Small molecule-drug conjugates cleavable in tumor microenvironment

Abstract

The present invention relates to novel pharmaceutical compounds comprising one or more binding molecules (e.g., a protein of interest binding agent T) capable of binding a protein of interest expressed on a tumor cell or on a cell present in the tumor microenvironment and linked to one or more payload molecules via a protease cleavable linker; a preparation method thereof; and their use for the treatment of diseases and conditions in humans and other mammals, including hyperproliferative disorders such as cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This international patent application claims the benefit of European Patent Application No. 22306696.0 filed on November 17, 2022, and European Patent Application No. 23305484.0 filed on April 3, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] The present application relates to small molecule-drug conjugates (or "Compounds"), pharmaceutical compositions, methods for their preparation, and their use for treating, preventing, or managing diseases and conditions in humans and other mammals, including hyperproliferative disorders such as cancer. Summary of the Invention

[0004] Provided herein are compounds comprising one or more active agents (i.e., payloads) having therapeutic activity (e.g., cytotoxic or immunostimulatory activity), which are conjugated to one or more target protein binding agents, preferably small molecule binding agents, via one or more linking units. The compounds provided herein may include one or more enzymatically cleavable peptide linkers that enable targeted release of the payload in the tumor microenvironment. The peptide linker can be cleaved, for example, by neutrophil elastase (a protease secreted by neutrophils), particularly in response to inflammation and / or disease. Neutrophil elastase can be overexpressed in the tumor microenvironment, thereby providing site-specific delivery of cytotoxic payloads to tumors. A particular advantage of use according to the present invention is a cell-permeable payload that can penetrate diseased cells (e.g., tumor cells) when released in the tumor microenvironment, thereby producing a cytotoxic / anti-tumor effect.

[0005] The compound may include one or more (e.g., one, two, etc.) payloads and / or one or more targeting groups conjugated via a linker, the linker including a cleavable peptide and optionally further including a solubility enhancing or tumor maintaining polymeric linker unit (e.g., polyethylene glycol, polyethyleneimine, polysarcosine, etc.) and / or a branching unit. The branching unit may be a trivalent or tetravalent group, which may optionally include an amino acid or a peptide. The payloads provided herein include, but are not limited to, PTEFb inhibitors, topoisomerase inhibitors, spindle kinesin inhibitors, tubulin inhibitors, and immune agonists. Specific examples include camptothecin and its derivatives (e.g., 7-ethylcamptothecin, exatecan and N-acetyl or N-alkyl exatecan derivatives, etc.), diterpenoids (e.g., triptolide, paclitaxel or its derivatives), auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F, etc.), toll-like receptor agonists (e.g., resiquimod, imiquimod), or other small molecule cytotoxins that can be cleaved by neutrophil elastase. In some embodiments, the therapeutic payload is a novel cytotoxin with favorable permeability and / or low efflux. Advantageously, the payloads provided herein have been shown to be cleaved in the tumor microenvironment without the use of self-immolative groups, which can result in unwanted off-target release of the payload. An additional advantage is that the compounds provided herein do not need to be internalized within the cell to produce an anti-tumor effect.

[0006] The compounds provided herein further include at least one and optionally two target protein binding agents, which further direct the compound to the desired site of action (e.g., a tumor or tumor microenvironment). The target protein binding agents disclosed herein are generally small molecules (molecular weight of about 1000 g / mol or less). The target protein to which the binding agent can bind includes but is not limited to α v β3 (avb3) integrin binders, fibroblast activation protein (FAP) binders, folate receptor (FR) binders, prostate-specific membrane antigen (PSMA) binders, and carbonic anhydrase IX (CAIX or CA9) binders, etc.

[0007] Small molecules and their conjugates may have certain advantages over their antibody counterparts (e.g., antibody-drug conjugates) due to their lower molecular weight, plasma protein binding, or other pharmacological or pharmacokinetic considerations. In addition, the conjugates provided herein may provide additional advantages over the payload itself, because not only is the payload directed to the desired cells or tissues, but the payload is non-selectively distributed throughout the subject, the conjugates provided herein may also have novel and favorable pharmacokinetic benefits (e.g., distribution, metabolism, clearance, secretion, half-life, AUC, etc.). In some embodiments, the half-life of the conjugates provided herein is more than 24 hours. Also provided herein are novel conjugate designs and / or arrangements of binding agents, payloads, and linkers. In some embodiments, the conjugates further include a pharmacokinetic modulator that can regulate one or more pharmacokinetic parameters of the conjugate. In some embodiments, the pharmacokinetic modulator includes a charged or ionizable group (e.g., a carboxylate group, a sulfonate group, a sulfinate group, a phosphonate group, an amine group, an imine group, a guanidine group, etc.).

[0008] In one aspect, provided herein is a compound or a pharmaceutically acceptable salt thereof, comprising one or more payloads conjugated to one or more target protein binding agents via a linker. In some embodiments, the compound or a pharmaceutically acceptable salt thereof has a structure of formula (I):

[0009] T––L––EL––P

[0010] Formula (I)

[0011] in:

[0012] T is the target protein binder;

[0013] P is the effective load;

[0014] EL is a peptide linker, which optionally further comprises a self-immolative group; and

[0015] L is a linker, which optionally further comprises a second T and / or a second EL-P.

[0016] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula (II) (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), or Formula (IX):

[0017]

[0018] in:

[0019] Each P is a valid load;

[0020] Each EL is a peptide linker, which optionally further comprises a self-immolative group;

[0021] Each L 1 , L 2 , L 3 and L 4 are independently a divalent linker,

[0022] A 1 is a trivalent linker;

[0023] A 2 is a tetravalent linker;

[0024] Each T is a target protein binder; and

[0025] MOD is the pharmacokinetic modulator group.

[0026] In some embodiments, the target protein binding agent is a binding agent for avb3 integrin, avb6 integrin, PSMA, CAIX, FAP, folate, Hsp90, somatostatin, GLUT1, APN, LRP1, bombesin, GnRH, LHRH, MT1-MMP, P32, phosphatidylserine, or sortilin. In some embodiments, the payload is a tubulin regulator, a DNA regulator, an RNA regulator, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, or an immunomodulator. In some embodiments, the bivalent linker comprises 1 to 12 (e.g., 3 to 6) PEG or PEI units; and the compound may comprise multiple bivalent linkers. In some embodiments, provided herein is a pharmaceutical composition comprising a compound or salt of Formula (II) (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), or Formula (IX), and a pharmaceutically acceptable excipient.

[0027] In yet another aspect, provided herein is a compound or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutical composition thereof, for use as a medicament. In yet another aspect, described herein is a compound or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof, or a pharmaceutical composition thereof, for use in a method of treating a disease or condition disclosed herein. In some embodiments, the disease or condition is a hyperproliferative disorder. In some embodiments, the disease or condition is cancer.

[0028] Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0029] References incorporated

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent such incorporated by reference publications, patents, or patent applications contradict the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The features and advantages of the present invention will be better understood by referring to the following detailed description, which describes illustrative embodiments utilizing the principles of the invention, and the accompanying drawings (also referred to herein as "Figures" or "FIG."), wherein:

[0032] Figure 1 Shown are cytokine release from freshly prepared PBMCs from three healthy donors following treatment with the indicated compounds (1 μM). DETAILED DESCRIPTION

[0033] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Many variations, changes, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0034] In some embodiments, provided herein are novel pharmaceutical compounds comprising one or more binding molecules that bind to a target molecule expressed on a tumor cell or on a cell present in the tumor microenvironment and are linked to one or more payload molecules via a protease-cleavable linker; methods for their preparation; and their use for treating, preventing, or managing diseases and conditions in humans and other mammals, including hyperproliferative disorders such as cancer.

[0035] Examples of binding molecules include those for cell adhesion proteins (e.g., integrins, such as α v β3 integrin, α vβ6 integrin, prostate-specific membrane antigen (PSMA), fibroblast activation protein (FAP), carbonic anhydrase IX (CAIX)) binders, chaperone protein heat shock protein 90 (Hsp 90) binders, folate receptor binders, glucose transporter 1 binders, somatostatin receptor binders, aminopeptidase N (APN) binders, low-density lipoprotein receptor-related protein 1 (LRP1) binders, bombesin receptor binders, gonadotropin-releasing hormone (GnRH or LHRH) receptor binders, P32 binders, membrane type 1 matrix metalloproteinase (MT1-MMP) binders, sortilin binders, and nectin-4 binders.

[0036] Protease cleavable linkers include peptide sequences that can be cleaved by tumor-associated enzymes present in the tumor microenvironment. Many of these enzymes have been shown to be part of a protease family of enzymes that primarily regulates the mobility of tumor stroma and tumor cells. Tumor microenvironment shaping proteases are, for example, serine proteases such as plasmin activators, seprase, type II transmembrane serine proteases (hepsin), or kallikreins. Another protease family in the TME is a cysteine ​​protease (such as cathepsin B and cathepsin K) or an aspartic protease (such as cathepsin D and cathepsin E), but other proteases (such as heparanase, endoglycosidase, and hyaluronidase) have been shown to be upregulated and activated within the TME. Such enzymes may be proteases, such as matrix metalloproteinases and neutrophil elastases.

[0037] The effective load employed in such conjugates may be a cytotoxic agent or an immunostimulatory agent. In some preferred embodiments, provided herein are compounds having an effective load such as a camptothecin derivative, an auristatin derivative, a CDK9 / PTEFb derivative, a spindle kinesin inhibitor derivative, or a toll-like receptor 7 and / or 8 ("TLR7 / 8") agonist derivative.

[0038] Cancer chemotherapy is often associated with severe side effects due to the toxic effects of the chemotherapeutic agents on proliferating cells of tissue types other than tumor tissue.For many years, scientists have been busy solving the problem of improving the selectivity of the active compounds employed.

[0039] 20(S)-camptothecin is a pentacyclic alkaloid isolated by Wall et al. (J. Am. Chem. Soc. 88, 3888 (1966)) in 1966. It has high anti-tumor potential in many in vitro and in vivo tests. Unfortunately, however, due to toxicity and solubility issues, the promising potential was not realized during the clinical investigation stage.

[0040] A large number of camptothecin derivatives have been investigated in preclinical and clinical studies; among them, irinotecan, topotecan, and belotecan have been successfully approved (Li et al., Am J Cancer Res 2017;7(12):2350-2394). Some of these derivatives (AW) are listed below.

[0041]

[0042]

[0043] Improving the therapeutic window of cytotoxic agents such as camptothecin and its derivatives remains a challenge.

[0044] Integrins are heterodimeric transmembrane proteins expressed on the cell surface that play an important role in cell attachment to the extracellular matrix. They recognize extracellular glycoproteins on the extracellular matrix, such as fibronectin or vitronectin, via the RGD sequence present in these proteins (RGD is the single letter code for the amino acid sequence Arginine-Glycine-Aspartic Acid). Typically, integrins (such as, for example, vitronectin receptors, which are also known as α v β3 receptor or alternatively called α v β5 receptors or GpIIb / IIIa receptors) play an important role in biological processes such as cell migration, angiogenesis and cell-matrix adhesion and therefore play an important role in diseases in which these processes are key steps. Examples include cancer, osteoporosis, arteriosclerosis, restenosis and ophthalmitis.

[0045] α v β3 receptors are present in large numbers on growing endothelial cells, for example, and make it possible for them to adhere to the extracellular matrix. v The β3 receptor therefore plays an important role in angiogenesis (ie, the formation of new blood vessels), a key prerequisite for tumor growth and metastasis formation in cancerous conditions.

[0046] It is possible to show that blocking the above-mentioned receptors is an important starting point for the treatment of this type of disease. If the adhesion of growing endothelial cells to the extracellular matrix is ​​inhibited by blocking their corresponding integrin receptors (for example by cyclic peptides or monoclonal antibodies), angiogenesis will not occur, leading to cessation or regression of tumor growth. Although strong preclinical results suggest that the inhibition of integrins has therapeutic potential, clinical trials using integrin inhibitors targeting those integrins have repeatedly failed to show therapeutic benefit in cancer patients.

[0047] The conjugates provided herein can be selectively concentrated in tumor tissue by introducing one or more binding molecules (i.e., target protein binding agents), and the binding molecules can be combined with target proteins (e.g., receptors) expressed in tumor tissue, preferably overexpressed. The conjugates provided herein may include one or more protease cleavable payloads (e.g., therapeutic payloads) that exhibit cytotoxicity or immunostimulatory properties when cleaved by protease. In order to reduce off-target cytotoxicity or immunostimulatory properties, the payload can be bound to the conjugate (as provided herein) via a protease cleavable linker, wherein the protease is selectively expressed (e.g., overexpressed) by tumor cells or by cells present in the tumor microenvironment. When the release of payload occurs near tumor tissue (immediate vicinity) (i.e., within the tumor microenvironment) or in tumor cells, the targeted cytotoxicity or immunostimulatory effects of the conjugates provided herein (or the therapeutic payload conjugated thereto) can be further enhanced. Additional advantages and practicality are therapeutic payloads that can penetrate tumor cells when released from the conjugate. Preferably, the therapeutic payload is not effluent or transported to the extracellular space.

[0048] WO 2000 / 069472 discloses enzyme-activated anti-tumor prodrug compounds that are specifically cleavable by collagenase (IV) and elastase. Regarding the linker unit that can be cleaved by elastase, the present application describes the specific tetrapeptide sequences Ala-Ala-Pro-Val and Ala-Ala-Pro-Nva as suitable. In addition, in this reference, there is no mention of including anti-α v Y.Liu et al. (Mol.Pharmaceutics 2012, 9, 168) described a conjugate of a portion of the β3 integrin receptor and a cytostatic. v Conjugate of auristatin with a β3 integrin targeting moiety.

[0049] EP1238678 discloses conjugates with cytotoxic agents that target α v β3 integrin and having a peptide linker specifically cleavable by elastase. Regarding the linker unit cleavable by elastase, the present application describes peptide sequences comprising Pro-Val and Pro-Leu, as exemplified by the toxophore moieties camptothecin and quinolone carboxylic acid.

[0050] Specific challenges for this type of conjugate include:

[0051] Sufficient solubility to enable intravenous administration in an appropriate vehicle,

[0052] High tumor penetration of the intact conjugate,

[0053] High stability in plasma to avoid systemic deconjugation,

[0054] Effectively binds to target receptors in the tumor microenvironment,

[0055] Efficiently cleaved by enzymes present in the tumor microenvironment,

[0056] The high cell permeability and low efflux ratio of the lysed toxin-producing fraction enhances tumor cell uptake and redistribution.

[0057] Therefore, the goal is to develop conjugates that include a moiety directed against a tumor target and a payload that is preferably released from the conjugate in the tumor microenvironment, wherein the moiety in the conjugate that is directed against a receptor in tumor tissue retains its ability to bind to the receptor and thus provides tissue selectivity for such compounds. In addition, the cleavage of the conjugate and drug release should be mediated by enzymes present and active in the tumor environment (such as neutrophil elastase). Finally, the profile of the toxin bolus should be suitable for extracellular cleavage and release mechanisms. The toxin bolus should be highly permeable to tumor cells and tissues and not a substrate for drug transporters.

[0058] WO2020094471 describes α v A conjugate of β3 integrin with 7-ethylcamptothecin, and in WO2020094471, a negatively charged carboxyl group and a PEG unit were introduced into the linker to address solubility. Many camptothecin derivatives have been studied as payloads in antibody-drug conjugates, such as DxD in Enhertu (Modi et al., N. Engl. J. Med. 382, ​​610-621) and SN38 in Trodelvy (Rugo et al., Future Oncol. 16, 705-715), both of which have recently been approved.

[0059] Since small molecule drug conjugates can show advantageous features over ADC, such as higher tumor penetration (Cazzamalli et al., J.Am.Chem.Soc.2018, 140, 1617), the present invention includes small molecule drug conjugates with selected camptothecin derivatives. Preferred camptothecin derivatives should show high efficacy, high membrane penetration properties and low efflux ratio. Exemplary camptothecin derivatives described herein include 7-ethylcamptothecin ("7EC"), 10,11-methylenedioxy-camptothecin ("FL118") and exotecan or its derivatives (e.g., N-alkyl or N-acyl derivatives of exotecan provided herein).

[0060] In order to improve tumor targeting and accumulation in the tumor microenvironment, the present invention relates to conjugates comprising one or more tumor binding molecules, a linker unit that can be selectively cleaved by a tumor-associated enzyme (such as neutrophil elastase), and one or more payload molecules. The special structural features of such conjugates may be, but are not limited to, polyalkylamine spacers, multimeric binding achieved by two or more binding molecules in the conjugate through a dendrimer approach, and / or slow release of prodrug residues to modulate (i) the PK of the conjugate, (ii) drug release from the conjugate, and / or (iii) the PK of the released free drug. Such conjugates have tumor-specific effects because they are linked to tumor homing molecules (such as binding agents) via preferred linkers that can be selectively cleaved by tumor-associated enzymes (such as neutrophil elastase). Preferred linkers provide sufficient stability of the compound / conjugate in biological media (e.g., culture medium or serum) and at the same time, due to their specific enzymatic or hydrolytic cleavability, the drug moiety is released, resulting in the desired intracellular effect within the tumor tissue.

[0061] In particular, the compounds of the present invention exhibit one or more of the following characteristics:

[0062] The modified alkyl spacer is interrupted by one or more N-alkylamino groups, which has a beneficial effect on retention in the tumor microenvironment with acidic pH, and also allows for high solubility

[0063] One or more tumor-binding moieties enable high or moderate affinity

[0064] One or more payload moieties enable high anti-proliferative potency

[0065] Despite significant steric challenges, these novel linkers appear to be highly susceptible to cleavage by enzymes present in the tumor stroma, such as neutrophil elastase

[0066] High stability of the conjugate in plasma and increased cytotoxic activity in the presence of elastase

[0067] definition

[0068] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from what is generally understood in the art.

[0069] Throughout this application, various embodiments can be presented in range format. It should be understood that the description of range format is only for convenience and simplicity and should not be construed as an unchangeable restriction to the scope of the present disclosure. Therefore, the description of the scope should be considered as each numerical value in all possible subranges disclosed specifically and the scope. For example, the description of a scope such as 1 to 6 should be considered as each numerical value (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and the scope. Regardless of the breadth of the scope, this is all applicable.

[0070] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes plural samples, including mixtures thereof.

[0071] The terms "determine," "measure," "evaluate," "assess," "assay," and "analyze" are generally used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present (e.g., detecting). These terms may include quantitative, qualitative, or both quantitative and qualitative determinations. Assessments may be relative or absolute. Depending on the context, "detecting the presence of" may include determining the amount of something present in addition to determining whether something is present.

[0072] The terms "subject," "individual," or "patient" are generally used interchangeably herein. A "subject" can be a biological entity containing expressed genetic material. A biological entity can be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, cell, or progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. A mammal can be a human. A subject can be diagnosed or suspected of being at high risk for a disease. In some cases, a subject may not necessarily be diagnosed or suspected of being at high risk for a disease.

[0073] As used herein, the term "about" a number refers to the stated number plus or minus 15% of the stated number. The term "about" a range refers to the stated range minus 15% of its minimum value and plus 15% of its maximum value.

[0074] As used herein, the terms "treatment" or "treating" are used in reference to a drug or other intervention regimen for obtaining a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit may refer to the eradication or mitigation of the symptoms or underlying condition being treated. Additionally, therapeutic benefit may be achieved by eradicating or mitigating one or more physiological symptoms associated with the underlying condition so that an improvement is observed in the subject, although the subject may still be suffering from the underlying condition. Prophylactic effects include delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, preventing, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease or a subject reporting one or more physiological symptoms of a disease may be treated, even though a diagnosis of the disease may not yet have been performed.

[0075] As used herein, the term "payload" or "therapeutic payload" generally refers to a small molecule (i.e., non-protein) compound with therapeutic (e.g., anticancer) activity in cells and / or tissues. Preferably, the therapeutic activity is exhibited after separation from the cleavable group. In some embodiments, the cleavable group is an enzymatically cleavable group. In some embodiments, the therapeutic payload is activated after cleavage by a tumor-associated protein (such as neutrophil elastase). The therapeutic payload may be, for example, a drug. In some embodiments, the therapeutic payload is a cytotoxic compound, a cell growth inhibitory compound, or an immunomodulatory compound. In some embodiments, the therapeutic payload can effectively kill cancer cells or slow the growth of cancer cells. In some embodiments, the therapeutic payload is a spindle kinesin inhibitor, camptothecin, or a derivative thereof, a CDK9 inhibitor, etc., as described herein.

[0076] As used herein, the term "target protein" generally refers to a protein expressed on the surface of a cell (e.g., a cancer cell) that can effectively bind to a small molecule binding agent. As used herein, effective load generally refers to a compound with micromolar potency or better (e.g., submicromolar, nanomolar, subnanomolar, etc., as used in the art). Examples of target proteins are as defined herein.

[0077] As used herein, the term "non-cleavable linker" refers to a linking unit of atoms that are not yet known to be chemically or biologically unstable (e.g., 1 to 200 atoms selected from C, H, N, O, S, and halogens). The term "non-cleavable linker" is intended to be distinguished from cleavable linkers (e.g., protease-cleavable linkers, self-degradable linkers, pH-sensitive linkers, etc.). The non-cleavable linker may be an alkyl or heteroalkyl linker, which is optionally interrupted by one or more cyclic or heterocyclic groups (e.g., a click partner or an artifact produced therefrom). The non-cleavable linker may include a polymeric portion (e.g., PEG, PEI, polysarcosine, etc.) and / or an alkyl portion.

[0078] The non-cleavable linker may further include another functional group, such as a small molecule target protein binder, a pharmacokinetic modulator (e.g., a -COOH group), and / or one or more therapeutic payloads. The non-cleavable linker may be combined with a protease cleavable linker. In such cases, the protease cleavable linker can be cleaved by a protease (e.g., in a tumor microenvironment) to release the therapeutic payload and stably bind the non-cleavable linker to the antibody. In some embodiments, the non-cleavable linker and / or protease cleavable linker disclosed herein does not include a self-degradable linker group, which can further enhance the stability of the ADC disclosed herein (e.g., reduce off-target release).

[0079] As used herein, C1-C x Including C1-C2, C1-C3...C1-C x By way of example only, a group designated as "C1-C6" indicates that there are from one to six carbon atoms in the moiety, e.g., groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Thus, by way of example only, "C1-C4 alkyl" indicates that there are from one to four carbon atoms in the alkyl group, e.g., an alkyl group selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0080] "Alkyl" refers to an aliphatic hydrocarbon group. An alkyl group is a branched or straight chain. In some embodiments, an "alkyl" group has 1 to 10 carbon atoms, for example, C1-C 10Alkyl. Whenever a numerical range such as "1 to 10" appears herein, it refers to each integer within the given range; for example, "1 to 10 carbon atoms" means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although this definition also encompasses the presence of the term "alkyl" where no numerical range is specified. In some embodiments, the alkyl group is a C1-C6 alkyl group. In one aspect, the alkyl group is a methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl group. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, or hexyl group.

[0081] An "alkylene" group refers to a divalent alkyl group. Any of the above-mentioned monovalent alkyl groups may be an alkylene group formed by abstracting the second hydrogen atom from an alkyl group. In some embodiments, the alkylene group is a C1-C6 alkylene group. In other embodiments, the alkylene group is a C1-C4 alkylene group. Typical alkylene groups include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2-. In some embodiments, the alkylene group is -CH2-.

[0082] An "alkoxy" group refers to an -O(alkyl) group, wherein alkyl is as defined herein. Examples of alkoxy groups include -OCH3, -OCH2CH3, -OCH2CH2CH3, and -OC(CH3)3, among others.

[0083] "Hydroxyalkyl" refers to an alkyl group in which one hydrogen atom is replaced by a hydroxyl group. In some embodiments, the hydroxyalkyl group is a C1-C4 hydroxyalkyl group. Typical hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, and -C(CH3)2OH.

[0084] The term "alkylamine" refers to -N(alkyl) x H y A group wherein x is 0 and y is 2, or wherein x is 1 and y is 1, or wherein x is 2 and y is 0.

[0085] "Aminoalkyl" refers to an alkyl group in which one hydrogen atom is replaced by an amino group. In some embodiments, the aminoalkyl group is a C1-C4 aminoalkyl group. Typical aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and -C(CH3)2NH2.

[0086] The term "heteroalkyl" generally refers to a straight and / or branched hydrocarbon chain having 1 to 30 carbon atoms and which may be interrupted one or more times by one or more of the following groups: -O-, -S-, -C(=O)-, -S(=O)-, -S(=O)2-, -NR y -、-NR y C(=O)-, -C(=O)-NR y -、-NR y NR y -、-S(=O)2-NR y NR y -, -C(=O)-NR y NR y -and-CR x =NO-, and the hydrocarbon chain including the side chain (if present) may be substituted by: -NH-C(=O)-NH2, -C(=O)-OH, -OH, -NH2, -NH-C(=NNH2)-, sulfonamide, sulfone, sulfoxide, sulfonic acid, sulfamide, or a combination thereof. In this context, R y In each case -H, phenyl, C1-C 10 -alkyl, C2-C 10 -alkenyl or C2-C 10 -alkynyl, which in turn may be substituted in each case by -NHC(O)NH2, -COOH, -OH, -NH2, -NH-C(=NNH2)-, sulfonamide, sulfone, sulfoxide, sulfonic acid, sulfamide, or a combination thereof. In this context, R x is -H, C1-C3 alkyl or phenyl. As used herein, the terms "heteroalkyl-aryl" and "heteroalkyl-heteroaryl" generally refer to heteroalkyl (as defined above) substituted with an aromatic carbocycle or aromatic heterocycle, respectively; and each of which is optionally substituted.

[0087] The term "aromatic" refers to a planar ring having a delocalized π-electron system containing 4n+2π electrons, where n is an integer. The term "aromatic" includes both carbocyclic aromatic ("aryl," e.g., phenyl) and heterocyclic aromatic (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (or rings sharing adjacent pairs of carbon atoms) groups.

[0088] The term "carbocyclic" or "carbocycle" refers to a ring or ring system in which the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocycle from a "heterocyclic" ring or "heterocycle" in which the ring backbone contains at least one atom other than carbon. In some embodiments, at least one of the two rings of a bicyclic carbocycle is aromatic. In some embodiments, both rings of a bicyclic carbocycle are aromatic. Carbocycles include aryl and cycloalkyl groups.

[0089] As used herein, the term "aryl" refers to an aromatic ring wherein each atom forming the ring is a carbon atom. In one aspect, aryl is phenyl or naphthyl. In some embodiments, aryl is phenyl. In some embodiments, aryl is phenyl, naphthyl, dihydroindenyl, indenyl, or tetrahydronaphthyl. In some embodiments, aryl is C6-C 10 Aryl. Depending on the structure, an aryl group is a monovalent or divalent group (or an arylene group). As used herein, the term "aralkyl" generally refers to a C 1-4 A monocyclic aromatic carbocyclic ring (e.g., phenyl) to which an alkyl group is bonded. Illustrative aralkyl groups include benzyl and ethylphenyl.

[0090] The term "cycloalkyl" refers to a monocyclic or polycyclic aliphatic, non-aromatic group, wherein each of the atoms (or skeletal atoms) forming the ring is a carbon atom. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is optionally fused to an aromatic ring, and the point of attachment is at a carbon that is not an aromatic ring carbon atom. The cycloalkyl group includes a group with 3 to 10 ring atoms. In some embodiments, the cycloalkyl group is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, spiro [2.2] pentyl, norbornyl and bicyclo [1.1.1] pentyl. In some embodiments, the cycloalkyl is a C3-C6 cycloalkyl. In some embodiments, the cycloalkyl is a C3-C4 cycloalkyl.

[0091] The term "halo" or alternatively "halogen" or "halide" means fluoro, chloro, bromo or iodo. In some embodiments, halo is fluoro, chloro or bromo.

[0092] The term "fluoroalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by fluorine atoms. In one aspect, the fluoroalkyl group is a C1-C6 fluoroalkyl group.

[0093] The term "optionally substituted" or "substituted" means that the referenced group is optionally substituted with one or more additional groups individually and independently selected from the group consisting of halogen, -CN, -NH2, -NH(alkyl), -N(alkyl)2, -OH, -C02H, -C02alkyl, -C(=O)NH2, -C(=O)NH(alkyl), -C(=O)N(alkyl)2, -S(=O)2NH2, -S(=O)2NH(alkyl), -S(=O)2N(alkyl)2, alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from: halogen, -CN, -NH2, -NH(CH3), -N(CH3)2, -OH, -CO2H, -CO2(C1-C4 alkyl), -C(=O)NH2, -C(=O)NH(C1-C4 alkyl), -C(=O)N(C1-C4 alkyl)2, -S(=O)2NH2, -S(=O)2NH(C1-C4 alkyl), -S(=O)2N(C1-C4 alkyl)2, C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 fluoroalkyl, C1-C4 heteroalkyl, C1-C4 alkoxy, C1-C4 fluoroalkoxy, -SC1-C4 alkyl, -S(=O)C1-C4 alkyl and -S(=O)2C1-C4 alkyl. In some embodiments, the optional substituents are independently selected from the group consisting of halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CHF2, -CF3, -OCH3, -OCHF2, and -OCF3. In some embodiments, the substituted groups are substituted with one or both of the preceding groups. In some embodiments, the optional substituents on aliphatic carbon atoms (acyclic or cyclic) include oxo (=O).

[0094] As used herein, "small molecule" generally refers to any molecule with a molecular weight of about 1000 atomic mass units (Daltons) or less. In some embodiments, a portion within a compound described herein is considered a small molecule, meaning that the portion has a molecular weight of about 1000 Da or less. As used herein, small molecules do not include proteins or antibodies, but may include peptides or amino acids. In some cases, the compounds described herein are conjugates of two small molecule moieties. Therefore, the compounds described herein may be referred to as "small molecule drug conjugates" (SMDC) or "small molecule prodrug conjugates" (SMPC). For example, SMPC may include a cleavable group (e.g., enzymatic cleavage or chemical cleavage), such as an ester, which produces an SMDC when cleaved. In some instances, an SMPC described herein (sometimes referred to as a prodrug) may first be cleaved (e.g., in plasma) before an enzyme can effectively recognize and cleave the SMDC, thereby releasing the active agent in two steps. In some embodiments, SMPC achieves a slow conversion to SMDC, which is cleaved more rapidly (i.e., in the presence of a suitable enzyme (e.g., a tumor-associated enzyme such as elastase, legumin, or cathepsin)), thereby enhancing the therapeutic window or reducing side effects.

[0095] As used herein, "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively nontoxic at the concentrations or amounts employed, e.g., when administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any component of a composition in which it is contained.

[0096] The term "pharmaceutically acceptable salt" refers to a form of a therapeutically active agent consisting of a cationic form of the therapeutically active agent in combination with a suitable anion, or in alternative embodiments, a form of the therapeutically active agent consisting of an anionic form of the therapeutically active agent in combination with a suitable cation. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. SM Berge, LD Bigley, DC Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. PH Stahl and CG Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich: Wiley-VCH / VHCA, 2002. Pharmaceutical salts are generally more soluble and dissolve more rapidly in gastric and intestinal fluids than non-ionic species and are therefore suitable for use in solid dosage forms. Furthermore, since their solubility is generally a function of pH, selective solubility in one part of the digestive tract or another is possible and this ability can be manipulated as an aspect of delayed and sustained release behavior. Furthermore, since salt-forming molecules can be equilibrated in neutral form, delivery across biological membranes can be modulated.

[0097]

[00146] The term "acceptable," with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0098] As used herein, the terms "administer," "administering," and "administration" refer to methods that can be used to achieve delivery of a compound or composition to a desired biological site of action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), topical, and rectal administration. Those skilled in the art are familiar with the administration techniques that can be employed for the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0099] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or compound administered that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated. Results include alleviation or relief of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant reduction in disease symptoms. Techniques such as dose escalation studies are optionally used to determine an appropriate "effective" amount in any individual case.

[0100] As used herein, the terms "enhance" or "enhancing" mean to increase or prolong either the potency or duration of a desired effect. Thus, with respect to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong either the potency or duration of the effect of other therapeutic agents on a system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of other therapeutic agents in a desired system.

[0101] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0102] Small molecule-drug conjugates

[0103] Cancer cells or tumor microenvironments overexpress certain enzymes, including but not limited to neutrophil elastase. The conjugates described herein can generally be enzymatically cleaved by neutrophil elastase. In some embodiments, the conjugates described herein are selectively cleavable in the microenvironment of cancer cells, with less cleavage occurring in the circulation or in healthy tissues. In some embodiments, the conjugates described herein are cytotoxic or immunostimulatory after activation by tumor-related enzymes (such as neutrophil elastase and / or legumin). In some embodiments, the conjugates described herein are non-toxic at therapeutic concentrations in the absence of the activation. The cytotoxic agents described herein are topoisomerase (topoisomerase I) inhibitors, cyclin-dependent kinase 9 (CDK9) / positive transcription elongation factor (P-TEFb) inhibitors, spindle kinesin (KSP) inhibitors, or tubulin polymerization inhibitors. In some embodiments, the cytotoxic agent is a small molecule, peptide, peptide derivative (e.g., auristatin). In some embodiments, the conjugates described herein are immunostimulatory upon activation by neutrophil elastase (e.g., via activation of a toll-like receptor (TLR), which may include TLR 7 and / or TLR 8 (TLR7 / 8)). The cytotoxic agent or immunostimulatory agent may also be conjugated to a protein-binding moiety, e.g., via an enzymatically cleavable linker or spacer, or to a multivalent linker attached to two or more protein-binding agents. In some embodiments, the protein of interest is αv β3 (avb3) integrin, α v beta6 (avb6) integrin, fibroblast activation protein (FAP), folate receptor (FR), prostate-specific membrane antigen (PSMA), or carbonic anhydrase IX (CAIX or CA9).

