Novel linker drugs comprising phosphate antigens, novel conjugates and their use in therapy

Abstract

The present invention relates to novel linker pharmaceutical compounds based on a specific phosphate antigen (pAg) having the general structure of formula (I) wherein L represents a linking moiety (linker) and W1, W2, X1 to 5, x, m, n and R1 to 4 are as defined in the description. Also provided are conjugates comprising a targeting moiety, preferably a tumor-targeting antibody or antigen-binding fragment thereof, covalently linked to a linker drug compound according to the invention. Such conjugates are useful, for example, in the treatment of diseases such as cancer, infections or autoimmune diseases. # imgabs0 #

Description

TECHNICAL FIELD

[0001] The present invention relates to novel linker-drug compounds for the preparation of conjugates, said linker-drug compounds comprising one or more phosphoantigen moieties covalently bound to a linking moiety.

[0002] The present invention also relates to conjugates comprising a targeting moiety (e.g., an antibody or a binding fragment thereof) linked to said linker-drug compound, pharmaceutical compositions comprising said conjugates, and their use in the treatment of diseases (e.g., cancer, infectious diseases, and autoimmune diseases), said conjugates optionally in combination with additional therapeutic agents. BACKGROUND OF THE INVENTION

[0003] Conventional methods for treating cancer involve surgery, radiotherapy, and chemotherapy using cytotoxic agents, or combinations of these treatments. Due to their toxic and non-specific nature, treatment with cytotoxic agents or radiation often results in severe side effects. Since the discovery that the immune system plays an important role in eradicating neoplastic cells, more recent cancer treatments have aimed to use components of the immune system as tools for treating cancer.

[0004] One approach used in cancer immunotherapy is to target "immune checkpoints", such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1), with the aim of activating an anti-tumor immune response in patients with cancer. Both CTLA-4 and PD-1 are proteins involved in negative feedback systems, the function of which is to inhibit immune cell activation. Tumor cells can escape from the immune system by overexpressing immune checkpoint ligands on their surface and thereby "abusing" this inhibitory mechanism to protect themselves from attack by immune system cells. Immune checkpoints are activated by interaction with their ligands, leading to inactivation and exhaustion of T cells. Immune checkpoint inhibitors, such as antibodies against immune checkpoints or their ligands, are a new class of anti-cancer drugs that block overexpressed immune checkpoints on cancer cells. Some examples of approved immune checkpoint inhibitors are ipilimumab (blocks CTLA-4, trade name manufactured by BMS), which was approved in 2011 for the treatment of melanoma; the PD-1 antibody nivolumab (sold under the trade name and developed by BMS); and pembrolizumab (trade name another PD-1 inhibitor, manufactured by Merck). Although checkpoint inhibitors can reinvigorate the anti-tumor response, activated immune cells can also attack normal tissues, resulting in immunological adverse side effects.

[0005] Another approach to cancer treatment involves the use of Antibody-Drug Conjugates (ADCs). An ADC combines the specificity of a monoclonal antibody against a tumor-specific antigen with the cell-killing activity of a chemical cytotoxic agent. The antibody of the ADC serves as a targeting agent and a carrier for the cytotoxic payload. Binding of the antibody to its target enables efficient uptake of the ADC and its cytotoxic payload into the target tumor cells. The cytotoxic payload can be an inactive precursor (prodrug) of a cytotoxic agent, which is grafted onto the antibody via a linker that is stable in the circulation and cleaved after internalization into the tumor cells, for example, by intracellular proteases. Cleavage of the linker triggers the release of the active, cytotoxic form of the payload in the tumor cells. ADCs have the advantage of significantly reducing toxicity and non-specific side effects on healthy tissues. Clinically approved ADCs include gemtuzumab ozogamicin (anti-CD33) ( Wyeth Pharmaceuticals, a subsidiary of Pfizer), brentuximab vedotin (anti-CD30) ( Seattle Genetics / Millennium Pharmaceuticals), (ado-)trastuzumab emtansine (anti-HER2) ( Genentech / Roche), inotuzumab ozogamicin (anti-CD22) ( Wyeth Pharmaceuticals, a subsidiary of Pfizer), enfortumab vedotin (anti-Connexin-4) (Padcev TM ; Astellas Pharma / Seattle Genetics), fam-trastuzumab deruxtecan ( Daiichi Sankyo / AstraZeneca), polatuzumab vedotin (anti-CD79b) (Polivy TM ; Genentech / Roche) and sacituzumab govitecan (anti-TROP-2) (Trodelvy TM ; Immunomedics). More are in clinical development.

[0006] Another approach to cancer treatment is immunotherapy using therapeutic compounds that activate the immune system, particularly T cells, to attack and destroy tumor cells. Such therapeutic compounds can be agonists of immune cell receptors and can be macromolecules or relatively small chemical structures. An example of such a compound is a ligand that activates Toll-like receptor (TLR). Several TLR ligands have been approved for cancer treatment. The first approved TLR ligand (TLR agonist) is part of an attenuated strain of Mycobacterium bovis called Bacillus Calmette-Guérin (BCG). As an initially developed tuberculosis vaccine, BCG contains active TLR2 / 4 ligands and has been used as a therapeutic agent for bladder cancer. Other approved TLR ligands are the TLR4 ligand monophosphoryl lipid A (MPLA) and the small molecule TLR7 agonist imiquimod (an imidazoquinoline).

[0007] TLR ligands have also been used in immunoconjugates. Such immunoconjugates contain antibodies to tumor antigens that are specific for a targeting carrier that is a TLR ligand, with the aim of inducing local activation of cells of the immune system in the tumor microenvironment. An immunoconjugate for the treatment of breast cancer in which a TLR agonist is conjugated to an anti-HER antibody is described in WO2017 / 072662 (Novartis A.G.). Another anti-HER conjugate (ImmunoTAC TM SBT6050) with a TLR8 agonist payload has been developed by Silverback Therapeutics. TLR immunoconjugates are also described by Bolt Therapeutics (WO2020 / 047187) and Ackerman et al., 2021, Nature Cancer, vol. 2(8), 18–33, which contain a tumor-targeting monoclonal antibody conjugated to a TLR 7 / 8 agonist (T785) via a non-cleavable linker. The tumor-targeting antibody that binds to the tumor antigen activates antigen-presenting cells present in the tumor microenvironment (TME) through Fc effector functions, while the conjugated TLR agonist directly stimulates the APC through its TLR receptor, thereby promoting anti-tumor immunity.

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

[0009] In contrast to TLR agonists, phosphoantigens do not directly act on receptors presented on myeloid cells or T cells. It is thought that phosphoantigens bind intracellularly (e.g., within cancer cells) to the intracellular domain of the cell surface molecule butyrophilin 3A1 (BTN3A1), resulting in conformational changes associated with the extracellular portion of the BTN3A1 complex, which also includes an effect on BTN2A1 (Sandstrom A, et al., 2014, Immunity, 40(4), 490-500, doi:10.1016 / j.immuni.2014.03.003).

[0010] Conformational changes in the extracellular BTN3A1 / BTN2A1 complex lead to binding to γδTCR, which in turn leads to cytokine production and killing of tumor / pathogenic cells by activated γδT cells (Rigau et al., Science, 2020, 367, 642). Thus, the use of phosphoantigens as therapeutic agents to activate γδT cells is indirect, with phosphoantigens acting intracellularly (e.g., in tumor cells or infected cells) to effect conformational changes in the extracellular BTN3A1 / BTN2A1 complex on the surface of said cells, which in turn provides an activation signal to γδTCR on γδT cells. The γδT cells will then exert their cell-killing effect on tumor cells or infected cells.

[0011] Because of the poor pharmacokinetic properties of the pyrophosphate / ester HMBPP (which is rapidly hydrolyzed in plasma), (nitrogen-containing) bisphosphonate / ester analogues, as well as (monophosphate / ester) prodrug forms, which are converted to the active phosphoantigen after administration to a subject, have been developed. In phosphoantigen-prodrugs, the negatively charged non-bridging oxygen atoms of the phosphonate / ester group are protected with a neutral group to enhance, for example, diffusion across cell membranes. Once inside the cell, the protecting group is removed to release the active phosphoantigen. Another method for increasing the circulating half-life of phosphoantigens (especially bisphosphonate / ester phosphoantigens) is described in WO2012 / 042024. The phosphoantigen is complexed with inorganic and lipid-based nanocarriers into nanoparticles that act as delivery vehicles for the phosphoantigen. It is thought that the resulting nanoparticles can be coated on their surface with targeting ligands to target specific cells. Examples mentioned include molecules that induce targeting to cancer cells, such as antibodies. The use of human transferrin is illustrated.

[0012] Compounds with cellular pAg activity are thought to directly display their activity by binding to the pAg receptor in the target cell (“direct pAg”). This receptor is thought to be the intracellular domain of the cell surface molecule butyrophilin 3A1 (BTN3A1). Natural phosphoantigens include pyrophosphates / esters (diphosphates / esters), such as HMBPP and IPP. Known analogues of natural phosphoantigens include bromohydrin pyrophosphate (BrHPP) and pyrophosphonates / esters, such as C-HMBPP, which is a pyrophosphonate / ester equivalent of naturally occurring HMBPP. The natural pAg HMBPP is produced by pathogenic bacteria. It has been found that the allyl alcohol in natural pAg (such as HMBPP) is important for BTN3A1 binding and maximizing pAg activity. Direct pAg, such as HMBPP, binds directly to BTN3A1 in its intracellular B30.2 domain. Other analogues of HMBPP (such as halohydrins such as BrHPP, IHPP and ClHPP) are also known in the art (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030–1039).

[0013] Phosphoantigen analogue phosphonamidates with claimed enhanced efficacy, such as N-HDMAPP, in which the isoprene unit present in natural HMBPP is linked to the pyrophosphate / ester via an NH group, are described in WO2005 / 05258 (Innate Pharma).

[0014] Other compounds show indirect pAg activity through the accumulation of IPP. Such compounds may be referred to as “indirect pAg”. Indirect pAg acts on the pathway to increase the cellular level of (endogenous) direct pAg (such as IPP) and simultaneously activate Vγ9Vδ2 T cells. In contrast to direct pAg, indirect pAg does not interact directly with the butyrophilin receptor in the target cell, nor is it a pAg precursor (a compound that is enzymatically or chemically converted to direct pAg). Indirect pAg can be, for example, a compound that inhibits a downstream enzyme (such as farnesyl pyrophosphate synthase (FPPS)). Inhibition of FPPS blocks the use of IPP and leads to the accumulation of IPP in the cell. A known FPPS inhibitor is aminobisphosphonate (N-BP), such as zoledronate (Wiemer et al., 2020, Chem. Med. Chem., 15, 1030–1039; Park et al., 2021, Frontiers in Chemistry, Volume 8, Article number 612728).

[0015] Other examples of bisphosphonates / esters having nitrogen or an amino group in a substituent on the central carbon atom, which are considered to enhance the efficacy of bisphosphonates / esters (Drake et al., Mayo Clin. Proc., 2008, 83(9), 1032-1045) include alendronate, risedronate, ibandronate, pamidronate, neridronate, and olpadronate.

[0016] Aminobisphosphonates / esters (N-BPs) (e.g., zoledronate, pamidronate, and alendronate) are also referred to as "bone-targeting agents" due to their ability to specifically bind to hydroxyapatite (HA) (Farrell et al., 2018, Bone Reports, 9, 47-60). Alendronate has also been conjugated to trastuzumab with the aim of using alendronate as a bone-targeting agent to target trastuzumab to bone metastases (Tian et al., 2021, Sci. Adv., 7, 2-11). Since alendronate is negatively charged, it has a high affinity for HA, resulting in preferential binding to bone. Therefore, Tian et al. proposed using negatively charged aminobisphosphonates / esters such as alendronate as targeting agents for antibodies used to treat bone-related diseases.

[0017] The use of phosphoantigens has been tested for use in cancer treatment with the aim of promoting the cytotoxic effect of γδ T cells on tumor cells by expanding γδ T cells and antigen-presenting cells in vivo or in vitro for administration to a subject. Synthetic phosphoantigens such as BrHPP (Phosphostim, manufactured by Innate Pharma) and zoledronate (Novartis) have been the subject of clinical testing in patients with cancer. The phosphoantigens that have been the subject of clinical testing have shown an acceptable safety profile. However, their potency is generally insufficient (Sebestyen et al., Nature Reviews Drug Discovery, 2020, 19(3), 169-184).

[0018] It has been found that delivering phosphoantigens to cells (e.g., tumor or pathogenic cells) that (over)express the butyrophilin (BTN3A1 / BTN2A1) complex in a more robust, selective, and efficient manner can greatly improve the acceptable therapeutic window of such treatments. SUMMARY OF THE INVENTION

[0019] The present invention provides a more effective and more selective method for treating, for example, cancer using phosphoantigens. The present invention relates to linker-drug compounds having the general structure shown in formula (I):

[0020]

[0021] wherein L represents a linking moiety,

[0022] W 1 is N, CH or CF, preferably CH;

[0023] W 2 is CH2, CHF, CF2 or O;

[0024] X 1 is O, S, NH, CH2, CHF or CF2;

[0025] X 2 is O, CH2, CHF or CF2;

[0026] X 3 is absent, or is O or NH;

[0027] X 4a至4d each of which is independently selected from O and S;

[0028] X 5 is

[0029] -H, halogen (F, Cl, Br, I) or nitrile (CN), or

[0030] -vinyl, ethynyl, ethyl, which is optionally substituted with one or more of the following groups: F, CH3, CH2F, CHF2, CF3, or

[0031] -C3 to C4 cycloalkyl, C3 to C4 cycloalkenyl, allyl, propargyl, O-CH3, S-CH3, which is optionally substituted with one or more fluorine substituents, or

[0032] -CHR 1 OR 2 、CHR 1 SR 2 、CHO、CO2R 1 、CONR 1 R 2 wherein R 1 and R 2 are independently selected from H, CH3, CH2F, CHF2, CF3, CH2CH3;

[0033] x is an integer from 1 to 5;

[0034] m is 1, 2 or 3;

[0035] n is 0, 1 or 2;

[0036] R 1 is H or a connection part of the linking part (L), or a prodrug part;

[0037] R 2 is H or a connection part of the linking part (L), or Cat+ or a prodrug part;

[0038] R 3 is H or a connection part of the linking part (L), or Cat+ or a prodrug part;

[0039] R 4 is H or a connection part of the linking part (L), or Cat+ or a prodrug part;

[0040] Alternatively, when n is 0, R 3 and R 2 are connected through C 1-6 (hetero)alkyl, or

[0041] when n is 1 or 2, R 3 and R 4 are connected through C 1-6 (hetero)alkyl.

[0042] Preferably, X 5 is H or a halogen (Cl, F, I or Br), most preferably Br or Cl.

[0043] When W 1 is CH, R 1 is preferably H or a connection part of the linking part (L). When X 3 is O, R 3 is preferably a connection part of the linking part, and R 1 is H. In the linker-drug compound according to the present invention, when W 2 is CH2, preferably m is 1. X 1 is preferably CH2.

[0044] In a preferred embodiment, X 3 is O, R 3 is a connection part of the linking part, W 1 is CH, R 1 is H, W 2 is CH2 and m is 1, and X 1 is CH2.

[0045] In the linker-drug compound according to the present invention, preferably, n is 0 or 1, X 4a至4band X 4c至4d (when present) is O, and wherein R 2 and R 4 (when present) is H.

