Novel linker-drug conjugates containing phosphoantigens, novel conjugates and their use in therapy

Abstract

The present invention relates to a compound of formula (I) [Formula 1] JPEG2026502845000045.jpg42170 (wherein L represents a linker, W 1 , W 2 , X 1-5 , x, m, n and R 1-4 The present invention relates to novel linker-drug compounds based on specific phosphoantigens (pAgs) having the general structure reflected in (wherein R is as defined herein). Also provided are conjugates comprising a targeting moiety, preferably a tumor-targeting antibody or antigen-binding fragment thereof, covalently attached to the linker-drug compounds of the invention. The conjugates can be used, for example, to treat diseases such as cancer, infectious diseases, and autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to novel linker-drug compounds comprising one or more phosphoantigen moieties covalently attached to a linking moiety for use in preparing conjugates.

[0002] The present invention also relates to conjugates of said linker-drug compounds comprising a targeting moiety, such as an antibody or a binding fragment thereof, pharmaceutical compositions containing said conjugates, and their use in the treatment of diseases such as cancer, infectious diseases and autoimmune diseases, optionally in combination with other therapeutic agents. [Background technology]

[0003] Conventional methods for treating cancer include surgery, cytotoxic chemotherapy, and radiation therapy, or a combination thereof. Treatment with cytotoxic drugs or radiation often results in severe side effects due to their toxicity and nonspecific nature. Since the discovery that the immune system plays an important role in eradicating neoplastic cells, modern cancer therapies aim to use components of the immune system as tools to treat cancer.

[0004] One approach in cancer immunotherapy is to target "immune checkpoints," such as T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death protein 1 (PD1), to activate antitumor immune responses in cancer patients. CTLA4 and PD1 are both proteins involved in negative feedback systems that function to limit immune cell activation. Tumor cells can "exploit" this inhibitory mechanism by overexpressing immune checkpoint ligands on their surface, thereby protecting themselves from attack by immune system cells and evading the immune system. When immune checkpoints interact with their ligands and become activated, they result in the inactivation and exhaustion of T cells. Immune checkpoint inhibitors, such as antibodies that bind to immune checkpoints or their ligands, are novel anticancer drugs that block overexpressed immune checkpoints on cancer cells. Examples of approved immune checkpoint inhibitors are ipilimumab (a CTLA4 inhibitor; trade name Yervoy®, manufactured by BMS), which was approved in 2011 for the treatment of melanoma, the PD1 antibody nivolumab (marketed under the trade name Opdivo®, developed by BMS), and pembrolizumab (trade name Keytruda®, another PD1 inhibitor, manufactured by Merck). Although checkpoint inhibitors can reactivate anti-tumor responses, activated immune cells may also attack normal tissues, leading to adverse immunological side effects.

[0005] Another approach to cancer treatment involves the use of antibody-drug conjugates (ADCs). ADCs combine the specificity of monoclonal antibodies for tumor-specific antigens with the cell-killing activity of chemical cytotoxic agents. The antibody in the ADC acts as a targeting agent and a carrier for the cytotoxic payload. Upon binding, the ADC, coupled with the cytotoxic payload, is efficiently taken up by the targeted tumor cells. The cytotoxic payload may be an inactive precursor (prodrug) of the cytotoxic agent attached to the antibody via a stable linker in the blood. After internalization by tumor cells, the prodrug may be cleaved, for example, by intracellular proteases. Cleavage of the linker may release the active cytotoxic payload in tumor cells. ADCs have the advantage of significantly reducing toxicity to healthy tissues and nonspecific side effects. Clinically approved ADCs include gemtuzumab (anti-CD33) ozogamicin (Mylotarg®; Wyeth Pharmaceuticals, a subsidiary of Pfizer), brentuximab (anti-CD30) vedotin (Adcetris®; Seattle Genetics / Millennium Pharmaceuticals), (ad)trastuzumab (anti-HER2) emtansine (Kadcyla®; Genentech / Roche), inotuzumab (anti-CD22) ozogamicin (Besponsa®; Wyeth Pharmaceuticals, a subsidiary of Pfizer), enfortumab (anti-nectin 4) vedotin (Padcev®; Astellas Pharma / Seattle Genetics), and fam-trastuzumab deruxtecan (Enhertz®; Daiichi Sankyo). These include polatuzumab (anti-CD79b) vedotin (Polivy®; Genentech / Roche), and sacituzumab (anti-TROP2) govitecan (Trodelvy®; Immunomedics). Many more are in clinical development.

[0006] Yet another approach to cancer treatment is immunotherapy, which uses therapeutic compounds to activate the immune system, particularly T cells, to attack and destroy tumor cells. Such therapeutic compounds may be agonists of immune cell receptors and may be large molecules or relatively small chemical structures. Examples of such compounds are ligands that activate Toll-like receptors (TLRs). Several TLR ligands have been approved for the treatment of cancer. The first approved TLR ligand (TLR agonist) was part of an attenuated strain of Mycobacterium bovis called Bacillus Calmette-Guérin (BCG). Originally developed as a tuberculosis vaccine, BCG contains active TLR2 / 4 ligands and is used to treat bladder cancer. Other approved TLR ligands are the TLR4 ligand monophosphoryl lipid A (MPLA) and the small molecule TLR7 agonist imidazoquinoline imiquimod.

[0007] TLR ligands have also been used in immunoconjugates. These immunoconjugates contain tumor antigen-specific antibodies as targeting vehicles for the TLR ligands, with the aim of inducing local activation of immune system cells in the tumor microenvironment. An immunoconjugate combining a TLR agonist and an anti-HER antibody for breast cancer treatment is described in WO 2017 / 072662 (Novartis AG). Additionally, an anti-HER conjugate carrying a TLR8 agonist payload has been developed by Silverback Therapeutics (ImmunoTAC™ SBT6050). Bolt Therapeutics (WO 2020 / 047187) and Ackerman et al., 2021, Nature Cancer, , Vol. 2(8), 18-33 also describe TLR immunoconjugates comprising a tumor-targeting monoclonal antibody conjugated via a non-cleavable linker to a TLR7 / 8 agonist (T785); the tumor-targeting antibody bound to the tumor antigen activates antigen-presenting cells present in the tumor microenvironment (TME) through Fc effector function, and the TLR agonist conjugated to the antibody directly stimulates APCs via the TLR receptor, promoting anti-tumor immunity.

[0008] A specific subset of T cells known to exhibit cytotoxicity against cancer cells is the γδ T cell (a T cell with a T cell receptor (TCR) consisting of γ and δ chains). γδ T cells are considered a unique subset of T lymphocytes due to their ability to mount rapid, innate-like immune responses against infections and tumor cells. Tumor-infiltrating γδ T cells have been found in many different malignancies (Gentles et al., Nature Medicine, 2015, 21(8), 938-945). γδ T cells, more specifically Vγ9Vδ2 T cells, which form the major subset of γδ T cells, are activated by a specific set of antigens known as "phosphoantigens." Naturally occurring phosphoantigens are small alkyl pyrophosphates, such as 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP). These natural phosphoantigens are produced by pathogenic cells, and HMBPP is the direct precursor of IPP (a pathogenic phosphoantigen not present in humans). Bacteria and parasites can produce isoprenoid precursors and biosynthesize IPP via the non-mevalonate pathway (MEP pathway) or the 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP / DOXP) pathway. In humans, pAg production is driven by the mevalonate pathway.

[0009] In contrast to TLR agonists, phosphoantigens do not directly interact with receptors displayed on myeloid or T cells. Intracellular (e.g., cancer) binding of phosphoantigens to the intracellular domain of the cell surface molecule butyrophilin 3A1 (BTN3A1) induces a conformational change in the extracellular portion of the BTN3A1 complex, possibly involving BTN2A1 (Sandstrom A, et al., 2014, Immunity, 40(4), 490-500, doi: 10.1016 / j.immuni.2014.03.003).

[0010] The conformational change of the extracellular BTN3A1 / BTN2A1 complex results in binding to the γδ TCR, leading to cytokine production by activated γδ T cells and killing of tumor / pathogenic cells (Rigau et al., Science, 2020, 367, 642). Thus, activation of γδ T cells using phosphoantigens as therapeutic agents is indirect; phosphoantigens act from within cells (e.g., tumor cells or infected cells) to induce conformation of the extracellular BTN3A1 / BTN2A1 complex on the cell surface, transmitting an activation signal to the γδ TCR on the γδ T cell. The γδ T cell then exerts its killing effect on tumor or infected cells.

[0011] Because pyrophosphate HMBPP has poor pharmacokinetic properties (rapid hydrolysis in plasma), in addition to (nitrogen-containing) bisphosphonate analogs, (monophosphate-type) prodrugs have been developed that are converted to active phosphoantigens after administration to a subject. In phosphoantigen prodrugs, the negatively charged, unbonded oxygen atom of the phosphonate group is protected with a neutral group, for example, to facilitate diffusion across cell membranes. The protecting group is removed upon entry into the cell, releasing the active phosphoantigen. Another approach to improving the half-life of phosphoantigens (particularly bisphosphonate phosphoantigens) is described in WO 2012 / 042024. Phosphoantigens were complexed with inorganic nanoparticles and lipid nanovectors that function as delivery vehicles. The resulting nanoparticles were described as capable of being coated with targeting ligands to target specific cells. Examples include molecules, such as antibodies, that induce targeting to cancer cells. The use of human transferrin was demonstrated.

[0012] Compounds with cellular pAg activity are believed to exert their activity directly by binding to the pAg receptor on target cells ("direct pAg"). This receptor is believed to be the intracellular domain of the cell surface molecule butyrophilin 3A1 (BTN3A1). Naturally occurring phosphoantigens include pyrophosphates (diphosphates) such as HMBPP and IPP. Known analogs of natural phosphoantigens include bromohydrin pyrophosphate (BrHPP) and pyrophosphonates such as C-HMBPP, the pyrophosphonate equivalent of naturally occurring HMBPP. Naturally occurring pAg, HMBPP, is produced by pathogenic bacteria. The allylic alcohol in natural pAgs, such as HMBPP, has been found to be important for maximizing BTN3A1 binding and pAg activity. Direct pAgs, such as HMBPP, bind directly to the intracellular B30.2 domain of BTN3A1. Other analogs of HMBPP, such as halohydrins BrHPP, IHPP, and ClHPP, are also known in the art (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030-1039).

[0013] Phosphoramidite esters, analogs of phosphoantigens that are said to have increased potency, are described in WO 2005 / 05258 (Innate Pharma), such as N-HDMAPP, in which the isoprene unit in native HMBPP is linked to pyrophosphate via its NH group.

[0014] Other compounds exhibit indirect pAg activity through the accumulation of IPP. Such compounds are referred to as "indirect pAgs." Indirect pAgs act on pathways that increase cellular levels of (endogenous) direct pAgs (e.g., IPP) and coactivation of Vγ9Vδ2 T cells. In contrast to direct pAgs, indirect pAgs do not directly interact with the butyrophilin receptor on target cells, nor are they pAg precursors (compounds that are enzymatically or chemically converted to direct pAg). Indirect pAgs can be compounds that inhibit downstream enzymes, such as farnesyl pyrophosphate synthase (FPPS). Inhibition of FPPS prevents the utilization of IPP, leading to its accumulation in cells. Known FPPS inhibitors are aminobisphosphonic acids (N-BPs) such as zoledronate (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030-1039; Park et al., 2021, Frontiers in Chemistry, Vol. 8, Article 612728).

[0015] Other examples of bisphosphonic acids with nitrogen or amino groups as substituents on the central carbon atom, which are thought to increase the efficacy of the bisphosphonic acid (Drake et al., Mayo Clin. Proc., 2008, 83(9), 1032-1045), include alendronate, risedronate, ibandronate, pamidronate, neridronate, and olpadronate.

[0016] Aminobisphosphonates (N-BPs), such as zoledronate, pamidronate, and alendronate, are also known as "bone-targeting agents" due to their ability to specifically bind to hydroxyapatite (HA) (Farrell et al., 2018, Bone Reports, 9, 47-60). Furthermore, alendronate was conjugated with trastuzumab to target bone metastases (Tian et al., 2021, Sci. Adv., 7, 2-11). Due to its negative charge, alendronate has a high affinity for HA and preferentially binds to bone. Therefore, Tian et al. proposed the use of negatively charged aminobisphosphonates, such as alendronate, as antibody targeting agents for the treatment of bone-related diseases.

[0017] Phosphoantigens are being tested for use in cancer therapy, aiming to enhance the cytotoxicity of γδ T cells against tumor cells by expanding them in vivo or in vitro with antigen-presenting cells for administration to subjects. Synthetic phosphoantigens, such as BrHPP (phosphostim, Innate Pharma) and zoledronate (Novartis), are the subject of clinical trials in cancer patients. The phosphoantigens tested have demonstrated acceptable safety profiles. However, their efficacy has generally been limited (Sebestyen et al., Nature Reviews Drug Discovery, 2020,19(3), 169-184).

[0018] Finding an acceptable therapeutic window / therapeutic concentration window for such treatments could be greatly improved by more potent, selective, and effective methods for delivering phosphoantigens to cells (over)expressing the butyrophilin (BTN3A1 / BTN2A1) complex, e.g., tumor or pathogenic cells. Summary of the Invention

[0019] The present invention provides an effective and selective method of using phosphoantigens, for example in the treatment of cancer. The present invention provides a method of treating cancer comprising administering to a subject a phosphoantigen of formula (I):

[0020] [ka]

[0021] (wherein L represents a linking moiety, W 1 is N, CH or CF, preferably CH; W 2 is CH2, CHF, CF2 or O; X 1 is O, S, NH, CH2, CHF or CF2; X 2 is O, CH2, CHF or CF2; X 3 is absent, O or NH; X 4a-d each is independently selected from O and S; X 5 teeth, - H, halogen (F, Cl, Br, I) or nitrile (CN), ethenyl, ethynyl, ethyl, optionally substituted with one or more of F, CH3, CH2F, CHF2, CF3; - C3-C4 cycloalkyl, C3-C4 cycloalkenyl, allyl, propynyl, O-CH3, S-CH3, or optionally substituted by one or more fluorine substituents; -CHR 1 OR 2 , CHR 1 SR 2 , CHO, CO2R 1 ,CONR 1 R 2 (where R 1 and R 2 are independently selected from H, CH3, CH2F, CHF2, CF3, CH2CH3; and; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R 1 is H, a connector to a linking moiety (L) or a prodrug moiety; R 2 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; R 3 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; R 4 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; Or, when n is 0, R 3 and R 2 is C 1-6 connected by a (hetero)alkyl group; or When n is 1 or 2, R 3 and R 4 is C 1-6 (Hetero)alkyl groups The present invention relates to linker-drug compounds having the general structure reflected in:

[0022] Preferably, X 5 is H or a halogen (Cl, F, I or Br), most preferably Br or Cl.

[0023] W 1 If CH, then R 1 is preferably H or a connecting portion to the linking portion (L). 3 If O, then R 3 is preferably a connection part with the linking part, and R 1 is H. In the linker-drug compounds of the invention, W 2 When X is CH2, m is preferably 1. 1 is preferably CH2.

[0024] In a preferred embodiment, X 3 is O and R 3 is the connection part with the connecting part, and W 1 is CH and R 1 is H and W 2 is CH2, m is 1, and X1 is CH2.

[0025] In the linker-drug compounds of the present invention, preferably n is 0 or 1 and X 4a-b and X 4c-d (if present) is O and R 2 and R 4 (if present) is H.

[0026] R 2 , R 3 and R 4 (if present) - pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) groups, - substituted or unsubstituted (hetero)aryl groups, - a structure represented by formula IV or V

[0027] [ka]

[0028] (In the formula, R a and R a’ are independently H, an optionally substituted amino acid side chain, and an optionally substituted C. 1-14 a non-polar side chain having an alkyl chain; R b is H, benzyl, or substituted or unsubstituted (C 1-8 ) alkyl, R c and R c’ are independently selected from H and optionally substituted C1-C6 alkyl, C3-C6 cycloalkyl, aryl or heteroaryl. It may also be a prodrug moiety selected from the group consisting of:

[0029] R 2 , R 3 and R 4 (if present) is a prodrug moiety, and R 2 , R 3and R 4 may be independently selected from POM and POC groups.

[0030] R 2 and R 3 is the prodrug moiety and n is 0, then R 2 is a substituted or unsubstituted 5- or 6-membered (hetero)aryl group, R 3 may be a structure represented by formula IV or V, or vice versa.

[0031] The linking moiety (L) is preferably a cleavable linking moiety. The linking moiety (L) is preferably a linking moiety having the structure represented by formula VI or VII.

[0032] [ka]

[0033] (In the formula, m is an integer from 1 to 10, preferably 5; AA is an amino acid, preferably a naturally occurring amino acid; p is 0, 1, 2, 3 or 4; q is an integer of 1 to 12, preferably 2; ES does not exist or

[0034] [ka]

[0035] (where, R 5 H, halogen, CF3, C 1-4 Alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, C 1-4 Alkoxy or C 1-4 alkylthio, preferably H, F, CH3, CF3, more preferably H or F, and V is H, ethyl, -(CH2CH2O) p-OMe, CH2CH2SO2Me, or CH2CH2N(Me), and p is an integer from 1 to 12. may also include:

[0036] The linker-drug compounds of the present invention may be used to prepare conjugates. Conjugates are also provided that include a targeting moiety, preferably a tumor-targeting antibody or an antigen-binding fragment thereof, covalently linked to the linker-drug compounds of the present invention. In such conjugates, the linking moiety preferably includes a cleavable linker. The conjugates of the present invention may be used as pharmaceuticals. Pharmaceutical compositions containing the conjugates of the present invention and one or more pharmaceutical additives are also provided.

[0037] Such complexes can be used to activate γδ T cells, for example, in the treatment of diseases such as cancer, infectious diseases or autoimmune diseases. The complexes of the invention can be used alone or in combination with other therapeutic agents.

[0038] Preferably, the conjugate of the present invention is an immunoconjugate comprising a tumor-targeting antibody or an antigen-binding fragment thereof as a targeting moiety. Such immunoconjugates of the present invention comprising a tumor-targeting antibody as a targeting moiety can be used to specifically deliver phosphoantigens to localized tumor cells, which may be internalized after the antibody or antigen-binding fragment thereof binds to a tumor-specific or tumor-associated antigen (TAA). [Brief explanation of the drawings]

[0039] [Figure 1] CD107a (A, C) and IFNγ (B) production by gated Vδ2γδ T cells (A, B) or NK cells (C) following co-culture of PBMCs with Raji cells pretreated with various concentrations of pAg ADC. The level of activation is shown as the percentage of CD107a- or IFNγ-positive immune cell subsets. Each compound was measured in duplicate per donor per experiment. [Figure 2]CD107a (A) and IFNγ (B) production by gated Vδ2γδ T cells after co-culture of PBMCs with Raji cells pre-treated with various concentrations of pAg ADC. The level of activation is shown as the percentage of CD107a- or IFNγ-positive immune cell subsets. [Figure 3] Binding of pAg ADC and rituximab to Raji cells was detected using a fluorochrome-conjugated goat anti-human antibody. Results from two independent experiments are shown as mean median fluorescence intensity (MFI) ± standard deviation. [Figure 4] CD107a (A, C) and IFNγ (B) production by gated Vδ2γδ T cells (A, B) or NK cells (C) after co-culture of PBMCs with Raji cells pretreated with various concentrations of pAg ADC or rituximab. The level of activation is shown as the percentage of CD107a- or IFNγ-positive immune cell subsets. Each compound was measured in duplicate per donor per experiment. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description of the Invention The present invention provides linker-drug compounds and conjugates containing a phosphoantigen (pAg) moiety.

[0041] Linker-drug compounds The present invention relates to a compound of formula (I)

[0042] [ka]

[0043] (In the formula, L represents a linking portion, W 1 is N, CH or CF, preferably CH; W 2 is CH2, CHF, CF2 or O; X 1 is O, S, NH, CH2, CHF or CF2; X 2is O, CH2, CHF or CF2; X 3 is absent, O or NH; X 4a-d each is independently selected from O and S; X 5 teeth, - H, halogen (F, Cl, Br, I) or nitrile (CN), ethenyl, ethynyl, ethyl, optionally substituted with one or more of F, CH3, CH2F, CHF2, CF3; - C3-C4 cycloalkyl, C3-C4 cycloalkenyl, allyl, propynyl, O-CH3, S-CH3, or optionally substituted by one or more fluorine substituents; -CHR 1 OR 2 , CHR 1 SR 2 , CHO, CO2R 1 ,CONR 1 R 2 (where R 1 and R 2 are independently selected from H, CH3, CH2F, CHF2, CF3, and CH2CH3 and; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R 1 is H, a connector to a linking moiety (L) or a prodrug moiety; R 2 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; R 3 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; R 4 is H, a connection to a linking moiety (L), Cat+, or a prodrug moiety; Or, when n is 0, R 3 and R 2 is C 1-6 connected by a (hetero)alkyl group; or When n is 1 or 2, R3 and R 4 is C 1-6 (Hetero)alkyl groups The present invention provides linker-drug compounds having the general structure reflected in:

[0044] X 5 When is ethenyl, ethynyl or ethyl, X 5 may have one or more of the following substituents: F, CH3, CH2F, CHF2, CF3. Thus, the ethenyl, ethynyl or ethyl may be substituted with one or more fluorine substituents. X 5 is ethenyl, ethynyl or ethyl substituted with one or more (fluorinated) methyl groups (CH3, CH2F, CHF2 or CF3), the number of (fluorinated) methyl groups is preferably 1. This includes 5 is (iso)propyl.

[0045] C3-C4 cycloalkyl includes cyclopropyl, methylcyclopropyl, and cyclobutyl. C3-C4 cycloalkenyl includes cyclopropenyl, methylcyclopropenyl, and cyclobutenyl. X 5 is C3-C4 cycloalkyl, C3-C4 cycloalkenyl, allyl, propynyl, O-CH3, S-CH3, X 5 may be substituted with one or more fluorine substituents. 5 The number of fluorine substituents in X 5 It depends on the structure of X 5 can have multiple fluorine substituents on different C atoms, on the same C atom or on both.

[0046] Preferably, X 5 is H, Cl, F, I or Br, most preferably Cl or Br.

[0047] The linker-drug compounds of the present invention are modifications of the linker-drug compounds disclosed in patent application WO 2023 / 275025 (applicant Byondis BV), 5 The definition is different from this.

[0048] The linker-drug compounds of the invention comprise at least one phosphoantigen moiety (pAg or "drug"), as shown in the structural formula within the outer brackets of Formula I, attached to a linking moiety (L or "linker"). The number of pAg moieties per linker is designated "x." When x is greater than 1, multiple pAg moieties are attached to a single (branched) linker moiety.

[0049] Preferably, n is 0 or 1, and most preferably 0. When n is 1 or 2, X 2 is preferably O. Similarly, when n is 1 and X 2 is CH2, or n is 1 and X 2 Also part of the invention are linker-drugs having phosphoantigen moieties where each X is O. 4a-d is preferably O. In such linker-drug compounds, R 3 or R 1 may represent a connection part with the linking part, and preferably R 3 indicates the connection part with the connecting part.

[0050] If n is 2, then X 2 occurs twice in formula I, and X 2a and X 2b and are independently selected from O, CH2, CHF, and CF2. When n is 2, R 4 Also appears twice, R 4a and R 4b and are independently selected from H, a linking moiety (L), Cat+, and a prodrug moiety. 4c and X 4d Similarly, they occur twice (X 4c , X 4ci , X 4d and X 4di ), may be independently selected from O and S.

[0051] When m is 2 or 3, W 2 appears multiple times in formula I, and each W 2 are independently selected from CH2, CHF, CF2 or O. Preferably, W2 is CH2. In a preferred embodiment, m is 1, and when m is 1, W 2 is most preferably CH2.

[0052] Cat+ denotes a proton-containing (organic or inorganic) cation.

[0053] X 1 is preferably CH2, O or S, most preferably CH2.

[0054] each X 4a-d (when present) is preferably O. When n is 1 or 0, X 4a-b and X 4c-d (if present) is O and R 2 and R 4 Compounds in which (when present) is preferably H are part of the present invention.

[0055] Preferably, n is 0 and X 4a and X 4b is O and R 2 is preferably H.

[0056] Preferably W 1 is CH or CF, most preferably CH. 1 is preferably H or a connecting portion to the linking moiety (L), and is most preferably H.

[0057] Preferably, W 1 is CH and X 1 is CH2 and R1 is H, resulting in a linker-drug molecule having a pAg moiety containing an allylic alcohol group. 1 If CH, then X 1 is CH2, R1 is H, W2 is CH2, and m is 1.

[0058] Preferably, X 3 is O and R 3 is the connection with the disconnectable link, W 1 is CH, R 1 is H and W 2is CH2, m is 1, and X 1 is CH2.

[0059] In such compounds, R 2 , R 3 and / or R 4 may be a prodrug moiety alone, or X 4b , X 4d and / or X 3 (X 3 When present, each of the -X 4b -R 2 , -X 4d -R 4 and / or -X 3 -R 3 (It is).

[0060] Preferably, W 1 is CH, W 2 is CH2, X 4a-d is O and R 2 and R 4 is H and m is 1.

[0061] In a preferred embodiment, W 1 is CH, W 2 is CH2, n is 0, and X 4a-b is O and m is 1.

[0062] In Formula I, x indicates the number of phosphoantigen moieties (pAg) per linking moiety (L), and the structure in parentheses is the structure of a phosphoantigen moiety preferably used in the linker-drug compounds of the present invention. X can be an integer from 1 to 5 (each linking moiety has 1 to 5 pAg). Preferably, a linking moiety has one or two pAg moieties. In many cases, it may be sufficient for each linking moiety to have one pAg.

[0063] The connection part with the connecting part is R 1 (part of) or the connecting part is R 2 , R 3 or R 4Preferably, R 1 or R 3 One of these is the connection part with the connecting part, and R 3 More preferably, the linking moiety is R 3 If they are connected by X 3 is preferably O. 3 If is the connection part with the connecting part, X 3 is preferably O, and R 1 is preferably H.

[0064] The connecting part is R 2 or R 4 If the bond is at the X position, 4b or X 4d is preferably O.