[0104] In some embodiments, provided herein are small molecule-drug conjugates (SMDCs) designed for payload release and activation in the tumor microenvironment (TME). The linkers disclosed herein have been optimized to substrate sequences for enzymes that are upregulated in the tumor microenvironment. Many of these enzymes are part of a protease family of enzymes that are involved in regulating the motility of tumor stroma and tumor cells. Some of these tumor microenvironment-forming proteases are, for example, serine proteases, such as plasmin activators, separases, type II transmembrane serine proteases, or kallikreins. Another protease family in the TME is a cysteine ​​protease (such as cathepsin B and cathepsin K) or an aspartic protease (such as cathepsin D and cathepsin E), but other proteases (such as heparanase, endoglycosidase, and hyaluronidase) have been shown to be upregulated and activated in the TME. Such enzymes may be proteases, such as matrix metalloproteinases, cathepsins, and neutrophil elastases.

[0105] Another key aspect of SMDC performance is the physicochemical properties of the released payload. The payload cleaved extracellularly by the SMDC disclosed in the present invention should be membrane permeable and effectively penetrate into tumor tissue in the TME. The release of cell-permeable payloads is associated with a bystander killing effect, which is believed to be particularly beneficial for the treatment of tumors with heterogeneous target expression.

[0106] Disclosed herein are conjugates comprising one or more small molecule target protein binders connected via a linker and one or more therapeutic payloads connected via a protease cleavable linker. The linker may further include a non-cleavable linker. The non-cleavable linker described herein may be divalent or multivalent. For example, in some embodiments, a conjugate is provided herein comprising a non-cleavable linker that links a target protein binder to multiple therapeutic payloads, wherein each therapeutic payload is linked to the non-cleavable linker via a protease cleavable linker. In other embodiments, the non-cleavable linker links a single target protein binder to a single therapeutic payload, but a given conjugate may include multiple non-cleavable linkers, each of which is linked to a therapeutic payload via a protease cleavable linker. The non-cleavable linker may include functional elements, including physicochemical regulatory elements (e.g., solubility enhancers), pharmacokinetic regulatory elements (e.g., tumor targeting or tumor retention groups), proximity regulatory elements (e.g., spacers), or any combination thereof. In some embodiments, the non-cleavable linker disclosed herein (e.g., L 1 , L 2 ) is linked to a protease-cleavable linker (e.g., P 1 、P 1a ).

[0107] The therapeutic payload used according to the present invention can be, for example, a drug. Therefore, conjugates are generally referred to as SMDCs in this article. However, the term SMDC used herein may refer to any conjugate including a target protein binding agent and a therapeutic payload, and is not particularly limited to therapeutic payloads that must be defined as "drugs". The therapeutic payload can be, for example, a cytotoxic agent or an immunostimulant. Preferably, the therapeutic payload can permeate cell membranes (e.g., tumor cell membranes). More preferably, the therapeutic payload can penetrate into tumor cells and produce cytotoxic or antiproliferative effects in cells. The therapeutic payload can be configured to be released extracellularly in the tumor microenvironment (e.g., via cleavage of a protease cleavable linker by an extracellular tumor-associated protein (e.g., neutrophil elastase)). The therapeutic payload released extracellularly (e.g., tumor penetrating therapeutic payload) can enter tumor cells and produce potent cytotoxic or immunostimulatory effects. In some cases, the therapeutic payload is a microtubule toxin, a DNA toxin, a transcriptional toxin, or an immunostimulant. In some cases, the microtubule toxin is a maytansinoid, an auristatin, an epithilone, a taxoid, a tubulysin, an eribulin alkaloid, a vinca alkaloid, eribulin, or any combination thereof. In some cases, the DNA toxin is an anthracycline, a topoisomerase I inhibitor, a duacarmycin or its analog, a calicheamicin, a DNA cross-linking agent, bleomycin A2, dactinomucin, mitomycin C, or any combination thereof. In some cases, the transcriptional toxin is an amatoxin, thailanstatin A, an oxidative phosphorylation inhibitor, a protein kinase inhibitor, a dihydrofolate reductase (DHFR) inhibitor, or a histone deacetylase inhibitor.

[0108] The present invention may further include one or more small molecule binders (SMBs) to a target protein. In some cases, the target protein is a target protein on a tumor cell. In some embodiments, the target protein is selected from α-vβ-3 ("α v β3" or "avβ3") integrin, α-vβ-6 ("α vβ6 or “avβ6”) integrin, carbonic anhydrase IX (“CA9” or “CAIX”), fibroblast activation protein (“FAP”), prostate-specific membrane antigen (“PSMA”), heat shock protein 90 (“Hsp90”), folate receptor, glucose transporter 1 (GLUT1), somatostatin receptor, aminopeptidase N (APN), low-density lipoprotein receptor-related protein 1 (LRP1), bombesin receptor, gonadotropin-releasing hormone (GnRH) receptor, luteinizing hormone-releasing hormone (LHRH) receptor, p32, membrane type 1 matrix metalloproteinase (MT1-MMP), sortilin or adhesion protein-4. Other target proteins are B lymphocyte antigen CD20 (“CD20”), complement receptor type 2 (“CD21”), Lyb-2 (“CD72”), programmed cell death ligand 1 (“PD-L1”), carcinoembryonic antigen cell adhesion molecule ("CEACAM5"), galectin-3 binding protein ("Gal-3-BP"), leucine-rich alpha-2-glycoprotein 1 ("LRG1"), matrix metallopeptidase 9 ("MMP9"), tumor-associated glycoprotein 72 ("TAG72"), fibronectin 1 ("FN1"), tenascin-C ("TN-C"), type XI collagen alpha 1 chain ("COL11A1"), type XII collagen alpha 1 chain ("COL12A1"), type I collagen alpha 1 chain ("COL1A1"), type I collagen alpha 2 chain ("COL1A2"), type III collagen COL3A1, COL5A1, COL5A2, COL6A3, COL8A1 ... , anterior gradient protein 3 ("AGR3"), asporin ("ASPN"), BMP / retinoic acid-inducible nerve-specific protein 3 ("BRINP3"), chitinase 3-like protein 1 ("CHI3L1"), cartilage intermediate layer protein ("CILP"), type X collagen alpha 1 chain ("COL10A1"), cartilage oligomeric matrix protein ("COMP"), cystatin SN ("CST1"), collagen triple helical repeat-containing protein 1 ("CTHRC1"), epiphycan ("EPYC"), follicular dendritic cell secretory protein ("FDCSP"),Gastrin-releasing peptide ("GRP"), integrin-binding sialoprotein ("IBSP"), interleukin-4-induced protein 1 ("IL4I1"), lumican ("LUM"), matrix protein 3 ("MATN3"), midkine ("MDK"), microfibril-associated protein 2 ("MFAP2"), matrix Gla protein ("MGP"), matrix metallopeptidase 1 ("MMP1"), matrix metallopeptidase 11 ("MMP11"), matrix metallopeptidase 12 ("MMP13"), matrix metallopeptidase 13 ("MMP14"), matrix metallopeptidase 14 ("MMP15"), matrix metallopeptidase 15 ("MMP16"), matrix metallopeptidase 16 ("MMP17"), matrix metallopeptidase 17 ("MMP18"), matrix metallopeptidase 18 ("MMP19"), matrix metallopeptidase 19 ("MMP11"), matrix metallopeptidase 20 ("MMP12"), matrix metallopeptidase 21 ("MMP13"), matrix metallopeptidase 22 ("MMP1 "MMP12"), matrix metallopeptidase 13 ("MMP13"), matrix metallopeptidase 3 ("MMP3"), matrix metallopeptidase 7 ("MMP7"), mucin-like protein 1 ("MUCL1"), matrix remodeling-associated protein 5 ("MXRA5"), signal peptide CUB domain and EGF-like domain containing protein 2 ("SCUBE2"), secreted frizzled-related protein 4 ("SFRP4"), stanniocalcin 2 ("STC2"), and zymogen granule protein 16B ("ZG16B"). Further examples of relevant target proteins can be found in F. Fauteux, Oncotarget, 2015, 7, 3, 2555 and N. Ashman, Chem Soc Rev, 2022, Advance Article.

[0109] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof, comprising one or more payloads conjugated to one or more target protein binding agents via a linker. In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof, having a structure of Formula (I):

[0110] T––L––EL––P

[0111] Formula (I)

[0112] in:

[0113] T is the target protein binder;

[0114] L is a linker, which optionally further comprises a second T, a second EL-P and / or a pharmacokinetic regulator.

[0115] Section group (MOD);

[0116] EL is a peptide linker; and

[0117] P is a payload. In some embodiments, EL and P are bound via a self-immolative linker group. In some embodiments, EL and P are bound without a self-immolative linker.

[0118] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula (II) (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), or Formula (IX):

[0119]

[0120] in:

[0121] P is the effective load;

[0122] EL is a cleavable peptide linker, which optionally further comprises a self-immolative group;

[0123] Each L 1 , L 2 , L 3 and L 4 are independently a divalent linker,

[0124] A 1 is a trivalent linker;

[0125] A 2 is a tetravalent linker;

[0126] T is a target protein binder; and

[0127] MOD is the pharmacokinetic modulator group.

[0128] In some embodiments, each protein of interest is independently selected from the group consisting of: integrin (e.g., α-v-β-3), integrin (e.g., α-v-β-6), PSMA, CAIX, FAP, folate, Hsp90, somatostatin, GLUT1, APN, LRP1, bombesin, GnRH, LHRH, MT1-MMP, P32, phosphatidylserine, or sortilin.

[0129] In some embodiments, the protein-binding agent of interest is an integrin (e.g., α-v-β-3 integrin) binding agent, an integrin (e.g., α-v-β-6 integrin) binding agent, a PSMA binding agent, a CAIX binding agent, a FAP binding agent, a folate receptor (FR) binding agent, an Hsp90 binding agent, a somatostatin binding agent, a GLUT1 binding agent, an APN binding agent, an LRP1 binding agent, a bombesin binding agent, a GnRH binding agent, a LHRH binding agent, an MT1-MMP binding agent, a P32 binding agent, a phosphatidylserine binding agent, or a sortilin binding agent. In some embodiments, the protein-binding agent of interest (T) is a PSMA binding agent, a small molecule α vβ3 integrin binders, CAIX (CA9) binders, FAP binders, folate binders, or Hsp90 binders. In some embodiments, the target protein binder (T) is a small molecule α v β3 (α-v-β-3) integrin binding agent. In some embodiments, the target protein binding agent (T) is a FAP binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent. In some embodiments, the target protein binding agent (T) is a CAIX (CA9) binding agent. In some embodiments, the target protein binding agent (T) is a folate receptor (FR) binding agent. In some embodiments, the target protein binding agent (T) is an Hsp90 binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent, a CAIX (CA9) binding agent, a FAP binding agent, a folate binding agent, or an Hsp90 binding agent.

[0130] In some embodiments, the target protein binding agent (T) of any of Formula I, II, III, IV, V, VI, VII, VIII, or IX is:

[0131]

[0132]

[0133] In some embodiments, P is a cytotoxic drug or an immunostimulant. In some embodiments, P is a cytotoxic compound. In some embodiments, P is an immunostimulant. In some embodiments, P is a topoisomerase inhibitor, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a tubulin inhibitor, an epidermal growth factor receptor inhibitor, a taxane, a diterpenoid, or an agonist of toll-like receptor 7 and / or 8. In some embodiments, P is a cell-permeable topoisomerase inhibitor, a cell-permeable spindle kinesin inhibitor, a cell-permeable cyclin-dependent kinase 9 inhibitor, a cell-permeable tubulin inhibitor, or a cell-permeable agonist of toll-like receptor 7 and / or 8. In some embodiments, P is a topoisomerase inhibitor provided herein. In some embodiments, P is a CDK9 (e.g., PTEFb) inhibitor provided herein. In some embodiments, D 1In some embodiments, P is an auristatin (e.g., auristatin E or monomethyl auristatin E). In some embodiments, P is a cell-permeable spindle kinesin inhibitor. In some embodiments, P is a toll-like receptor (e.g., TLR7 and / or TLR8) agonist (e.g., resiquimod). In some embodiments, P is biologically active (i.e., cytotoxic or immunostimulatory) upon activation by a protease such as neutrophil elastase. In some embodiments, P is biologically inactive prior to activation via cleavage by neutrophil elastase.

[0134] In some embodiments, the payload (P) is selected from the group consisting of:

[0135]

[0136]

[0137] or a pharmaceutically acceptable salt thereof, wherein:

[0138] Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide;

[0139] R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl;

[0140] R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC 1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace;

[0141] R 10is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3;

[0142] R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH; or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring;

[0143] R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C 1-6 Alkylamine; or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle;

[0144] Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl or -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group;

[0145] R 16 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0146] R 17 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3; or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 20 replace;

[0147] R 18 For hydrogen, halogen, C 1-6 Alkyl, C1-6 Haloalkyl, -OH, -OCH3 or -OCF3; or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 20 replace;

[0148] R 19 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0149] R 20 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH;

[0150] R 21 is hydrogen or C 1-6 alkyl;

[0151] R 22 is hydrogen or C 1-6 Alkyl, where C 1-6 The alkyl group is unsubstituted or R 24 replace;

[0152] R 23 For hydrogen, C 1-6 Alkyl or benzyl, where C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution;

[0153] R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl);

[0154] R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl);

[0155] Each Y 1 、Y 2 、Y 3 and Y 4 are independently -CH, -CF or N;

[0156] Y 5 is CH2, NH or O;

[0157] q is 0, 1, 2, 3, 4, or 5; and

[0158] r is 0, 1, 2, 3, 4 or 5.

[0159] In some embodiments, provided herein is a small molecule drug conjugate having a structure of Formula (XA) or Formula (XB):

[0160]

[0161] or a pharmaceutically acceptable salt thereof, wherein:

[0162] # indicates that the 1 、A 2 or the key of T;

[0163] P is the therapeutic payload;

[0164] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0165] R 2 is -CH3, -CH(CH3)2 or -CH(CH3)(CH2-CH3);

[0166] R a is hydrogen or -CH3;

[0167] R b is independently hydrogen or CH3 in each occurrence;

[0168] or two R b Together with the carbon to which they are attached, they form a carbonyl group; and

[0169] R c It is hydrogen or -CH3.

[0170] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VII):

[0171]

[0172] in:

[0173] P is the effective load;

[0174] EL is a cleavable peptide linker;

[0175] Each L 1 , L 2 and L 3 are independently a divalent linker,

[0176] A 1 is a trivalent linker;

[0177] A 2 is a tetravalent linker;

[0178] T is a target protein binder; and

[0179] MOD is the pharmacokinetic modulator group.

[0180] In some embodiments, P is a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor, or an immunomodulator.

[0181] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having a structure of Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VII), wherein:

[0182] Each P is a tubulin polymerization inhibitor, a spindle kinesin inhibitor, a cyclin-dependent kinase inhibitor, an immunomodulator, an epidermal growth factor receptor inhibitor, or a microtubule inhibitor;

[0183] Each EL is a cleavable peptide linker;

[0184] Each L 1 , L 2 and L 3 are independently a divalent linker,

[0185] A 1 is a trivalent linker;

[0186] A 2 is a tetravalent linker;

[0187] each T is a target protein binder (e.g., a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, an αvβ6 integrin binder, or an αvβ3 integrin binder); and

[0188] MOD is the pharmacokinetic modulator group.

[0189] In some embodiments, P is auristatin or an auristatin derivative, a kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor, or a taxane.

[0190] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having a structure of Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VII), wherein:

[0191] Each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder;

[0192] Each L 1 , L 2 and L 3 are independently bivalent linkers;

[0193] A 1 is a trivalent linker;

[0194] A 2 is a tetravalent linker;

[0195] Each EL is a cleavable peptide linker; and

[0196] Each P is a payload (eg, a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor, or an immunomodulator).

[0197] In some embodiments, each P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor, or a taxane.

[0198] In some embodiments, provided herein is a compound having the structure of Formula II:

[0199]

[0200] in:

[0201] P is the effective load;

[0202] EL is a peptide linker;

[0203] L 1 is a divalent linker, and

[0204] T is the target protein binder.

[0205] In some embodiments, the effective load (P) of formula II is a cytotoxic drug. In some embodiments, a compound of formula II is provided herein, wherein the effective load (P) is a topoisomerase inhibitor (Topo-i). In some embodiments, a compound of formula II is provided herein, wherein the effective load (P) is camptothecin, exitecan, deruxtecan or a derivative thereof (e.g., 7-ethylcamptothecin, exitecan, N-alkyl exitecan, N-acyl exitecan or 5,6-methylenedioxycamptothecin ("FL118"), etc.). In some embodiments, the effective load (P) is 7-ethylcamptothecin. In some embodiments, the effective load of formula (II) is exitecan or FL118. In some embodiments, a compound of formula II is provided herein, wherein the effective load (P) is a spindle kinesin inhibitor (KSPi). In some embodiments, a compound of formula II is provided herein, wherein the effective load (P) is auristatin. In some embodiments, the payload (P) of Formula II is an auristatin selected from auristatin E and monomethyl auristatin E (MMAE). In some embodiments, the payload of Formula II is monomethyl auristatin E (MMAE). In some embodiments, the payload of Formula II is an inhibitor of cyclin-dependent kinase 9 (CDK9) and / or positive transcription elongation factor (P-TEFb). In some embodiments, the payload of Formula II is an inhibitor of epidermal growth factor receptor (EGFR). In some embodiments, the payload of Formula II is a tubulin inhibitor (e.g., a tubulin polymerization inhibitor such as auristatin E (or monomethyl auristatin E) or a tubulin depolymerization inhibitor (e.g., paclitaxel)). In some embodiments, the payload of Formula II is auristatin. In some embodiments, the payload of Formula II is monomethyl auristatin E. In some embodiments, the payload of Formula II is a taxane. In some embodiments, the payload of Formula II is paclitaxel. In some embodiments, the payload of Formula II is triptolide. In some embodiments, the payload of Formula II is erlotinib.

[0206] In some embodiments, the payload (P) of Formula II is an immune agonist. In some embodiments, the immune agonist of Formula II is a toll-like receptor agonist. In some embodiments, the payload (P) of Formula (II) is a toll-like receptor 7 and / or 8 ("TLR7 / 8) agonist. In some embodiments, the payload (P) of Formula II is resiquimod. Or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof.

[0207] In some embodiments, provided herein is a compound having the structure of Formula III:

[0208]

[0209] in:

[0210] P is the effective load;

[0211] EL is a peptide linker;

[0212] Each L 2 and L 3 are independently a divalent linker,

[0213] A 1 is a trivalent linker;

[0214] Each T is a target protein binder.

[0215] In some embodiments, the payload (P) of Formula III is a cytotoxic drug or immune agonist as described herein. In some embodiments, provided herein is a compound of Formula III, wherein the payload (P) is a topoisomerase inhibitor (Topo-i). In some embodiments, provided herein is a compound of Formula III, wherein the payload (P) is camptothecin, exitecan, derunotecan, or a derivative thereof (e.g., 7-ethylcamptothecin, exitecan, N-alkyl exitecan, N-acyl exitecan, or 5,6-methylenedioxycamptothecin ("FL118"), etc. In some embodiments, provided herein is a compound of Formula III, wherein the payload (P) is a spindle kinesin inhibitor (KSPi). In some embodiments, provided herein is a compound of Formula III, wherein the payload (P) is auristatin or an auristatin derivative (e.g., an auristatin selected from auristatin E, auristatin F, and monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)). In some embodiments In some embodiments, the payload of formula III is an inhibitor of cyclin-dependent kinase 9 (CDK9) and / or positive transcription elongation factor (P-TEFb). In some embodiments, the payload (P) of formula (III) is a toll-like receptor 7 and / or 8 ("TLR7 / 8) agonist. In some embodiments, the payload (P) of formula (III) is resiquimod. In some embodiments, the payload (P) is an EGFR inhibitor. In some embodiments, the payload (P) of formula (III) is erlotinib. In some embodiments, the payload (P) of formula (III) is auristatin. In some embodiments, the payload (P) of formula (III) is auristatin F or monomethyl auristatin E (MMAE).

[0216] In some embodiments, the protein-binding agent of interest (T) is an integrin (e.g., α-v-β-3 integrin) binding agent, α-v-β-6 integrin) binding agent, PSMA binding agent, CAIX binding agent, FAP binding agent, folate receptor (FR) binding agent, Hsp90 binding agent, somatostatin binding agent, GLUT1 binding agent, APN binding agent, LRP1 binding agent, bombesin binding agent, GnRH binding agent, LHRH binding agent, MT1-MMP binding agent, P32 binding agent, phosphatidylserine binding agent, or sortilin binding agent. In some embodiments, the protein-binding agent of interest (T) is a PSMA binding agent, a small molecule α v β3 integrin binders, CAIX (CA9) binders, FAP binders, folate binders, or Hsp90 binders. In some embodiments, the target protein binder (T) is a small molecule α v β3 (α-v-β-3) integrin binding agent. In some embodiments, the target protein binding agent (T) is a FAP binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent. In some embodiments, the target protein binding agent (T) is a CAIX (CA9) binding agent. In some embodiments, the target protein binding agent (T) is a folate receptor (FR) binding agent. In some embodiments, the target protein binding agent (T) is an Hsp90 binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent, a CAIX (CA9) binding agent, a FAP binding agent, a folate binding agent, or an Hsp90 binding agent.

[0217] In some embodiments, provided herein is a compound having the structure of Formula IV:

[0218]

[0219] in:

[0220] Each P is a valid load;

[0221] Each EL is a peptide linker;

[0222] Each L 2 and L 3 are independently a divalent linker,

[0223] A 1 is a trivalent linker;

[0224] T is the target protein binder.

[0225] In some embodiments, provided herein is a compound having the structure of Formula V:

[0226]

[0227] in:

[0228] Each P is a valid load;

[0229] Each EL is a peptide linker;

[0230] Each L 2 and L 3 are independently a divalent linker,

[0231] A 2 is a tetravalent linker;

[0232] Each T is a target protein binder.

[0233] In some embodiments, provided herein is a compound having the structure of Formula VII:

[0234]

[0235] in:

[0236] P is the effective load;

[0237] EL is a peptide linker;

[0238] Each L 2 and L 3 are independently a divalent linker,

[0239] A 1 is a trivalent linker;

[0240] T is a target protein binder; and

[0241] MOD is the pharmacokinetic modulator group.

[0242] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI:

[0243]

[0244] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof;

[0245] in:

[0246] T is the target protein binder;

[0247] L 1 is a bivalent linker (e.g., a non-cleavable linker);

[0248] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0249] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0250] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0251] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、

[0252] -SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5 )C(O)R 5 、

[0253] -S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0254] R 5 is independently hydrogen or C 1-6 alkyl; and

[0255] P is the payload (eg, therapeutic payload).

[0256] In some embodiments, L 1 is a divalent linker comprising 2 to 20 polyethylene glycol groups.

[0257] In some embodiments, the compound of Formula XI has the following structure:

[0258]

[0259] in:

[0260] T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder;

[0261] R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH;

[0262] R 2 is-CH3, -CH(CH3)2, -CH2CH(CH3)2, or -CH(CH3)CH2CH3; and

[0263] P is the effective load.

[0264] In some embodiments,

[0265] T is an αvβ3 integrin binder, and

[0266] P is a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor or an immunomodulator.

[0267] In some embodiments,

[0268] T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, an αvβ6 integrin binder, or an αvβ3 integrin binder, and

[0269] P is auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

[0270] In some embodiments,

[0271] T is a prostate-specific membrane antigen-binding agent, a carbonic anhydrase 9-binding agent, a fibroblast activation protein-binding agent, a folate receptor-binding agent, a heat shock protein 90-binding agent, or an αvβ6 integrin-binding agent, and

[0272] P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

[0273] In some embodiments, P is camptothecin or a camptothecin derivative. In some embodiments, P is auristatin or an auristatin derivative.

[0274] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XII:

[0275]

[0276] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0277] T is the target protein binder;

[0278] L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0279] A means A 1 or A 2 ,

[0280] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0281] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0282] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0283] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0284] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、

[0285] -SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5)C(O)R 5 、

[0286] -S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0287] R 5 is independently hydrogen or C 1-6 alkyl;

[0288] P is the payload (e.g., therapeutic payload);

[0289] m is 1 or 2; and

[0290] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0291] In some embodiments, L 2 and L 3 Each of is a divalent linker comprising 2 to 20 polyethylene glycol groups. In some embodiments, the compound of Formula XII has the following structure:

[0292]

[0293] in:

[0294] Each T is a target protein binding agent (e.g., a prostate-specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, or an αvβ6 integrin binding agent);

[0295] R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH;

[0296] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; and

[0297] P is the effective load.

[0298] In some embodiments,

[0299] Each T is an αvβ3 integrin binder, and

[0300] P is a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor or an immunomodulator.

[0301] In some embodiments,

[0302] each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, an αvβ6 integrin binder, or an αvβ3 integrin binder, and

[0303] P is auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

[0304] In some embodiments,

[0305] each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder, and

[0306] P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

[0307] In some embodiments, P is camptothecin or a camptothecin derivative. In some embodiments, P is auristatin or an auristatin derivative.

[0308] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-KSP or XII-KSP:

[0309]

[0310] Formula XII-KSP

[0311] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0312] T is the target protein binder;

[0313] L 1 , L 2 and L 3Each is a divalent linker (e.g., a non-cleavable linker);

[0314] A means A 1 or A 2 ,

[0315] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0316] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0317] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0318] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0319] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、-SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5 )C(O)R 5 、-S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0320] R 5 is independently hydrogen or C 1-6 alkyl;

[0321] Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide;

[0322] R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl;

[0323] R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NH-C 1-6 Alkyl-C(O)NH-C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)-C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace;

[0324] R 10 is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3;

[0325] m is 1 or 2;

[0326] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH);

[0327] q is 0, 1, 2, 3, 4, or 5; and

[0328] r is 0, 1, 2, 3, 4 or 5.

[0329] In some embodiments, each T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, an αvβ6 integrin binding agent, or an αvβ3 integrin binding agent.

[0330] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-KSP' or XII-KSP':

[0331]

[0332] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0333] T is the target protein binder;

[0334] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0335] A means A 1 or A 2 ,

[0336] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0337] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0338] R 2 is -CH3 or -CH(CH3)2,

[0339] R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NH-C 1-6 Alkyl-C(O)NH-C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)-C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace;

[0340] R 10 is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3;

[0341] m is 1 or 2; and

[0342] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0343] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TOPO or XII-TOPO:

[0344]

[0345]

[0346] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0347] T is the target protein binder;

[0348] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0349] A means A 1 or A 2 ,

[0350] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0351] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0352] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0353] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0354] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、-SR5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5 )C(O)R 5 、-S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0355] R 5 is independently hydrogen or C 1-6 alkyl;

[0356] R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH;

[0357] or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring;

[0358] R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C 1-6 Alkylamines;

[0359] or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle;

[0360] Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl or

[0361] -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group;

[0362] m is 1 or 2; and

[0363] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0364] In some embodiments, T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, or an αvβ6 integrin binding agent.

[0365] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TOPO' or XII-TOPO':

[0366]

[0367] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0368] T is the target protein binder;

[0369] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0370] A means A 1 or A 2 ,

[0371] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0372] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0373] R 2 is -CH3 or -CH(CH3)2,

[0374] R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH;

[0375] or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring;

[0376] R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C1-6 Alkylamines;

[0377] or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle;

[0378] Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl or

[0379] -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group;

[0380] m is 1 or 2; and

[0381] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0382] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-CDK9 or XII-CDK9:

[0383]

[0384]

[0385] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0386] T is the target protein binder;

[0387] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0388] A means A 1 or A 2 ,

[0389] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0390] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR3 ;

[0391] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0392] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0393] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、-SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5 )C(O)R 5 、-S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0394] R 5 is independently hydrogen or C 1-6 alkyl;

[0395] R 16 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0396] R 17 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0397] R 18 For hydrogen, halogen, C 1-6 Alkyl, C1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0398] or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-;

[0399] or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-;

[0400] R 19 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0401] Each Y 1 、Y 2 、Y 3 and Y 4 are independently -CH, -CF or N;

[0402] Y 5 is CH2, NH or O;

[0403] m is 1 or 2; and

[0404] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0405] In some embodiments, T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, or an αvβ6 integrin binding agent.

[0406] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-CDK9' or XII-CDK9':

[0407]

[0408] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0409] T is the target protein binder;

[0410] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0411] A means A 1 or A 2 ,

[0412] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0413] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0414] R 2 is -CH3 or -CH(CH3)2,

[0415] R 16 For hydrogen,

[0416] R 17 is hydrogen, -OH, -OCH3 or -OCF3;

[0417] R 18 is hydrogen;

[0418] or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-;

[0419] or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-;

[0420] Y 3 is CH or N;

[0421] Y 4 is CH or N;

[0422] m is 1 or 2; and

[0423] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0424] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TP or XII-TP:

[0425]

[0426]

[0427] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0428] T is the target protein binder;

[0429] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0430] A means A 1 or A 2 ,

[0431] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0432] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0433] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0434] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0435] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、

[0436] -SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R 5 )、-S(O)2N(R 5 )C(O)R5 、

[0437] -S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0438] R 5 is independently hydrogen or C 1-6 alkyl;

[0439] R 21 is hydrogen or -CH3;

[0440] m is 1 or 2; and

[0441] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0442] In some embodiments, T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, or an αvβ6 integrin binding agent.

[0443] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TP' or XII-TP':

[0444]

[0445] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0446] T is the target protein binder;

[0447] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0448] A means A 1 or A 2 ,

[0449] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0450] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0451] R 2 is -CH3 or -CH(CH3)2,

[0452] R 21 is hydrogen or -CH3;

[0453] m is 1 or 2; and

[0454] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0455] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TP' or XII-TP':

[0456]

[0457] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0458] T is the target protein binder;

[0459] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0460] A means A 1 or A 2 ,

[0461] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0462] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0463] R 2 is -CH3 or -CH(CH3)2,

[0464] R 21 is hydrogen or -CH3;

[0465] SIL is a self-cleaving linker (e.g. C 2-18 heteroalkylene)

[0466] m is 1 or 2; and

[0467] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0468] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TLR or XII-TLR:

[0469]

[0470] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0471] T is the target protein binder;

[0472] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0473] A means A 1 or A 2 ,

[0474] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0475] R 1 For hydrogen, C 1-6 Alkyl, -CH2CONH2, -CH2COOH or -CH2COOR 3 ;

[0476] R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)(CH2CH3);

[0477] R 3 0 to 3 R 4 The instance of C is replaced by 1-12 alkyl;

[0478] R 4 is hydrogen, halogen, -C(O)OR 5 、-C(O)N(R 5 )2、-N(R 5 )2、-N(R 5 )3 + 、-OR 5 、

[0479] -SR 5 、-S(O)R 5 、-S(O)2R 5 、-S(O)2N(R5 )、-S(O)2N(R 5 )C(O)R 5 、

[0480] -S(O)2N(R 5 )C(O)OR 5 、-N(R 5 )S(O)2N(R 5 )2、-N(R 5 )S(O)2N(R 5 )C(O)R 5 or -N(R 5 )S(O)2N(R 5 )C(O)OR 5 ;

[0481] R 5 is independently hydrogen or C 1-6 alkyl;

[0482] R 22 is hydrogen or C 1-6 Alkyl, where C 1-6 The alkyl group is unsubstituted or R 24 replace;

[0483] R 23 For hydrogen, C 1-6 Alkyl or benzyl, where C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution;

[0484] R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl);

[0485] R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2,

[0486] -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl);

[0487] m is 1 or 2; and

[0488] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0489] In some embodiments, T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, or an αvβ6 integrin binding agent.

[0490] In some embodiments, provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula XI-TLR' or XII-TLR':

[0491]

[0492]

[0493] or a stereoisomer thereof; or a pharmaceutically acceptable salt thereof; wherein:

[0494] T is the target protein binder;

[0495] L 1 , L 2 and L 3 Each is a divalent linker (e.g., a non-cleavable linker);

[0496] A means A 1 or A 2 ,

[0497] Among them A 1 is a trivalent linker, and A 2 is a tetravalent linker;

[0498] R 1 is hydrogen, -CH2CONH2 or -CH2COOH;

[0499] R 2 is -CH3 or -CH(CH3)2,

[0500] R 22 is hydrogen or C 1-6 Alkyl, where C 1-6 The alkyl group is unsubstituted or R 24 replace;

[0501] R 23 For hydrogen, C 1-6 Alkyl or benzyl, where C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution;

[0502] R24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl);

[0503] R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2,

[0504] -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl);

[0505] m is 1 or 2; and

[0506] n is 1 or 2; wherein if n is 2, one instance of T is optionally replaced with MOD; wherein MOD is a pharmacokinetic modulating group (eg, COOH).

