), 0.92 (t, J = 7.3 Hz, 3H), 0.82 (dq, J = 10.1, 6.5, 5.0 Hz, 9H). LCMS: m / zC 106 H 173 N 15 O 26 2073.63 found 692.29 [M+3H] 3+ .

[0241] Example 28. A general coupling method for ADC preparation and characterization Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl, 0.55 mM, 2.5-6.5 equivalents, PBS solution, pH 7.4) was added to the antibody solution. The resulting solution was gently vortexed and incubated at 25-37 °C for 224 hours. The linker-loaded substance (615 equivalents, dissolved in DMSO, 25 mM) was added to the partially reduced antibody, and the solution was gently vortexed and kept at 25 °C for 1-24 hours. The crude sample was purified using an illustra™ NAP column (GE Healthcare) to remove excess linker-loaded substance and exchanged into PBS buffer (pH 7.4) to obtain the purified conjugate. The purified ADC was filtered through a 0.25 μm sterile filter and then stored in solution at -80 °C or as a lyophilized powder at -20 °C until use. All ADCs were analyzed by hydrophobic interaction chromatography (HIC) to measure DAR (drug-antibody ratio) and UmAb% (unconjugated antibody percentage), by size exclusion chromatography (SEC) to determine Agg% (aggregation percentage), and by UV absorbance analysis to calculate ADC concentration and DAR.

[0242] HIC conditions: Instrument: Agilent 1100 HPLC system equipped with a UV detector and autosampler Chromatographic column: TSKgel Butyl-NPR, 4.6 mm inner diameter x 3.5 cm, 2.5 μm Mobile phase A: An aqueous solution of 1.5 M (NH4)2SO4 and 50 mM K2HPO4, pH 7.0 Mobile phase B: 50 mM K3PO4 and 20% IPA aqueous solution, pH 7.0 Flow rate: 0.7 mL / min Detector: UV at 280 nm Runtime: 22 min Gradient procedure: SEC conditions: Instrumentation: Agilent 1100 HPLC system equipped with a UV detector and autosampler; Column: YMC-Pack Diol 200, 300 x 8.0 mm inner diameter, S-5 μm, 20 nm Mobile phase: aqueous solution of 40 mM NaCl and 20 mM K₂HPO₄, pH 7.0 Flow rate: 0.8 mL / min Detector: UV at 280 nm Runtime: 22 min Table 1 summarizes the ADCs synthesized from a series of TLR agonist-derived linkers-payloads and various antibodies, including trastuzumab, avelumab, mouse anti-human HER2 antibody, anti-mouse PD-L1 (B7-H1) and atezolizumab.

[0243] Table 1: An exemplary ADC fabricated using a TLR agonist-derived linker-payload Example 29. TLR7 / 8 agonists induce the release of IFN-γ and TNF-α in human PBMCs. This system was used to assess the release of cytokines. Activity was measured based on the amount of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) secreted into the culture medium.

[0244] PBMC separation Dilute fresh human blood with an equal volume of PBS. Add 15 mL of Lymphoprep to a Sepmate tube, then gently add 30 mL of diluted blood on top, being careful not to disturb the interface.

[0245] Centrifuge the Sepmate tube at 1000x g for 25 minutes at room temperature and then brake off. Collect the leukocyte layer containing peripheral blood mononuclear cells (PBMCs) from the Sepmate tube and transfer it to a new tube. Wash the cells twice with 40 mL PBS and centrifuge at 350x g for 5 minutes.

[0246] PBMCs were resuspended in complete culture medium at a density of 2E6 / ml.

[0247] Compound preparation The compound was dissolved in dimethyl sulfoxide (DMSO) and diluted to the specified concentration with complete culture medium.

[0248] The final test concentrations of the compounds were 100 μM, 33.3 μM, 11.1 μM, 3.7 μM, 1.23 μM, 0.41 μM, 0.137 μM, 0.0457 μM and 0.0152 μM.

[0249] Incubation 2*10 5 One PBMC (in 100 µL) was added to each well of a 96-well plate. 100 μL of a 3-fold serially diluted compound (2 times the final concentration) was added to each well to bring the final volume to 200 µL.