[0065] Preferred linker-drug compounds are 3 is O and R 3 is at the connection with the cleavable linking moiety, and preferably W 1 is CH, R 1 is H and W 2 is CH2, m is 1, and X 1 is CH2. In such compounds, n is preferably 0.

[0066] "Junction with the linking moiety" refers to the position within the molecule where the linker connects to the phosphoantigen moiety. 1 , R 2 , R 3 or R 4 (depending on the site of attachment of the linker) is not meant to indicate the actual (remaining) structural elements of the linker-drug compound between the linker and the remainder of the phosphoantigen moiety. For example, depending on the linker used, R 1 When R is the connection to the linking moiety, it also includes the situation where the linker is directly connected to an oxygen atom of the phosphoantigen moiety in the linker-drug molecule. 1 is the connection part with the connecting part (L). R 1 is the connecting portion with the connecting portion (L), preferably, W 1is CH, W 2 is CH2, m is 1, and X 1 When such a linker-drug molecule is incorporated into a conjugate of the invention, cleavage of the linker after administration leaves an allylic alcohol group (R 1 (is H) can be (re)formed. 1 may be a prodrug moiety. Suitable alcohol prodrug moieties are known in the art. For example, an alcohol can be masked by an ester-based prodrug group. The generation of the activated alcohol relies on hydrolysis of the ester bond by (cellular) esterases, metabolically regenerating the alcohol (drug) and carboxylic acid (leaving group).

[0067] R 2 , R 3 and R 4 can each independently be H, a connection to a linking moiety (L), Cat+, or a prodrug moiety. In a preferred embodiment, the linker-drug compounds of the present invention are monophosphonic acids (n is 0), and therefore R 4 does not exist.

[0068] Cat+ denotes a cation (organic or inorganic) that contains a proton (and may be exchanged in the formulation buffer or plasma). 2 , R 3 and / or R 4 When R is Cat+, the Cat+ may be the same or different. 2 , R 3 and / or R 4 If is Cat+, then X 4b , X 4d (present, i.e., if n is not 0), and / or X 3 is O, and O - Cat + This becomes:

[0069] In another embodiment of this invention where n is 0, R 3 and R 2 is C 1-6(hetero)alkyl groups. In this case, R 3 and R 2 are taken together to form a substituted or unsubstituted 5- to 8-membered ring. In such embodiments, the linking moiety is R 1 In another embodiment where n is not 0, R 3 and R 4 Similarly, C 1-6 It may be connected by a (hetero)alkyl group.

[0070] R 2 , R 3 and / or R 4 can be the prodrug moiety alone, or X 4b , X 4d and / or X 3 (X 3 When present, each of the -X 4b -R 2 , -X 4d -R 4 and / or -X 3 -R 3 (It is).

[0071] A "prodrug moiety" can be a group that can be cleaved (to release an active compound) either enzymatically or non-enzymatically. A "prodrug moiety" may induce the release of a second prodrug moiety at another site in the molecule after the conjugate of the invention is administered to a subject. Preferably, the phosphoantigen moiety in prodrug form is converted to a functionally active phosphoantigen in target cells (e.g., tumor cells), for example, by enzymatic removal of the prodrug moiety.

[0072] Examples of prodrug technology known in the art include the use of pivaloyloxymethyl (POM) or isopropyloxycarbonyloxymethyl (POC) groups. In preferred embodiments, at least R 2 and R 3 are independently selected from a POM group or a POC group (e.g., when n is 0). When n is 1 or 2, R 4The alkyl group may also be a POM group or a POC group.

[0073] Phosphoantigen prodrugs of this type are described, for example, in WO 2019 / 182904.

[0074] Alternatively, a combination of free radicals can be used. An example of such a prodrug technology is the "ProTide" technology, which was developed for the intracellular delivery of monophosphates and monophosphonates. The hydroxyl of the monophosphate or monophosphonate in the ProTide prodrug is masked (or replaced) by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved in the cell to release the free monophosphate and monophosphonate (Mehellou et al. 2018, Journal of Medicinal Chemistry, 61(6), 2211-2226).

[0075] Therefore, if the phosphoantigen is a monophosphate or monophosphonate, and R 2 and R 3 Linker-drug compounds and conjugates of the invention in which R is a "ProTide" radical combination are also part of the invention. In those cases where the phosphoantigen moiety is a ProTide prodrug of the phosphoantigen, R 2 is the aromatic part and R 3 is an amino acid ester moiety, or vice versa. In a preferred embodiment of the present invention, when n is 0, R 2 or R 3 may be a substituted or unsubstituted (hetero)aryl group, (R 2 or R 3 The other is represented by the structures IV and V

[0076] [ka]

[0077] (In the formula, R a and R a’are independently H, an optionally substituted amino acid side chain, and an optionally substituted C. 1-14 a non-polar side chain having an alkyl chain; R b is H, benzyl, or substituted or unsubstituted (C 1-8 ) alkyl, R c and R c’ are independently H or optionally substituted (C 1-6 ) alkyl, (C 3-6 ) cycloalkyl, aryl, or heteroaryl, or R c and R c’ may, together with the nitrogen to which they are attached, form an optionally substituted ring, for example, an aziridino, azetidino, morpholino, piperazino, pyrrolidino, or piperidino ring. may be selected from:

[0078] R c and / or R c’ The substituents are carboxylic acid bioisosteres, amino, tetrazole, sulfonate, hydroxyl, halo, or alkyl.

[0079] R b When is a substituted alkyl, the substituents are hydroxy, amino, halo, nitro, cyano, carboxy, NR x R y , (C 1-6 ) alkoxy, (C 1-6 ) alkanoyl, (C 1-6 ) alkoxycarbonyl, (C 1-6 ) alkylthio and (C 2-6 ) alkanoyloxy; R x and R y is H, (C 1― C6) alkyl, (C 3-6 ) cycloalkyl and (C 3-6 ) Cycloalkyl(C 1-6 ) alkyl; or R x and R ytogether with the nitrogen to which they are attached form an aziridino, azetidino, morpholino, piperazino, pyrrolidino, or piperidino group.

[0080] connecting part The linking moiety (or "linker") used in the conjugates or linker-drug compounds of the invention is preferably a synthetic linker. The linker's structure is such that it can be readily chemically coupled to a low molecular weight effector molecule (phosphoantigen), and the resulting linker-drug compound can be readily conjugated to another substance, such as a polypeptide (e.g., an antibody). The choice of linker can affect the stability of the conjugate in blood and, if any, the manner in which the low molecular weight effector compound (phosphoantigen) is released. Suitable linkers are described, for example, in Ducrry et al., 2010, Bioconjugate Chem., 21, 5-13; King and Wagner, 2014, Bioconjugate Chem., 25, 825-839; Gordon et al., 2015, Bioconjugate Chem., 26, 2198-2215; Tsuchikama and An, 2018, Protein & Cell, 9, 33-46 DOI: 10.1007 / s13238-016-0323-0; Polakis, 2016, Pharmacological Reviews, 68 (1), 3-19, DOI: 10.1124 / pr.114.009373; Bargh et al., 2019, Chem. Soc. Rev., 48, 4361-4374, DOI: 10.1039 / c8cs00676h; WO 02 / 83180, WO 2004 / 043493, WO 2010 / 062171, WO 2011 / 133039, WO 2015 / 177360, and WO 2018 / 069375. Linkers may be cleavable or non-cleavable, for example, as described in van Delft, F and Lambert, JM, 2021, Chemical Linkers in Antibody-Drug Conjugates (ADCs), 1st Ed. Royal Society of Chemistry, ISBN-10: 1839162635.Another method for linker-drug conjugation to antibodies is to use transpeptidases (e.g., bacterial sortase, plant asparaginyl endopeptidase) to site-specifically conjugate chemical moieties to appropriate synthetic peptides. Sortase A (Sort-A) recognizes the C-terminal peptide sequence (LPXTG) and creates a bond between the threonine in this sequence and the N-terminal glycine of the conjugation partner (e.g., a glycine-tagged payload, an ADC) (Combs et al., 2015, the AAPS Journal, Vol. 17, No. 2, 339-351, DOI: 10.1208 / s12248-014-9710-8). Antibody-drug conjugation can also be achieved by site-specific glycoengineering, for example, using endo-β-N-acetylglucosaminidase (ENGase) and monosaccharyltransferase mutants (Manabe et al., 2021, Chem Rec, (11), 3005-3014, doi: 10.1002 / tcr.202100054; Wang et al., 2019, Annu Rev Biochem, 20; 88, 433-459, doi: 10.1146 / annurev-biochem-062917-012911).

[0081] The use of a cleavable linker in the conjugates of the present invention is preferred. The cleavable linker comprises a moiety that is cleaved, for example, by a lysosomal protease or when exposed to an environment of acidic pH or high reduction potential. Suitable cleavable linkers are known in the art and include, for example, mono-, di-, tri-, or tetrapeptides, i.e., 1, 2, 3, or 4 amino acid residues. Furthermore, the cleavable linker may comprise a self-immolative moiety, such as an ω-aminoaminocarbonyl cyclization spacer (see Saari et al., 1990, J. Med. Chem., 33, 97-101) or an -NH-CH2-O- moiety. Other cleavable linkers known in the art are, for example, β-glucuronide linkers, as disclosed in Jeffrey et al., 2006, Bioconjugate Chem. 2006, 17, 831-840. Cleavage of the linker makes the immunomodulatory effector moiety (phosphoantigen or "pAg" moiety) in the conjugate of the invention available to its surrounding environment. Non-cleavable linkers also effectively release the (active derivative of) the phosphoantigen moiety from the immunoconjugate of the invention, for example, when the conjugated polypeptide (antibody) is degraded in the lysosome. Non-cleavable linkers include, for example, succinimidyl-4-(N-maleimidomethyl(cyclohexane)-1-carboxylate and maleimidocaproic acid and analogs thereof.

[0082] To enable conjugation of a linking moiety or linker-drug compound to a polypeptide such as an antibody, the linking moiety (covalently) bound to the antibody typically has a functional group capable of reacting with an amino acid residue of the antibody under relatively mild conditions. This functional group is referred to herein as the reactive moiety (RM). Examples of reactive moieties include, but are not limited to, carbamoyl halides, acyl halides, active esters, anhydrides, α-haloacetyls, α-haloacetamides, maleimides, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, hydrazides, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyls, methyl sulfonates, cyclooctynes, and trans-cyclooctene (TCO). The amino acid residue reactive with the functional group may be a natural or unnatural amino acid residue, or a (un)natural glycan (Manabe et al., Wang et al., supra). In this specification, the term "unnatural amino acid" is intended to denote a (synthetically) modified amino acid or a D-stereoisomer of a naturally occurring amino acid. Preferably, the amino acid residue with which the functional group is reacted is a natural amino acid.

[0083] The linking moiety (L) used in the conjugates or linker-drug compounds of the present invention can be represented by Formula VI or VII

[0084] [ka]

[0085] (wherein m is an integer of 1 to 10, preferably 5; A is an amino acid, preferably a natural amino acid; and p is 0, 1, 2, 3, or 4.) When p is 2 or more, the amino acids may be the same or different.

[0086] Suitable amino acid combinations are known in the art and include amino acids selected from the group consisting of alanine, glycine, lysine, phenylalanine, valine, and citrulline. Preferably, p is 2. When p is 2, AA2 may be, for example, phenylalanyl-lysine, valylalanine, valylcitrulline, or valyllysine. When p is 2, AA2 is preferably valylalanine or valylcitrulline. When p is 3, AA3 may be, for example, alanyl-phenylalanyl-lysine, and when p is 4, AA4 may be, for example, glycyl-glycyl-phenylalanyl-glycine.

[0087] "q" is an integer from 1 to 12, preferably 2; ES is absent or

[0088] [ka]

[0089] (where R 5 is H, halogen, CF, C alkyl, C alkenyl, C alkynyl, C alkoxy or C alkylthio, preferably H, F, CH or CF, more preferably H or F; V is H, ethyl, -(CHCHO) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, where p is an integer from 1 to 12.

[0090] If an ES is present, the ES is preferably

[0091] [ka]

[0092] (In the formula, R 5is H, halogen, CF, C alkyl, C alkenyl, C alkynyl, C alkoxy or C alkylthio, preferably H, F, CH or CF, more preferably H or F; V is H, ethyl, -(CHCHO) p -OMe, CH2CH2SO2Me, or CH2CH2N(Me)2, and p is an integer from 1 to 12. is selected from.

[0093] ES,

[0094] [ka]

[0095] (In the formula, R 5 is H, halogen, CF, C alkyl, C alkenyl, C alkynyl, C alkoxy or C alkylthio, preferably H, F, CH or CF, more preferably H or F; V is H, ethyl, -(CHCHO) p -OMe, CH2CH2SO2Me, or CH2CH2N(Me)2, and p is an integer from 1 to 12. If selected from AA p is preferably absent (p means 0).

[0096] Linking moieties may be branched, in which case one linking moiety can have multiple phosphoantigen moieties. Examples of branched linking moieties include:

[0097] [ka]

[0098] is.

[0099] These branched linker moieties can be used to create conjugates with relatively high pAg to targeting moiety ratios ("DAR"). Using such branched linkers, conjugates with DARs of 16 and even 20 or more can be synthesized. Antibody-based conjugates of the invention may have a DAR of only about 2. However, for antibodies against tumor-specific targets known to be relatively poorly expressed in target tumor cells, conjugates with high pAg to targeting moiety ratios may be preferred. Linker-drug compounds used in the linker-drug compounds of the invention may have a linking moiety selected from, for example, the following:

[0100] [ka]

[0101] [ka]

[0102] The connector (L) may be conjugated to a pAg moiety to provide a linker-drug compound of the invention having the general formula shown in Formula I.

[0103] The linker-drug compounds of the invention can be conjugated to a targeting moiety to form the conjugates of the invention. A preferred conjugate of the invention comprises a tumor-targeting antibody or antigen-binding fragment thereof conjugated to a linker-drug compound of the invention.

[0104] In a particular embodiment of the invention, the phosphoantigen moiety as part of the conjugate of the invention is a monophosphonate prodrug, in which the negatively charged non-linking oxygen atom of the phosphonate group is protected by a combination of a ProTide moiety (a (hetero)aryl group and an amino ester radical) or one or more prodrug moieties such as POM or POC, while the cleavable linking moiety may be attached to an isoprene unit in the phosphoantigen molecule that is converted to an allylic alcohol present in the phosphoantigen, such as HMBPP, upon cleavage of the linker.

[0105] Examples of the synthesis of linker-drug molecules of the present invention are further illustrated in the Examples. Examples of preferred linker-drug compounds of the present invention are shown in Table 1 in the Examples section of this application.

[0106] It should be understood that a linker-drug compound comprising at least one phosphoantigen moiety covalently attached to a linking moiety of the invention, when included in a conjugate of the invention, may lose or gain a certain atom or group of atoms, e.g., it may lose a hydrogen atom compared to the same linker-drug compound of the invention when not included in a conjugate. This may be, for example, because the linker-drug compound of the invention is conjugated to a polypeptide, e.g., by esterification of a hydroxyl moiety.

[0107] For example, when the targeting moiety is an antibody or antigen-binding fragment thereof, one or more linker-drug compounds of the invention can be conjugated to the targeting antibody to form a conjugate of the invention.

[0108] Complex The invention provides conjugates comprising a targeting moiety (Tm) covalently attached to one or more linker-drug compounds of the invention.

[0109] The conjugates of the present invention comprise a targeting moiety that specifically binds to a target cell. Preferably, the targeting moiety is a tumor-targeting antibody or an antigen-binding fragment thereof. The targeting moiety serves as a delivery vehicle; it delivers the pAg moiety, covalently linked to the targeting moiety, to the target cell. The pAg may be directly linked, for example, to an amino acid side chain in the (polypeptide) targeting moiety. Preferably, the pAg is conjugated to the targeting moiety via a linking moiety.

[0110] Preferred conjugates of the present invention have the general formula II: Tm-(L-(pAg) x ) y (II) where Tm represents a targeting moiety, preferably an antibody or antigen-binding fragment thereof; L represents a linking moiety, corresponding to the "L" portion of Formula I; pAg represents a phosphoantigen moiety, corresponding to the structural formula in the outer parentheses of Formula I; x (as in Formula I) represents the number of phosphoantigen moieties per linking moiety, ranging from 1 to 5; and y represents the number of L-(pAg) per Tm. x The number of pAgs per conjugate in Formula II (the ratio of pAg to Tm) is x multiplied by y. The average ratio of pAg to Tm can be from 1 to 16, or 20, or even more. The ratio of pAg units per targeting moiety can be varied, for example, depending on the structural or functional characteristics of either the phosphoantigen moiety or the targeting moiety. In fact, even a number as low as 2-8, 2-6, or even 2 pAgs per targeting moiety may provide sufficient therapeutic effect. Preferably, the linking moiety has one or two pAgs. In most cases, one pAg per linking moiety may be sufficient. Preferably, the ratio of target pAg to Tm is 2 (x is 1 and y is 2).

[0111] The linker moiety is preferably a cleavable linker moiety. Linear or branched linker moieties may be used in the conjugates of the present invention. When multiple phosphoantigen moieties are linked to a single targeting moiety, each phosphoantigen moiety may be covalently linked to the targeting moiety by a separate linking moiety. Indeed, when the targeting moiety is an antibody and the linkage occurs via reduced interchain disulfides, there may be up to eight separate linking moieties (linker-drug moieties) linked to a single targeting moiety, resulting in eight phosphoantigen moieties per targeting moiety when each phosphoantigen moiety is carried by its own linking moiety. Alternatively, a branched linker moiety may contain 1 to 5 phosphoantigen moieties (x=1, 2, 3, 4, or 5) per linking moiety.

[0112] Branched linkers are particularly preferred when a high pAg to Tm ratio is desired or when the available conjugation sites on the targeting moiety are limited. For example, a branched linker with two pAgs (x=2) can be used to increase the number of phosphoantigen moieties per targeting moiety. Using such linking moieties, for example, 16 phosphoantigen moieties can be conjugated to a targeting moiety with only eight linking moieties. Antibodies can be modified to include additional cysteines in their amino acid sequence that can form disulfide bonds and be reduced for conjugation with a linker-drug molecule. For example, an additional cysteine ​​can be introduced at position 41C, as disclosed in WO 2015 / 177360. For antibodies with up to 10 available cysteines for conjugation to which linking moieties can be attached, a DAR of 20 (x=2 and y=10) or greater can be achieved using a branched linker with two pAgs (x=2) per linker. Under optimal conditions, all binding sites on the targeting moiety are occupied by linking moieties. In practice, a mixture of complexes may be produced, in which case the exact number of phosphoantigen moieties per targeting moiety may vary slightly depending on reaction conditions, and the y value is an average number.

[0113] The conjugates of the present invention may be used in combination with other pharmaceutically active compounds, which can be administered simultaneously or sequentially to a subject in need thereof. Furthermore, the targeting moiety may have a combination of different payloads and the linker-drug compounds of the present invention. The advantage of such a "multiple payload" approach is that different active substances can be targeted by the same targeting moiety. The ratio between the payloads, as well as the reaction conditions and binding sites (for conjugation), must be appropriately determined. Separate linker-drug compounds for each payload may be conjugated, for example, to different binding sites on the targeting moiety (e.g., different types of amino acids) and / or by different conjugation methods and / or linkers of different chemical natures to adjust the binding, distribution, and drug-antibody ratio (DAR) of the different payloads. Antibody-drug conjugates (ADCs) with multiple cytotoxic payloads are known in the art. The conjugates of the present invention may combine a phosphoantigen moiety with a cytotoxic payload or another immunomodulatory payload, for example, designed to enhance the overall desired therapeutic effect. Therefore, nonspecific binding and / or effects of phosphoantigens at non-target sites on non-target tissues are reduced.

[0114] As is well known in the art, drug binding in ADCs can be determined, for example, by hydrophobic interaction chromatography (HIC) or reverse-phase high performance liquid chromatography (RP-HPLC). HIC is particularly suitable for determining the average DAR (ratio of pAg to Tm in conjugates of the invention).

[0115] Targeting part The targeting moiety specifically or preferentially binds to the target cell and may be a targeting antibody or antigen-binding fragment thereof or another targeting moiety such as a nucleic acid (aptamer) or a (poly)peptide, which may be an enzyme inhibitor, enzyme substrate, receptor ligand and / or fusion protein. Small molecule inhibitors can also be used as targeting moieties (resulting in small molecule drug conjugates (SMDCs)). The binding specificity (and affinity) of the targeting moiety for the target determines the site in the body where the conjugate of the invention exerts its therapeutic effect.

[0116] Thus, selection of an appropriate targeting moiety ensures that the phosphoantigen is delivered to the site where it must exert its therapeutic effect.

[0117] Preferably, the targeting moiety in the conjugates of the present invention is an antibody or an antigen-binding fragment thereof. When the targeting moiety is an antibody or an antigen-binding fragment thereof, the conjugate is generally called an immunoconjugate or antibody-drug conjugate (ADC). The targeting moiety is an antibody that recognizes an antigen expressed in target cells, such as a tumor-associated antigen, with high specificity. The specificity of the antibody or its fragment for the antigen allows for the specific delivery of the effector molecule (or "payload") to the target cell, with minimal effect on healthy tissue. The effector molecule is covalently attached to the antibody via a linker, which ensures that the effector molecule remains attached to the antibody at least until the antibody reaches the target cell, e.g., a cancer cell. The effector molecule exerts its effect on or in the target cell (if the conjugate is internalized) when the antibody binds to the target. The effector molecule can be a cytotoxic agent, a radioisotope, or an immunomodulatory moiety. In the conjugates of the present invention, the effector molecule is a phosphoantigen moiety.

[0118] antibody As used herein, the term "antibody" preferably refers to an antibody comprising two heavy chains and two light chains. Generally, an antibody or an antigen-binding fragment thereof has therapeutic activity, but such independent efficacy is not necessarily required, as is known in the field of ADCs. The antibody that can be used in the present invention may be of any isotype, for example, IgA, IgE, IgG, or IgM. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. The antibody may be a chimeric, humanized, or human antibody. Preferably, the antibody is a humanized or human antibody. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG1 monoclonal antibody. The antibody may have a κ or λ light chain, preferably a κ light chain, i.e., a humanized or human IgG1-κ antibody.

[0119] As used herein, the term "antigen-binding fragment" includes Fab, Fab', F(ab')2, Fv, scFv, or reduced IgG (rIgG) fragments, single-chain (sc) antibodies, single-domain (sd) antibodies, diabodies, or minibodies.

[0120] A "humanized" non-human (e.g., rodent) antibody is an antibody with minimal sequence derived from a non-human antibody (e.g., a non-human-human chimeric antibody). Various methods for humanizing non-human antibodies are known in the art. For example, the antigen-binding complementarity-determining regions (CDRs) in the variable regions (VRs) of the heavy chain (HC) and light chain (LC) are derived from antibodies of a non-human species, typically mouse, rat, or rabbit. These non-human CDRs are combined with human framework regions (FRs, i.e., FR1, FR2, FR3, and FR4) of the HC and LC variable regions such that the functional properties of the antibody, such as binding affinity and specificity, are at least partially retained. To further refine antibody performance, for example, to improve binding affinity while maintaining low immunogenicity, selected amino acids in the human FRs may be replaced with corresponding amino acids from the original non-human species. The variable regions thus humanized are typically combined with human constant regions. A typical method for humanizing non-human antibodies is the method of Winter and coworkers (Jones et al., 1986, Nature, 321, 522-525; Riechmann et al., 1988, Nature, 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Alternatively, non-human antibodies can be humanized by modifying their amino acid sequences to increase their similarity to antibody variants naturally produced in humans. For example, selected amino acids in the original non-human species FR can be replaced with their corresponding human amino acids to reduce immunogenicity while retaining the antibody's binding affinity. For details, see Jones et al., supra; Riechmann et al., supra, and Presta, 1992, Curr. Op. Struct. Biol. 2, 593-596.See also the following reviews and references cited therein: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma and Immunol., 1, 105-115; Harris, 1995, Biochem. Soc. Transactions, 23, 1035-1038; and Hurle and Gross, 1994, Curr. Op. Biotech., 5, 428-433.

[0121] CDRs can be determined by the method of Kabat (in Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, NIH publication no. 91-3242, pp. 662, 680, 689), Chothia (et al., 1989, Nature, 342, 877-883) or IMGT (Lefranc, 1999, The Immunologist, 7, 132-136).

[0122] Typically, antibodies are monospecific (specific for one antigen; such antigen may be common between species or may have similar amino acid sequences between species) or bispecific (specific for two different antigens in a species) antibodies that contain at least one HC and LC variable region that binds to an antigen target, preferably a membrane-bound antigen target that may or may not be internalized. Preferably, after binding to its (antigen) target, the antibody is internalized into the target cell, after which the active effector molecule, which in the complexes of the invention is a phosphoantigen, is released intracellularly.

[0123] Targeting antibodies that may be used in conjugates of the invention for use in cancer therapy may be tumor-targeting antibodies that selectively bind to tumor-specific or tumor-associated antigens. Tumor-specific antigens are present only on tumor cells, while tumor-associated antigens are antigens that are expressed at higher levels (e.g., overexpressed) in cancer cells compared to normal (healthy) cells.

[0124] Antigen targets to which the antibodies or antigen-binding fragments of the conjugates of the invention bind include, for example, annexin A1, B7H3, B7H4, BCMA, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242 (cancer antigen 242), CAIX, CCR2, CCR5, CD2, CD19, CD20, CD22, CD24, CD30 (tumor necrosis factor 8), CD33, CD37, CD38 (cyclic ADP-ribose hydrolase), CD40, CD44, CD47 (integrin-associated protein), CD56 (neural cell adhesion molecule), CD70, CD71, CD73, CD74, CD79, CD115 (colony-stimulating factor 1 receptor), and the like. receptor), CD123 (interleukin-3 receptor), CD138 (syndecan-1), CD203c (ENPP3), CD303, CD333, CDCP1, CEA, CEACAM, claudin-4, claudin-7, CLCA1 (C-type lectin-like molecule 1), CLL1, cMET (hepatocyte growth factor receptor), Cripto, DLL3, EGFL, EGFR, EPCAM, EphA2, EPhB3, ETBR (endothelin type B receptor), FAP, FcRL5 (Fc receptor-like protein 5, CD307), FGFR3, FOLR1 (folate receptor alpha), FRbeta, GCC (guanylyl cyclase C), GD2, GITR, GLOBO The antigen may be selected from the group consisting of H, GPA33, GPC3, GPNMB, HER2, p95HER2, HER3, HMW-MAA (high molecular weight melanoma-associated antigen), integrin α (e.g., αvβ3 and αvβ5), IGF1R, TM4SF1 (L6), Lewis A-like carbohydrate chain, Lewis X, Lewis Y (CD174), LGR5, LIV1, mesothelin (MSLN), MN (CA9), MUC1, MUC16, NaPi2b, nectin-4, Notch3, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4 (or TPBG, trophoblast glycoprotein), TF (tissue factor, thromboplastin, CD142), TF-Ag, Tag72, TNFα, TNFR, TROP2 (tumor-associated calcium signal transducer 2), uPAR, VEGFR, and VLA.