[0507] In some embodiments, the target protein binding agent (T) is α v β3 integrin (e.g., α-v-β-3 integrin) binding agents, α v β6 (avb6) integrin binders, PSMA binders, CAIX binders, FAP binders, folate receptor (FR) binders, Hsp90 binders, somatostatin binders, GLUT1 binders, APN binders, LRP1 binders, bombesin binders, GnRH binders, LHRH binders, MT1-MMP binders, P32 binders, phosphatidylserine binders, or sortilin binders. In some embodiments, the protein-binding agent of interest (T) is a PSMA binder, a small molecule α v β3 integrin binders, CAIX (CA9) binders, FAP binders, folate binders, or Hsp90 binders. In some embodiments, the target protein binder (T) is a small molecule α v β3 (α-v-β-3) integrin binding agent. In some embodiments, the target protein binding agent (T) is a small molecule α vβ6 (avb6) integrin binding agent. In some embodiments, the target protein binding agent (T) is a FAP binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent. In some embodiments, the target protein binding agent (T) is a CAIX (CA9) binding agent. In some embodiments, the target protein binding agent (T) is a folate receptor (FR) binding agent. In some embodiments, the target protein binding agent (T) is an Hsp90 binding agent. In some embodiments, the target protein binding agent (T) is a PSMA binding agent, a CAIX (CA9) binding agent, a FAP binding agent, a folate binding agent, or an Hsp90 binding agent.

[0508] In some embodiments, T is:

[0509]

[0510]

[0511] In some embodiments, each T is:

[0512]

[0513]

[0514] In some embodiments, the linker (eg, L 1 , L 2 or L 3 In some embodiments, L 1 , L 2 and / or L 3 The polyamine or polyamide linker is substituted with one or more carbonyl groups. 1 , L 2 and / or L 3 The polyamine or polyamide linker forms an ammonium ion (or optionally multiple ammonium ions) in the acidic tumor microenvironment. In some embodiments, L 1 , L 2 and / or L 3 The polyamine or polyamide linker is selectively retained in the tumor microenvironment. In some embodiments, L 1 , L 2 and / or L 3 In some embodiments, L 1 , L 2 and / or L 3 The PEG linker enhances the solubility of the conjugate and / or the payload conjugated thereto.

[0515] In some embodiments, each L 1 , L 2 and L 3 is a divalent linker having a structure represented by the following formula:

[0516] (i)-(CO) r (CH2) s (OC 2-6 alkyl) t (NH) u (CO) v -;

[0517] (ii)-(CO) r (CH2) s (NR c C 1-6 alkyl) t (NR a ) u (CO) v -;

[0518] (iii)-(CO) r (CH2) s (NR c C(O)C 1-6 alkyl) t (NR a ) u (CO) v -;

[0519] (iv)-(CO) r (CH2) s (NR c C 1-6 alkyl) t (NR c C(O)C 1-6 Alkyl)(NR c C 1-6 alkyl) t (NR a ) u (CO) v -;

[0520] (v)-(CO) r (CH2) s (NH) u (CO) v -;

[0521] (vi)-(CO) r (CH2) s (OC 2-6 alkyl) t(NH) u (CO) v -(NH) u (CH2) s (OC 2-6 alkyl) t

[0522] in:

[0523] R a is independently selected at each occurrence from hydrogen or C 1-3 alkyl;

[0524] R c is independently selected at each occurrence from hydrogen or C 1-3 alkyl;

[0525] r is 0 or 1; s is 0 to 10; t is 1 to 10; u is 0 or 1; and v is 0 or 1.

[0526] In some embodiments, each L 1 , L 2 and L 3 is a divalent linker having a structure represented by the following formula:

[0527] (i)-(CO) r (CH2) s (OC 2-6 alkyl) t (NH) u (CO) v -;

[0528] (ii)-(CO) r (CH2) s (NR c C 1-6 alkyl) t (NR a ) u (CO) v -;or

[0529] (v)-(CO) r (CH2) s (NH) u (CO) v -;

[0530] (vi)-(CO) r (CH2) s (OC 2-6 alkyl) t (NH) u (CO) v -(NH) u (CH2) s (OC2-6 alkyl) t

[0531] Among them, each R a and R c are independently hydrogen or -CH3;

[0532] r is 0 or 1;

[0533] s is 1 to 4;

[0534] t is 1 to 10;

[0535] u is 0 or 1; and

[0536] v is 0 or 1.

[0537] In some embodiments, each L 1 , L 2 and L 3 A divalent linker independently of the formula:

[0538] -(CO) 0-1 -(CH2) 2-4 -[OC 2-6 alkyl] 1-8 -NH-(CO) 0-1 -;

[0539] -(CO) 0-1 -(CH2) 2-4 -[OC 2-6 alkyl] 1-8 -NHCONH-[OC 2-6 alkyl] 1-8 ;

[0540] -(CO) 0-1 -(CH2) 1-4 -[N(CH3)-C 1-6 alkyl] 1-8 -NH-(CO) 0-1 -,

[0541] -(CO) 0-1 -(CH2) 1-4 -[N(CH3)-C 1-6 alkyl] 1-8 -N(CH3)-(CO) 0-1 -;or

[0542] -(NH) 0-1 -(CH2) 1-10 -.

[0543] In some embodiments, L 1 , L 2 and / or L3 for:

[0544]

[0545] In some embodiments, L 1 for:

[0546]

[0547] In some embodiments, L 3 for:

[0548]

[0549] In some embodiments,

[0550] Each L 1 For: -CO-(CH2) 2-4 -[OCH2CH2] 1-8 -NHCONH-[OCH2CH2] 1-8 ,

[0551] -CO-(CH2) 2-4 -(OCH2CH2) 1-8 -NH-,

[0552] -CO-(CH2) 2-4 -(N(CH3)CH2CH2) 1-8 -N(CH3)-, or

[0553] -CO-(CH2) 2-4 (N(CH3)CH2CH2) 1-8 -NH-;

[0554] Each L 2 For: -CO-(CH2) s -(OCH2CH2) 1-8 -NH-; and

[0555] Each L 3 For: -CO-(CH2) s -(OCH2CH2) 1-8 -NH- or -NH-(CH2) 1-10 .

[0556] In some embodiments, L 1 , L 2 and / or L 3 is a divalent linker comprising 2 to 20 polyethylene glycol groups.

[0557] On the other hand, there is provided herein a compound of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI or XII (including its subformula), which is used to manufacture a medicine for treating a disease. On the other hand, there is provided herein a method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI or XII (including its subformula) disclosed in any one or more thereof and a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the disease or condition is a hyperproliferative disease or condition. In some embodiments, there is provided herein an antibody-drug conjugate, which is used as a medicine. In some embodiments, the medicine is used to treat a disease (such as a hyperproliferative disease or condition). In some embodiments, the hyperproliferative disease or condition is cancer.

[0558] Payload (P)

[0559] As used herein, the term "payload" or "therapeutic payload" generally refers to a chemical group, typically a small molecule (i.e., non-protein) group, that has therapeutic activity. Preferably, the therapeutic activity is enhanced (e.g., activated) after separation from the cleavable group. In some embodiments, the cleavable group is an enzymatically cleavable group. In some embodiments, the payload is activated after cleavage by a tumor-associated protein (such as neutrophil elastase). The therapeutic payload may be, for example, a drug. In some embodiments, the therapeutic payload is a cytotoxic compound, a cell growth inhibitory compound, or an immunomodulatory compound. In some embodiments, the payload can effectively kill cancer cells or slow the growth of cancer cells. In some embodiments, the payload is a spindle kinesin inhibitor, camptothecin or a derivative thereof, a CDK9 inhibitor, an auristatin, or a taxane as described herein. In some embodiments, the payload is a protease-cleavable spindle kinesin inhibitor (KSPi) disclosed herein, a protease-cleavable camptothecin derivative, a protease-cleavable CDK9 inhibitor, a protease-cleavable auristatin, a protease-cleavable taxane, or a protease-cleavable cytotoxic or immunostimulatory payload.

[0560] The payload used according to the present invention may be, for example, a drug. Therefore, the conjugate is generally referred to herein as a small molecule drug conjugate (SMDC). The payload may be, for example, a cytotoxic agent or an immunostimulant. Preferably, the therapeutic payload can penetrate the cell membrane (e.g., a tumor cell membrane). More preferably, the therapeutic payload penetrates the tumor cell and produces a cytotoxic or anti-proliferative effect in the cell. The therapeutic payload can be configured to be released extracellularly in the tumor microenvironment (e.g., via a cleavage protease cleavable linker by an extracellular tumor-associated protein (e.g., neutrophil elastase)). The therapeutic payload released extracellularly (e.g., a tumor-penetrating therapeutic payload) can enter the tumor cell and produce a potent cytotoxic or immunostimulatory effect.

[0561] In some embodiments, the payload is a microtubule toxin (e.g., maytansinoids, maytansin, DM1, DM4, DM21, DM23), an auristatin (MMAE, MMAF, auristatin F, dolastatin, PF-06380101, amberstatin 269, auristatin F-HPA, auristatin W analog, duostatin 5.2, duostatin 5, MMAD, SHR152852, combretastatin (C

[0015] In some embodiments, the present invention relates to a pharmaceutical composition comprising the following: a) selenostatin (selenostatin A (selenostatin B), selenostatin B (selenostatin B), selenostatin D (selenostatin D), selenostatin A (selenostatin B ...

[0562] In some embodiments, the payload is a DNA toxin. In some embodiments, the therapeutic payload is an anthracycline (e.g., doxorubicin, daunorubicin, epibucricin, PNU-159682, panobinostat), a topoisomerase I inhibitor (e.g., AZ'0133, camptothecin, belotecan, irinotecan, topotecan, DXd / DX8951, exitecan, FL-118, SN-38, VIP126), a duocarmycin or its analog (e.g., duocarmycin, duocarmycin-hydroxybenzamide azaindole (DUBA), MED-A / DNAMGBA toxin), a calicheamicin, a DNA cross-linking agent (e.g., PBD-dimer-FGX20-75, SC-DR003, SG2000, SG3199, SG1882, FGX2-62, indolin-benzodiazepine (indolino-benzodiazepine) dimer-DGN462, DGN549, IGN-P1-, cyclopropylpyrroloindole, isoquinoline-benzodiazepine (isoquinolidinobenzodiazepine)-D211), bleomycin A2, actinomycin D, and mitomycin C.

[0563] In some embodiments, the payload is a transcriptional toxin. In some embodiments, the therapeutic payload is a toxic amatoxin (e.g., targeting RNA polymerase II, such as amanitin), talanstatin A (e.g., targeting the spliceosome), an oxidative phosphorylation inhibitor (e.g., oligomycin), a protein kinase inhibitor (e.g., an inhibitor of protein kinase B (Akt), such as ipatasertib), an EGFR inhibitor (e.g., erlotinib), a VEGFR inhibitor (e.g., sorafenib, sunitinib, bevacizumab), or a combination thereof. b), lenvatinib, vandetanib, pazopanib, axitinib, cabozantinib, regorafenib, nintedanib, apatinib), PDGFR inhibitors, dihydrofolate reductase (DHFR) inhibitors, such as methotrexate, aminopterin, histone deacetylase inhibitors (such as HST746AA1), or kinesin spindle inhibitors (KSPi).

[0564] In some cases, the payload is a microtubule toxin, a DNA toxin, a transcriptional toxin or an immunostimulant. In some cases, the microtubule toxin is a maytansine, auristatin, epothilone, taxanes, terpilexin, eribulin alkaloids, vinca alkaloids, eribulin or any combination thereof. In some cases, the DNA toxin is an anthracycline, a topoisomerase I inhibitor, a duocarmycin or its analog, calicheamicin, a DNA cross-linking agent, bleomycin A2, actinomycin D, mitomycin C or any combination thereof. In some cases, the transcriptional toxin is a phalloidin, tyranstatin A, an oxidative phosphorylation inhibitor, a protein kinase inhibitor, a dihydrofolate reductase (DHFR) inhibitor or a histone deacetylase inhibitor.

[0565] In some preferred embodiments, provided herein are compounds having payloads such as camptothecin derivatives, auristatin derivatives, CDK9 / PTEFb derivatives, spindle kinesin inhibitor derivatives. In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is represented by one of the following structures:

[0566]

[0567]

[0568] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0569] Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide;

[0570] R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl;

[0571] R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -CH2C(O)NH2, -CH2C(O)NHC 1-6 Alkyl, -CH2C(O)N(C 1-6 Alkyl)2, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC 1-6Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace;

[0572] R 10 is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3;

[0573] R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH;

[0574] or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring;

[0575] R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C 1-6 Alkylamines;

[0576] or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle;

[0577] Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl or -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group;

[0578] R 16 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0579] R 17For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0580] or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 21 Replacement; R 18 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0581] or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 20 Replacement; R 19 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0582] R 20 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or OH;

[0583] R 21 is hydrogen or methyl;

[0584] R 22 is hydrogen or C 1-6 Alkyl, where C 1-6 The alkyl group is unsubstituted or R 24 replace;

[0585] R 23 For hydrogen, C 1-6 Alkyl or benzyl, where C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution;

[0586] R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6Alkyl)2, -SH or -S(C 1-6 alkyl);

[0587] R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl);

[0588] Each Y 1 、Y 2 、Y 3 and Y 4 are independently -CH, -CF or N;

[0589] Y 5 is CH2, NH or O;

[0590] q is 0, 1, 2, 3, 4 or 5; and r is 0, 1, 2, 3, 4 or 5.

[0591] In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is:

[0592]

[0593] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0594] Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide;

[0595] R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl;

[0596] R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace;

[0597] R 10 is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3;

[0598] q is 0, 1, 2, 3, 4, or 5; and

[0599] r is 0, 1, 2, 3, 4 or 5.

[0600] In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is:

[0601]

[0602] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0603] R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH;

[0604] or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring;

[0605] R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C 1-6 Alkylamines;

[0606] or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle;

[0607] Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6Alkyl, -C(O)NHC 1-6 Alkyl or

[0608] -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group (eg, benzyl ester).

[0609] In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is:

[0610]

[0611] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0612] R 16 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0613] R 17 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0614] or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 21 replace;

[0615] R 18 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0616] or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein heteroalkylene is optionally substituted by R 21 replace;

[0617] R 19For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3;

[0618] R 21 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or OH;

[0619] Each Y 1 、Y 2 、Y 3 and Y 4 are independently -CH, -CF or N; and

[0620] Y 5 is CH2, NH or O.

[0621] In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is:

[0622]

[0623] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0624] R 21 is hydrogen or methyl.

[0625] In some embodiments, the SIL is a self-immolative linker group. In some embodiments, the SIL is C 2-18 In some embodiments, SIL is -(NH) 0-1 -(O) 0-1 -(CR d 2) 1-4 -(CO) 0-1 -*; where each R d are independently hydrogen or -CH3; * represents a bond to P. In some embodiments, SIL is -(NH-(CR d 2) 1-4 -CO*. In some embodiments, the SIL is: where * represents a bond to P.

[0626] In some embodiments, R 21 In some embodiments, R 21 In some embodiments, P is:

[0627]

[0628] or a pharmaceutically acceptable salt thereof.

[0629] In some embodiments, the payload (P) of Formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, or XII is:

[0630]

[0631] or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

[0632] R 22 is hydrogen or C 1-6 Alkyl, where C 1-6 The alkyl group is unsubstituted or R 24 replace;

[0633] R 23 For hydrogen, C 1-6 Alkyl or benzyl, where C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution;

[0634] R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl);

[0635] R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2,

[0636] -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl).

[0637] Examples of payload (P) include, but are not limited to:

[0638]

[0639]

[0640]

[0641] In some embodiments, the conjugate includes a payload (P) having the following structure:

[0642]

[0643]

[0644] In some embodiments, provided herein are the following small molecule drug conjugates:

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662] Non-cleavable linker (L 1 , L 2 and / or L 3 )

[0663] Provided herein are compounds with linkers or spacers (used interchangeably) that are used to create physical space between one or more elements of a compound or conjugate. In some embodiments, the linker or spacer provides additional uses (e.g., functional linkers). For example, in some embodiments, the linker has a specific length that enables enhanced cleavage of adjacent enzymatically cleavable moieties (e.g., EL). In some embodiments, the linker has a specific length that reduces steric hindrance and / or enables greater target binding. In some embodiments, a linker of a specific length is provided so that the integrin binding agent can be 1.0 -9 M or less (for example, 9E -10 , 8E -10 , 7E -10 、6E -10 , 5E -10 、4E -10 、3E -10 , 2E -10 , 1E -10 The effectiveness of the following (ie, IC 50 ) and integrin receptors (e.g. α v In some embodiments, a linker of a specific length is provided so that the integrin binding agent can bind to 1.0 -10 M or less (for example, 9E -11 , 8E -11 , 7E -11 、6E -11 , 5E -11 、4E -11 、3E -11 , 2E -11 , 1E -11 The effectiveness of the following (ie, IC 50 ) and integrin receptors (e.g. α v In some embodiments, a linker of a specific length is provided so that the integrin binding agent can bind to 1.0 -11 M or less (for example, 9E -12 , 8E -12 , 7E -12 、6E -12 , 5E -12 、4E -12 、3E -12 , 2E -12 , 1E -12 The effectiveness of the following (ie, IC 50 ) and integrin receptors (e.g. α v β3 integrin receptor) binding.

[0664] In some embodiments, provided herein is a method comprising a linker (e.g., L1 , L 2 and / or L 3 ) compound, wherein the linker enhances retention of the compound within the tumor microenvironment. In some embodiments, the linkers disclosed herein (e.g., polyamine or polyamide linkers) increase the tumor to plasma ratio of the compound compared to a reference compound comprising an alkyl or PEG linker. In some embodiments, provided herein is a compound comprising a trivalent linker (A 1 ) and two linker-binding agent groups (L 2 IN、L 3 IN) or linker-binder and linker-MOD group (L 2 IN)(L 3 MOD) compound, wherein, compared to the compound comprising a divalent linker, L 3 IN or L 3 Compounds comprising a MOD group have increased half-life and / or increased AUC. In some embodiments, provided herein is a compound comprising a trivalent linker and two integrin-binding groups, wherein the compound comprising a trivalent linker and two integrin-binding groups has increased half-life and / or increased AUC compared to a compound comprising a bivalent linker.

[0665] In some embodiments, the linker is a branched or straight chain of atoms selected from C, N, O, or S (each of which is substituted with hydrogen or a bond to satisfy standard valences). In some embodiments, the linker is a carbonyl group (-C(O)-). In some embodiments, the linker comprises six or fewer (non-hydrogen) atoms. In some embodiments, the linker comprises about 10 to about 20 (non-hydrogen) atoms. In some embodiments, the linker comprises about 10 to about 20 (non-hydrogen) atoms arranged in a straight chain. In some embodiments, the linker comprises about 20 to about 30 (non-hydrogen) atoms. In some embodiments, the linker comprises about 20 to about 30 (non-hydrogen) atoms arranged in a straight chain. In some embodiments, the linker comprises about 30 to about 40 (non-hydrogen) atoms. In some embodiments, there are multiple linkers, each of which has a different length. In some embodiments, the linker comprises a cyclic or branched portion. In some embodiments, the linker is substituted with one or more alkyl, oxo, amino, or amide groups.

[0666] In some embodiments, L 1 , L 2 and / or L 3Each of the following comprises or is terminally substituted with: one or more carbonyl groups (-C(O)-), amine groups (e.g., -NH- or -N(CH3)-), or amide groups (e.g., -C(O)NH-, -C(O)N(CH3)-, -NHC(O)-, or N(CH3)C(O)-).

[0667] In some embodiments, L 1 , L 2 and / or L 3 Each of which is substituted or unsubstituted C 2-20 An alkyl chain, which is optionally interrupted one or more times by a group selected from: -O-, -NH-, -N(CH3)-, -C(O)-, -C(O)NH-, -C(O)N(CH3)-, -C(O)O-, -NHC(O)-, -N(CH3)C(O)-, -NHC(O)NH-, -S-, -S(O)-, -S(O)2-, or any combination thereof. In some embodiments, L 1 , L 2 and / or L 3 is substituted or unsubstituted C 2-20 An alkyl chain optionally interrupted one or more times by a group selected from: -C(O)-, -C(O)NH-, -C(O)N(CH3)-, -C(O)O-, -NH-, -N(CH3)-, -NHC(O)-, -N(CH3)C(O)-, -NHC(O)NH-, -NHS(O)2NH-, -NHS(O)2NHC(O)-, -NHS(O)2NHC(O)O-, -O-, -S-, -S(O)-, -S(O)2-, -S(O)2NH-, -S(O)2NHC(O)-, -S(O)2NHC(O)NH-, -S(O)2NHC(O)O-, carbocyclyl, heterocyclyl, aralkyl, heteroaralkyl, or any combination thereof. In some embodiments, L 1 , L 2 and / or L 3 is the linker disclosed in WO2016207089, which is incorporated herein by reference in its entirety. In some embodiments, the linker comprises C 1-30 Alkyl or heteroalkyl groups (optionally substituted), optionally interrupted by aryl or heteroaryl groups (e.g., triazole, dibenzocyclooctyne, or derivatives thereof). For example, the linker may comprise a dibenzylcyclooctyne (DBCO) derivative (such as DBCO NHS ester) or a chemical group formed therefrom. For example, the interrupting group may comprise:

[0668] or derivatives thereof, wherein the intervening groups are substituted at each end to form a linker (e.g., L1 , L 2 and / or L 3 In some embodiments, the intervening groups may include:

[0669] -NHS(O)2NH-, -NHS(O)2NHC(O)-, -NHS(O)2NHC(O)O- or (wherein y is 1-20), or any combination thereof. In some embodiments, L 1 , L 2 and / or L 3 is a thioamide linker comprising one or more (e.g., one, two, three, or four) simple spacers as defined herein, optionally interrupted (i.e., linked) with thioamide groups (e.g., -NHS(O)2NH-, -NHS(O)2NHC(O)-, or -NHS(O)2NHC(O)O-). In some embodiments, L 1 , L 2 and / or L 3 Including sulfamide groups (-NHS(O)2NH-). In some embodiments, L 1 , L 2 and / or L 3 is a thioamide linker consisting of one or more (e.g., one, two, three, or four) of the following groups: -NHS(O)2NH-, -NHS(O)2NHC(O)-, -NHS(O)2NHC(O)O-, and (wherein y is 1-20), and wherein the thioamide linker is optionally linked to the adjacent group via a spanner (as defined herein).

[0670] In some embodiments, the linker ionizes in a biological system (e.g., within an acidic tumor microenvironment). In some embodiments, the ionized or partially charged (e.g., partially positively charged or partially negatively charged) moiety within the linker enhances localization of the compound to a preferred site (e.g., extracellularly, within a tumor microenvironment).

[0671] In some embodiments, the linker (eg, L 1 , L 2 and / or L 3 One or more of ) is an optionally substituted polyamine linker or an optionally substituted polyamide linker.

[0672] In some embodiments, polyamine or polyamide linker is a functional linker. In some embodiments, the functional linker is a polyamide or sulfamide linker, wherein the functional linker increases the retention of the compound in the tumor microenvironment (ie, relative to an alkyl or PEG linker) and / or reduces liver exposure. In some embodiments, a functional linker (eg, a polyamide linker) reduces the immunogenic response to the compound containing the functional linker (ie, relative to a PEG linker). In some embodiments, polyamide or sulfamide linkers disclosed herein improve one or more pharmacokinetic parameters. In some embodiments, polyamide or sulfamide linkers disclosed herein reduce the liver toxicity of the compound containing the functional linker, increase tumor and liver exposure and / or increase liver clearance. In some embodiments, linkers disclosed herein increase stability (ie, reduce the off-target release of the payload). In some embodiments, biodistribution, AUC and / or solubility are affected (eg, favorably affected) by functional linkers disclosed herein.

[0673] In some embodiments, L 1 , L 2 and / or L 3 In some embodiments, one or more of the polyamine or polyamide linkers are substituted with one or more carbonyl groups. 1 , L 2 and / or L 3 In some embodiments, one or more polyamine linkers in L form an ammonium ion (or optionally multiple ammonium ions) in the acidic tumor microenvironment. 1 , L 2 and / or L 3 One or more polyamine or polyamide linkers are selectively retained in the tumor microenvironment.

[0674] In some embodiments, the present invention provides a compound having a structure of the formula disclosed herein or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein L 1 , L 2 and / or L 3 Each of 1-30 In some embodiments, L 1 , L 2 and / or L 3 Each of the 2-30 alkyl, or substituted or unsubstituted heteroalkyl.

[0675] In some embodiments, L 1 , L2 and / or L 3 Each of which is substituted or unsubstituted C 2-20 an alkyl chain, which is optionally interrupted one or more times by groups each independently selected from the group consisting of: -O-, -S-, -NH-, -N(CH3)-, -C(O)-, -C(O)NH-, -C(O)N(CH3)-, -C(O)O-, -NHC(O)-, N(CH3)C(O)- or -NHC(O)NH-, or any combination thereof.

[0676] In some embodiments, L 1 , L 2 and / or L 3 Each of the comprises (e.g., with terminal substitution) one or more carbonyl groups (-C(O)-), amine groups (e.g., -NH- or -N(CH3)-), or amide groups (e.g., -C(O)NH-, -C(O)N(CH3)-, -NHC(O)-, or N(CH3)C(O)-). In some embodiments, such as L 1 , L 2 and / or L 3 The equal spacer comprises one or more polymeric units selected from the group consisting of: In some embodiments, the spacer comprises one to twenty polymerized units (i.e., y is 1-20). In some embodiments, y is between 2 and 6 (e.g., y is 2 to 6, 2 to 4, 2 or 3, 3 to 6, 3 or 4, etc.). In some embodiments, y is between 6 and 12 (e.g., y is 6 to 12, 6 to 10, 6 to 8, 8 to 12, 8 to 10, etc.). In some embodiments, y is 2, 3, or 4. In some embodiments, y is 8, 9, or 10. In some embodiments, L 1 , L 2 and / or L 3 One or more of are optionally substituted polyamine linkers or optionally substituted polyamide linkers.

[0677] In some embodiments, L 1 , L 2 and / or L 3 is a simple spacer selected from the group consisting of:

[0678] -O--S-

[0679] -S(O)2--C(O)-

[0680] -C 1-30 Alkyl-, -C(O)-C 1-30 alkyl

[0681] -C 1-30Alkyl-C(O)-, -C(O)-C 1-30 Alkyl-C(O)-,

[0682] -C 1-30 Alkyl-C(O)NH-, -C(O)-C 1-30 Alkyl-C(O)NH-,

[0683] -C 1-30 Alkyl-C(O)N(CH3)-, -C(O)-C 1-30 Alkyl-C(O)N(CH3)-,

[0684] -C 1-30 Alkyl-NH-, -C(O)-C 1-30 Alkyl-NH-,

[0685] -C 1-30 Alkyl-NHC(O)-, -C(O)-C 1-30 Alkyl-NHC(O)-,

[0686] -C 1-30 Alkyl-N(CH3)-, -C(O)-C 1-30 Alkyl-N(CH3)-,

[0687] -C 1-30 Alkyl-N(CH3)C(O)-, -C(O)-C 1-30 Alkyl-N(CH3)C(O)-,

[0688] -C(O)NH-, -C(O)N(CH3)-

[0689] -C(O)NH-C 1-30 Alkyl-, -C(O)N(CH3)-C 1-30 alkyl

[0690] -C(O)NH-C 1-30 Alkyl-C(O)-, -C(O)N(CH3)-C 1-30 Alkyl-C(O)-,

[0691] -C(O)NH-C 1-30 Alkyl-C(O)NH-, -C(O)N(CH3)-C 1-30 Alkyl-C(O)NH-,

[0692] -C(O)NH-C 1-30 Alkyl-C(O)N(CH3)-, -C(O)N(CH3)-C 1-30 Alkyl-C(O)N(CH3)-,

[0693] -C(O)NH-C1-30 Alkyl-NH-, -C(O)N(CH3)-C 1-30 Alkyl-NH-,

[0694] -C(O)NH-C 1-30 Alkyl-NHC(O)-, -C(O)N(CH3)-C 1-30 Alkyl-NHC(O)-,

[0695] -C(O)NH-C 1-30 Alkyl-N(CH3)-, -C(O)N(CH3)-C 1-30 Alkyl-N(CH3)-,

[0696] -C(O)NH-C 1-30 Alkyl-N(CH3)C(O)-, -C(O)N(CH3)-C 1-30 Alkyl-N(CH3)C(O)-,

[0697] -NH-, -NHC(O)-,

[0698] -NH-C 1-30 Alkyl-, -NHC(O)-C 1-30 alkyl-,

[0699] -NH-C 1-30 Alkyl-C(O)-, -NHC(O)-C 1-30 Alkyl-C(O)-,

[0700] -NH-C 1-30 Alkyl-C(O)NH-, -NHC(O)-C 1-30 Alkyl-C(O)NH-,

[0701] -NH-C 1-30 Alkyl-C(O)N(CH3)-, -NHC(O)-C 1-30 Alkyl-C(O)N(CH3)-,

[0702] -NH-C 1-30 Alkyl-NH-, -NHC(O)-C 1-30 Alkyl-NH-,

[0703] -NH-C 1-30 Alkyl-NHC(O)-, -NHC(O)-C 1-30 Alkyl-NHC(O)-,

[0704] -NH-C 1-30 Alkyl-N(CH3)-, -NHC(O)-C 1-30 Alkyl-N(CH3)-,

[0705] -NH-C 1-30 Alkyl-N(CH3)C(O)-, -NHC(O)-C 1-30 Alkyl-N(CH3C(O)-,

[0706] -N(CH3)-, -N(CH3)C(O)-,

[0707] -N(CH3)-C 1-30 Alkyl-, -N(CH3)C(O)-C 1-30 alkyl-,

[0708] -N(CH3)-C 1-30 Alkyl-C(O)-, -N(CH3)C(O)-C 1-30 Alkyl-C(O)-,

[0709] -N(CH3)-C 1-30 Alkyl-C(O)NH-, -N(CH3)C(O)-C 1-30 Alkyl-C(O)NH-,

[0710] -N(CH3)-C 1-30 Alkyl-C(O)N(CH3)-, -N(CH3)C(O)-C 1-30 Alkyl-C(O)N(CH3)-,

[0711] -N(CH3)-C 1-30 Alkyl-NH-, -N(CH3)C(O)-C 1-30 Alkyl-NH-,

[0712] -N(CH3)-C 1-30 Alkyl-NHC(O)-, -N(CH3)C(O)-C 1-30 Alkyl-NHC(O)-,

[0713] -N(CH3)-C 1-30 Alkyl-N(CH3)-, -N(CH3)C(O)-C 1-30 Alkyl-N(CH3)-,

[0714] -N(CH3)-C 1-30 Alkyl-N(CH3)C(O)-, and -N(CH3)C(O)-C 1-30 Alkyl-N(CH3C(O)-.

[0715] In some embodiments, L 1 , L 2 and / or L 3is a compound linker comprising two or more (e.g., one, two, three, or four) simple spacer elements as defined above. In some embodiments, the two or more elements in the compound linker are linked by an intervening group, such as a sulfamide group (e.g., -NHS(O)2NHC(O)-, or -NHS(O)2NHC(O)O-), or a heteroaryl or heteroarylalkyl group (e.g., a substituted triazole, a substituted DBCO, or a combination or derivative thereof). In some embodiments, the compound linker further comprises one or more units selected from the group consisting of: (eg, where y is 1 to 20).

[0716] In some embodiments, L 1 , L 2 and / or L 3 is a combination of two or three simple spacers. 1 , L 2 and / or L 3 is a combination of two or three simple spacers interspersed with (e.g., linked via) amino acids, dipeptides, or tripeptides. 1 , L 2 and / or L 3 is a combination of two or three simple spacers, interrupted by (e.g., linked via) a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group. In some embodiments, the intervening group is a substituted triazole. In some embodiments, the intervening group is an amino acid. In some embodiments, the intervening group is: -S-, -S(O)-, -S(O)2-, -NHS(O)2NH-, -NHS(O)2NHC(O)-, -NHS(O)2NHC(O)O-, (where y is 1-20),

[0717] In some embodiments, L 1 , L 2 and / or L 3 is substituted or unsubstituted C 2-20 An alkyl chain, which is optionally interrupted one or more times by a group selected from: -C(O)-, -C(O)NH-, -C(O)N(CH3)-, -C(O)O-, -NH-, -N(CH3)-, -NHC(O)-, -N(CH3)C(O)-, -NHC(O)NH-, -O-, -S-, -S(O)-, -S(O)2-, carbocyclyl, heterocyclyl, aralkyl, heteroaralkyl, or any combination thereof. In some embodiments, L 1 , L 2 and / or L3 is a linker disclosed in WO2016207089, which is incorporated herein by reference in its entirety. In some embodiments, the linker comprises two simple spacers linked via an aryl or heteroaryl group (e.g., a triazole, dibenzocyclooctyne, or a derivative thereof). For example, the linker may comprise a dibenzylcyclooctyne (DBCO) derivative (such as DBCO NHS ester) or a chemical group formed therefrom. For example, the intercalating group may comprise:

[0718] or a derivative thereof, wherein an intervening group is substituted on each end to form a linker.