[0250] Cover the plate with a sterile cap, mix gently, and then incubate at 37 °C in a 5% CO2 incubator for 24 hours.

[0251] Separate the supernatant After incubation, centrifuge the plate at 400 x g for 5 minutes. Transfer the cell-free culture supernatant to a non-sterile polypropylene plate. Store the samples at -80 °C until analysis. Analyze the TNF-α and IFN-γ levels in the samples using ELISA, following the manufacturer's instructions.

[0252] TNF-α and IFN-α were analyzed by ELISA. IFN-α concentrations were determined by ELISA using the Human IFN-α ELISA Kit from R&D Systems (catalog number #41100-2) and read on a VICTOR Nivo™ from PerkinElmer. Results are expressed in pg / mL. TNF-α concentrations were determined by ELISA using the Human TNF-α ELISA MAX™ Deluxe from BioLegend (catalog number #430205) and read on a VICTOR Nivo™ from PerkinElmer. Results are expressed in pg / mL.

[0253] The data were analyzed to determine the minimum effective concentration (MEC) at which each compound was observed to induce a specific cytokine in the application. Specifically, the MEC (micromolar) of each compound was determined as the minimum compound concentration required to induce a measured cytokine response level (pg / mL) that was at least twice the level observed in the negative control wells. The results are shown in Table 2.

[0254] Table 2 Example 30. Antitumor effect of immunostimulatory antibody-drug conjugate (iADC) in C57BL / 6 mouse MC38 colorectal cancer model. Animals and materials This study used 63 female C57BL / 6 inbred mice aged 6-8 weeks. The mice were purchased from Charles River Laboratories. Upon arrival, they were kept in an SPF (specific pathogen-free) environment. Specifically, they were housed on corncob bedding (ScottPharma) from the Innovive cage system, with a 12-hour light-dark cycle (07:00-19:00), a temperature of 68-74 °F, and humidity of 30-70%. They were allowed continuous free access to water and a standard rodent diet (Purine 5001). Animal handling and procedures were performed according to protocols and / or guidelines approved by the Institutional Animal Care and Application Committee (IACUC).

[0255] All tested compounds were clear solutions stored at 4 °C.

[0256] Implantation of mouse CRC MC38 cells Revive one tube of previously frozen MC38 cells. Expand the cells in complete DMEM (high glucose) medium containing 10% fetal bovine serum (inactivated at 56 °C), 2 mM glutamine, and 0.5% penicillin / streptomycin, and incubate at 37 °C with a 5% CO2 supply. Change the medium twice a week until the day of inoculation. Cells selected for inoculation typically meet the following four criteria: 1) in a rapid growth phase (usually reaching approximately 80% to 90% confluence in the culture flask); 2) low passage number, typically between passage 2 and 3; 3) medium changed the day before inoculation; 4) high viability (approximately 92%).

[0257] On the day of inoculation, MC38 cells were harvested according to the standard operating procedure (SOP) of the cell culture program, washed, and counted. The final step involved resuspending the cells in PBS as a single-cell solution at a concentration of 5 × 10⁻⁶. 6 Cells / mL. Immediately suspend 500,000 cells (5 × 10⁶ cells / mL) in 0.1 mL of PBS. 5 MC38 cells were subcutaneously injected into the right abdomen of 80 C57BL / 6 mice.

[0258] Research Design Before initiating treatment, the growth of MC38 tumors in mice was closely monitored. In short, once the tumor became palpable, its size was measured in millimeters (mm) in two dimensions using calipers, and its volume was calculated using the following formula: Tumor volume (mm) 3= l * w * w * 0.5236, (l = length, w = width) In this study, tumors reached the expected size on day 9 post-inoculation. Each group received one dosing regimen, with the predetermined dosage and schedule detailed in Table 3.

[0259] Preparation of drug delivery solution The dosing solution is freshly prepared on each dosing day. The formulation (carrier) used is PBS.

[0260] The compound administration solution was prepared immediately prior to treatment. All test items were administered intravenously in a volume of 10 mL / kg.