[0046] R 2 and R 3 , and R 4 (when present) may be a prodrug moiety selected from the following:

[0047] - pivaloyloxymethyl (POM) group and isopropyloxycarbonyloxymethyl (POC) group,

[0048] - substituted or unsubstituted (hetero)aryl, and

[0049] - a structure according to Formula IV or V:

[0050]

[0051] wherein:

[0052] R a and R a’ are independently selected from H, optionally substituted amino acid side chains, and nonpolar side chains containing an optionally substituted C 1-14 alkyl chain,

[0053] R b is H, benzyl, or substituted or unsubstituted (C 1-8 )alkyl,

[0054] R c and R c’ are independently selected from H and optionally substituted C1 to C6 alkyl, C3 to C6 cycloalkyl, aryl, or heteroaryl.

[0055] When R 2 , R 3 and R 4 (when present) is a prodrug moiety, R 2 , R 3 and R 4 may independently be selected from the POM group and the POC group.

[0056] When R 2 and R 3 are prodrug moieties and n is 0, R 2 may be a substituted or unsubstituted 5 - or 6 - membered (hetero)aryl, and R 3 may be a structure according to Formula IV or V, or vice versa.

[0057] The linking moiety (L) is preferably a cleavable linking moiety.

[0058] The linking moiety (L) can comprise a structure according to Formula VI or VII:

[0059]

[0060] wherein:

[0061] m is an integer from 1 to 10, preferably 5;

[0062] AA is an amino acid, preferably a natural amino acid; and

[0063] p is 0, 1, 2, 3 or 4;

[0064] q is an integer from 1 to 12, preferably 2;

[0065] ES is absent or is a spacer selected from the following:

[0066]

[0067] wherein:

[0068] R 5 is 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 wherein V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, and wherein p is an integer from 1 to 12.

[0069] The linker-drug compounds according to the invention can be used for the preparation of conjugates. Conjugates are also provided which comprise a targeting moiety covalently linked to a linker-drug compound according to the invention, preferably a tumor-targeting antibody or an antigen-binding fragment thereof. In such conjugates, the linking moiety preferably comprises a cleavable linker. The conjugates according to the invention can be used as medicaments. Pharmaceutical compositions are also provided which comprise a conjugate according to the invention and one or more pharmaceutical excipients.

[0070] Such conjugates can be used for the activation of γδ T cells, for example in the treatment of diseases such as cancer, infection or autoimmune diseases. The conjugates according to the invention can be used alone or in combination with additional therapeutic agents.

[0071] Preferably, the conjugate according to the present invention is an immunoconjugate comprising a tumor-targeting antibody or an antigen-binding fragment thereof as a targeting moiety. Such an immunoconjugate according to the present invention comprising a tumor-targeting antibody as a targeting moiety can be used to specifically deliver a phosphoantigen to local tumor cells, where the immunoconjugate can be internalized into tumor cells after the antibody or its antigen-binding fragment binds to its tumor-specific antigen or tumor-associated antigen (TAA). BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 (A-C): CD107a (A, C) and IFNγ (B) production of gated Vδ2 γδT cells (A, B) or NK cells (C) after co-culturing PBMC with Raji cells pre-treated with pAg ADC over a certain concentration range. The activation level is represented by the proportion of immune cell subsets that are positive for CD107a or IFNγ. The measurement of each compound was performed in 2 independent experiments, with one donor in each experiment.

[0073] Figure 2 (A-B): CD107a (A) and IFNγ (B) production of gated Vδ2 γδT cells after co-culturing PBMC with Raji cells pre-treated with pAg ADC over a certain concentration range. The activation level is represented by the proportion of immune cell subsets that are positive for CD107a or IFNγ.

[0074] Figure 3 : Binding of pAg ADC and rituximab to Raji cells revealed using fluorescent dye-labeled goat anti-human antibodies. The results show the mean + / − standard deviation of the median fluorescence intensity (MFI) from two independent experiments.

[0075] Figure 4 (A-C): CD107a (A, C) and IFNγ (B) production of gated Vδ2 γδT cells (A, B) or NK cells (C) after co-culturing PBMC with Raji cells pre-treated with pAg ADC or rituximab over a certain concentration range. The activation level is represented by the proportion of immune cell subsets that are positive for CD107a or IFNγ. The measurement of each compound was performed in two independent experiments, with one donor in each experiment. DETAILED DESCRIPTION

[0076] The present invention provides linker-drug compounds and conjugates comprising a phosphoantigen (pAg) moiety.

[0077] Linker-drug compounds

[0078] The present invention provides linker-drug compounds having the general structure shown in formula (I):

[0079]

[0080] wherein L represents a linking moiety,

[0081] W 1 is N, CH or CF, preferably CH;

[0082] W 2 is CH2, CHF, CF2 or O;

[0083] X 1 is O, S, NH, CH2, CHF or CF2;

[0084] X 2 is O, CH2, CHF or CF2;

[0085] X 3 is absent, or is O or NH;

[0086] X 4a至4d each independently selected from O and S;

[0087] X 5 is

[0088] -H, halogen (F, Cl, Br, I) or nitrile (CN), or

[0089] -vinyl, ethynyl, ethyl, optionally substituted by one or more of the following groups: F, CH3, CH2F, CHF2, CF3, or

[0090] -C3 to C4 cycloalkyl, C3 to C4 cycloalkenyl, allyl, propargyl, O-CH3, S-CH3, optionally substituted by one or more fluorine substituents, or

[0091] -CHR 1 OR 2 、CHR 1 SR 2 、CHO、CO2R 1 、CONR 1 R 2 wherein R 1 and R 2 are independently selected from H, CH3, CH2F, CHF2, CF3, CH2CH3;

[0092] x is an integer from 1 to 5;

[0093] m is 1, 2 or 3;

[0094] n is 0, 1 or 2;

[0095] R 1 is H or a linking moiety with the linking portion (L), or a prodrug moiety;

[0096] R 2 is H or a linking moiety with the linking portion (L), or Cat+ or a prodrug moiety;

[0097] R 3 is H or a linking moiety with the linking portion (L), or Cat+ or a prodrug moiety;

[0098] R 4 is H or a linking moiety with the linking portion (L), or Cat+ or a prodrug moiety;

[0099] Alternatively, when n is 0, R 3 and R 2 are linked through C 1-6 (hetero)alkyl, or

[0100] when n is 1 or 2, R 3 and R 4 are linked through C 1-6 (hetero)alkyl.

[0101] When X 5 is vinyl, ethynyl or ethyl, X 5 may be substituted by one or more of the following groups: F, CH3, CH2F, CHF2, CF3. Thus, the vinyl, ethynyl or ethyl may be substituted by one or more fluorine substituents. When X 5 is vinyl, ethynyl or ethyl substituted by one or more (fluorinated) methyl groups (CH3, CH2F, CHF2 or CF3), the number of (fluorinated) methyl groups is preferably 1. This includes compounds in which X 5 is (iso)propyl.

[0102] C3 to C4 cycloalkyl includes cyclopropyl, methylcyclopropyl and cyclobutyl. C3 to C4 cycloalkenyl includes cyclopropenyl, methylcyclopropenyl and cyclobutenyl. When X 5 is C3 to C4 cycloalkyl, C3 to C4 cycloalkenyl, allyl, propargyl, O-CH3, S-CH3, X 5 may also be substituted by one or more fluorine substituents. The number of fluorine substituents on X 5 depends on the structure of X 5 . X 5 may carry multiple fluorine substituents, the fluorine substituents on separate C atoms or on the same C atom, or on both.

[0103] Preferably, X5 is H, Cl, F, I or Br, most preferably Cl or Br.

[0104] The linker-drug compound according to the invention is a modification of the linker-drug compound disclosed in co-pending patent application number WO2023 / 275025 filed in the name of Byondis B.V., and differs therefrom in the definition of X 5 .

[0105] The linker-drug compound according to the invention comprises at least one phosphoantigen moiety (pAg or "drug") represented by the structural formula between the outer brackets in formula I, which is linked to a linking moiety (L or "linker"). The number of pAg moieties per linker is represented by "x". When x is greater than 1, there are multiple pAg moieties linked to one (branched) linker moiety.

[0106] Preferably, n is 0 or 1, most preferably 0. When n is 1 or 2, X 2 is preferably O. Linker-drug compounds having a phosphoantigen moiety in which n is 1 and X 2 is CH2 or in which n is 1 and X 2 is O are also part of the invention. In this case, each of the X 4a至4d is preferably O. In such linker-drug compounds, R 3 or R 1 may represent the connecting portion to the linking moiety, preferably R 3 represents the connecting portion to the linking moiety.

[0107] When n is 2, X 2 will occur twice in formula I and may be referred to as X 2a and X 2b , which may independently be selected from O, CH2, CHF and CF2. When n is 2, R 4 will also occur twice and may be referred to as R 4a and R 4b , which may independently be selected from H, the connecting portion to the linking moiety (L), Cat+ and a prodrug moiety. When n is 2, X 4c and X 4d are also the case. Both occur twice (X 4c , X 4ci , X 4d and X 4di ), and may independently be selected from O and S.

[0108] When m is 2 or 3, W 2 will occur multiple times in formula I, and each W 2 may independently be selected from CH2, CHF, CF2 or O. Preferably, W2 is CH2. In a preferred embodiment, m is 1, and most preferably, when m is 1, W 2 is CH2.

[0109] Cat+ represents (organic or inorganic (mineral)) cations, including protons.

[0110] X 1 is preferably CH2, O or S, most preferably CH2.

[0111] X 4a至4d each of which (when present) is preferably O. Part of the invention is such compounds: where n is 1 or 0, and where X 4a至4b and X 4c至4d (when present) is O, and where R 2 and R 4 (when present) is preferably H.

[0112] Preferably, n is 0, and X 4a as well as X 4b is O, and R 2 is preferably H.

[0113] Preferably, W 1 is CH or CF, most preferably CH. R 1 is preferably H or the connecting part to the linking moiety (L), most preferably H.

[0114] Preferably, W 1 is CH, X 1 is CH2 and R1 is H, resulting in a linker-drug molecule carrying the pAg moiety with an allyl alcohol group. Preferably, when W 1 is CH, X 1 is CH2 and R 1 is H, then W2 is CH2 and m is 1.

[0115] Preferably, X 3 is O, R 3 is the connecting part to a cleavable linking moiety, W 1 is CH, R 1 is H, W 2 is CH2 and m is 1, and X 1 is CH2.

[0116] In such compounds, R 2 、R 3 and / or R 4 can be separate or are respectively linked to X 4b 、X 4d and / or X 3 (when X3 in the presence of) the prodrug moiety of the combination (the prodrug moiety is -X 4b -R 2 、-X 4d -R 4 and / or -X 3 -R 3 ).

[0117] Preferably, W 1 is CH, W 2 is CH2, X 4a至4d is O, R 2 and R 4 are H, and m is 1.

[0118] In a preferred embodiment, W 1 is CH, W 2 is CH2, n is 0, X 4a至4b is O, and m is 1.

[0119] In formula I, x represents the number of phosphoantigen moieties (pAg) of each linking moiety (L), thus, the structure between the brackets is preferably the structure representation of the phosphoantigen moiety for the linker-drug compounds according to the present invention. x can be an integer from 1 to 5 (each linking moiety carries 1 to 5 pAg). Preferably, the linking moiety carries 1 or 2 pAg moieties. In most cases, it may be sufficient for each linking moiety to carry 1 pAg.

[0120] The connection to the linking moiety can be R 1 (part of), or, in an alternative, the linking moiety can (be connected to) R 2 、R 3 or R 4 . Preferably, R 1 or R 3 is the connection to the linking moiety, more preferably R 3 . When the linking moiety is connected at R 3 position, X 3 is preferably O. When R 3 is the connection to the linker moiety, preferably X 3 is O, and R 1 is preferably H.

[0121] When the linking moiety is connected at R 2 or R 4 position, X 4b or X 4d are respectively preferably O.

[0122] A preferred linker-drug compound is a linker-drug compound wherein X 3 is O and R3 is a connecting part to the cleavable linking part, where preferably W 1 is CH, R 1 is H, W 2 is CH2 and m is 1, and X 1 is CH2. In such a compound, n is preferably 0.

[0123] By "connecting part to the linking part" is meant the position in the molecule where the linker is attached to the phosphoantigen moiety. "Connecting part" does not necessarily mean R 1 、R 2 、R 3 or R 4 (depending on the position of linker attachment) represents the actual (remaining) structural element of the linker-drug compound between the linker and the remainder of the phosphoantigen moiety. For example, depending on the linker chemistry used, when R 1 represents the connecting part to the linking part, this also includes the case where the linker is directly attached to the oxygen atom of the phosphoantigen moiety in the linker-drug molecule. In an alternative, R 1 is the connecting part to the linking part (L). In such a case where R 1 is the connecting part to the linking part (L), preferably W 1 is CH, W 2 is CH2, m is 1, and X 1 is CH2. When such a linker-drug molecule is incorporated into a conjugate according to the invention, cleavage of the linker after administration can lead to the (re)formation of an allyl alcohol group (in the actual functionally active phosphoantigen moiety released from the conjugate, R 1 is H). R 1 can also be a prodrug moiety. Suitable alcohol prodrug moieties are known in the art. For example, the alcohol can be masked by an ester-based prodrug group. Generation of the active alcohol depends on the hydrolysis of the ester bond by (cellular) esterases, resulting in the metabolic regeneration of the alcohol (drug) and the carboxylic acid (leaving group).

[0124] R 2 、R 3 and R 4 can each independently be H or a connecting part to the linking part (L), or Cat+ or a prodrug moiety. In a preferred embodiment, the compound according to the invention is a monophosphate / ester (n is 0) and thus R 4 is absent.

[0125] Cat+ represents an (organic or inorganic) cation, including a proton (and can be exchanged in the formulation buffer or plasma). When R 2 、R 3 and / or R 4When it is Cat+, the Cat+ can be the same or different. Preferably, when R 2 , R 3 and / or R 4 is Cat+, X 4b and X 4d (when present, i.e., n is not 0) and / or X 3 is O, generating O - Cat + .

[0126] In another embodiment of the present invention, where n is 0, R 3 and R 2 are linked by C 1-6 (hetero)alkyl. In this case, R 3 and R 2 together form a substituted or unsubstituted 5- to 8-membered ring. In such an embodiment, the linking moiety is preferably linked at the R 1 position. In an alternative embodiment where n is not 0, R 3 and R 4 can be linked in a similar manner by C 1-6 (hetero)alkyl.

[0127] R 2 , R 3 and / or R 4 can also be separate or combined with X 4b , X 4d and / or X 3 (when X 3 is present) to form prodrug moieties (the prodrug moieties are -X 4b -R 2 , -X 4d -R 4 and / or -X 3 -R 3 ).

[0128] The "prodrug moiety" can be a group that can be cleaved non-enzymatically or enzymatically (releasing the active compound). After administering the conjugate according to the present invention to a subject, the "prodrug moiety" can induce the release of a second prodrug moiety at another position in the molecule. Preferably, the prodrug form of the phosphoantigen moiety is converted into a functionally active phosphoantigen inside the target cells (such as tumor cells), for example, by enzymatic removal of the prodrug moiety.

[0129] Some examples of prodrug techniques known in the art include using a pivaloyloxymethyl (POM) group or an isopropoxycarbonyloxymethyl (POC) group. In a preferred embodiment, at least R 2 and R 3Independently selected from POM groups or POC groups (e.g., when n is 0). When n is 1 or 2, R 4 can also be a POM group or a POC group.