[0719] In some embodiments, L 1 , L 2 and / or L 3 is a linker having a structure represented by the following formula (i), (ii), (iii), (iv) or (v):

[0720] (i)-(CO) r (CH2) s (OC 2-6 alkyl) t (NH) u (CO) v -;

[0721] (ii)-(CO) r (CH2) s (NR c C 1-6 alkyl) t (NR a ) u (CO) v -;

[0722] (iii)-(CO) r (CH2) s (NR c C(O)C 1-6 alkyl) t (NR a ) u (CO) v -;

[0723] (iv)-(CO) r (CH2) s (NR c C 1-6 alkyl) t (NR c C(O)C 1-6 Alkyl)(NR c C 1-6 alkyl)t (NR a ) u (CO) v -;

[0724] (v)-(CO) r (CH2) s (NH) u (CO) v -;

[0725] in:

[0726] R a is independently selected at each occurrence from hydrogen or C 1-3 alkyl;

[0727] R c is independently selected at each occurrence from hydrogen or C 1-3 alkyl;

[0728] r is 0 or 1; s is 0 to 10; t is 1 to 10; u is 0 or 1; and v is 0 or 1.

[0729] In some embodiments, L 1 , L 2 and / or L 3 is a polymeric linker selected from the group consisting of:

[0730] -C(O)-C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -NHC(O)-,

[0731] -C(O)-C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0732] -C(O)-C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -NHC(O)-,

[0733] -C(O)-C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0734] -C(O)-C 1-6 Alkyl-[NHC(O)-C 1-6 alkyl] 1-8 -NHC(O)-,

[0735] -C(O)-C 1-6Alkyl-[NHC(O)-C 1-6 alkyl] 1-8 -N(CH3)C(O)-,

[0736] -C(O)-C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -NHC(O)-,

[0737] -C(O)-C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0738] -C(O)-C 1-6 Alkyl-[N(CH3)C(O)-C 1-6 alkyl] 1-8 -NHC(O)-,

[0739] -C(O)-C 1-6 Alkyl-[N(CH3)C(O)-C 1-6 alkyl] 1-8 -N(CH3)C(O)-,

[0740] -C(O)-C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -NH-,

[0741] -C(O)-C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -N(CH3)-,

[0742] -C(O)-C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -NH-,

[0743] -C(O)-C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -N(CH3)-,

[0744] -C(O)-C 1-6 Alkyl-[NHC(O)-C 1-6 alkyl] 1-8 -NH-,

[0745] -C(O)-C 1-6 Alkyl-[NHC(O)-C 1-6 alkyl] 1-8 -N(CH3)-,

[0746] -C(O)-C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -NH-,

[0747] -C(O)-C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -N(CH3)-,

[0748] -C(O)-C 1-6 Alkyl-[N(CH3)C(O)-C 1-6 alkyl] 1-8 -NH-,

[0749] -C(O)-C 1-6 Alkyl-[N(CH3)C(O)-C 1-6 alkyl] 1-8 -N(CH3)-,

[0750] -C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -NHC(O)-,

[0751] -C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0752] -C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -NHC(O)-,

[0753] -C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0754] -C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -NHC(O)-,

[0755] -C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -N(CH3)C(O)-,

[0756] -C 1-6 Alkyl-[OC 2-6 alkyl] 1-8-NH-,

[0757] -C 1-6 Alkyl-[OC 2-6 alkyl] 1-8 -N(CH3)-,

[0758] -C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -NH-,

[0759] -C 1-6 Alkyl-[NH-C 2-6 alkyl] 1-8 -N(CH3)-,

[0760] -C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -NH-, and

[0761] -C 1-6 Alkyl-[N(CH3)-C 2-6 alkyl] 1-8 -N(CH3)-.

[0762] In some embodiments, L 1 Selected from:

[0763]

[0764]

[0765] in:

[0766] #EL is the bond to the peptide linker (EL); and

[0767] #T is the bond to the target protein binder (T).

[0768] In some embodiments, L 2 Selected from:

[0769]

[0770] in:

[0771] #EL is the bond to EL; and

[0772] #A is a trivalent or tetravalent linker A (i.e., A 1 or A 2 ) key.

[0773] In some embodiments, L 3 Selected from:

[0774]

[0775] in:

[0776] #A is a trivalent or tetravalent linker A (i.e., A 1 or A 2 ) key; and

[0777] #T is the bond to the target protein binder (T).

[0778] Self-Igniting Linker (SIL)

[0779] In order to ensure efficient release of the free drug, it is optionally also possible to introduce a so-called self-immolative linker element (SIL) between the enzymatic cleavage site and the drug (Anticancer Agents in Medicinal Chemistry, 2008, 8, 618-637). The drug can be released by various mechanisms, for example by subsequent elimination via an electronic cascade after the initial enzymatic release of the nucleophile group (Bioorg. Med. Chem., 1999, 7, 1597; J. Med. Chem., 2002, 45, 937; Bioorg. Med. Chem., 2002, 10, 71) or by cyclization of the corresponding linker element (Bioorg. Med. Chem., 2003, 11, 2277; Bioorg. Med. Chem., 2007, 15, 4973; Bioorg. Med. Chem. Lett., 2007, 17, 2241, Org. Biomol. Chem., 2011, 9, 1846-1854) or by a combination of both (Angew. Chem. Inter. Ed., 2005, 44, 4378). Examples of such linker elements are shown in Scheme 1:

[0780] Scheme 1: Proposed cleavage mechanism of the self-immolative linker

[0781]

[0782] In some embodiments, the SIL is a para-aminocarbamate (PABC) group. In some embodiments, the SIL is: In some embodiments, the SIL is absent (i.e., the SIL is a bond). In some embodiments, the SIL is where * indicates a bond to the payload nitrogen and unlabeled groups form a bond to the enzymatically cleavable peptide.

[0783] Trivalent linker (A 1 )

[0784] In some embodiments, the trivalent linker A 1 is a moiety containing 1 to 100 atoms selected from H, C, N, O and S, which is configured to bind to the linker L 2 and L 3 In some embodiments, A 1 In some embodiments, the central atom or group (Y) comprises a N or CH. In some embodiments, the central atom or group (Y) has one or more arms (e.g., an alkyl or heteroalkyl chain, which is optionally substituted with or interrupted by one or more groups independently selected from the following: carbonyl (-C(O)-), ether (-O-), amine (e.g., -NH- or -N(CH3)-), or amide (e.g., -C(O)NH-, -C(O)N(CH3)-, -NHC(O)-, or -N(CH3)C(O)-, alternatively referred to as an amide linker). In some embodiments, A 1 In some embodiments, A 1 For an arm having an amide linker (e.g., B 1 、B 2 、B 3 ) of a trivalent linker. In some embodiments, A 1 is a trivalent heteroaryl, heteroaralkyl, aryl, aralkyl, heteroalkyl-aryl, heteroalkyl-heteroaryl group. In some embodiments, A 1 The invention comprises a central atom or group (Y) which is a carbocyclic ring (e.g., cycloalkyl or aryl) or a heterocyclic ring (e.g., heterocycloalkyl or heteroaryl). In some embodiments, (Y) is phenyl, triazinyl, or triazolyl, each of which is optionally substituted with 1 to 3 heteroalkyl arms. In some embodiments, the linker (e.g., L) is substituted with 1 to 3 heteroalkyl arms. 2 or L 3 ) is or includes alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkyl-heterocyclyl, heteroalkyl-heterocyclyl, alkyl-aralkyl, heteroalkyl-aralkyl, alkyl-heteroaralkyl, heteroalkyl-heteroaralkyl, alkyl-heterocycloalkyl, or any combination thereof. In some embodiments, the linker (e.g., L 1 , L 2 or L 3 ) is a heteroalkyl or alkyl linker, optionally interrupted by aryl, heteroaryl, cycloalkyl, or heterocyclyl groups. In some embodiments, the linker is interrupted by a click group (e.g., a substituted triazole).

[0785] In some embodiments, A or A 1 is a trivalent linker comprising arm B 1 、B 2 and B 3and optionally further comprising an extension S 1 、S 2 and S 3 , wherein the trivalent linker is configured to be attached to an adjacent group (e.g., L 2 or L 3 ) to form an amide bond. In some embodiments, A 1 In this context, a trivalent amide linker may be a chemical group having three valencies (or capable of forming bonds with three reagents), typically consisting of an alkyl group, a carbonyl group, and an amine group, and preferably capable of forming bonds with adjacent groups (e.g., L 2 , L 3 ) forms an amide bond. In some embodiments, the trivalent amide linker includes a central amino acid (e.g., lysine, glutamine, glutamic acid, etc.), wherein the amino acid is connected to the adjacent group (e.g., L 2 , L 3 ) forms a bond, optionally via a linker (e.g., B 1 、B 2 、B 3 ) and / or expanders (i.e., S 1 、S 2 or S 3 ) bridged. Preferably, the trivalent group described herein links a payload (e.g., a protease-cleavable payload (P)) to two target protein binding agents or to a target protein binding agent and a group MOD, and wherein the payload, target protein binding agent, and MOD are bridged via a linker (L 2 or L 3 )link.

[0786] In some embodiments, S 1 、S 2 and S 3 In some embodiments, one or more of S 1 、S 2 and S 3 One or more of them is -C(O)-C 2-6 Alkyl-C(O)-, -N(CH3)-C 1-6 Alkyl-N(CH3)-, -NH-C 1-6 Alkyl-NH- or -NH-C 1-6 Alkyl-N(CH3)-. In some embodiments, S 1 、S 2 and / or S 3 -C(O)-C 2-6 Alkyl-C(O)- or -NH-C 1-6 Alkyl-NH-.

[0787] In some embodiments, each B 1 、B2 and B 3 is an amide linker (e.g., an alkylamide and / or polyamide linker). In some embodiments, each B 1 、B 2 and B 3 is an amide linker comprising 1 to about 32 atoms. In some embodiments, the 1 to 32 atoms are selected from carbon, nitrogen, and oxygen.

[0788] In some embodiments, each B 1 、B 2 and B 3 Selected from:

[0789] -C(O)-;

[0790] -C(O)NH-;

[0791] -C(O)N(CH3)-;

[0792] -C(O)NH-C 0-6 Alkyl-C(O)-;

[0793] -C(O)NH-C 1-6 Alkyl-C(O)NH-;

[0794] -C(O)NH-C 1-6 Alkyl-C(O)N(CH3)-;

[0795] -C(O)NH-C 1-6 Alkyl-NH-;

[0796] -C(O)NH-C 1-6 Alkyl-N(CH3)-;

[0797] -C(O)NH-C 1-6 Alkyl-NHC(O)-;

[0798] -C(O)NH-C 1-6 Alkyl-N(CH3)C(O)-;

[0799] -C(O)N(CH3)-C 1-6 Alkyl-C(O)-;

[0800] -C(O)N(CH3)-C 1-6 Alkyl-C(O)NH-;

[0801] -C(O)N(CH3)-C 1-6 Alkyl-C(O)N(CH3)-;

[0802] -C(O)NH-C 1-6 Alkyl-NH-;

[0803] -C(O)NH-C 1-6 Alkyl-N(CH3)-;

[0804] -C(O)N(CH3)-C 1-6 Alkyl-NHC(O)-;

[0805] -C(O)N(CH3)-C 1-6 Alkyl-N(CH3)C(O)-;

[0806] -C(O)-C 1-6 Alkyl-C(O)-;

[0807] -C(O)-C 1-6 Alkyl-C(O)NH-;

[0808] -C(O)-C 1-6 Alkyl-C(O)N(CH3)-;

[0809] -C(O)-C 1-6 Alkyl-NH-;

[0810] -C(O)-C 1-6 Alkyl-N(CH3)-;

[0811] -C(O)-C 1-6 Alkyl-NHC(O)-;

[0812] -C(O)-C 1-6 Alkyl-N(CH3)C(O)-;

[0813] -C 0-6 Alkyl-C(O)-;

[0814] -C 0-6 Alkyl-C(O)NH-;

[0815] -C 0-6 Alkyl-C(O)N(CH3)-;

[0816] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-C(O)-;

[0817] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-C(O)NH-;

[0818] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-C(O)N(CH3)-;

[0819] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-NH-;

[0820] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-N(CH3)-;

[0821] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-NHC(O)-;

[0822] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-N(CH3)C(O)-;

[0823] -C 0-6 Alkyl-C(O)N(CH3)-C 1-6 Alkyl-C(O)-;

[0824] -C 0-6 Alkyl-C(O)N(CH3)-C 1-6 Alkyl-C(O)NH-;

[0825] -C 0-6 Alkyl-C(O)N(CH3)-C 1-6 Alkyl-C(O)N(CH3)-;

[0826] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-NH-;

[0827] -C 0-6 Alkyl-C(O)NH-C 1-6 Alkyl-N(CH3)-;

[0828] -C 0-6 Alkyl-C(O)N(CH3)-C 1-6 Alkyl-NHC(O)-;

[0829] -C 0-6 Alkyl-C(O)N(CH3)-C 1-6 Alkyl-N(CH3)C(O)-;

[0830] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-C(O)-;

[0831] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-C(O)NH-;

[0832] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-C(O)N(CH3)-;

[0833] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-NH-;

[0834] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-N(CH3)-;

[0835] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-NHC(O)-;

[0836] -C 0-6 Alkyl-C(O)-C 1-6 Alkyl-N(CH3)C(O)-;

[0837] -C 0-6 Alkyl-NH-;

[0838] -C 0-6 Alkyl-NHC(O)-;

[0839] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-C(O)-;

[0840] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-C(O)NH-;

[0841] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-C(O)N(CH3)-;

[0842] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-NH-;

[0843] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-N(CH3)-;

[0844] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-NHC(O)-;

[0845] -C 0-6 Alkyl-NHC(O)-C 1-6 Alkyl-N(CH3)C(O)-;

[0846] -C 0-6 Alkyl-N(CH3)-;

[0847] -C 0-6 Alkyl-N(CH3)C(O)-;

[0848] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-C(O)-;

[0849] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-C(O)NH-;

[0850] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-C(O)N(CH3)-;

[0851] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-NH-;

[0852] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-N(CH3)-;

[0853] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-NHC(O)-; and

[0854] -C 0-6 Alkyl-N(CH3)C(O)-C 1-6 Alkyl-N(CH3)C(O)-.

[0855] In some embodiments, A 1 is an amino acid or a derivative thereof. 1 is Lys (e.g., L-Lys or D-Lys), Glu (L-Glu or D-Glu), or Asp (L-Asp or D-Asp), or a derivative thereof (e.g., via one or more arms (e.g., B 1 、B 2 or B 3 )replace).

[0856] In some embodiments, A 1 Has one of the following structures:

[0857]

[0858]

[0859] Tetravalent linker (A 2 )

[0860] In some embodiments, the tetravalent linker A 2 is a moiety containing 1 to 100 atoms selected from H, C, N, O and S, which is configured to be connected to four linkers (e.g., two L 2 and two L 3 In some embodiments, A 2 is a diamino acid or a dipeptide. In some embodiments, A 2 is an amino acid or a peptide. In some embodiments, A 2 In some embodiments, A 2 is an amino acid or diamino acid substituted with one or more arms (e.g., an alkyl or heteroalkyl chain, optionally substituted with or interrupted by one or more groups independently selected from carbonyl (—C(O)—), ether (—O—), amine (e.g., —NH— or —N(CH 3 )—), or amide (e.g., —C(O)NH—, —C(O)N(CH 3 )—, —NHC(O)—, or —N(CH 3 )C(O), alternatively referred to as an amide linker). In some embodiments, A 2 In some embodiments, A 2 is a tetravalent heteroaryl, heteroaralkyl, aryl, aralkyl, heteroalkyl-aryl, or heteroalkyl-heteroaryl group.

[0861] In some embodiments, A 2 To include arm B 1 、B 2 、B 3 and / or B 4 A tetravalent linker, wherein the tetravalent linker is configured to connect to four adjacent groups (e.g., L 2 or L 3 In some embodiments, the tetravalent amide linker can be a chemical group having four valencies (or capable of forming bonds with four linkers), typically consisting of an alkyl group, a carbonyl group, and an amine group, and preferably capable of forming bonds with adjacent groups (e.g., L 2 , L 3 ) to form an amide bond. In some embodiments, the tetravalent amide linker comprises one or two central amino acid residues (e.g., lysine, glutamine, glutamic acid, or a combination thereof), wherein the amino acid is bound to an adjacent group (e.g., L 2 , L 3) forms a bond, which is optionally bridged via a linker and / or an extender. Preferably, the tetravalent groups described herein link two payloads (e.g., two protease-cleavable payloads (P)) to two target protein binding agents; or link two payloads to a target protein binding agent and a MOD; or link a payload and a MOD to two target protein binding agents.

[0862] In some embodiments, A 2 Has the following structure:

[0863] Physicochemical or pharmacokinetic modulators (MODs)

[0864] In some embodiments, the present disclosure provides conjugates that include a physicochemical or pharmacokinetic modulator ("MOD"). In some embodiments, the MOD is a physicochemical modulator. In some embodiments, the MOD is a pharmacokinetic modulator. Generally, unless otherwise specified, these terms are used interchangeably. The group MOD can be linked to the rest of the conjugate via a linker (e.g., a stabilizing linker). In some embodiments, the MOD is linked to the rest of the conjugate via a linker (e.g., an L 4 ) is linked to EL. In some embodiments, MOD is linked to EL via a linker (e.g., L 2 , L 3 ) bonded to A 1 In some embodiments, MOD is a charged or polar group. Examples of charged or polar groups include hydroxides and alcohols, carboxylates and carboxylic acids, ammonium and amines, guanidinium and guanidine, etc. In some embodiments, MOD is or includes an amine. In some embodiments, MOD is a polyamine group. In some embodiments, MOD is -NR2 or -NR3 + In some embodiments, MOD is -C 1-6 Alkyl-NR2 or -C 1-6 Alkyl-NR3 + (e.g. -C 1-6 Alkyl-NH2, -C 1-6 Alkyl-NHCH3, -C 1-6 Alkyl-N(CH3)2 or -C 1-6 Alkyl-N(CH3)3 + In some embodiments, MOD is -C 1-6 Alkyl-NHC(=NH + )NH2. In some embodiments, MOD is a polyacid group. In some embodiments, MOD is -COOH or -COO - In some embodiments, MOD is -C 1-6 Alkyl-COOR (e.g. -C 1-6 Alkyl-COOH, -C1-6 Alkyl-COO - 、-C 1-6 Alkyl-COO - Na + or -C 1-6 In some embodiments, MOD is a group that is converted to a polar or charged group in vivo (e.g., MOD is an alkyl ester that is cleaved in vivo to an alkyl acid, thereby reducing membrane permeability). In some embodiments, MOD is -OH or -O - In some embodiments, MOD is an amino acid. In some embodiments, MOD is a basic amino acid. In some embodiments, MOD is Arg, His, or Lys. In some embodiments, MOD is an acidic amino acid. In some embodiments, MOD is Glu or Asp. In some embodiments, MOD is an unnatural amino acid (e.g., D-Glu or D-Asp). In some embodiments, MOD is a polar amino acid (e.g., Ser, Thr, Asn, Gln). In some embodiments, MOD is a PEG or polysarcosine group.

[0865] Pharmaceutically acceptable salts

[0866] In one aspect, the compounds described herein are in the form of pharmaceutically acceptable salts. Likewise, active metabolites of these compounds having the same type of activity are included within the scope of this disclosure. In addition, the compounds described herein may exist in unsolvated form as well as in solvated form with pharmaceutically acceptable solvents such as water and ethanol. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0867] In some embodiments, a pharmaceutically acceptable salt is obtained by reacting a conjugate described herein with an acid. In some embodiments, the conjugate described herein (e.g., in the free base form) is alkaline and reacts with an organic or inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid. Organic acids include, but are not limited to, 1-hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid; 2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor-10-sulfonic acid (+); decanoic acid; caproic acid; octanoic acid; caprylic acid; octanoic acid; benzoic acid; benzoic acid; camphoric acid (+); camphor-10-sulfonic acid (+); decanoic acid; caproic acid; octanoic acid; benzo ... acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecylsulfuric acid; ethane-1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; glycerophosphate; glycolic acid; hippuric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (-L); malonic acid; mandelic acid (DL); methanesulfonic acid; naphthalene-1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; propionic acid; pyroglutamic acid (-L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+L); thiocyanic acid; toluenesulfonic acid (p); and undecylenic acid.

[0868] In some embodiments, pharmaceutically acceptable salts of the compounds of the present invention include, inter alia, acid addition salts of mineral acids, carboxylic acids, and sulfonic acids, such as salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalene disulfonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, succinic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, benzoic acid, and pamoic acid.

[0869] In some embodiments, the conjugates described herein are prepared as chloride, sulfate, bromide, methanesulfonate, maleate, citrate, or phosphate salts.

[0870] In some embodiments, pharmaceutically acceptable salts are obtained by reacting a conjugate described herein with a base. In some embodiments, the conjugate described herein is acidic and reacts with a base. In such cases, the acidic protons of the conjugate described herein are replaced by metal ions (e.g., lithium, sodium, potassium, magnesium, calcium, or aluminum ions). In some cases, the compounds described herein are coordinated with organic bases such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, meglumine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, the compounds described herein form salts with amino acids such as, but not limited to, arginine and lysine. Acceptable inorganic bases for forming salts with compounds comprising acidic protons include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, and lithium hydroxide. In some embodiments, provided herein are compounds prepared as sodium salts, calcium salts, potassium salts, magnesium salts, meglumine salts, N-methylglucamine salts, or ammonium salts. In some embodiments, the compounds provided herein are prepared as sodium salts, such as monosodium salts, disodium salts, trisodium salts, or tetrasodium salts. In some embodiments, the salt is disodium salts or trisodium salts. In some embodiments, the salt is trifluoroacetate (TFA), such as mono-TFA, di-TFA, tri-TFA, or tetra-TFA salts.

[0871] In some embodiments, pharmaceutically acceptable salts of the compounds of the present invention also include salts derived from conventional bases, such as alkali metal salts (e.g., sodium salts and potassium salts), alkaline earth metal salts (e.g., calcium salts and magnesium salts), zinc salts, and ammonium salts derived from ammonia or organic amines having 1 to 20 carbon atoms, such as ethylamine, diethylamine, triethylamine, N,N-ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, choline, benzalkonium chloride, procaine, dibenzylamine, dicyclohexylamine, N-methylmorpholine, N-methylpiperidine, arginine, lysine, and 1,2-ethylenediamine.

[0872] It should be understood that references to pharmaceutically acceptable salts include solvent addition forms. In some embodiments, solvates comprise stoichiometric or non-stoichiometric amounts of solvent and are formed during the crystallization process with pharmaceutically acceptable solvents such as water and ethanol. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein optionally exist in unsolvated as well as solvated forms.

[0873] In some embodiments, the present invention describes a form of the compound of the invention that forms a solid or liquid complex by coordination with a solvent molecule. Hydrates are a specific form of solvates in which coordination occurs with water. In the context of the present invention, solvates are preferably hydrates.

[0874] The methods and formulations described herein include the use of N-oxides (where appropriate) or pharmaceutically acceptable salts of compounds having the structure of any of the formulae disclosed herein, as well as active metabolites of these compounds having the same type of activity.

[0875] In some embodiments, sites on the organic groups (e.g., alkyl groups, aromatic rings) of the compounds of the formulae disclosed herein are susceptible to various metabolic reactions. The introduction of appropriate substituents onto the organic groups will reduce, minimize, or eliminate these metabolic pathways. In certain embodiments, suitable substituents that reduce or eliminate the susceptibility of the aromatic ring to metabolic reactions are, by way of example only, halogens, deuteriums, alkyl groups, haloalkyl groups, or deuterated alkyl groups.

[0876] In another embodiment, the compounds described herein are isotopically labeled (e.g., with a radioisotope) or labeled by other means including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0877] The compounds described herein include isotopically labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, iodine, phosphorus, such as, for example 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F. 36 Cl, 123 I. 124 I. 125 I. 131 I. 32 P and 33 In one aspect, an isotopically labeled compound described herein (e.g., into which a 3 H and 14In one aspect, substitution with isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

[0878] In some embodiments, the present invention also encompasses all suitable isotopic variants of the compounds of the present invention. It is understood herein that an isotopic variant of a compound of the present invention refers to a compound in which at least one atom in the compound of the present invention has been exchanged for another atom of the same atomic number but having an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention are isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, such as 2 H (deuterium), 3 H (tritium), 13 C. 14 C. 15 N. 17 O. 18 O. 32 P. 33 P. 33 S. 34 S. 35 S. 36 S. 18 F. 36 Cl, 82 Br, 123 I. 124 I. 129 I and 131 I. Certain isotopic variants of the compounds of the invention, especially those into which one or more radioactive isotopes have been incorporated, may be useful, for example, for examining the mechanism of action or the distribution of the active ingredient in vivo; they are particularly useful due to their relative ease of preparation and detectability. 3 H. 14 C or 18 Compounds labeled with an F isotope are suitable for the purposes described. In addition, the introduction of isotopes such as deuterium can provide specific therapeutic benefits due to the greater metabolic stability of the compound, such as extending the in vivo half-life or reducing the required active dose; therefore, such modifications of the compounds of the present invention may also constitute preferred embodiments of the present invention. Isotopic variants of the compounds of the present invention can be prepared by conventional procedures known to those skilled in the art, for example, by using corresponding isotopic modifications of the reagents or starting compounds, as described in the methods further below and in the working examples.

[0879] In some embodiments, the compounds disclosed herein have one or more stereo centers, and each stereo center exists independently in R or S configuration. In some embodiments, the conjugates described herein exist in R configuration. In some embodiments, the conjugates described herein exist in S configuration. The compounds presented herein include all diastereomers, single enantiomers, atropisomers and epimer forms, and suitable mixtures thereof. The compounds and methods provided herein include all cis, trans, same (syn), right (anti), different side (E) and same side (Z) isomers, and suitable mixtures thereof.

[0880] As needed, individual stereoisomers are obtained by methods such as stereoselective synthesis or separation of stereoisomers by chiral chromatographic columns, or separation of diastereomers by achiral or chiral chromatographic columns, or crystallization and recrystallization in appropriate solvents or mixtures of solvents. In certain embodiments, the compounds disclosed herein are prepared as individual stereoisomers thereof by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds / salts, separating the diastereomers and recovering optically pure individual enantiomers. In some embodiments, single enantiomers are resolved using covalent diastereomeric derivatives of the compounds described herein. In another embodiment, diastereomers are separated by separation / resolution techniques based on differences in solubility. In other embodiments, separation of stereoisomers is performed by chromatography or by forming diastereomeric salts and separating by recrystallization or chromatography, or any combination thereof. Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981. In some embodiments, stereoisomers are obtained by stereoselective synthesis.

[0881] Prodrug

[0882] In some embodiments, the compounds described herein are prepared as prodrugs. "Prodrug" refers to an agent that is converted into a parent drug in vivo. Prodrugs are generally useful because they are easier to administer than the parent drug in some cases. They are, for example, bioavailable by oral administration, while the parent drug is not. Additionally or alternatively, compared to the parent drug, the prodrug also has improved solubility in the pharmaceutical composition. In some embodiments, the design of the prodrug increases effective water solubility. Examples of prodrugs (not limited to) are compounds described herein, which are administered as esters ("prodrugs"), but are then metabolically hydrolyzed to provide an active entity. Another example of a prodrug is a short peptide (polyamino acid) bonded to an acid group, wherein the peptide is metabolized to display an active portion. In certain embodiments, in the case of in vivo administration, the prodrug is chemically converted into a biologically, pharmaceutically, or therapeutically active form of the compound. In certain embodiments, the prodrug is enzymatically metabolized to a biologically, pharmaceutically, or therapeutically active form of the compound by one or more steps or processes.

[0883] In some embodiments, the prodrug is activated when an enzyme cleaves the prodrug (e.g., a tumor-associated enzyme such as neutrophil elastase). In some embodiments, the prodrug comprises a peptide sequence recognized by a given enzyme. In some embodiments, the prodrug is selectively cleaved by a specific enzyme.

[0884] In some embodiments, the prodrug further comprises a non-peptide prodrug moiety (e.g., an ester) that is released in vivo. The non-peptide prodrug moiety indicates that the bond that is metabolized or broken to release the active agent is not a peptide bond (-C(O)--NH-). In some embodiments, the non-peptide prodrug moiety is an alkyl ester (including substitutions, such as alkyl substitutions on alkyl groups). In some embodiments, the non-peptide prodrug moiety is an ester of an amino acid, such as an aspartic acid or glutamic acid residue. In some embodiments, the alkyl ester prodrug is represented by Asp* or Glu*, indicating that the alkyl (e.g., substituted alkyl) group masks the carboxylic acid portion of the side chain. In some embodiments, provided herein are prodrug compounds wherein the alkylamine (-C 1-6 Alkyl-NR2) or alkylammonium (-C 1-6 Alkyl-NR3 + ) group in vivo (wherein "R" as used herein is hydrogen or C 1-6In some embodiments, the non-peptide prodrug portion (e.g., an ester (e.g., an alkylamine or alkylammonium ester)) is released from an amino acid ester (e.g., Asp* or Glu*) to liberate the acid portion. In some embodiments, the non-peptide prodrug portion (e.g., an ester (e.g., an alkylamine or alkylammonium ester)) is released (i.e., cleaved) in plasma, while the enzymatically cleavable portion remains intact (e.g., when the cleavage enzyme is absent or sufficient). In some embodiments, the prodrug ester is slowly cleaved in plasma, and there is no detectable release of the enzymatically cleavable portion or the payload (e.g., a cytotoxic or cytostatic ligand) attached thereto. In some embodiments, the non-peptide (i.e., ester) prodrug portion is cleaved before the peptide prodrug portion. In some embodiments, the non-peptide (i.e., ester) prodrug portion is cleaved independently of protease activity (e.g., non-proteolytic cleavage). In some embodiments, the non-peptide (i.e., ester) prodrug portion is slowly cleaved to an extent of about 50% (± 5%). As used herein, "slowly" means that the prodrug is cleaved to an extent of about 50% after at least two hours in plasma. In some embodiments, slow cleavage constitutes about 50% prodrug release (i.e., release of the ester portion to produce the active carboxylic acid) in plasma after about 2, about 3, about 4, about 5, about 6, about 8, about 10, or about 12 hours. In some embodiments, slow cleavage constitutes about 50% prodrug release in plasma after about 2 to about 12 hours. In some embodiments, the ester prodrug is cleaved to about 50% in plasma after about 2 to about 6 hours. In some embodiments, the ester prodrug is cleaved to about 50% in plasma after about 2 to about 4 hours. As previously described, the non-peptide (i.e., ester) prodrug portion is cleaved independently of (e.g., in the absence or presence of) proteolytic enzymes (such as cathepsin B, legumin, or neutrophil elastase). As shown in Figures 2 and 3, in some embodiments, the ester prodrug portion is cleaved to about 50% after about 3 or 4 hours of in vivo administration (and within about 6 hours). In some embodiments, the degradation product of such prodrugs is the parent conjugate without the alkyl ester, meaning that the enzymatically cleavable moiety, the cytotoxic or cytostatic moiety, the linker / spacer, and the integrin binding agent remain intact. In some embodiments, enzymatic cleavage occurs after the release of the ester prodrug moiety. In some embodiments, the protease responsible for cleaving the enzymatically cleavable moiety recognizes free Asp or Glu residues rather than the Asp* or Glu* prodrug ester.

[0885] Prodrugs of the compounds described herein include, but are not limited to, esters, ethers, carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl derivatives, N-alkoxyacyl derivatives, quaternary derivatives of tertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates, phosphates, and sulfonates. See, for example, Design of Prodrugs, Bundgaard, A. Ed., Elsevier, 1985, and Method in Enzymology, Widder, K. et al., eds.; Academic, 1985, vol. 42, p. 309-396; A Textbook of Bundgaard, H. "Design and Application of Prodrugs" in Drug Design and Development, Krosgaard-Larsen and H. Bundgaard, eds., 1991, Chapter 5, p. 113-191; and Bundgaard, H., Advanced Drug Delivery Review, 1992, 8, 1-38, each of which is incorporated herein by reference. In some embodiments, the hydroxyl groups in the compounds disclosed herein are used to form prodrugs, wherein the hydroxyl groups are introduced into acyloxyalkyl esters, alkoxycarbonyloxyalkyl esters, alkyl esters, aryl esters, phosphate esters, sugar esters, and ethers, etc. In some embodiments, the hydroxyl groups in the compounds disclosed herein are prodrugs, wherein the hydroxyl groups are subsequently metabolized in vivo to provide a carboxylic acid group. In some embodiments, the carboxyl group is used to provide an ester or amide (e.g., a prodrug), which is subsequently metabolized in vivo to provide a carboxylic acid group. In some embodiments, the compounds described herein are prepared as alkyl ester prodrugs.