[0125] Examples of suitable antibodies known in the art include blinatumomab (CD19), rituximab (CD20) or other anti-CD20 antibodies, such as ofatumumab, ublituximab or ocrelizumab, epratuzumab (CD22), iratumumab and brentuximab (CD30), gemtuzumab, vadastuximab (CD33), tetulumab (CD37), daratumumab, isatuximab (CD38), bivatuzumab (CD44), alemtuzumab (CD52), lorvotuzumab (CD56), borsetuzumab (CD70), milatuzumab ( These include antibodies against CD74), polatuzumab (CD79), rovalpituzumab (DLL3), futuximab (EGFR), oportuzumab (EPCAM), farletuzumab (FOLR1), glembatumumab (GPNMB), trastuzumab, pertuzumab, and margetuximab (HER2), etaracizumab (integrins), anetumab (mesothelin), pancomab (MUC1), enfortumab (nectin 4), H8, A1, and A3 (5T4), and antibodies against TROP2, such as sacituzumab, datopotamab, and PF-06664178. An example of a suitable antibody is the anti-CD20 antibody rituximab, which was specifically used in the present invention.

[0126] Since the pAg activity of the pAg moiety must be exerted intracellularly, an antibody that is internalized within cells is preferred.

[0127] The antibody or antigen-binding fragment thereof may, where applicable, comprise: (1) a constant region that has been engineered, i.e., one or more mutations may be introduced, e.g., to increase half-life, provide a linker-drug attachment site, and / or increase or decrease effector function; or (2) a variable region that has been engineered, i.e., one or more mutations may be introduced, e.g., to provide a linker-drug attachment site. The antibody or antigen-binding fragment thereof may be produced recombinantly, synthetically, or by other known suitable methods. Mutations that may decrease Fc-mediated effector function of the antibody are, for example, those described in Leabman et al., 2013, MAbs, 5(6):896-903 and Bruhns P, et al., 2015, Immunol Rev., 268(1):25-51. doi: 10.1111 / imr.12350. PMID: 26497511.

[0128] Conjugates of the invention may be wild-type or site-directed (meaning that specific conjugation sites, e.g., cysteines or unnatural amino acids, have been engineered into the antibody protein sequence), or a combination thereof, and can be produced by methods known in the art.

[0129] The immunoconjugates of the present invention have been found to deliver pAg payloads to antigen-presenting cells, such as cancer cells, very efficiently, resulting in active phosphoantigens within the antigen-presenting cells. Antigen-presenting cells may be tumor cells that express or overexpress specific tumor antigens on their surface. Such cells may also express or overexpress TCR activating molecules, such as BTN3A1 / BTN2A1 receptor complex molecules, that are involved in the indirect activation of γδ T cells by pAgs.

[0130] Phosphoantigen moiety (pAg) Throughout this specification, the term "phosphoantigen moiety" or "pAg" refers to a pAg moiety having the structural formula shown in parentheses outside Formula I. In Formula I, at least one pAg is conjugated to a linking moiety (L).

[0131] The phosphoantigen moiety comprises a relatively small mass non-peptide antigen that is capable of stimulating γδ T cells (more specifically Vγ9Vδ2 cells) in the presence of antigen-presenting cells.

[0132] A "phosphoantigen moiety" as part of a conjugate or linker-drug compound of the present invention does not necessarily comprise the phosphoantigen in its active form. The phosphoantigen moiety in a conjugate or linker-drug compound may comprise an inactive precursor form of the active phosphoantigen and / or may release the active phosphoantigen only after the conjugate binds to the target and is processed. Thus, the phosphoantigen moiety in its bound state as part of a conjugate or linker-drug compound may be structurally different from the active phosphoantigen released therefrom. For example, separation from or cleavage of the linking moiety may initiate structural rearrangements and / or chemical or enzymatic reactions that lead to the formation of functionally active phosphoantigen. Alternatively, removal or relocation of the prodrug moiety, e.g., in response to a change in the environment or as a result of enzymatic activity at the target site, may release the functionally active phosphoantigen.

[0133] In the conjugates of the invention, for example, specific binding between an antibody (the targeting moiety) and its specific binding partner (e.g., a tumor-specific antigen) directs the pAg moiety to its target site, but not vice versa (the pAg moiety is not the targeting moiety). In the conjugates of the invention, it is the binding specificity and affinity of the targeting moiety (e.g., antibody) that ensures that the phosphoantigen moiety is delivered to the site where it must exert its therapeutic effect.

[0134] The pAg moieties used in the linker-drug compounds and conjugates of the invention include allylic alcohols or prodrugs thereof (e.g., pAgs in which an allylic alcohol is generated after removal of the prodrug group or after cleavage of a linker moiety via or conjugated to an isoprene unit). Such compounds are considered examples of pAgs with direct pAg activity (pAgs that act as BTN3A1 ligands).

[0135] The activity of phosphoantigens on Vy9V52 T cells can be measured by a cell assay, as shown in the Examples. In the cell-based assay used, in a first step, target cells, e.g., tumor cells, e.g., cells from the CD20-positive Burkitt's lymphoma human tumor cell line Raji, are incubated (overnight) with the phosphoantigen or phosphoantigen-containing complex of the invention.

[0136] In this first step, the phosphoantigen or conjugate of the invention is taken up into the target (tumor) cell. After internalization (and, in the case of a conjugate, linker cleavage), the phosphoantigen binds to the intracellular domain of the BTN3A1 receptor and activates the BTN3A1 / BTN2A1 dimer.

[0137] The pretreated and washed tumor cells from the first step can be co-cultured with γδ T cells in the second step. When Vγ9Vδ2 T cells are activated, they produce cytokines to activate the immune system and release cytotoxic granules (degranulation) to kill target cells.

[0138] To assess the activity of phosphoantigens in γδ T cells, monensin and / or brefeldin A are added to co-cultures of γδ T cells and targets, which capture cytokines (e.g., interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα)) produced by activated cells. Staining with fluorescently labeled antibodies in the presence of saponin, which allows the anti-cytokine antibodies to enter the cells, identifies cytokine-producing cells. Fluorescently labeled antibodies against CD107a can also be added to the co-cultures to stain degranulating cells. Degranulation correlates with tumor cell killing (Aktas et al., 2009, Cell Immunol., 254(2),149-154).

[0139] Thus, by combining fluorescently labeled immune cell-specific markers with CD107a and cytokine markers, it is possible to determine the activation state of γδ T cells and / or other immune cell subsets after co-culture with pre-treated target cells.

[0140] The ability of γδ T cells to kill pre-treated tumor cells can be investigated by determining the percentage of dead tumor cells after co-culture. Tumor cells can be easily identified by fluorescent tags, and their dead cells can be determined as early as 1 hour after co-culture with γδ T cells.

[0141] Phosphoantigen analogs A (chemical) analog is a compound that differs from the native phosphoantigen in structural features but resembles the native phosphoantigen in functional biological activity (i.e., (indirect) immunostimulatory activity, particularly for γδ T cells). Analogs can be designed to improve one or more characteristics of the naturally occurring pAg, such as stability, potency, bioavailability, or binding to the linking moiety, in relation to their use in the immunoconjugates and linker-drug compounds of the invention.

[0142] Phosphoantigen Prodrugs With respect to prodrugs, an inactive precursor of a phosphoantigen is one that is converted to an active phosphoantigen after removal or conversion of a protecting group (e.g., a neutral protecting group on the negatively charged non-bonding oxygen atom of a phosphonate group). After a conjugate containing a phosphoantigen moiety of the present invention in prodrug form is administered to the body, the protecting group may be metabolically removed at the target site. A prodrug may be formed by conjugation of a phosphoantigen moiety with a linking moiety. In this case, the active phosphoantigen may be formed by cleavage of a linker in the conjugate used to connect the phosphoantigen prodrug moiety with the targeting moiety, releasing the active phosphoantigen, and / or by removal of a protecting group from the phosphoantigen moiety. Preferably, such conversion to release the active phosphoantigen occurs only after the conjugate of the present invention has reached the site where it must exert its therapeutic effect, e.g., after internalization by tumor cells, or at least within the tumor microenvironment, to prevent unwanted and nonspecific side effects of the phosphoantigen moiety in healthy and / or non-target tissues.

[0143] In the conjugates of the invention, the phosphoantigen is conjugated to a targeting moiety (e.g., a tumor-specific antibody). In the conjugates of the invention, it is the binding specificity of the targeting moiety that ensures that the phosphoantigen moiety is delivered to the site where it must exert its therapeutic effect.

[0144] Prodrug types include protecting groups known in the art, such as aryl ester, aryl amide, or pivaloyloxymethyl (POM) prodrugs. Analogs / prodrugs of C-HMBP (monophosphonate) phosphoantigens are described in WO 2019 / 182904. Aiming to synthesize phosphoantigen prodrugs as potent as natural phosphoantigens, such as HMBPP, aryloxytriester phosphoramidite prodrugs of (monophosphonate) phosphoantigens were synthesized as described in Davey et al., 2018, J. Med. Chem., 61, 2111-2117. In these prodrugs, the monophosphonate group is masked by an aryl motif and an amino acid ester moiety. These compounds ("HMBP ProPagens") exhibited significantly lower serum stability due to cleavage of the -PO- bond between the phosphate and isoprenoid moieties in the molecule. Similar "ProPagens" compounds, in which the oxygen in the -PO- bond is replaced by carbon, are described in WO 2020 / 008189. Proposed structure-activity relationships (SAR) of phosphoantigens (prodrugs) are described in Wiemer et al., 2020, Chem.Med.Chem., 15, 1030-1039.

[0145] The cleavable linking moiety may be covalently attached to the phosphoantigen via the alcohol group of the allylic alcohol, in which case the allylic alcohol is (re)formed intracellularly when the cleavable linking moiety is cleaved.

[0146] The prodrug moieties in a phosphoantigen prodrug as part of a conjugate of the invention can be the same or different, for example, all prodrug moieties can be POM groups, or the phosphoantigen moieties can include, for example, a "proTide" group, such as a combination of aryloxy and amino acid ester radicals, such as those described for phosphoantigen prodrugs in WO2020 / 008189 or WO2019 / 182904.

[0147] Suitable phosphonate prodrug technology and synthesis of phosphonate prodrugs are known in the art. Such prodrug technologies are further reviewed, for example, in Praderé et al., 2014, Chem. Rev., 114, 9154-9218, and include the use of carbonyloxymethyl prodrug moieties such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) derivatives, S-acyl-2-thioethyl (SATE) and S-[(2-hydroxyethyl)sulfidyl]-2-thioethyl (DTE)-based prodrugs, cyclosaligenyl (cycloSal) phosphate and phosphonate-based prodrugs and alkoxyalkyl monoesters (hexadecyloxypropyl-(HDP), octadecyloxyethyl-(ODE))-based prodrugs, phosphoramidite and phosphonamidite-based prodrugs (including aryloxyamino acid amidate (ProTide) prodrugs), and phosphorodiamidates and phosphonodiamidates.

[0148] Synthesis of the conjugates of the present invention To synthesize a conjugate of the invention, one or more linker-drug compounds of the invention may be conjugated to a suitable targeting moiety. When the targeting moiety is a polypeptide (antibody or binding fragment thereof), the linker-drug compound may be conjugated via a reactive natural amino acid residue, such as lysine or cysteine, present in the suitable polypeptide, or via the N-terminus or C-terminus. Alternatively, a natural or non-natural reactive amino acid residue may be genetically engineered into the suitable polypeptide, or a reactive group may be introduced by post-translational modification.

[0149] Conjugates of the invention may be prepared by conjugating a linker-drug compound of the invention to an antibody or antigen-binding fragment thereof, e.g., via the ε-amino group of a lysine on the antibody, preferably using an intermediate containing an amine-reactive group, e.g., an activated ester. Such methods are known for preparing conventional antibody-drug conjugates (ADCs).

[0150] Alternatively, immunoconjugates can be prepared by conjugating linkers via the free thiol on the side chain of cysteine, generated by reduction of the interchain disulfide bond, using methods and conditions known in the art (see, e.g., Doronina et al., 2006, Bioconjugate Chem., 17, 114-124). This preparation method involves partial reduction of the solvent-exposed interchain disulfide and modification of the resulting thiol with a Michael acceptor-containing linker (e.g., maleimide-containing linker, α-haloacetic acid amide, or ester). The cysteine ​​conjugation strategy allows for up to two linkers per reduced disulfide to be conjugated to the linker-drug.

[0151] The preferred antibody used as the targeting moiety in the conjugates of the invention is human IgG. Most human IgG molecules have four solvent-exposed disulfide bonds, which equates to an integer of 0 to 8 linked moieties per antibody. The exact number of phosphoantigen moieties attached per targeting moiety depends on the number of phosphoantigen moieties per linked moiety, the degree of disulfide reduction, and the molar equivalents of linker-drug containing linkers in the subsequent conjugation reaction. Complete reduction of all four disulfide bonds yields a homogeneous construct with eight linker moieties per antibody, while partial reduction typically results in a heterogeneous mixture with 0, 2, 4, 6, or 8 linked moieties per antibody.

[0152] In a preferred embodiment, the invention relates to a conjugate in which a linker-drug compound of the invention is conjugated to an antibody or antigen-binding fragment thereof via a cysteine ​​residue on the antibody or antigen-binding fragment thereof.

[0153] Site-specific conjugation with antibodies or antigen-binding fragments thereof Because antibodies contain many lysine residues and cysteine ​​disulfide bonds, conventional conjugation results in heterogeneous mixtures that present challenges for analytical characterization and manufacturing. Furthermore, the individual components of these mixtures exhibit different physicochemical properties and pharmacology with respect to their pharmacokinetics, efficacy, and safety profiles, hindering a rational approach to optimizing this modality.

[0154] To improve the homogeneity of the conjugates, the antibodies used in the (immuno)conjugates of the invention may be modified to allow site-specific conjugation of linkers. Methods for site-specific conjugation of drugs to antibodies are comprehensively reviewed in C.R. Behrens and B. Liu, 2014, mAbs, 6 (1), 1-8, and are described in WO 2015 / 177360, WO 2005 / 084390, and WO 2006 / 034488.

[0155] Site-specific immunoconjugates are preferably prepared by conjugating a linker-drug compound to an antibody or antigen-binding fragment thereof via the side chain of an engineered cysteine ​​residue at an appropriate position on the mutant antibody or antigen-binding fragment thereof. The engineered cysteine ​​is typically capped with another thiol, such as cysteine ​​or glutathione, to form a disulfide. These capped residues must be uncapped before linker-drug conjugation can be performed. Conjugation of the linker-drug to the engineered residue can be achieved by either (1) reducing both the native interchain disulfide and the mutant disulfide, followed by reoxidizing the native interchain cysteine ​​using a mild oxidizing agent, such as CuSO or dehydroascorbic acid, followed by standard conjugation of the uncapped engineered cysteine ​​with the linker-drug; or (2) using a mild reducing agent that reduces the mutant disulfide more quickly than the interchain disulfide bond, followed by standard conjugation of the uncapped engineered cysteine ​​with the linker-drug. Suitable methods for site-specific linker-drug conjugation can be found, for example, in WO 2015 / 177360, which describes reduction and reoxidation, WO 2017 / 137628, which describes a method using mild reducing agents, and WO 2018 / 215427, which describes a method for coupling a reduced interchain cysteine ​​with an uncapped engineered cysteine.

[0156] Pharmaceutical Composition The conjugates of the present invention are particularly intended for use as pharmaceuticals for treating cancer, autoimmune diseases or infectious diseases.

[0157] In another aspect, the present invention relates to a composition comprising the conjugate of the present invention, preferably a pharmaceutical composition, more preferably further comprising one or more pharmaceutically acceptable additives. Such a composition is hereinafter referred to as the composition of the present invention. The composition may be a liquid formulation, a lyophilized formulation, or in the form of a capsule or tablet.

[0158] Pharmaceutical compositions comprising the immunoconjugates of the invention are usually in the form of lyophilized cakes (lyophilized powders) that require dissolution (i.e., reconstitution) (in water) before intravenous infusion, or frozen (aqueous) solutions that require thawing before use. Thus, in a preferred embodiment, the invention relates to a lyophilized composition comprising the immunoconjugates of the invention, preferably a pharmaceutical composition, more preferably further comprising one or more pharmaceutically acceptable additives. In a further preferred embodiment, the invention relates to a frozen composition comprising water and the immunoconjugates of the invention, preferably a pharmaceutical composition, more preferably further comprising one or more pharmaceutically acceptable additives. In this context, the frozen solution is preferably at atmospheric pressure, and is preferably obtained by freezing a liquid composition of the invention at temperatures below 0°C. Suitable pharmaceutically acceptable excipients to be incorporated into the pharmaceutical composition of the present invention (before lyophilization) include buffers (e.g., aqueous solutions of salts of amino acids such as citric acid and histidine or succinic acid), lyoprotectants (e.g., sucrose and trehalose), osmolality adjusters (e.g., chloride salts such as sodium chloride), surfactants (e.g., polysorbates), and bulking agents (e.g., mannitol and glycine). Excipients used in lyophilized protein formulations are selected based on their ability to prevent protein denaturation during the lyophilization process and storage.

[0159] medical use In another aspect, the present invention provides a conjugate of the present invention or a composition of the present invention for use as a medicament, preferably for treating cancer, an autoimmune disease or an infectious disease, for example, to induce γδ T cell cytotoxicity against tumor cells and / or infected cells.

[0160] The complexes and compositions are hereinafter collectively referred to as products for use in the present invention.

[0161] In a first embodiment, the product of the present invention finds use in the treatment of solid tumors or hematological malignancies. In a second embodiment, the product of the present invention finds use in the treatment of autoimmune diseases. In a third embodiment, the product of the present invention finds use in the treatment of infectious diseases, such as bacterial, viral, fungal, parasitic or other infectious diseases.

[0162] The cancer in the present invention is preferably a tumor that expresses the antigen to which the product used in the present invention binds. Such tumors may be solid tumors or hematological malignancies. Examples of tumors or hematological malignancies that can be treated with the product used in the present invention described above include breast cancer; brain cancer (e.g., glioblastoma); head and neck cancer; thyroid cancer; parotid cancer; adrenal cancer (e.g., neuroblastoma, paraganglioma, or pheochromocytoma); bone cancer (e.g., osteosarcoma); soft tissue sarcoma (STS); eye cancer (e.g., uveal melanoma); esophageal cancer; gastric cancer; small intestine cancer; colorectal cancer; urothelial cell cancer (e.g., bladder, penile, ureter, or kidney cancer); ovarian cancer; uterine cancer; vaginal cancer, vulvar cancer, and cervical cancer; lung cancer (particularly non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)); melanoma; mesothelioma (particularly malignant pleural and peritoneal mesothelioma); and liver cancer. These include, but are not limited to, cancer (e.g., hepatocellular carcinoma); pancreatic cancer; skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, or dermatofibrosarcoma protuberans); testicular cancer; prostate cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); myelodysplastic syndromes (MDS); blastic plasmacytoid dendritic cell neoplasia (BPDCN); Hodgkin's lymphoma; non-Hodgkin's lymphoma (NHL) (including follicular lymphoma (FL), CNS lymphoma, and diffuse large B-cell lymphoma (DLBCL)); light chain amyloidosis; plasma cell leukemia; and multiple myeloma (MM).

[0163] The autoimmune disease of the present invention is preferably an autoimmune disease associated with the antigen to which the product used in the present invention binds. Autoimmune diseases refer to conditions resulting from an abnormal immune response against normal body cells and tissues. There are at least 80 different types of autoimmune diseases. Some diseases are organ-specific and limited to affecting certain tissues, while others resemble systemic inflammatory diseases that affect many tissues throughout the body. The appearance and severity of these signs and symptoms depend on the location and type of inflammatory response that occurs and may vary over time. Examples of autoimmune diseases that can be treated with the products used in the present invention described above include rheumatoid arthritis; juvenile dermatomyositis; psoriasis; psoriatic arthritis; lupus; sarcoidosis; Crohn's disease; eczema; nephritis; uveitis; polymyositis; neuritis, e.g., Guillain-Barré syndrome; encephalitis; arachnoiditis; systemic sclerosis; autoimmune-mediated musculoskeletal and connective tissue diseases; neuromuscular degenerative diseases, e.g., Alzheimer's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and major, medium, and minor encephalopathy. These include, but are not limited to, Kawasaki and Henoch-Schönlein vasculitis of the blood vessels; cold and warm agglutinin disease; autoimmune hemolytic anemia (AIHA); immune thrombocytopenic purpura (ITP), type 1 diabetes; Hashimoto's thyroiditis; Graves' disease; Graves' ophthalmopathy; adrenalitis; hypophysitis; pemphigus vulgaris; Addison's disease; ankylosing spondylitis; Behcet's syndrome; celiac disease; Goodpasture's syndrome; myasthenia gravis; sarcoidosis; scleroderma; primary sclerosing cholangitis, epidermolysis bullosa acquisita, and pemphigoid.

[0164] The infectious disease of the present invention is preferably an infectious disease associated with the antigen to which the product of the present invention binds. Such an infectious disease may be bacterial, viral, fungal, parasitic, or other infectious disease. Examples of infectious diseases that can be treated with the product of the present invention described above include, but are not limited to, malaria, toxoplasmosis, pneumocystis jirovecii, melioidosis, shigellosis, listeria, diseases caused by Cyclospora or Mycobacterium leprae, tuberculosis, and infections in immunocompromised individuals, such as HIV-positive individuals, individuals on immunosuppressive therapy, or individuals with congenital anomalies such as cystic fibrosis or benign proliferative disorders (e.g., hydatidiform mole or endometriosis).

[0165] The use of the products described herein may be for the manufacture of a medicament as described herein. The products used in the invention described herein are preferably for a method of treatment, wherein the product for use is administered to a subject, preferably a subject in need thereof, in a therapeutically effective amount. Thus, or in combination with other aspects, in one aspect, the invention relates to the use of the products of the invention for the manufacture of a medicament for treating cancer, autoimmune diseases or infectious diseases, particularly cancer. For non-limiting examples of cancer or other diseases that can be treated with the invention, see the preceding description.

[0166] Alternatively, in one aspect, in combination with other aspects, the present invention relates to a method for treating cancer, an autoimmune disease or an infectious disease, in particular cancer, comprising administering to a subject in need of said treatment a therapeutically effective amount of a product for use in the present invention. See above for non-limiting examples of cancer or other diseases that can be treated with the present invention.

[0167] The products used in the present invention are intended for administration to a subject. The products used in the present invention can be used in the treatment methods described above by administering an effective amount of the composition to a subject in need thereof. As used herein, the term "subject" refers to all animals classified as mammals, including, but not limited to, primates and humans. The subject is preferably a human. A "therapeutically effective amount" means an amount sufficient to produce a desired response or ameliorate symptoms or signs. The therapeutically effective amount for a particular subject may vary depending on factors such as the condition being treated, the subject's health, the method, route and dose of administration, and the severity of side effects.

[0168] Combination Use In another aspect, the present invention provides a product for use in the present invention, for use in combination with one or more other therapeutic agents, which may be used simultaneously or sequentially with one or more other therapeutic agents.

[0169] Suitable chemotherapeutic agents include alkylating agents such as nitrogen mustard, hydroxyurea, nitrosourea, tetrazines (e.g., temozolomide) and aziridines (e.g., mitomycin); drugs that interfere with the DNA damage response, such as PARP inhibitors, ATR and ATM inhibitors, CHK1 and CHK2 inhibitors, DNA-PK inhibitors, and WEE1 inhibitors; antimetabolites, such as antifolates (e.g., pemetrexed), fluoropyrimidines (e.g., gemcitabine), deoxynucleoside analogs, and thiopurines; microtubule inhibitors, such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics, such as anthracyclines and bleomycin; hypomethylating agents, such as decitabine and azacytidine; histone deacetylase inhibitors; all-trans retinoic acid; and arsenic trioxide. Suitable radiotherapeutic agents include radioisotopes, such as 131 I-metaiodobenzylguanidine (MIBG), as sodium phosphate 32 P, chloride 223 Ra, chloride 89 Sr and 153and Sm diaminetetramethylenephosphonic acid (EDTMP). Suitable substances for use as hormone therapeutic agents include inhibitors of hormone synthesis, such as aromatase inhibitors and GnRH analogs; hormone receptor antagonists, such as selective estrogen receptor modulators (e.g., tamoxifen and fulvestrant) and antiandrogens, such as bicalutamide, enzalutamide, and flutamide; CYP17A1 inhibitors, such as abiraterone; and somatostatin analogs.

[0170] Targeted therapeutic agents are therapeutic agents that interfere with specific proteins involved in tumor formation and growth, and may be small molecule drugs; proteins, such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids, such as ADCs. Examples of targeted small molecule drugs include TLR ligands, mTor inhibitors, such as everolimus, temsirolimus, and rapamycin; kinase inhibitors, such as imatinib, dasatinib, and nilotinib; VEGF inhibitors, such as sorafenib and regorafenib; EGFR / HER2 inhibitors, such as gefitinib, lapatinib, and erlotinib; and CDK4 / 6 inhibitors, such as palbociclib, ribociclib, and abemaciclib. Examples of peptide or peptide derivative targeted therapeutic agents include proteasome inhibitors, such as bortezomib and carfilzomib.