[0130] For example, this type of phosphoric acid antigen prodrug is described in WO2019 / 182904.

[0131] In an alternative, combinations of leaving groups can be used; an example of such a prodrug technology is the "ProTide" technology developed for the intracellular delivery of monophosphates / esters and monophosphonates / esters. The hydroxyl groups of the monophosphate / ester or monophosphonate / ester groups in ProTide prodrugs are masked (or replaced) by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved inside the cell to release the free monophosphates / esters and monophosphonates / esters (Mehellou et al. 2018, Journal of Medicinal Chemistry, 61(6), 2211 - 2226).

[0132] Thus, linker - drug compounds and conjugates according to the invention in which the phosphoric acid antigen moiety is a monophosphate / ester or a monophosphonate / ester and in which R 2 and R 3 are combinations of "ProTide" leaving groups are also part of the invention. In such cases where the phosphoric acid antigen moiety is a ProTide prodrug of a phosphoric acid antigen, R 2 is an aromatic moiety and R 3 is an amino acid ester moiety, or vice versa. In a preferred embodiment of the invention, when n is 0, one of R 2 or R 3 can be a substituted or unsubstituted (hetero)aryl, while the other (R 3 or R 2 ) is optionally selected from the structures according to Formulas IV and V:

[0133]

[0134] Wherein:

[0135] R a and R a’ are independently selected from H, an optionally substituted amino acid side chain, and a non - polar side chain comprising an optionally substituted C 1-14 alkyl chain,

[0136] R b is H, benzyl, or a substituted or unsubstituted (C 1-8 )alkyl,

[0137] R c and Rc’ independently selected from H, or optionally substituted (C 1-6 )alkyl, (C 3-6 )cycloalkyl, aryl or heteroaryl. R c and R c’ may also together with the nitrogen to which they are attached form an optionally substituted ring, such as an aziridine ring, an azetidine ring, a morpholine ring, a piperazine ring, a pyrrolidine ring or a piperidine ring.

[0138] R c and / or R c’ The optional substituents on are carboxylic acid bioisosteres, amino, tetrazole, sulfonate, hydroxyl, halogen or alkyl.

[0139] When R b is a substituted alkyl, the substituents may be one or more groups independently selected from the following: hydroxyl, amino, halogen, nitro, cyano, carboxyl, 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, wherein R x and R y are each independently selected from H, (C1 to C6)alkyl, (C 3-6 )cycloalkyl and (C 3-6 )cycloalkyl(C 1-6 )alkyl. In an alternative, R x and R y together with the nitrogen to which they are attached form an aziridinyl, azetidinyl, morpholinyl, piperazinyl, pyrrolidinyl or piperidinyl.

[0140] Linking moiety

[0141] The linker (or "spacer") in the conjugate or linker-drug compound according to the present invention is preferably a synthetic linker. The structure of the linker is such that the linker can be readily chemically linked to a small effector molecule (phosphoantigen moiety), and such that 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 such a final conjugate in circulation, and, if released, can affect the manner in which the small molecule effector compound (phosphoantigen) is released. Suitable linkers are described, for example, in Ducry 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 / 083180, WO2004 / 043493, WO2010 / 062171, WO2011 / 133039, WO2015 / 177360 and WO2018 / 069375. The linker can be cleavable or non-cleavable, as described, for example, in van Delft, F and Lambert, J.M., 2021, Chemical Linkers in Antibody-Drug Conjugates (ADCs), 1st Ed. Royal Society of Chemistry, ISBN-10: 1839162635. Another method of conjugating a linker-drug to an antibody is by using a transpeptidase, such as a bacterial sortase or a plant asparaginyl endopeptidase, to achieve site-specific immobilization of a chemical moiety linked to a suitable synthetic peptide.The Sortase A (Sort-A) recognizes the C-terminal peptide sequence (LPXTG) and generates a bond between the threonine within this sequence and a glycine provided at the N-terminus of the conjugate partner of the ADC, such as a glycine-labeled payload (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, by using endo-β-N-acetylglucosaminidase (ENGase) and monosaccharide transferase 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).

[0142] Preferably, cleavable linkers are used in the conjugates according to the invention. A cleavable linker contains a moiety that can be cleaved, for example, when exposed to lysosomal proteases or an environment with an acidic pH or a higher reduction potential. Suitable cleavable linkers are known in the art and include, for example, single peptides, dipeptides, tripeptides, or tetrapeptides, i.e., single, two, three, or four amino acid residues. Additionally, the cleavable linker can contain a self-immolative moiety, such as an ω-aminoaminocarbonyl cyclizing spacer, see Saari et al, 1990, J. Med. Chem., 33, 97–101, or -NH-CH2-O- moiety. Other cleavable linkers known in the art are β-glucuronide linkers, such as those 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 according to the invention available to the surrounding environment. Non-cleavable linkers can still effectively release the (active derivative of the) phosphoantigen moiety from the immunoconjugate according to 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 their analogs.

[0143] In order to be able to conjugate a linking moiety or linker-drug compound to a polypeptide (such as an antibody), the side of the linking moiety that will bind (covalently) to the antibody typically contains functional groups that can react with the amino acid residues of the antibody under relatively mild conditions. Such functional groups are referred to herein as reactive moieties (RM). Some examples of reactive moieties include, but are not limited to, carbamoyl halides, acyl halides, active esters, acid anhydrides, α-haloacetyls, α-haloacetamides, maleimides, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, acylhydrazines, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyls, methyl sulfonates, cyclooctynes, and trans-cyclooctene (TCO). Such amino acid residues with which the functional group reacts can be natural or unnatural amino acid residues, or (non)-natural glycans (Manabe et al., Wang et al., (see above)). The term "unnatural amino acid" as used herein is intended to mean an amino acid that has been (synthetically) modified or the D-stereoisomer of a naturally occurring amino acid. Preferably, the amino acid residues with which the functional group reacts are natural amino acids.

[0144] The linking moiety (L) for the conjugate or linker-drug compound according to the invention may comprise a structure according to formula VI or formula VII:

[0145]

[0146] wherein m is an integer from 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 greater than 1, the amino acids may be the same or different.

[0147] Suitable combinations of amino acids are known in the art and include amino acids selected from alanine, glycine, lysine, phenylalanine, valine, and citrulline. Preferably p is 2. When p is 2, AA2 can be, for example, phenylalanyllysine, valylalanine, valylcitrulline, or valyllysine. When p is 2, AA2 is preferably valylalanine or valylcitrulline. When p is 3, AA3 can be, for example, alanyl-phenylalanyllysine, and when p is 4, AA4 can be, for example, glycylglycylphenylalanylglycine.

[0148] "q" is an integer from 1 to 12, preferably 2; ES is absent, or is an extended spacer selected from:

[0149]

[0150] wherein R 5is H, halogen, CF3, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy or C1-4 alkylthio, preferably H, F, CH3 or CF3, more preferably H or F; and V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, where p is an integer from 1 to 12.

[0151] When ES is present, ES is preferably selected from:

[0152]

[0153] where R 5 is H, halogen, CF3, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy or C1-4 alkylthio, preferably H, F, CH3 or CF3, more preferably H or F; and V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, where p is an integer from 1 to 12.

[0154] When ES is selected from the following, AA p is preferably absent (meaning p is 0):

[0155]

[0156] where R 5 is H, halogen, CF3, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C1-4 alkoxy or C1-4 alkylthio, preferably H, F, CH3 or CF3, more preferably H or F; and V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, where p is an integer from 1 to 12.

[0157] The linking moiety can also be branched, which results in a linking moiety being able to carry multiple phosphoantigen moieties. Some examples of branched linking moieties are:

[0158]

[0159] These branched linker moieties can be used to generate conjugates having a relatively high ratio of pAg to targeting moiety (“DAR”). Using such branched linkers, conjugates with a DAR of 16 and even 20 or higher can be synthesized. Antibody-based conjugates according to the present invention may only require a DAR of about 2. However, for antibodies against tumor-specific targets that are known to be expressed at relatively low levels on target tumor cells, conjugates having a high pAg to targeting moiety ratio may be preferred. The linker-drug compounds used in the linker-drug compounds according to the present invention may for example comprise any linking moiety selected from the following:

[0160]

[0161]

[0162] The linker moiety (L) can be conjugated to the pAg moiety, yielding a linker-drug compound having the general formula shown in Formula I according to the present invention.

[0163] The linker-drug compounds according to the present invention can be conjugated to a targeting moiety to yield a conjugate according to the present invention. Preferred conjugates according to the present invention comprise a tumor-targeting antibody or an antigen-binding fragment thereof conjugated to a linker-drug compound according to the present invention.

[0164] In a specific embodiment of the present invention, the phosphoantigen moiety, as part of a conjugate according to the present invention, is a monophosphate / ester prodrug, wherein the negatively charged non-bonding oxygen atom of the phosphate / ester group is protected by a prodrug moiety (e.g., a combination of a ProTide moiety ((hetero)aryl and amino ester groups) or one or more POM or POC), and the cleavable linker moiety can be linked to the isoprenoid unit of the phosphoantigen molecule, which upon cleavage of the linker will be converted to an allyl alcohol, as seen in phosphoantigens such as HMBPP.

[0165] The synthesis of some examples of the linker-drug molecules according to the present invention is further illustrated in the Examples. Some examples of preferred linker-drug compounds according to the present invention are described in Table 1 in the “Examples” section of this patent application.

[0166] It should be understood that when included in a conjugate according to the present invention, a linker-drug compound comprising at least one phosphoantigen moiety covalently bound to a linking moiety according to the present invention may lack or acquire certain atoms or groups of atoms compared to the same linker-drug compound according to the present invention when not included in the conjugate. For example, it may lack hydrogen atoms. This can be, for example, because the linker-drug compound according to the present invention is conjugated to a polypeptide by, for example, esterification with a hydroxy moiety.

[0167] For example, when the targeting moiety is an antibody or an antigen-binding fragment thereof, one or more linker-drug compounds according to the present invention can be conjugated to the targeting antibody, thereby producing a conjugate according to the present invention.

[0168] Conjugate

[0169] The present invention provides a conjugate comprising a targeting moiety (Tm) covalently linked to one or more linker-drug compounds according to the present invention.

[0170] The conjugate according to the present invention comprises 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 acts as a delivery vehicle that delivers the pAg moiety covalently linked to the targeting moiety to the target cell. The pAg moiety can be directly coupled to, for example, an amino acid side chain in the (polypeptide) targeting moiety. However, preferably, the pAg is conjugated to the targeting moiety through a linking moiety.

[0171] A preferred conjugate according to the present invention can be represented by the general formula II:

[0172] Tm-(L-(pAg) x ) y (II),

[0173] wherein Tm represents the targeting moiety, preferably an antibody or an antigen-binding fragment thereof, L represents the linking moiety and corresponds to the "L" moiety in formula I, and pAg represents the phosphoantigen moiety and corresponds to the structural formula between the outer brackets in formula I, x represents the number of phosphoantigen moieties per linking moiety (as shown in formula I), and has a value of 1 to 5, and y represents the average number of L-(pAg) x (the linker-drug moiety per targeting moiety) for each Tm, and it is an integer from 1 to 10, preferably from 1 to 8. The number of pAg moieties (pAg to Tm ratio) per conjugate in formula II is x multiplied by y. The average pAg to Tm ratio can be from 1 to 16, or even 20, or higher. The ratio of pAg units per targeting moiety can vary, for example, based on the structural or functional characteristics of the phosphoantigen moiety or the targeting moiety. In practice, a pAg number of 2 to 8 or 2 to 6, or even as low as 2 per targeting moiety can provide sufficient therapeutic effect. Preferably, the linking moiety carries 1 or 2 pAg moieties. In most cases, it may be sufficient for each linking moiety to carry 1 pAg. Preferably, the target pAg to Tm ratio is 2 (x is 1 and y is 2).

[0174] The linker moiety is preferably a cleavable linker moiety. In the conjugates according to the invention, straight or branched linker moieties can be used. When multiple phosphoantigen moieties are linked to one targeting moiety, each phosphoantigen moiety can be covalently coupled to the targeting moiety via a separate linking moiety. In practice, when the targeting moiety is an antibody and is conjugated to a reduced interchain disulfide bond, one targeting moiety can be linked to up to 8 separate linking moieties (linker-drug moieties), resulting in 8 phosphoantigen moieties per targeting moiety when each phosphoantigen moiety is carried by its own linking moiety. In an alternative, a branched linker moiety can carry 1 to 5 phosphoantigen moieties / linking moiety (x is 1, 2, 3, 4 or 5).

[0175] Particularly when a higher pAg to Tm ratio is desired, or when only a limited number of binding positions are available on the targeting moiety, a branched linker is preferred. For example, a branched linker carrying 2 pAg (x = 2) can be used to increase the number of phosphoantigen moieties per targeting moiety to a higher value. By using such linking moieties, for example, 16 phosphoantigen moieties can be bound to the targeting moiety using only 8 linking moieties. Antibodies can be modified to introduce additional cysteines in addition to the number of cysteines in the antibody amino acid sequence, which form disulfide bonds and can be reduced and conjugated to linker-drug molecules. For example, as disclosed in WO2015177360, additional cysteines can be introduced at a position such as position 41C. For an antibody containing up to 10 cysteines available for conjugation to linking moieties that can be bound, when using a branched linker carrying two pAg moieties per linker (x = 2), the DAR can even reach 20 (x = 2, y = 10) or higher. Under optimal conditions, all binding sites in the targeting moiety will be occupied by the linking moieties. In practice, a conjugate mixture can be produced in which the exact number of phosphoantigen moieties per target moiety can vary to some extent depending on the reaction conditions, and the y value is an average.

[0176] The conjugates according to the invention can be used in combination with other pharmaceutically active compounds which can be administered simultaneously or sequentially to a subject in need thereof. Additionally, the targeting moiety can carry a combination of a linker-drug compound according to the invention and different payloads. The advantage of such a "multi-payload" approach is that the two active substances will be targeted by the same targeting moiety. Of course, the ratio between the payloads has to be set appropriately depending on the (conjugation) reaction conditions and the binding sites. For example, the individual linker-drug compounds for each payload can be conjugated to different binding sites (such as different types of amino acids) on the targeting moiety, and / or conjugated by different conjugation methods and / or different linker chemistries to control the binding, distribution and drug to antibody ratio (DAR) of the two payloads. Antibody-drug conjugates (ADCs) carrying multiple cytotoxic payloads are known in the art. The conjugates according to the invention can combine a phosphoantigen moiety, for example, with a cytotoxic payload or with another immunomodulatory payload designed to enhance the overall desired therapeutic effect. Thereby, any non-specific binding of the phosphoantigen to non-target tissues and / or the action on non-target tissues at non-target sites is reduced.

[0177] As is known in the art, the drug loading distribution in an ADC can be determined, for example, by using hydrophobic interaction chromatography (HIC) or reversed phase high-performance liquid chromatography (RP-HPLC). HIC is particularly suitable for determining the average DAR (the pAg to Tm ratio in the conjugates according to the invention).

[0178] Targeting moiety

[0179] The targeting moiety binds specifically or preferably to target cells and can be a targeting antibody or an antigen-binding fragment thereof, or can be another targeting moiety such as a nucleic acid (aptamer) or a (poly)peptide, which can be an enzyme inhibitor, an enzyme substrate, a receptor ligand and / or a 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 its target determines where in the body the conjugate according to the invention will exert its therapeutic effect.