[0886] Prodrug forms of the compounds described herein, wherein the prodrug as described herein is metabolized in vivo to produce the conjugates described herein are included within the scope of the claims.In some cases, some of the compounds described herein are prodrugs of another derivative or active compound.

[0887] In some embodiments, any of the hydroxyl group, amino group, or carboxylic acid group is functionalized in a suitable manner to provide a prodrug moiety. In some embodiments, the prodrug moiety is as described above.

[0888] Pharmaceutical composition

[0889] The present invention provides a pharmaceutical composition comprising a compound of the formula disclosed herein or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; and a pharmaceutically acceptable excipient.

[0890] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated into pharmaceutically useful preparations in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate the processing of the active compound. Suitable formulations depend on the chosen route of administration. An overview of the pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999), the disclosures of which are incorporated herein by reference.

[0891] In some embodiments, the compounds described herein are administered alone or in combination with a pharmaceutically acceptable carrier, excipient, or diluent in a pharmaceutical composition. The administration of the compounds described herein and compositions can be achieved by any method that enables the compound to be delivered to the site of action. These methods include, but are not limited to, delivery via: enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories, and rectal enemas), parenteral routes (injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and subcutaneous), inhalation, transdermal, transmucosal, sublingual, buccal, and topical (including epidermal, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration, although the most suitable approach may depend on, for example, the condition and illness of the recipient. By way of example only, the compounds described herein can be administered topically to the area in need of treatment, for example, by local infusion during surgery, topical application such as cream or ointment, injection, catheter, or implant. Administration can also be by direct injection at the site of the diseased tissue or organ.

[0892] In some embodiments, pharmaceutical compositions suitable for oral administration are presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. In some embodiments, the active ingredient is presented as a bolus, electuary, or paste.

[0893] Orally usable pharmaceutical compositions include tablets, push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Tablets can be manufactured by compressing or molding together with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, an inert diluent or lubricant, a surfactant or a dispersant. Molded tablets can be manufactured by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. In some embodiments, the tablets are coated or scored and formulated to provide a slow or controlled release of the active ingredient therein. All formulations for oral administration should be in a dosage suitable for such administration. Push-fit capsules can contain a mixture of the active ingredient and fillers such as lactose, binders such as starch, or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compound can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycol. In some embodiments, a stabilizer is added. The dragee core has a suitable coating. For this purpose, a concentrated sugar solution can be used, which can optionally include gum arabic, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol or titanium dioxide, lacquer liquid and a suitable organic solvent or solvent mixture. Dyes or pigments can be added to tablets or dragee coatings for identification or in order to characterize the different combinations of active compound dosages.

[0894] In some embodiments, the pharmaceutical composition is formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injectable formulations can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of preservatives. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous solvent, and can include preparatons such as suspending agents, stabilizers, or dispersants. The composition can be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in powder form or stored under freeze-dried (lyophilized) conditions requiring only the addition of a sterile liquid carrier (e.g., saline or sterile pyrogen-free water) immediately prior to use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type previously described.

[0895] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compound that may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also include suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions.

[0896] Pharmaceutical compositions can also be formulated as depot preparations. Such depot preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or with an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt.

[0897] For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner. Such compositions may include the active ingredient in a flavored base such as sucrose and acacia or tragacanth.

[0898] The pharmaceutical composition can be administered topically, i.e., by non-systemic administration. This includes external application of the compounds of the invention to the epidermis or buccal cavity and instillation of such compounds into the ears, eyes, and nose, so that the compounds do not significantly enter the bloodstream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal, and intramuscular administration.

[0899] Pharmaceutical compositions suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation, such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for application to the eyes, ears or noses. For topical administration, the active ingredient may comprise 0.001% to 10% w / w, for example 1% to 2%, by weight of the formulation.

[0900] The pharmaceutical composition for administration by inhalation is conveniently delivered by insufflator, nebulizer pressurized packaging or other convenient means of delivering aerosol spray. Pressurized packaging can include suitable propellants, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve for delivering the metered amount. Alternatively, for administration by suction or insufflation, the pharmaceutical preparation can take the form of a dry powder composition, such as a powder mixture of a compound and a suitable powder matrix (such as lactose or starch). Powder composition can be present in, for example, capsules, cartridges, gelatin or blister packs in unit dosage form, and powder can be administered by means of an insufflator or insufflator.

[0901] In addition to the ingredients particularly mentioned above, the compounds and compositions described herein may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

[0902] Compounds for treating diseases or conditions

[0903] In some embodiments, the present invention provides a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in the treatment of a disease or condition.

[0904] In some embodiments, the present invention provides a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in the manufacture of a medicament for treating a disease or condition described herein.

[0905] In some embodiments, the present invention provides a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in the treatment of a hyperproliferative disorder.

[0906] In some embodiments, the present invention provides a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in the treatment of cancer.

[0907] In some embodiments, the present invention provides a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in the treatment of an autoimmune disorder.

[0908] Dosage and treatment regimen

[0909] In some embodiments, the present invention provides a method of treating a disease or condition in a subject, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; or a pharmaceutical composition thereof. In some embodiments, the compound used in the method of treating a disease or condition is a compound of Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX), or a pharmaceutically acceptable salt thereof, or a stereoisomer or mixture of stereoisomers thereof.

[0910] In some embodiments, the present invention provides a method of treating a hyperproliferative disorder in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; or a pharmaceutical composition thereof.

[0911] In some embodiments, the present invention provides a method of treating cancer in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; or a pharmaceutical composition thereof.

[0912] In some embodiments, the present invention provides a method of treating an autoimmune disorder in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a compound of the formula disclosed herein, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; or a pharmaceutical composition thereof.

[0913] Example

[0914] As used above and throughout the specification of the present invention, unless otherwise indicated, the following abbreviations shall be understood to have the following meanings:

[0915] abbreviation

[0916] Abu: γ-aminobutyric acid

[0917] ACN: acetonitrile

[0918] Boc: tert-Butoxycarbonyl

[0919] Bzl: benzyl

[0920] DCM: dichloromethane

[0921] DIEA: N,N-diisopropylethylamine (Hünig's base)

[0922] DMAP: dimethylaminopyridine

[0923] DMF: dimethylformamide

[0924] DMSO: dimethyl sulfoxide

[0925] EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide

[0926] ee: enantiomeric excess

[0927] Fmoc: fluorenyl-9-methoxycarbonyl

[0928] HATU: 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0929] HBTU: Benzotriazole tetramethyluronium hexafluorophosphate

[0930] HPLC: High Performance Liquid Chromatography

[0931] MTBE: Methyl tert-butyl ether

[0932] NMP: N-methylpyrrolidone

[0933] RP: Reverse Phase

[0934] rt: room temperature

[0935] T3P: Propane phosphoric anhydride

[0936] TFA: trifluoroacetic acid

[0937] THF: Tetrahydrofuran

[0938] TLC: Thin layer chromatography

[0939] The following examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0940] Analytical method (LC-MS)

[0941] Method 1 (LC-MS): Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8μ 50 x 1mm; Eluent A: 1L water + 0.25mL 99% formic acid, Eluent B: 1L acetonitrile + 0.25mL 99% formic acid; Gradient: 0.0min 90% A→1.2min 5% A→2.0min 5% A; Oven: 50°C; Flow rate: 0.40mL / min; UV detection: 208-400nm.

[0942] Method 2 (LC-MS): System MS: Thermo Scientific FT-MS; System UHPLC+: ThermoScientific UltiMate 3000; Column: Waters, HSST3, 2.1 x 75 mm, C18 1.8 μm; Eluent A: 1 l water + 0.01% formic acid; Eluent B: 1 l acetonitrile + 0.01% formic acid; Gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; Oven: 50°C; Flow rate: 0.90 ml / min; UV detection: 210 nm / optimal integration path 210-300 nm.

[0943] Method 3 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters, HSST3, 2.1 x 50 mm, C18 1.8 μm; Eluent A: 1 l water + 0.01% formic acid; Eluent B: 1 l acetonitrile + 0.01% formic acid; Gradient: 0.0 min 2% B → 0.5 min 2% B → 7.5 min 95% B → 10.0 min 95% B; Oven: 50°C; Flow rate: 1.00 ml / min; UV detection: 210 nm.

[0944] Method 4 (LC-MS): System MS: Thermo Scientific FT-MS; System UHPLC+: ThermoScientific Vanquish; Column: Waters, HSST3, 2.1 x 75 mm, C18 1.8 μm; Eluent A: 1 l water + 0.01% formic acid; Eluent B: 1 l acetonitrile + 0.01% formic acid; Gradient: 0.0 min 10% B → 2.5 min 95% B → 3.5 min 95% B; Oven: 50°C; Flow rate: 0.90 ml / min; UV detection: 210 nm.

[0945] Method 5 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 1 mm; Eluent A: 1 l water + 0.100 ml 99% formic acid, Eluent B: 1 l acetonitrile + 0.100 ml 99% formic acid; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; Oven: 50°C; Flow rate: 0.40 ml / min; UV detection: 210 nm.

[0946] Method 6 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 1 mm; Eluent A: 1 l water + 0.100 ml 99% formic acid, Eluent B: 1 l acetonitrile + 0.100 ml 99% formic acid; Gradient: 0.0 min 95% A → 6.0 min 5% A → 7.5 min 5% A; Oven: 50°C; Flow rate: 0.35 ml / min; UV detection: 210 nm.

[0947] Method 7 (LC-MS): Instrument: Waters Single Quad MS system; Instrument Waters UPLC Acquity; Column: Waters BEH C18 1.7μ50x2.1mm; Eluent A: 1L water + 1.0mL (25% ig ammonia) / L, Eluent B: 1L acetonitrile; Gradient: 0.0 minute 92% A→0.1 minute 92% A→1.8 minutes 5% A→3.5 minutes 5% A; Oven: 50°C; Flow rate: 0.45mL / min; UV detection: 210nm.

[0948] Method 8 (LC-MS): Instrument: Waters ACQUITY SQD UPLC system; Column: Waters Acquity UPLC HSS T3 1.8 μm 50 x 1 mm; Eluent A: 1 l water + 0.25 ml 99% formic acid, Eluent B: 1 l acetonitrile + 0.25 ml 99% formic acid; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; Oven: 50 ° C; Flow rate: 0.40 ml / min; UV detection: 210 nm.

[0949] Method 9 (LC-MS): System MS: Waters TOF instrument; System UPLC: Waters Acquity I-CLASS; Column: Waters Acquity UPLC HSS T3, 2.1 x 150 mm, 1.8 μm; Eluent A: 1 L water + 0.100 mL 99% trifluoroacetic acid, Eluent B: 1 L acetonitrile + 0.100 mL 99% trifluoroacetic acid; Gradient: 0.0 min 5% B → 1 min 5% B → 13 min 95% B → 15 min 95% B; Oven: 50°C; Flow rate: 0.60 mL / min; UV detection: 210 nm.

[0950] Synthesis example

[0951] Building Blocks – Intermediates

[0952] Example S1: Preparation of (3R)-3-{[(4-aminophenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid (Intermediate 1)

[0953]

[0954] The synthesis of intermediate 1 is described in WO2020 / 094471.

[0955] 1 H-NMR (500MHz, D4-methanol): δ = 0.93 (t, 3H), 1.5 (m, 2H), 2.74 (d, 2H), 3.1 (dt, 2H), 5.15 (t, 1H), 6.68 (d, 2H), 6.85(d, 1H), 7.05(d, 1H), 7.1(d, 1H), 7.13(t, 1H), 7.28-7.4(m, 3H), 7.6(s, 1H), 7.66(d, 1H).

[0956] Example S2: Preparation of (3R)-3-{[(4-{[(4-nitrophenoxy)carbonyl]amino}phenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid (Intermediate 2)

[0957]

[0958] The synthesis of intermediate 2 is described in WO2020 / 094471.

[0959] Example S3: Preparation of (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (Intermediate 3)

[0960]

[0961] Intermediate 3 was synthesized using a classic method for peptide synthesis: Z-Asp(OtBu)-OH was first coupled with L-proline benzyl ester hydrochloride (1:1) in THF in the presence of T3P and DIPEA, followed by removal of the Z-protecting group and the benzyl ester by hydrogenolysis over Pd / C to afford (2S)-1-[(2S)-2-amino-4-tert-butoxy-4-oxobutanoyl]pyrrolidine-2-carboxylic acid. This partially protected dipeptide was acylated with tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate to afford the title compound. Tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate was previously prepared by reacting 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid with N-hydroxysuccinimide in dioxane in the presence of EDCI. LC-MS: R t =0.81 min; MS (ESIpos): m / z=590 [M+H] + .

[0962] Example S4: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl L-valine ester·trifluoroacetic acid (1:1) (Intermediate 4)

[0963]

[0964] 2.59 g (10.6 mmol) of N-(tert-butoxycarbonyl)-valine-N-carboxylic anhydride and 0.5 g of 4-(N,N-dimethylamino)-pyridine were added to a stirred suspension of 2 g (5.3 mmol) of (4S)-4,11-diethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione in 150 ml of anhydrous dichloromethane. The mixture was stirred at room temperature for 20 hours and then concentrated in vacuo. 8 ml of ACN was added to the residue, followed by 5 mL of diethyl ether. The mixture was filtered and the remaining residue was dried in vacuo. 2964 mg (92% yield) of the protected intermediate were obtained. LC-MS: Rt = 1.19 minutes; MS (ESIpos): m / z = 576 (M+H) +Next, 2964 mg (5.15 mmol) of the Boc-protected intermediate compound in 6 ml of dichloromethane and 60 ml of anhydrous trifluoroacetic acid were stirred at room temperature for 30 minutes and then sonicated for 1 hour. After vacuum concentration, the product was freeze-dried from an acetonitrile / water mixture. 3.622 g (quantitative) of intermediate 4 were obtained. LC-MS: Rt = 0.68 min; MS (ESIpos): m / z = 476 [M+H] + .

[0965] Example S5: Preparation of (19S)-19-[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (Intermediate 5)

[0966]

[0967] (2S)-1-[(19S)-19-(2-tert-Butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (Intermediate 3) (50.0 g, 84.8 mmol) was dissolved in 900 ml of DMF and the solution was cooled to 0°C. 1.1 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (17.0 g, 88.8 mmol) and 1.3 equivalents of ethyl cyanohydroxyiminoacetate (14.9 g, 105 mmol) and trifluoroacetic acid-(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl L-valine ester (1 / 1) (Intermediate 4) (47.6 g, 80.8 mmol) were added, followed by the dropwise addition of 3.0 equivalents (42 ml, 240 mmol) of N,N-diisopropylethylamine. The mixture was stirred at 0°C overnight. It is diluted with 4L EtOAc and with organic layer 10% citric acid aqueous solution (2x 2l), with 10%NaHCO the aqueous solution (2x 2l) and with saturated NaCl aqueous solution (2x 3l) washing.Subsequently, it is through Mg SO dry, filter and concentrate.Resistates is dissolved among the 250ml DCM and uses flash chromatography (DCM:MeOH 100: 1) purifying.Collect related fractions and vacuum evaporation, to obtain intermediate 5 (72.8g, 98% purity, 84% productive rate) as yellow foam.

[0968] Example S6: Preparation of 4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester trifluoroacetic acid (1:1) (Intermediate 6)

[0969]

[0970] 12 g (11.5 mmol) of (19S)-19-[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutane-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-acid tert-butyl ester (intermediate 5) was dissolved in 100 ml of dichloromethane and 40 ml of anhydrous trifluoroacetic acid was added, and the solution was stirred at room temperature for 2 days. After vacuum concentration, 50 ml of toluene was added and evaporated again. The residue was dissolved in 50 ml of DCM / MeOH and poured into 600 ml of diethyl ether. The precipitated product (Intermediate 6) was filtered, washed with diethyl ether and dried in vacuo (11.7 g, 95% purity, 97% yield).

[0971] Example S7: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-α-aspartyl-L-prolyl-L-valine ester (Intermediate 7)

[0972]

[0973] 1 g (995 μmol) of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester trifluoroacetic acid (1:1) (intermediate 6) was dissolved in 11 ml of DMF and 1-[(tert-butoxycarbonyl)oxy]pyrrolidine-2,5-dione (214 mg, 995 μmol) and 1 equivalent of N,N-diisopropylethylamine (170 μl, 1000 μmol) were added. The batch was stirred at room temperature for 20 hours. A further 1 / 2 equivalent of BOC-OSu and N,N-diisopropylethylamine was added and the mixture was further stirred at room temperature for 3 hours. The batch was then evaporated to dryness using a rotary evaporator. The residue was separated by HPLC. The relevant fractions were collected and evaporated in vacuo to give 744 mg (100% purity, 75% yield) of intermediate 7 as a yellow foam. LC-MS: R t=4.19 min; MS (ESIpos): m / z=991[M+H] + .

[0974] Example S8: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propionyl]-L-α-aspartyl-L-prolyl-L-valine ester·trifluoroacetic acid (1:1) (Intermediate 8)

[0975]

[0976] Step 1: (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-α-aspartyl-L-prolyl-L-valine ester (Intermediate 7) (500 mg, 498 μmol) was dissolved in 50 mL of DMF and 1.2 equivalents of 2,5-dioxopyrrolidin-1-yl N-(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-α-aspartyl-L-prolyl-L-valine ester was added. 2 ,N 6 -bis(tert-butoxycarbonyl)-L-lysine ester (265 mg, 597 μmol) and 260 μl N,N-diisopropylethylamine. After stirring at room temperature for 2 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC. The relevant fractions were collected and concentrated in vacuo to give Boc-intermediate 8 (506 mg, 98% purity, 81% yield) as a light yellow foam. LC-MS: R t =1.93 min; MS (ESIpos): m / z=1218 [M+H] + .

[0977] Step 2: 506 mg (415 μmol) of Boc-intermediate 8 was dissolved in 10 ml of DCM and 3 ml of TFA was added. The reaction mixture was stirred at room temperature for 30 minutes. It was concentrated in vacuo and the residue was dissolved in ACN / H2O and freeze-dried to give intermediate 8 (700 mg, (100% purity, quantitative) as a light yellow foam. LC-MS: R t =1.77 min; MS (ESIpos): m / z=510 [M+2H] ++ .

[0978] Example S9 (Intermediate 9): (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-{[N 2 ,N 6 Preparation of -bis(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester·trifluoroacetic acid (1 / 2) (Intermediate 9)

[0979]

[0980] Step 1: (4S)-4,11-Diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propionyl]-L-α-aspartyl-L-prolyl-L-valine ester (Intermediate 8) (70 mg, 62 μmol) was dissolved in 8 mL DMF and 2.2 equivalents of tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (56.9 mg, 136 μmol) and 65 μl N,N-diisopropylethylamine were added. After stirring at room temperature for 4 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC.The relevant fractions were collected and concentrated in vacuo to give (Boc-Intermediate 9) (60 mg, 87% purity, 52% yield) as a light yellow foam.

[0981] Step 2: 60 mg (37 μmol) of Boc-intermediate 9 was dissolved in 10 ml of DCM and 2 ml of TFA was added. The reaction mixture was stirred at room temperature for 30 minutes. It was concentrated in vacuo, and the residue was dissolved in ACN / H2O and freeze-dried to give intermediate 9 as a colorless foam (60 mg, 100% purity, 98% yield). LC-MS: R t =1.86 min; MS (ESIpos): m / z=1425 [M+H] + .

[0982] Example S10: Preparation of tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (Intermediate 10)

[0983]

[0984] To a suspension of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-acid (3.00 g, 9.33 mmol) in DCM (50 ml) was added 1-hydroxypyrrolidine-2,5-dione (1.61 g, 14.0 mmol), EDCI (2.15 g, 11.2 mmol) and DMAP (5.00 mg, 40.9 μmol). The reaction was stirred at room temperature for 1 hour and concentrated in vacuo. The residue was purified by preparative HPLC to obtain intermediate 10 (3.15 g, 90% purity, 72% yield) as a colorless oil. LC-MS (method 2): R t =1.38 min; MS (ESIpos): m / z=419 [M+H] + .

[0985] Example S11: (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-{18-[(2,5-dioxopyrrolidin-1-yl)oxy]-14,18-dioxo-4,7,10-trioxa-13-azaoctadecane-1-yl}-L-α-aspartyl-L-prolyl-L-valine ester (Intermediate 11)

[0986]

[0987] To a solution of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester.trifluoroacetic acid (1 / 1) (123 mg, 123 μmol) (Intermediate 6) in DMF (10 ml) was added 1,1'-[(1,5-dioxopentan-1,5-diyl)bis(oxy)]bis(pyrrolidine-2,5-dione) (120 mg, 368 μmol) and DIEA (64 μl, 370 μmol). The mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was purified by preparative HPLC to give intermediate 11 as a colorless foam (82.0 mg, 98% purity, 59% yield). LC-MS (Method 3): R t = 3.65 min; MS (ESIpos): m / z = 1102 [M+H] + .

[0988] Example S12: Bis(2,5-dioxopyrrolidin-1-yl)N 2 ,N 6 - Preparation of bis(tert-butoxycarbonyl)-L-lysyl-L-glutamate (Intermediate 12)

[0989]

[0990] To N 2 ,N 6 To a solution of 1-bis(tert-butoxycarbonyl)-L-lysyl-L-glutamate (50.0 mg, 82% purity, 86.3 μmol, CAS Nr = 1975207-44-3) in DMF (10 ml) was added 1-hydroxypyrrolidine-2,5-dione (149 mg, 1.29 mmol), HATU (164 mg, 432 μmol), and DIEA (75 μl, 430 μmol). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by preparative HPLC and then freeze-dried to give Intermediate 12 (20.5 mg, 90% purity, 32% yield) as an amorphous residue. LC-MS (Method 4): Rt = 1.79 min; MS (ESIpos): m / z = 670 [M+H]+.

[0991] Example S13: (3S,19S,37S)-19-{[(2S)-2,6-diaminohexanoyl]amino}-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1, Preparation of [2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-diacid-trifluoroacetic acid (1 / 2) (Intermediate 13)

[0992]

[0993] Step 1: To a solution of bis(2,5-dioxopyrrolidin-1-yl) N2,N6-bis(tert-butoxycarbonyl)-L-lysyl-L-glutamate (20.0 mg, 90% purity, 27.0 μmol) (Intermediate 12) in DMF (10 ml) was added (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester-trifluoroacetic acid (1 / 1) (57.0 mg, 56.7 μmol) (Intermediate 6) and DIEA (19 μl, 110 μmol). The mixture was stirred at room temperature for 5 hours. Then (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester-trifluoroacetic acid (1 / 1) (41.0 mg) (Intermediate 6) was added, and the mixture was stirred at room temperature for 72 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and then freeze-dried to give (3S,19S,37S)-19-({(2S)-2,6-bis[(tert-butoxycarbonyl)amino]hexanoyl}amino)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H- [pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (51 mg, 93% purity, 79% yield). LC-MS (Method 3): Rt = 4.91 min; MS (ESIpos): m / z = 1111 [M+2H] 2+ .

[0994] Step 2: To ((3S,19S,37S)-19-({(2S)-2,6-bis[(tert-butoxycarbonyl)amino]hexanoyl}amino)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26, To a solution of 29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (51.0 mg, 93% purity, 21.3 μmol) in DCM (10 ml) was added TFA (2.0 ml). The mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 13 (50 mg, 92% purity, 96% yield) as a yellow amorphous residue. LC-MS (Method 3): Rt = 3.28 min; MS (ESIpos): m / z = 1011 [M+2H] 2+ .

[0995] Example S14: (3S,19S,37S)-19-{[(2S)-19-amino-2-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionamido)-8-oxo-11,14,17-trioxa-7-azanonadecan-1-yl]amino}-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12, Preparation of 14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-diacid-trifluoroacetic acid (1 / 2) (Intermediate 14)

[0996]

[0997] Step 1: To (3S,19S,37S)-19-{[(2S)-2,6-diaminohexanoyl]amino}-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26, To a solution of 29,32-hexaoxa-4,17,23,36-tetraazanonatricarboxane-1,39-dioic acid-trifluoroacetic acid (1 / 2) (50.0 mg, 92% purity, 20.5 μmol) (Intermediate 13) in DMF (10 ml) were added tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (Intermediate 10) (22.9 mg, 90% purity, 49.1 μmol) and DIEA (18 μl, 100 μmol). The mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was purified by preparative HPLC and then freeze-dried to give (3S,19S,37S)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-19-({(23 ... -2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)amino]-2,2-dimethyl-4,17,24-trioxo-3,8,11,14-tetraoxa-5,18-diazatetracosan-24-yl}amino)-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (36 mg, 100% purity, 67% yield). LC-MS (Method 3): Rt = 4.84 min; MS (ESIpos): m / z = 1315 [M+2H] 2+ .

[0998] Step 2: To (3S,19S,37S)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-19-({(23S)-23-[(2,2-dimethyl-4,17-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl] To a solution of [3-(4-(2-(2-(2-(2-(2-(2-(2-(2-(2-dimethyl-4,17,24-trioxo-3,8,11,14-tetraoxa-5,18-diazatetracosan-24-yl)amino]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid) (36.0 mg, 13.7 μmol) in DCM (10 ml) was added TFA (1.5 ml). The mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was dissolved in ACN / HO and freeze-dried to give Intermediate 14 (36 mg, 100% purity, 99% yield) as a yellow amorphous residue. LC-MS (Method 3): Rt=3.24 min; MS (ESIpos): m / z=810 [M+3H] 3+ .

[0999] Example S15: Preparation of tert-butyl [2-(2-{2-[2-(L-lysinylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamate (Intermediate 15)

[1000]

[1001] Step 1: To N 2 ,N 6To a solution of bis[(benzyloxy)carbonyl]-L-lysine (50.0 mg, 121 μmol) in DMF (10 ml) was added tert-butyl (2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate (38.8 mg, 133 μmol), HATU (59.6 mg, 157 μmol), and N,N-diisopropylethylamine (84 μl, 480 μmol). The mixture was stirred at room temperature for 10 minutes and then concentrated under reduced pressure. The residue was purified by preparative HPLC to give benzyl [(19S)-19-{[(benzyloxy)carbonyl]amino}-2,2-dimethyl-4,18-dioxo-3,8,11,14-tetraoxa-5,17-diazatricosan-23-yl]carbamate (73 mg, 100% purity, 88% yield) as a resinous residue. LC-MS (Method 4): Rt = 2.04 min; MS (ESIpos): m / z = 689 [M+H] + .

[1002] Step 2: Benzyl [(19S)-19-{[(benzyloxy)carbonyl]amino}-2,2-dimethyl-4,18-dioxo-3,8,11,14-tetraoxa-5,17-diazatricosan-23-yl]carbamate (73.0 mg, 106 μmol) was dissolved in methanol (20 ml) and dichloromethane (5 ml). Pd / C 10% (10.0 mg) was added, and the reaction was hydrogenated at room temperature for 1 hour and filtered. The mother liquor was concentrated under reduced pressure and dried under high vacuum to give intermediate 15 as an amorphous residue (40 mg, 100% purity, 90% yield). LC-MS (Method 2): Rt = 0.48 min; MS (ESIpos): m / z = 421 [M+H] + .

[1003] Example S16: (3S,24S,49S)-24-[(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]-3,49-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[ Preparation of [1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,30,34,47-hexaoxo-8,11,14,38,41,44-hexaoxa-4,17,23,29,35,48-hexaazapentadecane-1,51-dioic acid-trifluoroacetic acid (1 / 1) (Intermediate 16)

[1004]

[1005] Step 1: To a solution of tert-butyl [2-(2-{2-[2-(L-lysinylamino)ethoxy]ethoxy}ethoxy)ethyl]carbamate (7.30 mg, 17.4 μmol) (Intermediate 15) in DMF (10 ml) was added (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizine. [1,2-b]quinolin-4-yl N-{18-[(2,5-dioxopyrrolidin-1-yl)oxy]-14,18-dioxo-4,7,10-trioxa-13-azaoctadec-1-yl}-L-α-aspartyl-L-prolyl-L-valine ester (41.0 mg, 98% purity, 36.5 μmol) (Intermediate 11) and DIEA (12 μl, 69 μmol). The mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was purified by preparative HPLC and then freeze-dried to give (3S,24S,49S)-3,49-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]- as an amorphous residue.

[00145] carbamoyl}pyrrolidine-1-carbonyl]-24-[(2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadec-16-yl)carbamoyl]-5,18,22,30,34,47-hexaoxo-8,11,14,38,41,44-hexaoxa-4,17,23,29,35,48-hexaazahexadecane-1,51-dioic acid (27 mg, 98% purity, 63% yield). LC-MS (Method 3): Rt = 4.46 min; MS (ESIpos): m / z = 1198 [M+2H] 2+ .

[1006] Step 2: To (3S,24S,49S)-3,49-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-24-[(2,2-dimethyl- To a solution of [4-oxo-3,8,11,14-tetraoxa-5-azahexadec-16-yl)carbamoyl]-5,18,22,30,34,47-hexaoxo-8,11,14,38,41,44-hexaoxa-4,17,23,29,35,48-hexaazapentadecane-1,51-dioic acid (27.0 mg, 98% purity, 11.0 μmol) in DCM (10 ml) was added TFA (1.0 ml). The mixture was stirred at room temperature for 30 minutes and then concentrated under reduced pressure. The residue was dissolved in ACN / HO and freeze-dried to give Intermediate 16 (28 mg, 92% purity, 98% yield) as a yellow amorphous residue. LC-MS (Method 3): Rt=3.59 min; MS (ESIpos): m / z=1147 [M+2H] 2+ .

[1007] Example S17: Preparation of (3S,19S,37S)-19-amino-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid-trifluoroacetic acid (1 / 1) (Intermediate 17)

[1008]

[1009] Step 1: To a solution of bis(2,5-dioxopyrrolidin-1-yl) N-(tert-butoxycarbonyl)-L-glutamate (6.59 mg, 14.9 μmol, CAS Nr = 246234-73-1) in DMF (1.5 ml) was added (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester-trifluoroacetic acid (1 / 1) (30.0 mg, 29.9 μmol) (Intermediate 6) and DIEA (16 μl, 90 μmol). The mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The residue was purified by preparative HPLC, then freeze-dried to obtain (3S, 19S, 37S) -19- [(tert-butoxycarbonyl) amino] -3, 37- bis [(2S) -2- { [(2S) -1- { [(4S) -4, 11- diethyl -3, 14- dioxo -3, 4, 12, 14- tetrahydro -1H- pyrans and [3 ', 4 ': 6, [7] indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (16.5 mg, 100% purity, 55% yield). LC-MS (Method 3): Rt = 4.53 min; MS (ESIpos): m / z = 997 [M+2H] 2+ .

[1010] Step 2: To (3S,19S,37S)-19-[(tert-butoxycarbonyl)amino]-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1- To a solution of [1,2-dioxobutan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (16.0 mg, 100% purity, 8.03 μmol) in DCM (1.5 ml) was added TFA (0.75 ml). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give Intermediate 17 (16.1 mg, 100% purity, 100% yield) as an amorphous residue. LC-MS (Method 3): Rt = 3.71 min; MS (ESIpos): m / z = 1893 [M+H] + .

[1011] Example S18: (3S,19S,37S)-19-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionamido)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indole Preparation of trifluoroacetic acid (1 / 1) (Intermediate 18)

[1012]

[1013] Step 1: To (3S,19S,37S)-19-amino-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexahydro- To a solution of hetero-4,17,23,36-tetraazanonatricarboxane-1,39-dioic acid-trifluoroacetic acid (1 / 1) (16.0 mg, 100% purity, 7.97 μmol) (Intermediate 17) in DMF (1.6 ml) were added tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (Intermediate 10) (4.00 mg, 9.57 μmol) and DIEA (5.6 μl, 32 μmol). The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and then dried under high vacuum to give (3S,19S,37S)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutane-2-

[00145] 1,39-dioic acid (12.8 mg, 100% purity, 73% yield) was added to the mixture of 1,3-dioxo-5,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)amino]-1,39-dioic acid (12.8 mg, 100% purity, 73% yield). LC-MS (Method 3): Rt = 4.59 min; MS (ESIpos): m / z = 1098 [M+2H] 2+ .

[1014] Step 2: To (3S,19S,37S)-3,37-bis[(2S)-2-{[(2S)-1-{[(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-19-[ To a solution of [2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)amino]-5,18,22,35-tetraoxo-8,11,14,26,29,32-hexaoxa-4,17,23,36-tetraazanonatriacontane-1,39-dioic acid (12.6 mg, 5.74 μmol) in DCM (2 ml) was added TFA (0.5 ml). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give Intermediate 18 (12.1 mg, 100% purity, 95% yield) as an amorphous residue. LC-MS (Method 3): Rt = 3.65 min; MS (ESIpos): m / z = 1048 [M+2H] 2+ .