[0171] Suitable anti-inflammatory agents include D-penicillamine, azathioprine and 6-mercaptopurine, cyclosporine, anti-TNF biologics (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab, or certolizumab pegol), leflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acitretin, and JAK inhibitors (e.g., tofacitinib, baricitinib, or upadacitinib).

[0172] Immunotherapeutic agents include substances that induce, enhance, or suppress immune responses, such as cytokines (IL2 and IFNα); immunomodulatory imidazole drugs, such as thalidomide, lenalidomide, pomalidomide, or imiquimod; therapeutic cancer vaccines, such as talimogene laherparepvec; cell-based immunotherapeutics, such as dendritic cell vaccines, adoptive T cells, or chimeric antigen receptor-modified T cells; and therapeutic (bispecific) antibodies or other ADCs that can trigger antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) via the Fc region when bound to membrane-bound ligands on cells.

[0173] In the present invention, treatment preferably refers to preventing, reversing, curing, ameliorating, and / or slowing the cancer, autoimmune disease, or infectious disease, which may mean reducing the severity of at least one symptom of the cancer, autoimmune disease, or infectious disease and / or improving at least one parameter associated with the cancer, autoimmune disease, or infectious disease.

[0174] In the present invention, a subject may be a survivor and / or considered disease-free. Alternatively, the disease or condition may be halted or delayed. In the present invention, improved quality of life and observed pain relief may mean that the subject may need less analgesic medication than at the start of treatment. In this context, "less" may mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, or 90% less. The subject may no longer require analgesic medication. These improved quality of life and observed pain relief may be seen, detected, or assessed in a subject after at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more of treatment, and may be compared to the quality of life and observed pain relief at the start of treatment for the subject.

[0175] General definition The conjugates and linker-drugs of the present invention may have one or more chiral centers and / or double bonds and therefore may exist as stereoisomers, e.g., double bond isomers (i.e., geometric isomers), positional isomers, enantiomers, or diastereomers. Therefore, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the exemplified and specified compounds, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation or chiral synthesis techniques well known to those skilled in the art. Compounds may exist in several tautomeric forms, including enol forms, keto forms, and mixtures thereof. Therefore, the chemical structures depicted herein encompass all possible tautomeric forms of the exemplified and specified compounds. It is also understood that some isomeric forms, such as diastereomers, enantiomers, and geometric isomers, can be separated by those skilled in the art using physical and / or chemical methods. While those skilled in the art will recognize chiral centers from the structural formula or substance name, when chirality is not indicated, all three are individually referenced for each chiral center: racemic mixture, pure R enantiomer, and pure S enantiomer. When the structure of a compound is shown as a specific enantiomer, it should be understood that this invention is not limited to that specific enantiomer. When two moieties are bonded together, this means that these moieties are not present as atoms, and valence compliance is met by replacing the electron bond. All of this is known in the art.

[0176] The compounds disclosed in this specification and claims may exist as exo and endo regioisomers. Unless otherwise indicated, the description of a compound in this specification and claims is intended to include both the individual exo and individual endo regioisomers of the compound, as well as mixtures thereof. Furthermore, the compounds disclosed in this specification and claims may exist as cis and trans isomers. Unless otherwise indicated, the description of a compound in this specification and claims is intended to include both the individual cis and individual trans isomers of the compound, as well as mixtures thereof. For example, if the structure of a compound is shown as a cis isomer, it should be understood that the corresponding trans isomer, or mixtures of cis and trans isomers, are not excluded from this invention.

[0177] In this specification and claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the items shown therein are included, but that items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of that element is present, unless the context clearly requires that there be one and only one of that element. Thus, the indefinite article "a" or "an" usually means "at least one."

[0178] "About" or "approximately" (eg, about 10) when used in conjunction with a numerical value preferably means that the value may be 1% more or less than the given value.

[0179] When parameters of a substance are discussed in this invention, unless otherwise specified, it is assumed that the parameters are determined, measured, or characterized at physiological conditions. Physiological conditions are known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, a pH of 6-8, room temperature (RT) to about 37°C (about 20°C to about 40°C), and appropriate concentrations of buffer salts or other components. It is understood that charge often involves balance. A moiety that is said to carry or possess a charge is one that is found in a state in which it possesses or carries such charge more often than in a state in which it does not possess or carry such charge. As such, as will be understood by those skilled in the art, atoms shown herein to be charged may be uncharged under certain conditions, and neutral moieties may become charged under certain conditions.

[0180] All patents and articles cited in this specification are hereby incorporated by reference in their entirety.

[0181] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0182] General Procedure solvent: Reagent-grade or HPLC-grade solvents from various vendors were used. NMR spectrum: NMR spectra were obtained using a Bruker AVANCE400 ( 1 400MHz for H; 13 C recorded at 101 MHz). Chemical shifts: Chemical shifts are given in ppm relative to the internal standard tetramethylsilane or residual non-deuterated solvent. UPLC determination of products: The products were measured on a Waters UPLC-MS (with SQD2 detector) equipped with a Waters ACQUITY UPLC BEH C18 column (1.7 μm particle size, 2.1 × 50 mm) at a flow rate of 0.4 mL / min (MeCN / water × 0.1% formic acid). HPLC purification: Preparative HPLC purification was performed on a Shimadzu Prominence 20AP system equipped with a Waters SunFire Prep C18 OBD 5 μm column (19×150 mm) at a flow rate of 17 ml / min.

[0183] General Procedure XXD: Pyrophosphate Formation The triethylamine salt of phosphoric acid XD36 (1.0 equiv.) was dissolved in DMF (0.15 M) under a N atmosphere, and CDI (2.1 equiv.) was added at room temperature. After stirring for 30 min, dry MeOH (1.0 equiv.) was added, and the mixture was stirred at room temperature for 15 min and then concentrated. The residue was co-evaporated with DMF to give crude product A.

[0184] In a separate flask, the monotriethylamine salt of phosphoric acid (1.2 equiv.) was coevaporated with DMF and then redissolved in DMF (0.36 M) under N2. The mixture was cannulated into the flask containing crude product A at room temperature. The flask was rinsed with an equal volume of DMF to complete the transfer. The mixture was stirred at room temperature under N2. After UPLC-MS analysis confirmed essentially complete conversion (typically 20-24 h), the reaction was concentrated and purified by preparative HPLC as described. Lyophilization of the product fractions afforded the product.

[0185] General Procedure XXE: Click Reaction Copper(II) sulfate pentahydrate (0.77 equiv.) in nitrogen-purged water (0.034 M) was added to a flask containing solid azide (1.0 equiv.) and alkyne (1.4 equiv.) at room temperature. An equal volume of THF was added to obtain a homogeneous water / THF (1:1) solution. The headspace of the flask was briefly purged with N2, and a nitrogen-purged solution of sodium ascorbate (1.5 equiv.) in water (0.13 M) was added. The reaction was stirred at room temperature until complete conversion was indicated by UPLC-MS analysis (typically 1-2 h). The majority of the THF was removed by brief rotary evaporation at room temperature, and the aqueous phase was dissolved in 25 mM NH4HCO3 in MeCN / Milli-Q water (1:9). Insoluble material was removed using a syringe filter, and the filtrate was purified by preparative HPLC as described. Lyophilization of the product fractions afforded the product.

[0186] General Procedure XXF: Synthesis of Alkyl Phosphates from XD34 and Allyl Alcohols To a room temperature solution of the alcohol (1.1 equiv.), 2,6-lutidine (3.3 equiv.), and 5-(ethylthio)-1H-tetrazole (1.0 equiv.) in MeCN (0.4 M) was added XD34 (1.0 equiv.) in DCM (0.4 M) dropwise. The reaction mixture was stirred for 1-3 h and then quenched with excess MeOH. The reaction mixture was concentrated and partitioned with 1 M hydrochloric acid and EtOAc / heptane (1:2). The organic phase was separated, and the aqueous phase was extracted with EtOAc / heptane (1:2). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography as described.

[0187] General Procedure XXG: TBDPS-Deprotection with HF·Pyridine TBDPS-ether (1.0 equiv.) was dissolved in THF / pyridine (1:1, 0.2 M) in a PFA tube under a N2 atmosphere. The solution was cooled to 0 °C, and HF·pyridine (13.3 equiv., 70% HF) was added slowly. The reaction mixture was stirred at 0 °C for 60–90 min and then carefully added to a mixture of saturated aqueous NaHCO3 / EtOAc (1:1) with stirring at 0 °C. After effervescence ceased, the phases were separated, and the aqueous phase was extracted with EtOAc (2×). The combined organic phases were washed with 1 M HCl and brine, dried over Na2SO4, and concentrated. The crude product was purified as described.

[0188] General Procedure XXH: Fluorenylmethyl-deprotection with triethylamine Difluorenylmethyl phosphate (1 equiv.) was dissolved in MeCN / THF (2:1, 0.13 M) and TEA (10 equiv.) was added at room temperature. The reaction mixture was stirred for 16–20 h. The mixture was allowed to settle, and the supernatant was discarded. The oily residue was dissolved in MeCN / MeOH (1:1, 1.0 mL), and EtO (10 mL) was added slowly with stirring. The mixture was stirred for 15 min, allowed to settle, and the supernatant was discarded. This process was repeated twice. The residue was evaporated with MeCN to give the alkyl phosphate as its triethylammonium salt.

[0189] General Procedure XXI: Stille Coupling with XS60 CuI (0.3 equiv.), Pd(PPh3)4 (0.15 equiv.), and tributyl(alkyl)tin (1.2 equiv.) were added to a solution of XS60 (1.0 equiv.) in toluene (0.11 M) and the reaction mixture was stirred at 80 °C for 3–20 h. The mixture was filtered and concentrated, and the crude product was purified by flash chromatography as described.

[0190] General Procedure XXJ: Suzuki Coupling with XS60 XS60 (1.0 equiv.) and Pd(PPh3)4 (0.1 equiv.) were purged with N2 (3x) and dissolved in THF (0.05 M). A suspension of the described zinc reagent (2.0–4.0 equiv.) was added, and the reaction mixture was stirred at room temperature for 3 h. Saturated aqueous NH4Cl was added to the reaction mixture, and the product was extracted with Et2O. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The crude product was filtered from heptane, and the filtrate was purified by flash chromatography as described.

[0191] General Procedure XXK: THP-Ether Deprotection THP-ether (1.0 equiv.) was dissolved in MeOH (0.1 M) and PPTS (0.1 equiv.) was added. The reaction mixture was stirred at 45-55 °C for 3-9 h and then added to saturated aqueous NaHCO3. The product was extracted with DCM (3x), washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography as described.

[0192] General Procedure XXL: Synthesis of Alkyl Phosphates from XD34 and Allyl Alcohols To a room temperature solution of the alcohol (1.0 equiv.), 2,6-lutidine (3.3 equiv.), and 5-(ethylthio)-1H-tetrazole (1.5 equiv.) in MeCN (0.4 M) was added XD34 (1.5 equiv.) in DCM (0.4 M) dropwise. The reaction mixture was stirred for 1-3 h and then quenched with excess MeOH. The reaction mixture was concentrated and partitioned with 1 M HCl and EtOAc / heptane (1:2). The organic phase was separated, and the aqueous phase was extracted with EtOAc / heptane (1:2). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography as described.

[0193] Example 1 Synthesis of Linker-Drug XD78 1A: Preparation of Chloride XD68

[0194] [ka]

[0195] (Z)-4-((tert-butyldiphenylsilyl)oxy)-3-chlorobut-2-en-1-ol (XD67) Prepared according to the method described by Nitelet, A. et al. Org. Lett. 2016, 18, 1904. A microwave-compatible vial was charged with CuI (227 mg, 1.19 mmol), Me4NCl (872 mg, 7.96 mmol), and iodide XD66 (1.80 g, 3.98 mmol, prepared according to Overman, LE, Tetrahedron, 2010, 66, 6514). The vial was purged with N2 and capped. Ethanol (8.0 mL) and (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (340 mg, 2.39 mmol) were added, and the vial was heated at 110 °C for 16 h. The reaction was diluted with EtOAc / heptane (1:1, 40 mL), and the suspension was filtered through a plug of silica gel. The filtrate was concentrated and the crude product was purified by flash chromatography (silica gel, 0-25% ether in heptane) to give alcohol XD67 (1.02 g, 78%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.73-7.61 (m, 4H), 7.47-7.37 (m, 6H), 6.14 (tt, J = 6.3, 1.6 Hz, 1H), 4.35 (tt, J = 6.2, 1.3 Hz, 2H), 4.21 (q, J = 1.3 Hz, 2H), 1.45-1.39 (m, 1H), 1.08 (s, 9H). MS (ESI + ) C 20 H 29 ClNO2Si + [M+NH4] + Calculated value: 378.2, measured value: 378.3.

[0196] (Z)-3-Bromo-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-ol (XS31) Iodide XD66 (2.00 g, 4.42 mmol, prepared according to Overman, LE, Tetrahedron, 2010, 66, 6514) was reacted with CuI (253 mg, 1.33 mmol), MeNBr (2.72 g, 17.7 mmol), and (1R,2R)-N,N-dimethylcyclohexane-1,2-diamine (377 mg, 2.65 mmol) in ethanol (8.9 mL) as in XD67. The crude product was purified twice by flash chromatography (silica gel, 0-20% EtOAc in heptane; silica gel, 0-30% EtO in heptane) to give alcohol XS31 (1.79 g, 51%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.67-7.62 (m, 4H), 7.52-7.42 (m, 6H), 6.35-6.30 (m, 1H), 5.04 (t, J = 5.6 Hz, 1H), 4.30 (d, J = 1.3 Hz, 2H), 4.13-4.08 (m, 2H), 1.03 (s, 9H). MS (ESI + ) C 20 H 24 BrOSi + [M+H-HO] + Calculated value: 387.1, measured value: 387.2.

[0197] (Z)-tert-butyl((2,4-dichlorobut-2-en-1-yl)oxy)diphenylsilane (XD68) N-Chlorosuccinimide (0.621 g, 4.65 mmol) was dissolved in dry DCM (15 mL) and the mixture was cooled to -40 °C. Dimethyl sulfide (0.424 mL, 5.73 mmol) was added dropwise with stirring, and the mixture was then stirred at 0 °C for 10 min. After cooling to -65 °C, alcohol XD67 (1.29 g, 3.58 mmol) in dry DCM (3 mL) was added. The reaction was allowed to warm to 0 °C over 2.5 h and then stirred at 0 °C for 90 min. Brine (30 mL) was added at 0 °C and the phases were separated. The aqueous phase was extracted with DCM (40 mL) and the combined organic phases were dried over Na SO . After filtration and concentration, the crude oil was purified by flash chromatography (silica gel, 0-20% DCM in heptane) to give chloride XD68 (1.31 g, 96%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.70-7.63 (m, 4H), 7.51-7.35 (m, 6H), 6.20 (tt, J = 7.6, 1.6 Hz, 1H), 4.28-4.24 (m, 2H), 4.24-4.21 (m, 2H), 1.08 (s, 9H).

[0198] Synthesis of linker-drug XD73 [ka]

[0199] (Z)-(5-((tert-butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl)phosphonic acid dimethyl ester (XD69) To a solution of dimethyl methylphosphonate (0.817 mL, 7.54 mmol) in THF (28 mL) was added n-BuLi (2.5 M in hexanes, 3.02 mL, 7.54 mmol) under a N atmosphere at −78 °C. The reaction was stirred at this temperature for 1 h and then warmed to −50 °C. Subsequently, CuI (718 mg, 3.77 mmol) was added, and the cloudy mixture was stirred at −50 °C to −40 °C for 1 h to obtain a clear solution. Chloride XD68 (1.30 g, 3.43 mmol) in THF (7 mL) was added at −40 °C. The reaction was allowed to warm to room temperature overnight and then quenched with saturated aqueous NH4Cl at 0 °C. The aqueous phase was extracted with EtOAc (2x), and the combined organic phases were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (silica gel, 0-100% EtOAc in heptane) afforded phosphonic acid XD69 (1.09 g, 68%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71-7.59 (m, 4H), 7.51-7.32 (m, 6H), 5.95-5.83 (m, 1H), 4.19 (d, J = 1.4 Hz, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 2.57-2.42 (m, 2H), 1.91-1.78 (m, 2H), 1.15-0.99 (m, 9H). MS (ESI + ) C 23 H 32 ClNaO4PSi + [M+Na] + Calculated value: 489.1, measured value: 489.4.

[0200] (Z)-(5-((tert-butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl)phosphonic acid bis(2-cyanoethyl)ester (XD70) Step 1: TMSBr (3.03 mL, 23.0 mmol) was added over 10 minutes to a cooled (0°C) solution of phosphonic acid XD69 (1.07 g, 2.30 mmol) in DCM (11 mL). After 30 minutes, the ice bath was removed and the reaction was stirred at room temperature for 3 hours and 30 minutes. The mixture was concentrated and the crude product was redissolved in DCM (11 mL) under a N2 atmosphere and cooled to 0°C.

[0201] Step 2: DMF (2 drops) was added, followed by oxalyl dichloride (0.591 mL, 6.89 mmol). After stirring for 30 min, the ice bath was removed and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated and co-evaporated with DCM (2×, 10 mL). The crude oil was dried at room temperature under high vacuum for 1 h.

[0202] Step 3: The crude oil was dissolved in DCM (16 mL) under a N atmosphere, and the mixture was cooled to 0 °C. To the mixture was added 5-(ethylthio)-1H-tetrazole (0.149 g, 1.15 mmol), followed by pyridine (0.742 mL, 9.18 mmol) and 3-hydroxypropanenitrile (0.627 mL, 9.18 mmol). After stirring at 0 °C for 2 h and at room temperature for 3 h, the reaction mixture was transferred to a separatory funnel and diluted with DCM (20 mL). The organic phase was washed with hydrochloric acid (1 M, 20 mL), and the aqueous phase was back-extracted with DCM (30 mL). The combined organic phase was washed with brine, dried over NaSO, filtered, and concentrated. Purification by flash chromatography (silica gel, 0-100% EtOAc in heptane) afforded phosphonic acid XD70 (0.811 g, 65%). 1 H NMR (400 MHz, CDCl3) ppm = 7.70-7.61 (m, 4H), 7.48-7.35 (m, 6H), 5.93-5.85 (m, 1H), 4.36-4.22 (m, 4H), 4.21 (d, J = 1.3 Hz, 2H), 2.76 (t, J = 6.1 Hz, 4H), 2.60-2.48 (m, 2H), 2.00-1.90 (m, 2H), 1.08 (s, 9H). MS (ESI + ) C 27 H 35ClN2O4PSi + [M+H] + Calculated value: 545.2, measured value: 545.4.

[0203] 2-Cyanoethyl (Z)-(5-((tert-butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl)phosphate triethylammonium salt (XD71) To a solution of phosphonic acid XD70 (811 mg, 1.49 mmol) in THF (13 mL) was added DBU (0.247 mL, 1.64 mmol) at room temperature. After 30 min, the reaction was concentrated to approximately 1 mL, diluted with MeOH (6.8 mL), and eluted from DOWEX 50WX8 using methanol (30 mL). Triethylamine (0.228 mL, 1.64 mmol) was added, and the mixture was concentrated and co-evaporated with MeCN (2×) to give phosphonic acid XD71 (840 mg, 99%) as a colorless oil. NMR analysis revealed a phosphonic acid:amine ratio of 1:0.8. 1 H NMR (400 MHz, CD3OD) ppm = 7.71-7.65 (m, 4H), 7.48-7.37 (m, 6H), 5.93-5.86 (m, 1H), 4.21 (d, J = 1.1 Hz, 2H), 4.05 (dt, J = 7.1, 6.2 Hz, 2H), 3.20 (q, J = 7.3 Hz, 5H), 2.77 (t, J = 6.1 Hz, 2H), 2.54-2.41 (m, 2H), 1.73-1.60 (m, 2H), 1.31 (t, J = 7.3 Hz, 7H), 1.06 (s, 9H). MS (ESI - ) C 24 H 30 ClNO4PSi - [MH] - Calculated value: 490.1, measured value: 490.4.

[0204] Phosphonic acid XD72 Step 1: The triethylamine salt XD71 (830 mg, 1.45 mmol) and Fmoc-Val-Ala-PAB-OH (898 mg, 1.74 mmol) were co-evaporated with dry DMF (3×, 8 mL). At room temperature under a N atmosphere, DMF (7 mL) was added, followed by PyBOP (906 mg, 1.74 mmol) and DIPEA (0.507 mL, 2.90 mmol). After 2 h, the reaction mixture was slowly added dropwise to ice-cold water (70 mL) with constant gentle stirring (to prevent gel formation). The white suspension was gently stirred for 5 min and then filtered. The solid was collected, and residual water was removed by co-evaporation with MeCN (2×). The crude solid was purified by flash chromatography (silica gel, 0-6% MeOH in DCM) to give the intermediate phosphonic acid (1.10 g, 77%).

[0205] Step 2: To a nitrogen-purged PFA vial containing the intermediate phosphonic acid (1.10 g, 1.11 mmol) was added THF (6.2 mL) and pyridine (3.1 mL). At 0 °C under N2 atmosphere, HF-pyridine (70% HF, 1.5 mL) was added via syringe, and the mixture was stirred at 0 °C for 90 min. The reaction mixture was transferred via cannula to cold (0 °C) saturated aqueous NaHCO3 (150 mL) and gently stirred. After stirring for 15 min, the suspension was filtered, and the white solid was washed with water (2 × 10 mL), collected from the filter, and co-evaporated with MeCN (2 × 15 mL). The solid was dried under vacuum and then purified by flash chromatography (silica gel, 0-8% MeOH in DCM) to afford phosphonic acid XD72 (516 mg, 62%) as a white foam. 1H NMR (400 MHz, CD3OD) ppm = 7.79 (dd, J = 7.5, 0.8 Hz, 2H), 7.70-7.59 (m, 4H), 7.43-7.35 (m, 4H), 7.33-7.28 (m, 2H), 5.88 (t, J = 7.1 Hz, 1H), 5.10 (d, J = 12.5 Hz, 1H), 5.06 (d, J = 12.5 Hz, 1H), 4.49 (q, J = 7.1 Hz, 1H), 4.44-4.34 (m, 2H), 4.25-4.07 (m, 3H), 4.06 (s, 2H), 3.95 (d, J = 7.0 Hz, 1H), 2.79 (t, J = 5.9 Hz, 2H), 2.47 (dq, J = 15.1, 7.4 Hz, 2H), 2.08 (dq, J = 13.6, 6.8 Hz, 1H), 2.01-1.89 (m, 2H), 1.44 (d, J = 7.1 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.8 Hz, 3H). MS (ESI + ) C 38 H 45 ClN4O8P + [M+H] + Calculated value: 751.3, measured value: 751.8.

[0206] 1B: Linker-drug XD73 Step 1: To a suspension of phosphonic acid XD72 (0.117 g, 0.156 mmol) in MeOH (2.3 mL) and water (0.26 mL) at 0 °C was added aqueous NaOH (2.0 M, 0.389 mL, 0.779 mmol). The cooling bath was removed and the mixture was stirred for 100 min. The reaction was cooled to 0 °C and AcOH (2 M in MeOH, 0.623 mL, 1.25 mmol) was added. The methanol was removed by rotary evaporation, and the suspension was diluted with water (2 mL) and filtered. The solid was washed with water, and the aqueous filtrate was lyophilized to give the crude amine as a glassy solid. The material was used directly without further purification.

[0207] Step 2: The crude amine was dissolved / suspended in DMF (1 mL). DIPEA (0.108 mL, 0.622 mmol) was added at room temperature, followed by 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid 2,5-dioxopyrrolidin-1-yl ester (0.096 g, 0.311 mmol). The reaction was stirred at room temperature for 2 h, then acetic acid (0.053 mL, 0.933 mmol) in DMF (1 mL) was added, and the mixture was concentrated. The residue was dissolved in aqueous MeCN / NHHCO (25 mM) (1:9), and the solution was washed with EtOAc (4×, 5 mL) and ether (1×, 5 mL). The aqueous phase was then directly purified by preparative RP-HPLC (25 mM NHHCO / MeCN in Milli-Q water, 90:10 to 40:60 gradient). Lyophilization of the product fractions gave linker-drug XD73 (60.5 mg, 57%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 9.96 (s, 1H), 8.18 (d, J = 6.8 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 6.95 (s, 2H), 5.87 (t, J = 7.0 Hz, 1H), 4.76 (d, J = 7.1 Hz, 2H), 4.35 (q, J = 7.1 Hz, 1H), 4.12 (d, J = 6.9 Hz, 1H), 3.92 (br s, 2H), 3.35 (t, J = 7.1 Hz, 2H), 2.35-2.22 (m, 2H), 2.21-2.05 (m, 2H), 2.02-1.87 (m, 1H), 1.62-1.50 (m, 2H), 1.50-1.38 (m, 4H), 1.29 (d, J = 7.1 Hz, 3H), 1.15 (quint, J = 7.6 Hz, 2H), 0.84 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H). MS (ESI - ) C 30 H 41 ClN4O9P - [MH]- Calculated value: 667.2, measured value: 667.7.

[0208] Example 2 Synthesis of Linker-Drugs XS54-XS58 2A. Synthesis of Alcohols XS3 and XS38 Synthesis of alcohol XS33

[0209] [ka]

[0210] (E)-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-ol (XS33) To a suspension of Red-Al (3.17 mL, 70% in toluene, 11.2 mmol) in EtO (26 mL) at 0 °C was added dropwise a solution of 4-((tert-butyldiphenylsilyl)oxy)but-2-yn-1-ol (1.75 g, 5.39 mmol, prepared as described in Trost and Livingston, J. Am. Chem. Soc. 2008, 130, 11970-11978) in EtO (5.2 mL). The reaction mixture was stirred at 0 °C for 90 min, and EtOAc (0.528 mL, 5.39 mmol) was added. After stirring was continued at 0 °C for 30 min, the reaction was quenched by the addition of 1 M aqueous potassium sodium tartrate (5.4 mL). The mixture was stirred at 0 °C for 30 min, diluted with water (5.4 mL), and added to heptane (30 mL). The organic phase was washed with brine (20 mL), dried over Na2SO4, and concentrated. Purification by flash chromatography (silica gel, 0-25% EtOAc in heptane) afforded the allylic alcohol XS33 (1.39 g, 79%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) ppm = 7.65-7.61 (m, 4H), 7.50-7.40 (m, 6H), 5.83 (dtt, J = 15.4, 4.9, 1.5 Hz, 1H), 5.72 (dtt, J = 15.4, 4.4, 1.5 Hz, 1H), 4.74 (t, J = 5.4 Hz, 1H), 4.22-4.17 (m, 2H), 3.99-3.92 (m, 2H), 1.00 (s, 9H). MS (ESI + ) C 20 H 25 OSi + [M+H-HO] + Calculated value: 309.2, measured value: 309.3.