[0261] Monitor tumor volume and body weight twice a week.

[0262] Primary endpoint Any animal shall be euthanized if it exhibits at least one of the following conditions: The average tumor size in the group exceeded 1,500 mm³. The animal becomes critically ill or morbid. The animal's weight decreased by more than 20% compared to its original weight. Data Analysis Statistical analyses between the control and treatment groups, or among the treatment groups themselves, were performed using one-way ANOVA, followed by Bonferroni multiple comparison tests. All analyses were conducted using the GraphPad Prism software suite (Prism 6 for Windows, version 6.0, GraphPad Software Inc., San Diego, CA). P A difference of <0.05 is considered statistically significant.

[0263] Research Results The findings are summarized in Table 3.

[0264] Table 3. Summary of in vivo research results Note: %MAX TGI = 100 x [(TVc-TVx) / TVc]; TVc: tumor volume in the control group; TVx: tumor volume in the treatment group; MST: mean survival time; A: >50%; B: 20–50%; C: <20%.

[0265] As used herein, the applicant has described its disclosure with reference to preferred embodiments in conjunction with the accompanying drawings, wherein the same reference numerals denote the same or similar elements. References to "an embodiment," "a particular embodiment," or similar terms throughout the specification mean that a specific feature, structure, or characteristic relating to the embodiment is included in at least one implementation of the invention. Therefore, the phrases "in one embodiment," "in a particular embodiment," and similar terms in this specification may or may not refer to the same embodiment.

[0266] The features, structures, or characteristics described in the applicant's disclosure may be combined in any suitable manner in one or more embodiments. Numerous specific details are set forth herein to provide a more complete understanding of embodiments of the invention. However, those skilled in the art will understand that the compositions and / or methods of this application may also be implemented without one or more of these specific details, or using other methods, components, materials, etc. In other instances, certain well-known structures, materials, or operations have not been described in detail to avoid obscuring aspects of this disclosure.

[0267] In this specification and the appended claims, the singular forms “1,” “the,” and “the” include plural references unless the context clearly indicates otherwise.

[0268] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While the contents of this disclosure may be practiced or tested using methods and materials similar to or equivalent to those described herein, preferred methods and materials are described hereafter. Except for the specific order disclosed, the methods described herein may be performed in any logically feasible order.

[0269] By incorporating via reference This disclosure references and mentions other documents, such as patents, patent applications, patent publications, journals, books, papers, and online content. All of the aforementioned documents are incorporated herein by reference in their entirety for all purposes. If any of the cited materials or portions thereof conflict with any existing definitions, statements, or other disclosures expressly provided herein, they will be incorporated only if there is no conflict between the cited materials and this disclosure. In the event of a conflict, the conflict will be resolved in a manner favorable to this application, and the disclosure favorable to this application will be considered the preferred embodiment.

[0270] equivalent The representative embodiments are intended to help illustrate the invention and are not intended to limit the scope of the invention, nor should they be construed as limiting the scope of the invention. In fact, in addition to the embodiments shown and described herein, various modifications and further embodiments of the invention will be readily apparent to those skilled in the art from the entirety of this document, including the embodiments and references to scientific and patent literature. These embodiments contain important additional information, examples, and guidance applicable to different embodiments of the invention and their equivalents.

Claims

1. A drug-linker conjugate, characterized in that, It has the structural formula (I): (I) in, L' is a group, including functional or reactive groups; D is the drug component, which has the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X is a single key or a connecting part; Or its pharmaceutically acceptable form or isotopic derivative.

2. The drug-linker conjugate according to claim 1, characterized in that, R 2 It's H.

3. The drug-linker conjugate according to claim 2, characterized in that, R 1 It is n-butyl and has the following structure: 。 4. The drug-linker conjugate according to claim 1, characterized in that, R2 is P(=O)(CH3)2.

5. The drug-linker conjugate according to claim 4, characterized in that, R 1 It is n-butyl and has the following structure: 。 6. The drug-linker conjugate according to any one of claims 1-5, characterized in that, n is 4.