[0180] Thus, by selecting a suitable targeting moiety, delivery of the phosphoantigen moiety to the site where it has to exert its therapeutic effect is ensured.

[0181] Preferably, the targeting moiety in the conjugate according to the present invention is an antibody or an antigen-binding fragment thereof. In the case where the targeting moiety is an antibody or an antigen-binding fragment thereof, the conjugate is generally referred to as an immunoconjugate or an antibody-drug conjugate (ADC). A targeting antibody is an antibody that specifically recognizes an antigen (such as a tumor-associated antigen) expressed by a target cell. The specificity of the antibody or fragment for its antigen allows the effector molecule (or "payload") to be specifically delivered to the target cell, leaving healthy tissues largely unaffected. The effector molecule is covalently coupled to the antibody via a linker that ensures the effector molecule remains attached to the antibody, at least until the antibody reaches the target cell, such as a cancer cell. When the antibody binds to its target, the effector molecule exerts its action on or in the target cell (when the conjugate is internalized). The effector molecule can be a cytotoxic agent, a radioisotope, or an immunomodulatory moiety. In the conjugate according to the present invention, the effector molecule is a phosphoantigen moiety.

[0182] antibody

[0183] As used herein, the term "antibody" preferably refers to an antibody comprising two heavy chains and two light chains. Generally, the antibody or any antigen-binding fragment thereof is an antibody or any antigen-binding fragment thereof having therapeutic activity, but as is known in the ADC art, such independent potency is not required. The antibody used according to the present invention can be of any isotype, such as IgA, IgE, IgG, or IgM antibodies. Preferably, the antibody is an IgG antibody, more preferably an IgG1 or IgG2 antibody. The antibody can be chimeric, humanized, or human. Preferably, the antibody is humanized or human. Even more preferably, the antibody is a humanized or human IgG antibody, more preferably a humanized or human IgG1 monoclonal antibody. The antibody can have a κ (kappa) or λ (lambda) light chain, preferably a κ (kappa) light chain, i.e., a humanized or human IgG1-κ antibody.

[0184] 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.

[0185] "Humanized" forms of non-human (e.g., rodent) antibodies are antibodies (e.g., non-human - human chimeric antibodies) that contain a minimal sequence derived from a non-human antibody. A variety of 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 from non-human species (usually mouse, rat, or rabbit). These non-human CDRs can be combined with the human framework regions (FRs, i.e., FR1, FR2, FR3, and FR4) of the variable regions of the HC and LC in such a way that at least some of the functional properties of the antibody, such as binding affinity and specificity, are retained. Selected amino acids in the human FRs can be replaced with the corresponding original non-human species amino acids to further improve antibody performance, such as improving binding affinity while maintaining low immunogenicity. Thus, the humanized variable regions are typically combined with human constant regions. An exemplary method for humanizing non-human antibodies is the method of Winter and his colleagues (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 that are naturally produced in humans. For example, selected amino acids of the original non-human species FRs are replaced with their corresponding human amino acids to reduce immunogenicity while retaining the binding affinity of the antibody. For more details, see Jones et al (see above); Riechmann et al (see above) and Presta, 1992, Curr.Op.Struct.Biol. 2, 593 - 596. See also the following review articles and the 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.

[0186] The CDRs can be determined using the methods of Kabat (Kabat, E.A. 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 (Chothia et al, 1989, Nature, 342, 877-883) or IMGT (Lefranc, 1999, The Immunologist, 7, 132-136).

[0187] Generally, the antibody is a monospecific (i.e., specific for one antigen; such an antigen can be shared between species or have a similar amino acid sequence between species) or bispecific (i.e., specific for two different antigens of a species) antibody that comprises at least one HC and LC variable region that binds to an antigen target, preferably a membrane-bound antigen target (which can be internalized or not internalized). Preferably, the antibody is internalized by the target cell after binding to the (antigen) target, and thereafter releases an active effector molecule intracellularly, which is a phosphoantigen in the conjugate according to the present invention.

[0188] The targeting antibody that can be used in the conjugate for cancer treatment according to the present invention can be a tumor targeting antibody that selectively binds to a tumor-specific or tumor-associated antigen. A tumor-specific antigen is only present on tumor cells, while a tumor-associated antigen is an antigen that is expressed at a higher level (e.g., overexpressed) in cancer cells compared to normal (healthy) cells.

[0189] Antigen targets that bind to the antibody or antigen-binding fragment of the conjugate according to the present invention can be selected, for example, from: 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), CD123 (Interleukin-3 Receptor), CD138 (Syndecan-1), CD203c (ENPP3), CD303, CD333, CDCP1, CEA, CEACAM, Claudin-4, Claudin-7, CLCA-1 (C-Type Lectin-Like Molecule-1), CLL 1, c-MET (Hepatocyte Growth Factor Receptor), Cripto, DLL3, EGFL, EGFR, EPCAM, EphA2, EphB3, ETBR (Endothelin B Receptor), FAP, FcRL5 (Fc Receptor-Like Protein 5, CD307), FGFR3, FOLR1 (Folate Receptor α), FRβ, GCC (Guanylate Cyclase C), GD2, GITR, GLOBO 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, Lewis X, Lewis Y (CD174), LGR5, LIV1, mesothelin (MSLN), MN (CA9), MUC1, MUC16, NaPi2b, Connexin-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.

[0190] Some 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), vorsetuzumab (CD70), milatuzumab (CD74), polatuzumab (CD79), rovalpituzumab (DLL3), futuximab (EGFR), oportuzumab (EPCAM), farletuzumab (FOLR1), glembatumumab (GPNMB), trastuzumab, pertuzumab, and margetuximab (HER2), etaracizumab (integrin), anetumab (mesothelin), pankomab (MUC1), enfortumab (connexin-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 is used to illustrate the present invention.

[0191] Since the pAg activity of the pAg moiety should be displayed in the cell, internalizing antibodies are preferred.

[0192] If applicable, the antibody or antigen-binding fragment thereof may comprise (1) a modified constant region, i.e., one or more mutations may be introduced to, for example, increase the half-life, provide a linker-drug attachment site, and / or increase or decrease effector function; or (2) a modified variable region, i.e., one or more mutations may be introduced to, for example, provide a linker-drug attachment site. The antibody or antigen-binding fragment thereof may be produced recombinantly, synthetically, or by other known suitable methods. For example, mutations that may reduce the Fc-mediated effector function of an antibody are those described, for example, 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.

[0193] The conjugate according to the invention can be wild-type or site-specific (meaning that a specific conjugation site, such as a cysteine or unnatural amino acid, has been engineered into the antibody protein sequence) or a combination thereof, and can be produced by any method known in the art.

[0194] It has been found that the immunoconjugates according to the invention are very effective in delivering their pAg payloads to antigen-presenting cells such as cancer cells, thereby generating active phosphoantigens within the antigen-presenting cells. The antigen-presenting cells can be tumor cells that express or overexpress certain tumor antigens on their surface. Such cells may also express or overexpress TCR activation molecules involved in the indirect activation of γδ T cells by pAg, such as the BTN3A1 / BTN2A1 receptor complex molecules.

[0195] Phosphoantigen moiety (pAg)

[0196] The term "phosphoantigen moiety" or "pAg" as used throughout this specification refers to the pAg moiety having the structural formula shown between the outer brackets in Formula I. In Formula I, at least one pAg is conjugated to a linking moiety (L).

[0197] The phosphoantigen moiety comprises a non-peptide antigen of relatively small mass that can stimulate γδ T cells (more specifically Vγ9Vδ2 cells) in the presence of antigen-presenting cells.

[0198] The "phosphoantigen moiety" as part of a conjugate or linker-drug compound according to the invention does not necessarily comprise the phosphoantigen in its active form. The phosphoantigen moiety in the 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 its target and is processed. Thus, the phosphoantigen moiety in bound state as part of the conjugate or linker-drug compound may be structurally different from the active phosphoantigen released therefrom. For example, cleavage from or of a linking moiety may trigger a structural rearrangement and / or a chemical or enzymatic reaction, resulting in the formation of the functionally active phosphoantigen. In addition, removal or rearrangement of a 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.

[0199] In a conjugate according to the invention, the specific binding of an antibody (targeting moiety) to its specific binding partner (e.g., a tumor-specific antigen), for example, will direct the pAg moiety to its target site, rather than the reverse (the pAg moiety is not the targeting moiety). In a conjugate according to the invention, it is the binding specificity and affinity of the targeting moiety (e.g., an antibody) that ensure delivery of the phosphoantigen moiety to the site where it must exert its therapeutic effect.

[0200] The pAg moiety for use in linker-drug compounds and conjugates according to the invention comprises allyl alcohol or a prodrug thereof (e.g., a pAg moiety in which allyl alcohol is generated after removal of the prodrug group or after cleavage of a linker moiety conjugated or conjugated via an isoprene unit to the isoprene unit). Such compounds are considered to be examples of pAg moieties that contain direct pAg activity (pAg acting as a ligand for BTN3A1).

[0201] As illustrated by way of example in the Examples, the activity of phosphoantigens on Vγ9Vδ2 T cells can be measured in a cell assay. 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 a phosphoantigen according to the invention or a conjugate bearing a phosphoantigen.

[0202] In this first step, the phosphoantigen or conjugate according to the invention will be internalized into the target (tumor) cells. It is assumed that after internalization (and cleavage of the linker in the case of a conjugate), the phosphoantigen will bind to the intracellular domain of the BTN3A1 receptor, which will result in the activation of the BTN3A1 / BTN2A1 dimer.

[0203] In a second step, the pre-treated, washed tumor cells from the first step can be co-cultured with γδ T cells. When Vγ9Vδ2 T cells are activated, they produce cytokines and release cytotoxic granules (degranulation), which lead to immune activation and target cell killing, respectively.

[0204] To assess the activity of phosphoantigens on γδ T cells, monensin and / or brefeldin A are added during the co-culture of γδ T cells and targets. This will trap the cytokines (such as interferon γ (IFNγ) and tumor necrosis factor α (TNFα)) produced in the activated cells. Staining with fluorescently labeled antibodies in the presence of saponin, which allows the anti-cytokine antibodies to enter the cells, will identify the cytokine-producing cells. Fluorescently labeled antibodies against CD107a can also be added during the co-culture, and the cells undergoing degranulation will be stained. Degranulation is associated with tumor cell killing (Aktas et al., 2009, Cell Immunol., 254(2), 149 - 154).

[0205] Thus, by combining fluorescently labeled immune cell-specific markers with the CD107a marker and cytokine markers, the activation status of γδ T cells and / or other immune cell subsets after co-culture with pre-treated target cells can be determined.

[0206] The ability of γδ T cells to kill pre-treated tumor cells can be detected by determining the proportion of dead tumor cells after co-culture. Tumor cells can be easily identified with a fluorescent tag, and their cell death can be determined as early as 1 hour after co-culture with γδ T cells.

[0207] Phosphoantigen analogs

[0208] (Chemical) analogs are compounds that differ in their structural characteristics from natural phosphoantigens but are similar in their functional biological activity to natural phosphoantigens. (That is, they exhibit (indirect) immunostimulatory activity, particularly on γδ T cells). Analogs can be designed to improve one or more characteristics of the naturally occurring pAg, such as improved characteristics regarding stability, efficacy, bioavailability, or linkage to the linker moiety, in the case of their use in immunoconjugates and linker-drug compounds according to the present invention.

[0209] Phosphoantigen prodrugs

[0210] A prodrug refers to an inactive precursor of a phosphoantigen moiety that is converted into an active phosphoantigen upon removal or transformation of a protecting group (e.g., a neutral protecting group on the negatively charged non-bonded oxygen atom of a phosphonate / ester group). After administration of a conjugate comprising a prodrug form of a phosphoantigen moiety according to the present invention to the body, the protecting group can be metabolically removed at the target site. A prodrug can also be formed by the binding of a linking moiety to the phosphoantigen moiety. In this case, an active phosphoantigen can be formed because the linker used to bind the phosphoantigen prodrug moiety to the targeting moiety in the conjugate is cleaved, resulting in the release of the active phosphoantigen, and / or because the protecting group is removed from the phosphoantigen moiety. Preferably, such a transformation that releases the active phosphoantigen occurs only after the conjugate according to the present invention reaches the site where it must exert its therapeutic effect (e.g., after it has been internalized by tumor cells) or at least in the tumor microenvironment to prevent unwanted and non-specific side effects of the phosphoantigen moiety in healthy and / or non-target tissues.

[0211] In the conjugate according to the present invention, the phosphoantigen is conjugated to a targeting moiety (e.g., a tumor-specific antibody). In the conjugate according to the present invention, the binding specificity of the targeting moiety ensures that the phosphoantigen moiety is delivered to the site where it must exert its therapeutic effect.

[0212] Prodrug forms include protecting groups known in the art, such as aryl esters, aryl amides, or pivaloyloxymethyl (POM) prodrug forms. C-HMBP (monophosphonate / ester) phosphoantigen analogues / prodrugs are described in WO2019 / 182904. To synthesize phosphoantigen prodrugs as effective as natural phosphoantigens such as HMBPP, aryloxy triester phosphoamidites of (monophosphonate / ester) phosphoantigens were synthesized as described in Davey et al., 2018, J. Med. Chem., 61, 2111-2117. In these prodrugs, the monophosphate ester group is masked by an aryl motif and an amino acid ester moiety. These compounds (“HMBP ProPagen”) still have relatively low serum stability due to the cleavage of the –P-O- bond between the phospho moiety and the isoprenoid moiety in the molecule. Similar “ProPagen” compounds in which carbon replaces the oxygen in the –P-O- bond are described in WO2020 / 008189. Wiemer et al., 2020, Chem. Med. Chem., 15, 1030–1039 describes the proposed structure activity relationship (SAR) for phosphoantigens (prodrugs).

[0213] The cleavable linking moiety can be conveniently conjugated to the phosphoantigen via the alcohol group of the allyl alcohol moiety. In this case, when the cleavable linking moiety is cleaved, allyl alcohol can be (re)-formed intracellularly.

[0214] The prodrug moieties in the phosphoantigen prodrugs that are part of the conjugates according to the invention can be the same or different. For example, all prodrug moieties can be POM groups, or the phosphoantigen moieties can comprise a combination of, for example, "proTide" groups such as aryloxy and amino acid ester groups, such as those described for phosphoantigen prodrugs in WO2020 / 008189 or WO2019 / 182904.

[0215] Suitable phosphonate / ester prodrug technologies and the synthesis of phosphonate / ester prodrugs are known in the art. Such prodrug technologies are further reviewed, for example, in Pradere et al., 2014, Chem. Rev., 114, 9154-9218, and include the use of carbonyloxymethyl prodrug moieties such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) derivatives, prodrugs based on S-acyl-2-thioethyl (SATE) and S-[(2-hydroxyethyl)thio]-2-thioethyl (DTE), prodrugs based on cycloSal phosphates / esters and phosphonate / esters, and prodrugs based on alkoxyalkyl monoesters (hexadecyloxypropyl-(HDP), octadecyloxyethyl-(ODE)), prodrugs based on phosphoramidites and phosphonamidites (including aryloxy amino acid amides (ProTide) prodrugs), and phosphoric diamide salts / esters and phosphonic diamide salts / esters.