[0211] Synthesis of alcohol XS38 [ka]

[0212] 2-(Trityloxy)acetaldehyde (XS34) Silica gel-supported NaIO (22.3 g, 15.2 mmol, prepared by the method described in Zong and Shing, J. Org. Chem. 1997, 62, 2622-2624) was purged with N (3x) and suspended in DCM (50 mL), and 3-(trityloxy)propane-1,2-diol (3.74 g, 11.2 mmol, prepared by the method described in Chinese Patent Publication No. 108478807) dissolved in DCM (25 mL) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through a silica gel plug and washed with DCM (100 mL). The filtrate was concentrated, and the crude product was purified by flash chromatography (silica gel, 0-25% EtOAc in heptane) to give aldehyde XS34 (2.88 g, 85%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 9.36 (s, 1H), 7.44-7.31 (m, 15H), 3.85 (s, 2H).

[0213] 2-Fluoro-4-(trityloxy)but-2-enoic acid ethyl ester (XS35) A flask containing aldehyde XS34 (2.77 g, 9.16 mmol) and MgSO4 (1.33 g, 11.1 mmol) was purged with N2 (3x) and MeCN (46 mL) was added. The mixture was cooled to 0 °C, and 2-(diethoxyphosphoryl)-2-fluoroacetic acid ethyl ester (2.29 mL, 11.3 mmol) was added, followed by DBU (1.38 mL, 9.16 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was concentrated, and the residue was dissolved in EtOAc (100 mL) and washed with 1 M HCl (2x, 50 mL), 2 M NaOH (2x, 50 mL), and brine (50 mL), dried over Na2SO4, and concentrated. Purification by flash chromatography (silica gel, 0-15% EtOAc in heptane) gave vinyl fluoride XS35 (2.92 g, 82%) as a mixture of Z:E isomers (2:1 ratio). MS (ESI + ) C 25 H 23 FNaO3 + [M+Na] + Calculated value: 413.2, measured value: 413.4.

[0214] (Z)-2-Fluoro-4-(trityloxy)but-2-en-1-ol (XS36) Ester XS35 (2.85 g, 7.30 mmol, Z:E ratio 2:1) was purged with N2 (3x) and dissolved in THF (24 mL). The solution was cooled to -78 °C, and DIBAL-H (21.9 mL, 1 M in toluene, 21.9 mmol) was added dropwise. After 15 min, the reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was cooled to 0 °C and quenched by the addition of 15% aqueous citric acid (25 mL). The mixture was diluted with water (75 mL), and the product was extracted with EtOAc (3x, 100 mL). The combined organic phases were washed with brine (100 mL), dried over Na2SO4, and concentrated. Purification by flash chromatography (silica gel, 0-35% EtOAc in heptane) afforded alcohol XS36 (1.54 g, 60%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) ppm = 7.41-7.32 (m, 12H), 7.30-7.24 (m, 3H), 5.32 (t, J = 5.9 Hz, 1H), 5.19 (dt, J = 37.8, 7.0 Hz, 1H), 3.94 (dd, J = 12.9, 5.6 Hz, 2H), 3.58 (dd, J = 6.8, 1.0 Hz, 2H). MS (ESI + ) C 23 H 21 FNaO2 + [M+Na] + Calculated 371.1, found 371.3. (E)-2-Fluoro-4-(trityloxy)but-2-en-1-ol (0.777 g, 31%) was also isolated. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.41-7.32 (m, 12H), 7.31-7.24 (m, 3H), 5.31 (dt, J = 20.1, 7.6 Hz, 1H), 5.17 (t, J = 5.6 Hz, 1H), 3.86 (dd, J = 22.1, 5.6 Hz, 2H), 3.56 (dd, J = 7.8, 1.0 Hz, 2H). MS (ESI + ) C 23 H 21 FNaO2 + [M+Na] + Calculated value: 371.1, measured value: 371.3.

[0215] (Z)-tert-butyl((2-fluoro-4-(trityloxy)but-2-en-1-yl)oxy)diphenylsilane (XS37) Alcohol XS36 (1.53 g, 4.38 mmol) was dissolved in DCM (29 mL) under a N atmosphere. The solution was cooled to 0 °C, and TEA (1.22 mL, 8.76 mmol) and imidazole (0.328 g, 4.82 mmol) were added, followed by the dropwise addition of TBDPS-Cl (1.69 mL, 6.57 mmol). After 15 min, the reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was added to water (30 mL), the organic phase was separated, and the aqueous phase was extracted with DCM (30 mL). The combined organic phases were washed (20 mL), dried over NaSO, and concentrated. Purification by flash chromatography (silica gel, 0-5% EtOAc in heptane) afforded the silyl ether XS37 (2.47 g, 96%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.67-7.59 (m, 4H), 7.53-7.40 (m, 6H), 7.39-7.31 (m, 12H), 7.30-7.23 (m, 3H), 5.17 (dt, J = 37.3, 6.9 Hz, 1H), 4.21 (d, J = 13.4 Hz, 2H), 3.59 (d, J = 6.9 Hz, 2H), 1.01 (s, 9H).

[0216] (Z)-4-((tert-butyldiphenylsilyl)oxy)-3-fluorobut-2-en-1-ol (XS38) Trityl ether XS37 (1.00 g, 1.70 mmol) was dissolved in DCM (8.5 mL) and MeOH (8.5 mL) under a N atmosphere. The solution was cooled to 0 °C, and TsOH·HO (0.486 g, 2.56 mmol) was added. The reaction mixture was stirred at 0 °C for 1 h. The reaction mixture was added to saturated aqueous NaHCO (50 mL), and the product was extracted with DCM (3 × , 50 mL). The combined organic phases were washed with brine (50 mL), dried over NaSO, and concentrated. Purification by flash chromatography (silica gel, 0–25% EtOAc in heptane) afforded the alcohol XS38 (0.462 g, 79%) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) ppm = 7.66-7.60 (m, 4H), 7.52-7.41 (m, 6H), 5.12 (dt, J = 38.0, 6.9 Hz, 1H), 4.78 (t, J = 5.6 Hz, 1H), 4.20 (d, J = 13.0 Hz, 2H), 4.05-3.99 (m, 2H), 1.01 (s, 9H). MS (ESI + ) C 20 H 29 FNO2Si + [M+H] + Calculated value: 362.2, measured value: 362.3.

[0217] 2B. Preparation of chlorophosphonic acid bis((9H-fluoren-9-yl)methyl) ester (XD34)

[0218] [ka]

[0219] Bis((9H-fluoren-9-yl)methyl)phosphonate (XD50) At room temperature under a N atmosphere, (9H-fluoren-9-yl)methanol (4.55 g, 23.2 mmol) was added to a solution of diphenyl phosphonate (2.13 mL, 10.6 mmol) in dry pyridine (20 mL), and the mixture was stirred for 2 h. The reaction was concentrated and dissolved in EtOAc (250 mL). The organic phase was washed with hydrochloric acid (2×, 1 M) and brine, dried over NaSO, filtered, and concentrated onto silica gel. Purification by flash chromatography (silica gel, 0-85% EtOAc / DCM (1:4) in heptane) afforded H-phosphonic acid XD50 (3.46 g, 75%) as a colorless wax. 1H NMR (400 MHz, CDCl3) ppm = 7.76-7.66 (m, 4H), 7.58-7.45 (m, 4H), 7.42-7.31 (m, 4H), 7.31-7.22 (m, 4H), 7.19-7.12 (m, 1H), 6.68 (d, J = 705.8 Hz, 1H), 4.34-4.21 (m, 4H), 4.15-4.08 (m, 2H). MS (ESI + ) C 28 H 24 O3P + [M+H] + Calculated value: 439.2, measured value: 439.3.

[0220] Chlorophosphonic acid bis((9H-fluoren-9-yl)methyl) ester (XD34) H-phosphonic acid XD50 (8.57 g, 19.6 mmol) was dissolved in toluene (98 mL) and the space was purged with N2. NCS (3.13 g, 23.5 mmol) was added at room temperature, and the reaction mixture was then stirred at 40 °C for 2 h. After cooling to room temperature, the reaction mixture was filtered and concentrated. The residue was co-evaporated with MeCN (10 mL) to give a white solid. All solids were dissolved in MeCN (25 mL) by gentle heating with a heat gun. The solution was gradually cooled to -30 °C, at which point a white solid began to precipitate. The flask was left overnight at -30 °C and allowed to warm to room temperature before filtration. The solid was washed with ice-cold MeCN (10 mL) to give chloride XD34 (8.03 g, 87% yield) as a white solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.76-7.71 (m, 4H), 7.56-7.48 (m, 4H), 7.43-7.36 (m, 4H), 7.33-7.25 (m, 4H), 4.46 (dt, J = 9.7, 7.1 Hz, 2H), 4.36-4.28 (m, 2H), 4.25-4.19 (m, 2H). MS (ESI + ) C 28 H 24 ClO4P + [M+NH4] + Calculated value: 490.1, measured value: 490.3.

[0221] 2C. Synthesis of Phosphate Esters XS44-XS48

[0222] [ka]

[0223] (4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS39) Following general procedure XXF, the allylic alcohol XS33 was reacted with XD34 (0.500 g, 1.06 mmol). The crude product was purified by flash chromatography (silica gel, 0-40% EtOAc in heptane) to afford alkyl phosphate XS39 (0.458 g, 57%) as a white sticky solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.84 (dd, J = 7.5, 4.1 Hz, 4H), 7.61-7.48 (m, 8H), 7.47-7.33 (m, 10H), 7.26 (tdd, J = 7.4, 5.2, 1.0 Hz, 4H), 5.76-5.62 (m, 2H), 4.29-4.13 (m, 8H), 4.13-4.09 (m, 2H), 0.95 (s, 9H). MS (ESI + ) C 48 H 48 O5PSi + [M+H] + Calculated value: 763.3, measured value: 763.6.

[0224] (4-((tert-butyldiphenylsilyl)oxy)-3-fluorobut-2-en-1-yl)phosphate (Z)-bis((9H-fluoren-9-yl)methyl) ester (XS40) Following general procedure XXF, allylic alcohol XS38 was reacted with XD34 (0.570 g, 1.21 mmol), and the crude product was purified by flash chromatography (silica gel, 0–40% EtOAc in heptane) to give alkyl phosphate XS40 (0.555 g, 59%) as a white sticky solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.83 (dd, J = 7.5, 3.8 Hz, 4H), 7.63-7.31 (m, 18H), 7.30-7.21 (m, 4H), 5.02 (dt, J = 35.8, 7.4 Hz, 1H), 4.28-4.19 (m, 6H), 4.18-4.09 (m, 4H), 0.95 (s, 9H). MS (ESI + ) C 48 H 47 FO5PSi + [M+H] + Calculated value: 781.3, measured value: 781.7.

[0225] (4-((tert-butyldiphenylsilyl)oxy)-3-chlorobut-2-en-1-yl)phosphate (Z)-bis((9H-fluoren-9-yl)methyl) ester (XS41) Following general procedure XXF, allylic alcohol XD67 was reacted with XD34 (0.378 g, 0.799 mmol), and the crude product was purified by flash chromatography (silica gel, 0–40% EtOAc in heptane) to afford alkyl phosphate XS41 (0.426 g, 67%) as a white sticky solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.84 (dd, J = 7.6, 3.8 Hz, 4H), 7.60-7.34 (m, 18H), 7.30-7.22 (m, 4H), 5.91 (t, J = 6.3 Hz, 1H), 4.35-4.21 (m, 6H), 4.19-4.13 (m, 4H), 0.96 (s, 9H). MS (ESI + ) C 48 H 47 ClO5PSi + [M+H] +Calculated value: 797.3, measured value: 797.9.

[0226] (3-Bromo-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)phosphate (Z)-bis((9H-fluoren-9-yl)methyl) ester (XS42) Following general procedure XXF, allylic alcohol XS31 was reacted with XD34 (0.500 g, 1.06 mmol), and the crude product was purified by flash chromatography (silica gel, 0–35% EtOAc in heptane) to give alkyl phosphate XS42 (0.890 g, 51%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.84 (dd, J = 7.5, 3.8 Hz, 4H), 7.62-7.57 (m, 4H), 7.53 (dd, J = 16.3, 7.5 Hz, 4H), 7.48-7.31 (m, 10H), 7.30-7.24 (m, 4H), 6.15 (t, J = 6.0 Hz, 1H), 4.33-4.19 (m, 8H), 4.19-4.13 (m, 2H), 0.96 (s, 9H).

[0227] (4-((tert-butyldiphenylsilyl)oxy)-3-iodobut-2-en-1-yl)phosphate (Z)-bis((9H-fluoren-9-yl)methyl) ester (XS43) Following general procedure XXF, allylic alcohol XD66 was reacted with XD34 (0.350 g, 0.740 mmol), and the crude product was purified by flash chromatography (silica gel, 0–35% EtOAc in heptane) to afford alkyl phosphate XS43 (0.430 g, 65%) as a white sticky solid. 1H NMR (400 MHz, DMSO-d6) ppm = 7.84 (dd, J = 7.5, 3.8 Hz, 4H), 7.61-7.50 (m, 8H), 7.47-7.33 (m, 10H), 7.31-7.22 (m, 4H), 6.12 (t, J = 5.8 Hz, 1H), 4.30-4.12 (m, 10H), 0.97 (s, 9H). MS (ESI + ) C 48 H 47 IO5PSi + [M+H] + Calculated value: 889.2, measured value: 889.7.

[0228] (E)-4-Hydroxybut-2-en-1-yl phosphate triethylammonium salt (XS44) Step 1: Following general procedure XXG, TBDPS-ether XS39 (0.455 g, 0.596 mmol) was reacted. The crude product was purified by flash chromatography (silica gel, 0-75% EtOAc in heptane) to afford the intermediate allylic alcohol (47 mg, 15%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.87 (t, J = 6.7 Hz, 4H), 7.53 (dd, J = 17.1, 7.5 Hz, 4H), 7.39 (dt, J = 11.8, 7.3 Hz, 4H), 7.34-7.25 (m, 4H), 5.74-5.65 (m, 1H), 5.59-5.50 (m, 1H), 4.79 (t, J = 5.4 Hz, 1H), 4.27-4.11 (m, 8H), 3.92-3.85 (m, 2H). MS (ESI + ) C 32 H 30 O5P + [M+H] + Calculated value: 525.2, measured value: 525.4.

[0229] Step 2: Following general procedure XXH, the intermediate (47 mg, 0.090 mmol) was reacted to give alkyl phosphate XS44 (14 mg, 61%) as the triethylamine salt. MS (ESI- ) C4H8O5P - [MH] - Calculated value: 167.0, measured value: 166.9.

[0230] (Z)-3-Fluoro-4-hydroxybut-2-en-1-yl phosphate triethylammonium salt (XS45) Step 1: Following general procedure XXG, TBDPS-ether XS40 (0.552 g, 0.707 mmol) was reacted. The crude product was purified by flash chromatography (silica gel, 0-60% EtOAc in heptane) to afford the intermediate allylic alcohol (0.355 g, 93%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.86 (t, J = 6.8 Hz, 4H), 7.53 (dd, J = 16.6, 7.5 Hz, 4H), 7.39 (dt, J = 12.1, 7.4 Hz, 4H), 7.34-7.24 (m, 4H), 5.37 (t, J = 5.9 Hz, 1H), 4.99 (dt, J = 36.1, 7.4 Hz, 1H), 4.29-4.19 (m, 6H), 4.19-4.13 (m, 2H), 3.91 (dd, J = 12.1, 5.9 Hz, 2H). MS (ESI + ) C 32 H 29 FO5P + [M+H] + Calculated value 543.2, measured value 543.2.

[0231] Step 2: Following general procedure XXH, the intermediate (0.350 g, 0.645 mmol) was reacted to give alkyl phosphate XS45 (0.115 g, 69%) as the triethylamine salt in a 1:0.7 ratio of phosphoric acid:Et3N. 1H NMR (400 MHz, DMSO-d6) ppm = 5.11 (dt, J = 37.9, 7.0 Hz, 1H), 4.30-4.24 (m, 2H), 3.93 (d, J = 13.1 Hz, 2H), 2.94 (q, J = 7.3 Hz, 4H), 1.15 (t, J = 7.3 Hz, 6H). MS (ESI - ) C4H7FO5P - [MH] - Calculated value 185.0, actual value 185.0.

[0232] (Z)-3-chloro-4-hydroxybut-2-en-1-yl phosphate monotriethylammonium salt (XS46) Step 1: Following general procedure XXG, TBDPS-ether XS41 (0.422 g, 0.529 mmol) was reacted. The crude product was suspended in DCM (10 mL), to which EtO (40 mL) and heptane (50 mL) were added. The suspension was gently heated, filtered, and the residue was washed with EtO (10 mL) to give the intermediate allylic alcohol (0.202 g, 68%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.86 (t, J = 6.6 Hz, 4H), 7.54 (dd, J = 17.3, 7.5 Hz, 4H), 7.39 (dt, J = 11.6, 7.3 Hz, 4H), 7.33-7.25 (m, 4H), 5.92-5.86 (m, 1H), 5.58 (t, J = 6.1 Hz, 1H), 4.33-4.21 (m, 6H), 4.19-4.13 (m, 2H), 3.96 (dd, J = 6.1, 1.1 Hz, 2H). MS (ESI + ) C 32 H 29 ClO5P + [M+H] + Calculated value: 559.1, measured value: 559.4.

[0233] Step 2: Following general procedure XXH, the intermediate (0.198 g, 0.354 mmol) was reacted to give alkyl phosphate XS46 (53 mg, 49%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, DMSO-d6) ppm = 6.03 (tt, J = 5.8, 1.3 Hz, 1H), 4.38-4.32 (m, 2H), 3.98 (d, J = 1.3 Hz, 2H), 2.94 (q, J = 7.0 Hz, 5H), 1.15 (t, J = 7.3 Hz, 8H). MS (ESI - ) C4H7ClO5P - [MH] - Calculated value: 201.0, measured value: 200.7.

[0234] (Z)-3-Bromo-4-hydroxybut-2-en-1-yl phosphate triethylammonium salt (XS47) Step 1: Following general procedure XXG, TBDPS-ether XS42 (0.445 g, 0.529 mmol) was reacted. The crude product was purified by flash chromatography (silica gel, 0-60% EtOAc in heptane) to afford the intermediate allylic alcohol (0.294 g, 92%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.86 (t, J = 6.6 Hz, 4H), 7.54 (dd, J = 16.9, 7.5 Hz, 4H), 7.39 (dt, J = 11.8, 7.4 Hz, 4H), 7.34-7.24 (m, 4H), 6.16-6.09 (m, 1H), 5.63 (t, J = 6.2 Hz, 1H), 4.32-4.22 (m, 6H), 4.20-4.14 (m, 2H), 4.02 (dd, J = 6.1, 1.4 Hz, 2H). MS (ESI + ) C 32 H 29 BrO5P + [M+H] + Calculated value 603.1, measured value 603.4.

[0235] Step 2: Following general procedure XXH, the intermediate (0.290 g, 0.481 mmol) was reacted to give alkyl phosphate XS47 (0.130 g, 78%) as the triethylamine salt in a 1:0.95 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 6.18 (t, J = 5.9 Hz, 1H), 4.47-4.39 (m, 2H), 4.15 (d, J = 0.9 Hz, 2H), 3.08 (q, J = 7.4 Hz, 6H), 1.15 (t, J = 7.4 Hz, 9H). MS (ESI - ) C4H7BrO5P - [MH] - Calculated value: 244.9, measured value: 245.1.

[0236] (Z)-4-Hydroxy-3-iodobut-2-en-1-yl phosphate monotriethylammonium salt (XS48) Step 1: Following general procedure XXG, TBDPS-ether XS43 (0.423 g, 0.476 mmol) was reacted. The crude product was purified by flash chromatography (silica gel, 0-60% EtOAc in heptane) to afford the intermediate allylic alcohol (0.262 g, 85%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.87 (t, J = 6.8 Hz, 4H), 7.54 (dd, J = 15.1, 7.4 Hz, 4H), 7.39 (dt, J = 12.1, 7.3 Hz, 4H), 7.33-7.25 (m, 4H), 6.13-6.08 (m, 1H), 5.64 (t, J = 6.1 Hz, 1H), 4.29-4.20 (m, 6H), 4.20-4.14 (m, 2H), 4.02 (dd, J = 6.2, 1.4 Hz, 2H). MS (ESI + ) C 32 H 29 IO5P + [M+H] + Calculated value 651.1, measured value 651.5.

[0237] Step 2: Following general procedure XXH, the intermediate (0.258 g, 0.397 mmol) was reacted to give alkyl phosphate XS48 (76 mg, 49%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, DMSO-d6) ppm = 6.24 (tt, J = 5.3, 1.4 Hz, 1H), 4.28-4.23 (m, 2H), 4.04 (d, J = 1.6 Hz, 2H), 2.95 (q, J = 7.1 Hz, 5H), 1.15 (t, J = 7.3 Hz, 8H). MS (ESI - ) C4H7IO5P - [MH] - Calculated value 292.9, measured value 292.9.

[0238] Preparation of 4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)benzyl phosphate (XD36) [ka]

[0239] (4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)benzyl)phosphate bis((9H-fluoren-9-yl)methyl)ester (XD35) Step 1: 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (142 mg, 0.574 mmol) was dissolved in DMF (1 mL). Val-Ala-PAB-OH (160 mg, 0.545 mmol) in DMF (3.0 mL) was added, followed by HATU (228 mg, 0.600 mmol) and DIPEA (0.143 mL, 0.818 mmol) at room temperature. The reaction was stirred for 30 minutes and then concentrated. The crude product was dissolved in MeOH (1 mL), and the solution was subjected to a short DOWEX 50WX8 column, pre-washed with methanol, to remove basic impurities. The product was eluted with methanol, and the crude product was concentrated onto silica gel. Purification by flash chromatography (silica gel, 0-8% MeOH in DCM) afforded the amide (262 mg, 92%) as a cream solid. MS (ESI) + ) C 24 H 39 N6O7 + [M+H] + Calculated value: 523.3, measured value: 523.6.

[0240] Step 2: To the amide (977 mg, 1.87 mmol) and 5-(ethylthio)-1H-tetrazole (19 mg, 0.15 mmol) in MeCN (3.7 mL) under a N atmosphere, 2,6-lutidine (719 μL, 6.17 mmol) was added at room temperature, followed by a solution of chloride XD34 (884 mg, 1.87 mmol) in DCM (3.7 mL). The mixture was stirred at room temperature. Additional portions of chloride XD34 were added after 80 min (88 mg, 0.187 mmol) and 140 min (177 mg, 0.374 mmol). After a total reaction time of 185 min, additional 2,6-lutidine (218 μL, 1.87 mmol) was added, and the reaction was continued for 2 h before being quenched with methanol (1 mL). The mixture was concentrated, and the crude product was dissolved in EtOAc (80 mL) and hydrochloric acid (40 mL, 1 M). A small amount of MeCN (4 mL) was added to dissolve the remaining solid, and the phases were separated. The aqueous phase was extracted with EtOAc (80 mL), and the combined organic phases were washed with brine and dried over Na2SO4. The crude product was purified by flash chromatography (silica gel, 0-5% MeOH in DCM) to give the phosphate ester XD35 (1.40 g, 66% yield). 1 H NMR (400 MHz, DMSO-d6) ppm = 9.94 (s, 1H), 8.18 (d, J = 7.0 Hz, 1H), 7.89-7.82 (m, 5H), 7.55 (d, J = 8.6 Hz, 2H), 7.52-7.44 (m, 4H), 7.42-7.34 (m, 4H), 7.30-7.24 (m, 4H), 7.09 (d, J = 8.6 Hz, 2H), 4.60 (d, J = 8.8 Hz, 2H), 4.40 (quint, J = 7.0 Hz, 1H), 4.25-4.17 (m, 5H), 4.15-4.11 (m, 2H), 3.62-3.56 (m, 4H), 3.55-3.46 (m, 8H), 3.39-3.36 (m, 2H), 2.50-2.36 (m, 2H), 2.02-1.93 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). MS (ESI +) C 52 H 59 NO 10 PNa + [M+H] + Calculated value: 981.4, measured value: 981.8.

[0241] 4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadecane-18-amido)benzyl phosphate (XD36) To a solution of the phosphate ester XD35 (160 mg, 0.167 mmol) in MeCN (1 mL) at room temperature, triethylamine (0.25 mL) was added, and the reaction was stirred for 24 h. The reaction was diluted with toluene (8 mL) and then concentrated. The crude product was suspended in ether (10 mL), filtered, and the solid was washed repeatedly with ether to give alkyl phosphate XD36 (108 mg, 92%) as the monotriethylamine salt. (Note: The product contained an impurity (m / z 606) likely formed by elimination of the phosphoric acid and trapping of the intermediate azaquinone methide with triethylamine. This impurity did not react in the next step, and no further purification was necessary.) MS (ESI - ) C 24 H 38 NO 10 P - [MH] - Calculated value 601.2, measured value 601.7.

[0242] Preparation of alkyne linker (XD43) [ka]

[0243] Prop-2-yn-1-ylcarbamic acid 2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ester (XD43) To PNP-carbonate XD53 (511 mg, 1.46 mmol, synthesized according to Elgersma, RC et al. Mol. Pharm. 2015, 12, 1813-1835) in THF (10 mL) at 0 °C was added propargylamine (0.093 mL, 1.46 mmol). The cold bath was removed, and the mixture was stirred at room temperature for 2 h. The mixture was concentrated, and the crude product was purified by flash chromatography (silica gel, 0-70% EtOAc in heptane) to give XD43 (265 mg, 68%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.60 (br t, J = 5.5 Hz, 1H), 7.02 (s, 2H), 4.07-3.96 (m, 2H), 3.74 (dd, J = 5.8, 2.4 Hz, 2H), 3.61-3.48 (m, 7H), 3.07 (t, J = 2.5 Hz, 1H).