7. The drug-linker conjugate according to any one of claims 1-6, characterized in that, L' includes para-aminobenzoic acid (PABA) and a dipeptide, and optionally, further includes a PEG unit.

8. The drug-linker conjugate according to any one of claims 1-6, characterized in that, L' includes Mal-Val-Cit-PABA, Mal-Val-Ala-PABA, or Mal-Val-Cit-PABA, Mal-Val-Ala-PABA, Mal-Gly-Gly-Phe-Gly, Mal-PEG2-Val-Cit-PABA, Mal-PEG8-Val-Ala-PABA, Mal-amide-PEG8-C2-acid, Py-MAA-Val-Cit-PAB, CL2A, SMCC, sulfonyl-SMCC sodium, SPDB, SPDP, DBCO, DBCO-NHCO-PEG4-amine, DBCO-PEG4-Gly-Gly-Phe-Gly, DBCO-PEG3-Val-Cit-PABA, DBCO-(PEG2-VC-PABA)2, or maleimide-DOTA.

9. The drug-linker conjugate according to any one of claims 1-6, characterized in that, L' is: 。 10. The drug-linker conjugate according to any one of claims 1-6, characterized in that, L' includes groups selected from the following: 。 11. The drug-linker conjugate according to any one of claims 1-10, characterized in that, X is selected from: 。 12. A drug-linker conjugate, characterized in that, Selected from: 。 13. An antibody-drug conjugate, characterized in that, Includes a drug moiety having the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 It is H or P(=O)(CH3)2; n Integers selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

14. An antibody-drug conjugate, characterized in that, It has structural formula (II): (II) in, Ab is the antigen-binding portion; L stands for connector; D represents the drug component, which has the following structure: in, R 1 It is a C1-C8 alkyl group; R 2 is H or P(=O)(CH3)2; n It is an integer selected from 1 to 8; X represents a single key or a connecting part; Or its pharmaceutically acceptable salt.

15. The antibody-drug conjugate according to any one of claims 13 or 14, characterized in that, R2 is H.

16. The antibody-drug conjugate according to claim 14, characterized in that, R 1 It is n-butyl and has the following structure: 。 17. The antibody-drug conjugate according to any one of claims 13 or 14, characterized in that, R 2 It is P(=O)(CH3)2.

18. The antibody-drug conjugate according to claim 17, characterized in that, R 1 It is n-butyl and has the following structure: 。 19. The antibody-drug conjugate according to any one of claims 13-18, characterized in that, n The value is 4.

20. The antibody-drug conjugate according to any one of claims 14-18, characterized in that, L represents a pyrolytic linker.

21. The antibody-drug conjugate according to claim 20, characterized in that, L is a linker sensitive to acid, lysosomal protease, β-glucuronide, or glutathione.

22. The antibody-drug conjugate according to any one of claims 14-21, characterized in that, L represents a non-split linker.

23. The antibody-drug conjugate according to any one of claims 14-21, characterized in that, X is selected from: 。 24. The antibody-drug conjugate according to any one of claims 13-23, characterized in that, Ab stands for antibody.

25. The antibody-drug conjugate according to claim 24, characterized in that, Ab is a monoclonal antibody.

26. The antibody-drug conjugate according to claim 24, characterized in that, Ab is a chimeric antibody.

27. The antibody-drug conjugate according to claim 24, characterized in that, Ab is a humanized antibody.

28. The antibody-drug conjugate according to claim 24, characterized in that, Ab represents an antibody fragment.

29. The antibody-drug conjugate according to claim 24, characterized in that, Ab is a polypeptide.

30. The antibody-drug conjugate according to claim 24, characterized in that, The antibody (Ab) is selected from: trastuzumab, avelumab, mouse anti-human HER2 antibody, anti-mouse PD-L1 (B7-H1), atezolizumab, or other antibodies targeting the following targets: HER2, CD30, Netin-4, Trop3, CD79b, CD22, BCMA, TF, CD33, CD22, EGFR, TSHR, FSHR, LHR, CD19, FAP, and FR-alpha.