[0216] Synthesis of the conjugates according to the invention

[0217] To synthesize the conjugates according to the invention, one or more linker-drug compounds according to the invention can be conjugated to a suitable target moiety. When the target moiety is a polypeptide (antibody or its binding fragment), the linker-drug compound can be conjugated via reactive native amino acid residues such as lysine or cysteine present in the suitable polypeptide, or via N-terminal or C-terminal conjugation. Alternatively, native or non-native reactive amino acid residues can be genetically engineered into the suitable polypeptide, or reactive groups can be introduced by post-translational modification.

[0218] The conjugates according to the invention can be produced by conjugating a linker-drug compound according to the invention with an antibody or an antigen-binding fragment thereof, for example via the lysine ε-amino group of the antibody, preferably using an intermediate comprising an amine-reactive group such as an activated ester. Such methods are known for producing conventional antibody-drug conjugates (ADCs).

[0219] Alternatively, the immunoconjugates can be produced as follows: Using methods and conditions known in the art, see for example Doronina et al, 2006, Bioconjugate Chem., 17, 114-124, the linker is conjugated via the free thiol of the cysteine side chain generated by the reduction of the interchain disulfide bond. The preparation method involves partial reduction of the interchain disulfide bond exposed to the solvent, followed by modification of the resulting thiol with a linker comprising a Michael acceptor such as a maleimide-containing linker, α-haloacetamide or ester. The cysteine ligation strategy results in a maximum of two linker-containing linker-drugs for each reduced disulfide bond.

[0220] The preferred antibodies used as targeting moieties in the conjugates according to the invention are of human IgG type. Most human IgG molecules have four disulfide bonds exposed to the solvent, which corresponds to an integer range of 0 to 8 linked linker moieties per antibody. The exact number of linked phosphoantigen moieties of each targeting moiety is determined by the number of phosphoantigen moieties of each linker moiety, the degree of disulfide bond reduction, and the molar equivalents of the linker-containing linker-drug in the subsequent conjugation reaction. Complete reduction of all four disulfide bonds yields a homogeneous construct of eight linker moieties per antibody, while partial reduction typically results in a heterogeneous mixture of zero, two, four, six, or eight linker moieties per antibody.

[0221] In a preferred embodiment, the invention relates to a conjugate wherein the linker-drug compound according to the invention is conjugated to an antibody or an antigen-binding fragment thereof via a cysteine residue of the antibody or antigen-binding fragment.

[0222] Site-specific conjugation to an antibody or an antigen-binding fragment thereof

[0223] Since antibodies contain many lysine residues and cysteine disulfide bonds, conventional conjugation usually results in a heterogeneous mixture, which poses challenges for analytical characterization and preparation. In addition, the individual components of these mixtures exhibit different physicochemical properties and pharmacologies for their pharmacokinetic, potency, and safety profiles, hindering a rational approach to optimizing this mode.

[0224] To improve conjugate homogeneity, the antibodies in the (immuno)conjugates according to the invention can be modified to allow site-specific conjugation of the linker. C.R. Behrens and B. Liu, 2014, mAbs, 6(1)1-8 comprehensively review methods for site-specific drug-antibody conjugation, and it can be found in WO2015 / 177360, WO2005 / 084390 and WO2006 / 034488.

[0225] A site-specific immunoconjugate is preferably generated by conjugating a linker-drug compound to an antibody or an antigen-binding fragment thereof through the side chain of a modified cysteine residue at a suitable position of a mutant antibody or an antigen-binding fragment thereof. The modified cysteine is typically capped with another thiol such as cysteine or glutathione to form a disulfide bond. These capped residues need to be uncapped before the linker-drug linkage can occur. The linker-drug can be linked to the modified residue by any of the following: (1) by reducing both the native interchain disulfide bond and the mutant disulfide bond, then re-oxidizing the native interchain cysteine using a mild oxidant (such as CuSO4 or dehydroascorbic acid), and subsequently performing standard conjugation of the uncapped modified cysteine with the linker-drug; or (2) by using a mild reducing agent that reduces the mutant disulfide bond at a higher rate than the interchain disulfide bond, and subsequently performing standard conjugation of the uncapped modified cysteine with the linker-drug. Suitable methods for site-specifically conjugating the linker-drug can be found in, for example, WO 2015 / 177360, which describes the reduction and re-oxidation process; WO 2017 / 137628, which describes the method using a mild reducing agent; and WO 2018 / 215427, which describes the method for conjugating both the reduced interchain cysteine and the uncapped modified cysteine.

[0226] Drug Compositions

[0227] The conjugates according to the invention are intended to be used as drugs, particularly for the treatment of cancer, autoimmune diseases or infections.

[0228] In another aspect, the invention provides a composition comprising a conjugate according to the invention, preferably wherein the composition is a pharmaceutical composition, more preferably the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Such a composition is hereinafter referred to as a composition according to the invention. The composition can be, for example, a liquid preparation, a lyophilized preparation or in the form of, for example, capsules or tablets.

[0229] Generally speaking, a pharmaceutical composition comprising an immunoconjugate according to the present invention takes the form of a lyophilized cake (lyophilized powder), which needs to be (aqueously) dissolved (i.e., reconstituted) before intravenous infusion; or takes the form of a frozen (aqueous) solution, which needs to be thawed before use. Thus, in some preferred embodiments, the present invention provides a lyophilized composition comprising an immunoconjugate according to the present invention, preferably wherein the composition is a pharmaceutical composition, more preferably the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. In some other preferred embodiments, the present invention provides a frozen composition comprising water and an immunoconjugate according to the present invention, preferably wherein the composition is a pharmaceutical composition, more preferably the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. In this case, the frozen solution is preferably at atmospheric pressure, and the frozen solution is preferably obtained by freezing a liquid composition according to the present invention at a temperature below 0 °C. Suitable pharmaceutically acceptable excipients for inclusion in the pharmaceutical composition according to the present invention (before lyophilization) include buffer solutions (e.g., citrate in water, amino acids such as histidine, or salts containing succinate), lyoprotectants (e.g., sucrose, trehalose), tonicity regulators (e.g., chloride salts such as sodium chloride), surfactants (e.g., polysorbate), and fillers (e.g., mannitol, glycine). The excipients for the protein formulation selected for lyophilization are because they can prevent protein denaturation during the lyophilization process and during storage.

[0230] Medical use

[0231] In another aspect, the present invention provides a conjugate according to the present invention or a composition according to the present invention for use as a medicament, preferably for the treatment of cancer, autoimmune diseases or infectious diseases. The conjugate according to the present invention can be used to induce the cytotoxic effect of γδ T cells on, for example, tumor cells and / or infected cells.

[0232] The conjugates and compositions are hereinafter collectively referred to as the products used according to the present invention.

[0233] In one embodiment, the product used according to the present invention is used for the treatment of solid tumors or malignant blood diseases. In a second embodiment, the product used according to the present invention is used for the treatment of autoimmune diseases. In a third embodiment, the product used according to the present invention is used for the treatment of infectious diseases, such as bacterial infections, viral infections, fungal infections, parasitic infections or other infections.

[0234] In the context of the present invention, the cancer is preferably a tumor that expresses the antigen targeted by the product used according to the present invention. Such a tumor can be a solid tumor or a malignant hematological disease. Some examples of tumors or malignant hematological diseases that can be treated with the product used according to the present invention as defined above may include, but are not limited to: breast cancer; brain cancer (e.g., glioblastoma); head and neck cancer; thyroid cancer; parotid gland cancer; adrenal gland 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 carcinoma (e.g., bladder cancer, penile cancer, ureteral cancer or kidney cancer); ovarian cancer; uterine cancer; vaginal cancer, vulvar cancer and cervical cancer; lung cancer (especially non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC)); melanoma; mesothelioma (especially malignant pleural and peritoneal mesothelioma); liver 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 lymphatic leukemia (CLL); acute lymphoblastic leukemia (ALL); myelodysplastic syndrome (MDS); blastic plasmacytoid dendritic cell neoplasia (BPDCN); Hodgkin 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).

[0235] In the context of the present invention, the autoimmune disease is preferably an autoimmune disease associated with the antigen targeted by the product used according to the present invention. An autoimmune disease represents a disorder caused by an abnormal immune response to normal body cells and tissues. There are at least 80 types of a wide variety of autoimmune diseases. Some diseases are organ-specific and limited to affecting certain tissues, while others are 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 the inflammatory response and can fluctuate over time. Some examples of autoimmune diseases that can be treated with the product used according to the present invention as defined above may include but are not limited to: rheumatoid arthritis; juvenile dermatomyositis; psoriasis; psoriatic arthritis; lupus; sarcoidosis; Crohn’s disease; eczema; nephritis; uveitis; polymyositis; neuritis, including Guillain-Barre syndrome; encephalitis; arachnoiditis; systemic sclerosis; autoimmune-mediated musculoskeletal and connective tissue diseases; neuromuscular degenerative diseases, including Alzheimer’s disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), neuromyelitis optica, and large, middle size, small vessel Kawasaki and Henoch Schonlein vasculitis; cold agglutinin disease; autoimmune hemolytic anemia (AIHA); immune thrombocytopenic purpura (ITP); type I diabetes; Hashimoto’s thyroiditis; Graves’ disease; Graves’ ophthalmopathy; adrenitis; hypophysitis; pemphigus vulgaris; Addison’s disease; ankylosing spondylitis; Behcet’s syndrome; celiac disease; Goodpasture’s syndrome; myasthenia gravis; sarcoidosis; scleroderma; primary sclerosing cholangitis, acquired epidermolysis bullosa, and bullous pemphigoid.

[0236] Infectious diseases in the context of the present invention are preferably infectious diseases associated with the antigens targeted by the products used according to the present invention. Such infectious diseases can be bacterial infections, viral infections, fungal infections, parasitic infections or other infections. Some examples of infectious diseases treatable with the products used according to the present invention as defined above may include but are not limited to: malaria; toxoplasmosis; pneumocystis jirovecii melioidosis; shigellosis; listeria; diseases caused by Cyclospora or mycobacterium leprae; tuberculosis; and prevention of infections in immunocompromised individuals (such as HIV-positive individuals, individuals receiving immunosuppressive therapy or individuals with congenital defects such as cystic fibrosis or benign proliferative diseases (such as molahydatidosa or endometriosis)).

[0237] The products used according to the present invention as described herein can be used to prepare the medicaments as described herein. The products used according to the present invention as described herein are preferably used in a treatment method, wherein the product used is administered to a subject, preferably to a subject in need thereof, in a therapeutically effective amount. Thus, alternatively, or in combination with any additional embodiments, in one embodiment, the present invention relates to the use of the products used according to the present invention in the preparation of a medicament for the treatment of cancer, autoimmune diseases or infectious diseases, particularly for the treatment of cancer. For illustrative, non-limiting cancers or other diseases to be treated according to the present invention: see above.

[0238] Alternatively, or in combination with any additional embodiments, in one embodiment, the present invention relates to a method for the treatment of cancer, autoimmune diseases or infectious diseases, particularly cancer, which method comprises administering to a subject in need of said treatment a therapeutically effective amount of the product used according to the present invention. For illustrative, non-limiting cancers or other diseases to be treated according to the present invention: see above.

[0239] The products used according to the present invention are for administration to a subject. The products used according to the present invention can be used in the above-described treatment method by administering an effective amount of the composition to a subject in need thereof. The term "subject" as used herein refers to all animals classified as mammals and includes but is not limited to primates and humans. The subject is preferably a human. The expression "therapeutically effective amount" means an amount sufficient to elicit a desired response or to improve symptoms or signs. The therapeutically effective amount for a particular subject can vary depending on a variety of factors such as the disorder being treated, the overall health of the subject, the method, route and dose of administration, and the severity of side effects.

[0240] Combined use

[0241] In other embodiments, the invention provides a product for use according to the invention, wherein the use is in combination with one or more additional therapeutic agents. The product for use according to the invention can be used simultaneously or sequentially with one or more additional therapeutic agents.

[0242] Suitable chemotherapeutic agents include alkylating agents such as nitrogen mustards, hydroxyurea, nitrosoureas, tetrazines (e.g., temozolomide) and aziridines (e.g., mitomycin); drugs that interfere with 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; antimicrotubule agents such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; cytotoxic antibiotics such as anthracyclines and bleomycin; hypomethylating agents such as decitabine and azacitidine; histone deacetylase inhibitors; all-trans retinoic acid; and arsenic trioxide. Suitable radiotherapeutic agents include radioisotopes such as 131 I-metaiodobenzylguanidine (MIBG), 32 P as sodium phosphate, 223 Ra chloride, 89 Sr chloride and 153 Sm ethylene diamine tetra methylene phosphonate / ester (EDTMP). Suitable agents to be used as hormonal therapeutic agents include hormone synthesis inhibitors 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.

[0243] Targeted therapeutic agents are therapeutic agents that interfere with specific proteins involved in tumorigenesis and proliferation, and they can be small molecule drugs; proteins, such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids, such as ADCs. Some 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. Some examples of peptide or peptide derivative targeted therapeutic agents include proteasome inhibitors such as bortezomib and carfilzomib.

[0244] Suitable anti-inflammatory drugs include D-penicillamine, azathioprine, and 6-mercaptopurine, cyclosporine, anti-TNF biologics (such as infliximab, etanercept, adalimumab, golimumab, certolizumab, or certolizumab pegol), leflunomide, abatacept, tocilizumab, anakinra, ustekinumab, rituximab, daratumumab, ofatumumab, obinutuzumab, secukinumab, apremilast, acetretin, and JAK inhibitors (such as tofacitinib, baricitinib, or upadacitinib).

[0245] Immunotherapeutic agents include substances that induce, enhance, or inhibit an immune response, such as cytokines (IL-2 and IFN-α); immunomodulatory imide drugs, such as thalidomide, lenalidomide, pomalidomide, or imiquimod; therapeutic cancer vaccines, such as talimogenelaherparepvec; cell-based immunotherapeutic agents, 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 cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC) through their Fc region when bound to a membrane-bound ligand on a cell.

[0246] In the context of the present invention, treatment preferably is prevention, reversal, cure, amelioration, and / or delay of cancer, autoimmune disease, or infectious disease. This can mean that the severity of at least one symptom of cancer, autoimmune disease, or infectious disease has been reduced, and / or at least one parameter associated with cancer, autoimmune disease, or infectious disease has been improved.

[0247] In the context of the present invention, the subject may be alive and / or may be considered disease-free. Alternatively, the disease or disorder may have stopped or been delayed. In the context of the present invention, an improvement in quality of life and an observed pain relief can mean that the subject may require less pain relief medication than at the start of treatment. In this case, "less" can mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less. The subject may no longer require any pain relief medication. Such an improvement in quality of life and observed pain relief can be observed, detected, or evaluated in the subject after treatment for at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer, compared to the quality of life and observed pain relief of the subject at the start of treatment.

[0248] General Definitions

[0249] The conjugates and linker-drug compounds according to the present invention may contain one or more chiral centers and / or double bonds, and may thus exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), regioisomers, enantiomers or diastereomers. Accordingly, the chemical structures described herein encompass all possible enantiomers and stereoisomers of the shown or identified compounds, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to those skilled in the art. Compounds may also exist in several tautomeric forms, including enol forms, keto forms and mixtures thereof. Accordingly, the chemical structures described herein encompass all possible tautomeric forms of the shown or identified compounds. It should also be understood that some isomeric forms, such as diastereomers, enantiomers and geometric isomers, can be separated by physical and / or chemical means by those skilled in the art. When those skilled in the art understand that a structural formula or chemical name has a chiral center but does not indicate chirality, all three of the racemic mixture, the pure R enantiomer and the pure S enantiomer are separately mentioned for each chiral center. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to that specific enantiomer. When it is said that two moieties form a bond together, this means that these moieties do not exist as atoms and satisfy the valences by replacement electron bonds. All of these are known in the art.