[0244] 2D. Synthesis of Linker-Drugs XS54–XS58

[0245] [ka]

[0246] Pyrophosphate ester XS49 According to general procedure XXD, alkyl phosphate XD36 (32 mg, 0.045 mmol) was reacted with phosphate ester XS44. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS49 (19.6 mg, 55%) as a white solid. MS (ESI + ) C 28 H 47 NO 14 P2 + [M+H] + Calculated value: 753.3, measured value: 753.8.

[0247] Pyrophosphate ester XS50 According to general procedure XXD, alkyl phosphate XD36 (55 mg, 0.078 mmol) was reacted with phosphate ester XS45. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) to give, after lyophilization, pyrophosphate ester XS50 (28.8 mg, 46%) as a white solid. 1 H NMR (400 MHz, D2O) ppm = 7.46-7.39 (m, 4H), 5.15 (dt, J = 35.8, 7.2 Hz, 1H), 4.94 (d, J = 6.8 Hz, 2H), 4.47 (t, J = 7.3 Hz, 2H), 4.39 (q, J = 7.2 Hz, 1H), 4.12 (d, J = 7.1 Hz, 1H), 4.03 (d, J = 15.9 Hz, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.67-3.56 (m, 10H), 3.45-3.40 (m, 2H), 2.64-2.48 (m, 2H), 2.12-1.98 (m, 1H), 1.43 (d, J = 7.3 Hz, 3H), 0.92 (d, J = 5.1 Hz, 3H), 0.90 (d, J = 5.1 Hz, 3H). MS (ESI + ) C 28 H 46 FN6O 14 P2 + [M+H] + Calculated value: 771.3, measured value: 771.9.

[0248] Pyrophosphate ester XS51 According to general procedure XXD, alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate ester XS46. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) to give, after lyophilization, pyrophosphate XS51 (62.4 mg, 54%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.36 (d, J = 1.8 Hz, 4H), 5.90 (t, J = 6.0 Hz, 1H), 4.86 (d, J = 7.0 Hz, 2H), 4.50-4.42 (m, 2H), 4.37-4.28 (m, 1H), 4.09-4.03 (m, 1H), 4.01 (d, J = 0.9 Hz, 2H), 3.66 (t, J = 6.0 Hz, 2H), 3.59-3.49 (m, 10H), 3.39-3.33 (m, 2H), 2.57-2.41 (m, 2H), 1.98 (dq, J = 13.7, 6.8 Hz, 1H), 1.36 (d, J = 7.3 Hz, 3H), 0.85 (d, J = 5.1 Hz, 3H), 0.84 (d, J = 5.1 Hz, 3H). MS (ESI + ) C 28 H 46 ClNO 14 P2 + [M+H] + Calculated value: 787.2, measured value: 787.6.

[0249] Pyrophosphate ester XS52 According to general procedure XXD, alkyl phosphate XD36 (70 mg, 0.099 mmol) was reacted with phosphate ester XS47. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) to give, after lyophilization, pyrophosphate XS52 (51.9 mg, 60%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.45-7.36 (m, 4H), 6.17 (t, J = 5.8 Hz, 1H), 4.95-4.88 (m, 2H), 4.46 (t, J = 6.5 Hz, 2H), 4.41-4.34 (m, 1H), 4.15-4.06 (m, 3H), 3.71 (t, J = 5.9 Hz, 2H), 3.65-3.53 (m, 10H), 3.45-3.37 (m, 2H), 2.65-2.42 (m, 2H), 2.13-1.95 (m, 1H), 1.41 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 5.3 Hz, 3H), 0.89 (d, J = 5.3 Hz, 3H). MS (ESI + ) C 28 H 46 BrNO 14 P2 + [M+H] + Calculated value: 831.2, measured value: 831.6.

[0250] Pyrophosphate ester XS53 According to general procedure XXD, alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate ester XS48. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS53 (66.4 mg, 51%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.46-7.39 (m, 4H), 6.12 (t, J = 5.6 Hz, 1H), 4.96-4.90 (m, 2H), 4.44-4.33 (m, 3H), 4.14-4.09 (m, 3H), 3.73 (t, J = 6.0 Hz, 2H), 3.66-3.54 (m, 10H), 3.45-3.39 (m, 2H), 2.65-2.47 (m, 2H), 2.11-1.98 (m, 1H), 1.43 (d, J = 7.1 Hz, 3H), 0.92 (d, J = 5.4 Hz, 3H), 0.90 (d, J = 5.5 Hz, 3H). MS (ESI + ) C 28 H 46 IN6O 14 P2 + [M+H] + Calculated value: 879.2, measured value: 879.6.

[0251] Linker-Drug XS54 According to general procedure XXE, azide XS49 (19.6 mg, 0.025 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS54 (11.7 mg, 45%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.92 (br s, 1H), 7.40 (s, 4H), 6.73 (s, 2H), 5.90-5.79 (m, 1H), 5.79-5.69 (m, 1H), 4.92 (s, 2H), 4.53 (t, J = 4.8 Hz, 2H), 4.41-4.33 (m, 3H), 4.33-4.26 (m, 2H), 4.14-4.04 (m, 3H), 4.01 (d, J = 4.9 Hz, 2H), 3.87 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.65-3.57 (m, 6H), 3.57-3.45 (m, 8H), 2.63-2.46 (m, 2H), 2.03 (dq, J = 13.7, 6.8 Hz, 1H), 1.41 (d, J = 7.1 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 40 H 61 N8O 19 P2 + [M+H] + Calculated value: 1019.4, measured value: 1020.0.

[0252] Linker-Drug XS55 According to general procedure XXE, azide XS50 (24.5 mg, 0.030 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) afforded the pyrophosphate XS55 (12.6 mg, 39%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.92 (br s, 1H), 7.47-7.35 (m, 4H), 6.74 (s, 2H), 5.15 (dt, J = 35.8, 7.0 Hz, 1H), 4.99-4.88 (m, 2H), 4.53 (t, J = 4.8 Hz, 2H), 4.50-4.45 (m, 2H), 4.38 (q, J = 7.2 Hz, 1H), 4.31 (t, J = 7.3 Hz, 2H), 4.15-4.06 (m, 3H), 4.03 (d, J = 15.9 Hz, 2H), 3.88 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.59 (m, 6H), 3.56-3.47 (m, 8H), 2.62-2.47 (m, 2H), 2.04 (dq, J = 13.7, 6.8 Hz, 1H), 1.42 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 40 H 60 FN8O 19 P2 + [M+H] + Calculated value: 1037.3, measured value: 1037.8.

[0253] Linker-Drug XS56 According to general procedure XXE, azide XS51 (59.2 mg, 0.072 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) afforded the pyrophosphate XS56 (43.4 mg, 55%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.93 (br s, 1H), 7.40 (s, 4H), 6.73 (s, 2H), 5.96 (t, J = 5.1 Hz, 1H), 4.92 (br s, 2H), 4.57-4.48 (m, 4H), 4.37 (q, J = 7.2 Hz, 1H), 4.30 (br s, 2H), 4.11 (d, J = 7.0 Hz, 1H), 4.07 (s, 4H), 3.87 (t, J = 4.8 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.58 (m, 6H), 3.57-3.42 (m, 8H), 2.61-2.46 (m, 2H), 2.04 (dq, J = 13.7, 6.8 Hz, 1H), 1.41 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 40 H 60 ClNO 19 P2 + [M+H] + Calculated value: 1053.3, measured value: 1053.9.

[0254] Linker-Drug XS57 According to general procedure XXE, azide XS52 (40.7 mg, 0.047 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) afforded the pyrophosphate XS57 (23.9 mg, 45%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.89 (br s, 1H), 7.39 (s, 4H), 6.72 (s, 2H), 6.17 (t, J = 5.4 Hz, 1H), 4.91 (br s, 2H), 4.51 (t, J = 4.9 Hz, 2H), 4.46 (br s, 2H), 4.36 (q, J = 7.2 Hz, 1H), 4.29 (br s, 2H), 4.14-4.02 (m, 5H), 3.86 (t, J = 4.9 Hz, 2H), 3.68 (t, J = 5.9 Hz, 2H), 3.64-3.57 (m, 6H), 3.54-3.45 (m, 8H), 2.61-2.45 (m, 2H), 2.09-1.95 (m, 1H), 1.40 (d, J = 7.3 Hz, 3H), 0.89 (d, J = 7.0 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 40 H 60 BrNO 19 P2 + [M+H] + Calculated value: 1097.3, measured value: 1097.6.

[0255] Linker-Drug XS58 Azide XS53 (62.6 mg, 0.069 mmol) was reacted with alkyne XD43 according to general procedure XXE. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 60:40 gradient) afforded the pyrophosphate XS58 (41.2 mg, 51%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.89 (br s, 1H), 7.39 (s, 4H), 6.72 (s, 2H), 6.11 (t, J = 5.0 Hz, 1H), 4.91 (br s, 2H), 4.51 (t, J = 4.8 Hz, 2H), 4.44-4.32 (m, 3H), 4.29 (br s, 2H), 4.15-4.02 (m, 5H), 3.86 (t, J = 4.9 Hz, 2H), 3.68 (t, J = 5.9 Hz, 2H), 3.64-3.57 (m, 6H), 3.55-3.44 (m, 8H), 2.52 (q, J = 5.8 Hz, 2H), 2.02 (dq, J = 13.7, 6.8 Hz, 1H), 1.40 (d, J = 7.1 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H). MS (ESI + ) C 40 H 60 IN8O 19 P2 + [M+H] + Calculated value: 1145.2, measured value: 1146.1.

[0256] Example 3 Synthesis of Linker-Drug Compounds XS100-XS107 3A. Preparation of Ether (XS60)

[0257] [ka]

[0258] (Z)-tert-butyl((2-iodo-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)diphenylsilane (XS60) Alcohol XD66 (1.00 g, 2.21 mmol) was dissolved in DCM (2.2 mL) and PPTS (56 mg, 0.22 mmol) was added. The reaction mixture was cooled to 0 °C and DHP (303 μL, 3.32 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. DCM was added, and the mixture was washed with saturated aqueous NaHCO, water, and brine, dried over NaSO, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give ether XS60 (1.05 g, 89%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.64 (dd, J = 7.8, 1.5 Hz, 4H), 7.52-7.41 (m, 6H), 6.30 (t, J = 5.7 Hz, 1H), 4.60 (t, J = 3.3 Hz, 1H), 4.30 (d, J = 1.3 Hz, 2H), 4.24-4.01 (m, 2H), 3.75 (ddd, J = 11.3, 8.3, 3.1 Hz, 1H), 3.49-3.41 (m, 1H), 1.76-1.57 (m, 2H), 1.55-1.39 (m, 4H), 1.03 (s, 9H). MS (ESI + ) C 25 H 33 INaO3Si + [M+Na] + Calculated value: 559.1, measured value: 559.5.

[0259] 3B. Preparation of Functionalized Alkenes (XS61-XS65), Trifluoroalkene (XS71), and Alkyne (XS72) Preparation of functionalized alkenes (XS61-XS62)

[0260] [ka]

[0261] (E)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-enenitrile (XS61) According to general procedure XXI, tributyltincarbonitrile XS60 (1.05 g, 1.96 mmol) was reacted with XS60. The crude product was purified by flash chromatography (silica gel, 0-25% EtO in heptane) and filtered from heptane. The filtrate was concentrated to give nitrile XS61 (0.882 g, quantitative) as a yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.65 (dd, J = 7.8, 1.4 Hz, 4H), 7.49-7.36 (m, 6H), 6.61-6.55 (m, 1H), 4.66 (t, J = 3.5 Hz, 1H), 4.53-4.46 (m, 1H), 4.36-4.29 (m, 1H), 4.27-4.22 (m, 2H), 3.90-3.81 (m, 1H), 3.58-3.51 (m, 1H), 1.91-1.70 (m, 2H), 1.68-1.58 (m, 2H), 1.55-1.50 (m, 1H), 1.43-1.29 (m, 1H), 1.08 (s, 9H). MS (ESI + ) C 26 H 34 NO3Si + [M+H] + Calculated value: 436.2, measured value: 436.4.

[0262] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-vinylbut-2-en-1-yl)oxy)silane (XS62) According to general procedure XXI, vinyltributyltin was reacted with XS60 (0.400 g, 0.746 mmol). The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give the diene XS62 (0.284 g, 87%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) ppm = 7.66-7.61 (m, 4H), 7.50-7.41 (m, 6H), 6.60 (dd, J = 17.7, 11.3 Hz, 1H), 5.87 (t, J = 6.7 Hz, 1H), 5.22-5.09 (m, 2H), 4.59 (t, J = 3.5 Hz, 1H), 4.36 (s, 2H), 4.33-4.18 (m, 2H), 3.75 (ddd, J = 11.2, 8.1, 3.1 Hz, 1H), 3.49-3.38 (m, 1H), 1.76-1.56 (m, 2H), 1.55-1.39 (m, 4H), 1.01 (s, 9H). MS (ESI + ) C 27 H 37 O3Si + [M+H] + Calculated value: 437.3, measured value: 437.4.

[0263] Preparation of functionalized alkenes (XS63) [ka]

[0264] (E)-tert-butyl((2-ethyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)diphenylsilane (XS63) According to general procedure XXJ, diethylzinc (1.0 M in hexanes, 1.92 mL) was reacted with XS60 (0.515 g, 0.960 mmol). The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give alkane XS63 (0.193 g, 46%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.70-7.66 (m, 4H), 7.45-7.34 (m, 6H), 5.69 (t, J = 6.8 Hz, 1H), 4.64 (t, J = 3.6 Hz, 1H), 4.29 (dd, J = 12.3, 6.2 Hz, 1H), 4.17-4.08 (m, 3H), 3.93-3.86 (m, 1H), 3.56-3.47 (m, 1H), 2.15-2.00 (m, 2H), 1.91-1.68 (m, 2H), 1.66-1.57 (m, 2H), 1.56-1.50 (m, 2H), 1.06 (s, 9H), 0.92 (t, J = 7.6 Hz, 3H). MS (ESI + ) C 27 H 38 NaO3Si + [M+H] + Calculated value: 461.2, measured value: 461.4.

[0265] Functionalized Alkene (XS64) [ka]

[0266] (E)-tert-butyldiphenyl((2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethylidene)pentyl)oxy)silane (XS64) According to Procedure XXJ, XS60 (0.770 g, 1.44 mmol) was reacted with isopropylchlorozinc (0.815 g, 5.74 mmol) (prepared fresh by adding i-PrMgCl (2.0 M in THF, 2.87 mL) to a solution of ZnCl (0.782 g, 5.74 mmol) in THF (4.8 mL) at 0 °C). The crude product was purified by flash chromatography (silica gel, 0-8% EtOAc in heptane) to give alkane XS64 (0.335 g, 52%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.70-7.66 (m, 4H), 7.44-7.33 (m, 6H), 5.74 (t, J = 6.9 Hz, 1H), 4.67-4.62 (m, 1H), 4.28 (dd, J = 12.2, 6.2 Hz, 1H), 4.17-4.12 (m, 1H), 4.11 (s, 2H), 3.94-3.85 (m, 1H), 3.55-3.48 (m, 1H), 2.10-1.96 (m, 2H), 1.90-1.68 (m, 2H), 1.64-1.56 (m, 2H), 1.54-1.49 (m, 2H), .36-1.28 (m, 2H), 1.06 (s, 9H), 0.83 (t, J = 7.4

[0267] Functionalized Alkenes (XS65) [ka]

[0268] (E)-tert-butyl((2-cyclobutyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)diphenylsilane (XS65) According to general procedure XXJ, cyclobutylchlorozinc (0.5 M in THF, 11.2 mL) was reacted with XS60 (0.750 g, 1.40 mmol). The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give cycloalkane XS65 (0.574 g, 88%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.72-7.67 (m, 4H), 7.46-7.34 (m, 6H), 5.67-5.61 (m, 1H), 4.65-4.60 (m, 1H), 4.25 (dd, J = 12.4, 6.2 Hz, 1H), 4.21 (s, 2H), 4.11 (dd, J = 12.3, 7.5 Hz, 1H), 3.94-3.86 (m, 1H), 3.55-3.48 (m, 1H), 3.33 (quint, J = 9.1 Hz, 1H), 2.06-1.95 (m, 4H), 1.90-1.79 (m, 2H), 1.77-1.64 (m, 2H), 1.64-1.53 ​​(m, 4H), 1.06 (s, 9H). MS (ESI + ) C 29 H 40 NaO3Si + [M+H] + Calculated value: 487.3, measured value: 487.5.

[0269] Trifluoroalkene (XS71)

[0270] Aldehyde (XS67) [ka]

[0271] 2-((tetrahydro-2H-pyran-2-yl)oxy)ethan-1-ol (XS66) Ethylene glycol (5.00 mL, 90.0 mmol) was suspended in THF (90 mL) and cooled to 0 °C. PTSA monohydrate (0.426 g, 2.24 mmol) was added, followed by the dropwise addition of DHP (2.05 mL, 22.4 mmol). The reaction mixture was stirred at 0 °C for 30 min, allowed to warm to room temperature, and stirred for 3 days. The reaction mixture was diluted with water, and the product was extracted with DCM (2x). The organic phase was washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-60% EtOAc in heptane) to give ether XS66 (2.24 g, 68%) as a colorless oil.1 H NMR (400 MHz, CDCl3) ppm = 4.60-4.55 (m, 1H), 3.98-3.90 (m, 1H), 3.82-3.66 (m, 4H), 3.61-3.50 (m, 1H), 2.85 (dd, J = 7.0, 4.5 Hz, 1H), 1.88-1.72 (m, 2H), 1.64-1.50 (m, 4H). MS (ESI + ) C7H 15 O3 + [M+H] + Calculated value: 147.1, measured value: 147.0.

[0272] 2-((tetrahydro-2H-pyran-2-yl)oxy)acetaldehyde (XS67) To a solution of oxalyl chloride (1.74 mL, 19.9 mmol) in DCM (140 mL) at −78° C., DMSO (2.80 mL, 39.4 mmol) was added dropwise. The reaction mixture was stirred at −78° C. for 1 h, after which a solution of alcohol XS66 (2.24 g, 15.3 mmol) in DCM (13 mL) was added dropwise. The reaction mixture was stirred at −78° C. for 30 min, after which TEA (13.7 mL, 98.0 mmol) was added dropwise. The reaction mixture was stirred at −78° C. for 45 min, allowed to warm to room temperature, and stirred for 45 min. Saturated aqueous NaHCO3 was added, and the mixture was stirred for 15 min. The organic phase was separated, and the aqueous phase was extracted with DCM (2×). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in heptane) to afford aldehyde XS67 (1.32 g, 60%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 9.76 (t, J = 1.0 Hz, 1H), 4.66 (dd, J = 4.0, 3.1 Hz, 1H), 4.24 (dd, J = 18.0, 1.2 Hz, 1H), 4.17 (dd, J = 18.0, 0.8 Hz, 1H), 3.92-3.82 (m, 1H), 3.59-3.48 (m, 1H), 1.91-1.67 (m, 4H), 1.58 (td, J = 4.2, 2.5 Hz, 2H).

[0273] Trifluoroalkene (XS71) [ka]

[0274] (E)-4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-enoic acid ethyl ester (XS69) To a solution of aldehyde XS67 (0.894 g, 6.20 mmol) in THF (4.1 mL) was added Wittig reagent XS68 (1.78 g, 4.14 mmol, synthesized according to U.S. Patent Application Publication No. 2007 / 0249723) in THF (4.1 mL). The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was diluted with EtO, dried over NaSO, and filtered through a silica plug. The filtrate was concentrated, and the crude product was purified by flash chromatography (silica gel, 0-20% EtOAc in heptane) to give the E-alkene XS69 (0.435 g, 36%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.20 (t, J = 6.0 Hz, 1H), 4.65 (t, J = 3.3 Hz, 1H), 4.47 (dd, J = 15.1, 5.7 Hz, 1H), 4.29-4.19 (m, 3H), 3.85 (ddd, J = 11.3, 8.4, 3.2 Hz, 1H), 3.58-3.51 (m, 1H), 3.28 (qd, J = 10.5, 1.8 Hz, 2H), 1.89-1.70 (m, 2H), 1.67-1.50 (m, 4H), 1.32 (t, J = 7.1Hz, 3H).

[0275] (E)-4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-en-1-ol (XS70) Ester XS69 (0.425 g, 1.43 mmol) was purged with N and dissolved in THF (7.2 mL). DIBAL-H (1.0 M in hexanes, 3.16 mL) was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. HCl (1.0 M) was then added, and the reaction mixture was stirred for 30 min. The aqueous phase was extracted with DCM (3 ×), and the combined organic phases were washed with brine, dried over Na SO , and concentrated. The crude product was purified by flash chromatography (silica gel, 0-40% EtOAc in heptane) to give alcohol XS70 (0.232 g, 64%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) ppm = 6.00 (t, J = 6.4 Hz, 1H), 4.63 (t, J = 3.4 Hz, 1H), 4.33 (dd, J = 13.0, 6.1 Hz, 1H), 4.17 (d, J = 6.4 Hz, 2H), 4.09 (dd, J = 13.1, 6.9 Hz, 1H), 3.86 (ddd, J = 11.2, 8.0, 3.4 Hz, 1H), 3.57-3.49 (m, 1H), 3.07-2.93 (m, 2H), 1.86-1.71 (m, 2H), 1.63-1.57 (m, 2H), 1.54-1.51 (m, 2H), 1.51-1.45 (m, 1H).

[0276] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-en-1-yl)oxy)silane (XS71) To a solution of alcohol XS70 (0.230 g, 0.905 mmol), TEA (0.252 mL, 1.81 mmol), and imidazole (68 mg, 1.0 mmol) at 0 °C, TBDPSCl (0.349 mL, 1.36 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. Water was added, and the product was extracted with DCM (3x). The combined organic phases were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give the silyl ether XS71 (0.378 g, 85%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.68-7.63 (m, 4H), 7.45-7.35 (m, 6H), 5.99 (t, J = 6.5 Hz, 1H), 4.62 (t, J = 3.4 Hz, 1H), 4.33-4.27 (m, 1H), 4.18-4.16 (m, 2H), 4.12 (dd, J = 13.1, 7.3 Hz, 1H), 3.90-3.82 (m, 1H), 3.55-3.49 (m, 1H), 3.06-2.85 (m, 2H), 1.90-1.67 (m, 2H), 1.66-1.54 (m, 4H), 1.06 (s, 9H).

[0277] Alkyne (XS72) [ka]

[0278] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-((trimethylsilyl)ethynyl)but-2-en-1-yl)oxy)silane (XS72) CuI (16 mg, 0.085 mmol) and Pd(PPh)Cl (60 mg, 0.085 mmol) were purged with N (3x) and a solution of XS60 (0.458 g, 0.854 mmol) in toluene (5.0 mL) was added, followed by the addition of trimethylsilylacetylene (0.126 g, 1.28 mmol) and DIPEA (0.298 mL, 1.71 mmol). The reaction mixture was stirred at room temperature for 6 h, concentrated, and dissolved in EtOAc. The organic phase was washed with 0.5 M aqueous KHSO, water, and brine, dried over NaSO, and concentrated. The crude residue was purified by flash chromatography (silica gel, 0-8% EtOAc in heptane) and filtered from heptane. The filtrate was concentrated to give the silyl ether intermediate (0.361 g, 84%) as an orange oil. The intermediate was dissolved in MeOH (7.1 mL) and K2CO3 (49 mg, 0.36 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc, washed with saturated aqueous NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0-10% EtOAc in heptane) to give alkyne XS72 (0.192 g, 62%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.69-7.65 (m, 4H), 7.46-7.34 (m, 6H), 6.40-6.32 (m, 1H), 4.66 (t, J = 3.6 Hz, 1H), 4.52-4.43 (m, 1H), 4.41-4.32 (m, 1H), 4.20 (d, J = 1.4 Hz, 2H), 3.95-3.86 (m, 1H), 3.57-3.49 (m, 1H), 3.12 (s, 1H), 1.92-1.69 (m, 2H), 1.67-1.56 (m, 2H), 1.54-1.49 (m, 2H), 1.07 (s, 9H).

[0279] 3C. Preparation of Phosphate Esters (XS87-XS93)

[0280] [ka]

[0281] (E)-2-(((tert-butyldiphenylsilyl)oxy)methyl)-4-hydroxybut-2-enenitrile (XS73) Following procedure XXK, ether XS61 (0.500 g, 1.15 mmol) was reacted at 45 °C. EtO was used instead of DCM for extraction. The crude product was purified by flash chromatography (silica gel, 0-60% EtO in heptane) to give alcohol XS73 (0.261 g, 65%) as a pale yellow oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.64 (dd, J = 7.8, 1.7 Hz, 4H), 7.54-7.41 (m, 6H), 6.62-6.53 (m, 1H), 5.29 (t, J = 5.7 Hz, 1H), 4.31 (d, J = 1.3 Hz, 2H), 4.19 (t, J = 5.9 Hz, 2H), 1.02 (s, 9H). MS (ESI + ) C 21 H 26 NO2Si + [M+H] + Calculated value: 352.2, measured value: 352.2.

[0282] (E)-3-(((tert-butyldiphenylsilyl)oxy)methyl)penta-2,4-dien-1-ol (XS74) Following procedure XXK, ether XS62 (0.284 g, 0.650 mmol) was reacted at 55° C. The crude product was purified by flash chromatography (silica gel, 0-50% EtO in heptane) to give alcohol XS74 (0.183 g, 80%) as a pale yellow oil, which was used directly in the next reaction step.

[0283] (E)-3-(((tert-butyldiphenylsilyl)oxy)methyl)pent-2-en-1-ol (XS75) Following procedure XXK, ether XS63 (0.193 g, 0.440 mmol) was reacted at 55 °C. The crude product was purified by flash chromatography (silica gel, 0-50% EtO in heptane) to give alcohol XS75 (0.112 g, 72%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.68 (dd, J = 7.8, 1.4 Hz, 4H), 7.46-7.35 (m, 6H), 5.71 (t, J = 6.9 Hz, 1H), 4.23-4.18 (m, 2H), 4.12 (s, 2H), 2.05 (q, J = 7.5 Hz, 2H), 1.26 (s, 1H), 1.07 (s, 9H), 0.92 (t, J = 7.6 Hz, 3H). MS (ESI + ) C 22 H 30 NaO2Si + [M+Na] + Calculated value: 377.2, measured value: 377.3.