[1015] Example S19: Preparation of pentafluorophenyl 4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino]butyrate (Intermediate 19)

[1016]

[1017] To a solution of 4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino]butanoic acid (100 mg, 357 μmol, CAS Nr: 78851-85-1) in pyridine (100 ml) was added pentafluorophenol (131 mg, 714 μmol) and EDCI (137 mg, 714 μmol; CAS Nr: 25952-53-8). The mixture was stirred at room temperature for 20 hours and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate and water and the two layers were separated. The organic phase was washed with water, dried over magnesium sulfate and concentrated under reduced pressure to give intermediate 19 (36 mg, 23% yield), which was used in the next step without further purification.

[1018] Example S20: Preparation of 3-[[6-chloro-4-(methylsulfanylmethyl)-2-pyridinyl]oxy]propan-1-ol (Intermediate 20)

[1019]

[1020] To a solution of propane-1,3-diol (10.97 g, 144.16 mmol, 10.45 mL, 2.5 equiv) in THF (144 mL) was added NaH (3.00 g, 74.96 mmol, 60% purity, 1.3 equiv) at 0 ° C under N2. The mixture was stirred at 20 ° C for 30 minutes, and then 2,6-dichloro-4-(methylsulfanylmethyl)pyridine (12 g, 57.66 mmol, 1 equiv) was added to the reaction mixture at 20 ° C under N2. The mixture was stirred at 70 ° C for 15.5 hours. The reaction mixture was poured into NH4Cl (100 mL). The aqueous phase was extracted with ethyl acetate (3x200 mL). The combined organic phase was washed with brine (2x200 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 26:1 to 10:1) to afford intermediate 20 (11 g, 44.40 mmol, 77% yield) as a colorless oil. 1 H NMR (400mHz, CDCl3) δ6.90 (s, 1H), 6.59 (d, J=1.0Hz, 1H), 4.48 (t, J=6.0Hz, 2H), 3.75 (t, J=5.8Hz, 2H), 3.54 (s, 2H), 2.45 (br s, 1H), 2.03-1.95 (m, 5H).

[1021] Example S21: Preparation of 4-[2-[3-[[6-chloro-4-(methylsulfanylmethyl)-2-pyridinyl]oxy]propoxy]-4-fluoro-phenyl]-5-fluoro-pyridin-2-amine (Intermediate 21)

[1022]

[1023] To a mixture of 3-[[6-chloro-4-(methylsulfanylmethyl)-2-pyridinyl]oxy]propan-1-ol (10.43 g, 42.08 mmol, 1.1 eq) and 2-(2-amino-5-fluoro-4-pyridinyl)-5-fluoro-phenol (8.5 g, 38.26 mmol, 1 eq) in toluene (100 mL) was added CMBP (27.70 g, 114.77 mmol, 3 eq) under N at 20° C. The mixture was stirred at 110° C. for 16 hours. The reaction mixture was poured into water (40 mL). The aqueous phase was extracted with ethyl acetate (3×50 mL). The combined organic phases were washed with brine (2×50 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 20:1 to 8:1) to afford intermediate 21 (16.7 g, 25.87 mmol, 68% yield, 70% purity) as a brown oil. 1H NMR (400mHz, CDCl3-d) δ7.94 (d, J=2.0Hz, 1H), 7.21 (dd, J=6.6, 8.8Hz, 1H), 6.88 (s, 1H), 6.77-6.64 (m, 2H), 6.55 (s, 1H), 6 .52-6.43 (m, 1H), 4.46 (s, 2H), 4.42-4.30 (m, 2H), 4.16-4.06 (m, 2H), 3.52 (s, 2H), 2.16 (quin, J=6.0Hz, 2H), 1.98 (s, 3H).

[1024] Example S22: Preparation of 5,22-difluoro-15-(methylsulfanylmethyl)-8,12-dioxa-18,20,24-triazatetracyclo[17.3.1.1^{13,17}.0^{2,7}]tetracosa-1(22),2,4,6,13,15,17(24),19(23),20-nonene (Intermediate 22)

[1025]

[1026] To a mixture of intermediate 21 (10 g, 15.49 mmol, 70% purity, 1 equivalent) in toluene (100 mL) and NMP (20 mL) was added K PO (16.44 g, 77.45 mmol, 5 equivalents), XPhos (738.41 mg, 1.55 mmol, 0.1 equivalents) and Xphos Pd G1 (572.16 mg, 774.48 umol, 0.05 equivalents) at 20° C. under N . The mixture was stirred at 110° C. for 3 hours. The reaction mixture was poured into water (50 mL). The aqueous phase was extracted with ethyl acetate (3x80 mL). The combined organic phases were washed with brine (2x100 mL), dried over anhydrous Na SO , filtered and concentrated in vacuo. The crude product was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 20:1 to 5:1) to afford intermediate 22 (5 g, 12.03 mmol, 77.70% yield) as a light yellow solid. 1 H NMR (400MHz, CDCl3) δ8.80 (d, J=6.0Hz, 1H), 8.17 (d, J=2.8Hz, 1H), 7.61 (ddd, J=3.8, 6.6, 8.4Hz, 1H), 7.26 (br s, 1H), 6.89-6.56 (m, 2H), 6.22 (s, 2H), 4.68-4.55 (m, 2H), 4.12-3.99 (m, 2H), 3.53 (s, 2H), 2.32-2.18 (m, 2H), 2.05 (s, 3H).

[1027] Example S23: (5,22-difluoro-8,12-dioxa-18,20,24-triazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(22),2,4,6,13,15,17(24),19(23),20-nonen-15-yl)methyl-imino-methyl-oxo-λ 6 Preparation of -sulfane (Intermediate 23, racemic mixture and Intermediates 23-a and 23-b, single enantiomers)

[1028]

[1029] To the mixture of intermediate 22 (400 mg, 962.80 umol, 1 equivalent) in DCM (10 mL) was added ammonia at 20 ° C: carbamic acid (751.66 mg, 9.63 mmol, 10 equivalents) and PhI (OAc) (775.28 mg, 2.41 mmol, 2.5 equivalents) under N2. The mixture was stirred at 20 ° C for 16 hours. 12 reactions were carried out in parallel. The reaction mixture was poured into water (50 mL). The aqueous phase was extracted with DCM (3x80 mL). The combined organic phases were washed with brine (2x80 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The reaction mixture was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1:1 to ethyl acetate: MeOH=50:1) to obtain intermediate 23 (2.1 g, 4.47 mmol, 38.6% yield, 95% purity) as a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ9.71 (s, 1H), 8.69 (s, 1H), 8.31 (d, J=2.4Hz, 1H), 7.57 (br s, 1H), 7.08 (dd, J=2.2, 11.4Hz, 1H), 6.90 (dt, J=2.4, 8.4Hz, 1H), 6.58 (s, 1H), 6.26 (s, 1H), 4.61–4.41(m, 2H), 4.34–4.22(m, 2H), 4.19–4.05(m, 2H), 3.73(s, 1H), 2.87(s, 3H), 2.10(br d, J=6.0Hz, 2H).

[1030]

[1031] Intermediate 23 was purified by preparative SPC (column: DAICEL CHIRALCEL OJ (250 mm*50 mm, 10 μm); mobile phase: [0.1% NH 3 H 2 O ETOH]; B%: 60%-60%, 14 min, R t 1=1.67,R t2=2.183) were isolated to give intermediate 23-a (750 mg, 1.52 mmol, 35.4% yield, 99.18% purity) as an off-white solid and intermediate 23-b (810 mg, 1.62 mmol, 37.7% yield, 97.77% purity) as a light yellow solid. Note: The R / S configuration of these two compounds was not confirmed.

[1032] Intermediate 23-a: 1 H NMR (400MHz, DMSO-d6) δ9.71 (s, 1H), 8.69 (d, J = 6.0Hz, 1H), 8.31 (d, J = 2.4Hz, 1H), 7.57 (dt, J = 2.8, 4.2Hz, 1H), 7.07 (dd, J = 2.2, 11.4Hz, 1H), 6.89 (dt, J=2.4, 8.4Hz, 1H), 6.58 (s, 1H), 6.26 (s, 1H), 4.59-4.42 (m, 2H), 4.2 8(d, J=2.2Hz, 2H), 4.19-4.05(m, 2H), 3.74(s, 1H), 2.88(s, 3H), 2.09(br d, J=6.2Hz, 2H). In R t The desired enantiomer (optical rotation -4.88) was obtained in 100% ee at 1.63-1.83 min. ° ±0.00 ° , 20C, 589nm). LC-MS: R t = 2.38 min; MS (ESIpos): m / z = 447 [M+H] + Single (-) isomer, absolute stereochemistry unknown.

[1033] Intermediate 23-b: 1 H NMR (400MHz, DMSO-d6) δ9.70 (s, 1H), 8.68 (br d, J=5.8Hz, 1H), 8.34-8.27 (m, 1H), 7.62-7.52 (m, 1H), 7.07 (br d, J=9.8Hz, 1H), 6.94-6.84 (m, 1H), 6.58 (s, 1H), 6.26 (s, 1H), 4.59-4.41 (m, 2H), 4.38-4.19 (m, 2H), 4.11 (br s, 2H), 3.74 (s, 1H), 2.88 (s, 3H), 2.18-2.01 (m, 2H). in R t The desired enantiomer (optical rotation 3.86) was obtained in 100% ee at 2.05-2.42 min. ° ±0.00 °, 20C, 589nm). LC-MS: R t = 2.38 min; MS (ESIpos): m / z = 447 [M+H] + Single (+) isomer, absolute stereochemistry unknown.

[1034] Example S24: N-[(1S)-1-[[(5,22-difluoro-8,12-dioxa-18,20-diazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl-methyl-oxo-λ 6 Preparation of tert-butyl]-sulfinyl]carbamoyl]-2-methyl-propyl]carbamate (Intermediate 24)

[1035]

[1036] (5,22-difluoro-8,12-dioxa-18,20,24-triazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(22),2,4,6,13,15,17(24),19(23),20-nonen-15-yl)methyl-imino-methyl-oxo-λ 6 -sulfane (300 mg, 671 μmol) (intermediate 23-b) was dissolved in DMF (30 mL). N-(tert-butoxycarbonyl)-L-valine (204 mg, 940 μmol), HATU (510 mg, 1.34 mmol) and DIEA (468 μl, 2.69 mmol) were added. The reaction was stirred at room temperature for 16 hours and then concentrated in vacuo. The residue was dissolved in DCM, washed with citric acid solution (5% in water) and concentrated. Water was then added, the resulting precipitate was filtered, washed with water and dried under high vacuum to give intermediate 24 (400 mg, 75.4% purity, 69.5% yield). LC-MS (Method 2): R t =2.42 min; MS (ESIpos): m / z=646[M+H] + .

[1037] Example S25: Trifluoroacetic acid. (2S)-2-amino-N-[(5,22-difluoro-8,12-dioxa-18,20-diazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl-methyl-oxo-λ 6 Preparation of [-sulfinyl]-3-methyl-butyramide (1 / 1) (Intermediate 25)

[1038]

[1039] N-[(1S)-1-[[(5,22-difluoro-8,12-dioxa-18,20-diazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl ... 6 [-sulfinyl]carbamoyl]-2-methyl-propyl] tert-butyl carbamate (400 mg, 75.4% purity, 467 μmol) (Intermediate 24) was dissolved in DCM (30 mL) and TFA (10 ml) was added. The reaction was stirred at room temperature for 30 minutes and then concentrated in vacuo. The residue was dissolved in ACN / water and freeze-dried. The residue was purified by preparative HPLC and freeze-dried to give Intermediate 25 (286 mg, 100% purity, 92.8% yield) as an amorphous residue. LC-MS (Method 2): R t =1.41 min; MS (ESIpos): m / z=546[M+H] + .

[1040] Example S26: (3S)-3-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propanoylamino]-4-[(2S)-2-[[(1S)-1-[[(5,22-difluoro-8,12-dioxa-18,20,24-triazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl-methyl-oxo-λ 6 Preparation of tert-butyl 4-oxo-butyrate (Intermediate 26)

[1041]

[1042] (2S)-2-amino-N-[(5,22-difluoro-8,12-dioxa-18,20-diazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl]-1-[ ... 6-sulfinyl]-3-methyl-butyramide (1 / 1) (266 mg, 403 μmol) (Intermediate 25) was dissolved in DMF (80 ml). (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosane-20-yl]pyrrolidine-2-carboxylic acid (Intermediate 3) (262 mg, 443 μmol), HATU (245 mg, 645 μmol) and DIEA (211 μl, 1.21 mmol) were added. The reaction was stirred at room temperature for 1 hour and 30 minutes and then concentrated in vacuo. The residue was separated by preparative HPLC to give Intermediate 26 (326 mg, 100% purity, 72.4% yield). LC-MS (Method 2): R t = 2.33 min; MS (ESIpos): m / z = 1117 [M+H] + .

[1043] Example S27: N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-[(R*)-{[15,19-difluoro-3,4-dihydro-2H,11H-10,6-(azeninyl)-12,16-(methylene)-1,5,11,13-benzodioxadiazacyclooctadecen-8-yl]methyl}(methyl)oxo-λ 6 Preparation of [-sulfinyl]-L-valinamide.trifluoroacetic acid (1 / 1) (Intermediate 27)

[1044]

[1045] (3S)-3-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propanoylamino]-4-[(2S)-2-[[(1S)-1-[[(5,22-difluoro-8,12-dioxa-18,20,24-triazatetracyclo[17.3.1.113,17.02,7]tetracosa-1(23),2(7),3,5,13(24),14,16,19,21-nonen-15-yl)methyl-1-[[(3S)-3-[3-[2-[2-[2-(tert-butoxycarbonylamino) ... 6[-sulfinyl]carbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidin-1-yl]-4-oxo-butyric acid tert-butyl ester (326 mg, 292 μmol) (Intermediate 26) was dissolved in DCM (80 ml) and then TFA (20 ml) was added. The reaction was stirred at room temperature for 3 hours and concentrated in vacuo. The residue was dissolved in ACN / water and freeze-dried to give Intermediate 27 as an amorphous residue (374 mg, 100% purity, quantitative). LC-MS (Method 5): R t =0.78 min; MS (ESIpos): m / z=960 [M+H] + .

[1046] Example S28: Preparation of benzyl [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]carbamate (Intermediate 28)

[1047]

[1048] Methanesulfonic acid-(1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1 / 1) (exitecan mesylate) (50.0 mg, 94.1 μmol) was dissolved in DMF (10.0 mL) and 1-{[(benzyloxy)carbonyl]oxy}pyrrolidine-2,5-dione (28.1 mg, 113 μmol) and DIEA (49 μl, 280 μmol) were added. The reaction was stirred at room temperature overnight. The reaction was concentrated in vacuo and the residue was mixed with ACN / H2O / DMF. A solid precipitated, which was filtered off, dried and purified by preparative HPLC to give intermediate 28 as a white foam (50.0 mg, 92% purity, 87% yield). LC-MS (Method 6): R t =3.21 min; MS (ESIpos): m / z=570 [M+H] + .

[1049] Example S29: Preparation of (1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl N-(tert-butoxycarbonyl)-L-valine ester (Intermediate 29)

[1050]

[1051] Benzyl [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]carbamate (Intermediate 28) (50.0 mg, 87.8 μmol) was added to DCM (20.0 mL) followed by tert-butyl (4S)-2,5-dioxo-4-(propan-2-yl)-1,3-oxazolidine-3-carbonate (51.2 mg, 211 μmol) and DMAP (19.3 mg, 158 μmol; CAS: 1122-58-3). The mixture was stirred at reflux for 7 hours. The reaction was concentrated in vacuo. Water was added and the product precipitated. The mixture was filtered and the filter residue was very poorly soluble but soluble in DMSO+ACN or MeOH / DCM. The compound was purified by preparative HPLC to give intermediate 29 (54.0 mg, 98% purity, 78% yield). LC-MS (Method 2): R t = 2.48 min; MS (ESIpos): m / z = 769 [M+H] + .

[1052] Example S30: Preparation of (1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl L-valine trifluoroacetate (1 / 1) (Intermediate 30)

[1053]

[1054] (1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl N-(tert-butoxycarbonyl)-L-valine ester (Intermediate 29) (44.0 mg, 98% purity, 55.9 μmol) was dissolved in DCM (6.3 mL), TFA (1.3 mL) was added and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated in vacuo and the residue was dissolved in ACN / H2O and freeze-dried to give Intermediate 30 (44.0 mg, 98% purity, 98% yield). LC-MS (Method 6): R t = 2.56 min; MS (ESIpos): m / z = 669 [M+H] + .

[1055] Example S31: Preparation of (19S)-19-[(2S)-2-{[(2S)-1-{[(1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (Intermediate 31)

[1056]

[1057] To (1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-9-yl L-valine trifluoroacetate (1 / 1) (44.0 mg, 98% purity, 54.9 μmol) (Intermediate 30) and (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3 To a solution of [5-[[(4-[ ... t =4.28 min; MS (ESIpos): m / z=1239[M+H] + .

[1058] Example S32: (19S)-19-[(2S)-2-{[(2S)-1-{[(1S,9S)-1-amino-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (Intermediate 32)

[1059]

[1060] (19S)-19-[(2S)-2-{[(2S)-1-{[(1S,9S)-1-{[(benzyloxy)carbonyl]amino}-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl]oxy}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (350 mg, 282 μmol) (Intermediate 31) was dissolved in ethanol (50 ml). Catalyst Pd / C 10% (50.0 mg) was added. The reaction was hydrogenated at standard pressure for 2 hours. The catalyst was filtered off and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 32 as a colorless foam (287 mg, 86% purity, 79% yield). LC-MS (Method 3): R t =3.59 min; MS (ESIpos): m / z=1107 [M+H] + .

[1061] Example S33: (1S,9S)-1-Amino-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester trifluoroacetic acid (1 / 1) (Intermediate 33)

[1062]

[1063] (19S)-19-[(2S)-2-{[(2S)-1-{[(1S,9S)-1-amino-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl]oxy}-3-methyl To the product was added tert-butyl 2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-ol (287 mg, 86% purity, 223 μmol) (Intermediate 32) which was dissolved in DCM (30 ml) and then TFA (5.0 ml) was added. The reaction was stirred at room temperature for 1 hour and then concentrated in vacuo. The residue was purified by preparative HPLC to give Intermediate 33 (216 mg, 95% purity, 87% yield) as a yellow foam. LC-MS (Method 3): R t = 2.04 min; MS (ESIpos): m / z = 950 [M+H] + .

[1064] Example S34: Preparation of trifluoroacetic acid, N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-1-phenyl-1-(L-valyloxy)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (1 / 1) (Intermediate 34)

[1065]

[1066] Step 1: To a solution of monomethyl auristatin E (N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide, 200 mg, 279 μmol) in DMF (28 ml) was added 1-{[(benzyloxy)carbonyl]oxy}pyrrolidine-2,5-dione (83.3 mg, 334 μmol) and DIEA (150 μl, 840 μmol). The mixture was stirred at room temperature for 20 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (224 mg, 100% purity, 94% yield). LC-MS (Method 2): R t = 2.29 min; MS (ESIpos): m / z = 853 [M+H] + .

[1067] Step 2: To a solution of N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (222 mg, 100% purity, 260 μmol) in DCM (40 ml) was added tert-butyl (4S)-4-methyl-2,5-dioxo-1,3-oxazolidine-3-carboxylate (190 mg, 781 μmol) and DMAP (63.6 mg, 521 μmol). The mixture was refluxed for 16 h and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-{[N-(tert-butoxycarbonyl)-L-valyl]oxy}-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (267 mg, 100% purity, 98% yield). LC-MS (Method 3): R t = 6.35 min; MS (ESIpos): m / z = 1052 [M+H] + .

[1068] Step 3: To a solution of N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S,2R)-1-{[N-(tert-butoxycarbonyl)-L-valyl]oxy}-1-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (323 mg, 100% purity, 307 μmol) in DCM (50 ml) was added TFA (5.0 ml). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 34 (339 mg, 100% purity, quantitative) as an amorphous residue. LC-MS (Method 2): R t =1.80 min; MS (ESIpos): m / z=952[M+H] + .

[1069] Example S35: Preparation of (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester. Trifluoroacetic acid (1 / 1) (Intermediate 35)

[1070]

[1071] Step 1: To trifluoroacetic acid, N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S,2R)-1-phenyl-1-(L-valyloxy)propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (1 / 1) (339 mg, 100% purity, To a solution of (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (206 mg, 350 μmol) (Intermediate 3), HATU (193 mg, 508 μmol) and DIEA (170 μl, 950 μmol) was added 1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (206 mg, 350 μmol) (Intermediate 3), HATU (193 mg, 508 μmol) and DIEA (170 μl, 950 μmol). The mixture was stirred at room temperature for 1 hour and 30 minutes and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give (19S)-19-[(2S)-2-({(2S)-1-[(1S,2R)-2-{[(2R,3R)-3-{(2S)-1-[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]-12-methoxy-4,10-dimethyl-3,6,9,14-tetraoxo-1-phenyl-5,8-di(propane-2

[0145] tert-Butyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-yl]-2-oxa-4,7,10-triazatetradec-14-yl]pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl]amino]-1-phenylpropoxy]-3-methyl-1-oxobutan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oate (447 mg, 100% purity, 92% yield). LC-MS (Method 3): R t =6.26 min; MS (ESIpos): m / z=1523 [M+H] + .

[1072] Step 2: To (19S)-19-[(2S)-2-({(2S)-1-[(1S,2R)-2-{[(2R,3R)-3-{(2S)-1-[(5S,8S,11S,12R)-11-[(2S)-butan-2-yl]-12-methoxy-4,10-dimethyl-3,6,9,14-tetraoxo-1-phenyl-5,8-di(propan-2-yl)-2-oxa-4,7,10-triazatetradec-14-yl]- To a solution of tert-butyl 2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-ate (447 mg, 100% purity, 294 μmol) in DCM (50 ml) was added TFA (5 ml). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 35 (497 mg, 93% purity, quantitative) as an amorphous residue. LC-MS (Method 3): R t =4.16 min; MS (ESIpos): m / z=1367 [M+H] + .

[1073] Example S36: Preparation of (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propanoyl]-L-α-aspartyl-L-prolyl-L-valine ester. trifluoroacetic acid (1 / 2) (Intermediate 36)

[1074]

[1075] Step 1: (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester. Trifluoroacetic acid (1 / 1) (245 mg, 93% purity, 153 μmol) (Intermediate 35) was dissolved in 25 mL of DMF and 2,5-dioxopyrrolidin-1-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester was added. 2 ,N 6 -bis(tert-butoxycarbonyl)-L-lysine ester (88.3 mg, 199 μmol) and 80 μL N,N-diisopropylethylamine. After stirring at room temperature for 2 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC to give (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-{[N 2 ,N 6 -bis(tert-butoxycarbonyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester (205 mg, 100% purity, 79% yield). LC-MS (Method 3): R t =5.73 min; MS (ESIpos): m / z=1695 [M+H] + .

[1076] Step 2: (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-{[N 2 ,N 6-bis(tert-butoxycarbonyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester (204 mg, 100% purity, 120 μmol) was dissolved in 20 ml of DCM and 5 ml of TFA was added. The reaction mixture was stirred at room temperature for 1 hour and 30 minutes and concentrated in vacuo. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 36 as an amorphous residue (205 mg, 100% purity, 99% yield). LC-MS (Method 3): R t =3.56 min; MS (ESIpos): m / z=1495 [M+H] + .

[1077] Example S37: (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-{[N 2 ,N 6 Preparation of -bis(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester. trifluoroacetic acid (1 / 2) (Intermediate 37)

[1078]

[1079] Step 1: (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propanoyl]-L-α-aspartyl-L-prolyl-L-valine ester, trifluoroacetic acid (1 / 2) (204 mg, 119 μmol) (Intermediate 36) was dissolved in 25 mL DMF was added with tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (119 mg, 284 μmol) and 170 μL N,N-diisopropylethylamine. After stirring at room temperature for 2 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC to give (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-{[N 2 ,N 6 -bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester (207 mg, 100% purity, 83% yield). LC-MS (Method 3): R t =5.48 min; MS (ESIpos): m / z=2101[M+H] + .

[1080] Step 2: (1S,2R)-2-({(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[{N-[(benzyloxy)carbonyl]-N-methyl-L-valyl-L-valyl}(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}amino)-1-phenylpropyl N-(3-{2-[2-(2-{[N 2 ,N 6-bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester (206 mg, 100% purity, 98.2 μmol) was dissolved in 20 ml DCM and 5 ml TFA was added. The reaction mixture was stirred at room temperature for 1 hour and concentrated in vacuo. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 37 (206 mg, 100% purity, 98% yield) as an amorphous residue. LC-MS (Method 3): R t =3.54 min; MS (ESIpos): m / z=1901[M+H] + .

[1081] Example S38: Preparation of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (1 / 1) (Intermediate 38)

[1082]

[1083] Step 1: To a solution of trifluoroacetic acid, N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1 / 1) (100 mg, 171 μmol, as described in WO2015096982) in DMF (25 ml) was added 2,5-dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl]-L-alaninate (110 mg, 343 μmol) and DIEA (120 μl, 690 μmol). The mixture was stirred at room temperature for 1 hour and concentrated under reduced pressure. The residue was purified by preparative HPLC to give N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (101 mg, 98% purity, 86% yield) as a colorless foam. LC-MS (Method 3): R t =5.43 min; MS (ESIpos): m / z=675[M+H] + .

[1084] Step 2: To a solution of benzyl [(2S)-1-({3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}amino)-1-oxopropan-2-yl]carbamate (101 mg, 150 μmol) in DCM (12.5 ml) and methanol (12.5 ml) was added Pd / C (20 mg). The reaction was hydrogenated at room temperature for 2 hours and filtered. The mother liquor was concentrated in vacuo and freeze-dried to give intermediate 38 (80 mg) as a colorless foam, which was used in the next step without further purification.

[1085] Example S39: Preparation of (2S)-1-[(2S)-4-amino-2-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionylamino]-4-oxo-butyryl]pyrrolidine-2-carboxylic acid (Intermediate 39)

[1086]

[1087] Intermediate 39 was synthesized using a classical peptide synthesis method: (2S)-4-amino-2-(tert-butoxycarbonylamino)-4-oxo-butanoic acid was first coupled with (2S)-pyrrolidine-2-carboxylic acid benzyl ester hydrochloride in DMF in the presence of N,N-diisopropylethylamine, followed by removal of the Boc-protecting group with TFA in DCM. The partially protected dipeptide was acylated with tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (Intermediate 10) in the presence of N,N-diisopropylethylamine in DMF. In the final step, the benzyl ester was removed by hydrogenolysis over 10% Pd / charcoal. LC-MS (Method 2): R t = 1.00 min; MS (ESIpos): m / z = 533 [M+H] + .

[1088] Example S40: Trifluoroacetic acid.N 2 Preparation of -(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (1 / 1) (Intermediate 40)

[1089]

[1090] Step 1: To a solution of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (40.0 mg, 74.0 μmol) (Intermediate 38) in DMF (10 ml) was added N 2 -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-proline (47.3 mg, 88.8 μmol) (Intermediate 39), HATU (36.6 mg, 96.2 μmol) and DIEA (39 μl, 220 μmol). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC to give N- ... 2 -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (62 mg, 96% purity, 77% yield). LC-MS (Method 3): R t =4.83 min; MS (ESIpos): m / z=1056 [M+H] + .

[1091] Step 2: To N 2 To a solution of -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-alaninamide (62.0 mg, 58.8 μmol) in trifluoroethanol (5 ml) was added zinc chloride (64.1 mg, 470 μmol). The mixture was stirred at 50° C. for 5 hours. EDTA (137 mg, 470 μmol) and water 0.1% TFA (4 ml) were then added, and the resulting mixture was purified by preparative HPLC to give Intermediate 40 (55 mg, 100% purity, 88% yield) as a colorless foam. LC-MS (Method 3): R t =3.46 min; MS (ESIpos): m / z=956 [M+H] + .

[1092] Example S41: Preparation of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (Intermediate 41)

[1093]

[1094] Step 1: To a solution of trifluoroacetic acid N-(3-aminopropyl)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1 / 1) (250 mg, 428 μmol, as described in WO2015096982) in DMF (20 ml) was added 2,5-dioxopyrrolidin-1-yl N-[(benzyloxy)carbonyl]-L-valine ester (298 mg, 857 μmol) and DIEA (300 μl, 1.7 mmol). The mixture was stirred at room temperature for 1 hour and then diluted with water and ethyl acetate. The layers were separated and the organic phase was concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-N-[ ... 2 -[(Benzyloxy)carbonyl]-L-valinamide (252 mg, 98% purity, 82% yield). LC-MS (Method 3): R t =5.72 min; MS (ESIpos): m / z=703 [M+H] + .

[1095] Step 2: To N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-N 2 To a solution of -[(benzyloxy)carbonyl]-L-valinamide (250 mg, 98% purity, 349 μmol) in DCM (15 ml) and methanol (15 ml) was added Pd / C (45 mg). The reaction was hydrogenated at room temperature for 2 h 30 min and filtered through celite. The mother liquor was concentrated in vacuo to give intermediate 41 (117 mg, 100% purity, 59% yield) as a colorless resin, which was used in the next step without further purification.

[1096] Example S42: Trifluoroacetic acid.N 2Preparation of -(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (1 / 1) (Intermediate 42)

[1097]

[1098] Step 1: To a solution of N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (115 mg, 202 μmol) (Intermediate 41) in DMF (20 ml) was added N 2 -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-proline (140 mg, 92% purity, 243 μmol) (Intermediate 39), HATU (100 mg, 263 μmol) and DIEA (110 μl, 610 μmol). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give N- ...). 2 -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (153 mg, 76% purity, 53% yield). LC-MS (Method 3): R t =5.01 min; MS (ESIpos): m / z=1084 [M+H] + .

[1099] Step 2: To N 2To a solution of -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (280 mg, 96% purity, 247 μmol) in trifluoroethanol (20 ml) was added zinc chloride (269 mg, 1.98 mmol). The mixture was stirred at 50° C. for 5 hours. Then EDTA (577 mg, 1.98 mmol) and water 0.1% TFA (6 ml) were added and the mixture was purified by preparative HPLC and freeze-dried to give intermediate 42 (192 mg, 100% purity, 71% yield) as an amorphous residue. LC-MS (Method 3): R t =3.62 min; MS (ESIpos): m / z=984 [M+H] + .

[1100] Example S43: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propionyl]-L-α-aspartyl-L-prolyl-L-valine ester·trifluoroacetic acid (1:1) (Intermediate 43)

[1101]

[1102] Step 1: Trifluoroacetic acid 2 -(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (1 / 1) (290 mg, 264 μmol) (Intermediate 42) was dissolved in 30 mL of DMF and 2,5-dioxopyrrolidin-1-yl N- 2 ,N 6 -bis[(benzyloxy)carbonyl]-L-lysine ester (185 mg, 95% purity, 344 μmol) and 92 μL N,N-diisopropylethylamine. After stirring at room temperature for 1 hour, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC to give N-bis[(benzyloxy)carbonyl]-L-lysine ester as an amorphous residue. 2 -[3-(2-{2-[2-({N 2 ,N6 -bis[(benzyloxy)carbonyl]-L-lysyl}amino)ethoxy]ethoxy}ethoxy)propionyl]-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (295 mg, 98% purity, 79% yield). LC-MS (Method 2): R t = 2.27 min; MS (ESIpos): m / z = 1379 [M+H] + .

[1103] Step 2: To N 2 -[3-(2-{2-[2-({N 2 ,N 6 To a solution of 4-[(benzyloxy)carbonyl]-L-lysyl}amino)ethoxy]ethoxy}ethoxy)propionyl]-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (295 mg, 98% purity, 209 μmol) in DCM (15 ml) and methanol (15 ml) was added Pd / C (50 mg). The reaction was hydrogenated at room temperature for 2 hours and filtered through celite. The mother liquor was concentrated in vacuo and freeze-dried to give intermediate 43 (205 mg, 92% purity, 81% yield) as a white amorphous residue. LC-MS (Method 3): R t = 3.32 min; MS (ESIpos): m / z = 1110 [M+H] + .

[1104] Example S44: Trifluoroacetic acid.N 2 -(3-{2-[2-(2-{[N 2 ,N 6 Preparation of -bis(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (2 / 1) (Intermediate 44)

[1105]

[1106] Step 1: Set (N 2-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propanoyl]-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (205 mg, 92% purity, 170 μmol) (Intermediate 43) was dissolved in 30 mL of DMF and 2.2 equivalents of tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (170 mg, 407 μmol) and 180 μL of N,N-diisopropylethylamine were added. After stirring at room temperature for 4 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC and freeze-dried to give N as an amorphous residue. 2 -(3-{2-[2-(2-{[N 2 ,N 6 -bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (250 mg, 93% purity, 80% yield). LC-MS (Method 3): R t =5.20 min; MS (ESIpos): m / z=1716 [M+H] + .