[0250] The compounds disclosed in this specification and the claims may also exist as exo and endo regioisomers. Unless otherwise indicated, the description of any compound in the specification and claims is meant to include both the individual exo and individual endo regioisomers of the compound, as well as mixtures thereof. In addition, the compounds disclosed in this specification and the claims may exist as cis and trans isomers. Unless otherwise indicated, the description of any compound in the specification and claims is meant to include both the individual cis and individual trans isomers of the compound, as well as mixtures thereof. For example, when the structure of a compound is described as a cis isomer, it should be understood that the corresponding trans isomer or a mixture of cis and trans isomers is not excluded from the invention of the present application.

[0251] In this document and its claims, the verb "comprise" and its variations are used in its non-limiting sense, meaning including the items after the word, but not excluding items not specifically mentioned. Additionally, unless the context clearly requires the presence of one and only one element, the mention of an element by an indefinite article does not exclude the possibility of there being more than one element. Thus, a noun not qualified by a quantifier generally means "at least one".

[0252] When used with a numerical value (e.g., about 10), the term "about" or "approximately" preferably means that the value can be the given value plus or minus 1% of that value.

[0253] Whenever a parameter of a substance is discussed in the context of the present invention, it is assumed that the parameter is determined, measured, or shown under physiological conditions, unless otherwise stated. Physiological conditions are known to those skilled in the art and include an aqueous solvent system, atmospheric pressure, a pH value of 6 to 8, a temperature from room temperature (RT) to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components. It should be understood that charge is generally related to equilibrium. A moiety that is said to carry or bear a charge is a moiety that exists in a state in which it carries or bears such a charge more often than it does not carry or bear such a charge. Thus, as understood by those skilled in the art, a charged atom shown in the present disclosure may be uncharged under certain conditions, and a neutral moiety may be charged under certain conditions.

[0254] All patents and literature references cited in this specification are hereby incorporated by reference in their entirety.

[0255] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0256] Examples

[0257] General procedures

[0258] Solvent:

[0259] All solvents used were reagent grade or HPLC grade from different suppliers.

[0260] NMR spectrum:

[0261] NMR spectra were recorded on a Bruker AVANCE400 (400 MHz for 1 H; 101 MHz for 13 C).

[0262] Chemical shift:

[0263] Chemical shifts were reported in ppm relative to tetramethylsilane as an internal standard or the residual undeuterated solvent.

[0264] UPLC characterization of the product:

[0265] The product was characterized on a Waters UPLC-MS equipped with a SQD 2 detector, using a Waters ACQUITY UPLC BEH C18 column (particle size 1.7 μm, 2.1×50 mm) at a flow rate of 0.4 mL / min. (MeCN / H2O×0.1% formic acid).

[0266] HPLC purification:

[0267] Purification was carried out by preparative HPLC using a Shimadzu Prominence 20AP system equipped with a Waters SunFire Prep C18 OBD 5μm column (19×150 mm) at a flow rate of 17 ml / min.

[0268] General operation XXD: Pyrophosphate formation

[0269] At RT, the triethylamine salt of the phosphate ester XD36 (1.0 equiv) was dissolved in DMF (0.15 M) under N2 and CDI (2.1 equiv) was added. After stirring for 30 min, anhydrous MeOH (1.0 equiv) was added and the mixture was stirred at RT for 15 min and then concentrated. The residue was co-evaporated with DMF to give crude A.

[0270] In a separate flask, under N2, the mono-triethylamine salt of the phosphate ester (1.2 equiv) was co-evaporated with DMF and then redissolved in DMF (0.36 M). The mixture was then cannulated into the flask containing crude A at RT. The flask was rinsed with the same volume of DMF to complete the transfer. The mixture was stirred at RT under N2 and once UPLC-MS analysis showed substantially complete conversion (usually 20 to 24 h), the reactants were concentrated and purified by preparative HPLC as indicated. The product fractions were lyophilized to give the product.

[0271] General operation XXE: Click-reaction

[0272] Under RT, 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). 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 solution of sodium ascorbate (1.5 equiv) in nitrogen-purged water (0.13 M) was added. The reactants were stirred at RT until UPLC-MS analysis indicated complete conversion (usually 1 to 2 h). Most of the THF was removed by brief rotary evaporation, and the aqueous phase was taken up in MeCN / 25 mM NH4HCO3 (1:9) in MilliQ. The insoluble material was filtered off using a syringe filter and the filtrate was purified by preparative HPLC as indicated. The product fractions were lyophilized to give the product.

[0273] General procedure XXF: Synthesis of alkyl phosphates from XD34 and allylic alcohols

[0274] To a RT solution of 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 dropwise XD34 (1.0 equiv) in DCM (0.4 M). The reaction mixture was stirred for 1 to 3 h and then quenched with an excess of MeOH. The reaction mixture was concentrated and partitioned between 1 M HCl (aqueous) and EtOAc / heptane (1:2). The organic layer was separated and the aqueous layer was extracted with EtOAc / heptane (1:2). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude was purified by flash chromatography as indicated.

[0275] General procedure XXG: TBDPS-deprotection with HF·pyridine

[0276] Under an N2 atmosphere, the TBDPS-ether (1.0 equiv) was dissolved in THF / pyridine (1:1, 0.2 M) in a PFA tube. 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 to 90 min and then carefully added to a saturated NaHCO3 aqueous solution / EtOAc (1:1) mixture with stirring at 0 °C. Once the effervescence stopped, the layers were separated and the aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with 1 M HCl (aqueous) and brine, dried over Na2SO4 and concentrated. The crude was purified as indicated.

[0277] General procedure XXH: Fluorenylmethyl-deprotection with triethylamine

[0278] Under RT, diphenylphosphoryl methyl ester (1 eq) was dissolved in MeCN / THF (2:1, 0.13 M) and TEA (10 eq) was added. The reaction mixture was stirred for 16 to 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 Et2O (10 mL) was slowly added with stirring. The mixture was stirred for 15 min and after settling, the supernatant was discarded. This process was repeated twice. The residue was co-evaporated with MeCN to afford the alkyl phosphate as the triethylammonium salt.

[0279] General Procedure XXI: Stille Coupling with XS60

[0280] CuI (0.3 eq), Pd(PPh3)4 (0.15 eq) and tributyl(alkyl)stannane (1.2 eq) were purged with N2 (3×). A solution of XS60 (1.0 eq) in toluene (0.11 M) was added and the reaction mixture was stirred at 80 °C for 3 to 20 h. The mixture was filtered, concentrated, and the crude product was purified by flash chromatography as indicated.

[0281] General Procedure XXJ: Suzuki Coupling with XS60

[0282] XS60 (1.0 eq) and Pd(PPh3)4 (0.1 eq) were purged with N2 (3×) and dissolved in THF (0.05 M). A suspension of the zinc reagent (2.0 to 4.0 eq) was added and the reaction mixture was stirred at RT for 3 h. Aqueous saturated NH4Cl solution was added to the reaction mixture and the product was extracted with Et2O. The combined organic layers 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 indicated.

[0283] General Procedure XXK: THP-Ether Deprotection

[0284] The THP-ether (1.0 eq) was dissolved in MeOH (0.1 M) and PPTS (0.1 eq) was added. The reaction mixture was stirred at 45 to 55 °C for 3 to 9 h and then added to aqueous saturated NaHCO3 solution. The product was extracted with DCM (3×) and washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography as indicated.

[0285] General Procedure XXL: Synthesis of Alkyl Phosphates from XD34 and Allyl Alcohol

[0286] To a RT solution of 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 dropwise XD34 (1.5 equiv) in DCM (0.4 M). The reaction mixture was stirred for 1 to 3 h and then quenched with an excess of MeOH. The reaction mixture was concentrated and partitioned between 1 M HCl (aqueous) and EtOAc / heptane (1:2). The organic layer was separated and the aqueous layer was extracted with EtOAc / heptane (1:2). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography as indicated.

[0287] Example 1: Synthesis of linker drug XD78

[0288] 1A: Preparation of chloride XD68

[0289]

[0290] (Z)-4-((tert-Butyldiphenylsilyl)oxy)-3-chlorobut-2-en-1-ol (XD67)

[0291] Prepared according to the procedure of Nitelet, A. et al. Org. Lett. 2016, 18, 1904. A microwave 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, L. E. 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 silica plug. The filtrate was concentrated and the crude was purified by flash chromatography (silica, 0% to 25% ether in heptane) to afford the alcohol XD67 (1.02 g, 78%) as a colorless oil.

[0292] 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).

[0293] For C 20 H 29 ClNO2Si + [M+NH4] + , MS(ESI + ) calculated value: 378.2, measured value: 378.3.

[0294] (Z)-3-Bromo-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-ol (XS31)

[0295] React iodide XD66 (2.00 g, 4.42 mmol, prepared according to Overman, L. E. Tetrahedron, 2010, 66, 6514) with CuI (253 mg, 1.33 mmol), Me4NBr (2.72 g, 17.7 mmol) and (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine (377 mg, 2.65 mmol) in ethanol (8.9 mL) in an operation similar to that for XD67. Purify the crude product twice by flash chromatography (silica gel, 0% to 20% EtOAc in heptane; silica gel, 0% to 30% Et2O in heptane) to obtain the alcohol XS31 (1.79 g, 51%) as a colorless oil.

[0296] 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).

[0297] For C 20 H 24 BrOSi + [M+H-H2O] + , MS(ESI + ) calculated value: 387.1, measured value: 387.2.

[0298] (Z)-tert-Butyl ((2,4-dichlorobut-2-en-1-yl)oxy)diphenylsilane (XD68)

[0299] N-Chlorosuccinimide (0.621 g, 4.65 mmol) was dissolved in anhydrous 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 minutes. After cooling to -65 °C, alcohol XD67 (1.29 g, 3.58 mmol) in anhydrous DCM (3 mL) was added. The reaction mixture was warmed to 0 °C over 2.5 hours and then stirred at 0 °C for 90 minutes. Brine (30 mL) was added at 0 °C and the layers were separated. The aqueous layer was extracted with DCM (40 mL) and the combined organic layers were dried over Na2SO4. After filtration and concentration, the crude oil was purified by flash chromatography (silica gel, 0% to 20% DCM in heptane) to give the chloride XD68 (1.31 g, 96%) as a colorless oil.

[0300] 1 1H 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).

[0301] Synthesis of linker-drug XD73

[0302]

[0303] (Z)-(5-((tert-Butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl)dimethyl phosphonate (XD69)

[0304] Under N2, n-BuLi (2.5 M in hexanes, 3.02 mL, 7.54 mmol) was added to a solution of dimethyl methylphosphonate (0.817 mL, 7.54 mmol) in THF (28 mL) at -78 °C. The reaction mixture 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 afford a clear solution. At -40 °C, chloride XD68 (1.30 g, 3.43 mmol) in THF (7 mL) was added. The reaction mixture was warmed to RT, stirred overnight, and then quenched with saturated aqueous NH4Cl at 0 °C. The aqueous layer was extracted with EtOAc (2×) and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) gave the phosphonate XD69 (1.09 g, 68%) as a colorless oil.

[0305] 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).

[0306] For C 23 H 32 ClNaO4PSi + [M+Na] + ,MS (ESI + ) Calcd: 489.1, Found: 489.4.

[0307] Bis(2-cyanoethyl) (Z)-(5-((tert-butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl) phosphonate (XD70)

[0308] Step 1: TMSBr (3.03 mL, 23.0 mmol) was added dropwise over 10 min to a cooled (0 °C) solution of phosphonate XD69 (1.07 g, 2.30 mmol) in DCM (11 mL). After 30 min, the ice bath was removed and the reaction mixture was stirred at RT for 3 h 30 min. The mixture was concentrated and the residue was redissolved in DCM (11 mL) under N2 and cooled to 0 °C.

[0309] Step 2: Add DMF (2 drops), then add oxalyl chloride (0.591 mL, 6.89 mmol). After stirring for 30 minutes, remove the ice bath and stir the mixture at RT for 16 hours. Concentrate the reaction mixture and co-evaporate with DCM (2 × 10 mL). Dry the crude oil under high vacuum at RT for 1 hour.

[0310] Step 3: Under N2, dissolve the crude oil in DCM (16 mL) and cool the mixture to 0 °C. Add 5-(ethylthio)-1H-tetrazole (0.149 g, 1.15 mmol) to the mixture, then add pyridine (0.742 mL, 9.18 mmol) and 3-hydroxypropionitrile (0.627 mL, 9.18 mmol). After stirring at 0 °C for 2 hours and at RT for 3 hours, transfer the reaction mixture to a separatory funnel and dilute with DCM (20 mL). Wash the organic phase with aqueous HCl solution (1 M, 20 mL), and back-extract the aqueous phase with DCM (30 mL). Wash the combined organic phases with brine, dry over Na2SO4, filter and concentrate. Purify by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to give phosphonate XD70 (0.811 g, 65%).

[0311] 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).

[0312] For C 27 H 35 ClN2O4PSi + [M+H] + , MS (ESI + ) Calcd: 545.2, Found: 545.4.

[0313] Triethylammonium 2-cyanoethyl (Z)-(5-((tert-butyldiphenylsilyl)oxy)-4-chloropent-3-en-1-yl)phosphonate (XD71)

[0314] Under RT, DBU (0.247 mL, 1.64 mmol) was added to a solution of phosphonate XD70 (811 mg, 1.49 mmol) in THF (13 mL). After 30 minutes, the reaction mixture was concentrated to about 1 mL, diluted with MeOH (6.8 mL), and eluted with methanol (30 mL) through a DOWEX 50WX8 plug. Triethylammonium (0.228 mL, 1.64 mmol) was added and the mixture was concentrated and co-evaporated with MeCN (2×) to give phosphonate XD71 (840 mg, 99%) as a colorless oil. NMR analysis indicated a phosphonate:amine salt ratio of 1:0.8.

[0315] 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).

[0316] For C 24 H 30 ClNO4PSi - [M - H] - , MS (ESI - ) calculated: 490.1, found: 490.4.

[0317] Phosphonate XD72

[0318] Step 1: The triethylammonium salt XD71 (830 mg, 1.45 mmol) and Fmoc - Val - Ala - PAB - OH (898 mg, 1.74) were co - evaporated with anhydrous DMF (3×8 mL). Under N2, DMF (7 mL) was added at RT, followed by PyBOP (906 mg, 1.74 mmol) and DIPEA (0.507 mL, 2.90 mmol). After 2 hours, with very gentle stirring (to inhibit gel formation), the reaction mixture was then slowly added dropwise to ice - cold water (70 mL). The white suspension was gently stirred for 5 minutes 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% to 6% MeOH in DCM) to give the intermediate phosphonate (1.10 g, 77%).

[0319] Step 2: To a nitrogen-flushed PFA vial containing the above-prepared intermediate phosphonate (1.10 g, 1.11 mmol) was added THF (6.2 mL) and pyridine (3.1 mL). At 0 °C, HF-pyridine (70% HF, 1.5 mL) was introduced via syringe under N2, and the mixture was stirred at 0 °C for 90 minutes. With gentle stirring, the reaction mixture was carefully transferred via cannula to cold (0 °C) saturated aqueous NaHCO3 (150 mL). After stirring for 15 minutes, 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 ON in vacuo and then purified by flash chromatography (silica gel, 0% to 8% MeOH in DCM) to afford the phosphonate XD72 as a white foam (516 mg, 62%).