[0284] (E)-3-(((tert-butyldiphenylsilyl)oxy)methyl)hex-2-en-1-ol (XS76) Following procedure XXK, ether XS64 (0.330 g, 0.729 mmol) was reacted at 55° C. The crude product was purified by flash chromatography (silica gel, 0-40% EtO in heptane) to give alcohol XS76 (0.241 g, 90%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71-7.64 (m, 4H), 7.46-7.33 (m, 6H), 5.77 (t, J = 7.0 Hz, 1H), 4.23-4.16 (m, 2H), 4.11 (s, 2H), 2.04-1.98 (m, 2H), 1.38-1.24 (m, 3H), 1.07 (s, 9H), 0.83 (t, J = 7.4 Hz, 3H). MS (ESI + ) C 23 H 31 OSi + [M+H-HO] +Calculated value: 351.2, measured value: 351.4.

[0285] (E)-4-((tert-butyldiphenylsilyl)oxy)-3-cyclobutylbut-2-en-1-ol (XS77) According to procedure XXK, ether XS65 (0.570 g, 1.23 mmol) was reacted at 50° C. The crude product was purified by flash chromatography (silica gel, 0-50% EtO in heptane) to give alcohol XS77 (0.414 g, 89%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.69 (dd, J = 7.8, 1.6 Hz, 4H), 7.46-7.36 (m, 6H), 5.66 (td, J = 6.8, 1.3 Hz, 1H), 4.22-4.16 (m, 4H), 3.30 (quint, J = 9.0 Hz, 1H), 2.03-1.96 (m, 4H), 1.92-1.79 (m, 1H), 1.72-1.63 (m, 1H), 1.26 (s, 1H), 1.07 (s, 9H). MS (ESI + ) C 24 H 32 NaO2Si + [M+Na] + Calculated value: 403.2, measured value: 403.3.

[0286] (E)-3-(((tert-butyldiphenylsilyl)oxy)methyl)-5,5,5-trifluoropent-2-en-1-ol (XS78) According to procedure XXK, ether XS71 (0.375 g, 0.761 mmol) was reacted at 55° C. The crude product was purified by flash chromatography (silica gel, 0-40% EtO in heptane) to give alcohol XS78 (0.272 g, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.68-7.63 (m, 4H), 7.46-7.36 (m, 6H), 5.98 (t, J = 6.8 Hz, 1H), 4.22 (t, J = 6.3 Hz, 2H), 4.17 (s, 2H), 2.92 (q, J = 11.0 Hz, 2H), 1.23 (t, J = 5.8 Hz, 1H), 1.07 (s, 9H).

[0287] (E)-3-(((tert-butyldiphenylsilyl)oxy)methyl)pent-2-en-4-yn-1-ol (XS79) Following procedure XXK, ether XS72 (0.190 g, 0.437 mmol) was reacted at 55° C. The crude product was purified by flash chromatography (silica gel, 0-50% EtO in heptane) to give alcohol XS79 (0.123 g, 80%) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.70-7.65 (m, 4H), 7.46-7.36 (m, 6H), 6.36 (tt, J = 6.6, 1.6 Hz, 1H), 4.45-4.39 (m, 2H), 4.20 (d, J = 1.4 Hz, 2H), 3.14 (s, 1H), 1.43 (t, J = 6.1 Hz, 1H), 1.08 (s, 9H). MS (ESI + ) C 22 H 27 O2Si + [M+H] + Calculated value: 351.2, measured value: 351.3.

[0288] (4-((tert-butyldiphenylsilyl)oxy)-3-cyanobut-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS80) Alcohol XS73 (0.261 g, 0.742 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in heptane) to give alkyl phosphate XS80 (0.339 g, 58%) as a colorless oil. 1 H NMR (400 MHz, DMSO-d6) ppm = 7.83 (dd, J = 7.5, 3.5 Hz, 4H), 7.66-7.57 (m, 4H), 7.57-7.32 (m, 14H), 7.30-7.23 (m, 4H), 6.34 (t, J = 6.3 Hz, 1H), 4.40 (dd, J = 9.1, 6.3 Hz, 2H), 4.34-4.23 (m, 6H), 4.20-4.14 (m, 2H), 0.98 (s, 9H). MS (ESI + ) C 49 H 47 NO5PSi + [M+H] + Calculated value: 788.3, ​​measured value: 788.5.

[0289] (3-(((tert-butyldiphenylsilyl)oxy)methyl)penta-2,4-dien-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS81) Alcohol XS74 (0.183 g, 0.519 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-30% EtOAc in heptane) to give alkyl phosphate XS81 (0.312 g, 76%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.3 Hz, 4H), 7.65-7.59 (m, 4H), 7.52 (dd, J = 15.6, 7.5 Hz, 4H), 7.41-7.29 (m, 10H), 7.26-7.21 (m, 4H), 6.41 (dd, J = 17.9, 11.1 Hz, 1H), 5.90 (br t, J = 6.9 Hz, 1H), 5.15-5.09 (m, 2H), 4.63 (t, J = 7.4 Hz, 2H), 4.30-4.22 (m, 6H), 4.15 (t, J = 6.6 Hz, 2H), 1.01 (s, 9H).

[0290] (3-(((tert-butyldiphenylsilyl)oxy)methyl)pent-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS82) Alcohol XS75 (0.110 g, 0.310 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in heptane) to give alkyl phosphate XS82 (0.195 g, 79%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.1 Hz, 4H), 7.62 (dd, J = 7.9, 1.4 Hz, 4H), 7.57-7.48 (m,4H), 7.42-7.30 (m, 10H), 7.28-7.20 (m, 4H), 5.67 (t, J = 7.1 Hz, 1H), 4.52 (t, J = 7.4 Hz, 2H), 4.26 (t, J =6.5 Hz, 4H), 4.20-4.11 (m, 2H), 4.06 (s, 2H), 1.96 (q, J = 7.7 Hz, 2H), 1.01 (s, 9H), 0.87-0.82 (m, 3H). MS (ESI + ) C 50 H 51 NaO5PSi + [M+Na] +Calculated value: 813.3, measured value: 813.5.

[0291] (3-(((tert-butyldiphenylsilyl)oxy)methyl)hex-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS83) Alcohol XS76 (0.239 g, 0.648 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-40% EtOAc in heptane) to give alkyl phosphate XS83 (0.347 g, 67%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.1 Hz, 4H), 7.62 (dd, J = 7.9, 1.4 Hz, 4H), 7.53 (dd, J = 15.5, 7.5 Hz, 4H), 7.42-7.29 (m, 10H), 7.25-7.21 (m, 4H), 5.73 (t, J = 7.1 Hz, 1H), 4.54 (t, J = 7.4 Hz, 2H), 4.25 (t, J = 6.5 Hz, 4H), 4.21-4.11 (m, 2H), 4.04 (s, 2H), 1.96-1.90 (m, 2H), 1.29-1.20 (m, 2H), 1.01 (s, 9H), 0.77 (t, J = 7.3 Hz, 3H).

[0292] (4-((tert-butyldiphenylsilyl)oxy)-3-cyclobutylbut-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS84) Alcohol XS77 (0.410 g, 1.08 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in heptane) to give alkyl phosphate XS84 (0.519 g, 59%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.3 Hz, 4H), 7.63 (dd, J = 7.9, 1.4 Hz, 4H), 7.57-7.49 (m, 4H), 7.42-7.30 (m, 10H), 7.25-7.20 (m, 4H), 5.63 (td, J = 7.0, 1.3 Hz, 1H), 4.51 (t, J = 7.3 Hz, 2H), 4.29-4.21 (m, 4H), 4.19-4.11 (m, 4H), 3.19 (quint, J = 8.8 Hz, 1H), 1.96-1.87 (m, 4H), 1.87-1.74 (m, 1H), 1.68-1.58 (m, 1H), 1.01 (s, 9H).

[0293] (3-(((tert-butyldiphenylsilyl)oxy)methyl)-5,5,5-trifluoropent-2-en-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS85) Alcohol XS78 (0.135 g, 0.330 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-50% EtOAc in heptane) to give alkyl phosphate XS85 (0.179 g, 64%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.3 Hz, 4H), 7.62-7.56 (m, 4H), 7.55-7.45 (m, 4H), 7.42-7.30 (m, 10H), 7.29-7.19 (m, 4H), 5.91 (t, J = 6.8 Hz, 1H), 4.43 (t, J = 7.5 Hz, 2H), 4.29-4.22 (m, 4H), 4.17-4.11 (m, 2H), 4.11-4.06 (m, 2H), 2.78 (q, J = 10.9 Hz, 2H), 1.01 (s, 9H).

[0294] (3-(((tert-butyldiphenylsilyl)oxy)methyl)pent-2-en-4-yn-1-yl)phosphate (E)-bis((9H-fluoren-9-yl)methyl) ester (XS86) Alcohol XS79 (0.120 g, 0.342 mmol) was reacted according to Procedure XXL. The crude product was purified by flash chromatography (silica gel, 0-40% EtOAc in heptane) to give alkyl phosphate XS86 (0.199 g, 74%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.71 (t, J = 7.4 Hz, 4H), 7.62 (dd, J = 7.9, 1.3 Hz, 4H), 7.53 (dd, J =14.3, 7.5 Hz, 4H), 7.43-7.29 (m, 10H), 7.29-7.21 (m, 4H), 6.28 (t, J = 6.8 Hz, 1H), 4.77 (t, J = 7.4 Hz, 2H), 4.32-4.23 (m, 4H), 4.20-4.12 (m, 4H), 3.09 (s, 1H), 1.03 (s, 9H). MS (ESI + ) C 50 H 48 O5PSi + [M+H] + Calculated value: 787.3, measured value: 787.6.

[0295] (E)-3-Cyano-4-hydroxybut-2-en-1-yl phosphate triethylamine salt (XS87) Step 1: Following general procedure XXG, TBDPS-ether XS80 (0.339 g, 0.430 mmol) was reacted. The crude intermediate was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.164 g, 69%) as a white solid. MS (ESI + ) C 33 H 29 NO5P + [M+H] + Calculated value: 550.2, measured value: 550.3.

[0296] Step 2: Following general procedure XXH, the intermediate (0.164 g, 0.298 mmol) was reacted to give alkyl phosphate XS87 (79 mg, 94%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 6.57 (t, J = 6.1 Hz, 1H), 4.57-4.50 (m, 2H), 4.12 (d, J = 0.9 Hz, 2H). MS (ESI - ) C5H7NO5P - [MH] - Calculated value 192.0, actual value 192.0.

[0297] (E)-3-(Hydroxymethyl)penta-2,4-dien-1-yl phosphate triethylamine salt (XS88) Step 1: Following general procedure XXG, TBDPS-ether XS81 (0.305 g, 0.387 mmol) was reacted. The crude intermediate was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.172 g, 81%) as a white solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.5 Hz, 4H), 7.58-7.48 (m, 4H), 7.43-7.33 (m, 4H), 7.31-7.25 (m, 4H), 6.41 (dd, J = 17.6, 11.3 Hz, 1H), 5.67 (t, J = 6.9 Hz, 1H), 5.33 (d, J = 17.6 Hz, 1H), 5.24 (d, J = 11.1 Hz, 1H), 4.57 (t, J = 7.8 Hz, 2H), 4.30-4.22 (m, 6H), 4.19-4.12 (m, 2H), 1.54-1.51 (m, 1H). MS (ESI + ) C 34 H 32 O5P + [M+H] + Calculated value: 551.2, measured value: 551.4.

[0298] Step 2: Following general procedure XXH, the intermediate (0.170 g, 0.309 mmol) was reacted to give alkyl phosphate XS88 (74 mg, 85%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 6.59 (dd, J = 17.8, 11.3 Hz, 1H), 5.73 (t, J = 6.8 Hz, 1H), 5.34 (d, J = 17.6 Hz, 1H), 5.25 (d, J = 11.4 Hz, 1H), 4.52 (t, J = 7.4 Hz, 2H), 4.22 (s, 2H). MS (ESI - ) C6H 10 O5P - [MH] - Calculated value: 193.0, measured value: 193.1.

[0299] (E)-3-(Hydroxymethyl)pent-2-en-1-yl phosphate triethylamine salt (XS89) Step 1: Following general procedure XXG, TBDPS-ether XS82 (0.191 g, 0.241 mmol) was reacted. The crude residue was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.105 g, 79%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.5 Hz, 4H), 7.59-7.48 (m, 4H), 7.43-7.33 (m, 4H), 7.31-7.22 (m, 4H), 5.49 (t, J = 7.0 Hz, 1H), 4.47 (t, J = 7.7 Hz, 2H), 4.26 (t, J = 6.3 Hz, 4H), 4.18-4.12 (m, 2H), 4.01 (s, 2H), 2.02 (q, J = 7.5 Hz, 2H), 0.94 (t, J = 7.6 Hz, 3H). MS (ESI + ) C 34 H 34 O5P + [M+H] +Calculated value: 553.5, measured value: 553.2.

[0300] Step 2: Following general procedure XXH, the intermediate (0.102 g, 0.185 mmol) was reacted to give alkyl phosphate XS89 (51 mg, 97%) as the triethylamine salt in a 1:0.7 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 5.51 (t, J = 7.0 Hz, 1H), 4.40 (t, J = 7.1 Hz, 2H), 4.00 (s, 2H), 2.06 (q, J = 7.6 Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H). MS (ESI - ) C6H 12 O5P - [MH] - Calculated value: 195.0, measured value: 195.1.

[0301] (E)-3-(Hydroxymethyl)hex-2-en-1-yl phosphate triethylamine salt (XS90) Step 1: Following general procedure XXG, TBDPS-ether XS83 (0.345 g, 0.429 mmol) was reacted. The crude residue was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.213 g, 88%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.5 Hz, 4H), 7.59-7.48 (m, 4H), 7.43-7.32 (m, 4H), 7.31-7.23 (m, 4H), 5.53 (t, J = 6.9 Hz, 1H), 4.48 (t, J = 7.7 Hz, 2H), 4.29-4.23 (m, 4H), 4.19-4.13 (m, 2H), 3.99 (s, 2H), 2.02-1.96 (m, 2H), 1.60-1.54 (m, 1H), 1.40-1.31 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). MS (ESI + ) C 35 H35 NaO5P + [M+Na] + Calculated value: 589.2, measured value: 589.5.

[0302] Step 2: Following general procedure XXH, the intermediate (0.211 g, 0.372 mmol) was reacted to give alkyl phosphate XS90 (61 mg, 55%) as the triethylamine salt in a 1:0.7 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 5.56 (t, J = 6.9 Hz, 1H), 4.40 (t, J = 7.1 Hz, 2H), 3.98 (s, 2H), 2.03 (t, J = 7.7 Hz, 2H), 1.34 (sextet, J = 7.5 Hz, 2H), 0.80 (t, J = 7.4 Hz, 3H). MS (ESI - ) C7H 14 O5P - [MH] - Calculated value 209.1, measured value 209.1.

[0303] (E)-3-Cyclobutyl-4-hydroxybut-2-en-1-yl phosphate triethylamine salt (XS91) Step 1: Following general procedure XXG, TBDPS-ether XS84 (0.515 g, 0.630 mmol) was reacted. The crude intermediate was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.281 g, 77%) as a colorless oil. 1H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.7 Hz, 4H), 7.58-7.49 (m, 4H), 7.38 (dt, J = 14.6, 7.4 Hz, 4H), 7.31-7.26 (m, 4H), 5.43 (td, J = 6.9, 1.4 Hz, 1H), 4.45 (t, J = 7.6 Hz, 2H), 4.30-4.21 (m, 4H), 4.18-4.10 (m, 2H), 4.10-4.07 (m, 2H), 3.20 (quint, J = 9.1 Hz, 1H), 2.06-1.94 (m, 4H), 1.94-1.81 (m, 1H), 1.77-1.66 (m, 1H). MS (ESI + ) C 36 H 36 O5P + [M+H] + Calculated value: 579.2, measured value: 579.5.

[0304] Step 2: Following general procedure XXH, the intermediate (0.275 g, 0.475 mmol) was reacted to give alkyl phosphate XS91 (0.113 g, 77%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 5.45 (td, J = 6.9, 1.1 Hz, 1H), 4.37 (t, J = 7.1 Hz, 2H), 4.07 (s, 2H), 3.34-3.24 (m, 1H), 3.12 (q, J = 7.3 Hz, 6H), 2.07-1.90 (m, 5H), 1.89-1.56 (m, 3H), 1.20 (t, J = 7.3 Hz, 8H). MS (ESI - ) C8H 14 O5P - [MH] - Calculated value 221.1, measured value 221.2.

[0305] (E)-5,5,5-trifluoro-3-(hydroxymethyl)pent-2-en-1-yl phosphate triethylamine salt (XS92) Step 1: Following general procedure XXG, TBDPS-ether XS85 (0.117 g, 0.209 mmol) was reacted. The crude intermediate was suspended in heptane and filtered to give the intermediate allylic alcohol (0.103 g, 75%) as a white solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.6 Hz, 4H), 7.57-7.46 (m, 4H), 7.38 (dt, J = 14.6, 7.3 Hz, 4H), 7.32-7.26 (m, 4H), 5.80 (t, J = 6.8 Hz, 1H), 4.44-4.34 (m, 2H), 4.27 (t, J = 6.4 Hz, 4H), 4.17-4.10 (m, 2H), 4.05 (s, 2H), 2.83 (q, J = 10.9 Hz, 2H). MS (ESI + ) C 34 H 31 F3O5P + [M+H] + Calculated value 607.2, measured value 607.4.

[0306] Step 2: Following general procedure XXH, the intermediate (0.119 g, 0.196 mmol) was reacted to give alkyl phosphate XS92 (27 mg, 41%) as the triethylamine salt in a 1:0.6 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 5.92 (t, J = 6.6 Hz, 1H), 4.43 (t, J = 7.3 Hz, 2H), 4.06 (s, 2H), 3.12 (q, J = 7.4 Hz, 4H), 3.02 (q, J = 11.4 Hz, 2H), 1.20 (t, J = 7.3 Hz, 6H). MS (ESI - ) C6H9F3O5P - [MH] - Calculated value 249.0, actual value 249.0.

[0307] (E)-3-(Hydroxymethyl)pent-2-en-4-yn-1-yl phosphate triethylamine salt (XS93) Step 1: Following general procedure XXG, TBDPS-ether XS86 (0.196 g, 0.249 mmol) was reacted. The crude intermediate was purified by flash chromatography (silica gel, 0-100% EtOAc in heptane) to afford the intermediate allylic alcohol (0.103 g, 75%) as a white solid. 1 H NMR (400 MHz, CDCl3) ppm = 7.73 (t, J = 7.8 Hz, 4H), 7.59-7.48 (m, 4H), 7.38 (dt, J = 14.7, 7.5 Hz, 4H), 7.31-7.23 (m, 4H), 6.04 (t, J = 6.6 Hz, 1H), 4.73-4.66 (m, 2H), 4.31-4.23 (m, 4H), 4.19-4.13 (m, 2H), 4.11-4.06 (m, 2H), 3.19 (s, 1H), 1.64 (t, J = 6.6 Hz, 1H). MS (ESI + ) C 34 H 30 O5P + [M+H] + Calculated value: 549.2, measured value: 549.4.

[0308] Step 2: Following general procedure XXH, the intermediate (0.100 g, 0.182 mmol) was reacted to give alkyl phosphate XS93 (43 mg, 84%) as the triethylamine salt in a 1:0.9 ratio of phosphoric acid:Et3N. 1 H NMR (400 MHz, D2O) ppm = 6.15 (t, J = 6.4 Hz, 1H), 4.56 (dd, J = 8.0, 7.0 Hz, 2H), 4.05 (d, J = 0.6 Hz, 2H), 3.60 (s, 1H). MS (ESI - ) C6H8O5P - [MH] - Calculated value 191.0, actual value 191.0.

[0309] 3D. Preparation of linker-drug (XS100-XS105)

[0310] [ka]

[0311] Pyrophosphate ester XS94 According to general procedure XXD, alkyl phosphate XD36 (79 mg, 0.11 mmol) was reacted with phosphate ester XS87. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS94 (41.0 mg, 45%) as a white solid. 1 H NMR (400 MHz, D2O) ppm = 7.42 (s, 4H), 6.60-6.52 (m, 1H), 4.93 (d, J = 6.9 Hz, 2H), 4.64-4.57 (m, 2H), 4.43-4.35 (m, 1H), 4.14-4.11 (m, 1H), 4.11-4.10 (m, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.66-3.57 (m, 10H), 3.44-3.40 (m, 2H), 2.62-2.48 (m, 2H), 2.11-1.98 (m, 1H), 1.43 (d, J = 7.3 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H). MS (ESI - ) C 29 H 44 N7O 14 P2 - [MH] - Calculated value: 776.2, measured value: 776.6.

[0312] Pyrophosphate ester XS95 According to general procedure XXD, alkyl phosphate XD36 (79 mg, 0.11 mmol) was reacted with phosphate ester XS88. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS95 (50.1 mg, 55%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.42 (s, 4H), 6.48 (dd, J = 17.6, 11.3 Hz, 1H), 5.69 (t, J = 6.8 Hz, 1H), 5.30 (d, J = 17.6 Hz, 1H), 5.20 (d, J = 11.4 Hz, 1H), 4.93 (d, J = 6.5 Hz, 2H), 4.57-4.50 (m, 2H), 4.40 (q, J = 7.2 Hz, 1H), 4.17 (s, 2H), 4.12 (d, J = 7.1 Hz, 1H), 3.73 (t, J = 6.0 Hz, 2H), 3.66-3.57 (m, 10H), 3.45-3.40 (m, 2H), 2.63-2.48 (m, 2H), 2.12-1.98 (m, 1H), 1.43 (d, J = 7.1 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6Hz, 3H). MS (ESI - ) C 30 H 47 NO 14 P2 - [MH] - Calculated value: 777.3, measured value: 777.4.

[0313] Pyrophosphate ester XS96 According to general procedure XXD, alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate ester XS89. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS96 (48.6 mg, 42%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.44 (s, 4H), 5.49 (t, J = 6.8 Hz, 1H), 4.99-4.92 (m, 2H), 4.48-4.36 (m, 3H), 4.13 (d, J = 7.1 Hz, 1H), 3.97 (s, 2H), 3.74 (t, J = 5.9 Hz, 2H), 3.69-3.57 (m, 10H), 3.48-3.40 (m, 2H), 2.64-2.49 (m, 2H), 2.12-2.03 (m, 1H), 2.00 (q, J = 7.6 Hz, 2H), 1.44 (d, J = 7.1 Hz, 3H), 0.95-0.85 (m, 9H). MS (ESI - ) C 30 H 49 NO 14 P2 - [MH] - Calculated value: 779.3, measured value: 779.5.

[0314] Pyrophosphate ester XS97 According to general procedure XXD, alkyl phosphate XD36 (54 mg, 0.077 mmol) was reacted with phosphate ester XS90. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS97 (37.3 mg, 59%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.42 (s, 4H), 5.52 (t, J = 6.8 Hz, 1H), 4.93 (d, J = 6.4 Hz, 2H), 4.45-4.37 (m, 3H), 4.12 (d, J = 7.0 Hz, 1H), 3.94 (s, 2H), 3.73 (t, J = 5.9 Hz, 2H), 3.65-3.56 (m, 10H), 3.45-3.40 (m, 2H), 2.63-2.48 (m, 2H), 2.10-2.00 (m, 1H), 1.95 (t, J = 7.6 Hz, 2H), 1.43 (d, J = 7.3 Hz, 3H), 1.28 (sxt, J = 7.5 Hz, 2H), 0.96-0.86 (m, 6H), 0.75 (t, J = 7.3 Hz, 3H). MS (ESI - ) C 31 H 51 NO 14 P2 - [MH] - Calculated value: 793.3, measured value: 793.5.

[0315] Pyrophosphate ester XS98 According to general procedure XXD, alkyl phosphate XD36 (105 mg, 0.149 mmol) was reacted with phosphate ester XS91. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS98 (64.1 mg, 51%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.43 (s, 4H), 5.43-5.37 (m, 1H), 4.94 (d, J = 6.6 Hz, 2H), 4.43-4.34 (m, 3H), 4.11 (d, J = 7.1 Hz, 1H), 4.01 (s, 2H), 3.73 (t, J = 6.0 Hz, 2H), 3.64-3.57 (m, 10H), 3.45-3.40 (m, 2H), 3.14 (quint, J = 9.0 Hz, 1H), 2.62-2.47 (m, 2H), 2.10-1.99 (m, 1H), 1.98-1.82 (m, 4H), 1.82-1.71 (m, 1H), 1.65-1.55 (m, 1H), 1.42 (d, J = 7.1 Hz, 3H), 0.91 (d, J = 6.5 Hz, 3H), 0.90 (d, J = 6.5 Hz, 3H). MS (ESI - ) C 32 H 51 NO 14 P2 - [MH] - Calculated value: 805.3, measured value: 805.5.

[0316] Pyrophosphate ester XS99 According to general procedure XXD, alkyl phosphate XD36 (43 mg, 0.061 mmol) was reacted with phosphate ester XS92. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS99 (12.3 mg, 23%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.40 (s, 4H), 5.85 (t, J = 6.3 Hz, 1H), 4.90 (br s, 2H), 4.47-4.40 (m, 2H), 4.40-4.34 (m, 1H), 4.14-4.07 (m, 1H), 3.98 (s, 2H), 3.71 (s, 2H), 3.64-3.53 (m, 10H), 3.43-3.37 (m, 2H), 2.91 (q, J = 11.4 Hz, 2H), 2.61-2.45 (m, 2H), 2.03 (dq, J = 13.7, 6.9 Hz, 1H), 1.40 (d, J = 7.3 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). MS (ESI - ) C 30 H 46 F3N6O 14 P2 - [MH] - Calculated value: 833.3, measured value: 833.6.