31. An antibody-drug conjugate, characterized in that, Including antibodies conjugated to the following linkers-payloads: 。 32. A pharmaceutical composition, characterized in that, Includes the antibody-drug conjugate according to any one of claims 13-31.

33. The pharmaceutical composition according to claim 32, characterized in that, It can effectively treat, prevent, or alleviate autoimmune diseases or related illnesses or conditions.

34. The pharmaceutical composition according to claim 32 or 33, characterized in that, It can effectively treat, prevent, or alleviate graft rejection or related diseases or symptoms.

35. The pharmaceutical composition according to claim 32 or 33, characterized in that, It can effectively treat, prevent, or alleviate allergies or related diseases or conditions.

36. The pharmaceutical composition according to claim 32 or 33, characterized in that, It can effectively treat, prevent, or alleviate immune deficiencies or related diseases or conditions.

37. The pharmaceutical composition according to claim 32 or 33, characterized in that, It can effectively treat, prevent, or alleviate infections, sepsis, or related diseases or conditions.

38. The pharmaceutical composition according to claim 32 or 33, characterized in that, It can effectively treat, prevent, or alleviate cancer or related diseases or symptoms.

39. A unit dosage form, characterized in that, The pharmaceutical composition comprising any one of claims 32-38.

40. A method for treating, alleviating, or preventing a disease or symptom, characterized in that, This includes administering the antibody-drug conjugate according to any one of claims 13-31 to a subject who requires it.

41. The method according to claim 40, characterized in that, The diseases or conditions mentioned are selected from: autoimmune diseases, graft rejection, allergies, immunodeficiency, infections, sepsis, cancer, and related diseases or conditions.

42. The method according to claim 41, characterized in that, The disease or condition mentioned is an autoimmune disease or a related disease or condition.

43. The method according to claim 41, characterized in that, The disease or condition mentioned is graft rejection or a related disease or condition.

44. The method according to claim 41, characterized in that, The disease or condition mentioned is an allergic or related disease or condition.

45. The method according to claim 41, characterized in that, The disease or condition mentioned is an immune deficiency or related disease or condition.

46. ​​The method according to claim 41, characterized in that, The disease or condition is an infection and / or sepsis, or a related disease or condition.

47. The method according to claim 41, characterized in that, The disease or condition mentioned is cancer or a related disease or condition.

48. A method for regulating an immune response, characterized in that, This includes administering the antibody-drug conjugate according to any one of claims 13-31 to a subject who requires it.

49. A method for regulating signal transduction mediated by TLR7 and / or TLR8, characterized in that, This includes administering the antibody-drug conjugate according to any one of claims 13-31 to a subject who requires it.

50. A method for treating or alleviating a disease or condition, said disease or condition being treated by modulating TLR7 and / or TLR8-mediated cell activity, characterized in that, This includes administering the antibody-drug conjugate according to any one of claims 13-31 to a subject who requires it.

51. An application characterized in that, It refers to the use of the antibody-drug conjugate according to any one of claims 13-31 in the treatment or relief of a disease or condition.

52. An application characterized in that, It is the drug-linker conjugate of any one of claims 1-12 or the antibody-drug conjugate of any one of claims 13-31, and the use of pharmaceutically acceptable excipients, carriers or diluents in the preparation of medicaments for treating or alleviating diseases or conditions.

53. The application according to claim 51 or 52, characterized in that, The disease or condition is associated with TLR7 and / or TLR8-mediated signal transduction.

54. The application according to claim 51 or 52, characterized in that, The disease or condition mentioned is selected from autoimmune diseases, graft rejection, allergies, immunodeficiency, infections, sepsis, cancer or related diseases or conditions.

55. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate autoimmune diseases or related diseases or conditions.

56. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate graft rejection or related diseases or conditions.

57. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate allergies or related diseases or conditions.

58. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate immune deficiencies or related diseases or conditions.

59. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate infection and / or sepsis, or related diseases or conditions.

60. The application according to claim 51 or 52, characterized in that, Used to treat or alleviate cancer or related diseases or conditions.

61. The application according to claim 51 or 52, characterized in that, The conjugate is used as a vaccine adjuvant.

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