[0320] 1 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).

[0321] For C 38 H 45 ClN4O8P + [M+H] + ,MS (ESI + ) Calcd: 751.3, Found: 751.8.

[0322] 1B: Linker-Drug XD73

[0323] Step 1: At 0 °C, an aqueous NaOH solution (2.0 M, 0.389 mL, 0.779 mmol) was added to a suspension of phosphonate XD72 (0.117 g, 0.156 mmol) in MeOH (2.3 mL) and water (0.26 mL). The cooling bath was removed and the mixture was stirred for 100 min. The reaction mixture was cooled to 0 °C and AcOH (2 M in MeOH, 0.623 mL, 1.25 mmol) was added. 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 filtrate aqueous solution was lyophilized to give the crude amine as a glassy solid. The material was carried forward to the next step without further purification.

[0324] Step 2: The crude amine was dissolved / suspended in DMF (1 mL). DIPEA (0.108 mL, 0.622 mmol) was added at RT, followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (0.096 g, 0.311 mmol). The reaction mixture was stirred at RT for 2 h, at which point acetic acid (0.053 mL, 0.933 mmol) in DMF (1 mL) was added and the mixture was concentrated. The residue was dissolved in 1:9 MeCN / NH4HCO3 aqueous solution (25 mM) and the solution was washed with EtOAc (4 × 5 mL) and diethyl ether (1 × 5 mL). The aqueous phase was then purified directly by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient 90:10 to 40:60). The product fractions were lyophilized to give the linker-drug XD73 as a white solid (60.5 mg, 57%).

[0325] 11H 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).

[0326] For C 30 H 41 ClN4O9P - [M - H] - , MS (ESI - ) Calculated: 667.2, Found: 667.7.

[0327] Example 2: Synthesis of Linker Drugs XS54 to XS58

[0328] 2A. Synthesis of Alcohols XS3 and XS38

[0329] Synthesis of Alcohol XS33

[0330]

[0331] (E)-4-((tert-Butyldiphenylsilyl)oxy)but-2-en-1-ol (XS33)

[0332] 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 Et2O (5.2 mL) was added dropwise to a 0 °C suspension of Red-Al (3.17 mL, 70% in toluene, 11.2 mmol) in Et2O (26 mL). The reaction mixture was stirred at 0 °C for 90 min and EtOAc (0.528 mL, 5.39 mmol) was added. Stirring was continued at 0 °C for 30 min, after which the reaction was quenched by addition of aqueous 1 M sodium potassium 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 layer was washed with brine (20 mL), dried over Na2SO4 and concentrated. Purification by flash chromatography (silica gel, 0% to 25% EtOAc in heptane) gave the allyl alcohol XS33 (1.39 g, 79%) as a colorless oil.

[0333] 1 H 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).

[0334] For C 20 H 25 OSi + [M + H - H2O] + ,MS (ESI + ) calcd: 309.2, found: 309.3.

[0335] Synthesis of alcohol XS38

[0336]

[0337] 2-(Trityloxy)acetaldehyde (XS34)

[0338] The silica-supported NaIO4 (22.3 g, 15.2 mmol, prepared as described in Zong and Shing, J. Org. Chem. 1997, 62, 2622 - 2624) was purged with N2 (3×) and suspended in DCM (50 mL), and 3-(trityloxy)propane-1,2-diol (3.74 g, 11.2 mmol, prepared as described in CN108478807) dissolved in DCM (25 mL) was added. The reaction mixture was stirred at RT for 2 h. The reaction mixture was filtered through a silica plug and washed with DCM (100 mL). The filtrate was concentrated and the crude product was purified by flash chromatography (silica, 0% to 25% EtOAc in heptane) to afford the aldehyde XS34 (2.88 g, 85%) as a colorless oil.

[0339] 1 1H NMR (400 MHz, DMSO-d6) ppm = 9.36 (s, 1H), 7.44 - 7.31 (m, 15H), 3.85 (s, 2H).

[0340] Ethyl 2-fluoro-4-(trityloxy)but-2-enoate (XS35)

[0341] A flask containing the aldehyde XS34 (2.77 g, 9.16 mmol) and MgSO4 (1.33 g, 11.1 mmol) was purged with N2 (3×) and MeCN (46 mL) was added. The mixture was cooled to 0 °C and ethyl 2-(diethoxyphosphoryl)-2-fluoroacetate (2.29 mL, 11.3 mmol) was added, followed by DBU (1.38 mL, 9.16 mmol). The reaction mixture was allowed to reach RT and stirred for 2 h. The reaction mixture was concentrated and the residue was dissolved in EtOAc (100 mL), washed with 1 M HCl (2×50 mL), 2 M NaOH (2×50 mL) and brine (50 mL), dried over Na2SO4 and concentrated. Purification by flash chromatography (silica, 0% to 15% EtOAc in heptane) gave the fluoroethylene XS35 (2.92 g, 82%) as a mixture of Z:E isomers (2:1 ratio). For C 25 H 23 FNaO3 + [M + Na] + , MS (ESI + ) calcd: 413.2, found: 413.4.

[0342] (Z)-2-Fluoro-4-(trityloxy)but-2-en-1-ol (XS36)

[0343] The ester XS35 (2.85 g, 7.30 mmol, 2:1 Z:E ratio) was purged with N2 (3×) 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 minutes, the reaction mixture was brought to RT and stirred for 2 hours. 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 (3×100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4 and concentrated. Purification by flash chromatography (silica gel, 0% to 35% EtOAc in heptane) gave the alcohol XS36 (1.54 g, 60%) as a colorless oil.

[0344] 1 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).

[0345] For C 23 H 21 FNaO2 + [M+Na] + ,MS (ESI + ) calcd: 371.1, found: 317.3. The (E)-2-fluoro-4-(trityloxy)but-2-en-1-ol (0.777 g, 31%) was also isolated.

[0346] 1 1H NMR (400 MHz, DMSO-d6) ppm = 7.41 - 7.32 (m, 12H), 7.31 - 7.24 (m, 3H), 531 (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).

[0347] For C 23 H 21 FNaO2 + [M+Na] + ,MS (ESI + ) calcd: 371.1, found: 317.3.

[0348] (Z)-tert-Butyl((2-fluoro-4-(trityloxy)but-2-en-1-yl)oxy)diphenylsilane (XS37)

[0349] Under N2, alcohol XS36 (1.53 g, 4.38 mmol) was dissolved in DCM (29 mL). 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 dropwise addition of TBDPS-Cl (1.69 mL, 6.57 mmol). After 15 minutes, the reaction mixture was brought to RT and stirred for 2 hours. The reaction mixture was added to water (30 mL), the organic layer was separated and the water was extracted with DCM (30 mL). The combined organic layers were washed with (20 mL), dried over Na2SO4 and concentrated. Purification by flash chromatography (silica gel, 0% to 5% EtOAc in heptane) gave the silyl ether XS37 (2.47 g, 96%) as a colorless oil.

[0350] 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).

[0351] (Z)-4-((tert-Butyldiphenylsilyl)oxy)-3-fluorobut-2-en-1-ol (XS38)

[0352] Under N2, the trityl ether XS37 (1.00 g, 1.70 mmol) was dissolved in DCM (8.5 mL) and MeOH (8.5 mL). The solution was cooled to 0 °C and TsOH·H2O (0.486 g, 2.56 mmol) was added. The reaction mixture was stirred at 0 °C for 1 hour. The reaction mixture was added to saturated aqueous NaHCO3 (50 mL) and the product was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4 and concentrated. Purification by flash chromatography (silica gel, 0% to 25% EtOAc in heptane) gave the alcohol XS38 (0.462 g, 79%) as a colorless oil.

[0353] 11H 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).

[0354] For C 20 H 29 FNO2Si + [M + H] + , MS (ESI + ) Calculated: 362.2, Found: 362.3.

[0355] 2B. Preparation of bis((9H-fluoren-9-yl)methyl) chlorophosphate (XD34)

[0356]

[0357] Bis((9H-fluoren-9-yl)methyl) phosphonate (XD50)

[0358] Under N2, at RT, (9H-Fluoren-9-yl)methanol (4.55 g, 23.2 mmol) was added to a solution of diphenyl phosphite (2.13 mL, 10.6 mmol) in anhydrous pyridine (20 mL), and the mixture was stirred for 2 h. The reaction mixture was concentrated and dissolved in EtOAc (250 mL). The organic phase was washed with aqueous HCl (2×, 1 M) and brine, dried over Na2SO4, filtered and concentrated on silica gel. Purification by flash chromatography (silica gel, 0% to 85% EtOAc / DCM (1:4) in heptane) gave the H-phosphonate XD50 as a colorless wax (3.46 g, 75%).

[0359] 1 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).

[0360] For C 28 H 24 O3P + [M + H] + ,MS (ESI +)Calculated value: 439.2, measured value: 439.3.

[0361] Bis((9H-fluoren-9-yl)methyl) chlorophosphate (XD34)

[0362] Dissolve H-phosphonate XD50 (8.57 g, 19.6 mmol) in toluene (98 mL) and purge the headspace with N2. At RT, add NCS (3.13 g, 23.5 mmol) and then stir the reaction mixture at 40 °C for 2 h. After cooling to RT, filter and concentrate the reaction mixture. Co-evaporate the residue with MeCN (10 mL) to give a white solid. Dissolve the solid in MeCN (25 mL) by using gentle heating with a heat gun to dissolve all the solid. Gradually cool the solution to -30 °C at which point the white solid starts to precipitate. Store the flask at -30 °C overnight and then warm it to RT and then filter. Wash the solid with ice-cold MeCN (10 mL) to give the chloride XD34 (8.03 g, 87% yield) as a white solid.

[0363] 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).

[0364] For C 28 H 24 ClO4P + [M+NH4] + , MS (ESI + ) Calculated value: 490.1, measured value: 490.3.

[0365] 2C. Synthesis of phosphonates XS44 to XS48

[0366]

[0367] (E)-Bis((9H-fluoren-9-yl)methyl)(4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)phosphonate (XS39)

[0368] Allyl alcohol XS33 was reacted with XD34 (0.500 g, 1.06 mmol) according to General Procedure XXF. The crude product was purified by flash chromatography (silica gel, 0% to 40% EtOAc in heptane) to afford the alkyl phosphate XS39 (0.458 g, 57%) as a white viscous solid.

[0369] 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).

[0370] For C 48 H 48 O5PSi + [M+H] + ,MS (ESI + ) Calcd for: 763.3, Found: 763.6.

[0371] (Z)-Bis((9H-fluoren-9-yl)methyl)(4-((tert-butyldiphenylsilyl)oxy)-3-fluorobut-2-en-1-yl) phosphate (XS40)

[0372] Allyl alcohol XS38 was reacted with XD34 (0.570 g, 1.21 mmol) according to General Procedure XXF. The crude product was purified by flash chromatography (silica gel, 0% to 40% EtOAc in heptane) to afford the alkyl phosphate XS40 (0.555 g, 59%) as a white viscous solid.

[0373] 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)

[0374] For C 48 H 47 FO5PSi + [M+H] + ,MS (ESI + ) Calcd for: 781.3, Found: 781.7.

[0375] (Z)-Bis((9H-fluoren-9-yl)methyl)(4-((tert-butyldiphenylsilyl)oxy)-3-chlorobut-2-en-1-yl)phosphate (XS41)

[0376] Allyl alcohol XD67 was reacted with XD34 (0.378 g, 0.799 mmol) according to General Procedure XXF. The crude product was purified by flash chromatography (silica gel, 0% to 40% EtOAc in heptane) to afford the alkyl phosphate XS41 (0.426 g, 67%) as a white viscous solid.

[0377] 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).

[0378] For C 48 H 47 ClO5PSi + [M+H] + ,MS (ESI + ) Calcd: 797.3, Found: 797.9.

[0379] (Z)-Bis((9H-fluoren-9-yl)methyl)(3-bromo-4-((tert-butyldiphenylsilyl)oxy)but-2-en-1-yl)phosphate (XS42)

[0380] Allyl alcohol XS31 was reacted with XD34 (0.500 g, 1.06 mmol) according to General Procedure XXF. The crude product was purified by flash chromatography (silica gel, 0% to 35% EtOAc in heptane) to afford the alkyl phosphate XS42 (0.890 g, 51%) as a colorless oil.

[0381] 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).

[0382] (Z)-Bis((9H-fluoren-9-yl)methyl)(4-((tert-butyldiphenylsilyl)oxy)-3-iodobut-2-en-1-yl) phosphate (XS43)

[0383] Allyl alcohol XD66 was reacted with XD34 (0.350 g, 0.740 mmol) according to General Procedure XXF. The crude product was purified by flash chromatography (silica gel, 0% to 35% EtOAc in heptane) to give the alkyl phosphate XS43 (0.430 g, 65%) as a white viscous solid.

[0384] 1 H 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).

[0385] For C 48 H 47 IO5PSi + [M+H] + , MS (ESI + ) Calcd: 889.2, Found: 889.7.

[0386] Triethylammonium (E)-4-hydroxybut-2-en-1-yl phosphate (XS44)

[0387] Step 1: TBDPS-ether XS39 (0.455 g, 0.596 mmol) was reacted according to General Procedure XXG. The crude product was purified by flash chromatography (silica gel, 0% to 75% EtOAc in heptane) to give the intermediate allyl alcohol (47 mg, 15%) as a white solid.

[0388] 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).

[0389] For C 32 H30 O5P + [M+H] + ,MS(ESI + ) Calculated value: 525.2, measured value: 525.4.

[0390] Step 2: React the intermediate (47 mg, 0.090 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS44 (14 mg, 61%) as the triethylamine salt. For C4H8O5P - [M-H] - ,MS(ESI - ) Calculated value: 167.0, measured value: 166.9.

[0391] Triethylammonium (Z)-3-fluoro-4-hydroxybut-2-en-1-yl phosphate (XS45)

[0392] Step 1: React TBDPS-ether XS40 (0.552 g, 0.707 mmol) according to General Procedure XXG. Purify the crude product by flash chromatography (silica gel, 0% to 60% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.355 g, 93%) as a colorless oil.

[0393] 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).

[0394] For C 32 H 29 FO5P + [M+H] + ,MS(ESI + ) Calculated value: 543.2, measured value: 543.2.

[0395] Step 2: React the intermediate (0.350 g, 0.645 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS45 (0.115 g, 69%) as the triethylamine salt in a phosphate:Et3N ratio of 1:0.7.

[0396] 11H 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).

[0397] For C4H7FO5P - [M-H] - , MS (ESI - ) Calculated: 185.0, Found: 185.0.

[0398] Triethylammonium (Z)-3-chloro-4-hydroxybut-2-en-1-yl hydrogen phosphate (XS46)

[0399] Step 1: TBDPS-ether XS41 (0.422 g, 0.529 mmol) was reacted according to General Procedure XXG. The crude product was suspended in DCM (10 mL), and Et2O (40 mL) and heptane (50 mL) were added thereto. After gentle heating, the suspension was filtered, and the residue was washed with Et2O (10 mL) to give the intermediate allyl alcohol (0.202 g, 68%) as a white solid.

[0400] 1 1H 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).

[0401] For C 32 H 29 ClO5P + [M+H] + , MS (ESI + ) Calculated: 559.1, Found: 559.4.