[0317] Linker-Drug XS100 According to general procedure XXE, azide XS94 (36 mg, 0.044 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS100 (15.5 mg, 33%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.90 (br s, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 6.56 (t, J = 5.9 Hz, 1H), 4.98-4.89 (m, 2H), 4.64-4.58 (m, 2H), 4.53 (t, J = 4.8 Hz, 2H), 4.38 (q, J = 7.2 Hz, 1H), 4.31 (br s, 2H), 4.16-4.03 (m, 5H), 3.88 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.59 (m, 6H), 3.57-3.47 (m, 8H), 2.61-2.47 (m, 2H), 2.11-1.97 (m, 1H), 1.41 (d, J = 7.1 Hz, 3H), 0.90 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI - ) C 41 H 58 N9O 19 P2 - [MH] - Calculated value: 1042.3, measured value: 1042.8.

[0318] Linker-drug XS101 According to general procedure XXE, azide XS95 (44 mg, 0.054 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS101 (19.8 mg, 34%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.92 (br s, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 6.48 (dd, J = 17.8, 11.3 Hz, 1H), 5.73-5.65 (m, 1H), 5.30 (d, J = 17.6 Hz, 1H), 5.20 (d, J = 11.1 Hz, 1H), 4.92 (td, J = 1.9, 1.0 Hz, 2H), 4.59-4.50 (m, 4H), 4.38 (q, J = 7.3 Hz, 1H), 4.31 (tdd, J = 4.2, 2.3, 1.3 Hz, 2H), 4.17 (s, 2H), 4.14-4.04 (m, 3H), 3.88 (t, J = 4.8 Hz, 2H), 3.71 (t, J = 5.9 Hz, 2H), 3.66-3.58 (m, 6H), 3.58-3.48 (m, 8H), 2.62-2.47 (m, 2H), 2.10-1.99 (m, 1H), 1.42 (d, J = 7.1 Hz, 3H), 0.91 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI - ) C 42 H 61 N8O 19 P2 - [MH] - Calculated value: 1043.4, measured value: 1043.6.

[0319] Linker-drug XS102 According to general procedure XXE, azide XS96 (42 mg, 0.052 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS102 (8.9 mg, 16%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.99-7.87 (m, J = 1.0 Hz, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 5.47 (br s, 1H), 4.93 (br s, 2H), 4.53 (br t, J = 4.6 Hz, 2H), 4.46-4.40 (m, 2H), 4.40-4.34 (m, 1H), 4.31 (d, J = 1.0 Hz, 2H), 4.15-4.04 (m, 3H), 3.95 (s, 2H), 3.87 (t, J = 4.6 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.62 (s, 6H), 3.58-3.46 (m, 8H), 2.62-2.46 (m, 2H), 2.10-1.93 (m, 3H), 1.41 (d, J = 7.1 Hz, 3H), 0.94-0.81 (m, 9H). MS (ESI - ) C 42 H 63 N8O 19 P2 - [MH] - Calculated value: 1045.4, measured value: 1045.6.

[0320] Linker-drug XS103 According to general procedure XXE, azide XS97 (32 mg, 0.038 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS103 (9.0 mg, 22%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.89 (br s, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 5.51 (t, J = 6.3 Hz, 1H), 4.92 (br s, 2H), 4.53 (br s, 2H), 4.46-4.35 (m, 3H), 4.31 (br s, 2H), 4.16-4.04 (m, 3H), 3.93 (s, 2H), 3.87 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.58 (m, 6H), 3.57-3.47 (m, 8H), 2.61-2.47 (m, 2H), 2.10-1.99 (m, 1H), 1.94 (t, J = 7.6 Hz, 2H), 1.41 (d, J = 7.3 Hz, 3H), 1.27 (sxt, J = 7.4 Hz, 2H), 0.90 (d, J = 7.3 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H), 0.74 (t, J = 7.3 Hz, 3H). MS (ESI - ) C 43 H 65 N8O 19 P2 - [MH] - Calculated value: 1059.4, measured value: 1059.8.

[0321] Linker-drug XS104 According to general procedure XXE, azide XS98 (56 mg, 0.067 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS104 (22.7 mg, 31%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.91 (br s, 1H), 7.42 (s, 4H), 6.74 (s, 2H), 5.41 (t, J = 5.4 Hz, 1H), 4.93 (br s, 2H), 4.53 (t, J = 4.9 Hz, 2H), 4.44-4.34 (m, 3H), 4.31 (br s, 2H), 4.10-4.10 (m, 1H), 4.12 (d, J = 7.0 Hz, 1H), 4.10-4.04 (m, 2H), 4.01 (s, 2H), 3.88 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.58 (m, 6H), 3.57-3.46 (m, 8H), 3.14 (quint, J = 9.0 Hz, 1H), 2.62-2.46 (m, 2H), 2.10-1.99 (m, 1H), 1.98-1.82 (m, 4H), 1.82-1.71 (m, 1H), 1.65-1.54 (m, 1H), 1.41 (d, J = 7.1 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI - ) C 44 H 65 N8O 19 P2 - [MH] - Calculated value: 1071.4, measured value: 1071.7.

[0322] Linker-drug XS105 According to general procedure XXE, azide XS99 (11 mg, 0.012 mmol) was reacted with alkyne XD43. Purification by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) afforded the pyrophosphate XS105 (3.3 mg, 24%) as a white solid after lyophilization. 1H NMR (400 MHz, D2O) ppm = 7.90 (br s, 1H), 7.41 (s, 4H), 6.74 (s, 2H), 5.87 (t, J = 5.5 Hz, 1H), 4.92 (br s, 2H), 4.53 (t, J = 4.8 Hz, 2H), 4.45 (br s, 2H), 4.42-4.34 (m, 1H), 4.31 (br s, 2H), 4.15-4.05 (m, 3H), 4.00 (s, 2H), 3.87 (t, J = 4.9 Hz, 2H), 3.70 (t, J = 5.9 Hz, 2H), 3.66-3.58 (m, 6H), 3.57-3.48 (m, 8H), 2.93 (q, J = 11.3 Hz, 2H), 2.61-2.46 (m, 2H), 2.11-1.97 (m, 1H), 1.41 (d, J = 7.3 Hz, 3H), 0.90 (d, J = 7.1 Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H). MS (ESI - ) C 42 H 60 F3N8O 19 P2 - [MH] - Calculated value: 1099.3, measured value: 1099.7.

[0323] Preparation of linker-drug (XS107) [ka]

[0324] Pyrophosphate ester XS106 According to general procedure XXD, alkyl phosphate XD36 (90 mg, 0.128 mmol) was reacted with phosphate ester XS93. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, pyrophosphate ester XS106 (50.1 mg, 48%) as a white solid. 1H NMR (400 MHz, D2O) ppm = 7.40 (s, 4H), 6.09 (t, J = 6.4 Hz, 1H), 4.93-4.88 (m, 2H), 4.60-4.54 (m, 2H), 4.37 (q, J = 7.1 Hz, 1H), 4.09 (d, J = 7.0 Hz, 1H), 3.98 (s, 2H), 3.71 (t, J = 6.0 Hz, 2H), 3.64-3.54 (m, 11H), 3.43-3.37 (m, 2H), 2.61-2.45 (m, 2H), 2.10-1.96 (m, 1H), 1.41 (d, J = 7.3 Hz, 3H), 0.89 (d, J = 6.6 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H). MS (ESI - ) C 30 H 45 NO 14 P2 - [MH] - Calculated value: 775.2, measured value: 775.4.

[0325] Linker-drug XS107 Azide XS106 (18 mg, 0.022 mmol) was dissolved in water (120 μL) and a solution of DBCO-PEG2-maleimide (18 mg, 0.030 mmol) was added. The reaction mixture was stirred at room temperature for 45 min and subsequently purified by preparative RP-HPLC (25 mM NH4HCO3 / MeCN in Milli-Q water, 90:10 to 50:50 gradient) to give, after lyophilization, the pyrophosphate ester XS107 (14.1 mg, 46%) as a white solid. MS (ESI) - ) C 62 H 79 N 10 O 21 P2 - [MH] - Calculated value: 1361.5, measured value: 1361.9.

[0326] Example 4 Synthesis of DAR2 Conjugates of Linker-Drug Compounds XD73, XS54-XS58, XS100-XS105, and XS107 with Rituximab To the antibody solution (10–12 mg / mL), TRIS (1% v / v, 1 M, pH 8), EDTA (4% v / v, 25 mM), and TCEP (5 mM in water) were added. The resulting solution was incubated at room temperature for 2 hours. The reduced antibody was then rebuffered with 4.2 mM histidine, 50 mM trehalose (pH 6), and treated with dimethylacetamide (DMA) and the linker-drug compound (LD) (10 mM in DMA, ≥1.5 equivalents per LD). The final DMA content was approximately 10% v / v. The resulting mixture was roller-mixed overnight at room temperature in the dark. Activated charcoal was added, and the suspension was roller-mixed in the dark for 1 hour, filtered, and washed with 4.2 mM histidine, 50 mM trehalose (pH 6). The solution was rebuffered with 4.2 mM histidine, 50 mM trehalose (pH 6), and sterile-filtered.

[0327] To bring the DAR (pAg-to-antibody ratio) of the conjugate closer to the target DAR of 2, alternative conjugation was performed using the hydrophobic seco-DUBA (SYD980, described in WO 2015 / 177360), which allows for easy analysis of the DAR by HIC. The resulting average DARs for conjugates with a target DAR of 2 are shown in Table 1.

[0328] [Table 1-1]

[0329] [Table 1-2]

[0330] [Table 1-3]

[0331] [Table 1-4]

[0332] Example 5 Activity of phosphoantigen (pAg) complexes (ADCs) on γδ T cells Several synthetic linker-drug compounds were conjugated to rituximab (anti-CD20), and the resulting ADCs were tested for their ability to bind to Raji cells and activate V52γδ T cells after overnight incubation with CD20-positive Raji cells. The linker-drug compounds conjugated to rituximab and the resulting ADCs are listed in Table 1. Pretreated Raji cells were cocultured with peripheral blood mononuclear cells (PBMCs), and V52γδ T cell and NK cell activation (IFNγ production) and degranulation (CD107a) were determined using multicolor flow cytometry.

[0333] Materials and Methods cell binding The CD20-positive Burkitt's lymphoma human tumor cell line Raji (DSMZ, the German collection of Microorganisms and cell cultures GmbH) was used for in vitro experiments. Raji cells were cultured in complete growth medium (CGM) (RPMI 1640 (Lonza) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS) (Gibco) and 80 U / mL penicillin-streptomycin solution (Lonza)). Raji cells were maintained in a humidified incubator at 37°C with 5% CO2 and passaged twice weekly. For cell binding in 96-well plates, 100,000 Raji cells per well were washed twice with ice-cold FACS buffer (PBS 1x, 0.1% v / w BSA, 0.02% v / v sodium azide) followed by the addition of 50 μL / well of anti-CD20 ADC, naked antibody (e.g., rituximab), or non-binding isotype control ADC diluted in ice-cold FACS buffer. After a 30-minute incubation at 4°C, the cells were washed twice with ice-cold FACS buffer. Then, 50 μL / well of APC-conjugated secondary F(ab')2 goat anti-human IgG (Fc fragment-specific, Jackson Immuno Research, 109-136-098, 1:6000) was added. After 30 minutes at 4°C, the cells were washed twice and resuspended in 150 μL of ice-cold FACS buffer. Fluorescence intensity was measured by flow cytometry using NovoCyte (Agilent) and median fluorescence intensity (MFI) was determined. Curves were fitted by nonlinear regression with variable slope (4 variables) in GraphPad Prism version 9. EC 50 Values ​​were calculated in GraphPad Prism as the concentration (μg / mL) of the response midway between the bottom and top of the curve. Binding experiments were performed three independent times.

[0334] Functional assay (determination of γδ T cell activity induced by various pAg complexes) For stimulation with the ADCs of the invention, Raji cells were harvested and cultured at 5×10 6The cells were diluted to 1000 cells / mL, and 50 μL of this cell suspension (equivalent to 250,000 cells / well) was seeded into a 96-well plate. Five-fold serial dilutions of 2x-concentrated ADC or rituximab were prepared in complete growth medium (CGM, RPMI-1640 (Lonza)) supplemented with 10% HI FBS (Gibco) and 80 U / mL penicillin-streptomycin solution (Lonza). The seeded Raji cells were incubated overnight with 50 μL / well of serially diluted compound (100 μL / well total) in a humidified incubator at 37°C and 5% CO2. The next day, the 96-well plates containing Raji cells and ADC or rituximab were washed with 100 μL of CGM per well to remove excess unbound compound, then centrifuged at 300 × g for 3 minutes at room temperature, and the supernatant was removed. As a source of immune cells, frozen PBMCs from healthy human donors were thawed, resuspended in CGM, and allowed to recover overnight in a humidified incubator at 37°C and 5% CO2. Recovered PBMCs were collected, counted, and diluted to 10 x 10 in CGM. 6 Dilute to 50 µL / well (0.5 x 10 cells / mL). 650 μL / well of a 2x concentrated anti-CD107a BV421 (BioLegend) solution was prepared in CGM containing GolgiStop (monensin) and GolgiPlug (brefeldin A) (BD Biosciences), and 50 μL / well was added to the Raji-PBMC cocultures. Samples were incubated for 6 hours in a humidified incubator at 37°C and 5% CO2. For staining of immune cell subsets, a multicolor antibody staining cocktail was prepared in Brilliant Stain Buffer (BD Biosciences) containing anti-CD3 BV711 (BioLegend), anti-CD56 BV510 (BioLegend), Fixable Viability Stain 780 (BD Biosciences), anti-CD16 FITC (BD Biosciences), FcR blocking reagent (Miltenyi Biotec), and anti-TCRVδ2 APC (BioLegend). After 6 h of incubation, the plate was centrifuged at 300 × g for 3 min at room temperature, and the supernatant was discarded. The pellet was resuspended in 50 μL of the antibody cocktail and incubated on ice for 30 min, protected from light. The plate was washed twice with 100 μL of ice-cold FACS buffer (PBS 1x, 0.1% v / w BSA, 0.02% v / v sodium azide), centrifuged at 300 × g for 3 minutes, and the supernatant was discarded. Cells were fixed and permeabilized with 100 μL / well of Cytofix / Cytoperm solution (BD biosciences) and incubated on ice for 20 minutes, protected from light. The cells were washed three times with 150 μL of BD Perm / wash solution containing saponin (10x BD Perm / Wash buffer was diluted with distilled water to prepare a 1x solution before use), centrifuged at 300 × g for 3 minutes, and the supernatant was discarded. Finally, the cells were resuspended in FACS buffer and placed in a refrigerator at 4 °C overnight, protected from light. On day 3, the stained PBMC / Raji cells were washed once with 150 μL of BD Perm / wash solution, centrifuged at 300 × g for 3 minutes, and the supernatant was discarded. The pellet was resuspended in 50 μL of a mixture of anti-IFNγ PE-Cy7 (BioLegend) diluted in Perm / Wash solution and incubated on ice for 30 minutes in the dark.After incubation, the plate was washed once with 150 μL of ice-cold FACS buffer, followed by centrifugation at 300 × g for 3 minutes, and the supernatant was discarded. The cell pellet was then resuspended in 100 μL of FACS buffer, and the samples were analyzed using NovoCyte (Agilent). Curves were fitted to nonlinear regression with variable slope (four variables) in GraphPad Prism version 9. EC. 50 Values ​​were calculated in GraphPad Prism as the concentration (μg / mL) of the response midway between the bottom and top of the curve. Each compound was tested in at least two experiments with separate donors.

[0335] Results / Conclusion Rituximab ADCs and non-binding controls were prepared at a drug-to-antibody ratio (DAR) of approximately 2. Their binding to Raji cells was comparable to rituximab alone (Table 2), and the non-binding isotype control showed no binding (data not shown).

[0336] The manufactured ADCs were incubated overnight with Raji cells and then cocultured with PBMCs containing V52γδ T cells for 6 hours before being tested for their ability to induce V52γδ T cell activation. Dose-response curves for V52γδ T cell degranulation (CD107a) and IFNγ production were generated. Linker-drug XD18 conjugated to rituximab (ADC-XD18-r) was previously described in WO 2023 / 275025 (applicant Byondis BV) and demonstrated superior potency and efficacy compared to rituximab. Linker-drug XD73 conjugated to rituximab (ADC-XD73-r) or a non-binding isotype control (ADC-XD73-i) were compared to ADC-XD18-r (Figures 1A and 1B and Table 3). Non-binding isotype control ADCs were less potent at activating Vδ2 γδ T cells and 50However, the potency and efficacy of ADC-XD73-r to activate γδ T cells were comparable to those of ADC-XD18-r. Linker-drug XD45 conjugated to rituximab (ADC-XD45-r) has previously been described in WO 2023 / 275025, and the potency and efficacy of ADC-XD45-r were superior to those of rituximab (WO 2023 / 275025). The linker-drugs XS58, XS56, XS54, and XS57 were conjugated to rituximab or a non-binding isotype control to generate the corresponding ADCs and compared them with ADC-XD45-r (Figures 1A, 1B, and Table 3). The non-binding isotype control ADC showed lower potency to activate Vδ2 γδ T cells and a lower EC 50 However, the potency and efficacy of ADC-XS56-r and ADC-XS-57-r to activate γδ T cells were comparable to those of ADC-XD45-r. ADC-XS58-r and ADC-XS54-r were also able to activate γδ T cells. γδ T cell activation induced by ADC-XS58-r and ADC-XS54-r was more effective than that induced by rituximab (Figure 2).

[0337] In WO 2023 / 275025, rituximab and rituximab-ADCs were shown to induce NK cell activation, most likely through FcγR, which is widely known to be expressed by NK cells (WO 2023 / 275025). Consistent with this, ADC-XD18-r, ADC-XD73-r, ADC-XD45-r, ADC-XS58-r, ADC-XS56-r, ADC-XS54-r, and ADC-XS57-r all induced NK cell degranulation (i.e., CD107a) in a similar manner (Figure 1C).

[0338] These results show that pretreatment of tumor cells with CD20-conjugated ADCs resulted in dose-dependent induction of IFNγ and degranulation of Vδ2γδ T cells. The ADCs possess an active Fc tail that activates NK cells, most likely through distinct interactions with FcγRs.

[0339] [Table 2]

[0340] [Table 3]

[0341] Example 6 Activity of additional phosphoantigen (pAg) conjugates (ADCs) on γδ T cells Many of the synthesized linker-drug compounds were conjugated to rituximab (anti-CD20) or a non-binding isotype control. Linker-drug compounds conjugated to rituximab include XS55, XS100-XS105, and XS107. Rituximab ADCs were prepared with a drug-antibody ratio (DAR) of approximately 2, as described in the Examples above. The linker-drug compounds and resulting ADCs are shown in Table 1.

[0342] These ADCs were tested for their ability to bind to Raji cells (by the binding assay described in Example 5) and to activate V52γδ T cells after overnight incubation with CD20-positive Raji cells. Pretreated Raji cells were co-cultured with peripheral blood mononuclear cells (PBMCs), and V52γδ T cell and NK cell activation (IFNγ production) and degranulation (CD107a) were determined using multicolor flow cytometry, as described in the functional assays in Example 5.

[0343] Results / Conclusion Binding of rituximab ADCs to Raji cells is shown in Figure 3; all compounds bound. The manufactured ADCs were incubated with Raji cells overnight and then cocultured with PBMCs containing V52 γδ T cells for 6 hours before testing their ability to induce V52 γδ T cell activation. Dose-response curves for V52 γδ T cell degranulation (CD107a) and IFNγ production were generated (Figure 4). ADC-XS55-r, ADC-XS100-r, ADC-XS101-r, ADC-XS102-r, and ADC-XS107-r induced V52 γδ T cell degranulation more effectively than rituximab. The corresponding nonbinding isotype control ADCs, ADC-XS55-i, ADC-XS100-i, ADC-XS101-i, ADC-XS102-i, and ADC-XS107-i, were less potent at activating V52 γδ T cells. ADC-XS103-r, ADC-XS104-r, and ADC-XS105-r demonstrated efficacy similar to that of rituximab. Consistent with this, the corresponding nonbinding isotype control ADCs, ADC-XS103-i, ADC-XS105-i, and ADC-XS104-I, also failed to induce V52 γδ T cell activation. All rituximab-ADCs tested (ADC-XS55-r, ADC-XS100-r, ADC-XS101-r, ADC-XS102-r, ADC-XS103-r, ADC-XS104-r, ADC-XS105-r, and ADC-XS107-r) induced NK cell activation, most likely via FcγR (WO 2023 / 275025), which is widely known to be expressed by NK cells (Figure 2C). Overall, these results indicate that pretreatment of tumor cells with ADC-XS55-r, ADC-XS100-r, ADC-XS101-r, ADC-XS102-r, and ADC-XS107-r resulted in greater IFNγ production and degranulation by Vδ2γδ T cells than unconjugated rituximab. All ADCs tested possess an active Fc tail that activates NK cells, most likely through distinct interactions with FcγR.

Claims

1. Formula (I) 【Chemistry 1】 (In the formula, L represents a linking moiety; W １ is N, CH or CF, preferably CH; W ２ is CH ２ , CHF, CF ２ or O; X １ are O, S, NH, CH ２ , CHF or CF ２ and X ２ is O, CH ２ , CHF or CF ２ and X ３ is absent, O or NH; X ４ａ－ｄ each is independently selected from O and S; X ５ teeth, - H, halogen (F, Cl, Br, I) or nitrile (CN), - F, CH ３ , C.H. ２ F, CHF ２ , C.F. ３ ethenyl, ethynyl, ethyl, optionally substituted with one or more of C optionally substituted with one or more fluorine substituents ３ -C ４ Cycloalkyl, C ３ -C ４ Cycloalkenyl, allyl, propynyl, O—CH ３ , S-CH ３ , or - CHR １ OR ２ , CHR １ SR ２ , CHO, CO ２ R １ , C.O.R. １ R ２ (where R １ and R ２ are independently H, CH ３ , C.H. ２ F, CHF ２ , C.F. ３ , C.H. ２ CH ３ ; selected from and x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R １ is H, a connecting moiety to the linking moiety (L) or a prodrug moiety; R ２ is H, a connecting portion to the linking portion (L), Cat+, or a prodrug portion; R ３ is H, a connecting portion to the linking portion (L), Cat+, or a prodrug portion; R ４ is H, a connecting portion to the linking portion (L), Cat+, or a prodrug portion; Or, when n is 0, R ３ and R ２ is C １－６ connected by a (hetero)alkyl group; or When n is 1 or 2, R ３ and R ４ is C １－６ (Hetero)alkyl groups) Linker-drug compounds having the general structure reflected in

2. X ５ The linker-drug of claim 1, wherein is H, Cl, F, I or Br.

3. W １ is CH, R １ is H or the connection point with said linking moiety (L).

4. X ３ is O and R ３ is the connection part with the connecting part, R １ The linker-drug compound of any one of claims 1 to 3, wherein is H.

5. W ２ is CH ２ and m is 1. The linker-drug compound of any one of claims 1 to 4, wherein

6. X １ is CH ２ The linker-drug compound of any one of claims 1 to 5, wherein

7. X ３ is O and R ３ is the connection part with the connecting part, W １ is CH, R １ is H and W ２ is CH ２ So, m is 1 and X １ is CH ２ The linker-drug compound of any one of claims 1 to 6, wherein

8. n is 0 or 1, and X ４ａ－ｂ and X ４ｃ－ｄ (if present) is O, R ２ and R ４ The linker-drug compound of any one of claims 1 to 7, wherein (when present) is H.

9. R ２ , R ３ and R ４ (if present) pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) groups, substituted or unsubstituted (hetero)aryl groups, and - a structure of formula IV or V 【Chemistry 2】 (In the formula; R ａ and R ａ’ are independently H, an optionally substituted amino acid side chain, and an optionally substituted C １－１４ a non-polar side chain having an alkyl chain; R ｂ is H, benzyl, or substituted or unsubstituted (C １－８ ) alkyl, R ｃ and R ｃ’ are independently H and optionally substituted C １ -C ６ Alkyl, C ３ -C ６ selected from cycloalkyl, aryl, or heteroaryl 2. The linker-drug compound of claim 1, which is a prodrug moiety selected from the group consisting of:

10. R ２ , R ３ and R ４ 10. The linker-drug compound of claim 9, wherein (if present) is independently selected from a POM group and a POC group.

11. n is 0 and R ２ or R ３ is a substituted or unsubstituted 5- or 6-membered (hetero)aryl group, R ３ The linker-drug compound of claim 9, wherein: is a structure represented by formula IV or V, or vice versa.

12. The linker-drug compound of any of claims 1 to 11, wherein the linking moiety (L) is a cleavable linking moiety.

13. The linking moiety (L) is a structure represented by formula VI or VII 【Transformation 3】 (In the formula, m is an integer from 1 to 10, preferably 5; AA is an amino acid, preferably a naturally occurring amino acid; p is 0, 1, 2, 3 or 4; q is an integer from 1 to 12, preferably 2; ES does not exist, 【Chemistry 4】 (where R ５ is H, halogen, CF ３ , C １－４ Alkyl, C ２－４ Alkenyl, C ２－４ Alkynyl, C １－４ Alkoxy or C １－４ Alkylthio, preferably H, F, CH ３ , C.F. ３ , more preferably H or F, and V is H, ethyl, —(CH ２ CH ２ O) ｐ -OMe, CH ２ CH ２ SO ２ Me or CH ２ CH ２ N (Me) ２ and p is an integer from 1 to 12.

13. The linker-drug compound of any of claims 1 to 12, comprising:

14. Use of a linker-drug compound according to any one of claims 1 to 13 in the manufacture of a conjugate.

15. A conjugate comprising a targeting moiety covalently attached to a linker-drug compound according to any one of claims 1 to 13.

16. 16. The conjugate of claim 15, wherein the targeting moiety is a tumor-targeting antibody or antigen-binding fragment thereof.

17. 17. The conjugate of claim 15 or 16, wherein the linking moiety comprises a cleavable linker.

18. The conjugate according to any one of claims 15 to 17 for use as a medicament.

19. 19. The conjugate of claim 18 for treating cancer, an autoimmune disease or an infectious disease.

20. A pharmaceutical composition comprising the conjugate according to any one of claims 15 to 19 and one or more pharmaceutical additives.

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