[1107] Step 2: To N 2 -(3-{2-[2-(2-{[N 2 ,N 6To a solution of 250 mg, 93% purity, 135 μmol, zinc chloride (148 mg, 1.08 mmol) was added to a solution of 17-bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]propyl}-L-valinamide (250 mg, 93% purity, 135 μmol) in trifluoroethanol (12 ml). The mixture was stirred at 50° C. for 2 hours and 30 minutes. The resulting mixture was diluted with acetonitrile / water, and EDTA (317 mg, 1.08 mmol) was added. The resulting mixture was filtered, purified by preparative HPLC and freeze-dried to give intermediate 44 as an amorphous residue (187 mg, 100% purity, 79% yield). LC-MS (Method 3): R t = 3.35 min; MS (ESIpos): m / z = 1516 [M+H] + .

[1108] Example S45: Preparation of trifluoroacetic acid L-prolyl-L-valine benzyl ester (2 / 1) (Intermediate 45)

[1109]

[1110] Intermediate 45 was synthesized using a classical peptide synthesis method: first, 1-(tert-butoxycarbonyl)-L-proline was coupled with 4-methylbenzene-1-sulfonic acid L-valine benzyl ester (1 / 1) in DMF in the presence of HATU and N,N-diisopropylethylamine, and then the Boc protecting group was removed by TFA in DCM. LC-MS (Method 2): R t =0.94 min; MS (ESIpos): m / z=305 [M+H] + .

[1111] Instance S46: N 2 Preparation of -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-L-valine (Intermediate 46)

[1112]

[1113] Intermediate 46 was synthesized by classical peptide synthesis: Intermediate 45 was first reacted with 2,5-dioxopyrrolidin-1-yl in the presence of N,N-diisopropylethylamine. 2-(tert-Butoxycarbonyl)-L-asparagine ester was coupled in DMF, followed by removal of the Boc protecting group with TFA in DCM. The partially protected tripeptide was acylated with tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (Intermediate 10) in the presence of N,N-diisopropylethylamine in DMF. In the final step, the benzyl ester was removed by hydrogenolysis over 10% Pd / charcoal. LC-MS (Method 2): R t = 1.10 min; MS (ESIpos): m / z = 632 [M+H] + .

[1114] Instance S47: N 2 Preparation of -(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-asparaginyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (Intermediate 47)

[1115]

[1116] Step 1: A solution of (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-N-methylbutanamide (63.0 mg, 120 μmol, CAS Nr: 1800460-13-2, as described in WO2015096982) in DMF (5 ml) was added with N 2 -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-L-valine (90.7 mg, 144 μmol) (Intermediate 46), HATU (59.1 mg, 156 μmol) and DIEA (63 μl, 360 μmol). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give N- ... as an amorphous residue. 2-(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (92 mg, 98% purity, 66% yield). LC-MS (Method 3): R t =4.93 min; MS (ESIpos): m / z=1141[M+H] + .

[1117] Step 2: To N 2 To a solution of -(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-asparaginyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (92.0 mg, 98% purity, 79.2 μmol) in trifluoroethanol (4 ml) was added zinc chloride (86.3 mg, 633 μmol). The mixture was stirred at 50° C. for 5 hours. The mixture was frozen at -20° C. for 4 days and then stirred again at 50° C. for 3 hours. EDTA (185 mg, 633 μmol) and water 0.1% TFA (3 ml) were then added and the resulting mixture was purified by preparative HPLC and freeze-dried to give intermediate 47 (84 mg, 96% purity, 97% yield) as an amorphous residue. LC-MS (Method 3): R t =3.44 min; MS (ESIpos): m / z=1041[M+H] + .

[1118] Example S48: Preparation of trifluoroacetic acid N-[(3RS)-3-amino-4,4-difluorobutyl]-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-hydroxyacetamide (1 / 1) (Intermediate 48)

[1119]

[1120] Step 1: To a solution of (1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropane-1-amine (159 mg, 448 μmol, as described in WO2015096982, CAS Nr: 1800455-85-9) in DCM (20 ml) was added sodium triacetoxyborohydride (133 mg, 627 μmol) and acetic acid (38 μl). The mixture was stirred at room temperature for 5 minutes, and then a solution of tert-butyl [(2RS)-1,1-difluoro-4-oxobutan-2-yl]carbamate (150 mg, 672 μmol) in DCM (5 ml) was added. The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed twice with saturated sodium carbonate solution and once with brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure to give tert-butyl [(2RS)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-1,1-difluorobutan-2-yl]carbamate (250 mg, 87% purity, 86% yield). LC-MS (Method 2): R t =1.74 min; MS (ESIpos): m / z=562[M+H] + .

[1121] Step 2: To a solution of tert-butyl [(2RS)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-1,1-difluorobutan-2-yl]carbamate (250 mg, 87% purity, 385 μmol) in DCM (50 ml) was added triethylamine (270 μl, 1.9 mmol) under argon. Ethyl 2-chloro-2-oxoacetate (140 μl, 1.3 mmol) was added at 0° C., the mixture was stirred at room temperature for 6 hours and concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with saturated citric acid solution, water, twice with saturated sodium bicarbonate solution and once with brine. The organic phase was dried over magnesium sulfate, filtered and evaporated under reduced pressure to give ethyl 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{(3RS)-3-[(tert-butoxycarbonyl)amino]-4,4-difluorobutyl}amino)-2-oxoacetate (326 mg, 77% purity, 99% yield) as a resinous residue. LC-MS (Method 2): R t = 2.59 min; MS (ESIpos): m / z = 662 [M+H] + .

[1122] Step 3: To a solution of ethyl 2-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}{(3RS)-3-[(tert-butoxycarbonyl)amino]-4,4-difluorobutyl}amino)-2-oxoacetate (326 mg, 77% purity, 379 μmol) in THF (30 ml) was added water (15 ml) and aqueous LiOH (1.9 ml, 2.0 M, 3.8 mmol). The mixture was stirred at room temperature for 3 hours and then neutralized with TFA. Ethyl acetate was then added. The organic phase was washed twice with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by preparative HPLC to give tert-butyl {(2RS)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1,1-difluorobutan-2-yl}carbamate (180 mg, 92% purity, 70% yield) as an amorphous residue. LC-MS (Method 2): R t = 2.58 min; MS (ESIpos): m / z = 619 [M+H] + .

[1123] Step 4: To a solution of tert-butyl {(2RS)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1,1-difluorobutan-2-yl}carbamate (180 mg, 92% purity, 267 μmol) in trifluoroethanol (15 ml) was added zinc chloride (219 mg, 1.60 mmol). The mixture was stirred at 50° C. for 1 hour and 30 minutes. EDTA (469 mg, 1.60 mmol) was added and the resulting mixture was diluted with water and acetonitrile, filtered, purified by preparative HPLC and freeze-dried to give intermediate 48 (76 mg, 100% purity, 45% yield) as an amorphous residue. LC-MS (Method 2): R t =1.66 min; MS (ESIpos): m / z=520 [M+H] + .

[1124] Example S51: Preparation of N-{5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxopentanoyl}-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycyl-N-methylglycine benzyl ester (Intermediate 51)

[1125]

[1126] Step 1: To N 6 -[(Benzyloxy)carbonyl]-N 2 To a solution of -(tert-butoxycarbonyl)-L-lysine (600mg, 1.58mmol) in DMF (15ml) was added tert-butyl 8-aminooctanoate (374mg, 1.73mmol), HATU (780mg, 2.05mmol) and N, N-diisopropylethylamine (410μl, 2.4mmol). The mixture was stirred at room temperature for 1 hour. Ethyl acetate was then added and the organic layer was washed with water and brine. The organic phase was then dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to obtain tert-butyl 8-({N6-[(benzyloxy)carbonyl]-N2-(tert-butoxycarbonyl)-L-lysinyl}amino)octanoate (940mg, 89% purity, 91% yield) as an amorphous residue. LC-MS (method 2): R t = 2.33 min; MS (ESIpos): m / z = 578 [M+H] + .

[1127] Step 2: tert-Butyl 8-({N6-[(benzyloxy)carbonyl]-N2-(tert-butoxycarbonyl)-L-lysinyl}amino)octanoate (938 mg, 1.62 mmol) was dissolved in methanol (25 ml) and dichloromethane (25 ml). Pd / C 10% (125 mg) was added and the reaction was hydrogenated at room temperature for 5 hours and filtered through celite. The mother liquor was concentrated under reduced pressure to give intermediate 51 (674 mg, 91% purity, 85% yield) as a colorless resin. LC-MS (Method 4): R t =1.41 min; MS (ESIpos): m / z=444[M+H] + .

[1128] Example S52: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[(24S)-24-amino-33-carboxy-14,18,25-trioxo-4,7,10-trioxa-13,19,26-triazatricarboxane-1-yl]-L-α-aspartyl-L-prolyl-L-valine ester-trifluoroacetic acid (1 / 2) (Intermediate 52)

[1129]

[1130] Step 1: To a solution of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-{18-[(2,5-dioxopyrrolidin-1-yl)oxy]-14,18-dioxo-4,7,10-trioxa-13-azaoctadecane-1-yl}-L-α-aspartyl-L-prolyl-L-valine ester (65.0 mg, 93% purity, 54.6 μmol) (Intermediate 11) in DMF (2.5 ml) was added 8-{[N 2 -tert-Butyl (tert-butoxycarbonyl)-L-lysyl]amino}octanoate (39.8 mg, 91% purity, 82.0 μmol) (Intermediate 51) and DIEA (38 μl, 220 μmol) The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-{(14S)-14-[(tert-butoxycarbonyl)amino]-2,2-dimethyl-4,13,20,24,37-pentaoxo-3,28,31,34-tetraoxa-12,19,25-triazaheptatriacont-37-yl}-L-α-aspartyl-L-prolyl-L-valine ester (66 mg, 92% purity, 78% yield) as a yellow foam. LC-MS (Method 3): R t =4.92 min; MS (ESIpos): m / z=1431[M+H] + .

[1131] Step 2: (4S) -4,11- diethyl -3,14- dioxo -3,4,12,14- tetrahydro -1H- pyrano [3 ', 4 ': 6,7] indolizino [1,2-b] quinolin-4-yl N- { (14S) -14- [(tert-butoxycarbonyl) amino] -2,2- dimethyl -4,13,20,24,37- pentaoxo -3,28,31,34- tetraoxa -12,19,25- triaza heptatriacont-37-yl} -L- α - aspartyl -L- prolyl -L- valine ester (65.0 mg, 92% purity, 41.8 μ mol) in DCM (2.5 ml) was added TFA (1.2 ml). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 52 (78 mg, 94% purity, 117%) as an amorphous residue. LC-MS (Method 3): R t= 3.08 min; MS (ESIpos): m / z = 1275 [M+H] + .

[1132] Example S53: Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-{(24S)-37-amino-24-[(7-carboxyheptyl)carbamoyl]-14,18,26-trioxo-4,7,10,29,32,35-hexaoxa-13,19,25-triazaheptatriacontanoyl}-L-α-aspartyl-L-prolyl-L-valine ester-trifluoroacetic acid (1 / 2) (Intermediate 53)

[1133]

[1134] Step 1: To (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[(24S)-24-amino-33-carboxyl-14,18,25-trioxo-4,7,10-trioxa-13,19,26-triazatricarboxan-1-yl]-L-α-aspartyl-L-prolyl To a solution of -L-valine ester-trifluoroacetic acid (1 / 2) (75.0 mg, 94% purity, 47.1 μmol) (Intermediate 52) in DMF (5 ml) were added tert-butyl {2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamate (47.3 mg, 113 μmol) and DIEA (66 μl, 380 μmol). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-{(19S)-19-[(7-carboxyheptyl)carbamoyl]-2,2-dimethyl-4,17,25,29,42-pentaoxo-3,8,11,14,33,36,39-heptaoxa-5,18,24,30-tetraazatetradodec-42-yl}-L-α-aspartyl-L-prolyl-L-valine ester (77 mg, 90% purity, 93% yield) as an amorphous residue. LC-MS (Method 3): R t =4.10 min; MS (ESIpos): m / z=1578[M+H] + .

[1135] Step 2: (4S) -4,11- diethyl -3,14- dioxo -3,4,12,14- tetrahydro -1H- pyrano [3 ', 4 ': 6,7] indolizino [1,2-b] quinolin-4-yl N- { (19S) -19- [(7- carboxyheptyl) carbamoyl] -2,2- dimethyl -4,17,25,29,42- pentaoxo -3,8,11,14,33,36,39- heptaoxa -5,18,24,30- tetraaza tetradodecane -42- yl} -L- α - aspartyl -L- prolyl -L- valine ester (76.0 mg, 90% purity, 43.4 μ mol) in DCM (2.5 ml) was added TFA (1.2 ml). The mixture was stirred at room temperature for 1 hour and then concentrated under reduced pressure. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 53 (88 mg, 90% purity, 107%) as an amorphous residue. LC-MS (Method 3): R t = 3.10 min; MS (ESIpos): m / z = 1478 [M+H] + .

[1136] Example S54: Preparation of (3S)-3-{[(4-{[(4-nitrophenoxy)carbonyl]amino}phenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid (Intermediate 54)

[1137]

[1138] The synthesis of intermediate 54 is described in WO2020 / 094471 LC-MS: R t =0.89 min; MS (ESIpos): m / z=720 [M+H] + .

[1139] Example S55: Preparation of N-{[methyl(2-{methyl[2-(methylamino)ethyl]amino}ethyl)amino]acetyl}-L-asparaginyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 55)

[1140]

[1141] Intermediate 56 (45.0 mg, 49.4 μmol) was dissolved in DCM (8.0 ml). TFA (1.50 ml) was added and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated in vacuo, dissolved in ACN / H2O and freeze-dried to give intermediate 55 (45.0 mg, 100% purity, 99% yield) as a white residue. LC-MS (Method 2): R t = 0.90 min; MS (ESIneg): m / z = 808 [MH] - .

[1142] Example S56: Preparation of N-(2,2,5,8,11-pentamethyl-4,13-dioxo-3-oxa-5,8,11-triazatridec-13-yl)-L-asparaginyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide (Intermediate 56)

[1143]

[1144] Intermediate 58 (38.0 mg, 51.4 umol) was added to DMF (10.0 ml), followed by addition of intermediate 57 (30.7 mg, 61.7 umol), HATU (39.1 mg, 102.9 umol) and DIEA (35.8 ul, 205.7 umol) and stirred at room temperature overnight. The reaction was then concentrated in vacuo. The residue was purified by preparative HPLC, concentrated and freeze-dried to give intermediate 56 (45.0 mg, 100% purity, 96% yield). LC-MS (method 2): R t = 1.20 min; MS (ESIneg): m / z = 908 [MH] - .

[1145] Example S57: Preparation of N-{2-[{2-[(tert-butoxycarbonyl)(methyl)amino]ethyl}(methyl)amino]ethyl}-N-methylglycine (Intermediate 57)

[1146]

[1147] Tert-butyl methyl(2-{methyl[2-(methylamino)ethyl]amino}ethyl)carbamate was first obtained from commercially available N,N′-dimethyl-N-[2-(methylamino)ethyl]ethane-1,2-diamine.

[1148] First, under argon, benzyl bromoacetate (609mg, 2.66mmol) is dissolved in acetonitrile (25mL) and potassium carbonate (734mg, 5.31mmol) is added. The solution is cooled to 0 DEG C and a solution of methyl (2-{ methyl [2- (methylamino) ethyl] amino} ethyl) tert-butyl carbamate (652mg, 2.66mmol) in acetonitrile is added. The batch is stirred at room temperature for 1 hour, then filtered. The filtrate is evaporated in vacuo and the remaining residue is purified by preparative HPLC. The relevant fractions are collected and evaporated to dryness. 665mg (97% purity, 62% yield) of the protected intermediate N-{2-[{2-[(tert-butoxycarbonyl) (methyl) amino] ethyl} (methyl) amino] ethyl}-N-methylglycine benzyl ester as a colorless oil is obtained. The intermediate (687mg, 1.75mmol) is dissolved in DCM / methanol and hydrogenated at room temperature for 2 hours through 10% palladium / charcoal. The catalyst was filtered off and the filtrate was concentrated in vacuo to give intermediate 57 (555 mg, 61% purity, 64% yield) as a colorless oil. LC-MS (Method 7): R t =0.74 min; MS (ESIpos): m / z=304 [M+H] + .

[1149] Example S58: Preparation of L-asparaginyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 58)

[1150]

[1151] Intermediate 59 (40.0 mg, 0.051 mmol) was dissolved in DCM (10.0 ml), TFA (1.0 ml) was added, and the reaction was stirred at room temperature for 45 minutes. The reaction was concentrated in vacuo, and the residue was dissolved in ACN / H2O and freeze-dried to give Intermediate 58 (38.0 mg, 100% purity, quantitative yield) as a white residue. LC-MS (Method 2): R t = 1.10 min; MS (ESIpos): m / z = 632 [M+H] + .

[1152] Instance S59: N 2 Preparation of -(tert-Butoxycarbonyl)-L-asparaginyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide (Intermediate 59)

[1153]

[1154] To a solution of Boc-Asn-OH (34.8 mg, 150.0 umol) in DMF (10.0 ml) was added HOBT (28.7 mg, 187.3 umol) and EDCI (35.9 mg, 187.3 umol). The reaction was stirred at room temperature for 10 minutes, and then intermediate 60 (80.0 mg, 124.9 umol) and DIEA (65.2 ul, 374.6 umol) were added and stirred at room temperature for 2 hours. The reaction was concentrated in vacuo. The residue was purified by preparative HPLC and freeze-dried to obtain intermediate 59 (40.0 mg, 92% purity, 41% yield) as a white residue. LC-MS (method 3): R t =3.18 min; MS (ESIpos): m / z=725 [M+H] + .

[1155] Example S60: Preparation of L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 60)

[1156]

[1157] Intermediate 61 (178.5 mg, 254.9 mmol) was dissolved in DCM (12.0 ml), TFA (3.0 ml) was added and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated in vacuo. The residue was dissolved in ACN / H2O and freeze-dried to give intermediate 60 (165.0 mg, 98% purity, quantitative yield). LC-MS (Method 2): R t = 1.10 min; MS (ESIpos): m / z = 509 [MH] - .

[1158] Example S61: Preparation of 1-(tert-Butoxycarbonyl)-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide (Intermediate 61)

[1159]

[1160] Intermediate 62 (172.0 mg, 0.326 mmol) was added to DMF (10.0 ml). Boc-Pro-OH (77.2 mg, 0.359 mmol), HATU (186.0 mg, 489.1 umol) and DIEA (126.4 mg, 978.1 umol) were added and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated in vacuo and the residue was purified by preparative HPLC, concentrated and freeze-dried to give intermediate 61 (178.0 mg, 87% purity, 78% yield) as a white foam. LC-MS (Method 2): R t =1.65 min; MS (ESIpos): m / z=611[M+H] + .

[1161] Example S62: Preparation of N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 62)

[1162]

[1163] Intermediate 63 (160 mg, 311.7 μmol) was dissolved in DCM (10.0 ml), TFA (2.0 ml) was added and the reaction was stirred at room temperature for 1.5 hours. The reaction was concentrated in vacuo, dissolved in ADC / H2O and freeze-dried to give intermediate 62 (172 mg, 100% purity, 67% yield) as a white foam. LC-MS (Method 2): R t =0.99 min; MS (ESIpos): m / z=414 [M+H] + .

[1164] Example S63: Preparation of tert-butyl [(2S)-1-{[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate (Intermediate 63)

[1165]

[1166] 1-(4-Amino-2-(ethoxymethyl)-1H(resiquimod) (80.0 mg, 0.254 mmol) was added to DCM (15.0 ml). Then, Boc-Val-NCA (123.8 mg, 0.509 mmol) and DMAP (31.1 mg, 0.254 mmol) were added. The mixture was heated and stirred at reflux for 2 hours. The reaction was concentrated in vacuo and the residue was purified by preparative HPLC, concentrated and freeze-dried to give intermediate 63 (160 mg, 96% purity, quantitative yield) as a white foam. LC-MS (Method 2): R t =1.64 min; MS (ESIpos): m / z=514 [M+H] + .

[1167] Example S64: Preparation of N-{14-[4-({[(1R)-2-carboxy-1-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}ethyl]carbamoyl}amino)anilino]-14-oxo-4,7,10-trioxa-13-azatetradec-1-yl}-L-α-aspartyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide (Intermediate 64)

[1168]

[1169] Intermediate 65 (60.9 mg, 64.6 μmol) was dissolved in DMF (10.0 ml). Intermediate 2 (46.5 mg, 64.6 μmol) and DIEA (225.0 μl, 1291.6 μmol) were added. The reaction was stirred at room temperature for 15 minutes and then concentrated in vacuo. The residue was separated by preparative HPLC to obtain intermediate 64 (58.0 mg, 97% purity, 62% yield) as a colorless foam. LC-MS (method 2): R t = 1.40 min; MS (ESIneg): m / z = 1407 [MH] - .

[1170] Example S65: Preparation of N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 65)

[1171]

[1172] Intermediate 66 (58.8 mg, 63.3 μmol) was dissolved in DCM (5.0 ml). TFA (1.0 ml) was added and the reaction was stirred at room temperature for 30 minutes. The reaction was concentrated via oil pump and the residue was dissolved in ACN / H2O and freeze-dried to give intermediate 65 (60.9 mg, 100% purity, quantitative yield) as a colorless foam. LC-MS (Method 2): R t =0.96 min; MS (ESIneg): m / z=827 [MH] - .

[1173] Example S66: Preparation of N-(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-α-aspartyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide (Intermediate 66)

[1174]

[1175] Intermediate 67 (69.0 mg, 0.093 mmol) was dissolved in DMF (8.0 ml). tBoc-N-amido-PEG3-NHS ester (46.8 mg, 0.112 mmol) and DIEA (48.7 ul, 279.8 umol) were added. The reaction was stirred at room temperature for 3 hours. The reaction was evaporated to dryness and the residue was separated by preparative HPLC to obtain intermediate 66 (58.8 mg, 100% purity, 68% yield) as a colorless foam. LC-MS (method 2): R t =1.45 min; MS (ESIpos): m / z=929[M+H] + .

[1176] Example S67: Preparation of L-α-aspartyl-L-prolyl-N-[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]-L-valinamide-trifluoroacetic acid (1 / 1) (Intermediate 67)

[1177]

[1178] Intermediate 68 (200 mg, 235 μmol) was dissolved in DCM (15 ml) and TFA (5.0 ml) was added. The reaction was stirred at room temperature for 4.5 hours and then concentrated in vacuo. The residue was dissolved in ACN / H2O and freeze-dried. The residue was redissolved in DCM / MeOH again and ether was added until turbidity. The emulsion was briefly cooled and then decanted. The remaining residue was redissolved in ACN / H2O and freeze-dried to give intermediate 67 (148 mg, 85% yield). LC-MS (method 8): R t = 0.53 min; MS (ESIneg): m / z = 624 [MH] - .

[1179] Example S68: Preparation of tert-butyl (3S)-3-[(tert-butoxycarbonyl)amino]-4-[(2S)-2-{[(2S)-1-{[2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidin-1-yl]-4-oxobutanoate (Intermediate 68)

[1180]

[1181] Intermediate 62 (160 mg, 303 μmol), intermediate 69 (141 mg, 364 μmol), HATU (150 mg, 394 μmol) and DIEA (160 μl, 910 μmol) were dissolved in DMF (15 ml) and stirred at room temperature for 10 minutes. The reaction was concentrated in vacuo and purified by preparative HPLC to give intermediate 68 (200 mg, 92% purity, 77% yield). LC-MS (Method 2): R t =1.89 min; MS (ESIpos): m / z=782 [M+H] + .

[1182] Example S69: Preparation of (2S)-1-{(2S)-4-tert-butoxy-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}pyrrolidine-2-carboxylic acid (Intermediate 69)

[1183]

[1184] Intermediate 70 (7.47 g, 15.7 mmol) was dissolved in methanol (1.0 L) and Pd / C 10% (1.30 g) was added. The reaction was hydrogenated at room temperature, filtered, concentrated, and extracted with ether. The compound was dried over a high vacuum line to give Intermediate 69 (5.92 g, 97% purity, 95% yield) as a foam. LC-MS (Method 2): Rt = 1.61 min; MS (ESIpos): m / z = 387 [M+H]+.

[1185] Example S70: Preparation of benzyl (2S)-1-{(2S)-4-tert-butoxy-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}pyrrolidine-2-carboxylate (Intermediate 70)

[1186]

[1187] Boc-Asp(OtBu)-OSu (0.390 g, 1.01 mmol) was dissolved in DMF (10 ml). H-Pro-OBzl.HCl (0.244 g, 1.01 mmol) and DIEA (0.352 ml, 2.02 mmol) were added and the reaction was stirred at room temperature overnight. The reaction was concentrated in vacuo, dissolved in DCM and extracted with H2O. The organic phase was purified by flash column chromatography (DCM / 3% MeOH). The product was concentrated in vacuo and dried over a high vacuum line to give intermediate 70 (0.41 g, 100% purity, 85% yield). LC-MS (Method 2): R t = 2.20 min; MS (ESIpos): m / z = 477 [M+H] + .

[1188] Example S71: Preparation of N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide trifluoroacetate (1:1) (Intermediate 71)

[1189]

[1190] Step 1: (2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-methylbutanamide trifluoroacetate (1:1) (100.0 mg, 0.155 mmol) (its synthesis is described in WO2015096982) was dissolved in DMF (2.5 ml). N-(tert-butoxycarbonyl)valine (40.5 mg, 0.187 mmol), HATU (88.7 mg, 0.233 mmol) and DIEA (81.2 μl, 0.466 mmol) were added and the reaction was stirred at room temperature for 3.5 hours. The residue was purified by preparative HPLC and then freeze-dried to give tert-butyl N-[(1S)-1-[[(1S)-3-[[(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)pyrrol-2-yl]-2,2-dimethyl-propyl]-(2-hydroxyacetyl)amino]-1-(methylcarbamoyl)propyl]carbamoyl]-2-methyl-propyl]carbamate (99.4 mg, 100% purity, 88% yield). LC-MS (Method 2): Rt = 2.43 min; MS (ESIpos): m / z = 724 [MH]-.

[1191] Step 2: Tert-butyl N-[(1S)-1-[[(1S)-3-[[(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)pyrrol-2-yl]-2,2-dimethyl-propyl]-(2-hydroxyacetyl)amino]-1-(methylcarbamoyl)propyl]carbamoyl]-2-methyl-propyl]carbamate (99.2 mg, 0.137 mmol) was dissolved in 2,2,2-trifluoroethanol (4.0 ml). ZnCl2 (111.8 mg, 0.82 mmol) was added and the reaction was stirred at 50° C. for 2 hours. EDTA (239.6 mg, 0.82 mmol) and H2O + 0.1% TFA (2 ml) were added, and the residue was purified by preparative HPLC and then freeze-dried to give intermediate 71 (94.6 mg, 100% purity, 94% yield). LC-MS (Method 2): Rt = 1.53 min; MS (ESIpos): m / z = 626 [M+H] + .

[1192] Example S72: Preparation of N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide trifluoroacetate (1:1)) (Intermediate 72)

[1193]

[1194] Step 1: To a solution of intermediate 71 (94.4 mg, 0.128 mmol) and intermediate 3 (82.8 mg, 0.14 mmol) in DMF (3.0 ml) was added HATU (77.6 mg, 0.204 mmol) and DIEA (66.7 ul, 0.383 mmol) The reaction was stirred at room temperature for 1.5 hours. The residue was purified by preparative HPLC and then freeze-dried to give (19S)-19-{[(2S)-2-{[(2S)-1-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidin-1-yl]carbonyl}-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (143.7 mg, 100% purity, 94% yield). LC-MS (method 2): Rt = 2.41 min; MS (ESIpos): m / z = 1197 [M+H] + .

[1195] Step 2: (19S)-19-{[(2S)-2-{[(2S)-1-{[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(ethanolyl)amino]-1-(methylamino)-1-oxobutan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamoyl}pyrrolidin-1-yl]carbonyl}-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneicosane-21-oic acid tert-butyl ester (143.4 mg, 0.120 mmol) was dissolved in 2,2,2-trifluoroethanol (4.0 ml). ZnCl2 (97.9 mg, 0.719 mmol) was added and the reaction was stirred at 50°C for 2 hours. 3 equivalents of ZnCl2 were further added and the reaction was stirred at 50°C for 1 hour. EDTA (210.0 mg, 0.719 mmol) and H2O+0.1% TFA (2 ml) were added. The residue was purified by preparative HPLC and then freeze-dried to give intermediate 72 (125.6 mg, 100% purity, 91% yield). LC-MS (Method 2): Rt=1.54 min; MS (ESIpos): m / z=1041 [M+H] + .

[1196] Example S73 Preparation of (3S)-3-[3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionylamino]-4-[(2S)-2-[[(1S)-1-[3-[[(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)pyrrol-2-yl]-2,2-dimethyl-propyl]-(2-hydroxyacetyl)amino]propylcarbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidin-1-yl]-4-oxo-butanoic acid; 2,2,2-trifluoroacetic acid (Intermediate 73)

[1197]

[1198] Step 1: To a solution of intermediate 41 (100 mg, 0.143 mmol) and intermediate 3 (100.9 mg, 0.171 mmol) in DMF (20 ml) were added HATU (70.4 mg, 0.185 mmol) and DIEA (74.5 ul, 0.428 mmol) The reaction was stirred at room temperature for 1 hour. The solvent was evaporated and the residue was purified by preparative HPLC and then freeze-dried to give (3S)-4-[(2S)-2-[[(1S)-1-[3-[[(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)pyrrol-2-yl]-2,2-dimethyl-propyl]-(2-hydroxyacetyl)amino]propylcarbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidin-1-yl]-3-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propanoylamino]-4-oxo-butyric acid tert-butyl ester (164 mg, 99% purity, 100% yield). LC-MS (Method 3): Rt = 5.87 min; MS (ESIpos): m / z = 1141 [M+H] + .

[1199] Step 2: (3S)-4-[(2S)-2-[[(1S)-1-[3-[[(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)pyrrol-2-yl]-2,2-dimethyl-propyl]-(2-hydroxyacetyl)amino]propylcarbamoyl]-2-methyl-propyl]carbamoyl]pyrrolidin-1-yl]-3-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]propionylamino]-4-oxo-butyric acid tert-butyl ester (25 mg, 0.022 mmol) was dissolved in 2,2,2-trifluoroethanol (5 ml). ZnCl2 (23.9 mg, 0.175 mmol) was added and the reaction was stirred at 50°C for 5 hours. EDTA (51.2 mg, 0.175 mmol) and water + 0.1% TFA (3 ml) were added. The mixture was purified by preparative HPLC and then freeze-dried to give intermediate 73 as an amorphous residue (20 mg, 98% purity, 81% yield). LC-MS (Method 3): Rt = 3.89 min; MS (ESIpos): m / z = 984 [M+H] + .

[1200] Example S74: Preparation of N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propanoyl]-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (Intermediate 74)

[1201]

[1202] Step 1: N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide. Trifluoroacetic acid (1 / 1) (355 mg, 307 μmol) (Intermediate 72) was dissolved in 12 mL of DMF and 2,5-dioxopyrrolidin-1-yl N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide. 2 ,N 6 -bis[(benzyloxy)carbonyl]-L-lysine ester (215 mg, 95% purity, 399 μmol) and 110 μL N,N-diisopropylethylamine. After stirring at room temperature for 4 hours, the mixture was concentrated in vacuo and the residue was purified by preparative HPLC to give N-[3-(2-{2-[2-({N2,N6-bis[(benzyloxy)carbonyl]-L-lysyl}amino)ethoxy]ethoxy}ethoxy)propanoyl]-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (412 mg, 100% purity, 93% yield) as a white foam. LC-MS (Method 2): R t =5.35 min; MS (ESIpos): m / z=1438[M+H] + .

[1203] Step 2: To N-[3-(2-{2-[2-({N 2 ,N 6To a solution of 410 mg (285 μmol) in DCM (10 ml) and methanol (10 ml) was added Pd / C (40 mg). The reaction was hydrogenated at room temperature for 4 hours and filtered through celite. The mother liquor was concentrated in vacuo to give intermediate 74 (354 mg, 100% purity, quantitative). LC-MS (Method 2): R t =1.28 min; MS (ESIpos): m / z=1168 [MH] - .

[1204] Example S75 Preparation of N-(3-{2-[2-(2-{[N2,N6-bis(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide. Trifluoroacetic acid (1 / 2) (Intermediate 75)

[1205]

[1206] Step 1: N-[3-(2-{2-[2-(L-lysylamino)ethoxy]ethoxy}ethoxy)propionyl]-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (352 mg, 301 μmol) (Intermediate 74) was dissolved in 12 mL The mixture was stirred at room temperature for 2 hours and 30 minutes, and the mixture was concentrated in vacuo, and the residue was purified by preparative HPLC and freeze-dried to give N-(3-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethyl}carbamic acid tert-butyl ester (302 mg, 722 μmol) and 310 μL N,N-diisopropylethylamine. After stirring at room temperature for 2 hours and 30 minutes, the mixture was concentrated in vacuo, and the residue was purified by preparative HPLC and freeze-dried to give N-(3-{2-[2-(2-{[N2 ,N 6 -bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (449 mg, 100% purity, 84% yield). LC-MS (Method 9): R t =9.77 min; MS (ESIpos): m / z=1177 [M+H] + .

[1207] Step 2: To a solution of N-(3-{2-[2-(2-{[N2,N6-bis(2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-yl)-L-lysyl]amino}ethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-[(2S)-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(hydroxyacetyl)amino]-1-(methylamino)-1-oxobutan-2-yl]-L-valinamide (447 mg, 252 μmol) in trifluoroethanol (8.0 ml) was added zinc chloride (206 mg, 1.51 mmol). The mixture was stirred at 50° C. for 3 hours. The resulting mixture was diluted in water and then EDTA (441 mg, 1.51 mmol) was added. The resulting mixture was filtered, purified by preparative HPLC and freeze-dried to give intermediate 75 (371 mg, 100% purity, 82% yield) as a white foam. LC-MS (Method 3): R t =3.18 min; MS (ESIpos): m / z=1576 [M+H] + .

[1208] Example S76 Preparation of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-[14-(4-nitrophenoxy)-14-oxo-4,7,10-trioxa-13-azatetradec-1-yl]-L-α-aspartyl-L-prolyl-L-valine ester (Intermediate 76)

[1209]

[1210] To a solution of (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propionyl)-L-α-aspartyl-L-prolyl-L-valine ester.trifluoroacetic acid (1 / 1) (20.0 mg, 19.9 μmol) intermediate 6 in THF (5.0 ml) was added 4-nitrophenyl chloroformate (6.42 mg, 31.8 μmol). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give intermediate 76 (6.0 mg, 100% purity, 29% yield) as a colorless foam. LC-MS (Method 2): R t =1.77 min; MS (ESIpos): m / z=1056 [MH] - .

[1211] Example S77 Preparation of (3S)-3-[4-[4-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]-1-naphthyl]phenyl]-3-[[2-[5-[(4-methyl-2-pyridinyl)amino]pentanoylamino]acetyl]amino]propionic acid methyl ester (Intermediate 77)

[1212]

[1213] Intermediate 77 was prepared using the method described in WO2022056273 for the synthesis of methyl (3S)-3-[4-[4-[2-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]-1-naphthyl]phenyl]-3-[[2-[5-[(4-methyl-2-pyridinyl)amino]pentanoylamino]acetyl]amino]propanoate (CAS Nr: 2763952-63-0).

[1214] Example S78 Preparation of (3S)-3-[4-[4-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]-1-naphthyl]phenyl]-3-[[2-[5-[(4-methyl-2-pyridinyl)amino]pentanoylamino]acetyl]amino]propionic acid (Intermediate 78)

[1215]

[1216] Intermediate 78 was prepared starting from intermediate 77 by reducing the azide using triphenylphosphine in a mixture of THF and water, followed by saponification of the methyl ester using lithium hydroxide in a mixture of THF and water. LC-MS (Method 2): Rt = 0.90 min; MS: m / z = 728 [MH] - .

[1217] Example S79 Preparation of (3S)-3-[[(2S)-3-amino-2-[5-[tert-butoxycarbonyl-(4-methyl-2-pyridyl)amino]pentanoylamino]propanoyl]amino]-3-(3,5-dichlorophenyl)propanoic acid (Intermediate 79)

[1218]

[1219] Intermediate 79 was prepared using the method described in WO2015179823 for the synthesis of methyl 3-[[(2S)-6-amino-2-[5-[tert-butoxycarbonyl(2-pyridyl)amino]pentanoylamino]hexanoyl]amino]-3-(3,5-dichlorophenyl)propanoate (CASNr: 1831059-47-2), followed by saponification of the methyl ester using lithium hydroxide. LC-MS (Method 2): Rt = 1.38 min; MS: m / z = 608 [MH] - .

[1220] Example S80 Preparation of (3S)-3-[(N-{5-[(tert-Butoxycarbonyl)(4-methylpyridin-2-yl)amino]pentanoyl}-3-{[(4-nitrophenoxy)carbonyl]amino}-L-alanyl)amino]-3-(3,5-dichlorophenyl)propanoic acid (Intermediate 80)

[1221]

[1222] To a solution of (3S)-3-[(3-amino-N-{5-[(tert-butoxycarbonyl)(4-methylpyridin-2-yl)amino]pentanoyl}-L-alanyl)amino]-3-(3,5-dichlorophenyl)propanoic acid (30.0 mg, 49.1 μmol) intermediate 79 in THF (5.0 ml) was added 4-nitrophenyl chloroformate (6.42 mg, 31.8 μmol). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give intermediate 80 (6.0 mg, 100% purity, 29% yield) as a colorless foam. LC-MS (Method 2): R t =1.77 min; MS: m / z=1056 [MH] - .

[1223] Example S81 Preparation of 3-[(N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanyl)amino]propyl N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-L-valine ester, trifluoroacetic acid (Intermediate 81)

[1224]

[1225] Step 1: To trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(2S)-1-oxo-3-phenyl-1-{[3-(L-valyloxy)propyl]amino}propan-2-yl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide (1 / 1) (25.0 mg, 24.6 μmol) (according to WO2015 To a solution of intermediate 3 (prepared by 054659, CASNr=1699749-70-6) and (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (17.4 mg, 29.5 μmol) in DMF (1.0 ml) were added HATU (12.2 mg, 32.0 μmol) and DIEA (13 μl, 74 μmol). The reaction was stirred at room temperature for 1 hour. The residue was purified by preparative HPLC and then freeze-dried to give (19S)-19-[(2S)-2-{[(3R,4R,7S,15S)-7-benzyl-3-{(2S)-1-[(3R,4S,5S)-4-{[(2S)-2-{[(2S)-2-(dimethylamino)-3-methylbutanoyl]amino}-3-methylbutanoyl](methyl)amino}-3-[ ... [methoxy-5-methylheptanoyl]pyrrolidin-2-yl]-4,16-dimethyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-15-yl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneconamide-21-oic acid tert-butyl ester (24 mg, 100% purity, 66% yield). LC-MS (Method 3): Rt = 4.15 min; MS (ESIpos): m / z = 1474 [M+H] + .

[1226] Step 2: To (19S)-19-[(2S)-2-{[(3R,4R,7S,15S)-7-benzyl-3-{(2S)-1-[(3R,4S,5S)-4-{[(2S)-2-{[(2S)-2-(dimethylamino)-3-methylbutanoyl]amino}-3-methylbutanoyl](methyl)amino}-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl}-4,16-dimethylamino] ...pyrrolidin- To a solution of tert-butyl]methyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-15-yl]carbamoyl}pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazaheneconamide-21-acid (23.0 mg, 15.6 μmol) in DCM (1.0 ml) was added TFA (130 μl). The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. The residue was dissolved in acetonitrile and water and then freeze-dried to give intermediate 81 (24 mg, 92% purity, 92% yield) as a colorless foam. LC-MS (Method 3): R t = 2.81 min; MS: m / z = 1318 [MH] - .

[1227] Example S82: Preparation of 3-aminopropyl 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoate (Intermediate 82)

[1228]

[1229] Step 1: To a solution of 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoic acid (144 mg, 621 μmol) in DMF (1 ml) was added TBTU (230 mg, 717 μmol) and DIEA (330 μl, 1.9 mmol) under argon. The mixture was stirred at 50 ° C for 3 hours. A solution of benzyl (3-hydroxypropyl)carbamate (100 mg, 478 μmol) in DMF (0.5 ml) was then added and the mixture was stirred at 50 ° C for 20 hours. The mixture was then purified by preparative HPLC and freeze-dried to give 3-{[(benzyloxy)carbonyl]amino}propyl 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoate (124 mg, 99% purity, 61% yield) as a white foam. LC-MS (method 2): R t = 2.09 min; MS (ESIpos): m / z = 423 [M+H] + .

[1230] Step 2: 3-{[(benzyloxy)carbonyl]amino}propyl 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoate (122 mg, 289 μmol) was dissolved in ethanol (20 ml). Pd / C 10% (12 mg) was added, and the reaction was hydrogenated at room temperature for 4 hours and filtered through celite. The mother liquor was concentrated in vacuo to give Intermediate 82 (82 mg, 100% purity, 98% yield).

[1231] Example S83 Preparation of trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-17-amino-7-benzyl-4,15,15-trimethyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide (2 / 1) (Intermediate 83)

[1232]

[1233] Step 1: To a solution of 3-aminopropyl 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoate (9.22 mg, 32.0 μmol) intermediate 82 in DMF (0.5 ml) under argon was added HATU (9.73 mg, 25.6 μmol) and DIEA (6.7 μl, 38 μmol). The mixture was stirred at 50° C. for 3 hours. Then, a solution of N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide trifluoroacetic acid (1 / 1) (11.0 mg, 12.8 μmol) in DMF (0.05 ml) was added. The mixture was stirred at 50° C. for 20 hours. The mixture was then purified by preparative HPLC and freeze-dried to give 3-{[(benzyloxy)carbonyl]amino}propyl 4-[(tert-butoxycarbonyl)amino]-2,2-dimethylbutanoate (14 mg, 100% purity, quantitative) as an amorphous residue. LC-MS (Method 2): R t =1.62 min; MS (ESIpos): m / z=1017 [M+H] + .

[1234] Step 2: To a solution of N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-4,15,15,21,21-pentamethyl-5,8,14,19-tetraoxo-2,13,20-trioxa-6,9,18-triazadocosan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (38.0 mg, 37.4 μmol) in DCM (4 ml) was added TFA (1 ml). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in acetonitrile and water and then freeze-dried to give intermediate 83 as a white foam (40 mg, 100% purity, 93% yield). LC-MS (Method 2): R t =1.04 min; MS (ESIneg): m / z=961 [M-H+HCOO] - .

[1235] Example S84 Preparation of trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-4,15,15-trimethyl-5,8,14-trioxo-17-(L-valylamino)-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide (1 / 1) (Intermediate 84)

[1236]

[1237] Step 1: To trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-17-amino-7-benzyl-4,15,15-trimethyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N- To a solution of methyl-L-valinamide (2 / 1) (39.0 mg, 34.1 μmol) (Intermediate 83) in DCM (1.0 ml) were added 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-L-valinate (21.4 mg, 68.2 μmol), 4-(dimethylamino)pyridine (4.16 mg, 34.1 μmol) and DIEA (12 μl, 68 μmol). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC and freeze-dried to give trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-17-{[N-(tert-butoxycarbonyl)-L-valyl]amino}-4,15,15-trimethyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide (1 / 1) (38 mg, 100% purity, 91% yield) as a colorless foam. LC-MS (Method 2): R t =1.66 min; MS (ESIpos): m / z=1116 [M+H] + .

[1238] Step 2: To a solution of trifluoroacetic acid, N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-17-{[N-(tert-butoxycarbonyl)-L-valyl]amino}-4,15,15-trimethyl-5,8,14-trioxo-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptane-4-yl]-N-methyl-L-valinamide (1 / 1) (37.0 mg, 30.1 μmol) in DCM (2 ml) was added TFA (500 μl). The mixture was stirred at room temperature for 2 hours and then concentrated under reduced pressure. The residue was dissolved in acetonitrile and water and freeze-dried to give intermediate 84 (33 mg, 90% purity, 87% yield). LC-MS (Method 2): R t=1.13 min; MS (ESIneg): m / z=1060 [M-H+CH3COOH] - .

[1239] Example S85 Preparation of N-(3-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}propanoyl)-L-α-aspartyl-L-prolyl-N-(4-{3-[(N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)-4-[(N,N-dimethyl-L-valyl-L-valyl)(methyl)amino]-3-methoxy-5-methylheptanoyl}pyrrolidin-2-yl]-3-methoxy-2-methylpropanoyl}-L-phenylalanyl)amino]propoxy}-3,3-dimethyl-4-oxobutyl)-L-valinamide. Trifluoroacetic acid (1 / 1) (Intermediate 85)

[1240]

[1241] Step 1: To a solution of trifluoroacetic acid N,N-dimethyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(3R,4R,7S)-7-benzyl-4,15,15-trimethyl-5,8,14-trioxo-17-(L-valylamino)-2,13-dioxa-6,9-diazaheptadecan-3-yl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (1 / 1) (33.0 mg, 90% purity, 26.3 μmol) intermediate 84 in DMF (1.3 ml) was added HATU (13.0 mg, 34.2 μmol) and DIEA (14 μl, 79 μmol). Then (2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5,18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid (18.6 mg, 31.6 μmol) intermediate 3 was added and the reaction was stirred at room temperature for 1 hour. The residue was purified by preparative HPLC and then freeze-dried to give trifluoroacetic acid as an amorphous residue.(19S)-19-[(2S)-2-{[(3R,4R,7S,20S)-7-benzyl-3-{(2S)-1-[(3R,4S,5S)-4-{[(2S)-2-{[(2S)-2-(dimethylamino)-3-methylbutanoyl]amino}-3-methylbutanoyl](methyl)amino}-3-methoxy-5-[ ... [methylheptanoyl]pyrrolidin-2-yl]-4,15,15,21-tetramethyl-5,8,14,19-tetraoxo-2,13-dioxa-6,9,18-triazadocosan-20-yl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethyl-4,17-dioxo-3,8,11,14-tetraoxa-5,18-diazahenecon-21-oic acid tert-butyl ester (1 / 1) (44 mg, 70% purity, 69% yield). LC-MS (Method 3): Rt = 4.23 min; MS (ESIpos): m / z = 1587 [M+H] + .

[1242] Step 2: To trifluoroaceti...

Claims

1. A compound having a structure of Formula (II), Formula (III), Formula (IV), Formula (V) or Formula (VII): in: Each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder; Each L 1 , L 2 and L 3 are independently bivalent linkers; A 1 is a trivalent linker; A 2 is a tetravalent linker; Each EL is a cleavable peptide linker; and Each P is a valid payload.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, which has a structure of formula (II) or formula (III): in: Each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder; Each L 1 , L 2 and L 3 are independently bivalent linkers; A 1 is a trivalent linker; EL is a cleavable peptide linker; and P is the effective load. 3 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by cathepsin B, legumin, or neutrophil elastase. 4 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by neutrophil elastase.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, which has the following structure: in: T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; and P is the effective load.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, which has the following structure: in: Each T is a prostate-specific membrane antigen binder, a carbonic anhydrase 9 binder, a fibroblast activation protein binder, a folate receptor binder, a heat shock protein 90 binder, or an αvβ6 integrin binder; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; and P is the effective load.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein each T is:

8. The compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, wherein P is a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor or an immunomodulator.

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

10. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein each P is independently selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein: Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide; R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl; R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC 1-6 alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace; R 10 is independently selected in each instance from the group consisting of hydrogen, halogen, CN, -COOH, -CONH2, -NH2, -NHCH3, -N(CH3)2, -OH, and -OCH3; R 11 and R 12 are independently hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH; or R 11 and R 12 Together they form a 5- or 6-membered heterocyclic ring; R 13 and R 14 are independently hydrogen, C 1-6 Alkyl or C 1-6 Alkylamine; or R 13 and R 14 Together they form ether-N(R 15 )2 substituted C6 carbocycle; Each R 15 are independently hydrogen, C 1-6 Alkyl, -C(O)C 1-6 Alkyl, -C(O)NHC 1-6 Alkyl or -C(O)OC 1-6 Alkyl; wherein R 15 C 1-6 Alkyl is optionally substituted with halogen, hydroxy, phenyl or heteroaryl; or R 15 is a cleavable prodrug group; R 16 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3; R 17 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3; or R 16 and R 17 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein the heteroalkylene group is optionally substituted by R 20 replace; R 18 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3; or R 16 and R 18 Together they form a heteroalkylene group of the formula: -OC 2-10 Alkylene-O-, -NH-C 2-10 Alkylene-O- or -NH-C 2-10 Alkylene-NH-; wherein the heteroalkylene group is optionally substituted by R 20 replace; R 19 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, -OH, -OCH3 or -OCF3; R 20 For hydrogen, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or -OH; R 21 is hydrogen or C 1-6 alkyl; R 22 is hydrogen or C 1-6 Alkyl, wherein the C 1-6 The alkyl group is unsubstituted or R 24 replace; R 23 For hydrogen, C 1-6 alkyl or benzyl, wherein the C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution; R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl); R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl); Each Y 1 、Y 2 、Y 3 and Y 4 are independently -CH, -CF or N; Y 5 is CH2, NH or O; q is 0, 1, 2, 3, 4, or 5; and r is 0, 1, 2, 3, 4 or 5.

11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein P is: or a pharmaceutically acceptable salt thereof.

12. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein P is: or a pharmaceutically acceptable salt thereof.

13. The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, which has the following structure: or a pharmaceutically acceptable salt thereof.

14. A compound having the structure of Formula (II), Formula (III), Formula (IV), Formula (V) or Formula (VII): in: Each P is a tubulin polymerization inhibitor, a spindle kinesin inhibitor, a cyclin-dependent kinase inhibitor, an immunomodulator, an epidermal growth factor receptor inhibitor, or a microtubule inhibitor; Each EL is a cleavable peptide linker; Each L 1 , L 2 and L 3 are independently a divalent linker, A 1 is a trivalent linker; A 2 is a tetravalent linker; Each T is a target protein binder; and MOD is the pharmacokinetic modulator group.

15. The compound according to claim 14 or a pharmaceutically acceptable salt thereof, which has a structure of formula (II) or formula (III): in: P is a tubulin polymerization inhibitor, a spindle kinesin inhibitor, a cyclin-dependent kinase inhibitor, an immunomodulator, an epidermal growth factor receptor inhibitor, or a microtubule inhibitor; EL is a cleavable peptide linker; Each L 1 , L 2 and L 3 are independently bivalent linkers; A 1 is a trivalent linker; and Each T is a target protein binder.

16. The compound according to any one of claims 14 to 15, or a pharmaceutically acceptable salt thereof, wherein P is auristatin, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor 7 and / or 8 agonist, an epidermal growth factor receptor inhibitor or a taxane.

17. The compound according to claim 16, or a pharmaceutically acceptable salt thereof, wherein the auristatin is auristatin E, monomethyl auristatin E, or a derivative thereof; and wherein the taxane is paclitaxel or a derivative thereof.

18. The compound according to any one of claims 14 to 17, or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by cathepsin B, legumin, or neutrophil elastase.

19. The compound according to any one of claims 14 to 18, or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by neutrophil elastase.

20. The compound according to any one of claims 14 to 19 or a pharmaceutically acceptable salt thereof, which has the following structure: in: P is auristatin, spindle kinesin inhibitor, toll-like receptor 7 and / or 8 agonist, epidermal growth factor receptor inhibitor, or taxane; R a is hydrogen or -CH3; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; and T is the target protein binder.

21. The compound according to any one of claims 14 to 19 or a pharmaceutically acceptable salt thereof, which has the following structure: in: P is auristatin, spindle kinesin inhibitor, toll-like receptor 7 and / or 8 agonist, epidermal growth factor receptor inhibitor, or taxane; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; and Each T is a target protein binder.

22. The compound according to any one of claims 14 to 21, or a pharmaceutically acceptable salt thereof, wherein P is: or a pharmaceutically acceptable salt thereof; in: Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide; R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl; R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC 1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace; R 21 is hydrogen or C 1-6 alkyl; R 22 is hydrogen or C 1-6 Alkyl, wherein the C 1-6 The alkyl group is unsubstituted or R 24 replace; R 23 For hydrogen, C 1-6 alkyl or benzyl, wherein the C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution; R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl); R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl); q is 0, 1, 2, 3, 4, or 5; and r is 0, 1, 2, 3, 4 or 5.

23. The compound according to any one of claims 14 to 20, or a pharmaceutically acceptable salt thereof, having the following structure: in: T is the target protein binder; L 1 is a divalent linker; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; R 21 is hydrogen or -CH3; and SIL is C 2-18 Heteroalkylene.

24. The compound according to claim 22 or 23, or a pharmaceutically acceptable salt thereof, wherein R 21 is hydrogen or -CH3; and SIL is -(NH-(CR d 2) 1-4 -CO*; each R d are independently hydrogen or -CH3; and * represents a bond to P.

25. The compound according to any one of claims 22 to 24, or a pharmaceutically acceptable salt thereof, wherein SIL is where * represents a bond to P.

26. A compound according to any one of claims 14 to 24, wherein P is: or a pharmaceutically acceptable salt thereof.

27. The compound of any one of claims 14 to 24, wherein each T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, an αvβ6 integrin binding agent, or an αvβ3 integrin binding agent.

28. The compound according to any one of claims 14 to 26, or a pharmaceutically acceptable salt thereof, wherein T is: or a pharmaceutically acceptable salt thereof.

29. The compound according to any one of claims 14 to 28, or a pharmaceutically acceptable salt thereof, having the following structure: or a pharmaceutically acceptable salt thereof.

30. A compound or a pharmaceutically acceptable salt thereof having a structure of Formula (IV), Formula (V) or Formula (VII): in: Each P is a valid load; Each EL is a cleavable peptide linker; Each L 2 and L 3 are independently a divalent linker, A 1 is a trivalent linker; A 2 is a tetravalent linker; Each T is a target protein binder; and MOD is the pharmacokinetic modulator group.

31. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein each T is a prostate specific membrane antigen binding agent, a carbonic anhydrase 9 binding agent, a fibroblast activation protein binding agent, a folate receptor binding agent, a heat shock protein 90 binding agent, an αvβ6 integrin binding agent, or an αvβ3 integrin binding agent.

32. The compound according to claim 30 or 31, or a pharmaceutically acceptable salt thereof, wherein each P is a tubulin polymerization inhibitor, a topoisomerase inhibitor, an oxidative phosphorylation inhibitor, a kinase inhibitor, a dihydrofolate reductase inhibitor, a histone deacetylase inhibitor, a microtubule inhibitor, or an immunomodulator.

33. The compound according to claim 30 or 31, or a pharmaceutically acceptable salt thereof, wherein P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

34. The compound of any one of claims 30 to 33, or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by cathepsin B, legumin, or neutrophil elastase.

35. The compound of any one of claims 30 to 34, or a pharmaceutically acceptable salt thereof, wherein each cleavable peptide linker is cleaved by neutrophil elastase.

36. A compound according to any one of claims 30 to 35, or a pharmaceutically acceptable salt thereof, having the following structure: in: A # Indicates A 1 or A 2 ; R a is hydrogen or -CH3; R 1 is hydrogen, -CH3, -CH2CH3, -CH2CH2CH3, -CH2C(O)NH2 or -CH2C(O)OH; R 2 is -CH3, -CH(CH3)2, -CH2CH(CH3)2 or -CH(CH3)CH2CH3; R 21 is hydrogen or -CH3; and P is camptothecin or a camptothecin derivative, auristatin or an auristatin derivative, a spindle kinesin inhibitor, a cyclin-dependent kinase 9 inhibitor, a toll-like receptor agonist, an epidermal growth factor receptor inhibitor or a taxane.

37. A compound according to any one of claims 30 to 36, or a pharmaceutically acceptable salt thereof, wherein each P is: or a pharmaceutically acceptable salt thereof; in: Each R 6 and R 7 are independently hydrogen, halogen, CN, -C 1-6 Alkyl or C 1-6 alkyl halide; R 8 For hydrogen, halogen, CN, -C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 3-6 Cycloalkyl or 5- to 7-membered heterocycloalkyl; R 9 For hydrogen, halogen, CN, C 1-6 Alkyl, -C(O)NH2, -C(O)NHC 1-6 Alkyl, -C(O)N(C 1-6 Alkyl)2, -C(O)NHC 1-6 Alkyl-C(O)NHC 1-6 Alkyl, -C(O)NHC 1-6 Alkyl-NHC(O)C 1-6 Alkyl, -NH2, -NHC 1-6 Alkyl, -N(C 1-6 Alkyl)2, -NHC(O)C 1-6 Alkyl, -OH or -OC 1-6 Alkyl; wherein each C 1-6 Alkyl with 0 to 5 R 10 replace; R 21 is hydrogen or C 1-6 alkyl; R 22 is hydrogen or C 1-6 Alkyl, wherein the C 1-6 The alkyl group is unsubstituted or R 24 replace; R 23 For hydrogen, C 1-6 alkyl or benzyl, wherein the C 1-6 Alkyl or benzyl is unsubstituted or substituted with one, two or three R 25 group substitution; R 24 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -SH or -S(C 1-6 alkyl); R 25 -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 Alkyl), -N(C 1-6 Alkyl)2, -NHS(O)2(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Aminoalkyl or OCH2CH2NHC(O)(C 1-6 aminoalkyl); q is 0, 1, 2, 3, 4, or 5; and r is 0, 1, 2, 3, 4 or 5.

38. The compound according to any one of claims 30 to 37, or a pharmaceutically acceptable salt thereof, wherein each T is: or a pharmaceutically acceptable salt thereof.

39. A compound according to any one of claims 30 to 38, or a pharmaceutically acceptable salt thereof, wherein A 1 including amino acids (e.g., lysine, glutamine, or glutamic acid), wherein the amino acids are 2 and L 3 Bond formation, optionally via C 1-12 Alkyl or C 1-12 The heteroalkyl linker arm is bridged.

40. A compound according to any one of claims 30 to 39, or a pharmaceutically acceptable salt thereof, wherein A 2 including one or two amino acids (e.g., lysine, glutamine, glutamic acid, or a combination thereof), wherein the amino acids are 2 and L 3 Bond formation, optionally via C 1-12 Alkylene or C 1-12 The heteroalkylene linker arm is bridged.

41. A compound according to any one of claims 30 to 40, or a pharmaceutically acceptable salt thereof, wherein A 1 for: A 2 for and MOD is -COOH.

42. A compound according to any one of claims 30 to 41 or a pharmaceutically acceptable salt thereof, having the following structure: or a pharmaceutically acceptable salt thereof.

43. A compound according to any one of claims 1 to 4, 7 to 13, 15 to 19, or 22 to 28, 30 to 35, or 37 to 41, or a pharmaceutically acceptable salt thereof, wherein each L 1 , L 2 and L 3 is a divalent linker having a structure represented by the following formula: (i)-(CO) r (CH2) s (OC 2-6 alkylene) t (NH) u (CO) v -; (ii)-(CO) r (CH2) s (NR c C 1-6 alkylene) t (NR a ) u (CO) v -; (iii)-(CO) r (CH2) s (NR c C(O)C 1-6 alkylene) t (NR a ) u (CO) v -; (iv)-(CO) r (CH2) s (NR c C 1-6 alkylene) t (NR c C(O)C 1-6 Alkylene)(NR c C 1-6 alkylene) t (NR a ) u (CO) v -; (v)-(CO) r (CH2) s (NH) u (CO) v -; (vi)-(CO) r (CH2) s (OC 2-6 alkylene) t (NH) u (CO) v -(NH) u (CH2) s (OC 2-6 alkylene) t in: R a is independently selected at each occurrence from hydrogen or C 1-3 alkyl; R c is independently selected at each occurrence from hydrogen or C 1-3 alkyl; r is 0 or 1; s is 0 to 10; t is 1 to 10; u is 0 or 1; and v is 0 or 1.

44. A compound according to any one of claims 1 to 4, 7 to 13, 15 to 19, or 22 to 28, 30 to 35, or 37 to 41, or a pharmaceutically acceptable salt thereof, wherein each L 1 , L 2 and L 3 is a divalent linker having a structure represented by the following formula: (i)-(CO) r (CH2) s (OC 2-6 alkylene) t (NH) u (CO) v -; (ii)-(CO) r (CH2) s (NR c C 1-6 alkylene) t (NR a ) u (CO) v -;or (v)-(CO) r (CH2) s (NH) u (CO) v -; (vi)-(CO) r (CH2) s (OC 2-6 alkylene) t (NH) u (CO) v -(NH) u (CH2) s (OC 2-6 alkylene) t Among them, each R a and R c are independently hydrogen or -CH3; r is 0 or 1; s is 1 to 4; t is 1 to 10; u is 0 or 1; and v is 0 or 1.

45. A compound according to any one of claims 1 to 4, 7 to 13, 15 to 19, or 22 to 28, 30 to 35, or 37 to 41, or a pharmaceutically acceptable salt thereof, wherein each L 1 , L 2 and L 3 A divalent linker independently of the formula: -(CO) 0-1 -(CH2) 2-4 -[OC 2-6 alkylene] 1-8 -NH-(CO) 0-1 -; -(CO) 0-1 -(CH2) 2-4 -[OC 2-6 alkylene] 1-8 -NHCONH-[OC 2-6 alkylene] 1-8 ; -(CO) 0-1 -(CH2) 1-4 -[N(CH3)-C 1-6 alkylene] 1-8 -NH-(CO) 0-1 -, -(CO) 0-1 -(CH2) 1-4 -[N(CH3)-C 1-6 alkylene] 1-8 -N(CH3)-(CO) 0-1 -;or -(NH) 0-1 -(CH2) 1-10 -。 46. ​​A compound according to any one of claims 1 to 4, 7 to 13, 15 to 19, or 22 to 28, 30 to 35, or 37 to 41, or a pharmaceutically acceptable salt thereof, wherein: Each L 1 For: -CO-(CH2) 2-4 -[OCH2CH2] 1-8 -NHCONH-[OCH2CH2] 1-8 、-CO-(CH2) 2-4 -(OCH2CH2) 1-8 -NH-, -CO-(CH2) 2-4 -(N(CH3)CH2CH2) 1-8 -N(CH3)- or -CO-(CH2) 2-4 (N(CH3)CH2CH2) 1-8 -NH-; Each L 2 For: -CO-(CH2) s -(OCH2CH2) 1-8 -NH-; and Each L 3 For: -CO-(CH2) s -(OCH2CH2) 1-8 -NH- or -NH-(CH2) 1-10 .

47. A compound according to any one of claims 1 to 4, 7 to 13, 15 to 19, or 22 to 28, 30 to 35, or 37 to 41, or a pharmaceutically acceptable salt thereof, wherein each L 1 , L 2 and L 3 is a divalent linker comprising 2 to 20 polyethylene glycol groups.

48. A pharmaceutical composition comprising a compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; and at least one pharmaceutically acceptable excipient.

49. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use as a medicament.

50. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; for use in a method of treating a disease or condition.

51. The compound of claim 50, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the disease or disorder is a hyperproliferative disorder.

52. The compound of claim 51 or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the hyperproliferative disorder is an autoimmune disorder.

53. The compound of claim 51 or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the hyperproliferative disorder is cancer.

54. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is a solid tumor.

55. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is a hematological malignancy.

56. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is a B-cell malignancy.

57. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is a MYC-driven cancer.

58. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is an MCL1-driven cancer.

59. The compound according to claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or a mixture of stereoisomers thereof; wherein the cancer is a tumor that overexpresses MYC, MYB or MCL1 mRNA; or a MYC, MCL1 or MYB protein associated therewith.

60. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is a transcriptionally addicted tumor.

61. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is aggressive non-Hodgkin lymphoma (NHL), double hit diffuse large B-cell lymphoma (DH-DLBCL), high-grade B-cell lymphoma (HGBCL), transformed follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or Richter syndrome (RS).

62. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is relapsed / refractory (r / r) aggressive non-Hodgkin lymphoma (r / r NHL), relapsed / refractory double-hit diffuse large B-cell lymphoma (r / rDH-DLBCL), relapsed / refractory high-grade B-cell lymphoma (r / r HGBCL), relapsed / refractory transformed follicular lymphoma (r / r FL), relapsed / refractory mantle cell lymphoma (r / r MCL), relapsed / refractory chronic lymphocytic leukemia (r / r CLL), relapsed / refractory small lymphocytic lymphoma (r / r SLL), or relapsed / refractory Richter syndrome (r / r RS).

63. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is ovarian cancer, breast cancer, or prostate cancer.

64. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is advanced ovarian cancer, triple-negative breast cancer, or castration-resistant neuroendocrine prostate cancer.

65. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is neuroblastoma.

66. The compound of claim 53, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cancer is osteosarcoma.

67. The compound of claim 50, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the disease or disorder is an ophthalmic condition.

68. The compound of claim 67, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the ophthalmic condition is macular degeneration.

69. The compound or composition of claim 50, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the disease or disorder is a cardiovascular condition.

70. The compound or composition of claim 69, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; wherein the cardiovascular condition is cardiac hypertrophy.

71. A method of treating a disease or condition in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 47, or a pharmaceutically acceptable salt thereof; or a stereoisomer or mixture of stereoisomers thereof; or a pharmaceutical composition according to claim 43.

72. The method of claim 71, wherein the disease or disorder is a hyperproliferative disorder.

73. The method of claim 72, wherein the hyperproliferative disorder is cancer.

74. The method of claim 73, wherein the cancer is a MYC-driven cancer, an MCL1-driven cancer, or a MYB-driven cancer.

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