[0402] Step 2: The intermediate (0.198 g, 0.354 mmol) was reacted according to General Procedure XXH to give the alkyl phosphate XS46 (53 mg, 49%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.9.

[0403] 11H 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).

[0404] For C4H7ClO5P - [M-H] - , MS (ESI - ) Calcd: 201.0, Found: 200.7.

[0405] Triethylammonium (Z)-3-bromo-4-hydroxybut-2-en-1-yl phosphate (XS47)

[0406] Step 1: TBDPS-ether XS42 (0.445 g, 0.529 mmol) was reacted according to General Procedure XXG. The crude product was purified by flash chromatography (silica gel, 0% to 60% EtOAc in heptane) to afford the intermediate allyl alcohol (0.294 g, 92%) as a white solid.

[0407] 1 1H 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).

[0408] For C 32 H 29 BrO5P + [M+H] + , MS (ESI + ) Calcd: 603.1, Found: 603.4.

[0409] Step 2: The intermediate (0.290 g, 0.481 mmol) was reacted according to General Procedure XXH to afford the alkyl phosphate XS47 (0.130 g, 78%) as the triethylamine salt in a ratio of phosphate:Et3N of 1:0.95.

[0410] 11H 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). For C4H7BrO5P - [M - H] - , MS (ESI - ) Calculated: 244.9, Found: 245.1.

[0411] Triethylammonium (Z)-4-hydroxy-3-iodobut-2-en-1-yl hydrogen phosphate (XS48)

[0412] Step 1: TBDPS-ether XS43 (0.423 g, 0.476 mmol) was reacted according to General Procedure XXG. The crude product was purified by flash chromatography (silica gel, 0% to 60% EtOAc in heptane) to afford the intermediate allyl alcohol as a white solid (0.262 g, 85%).

[0413] 1 1H NMR (400 MHz, DMSO-d6) ppm = 7.87 (t, J = 6.8 Hz, 4H), 754 (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).

[0414] For C 32 H 29 IO5P + [M + H] + , MS (ESI + ) Calculated: 651.1, Found: 651.5.

[0415] Step 2: The intermediate (0.258 g, 0.397 mmol) was reacted according to General Procedure XXH to afford the alkyl phosphate XS48 as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.9 (76 mg, 49%).

[0416] 11H 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).

[0417] For C4H7IO5P - [M-H] - , MS (ESI - ) calculated: 292.9, found: 292.9.

[0418] Preparation of benzyl dihydrogen ((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadec-18-yl)phosphate (XD36)

[0419]

[0420] Bis((9H-fluoren-9-yl)methyl) (4-((14S,17S)-1-azido-14-isopropyl-17-methyl-12,15-dioxo-3,6,9-trioxa-13,16-diazaoctadec-18-yl)benzyl) phosphate (XD35)

[0421] Step 1: Dissolve 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid (142 mg, 0.574 mmol) in DMF (1 mL). At RT, add Val-Ala-PAB-OH (160 mg, 0.545 mmol) in DMF (3.0 mL), then add HATU (228 mg, 0.600 mmol) and DIPEA (0.143 mL, 0.818 mmol). Stir the reaction mixture for 30 minutes and then concentrate. Dissolve the crude product in MeOH (1 mL) and remove basic impurities by passing the solution through a short plug of DOWEX 50WX8 that has been pre-washed with methanol. Elute the product with methanol and concentrate the crude product on silica gel. Purify by flash chromatography (silica gel, 0% to 8% MeOH in DCM) to give the resulting amide as a creamy solid (262 mg, 92%). For C 24 H 39 N6O7 + [M+H] + , MS (ESI + ) calculated: 523.3, found: 523.6.

[0422] Step 2: Under RT, under N2, add 2,6-lutidine (719 μL, 6.17 mmol) to the amide product (977 mg, 1.87 mmol) and 5-(ethylthio)-1H-tetrazole (19 mg, 0.15 mmol) in MeCN (3.7 mL), then add a solution of chloride XD34 (884 mg, 1.87 mmol) in DCM (3.7 mL), and stir the mixture at RT. After 80 minutes (88 mg, 0.187 mmol) and 140 minutes (177 mg, 0.374 mmol), add more chloride XD34. After a total reaction time of 185 minutes, add more 2,6-lutidine (218 μL, 1.87 mmol), and continue the reaction for 2 hours, then quench it with methanol (1 mL). Concentrate the mixture, and dissolve the crude product in EtOAc (80 mL) and aqueous HCl solution (40 mL, 1 M). Add a small amount of MeCN (4 mL) to dissolve the remaining solid, and separate the layers. Extract the aqueous layer with EtOAc (80 mL), and wash the combined organic layers with brine and dry over Na2SO4. Purify the crude product by flash chromatography (silica gel, 0% to 5% MeOH in DCM) to give phosphate XD35 (1.40 g, 66% yield).

[0423] 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).

[0424] For C 52 H 59 N6O 10 PNa + [M+H] + ,MS (ESI +)Calculated value: 981.4, measured value: 981.8.

[0425] Benzyl dihydrogen phosphate ester (XD36) of 4 - ((14S,17S)-1 - azido - 14 - isopropyl - 17 - methyl - 12,15 - dioxo - 3,6,9 - trioxa - 13,16 - diazaoctadecane - 18 - amide group)

[0426] Triethylamine (0.25 mL) was added to a solution of phosphate ester XD35 (160 mg, 0.167 mmol) in MeCN (1 mL) at RT, and the reactants were stirred for 24 hours. The reactants were diluted with toluene (8 mL) and then concentrated. The crude product was suspended in diethyl ether (10 mL), filtered, and the solid was washed repeatedly with diethyl ether to obtain the alkyl phosphate ester XD36 (108 mg, 92%) as the mono - triethylammonium salt. (Note: The product contains an impurity (m / z 606), which may be formed by elimination of the phosphate ester and capture of the intermediate azaquinone methide by triethylamine. This impurity is non - reactive in the next step and does not require further purification). For C 24 H 38 N6O 10 P - [M - H] - , MS (ESI - )Calculated value: 601.2, measured value: 601.7.

[0427] Preparation of alkyne linker (XD43)

[0428]

[0429] 2 - (2 - (2,5 - Dioxo - 2,5 - dihydro - 1H - pyrrol - 1 - yl)ethoxy)ethyl prop - 2 - yn - 1 - yl carbamate (XD43)

[0430] At 0 °C, propargylamine (0.093 mL, 1.46 mmol) was added to PNP - carbonate XD53 (511 mg, 1.46 mmol, synthesized according to Elgersma, R.C. et al. Mol. Pharm. 2015, 12, 1813 - 1835) in THF (10 ml). The cooling bath was removed and the mixture was stirred at RT for 2 hours. The mixture was concentrated and the crude product was purified by flash chromatography (silica gel, 0% to 70% EtOAc in heptane) to obtain XD43 (265 mg, 68%) as a white solid.

[0431] 11H 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)

[0432] 2D. Synthesis of linker-drugs XS54 to XS58

[0433]

[0434] Pyrophosphate XS49

[0435] Alkyl phosphate XD36 (32 mg, 0.045 mmol) was reacted with phosphate XS44 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to give pyrophosphate XS49 (19.6 mg, 55%) as a white solid after lyophilization. For C 28 H 47 N6O 14 P2 + [M + H] + ,MS (ESI + ) Calcd: 753.3, Found: 753.8.

[0436] Pyrophosphate XS50

[0437] Alkyl phosphate XD36 (55 mg, 0.078 mmol) was reacted with phosphate XS45 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) to give pyrophosphate XS50 (28.8 mg, 46%) as a white solid after lyophilization.

[0438] 11H 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 = 73 Hz, 3H), 0.92 (d, J = 5.1 Hz, 3H), 0.90 (d, J = 51 Hz, 3H).

[0439] For C 28 H 46 FN6O 14 P2 + [M + H] + , MS (ESI + ) Calculated: 771.3, Found: 771.9.

[0440] Pyrophosphate XS51

[0441] Alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate XS46 according to General Procedure XXD. The crude product was purified by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) to give pyrophosphate XS51 (62.4 mg, 54%) as a white solid after lyophilization.

[0442] 1 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).

[0443] For C 28 H 46 ClN6O 14 P2 + [M+H] + , MS(ESI + ) Calculated value: 787.2, measured value: 787.6.

[0444] Pyrophosphate XS52

[0445] Alkyl phosphate XD36 (70 mg, 0.099 mmol) was reacted with phosphate XS47 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) to give pyrophosphate XS52 (51.9 mg, 60%) as a white solid after lyophilization.

[0446] 1 H 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).

[0447] For C 28 H 46 BrN6O 14 P2 + [M+H] + , MS(ESI + ) Calculated value: 831.2, measured value: 831.6.

[0448] Pyrophosphate XS53

[0449] Alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate XS48 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to give pyrophosphate XS53 (66.4 mg, 51%) as a white solid after lyophilization.

[0450] 1 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).

[0451] For C 28 H 46 IN6O 14 P2 + [M + H] + , MS (ESI + ) calculated: 879.2, found: 879.6.

[0452] Linker - Drug XS54

[0453] The azide XS49 (19.6 mg, 0.025 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave the pyrophosphate XS54 (11.7 mg, 45%) as a white solid after lyophilization.

[0454] 11H 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).

[0455] For C 40 H 61 N8O 19 P2 + [M + H] + , MS (ESI + ) Calcd: 1019.4, Found: 1020.0.

[0456] Linker - Drug XS55

[0457] Azide XS50 (24.5 mg, 0.030 mmol) was reacted with alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) gave pyrophosphate XS55 as a white solid (12.6 mg, 39%) after lyophilization.

[0458] 11H 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).

[0459] For C 40 H 60 FN8O 19 P2 + [M + H] + , MS (ESI + ) Calcd: 1037.3, Found: 1037.8.

[0460] Linker - Drug XS56

[0461] Azide XS51 (59.2 mg, 0.072 mmol) was reacted with alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) followed by lyophilization gave pyrophosphate XS56 (43.4 mg, 55%) as a white solid.

[0462] 11H 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).

[0463] For C 40 H 60 ClN8O 19 P2 + [M + H] + , MS (ESI + ) Calcd: 1053.3, Found: 1053.9.

[0464] Linker - Drug XS57

[0465] The azide XS52 (40.7 mg, 0.047 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) afforded the pyrophosphate XS57 (23.9 mg, 45%) as a white solid after lyophilization.

[0466] 11H 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).

[0467] For C 40 H 60 BrN8O 19 P2 + [M + H] + , MS (ESI + ) Calcd: 1097.3, Found: 1097.6.

[0468] Linker - Drug XS58

[0469] The azide XS53 (62.6 mg, 0.069 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 60:40) gave the pyrophosphate XS58 (41.2 mg, 51%) as a white solid after lyophilization.

[0470] 11H 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, JJ = 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).

[0471] For C 40 H 60 IN8O 19 P2 + [M + H] + , MS (ESI + ) Calcd: 1145.2, Found: 1146.1.

[0472] Example 3: Synthesis of Linker - Drug Compounds XS100 to XS107

[0473] 3A. Preparation of Ether (XS60)

[0474]

[0475] (Z)-tert - Butyl ((2 - iodo - 4 - ((tetrahydro - 2H - pyran - 2 - yl)oxy)but - 2 - en - 1 - yl)oxy)diphenylsilane (XS60)

[0476] The 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 reach RT and stirred for 2 h. DCM was added and the mixture was washed with saturated aqueous NaHCO3, water and brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to afford the ether XS60 (1.05 g, 89%) as a colorless oil.

[0477] 11H 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).

[0478] For C 25 H 33 INaO3Si + [M + Na] + , MS (ESI + ) Calcd: 559.1, Found: 559.5.

[0479] 3B. Preparation of Functionalized Olefins (XS61 to XS65), Trifluoroolefins (XS71), and Alkynes (XS72)

[0480] Preparation of Functionalized Olefins (XS61 to XS62)

[0481]

[0482] (E)-2-(((tert-Butyldiphenylsilyl)oxy)methyl)-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-enenitrile (XS61)

[0483] Tributylstannanecarbonitril was reacted with XS60 (1.05 g, 1.96 mmol) according to General Procedure XXI. The crude product was purified by flash chromatography (silica gel, 0% to 25% Et2O in heptane) and filtered from heptane. The filtrate was concentrated to give the nitrile XS61 (0.882 g, quantitative) as a yellow oil.

[0484] 11H 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).

[0485] For C 26 H 34 NO3Si + [M + H] + , MS (ESI + ) Calculated: 436.2, Found: 436.4.

[0486] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-vinylbut-2-en-1-yl)oxy)silane (XS62)

[0487] Tributyl(vinyl)stannane was reacted with XS60 (0.400 g, 0.746 mmol) according to General Procedure XXI. The crude product was purified by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to afford the diene XS62 (0.284 g, 87%) as a yellow solid.

[0488] 1 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).

[0489] For C 27 H 37 O3Si + [M + H] + , MS (ESI+ )Calculated value: 437.3, measured value: 437.4.

[0490] Preparation of Functionalized Olefin (XS63)

[0491]

[0492] (E)-tert-Butyl ((2-ethyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)diphenylsilane (XS63)

[0493] React diethylzinc (1.0 M in hexane, 1.92 mL) with XS60 (0.515 g, 0.960 mmol) according to General Procedure XXJ. Purify the crude product by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to obtain alkane XS63 (0.193 g, 46%) as a colorless oil.

[0494] 1 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).

[0495] For C 27 H 38 NaO3Si + [M + H] + , MS (ESI + ) Calculated value: 461.2, measured value: 461.4.

[0496] Functionalized Olefin (XS64)

[0497]

[0498] (E)-tert-Butyldiphenyl ((2-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethylidene)pentyl)oxy)silane (XS64)

[0499] Isopropylzinc chloride (0.815 g, 5.74 mmol) (freshly prepared at 0 °C by adding i-PrMgCl (2.0 M in THF, 2.87 mL) to a solution of ZnCl2 (0.782 g, 5.74 mmol) in THF (4.8 mL)) was reacted with XS60 (0.770 g, 1.44 mmol) according to procedure XXJ. The crude product was purified by flash chromatography (silica gel, 0% to 8% EtOAc in heptane) to afford the alkane XS64 (0.335 g, 52%) as a colorless oil.

[0500] 1 H 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.

[0501] Functionalized alkene (XS65)

[0502]

[0503] (E)-tert-Butyl ((2-cyclobutyl-4-((tetrahydro-2H-pyran-2-yl)oxy)but-2-en-1-yl)oxy)diphenylsilane (XS65)

[0504] Cyclobutylzinc chloride (0.5 M in THF, 11.2 mL) was reacted with XS60 (0.750 g, 1.40 mmol) according to the general procedure XXJ. The crude product was purified by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to afford the cycloalkane XS65 (0.574 g, 88%) as a colorless oil.

[0505] 11H 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).

[0506] For C 29 H 40 NaO3Si + [M + H] + , MS (ESI + ) Calcd: 487.3, Found: 487.5.

[0507] Trifluoroolefin (XS71)

[0508] Aldehyde (XS67)

[0509]

[0510] 2 - ((Tetrahydro - 2H - pyran - 2 - yl)oxy)ethan - 1 - ol (XS66)

[0511] 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 dropwise addition of DHP (2.05 mL, 22.4 mmol). The reaction mixture was stirred at 0 °C for 30 min, allowed to reach RT and stirred for 3 days. The reaction mixture was diluted with water and the product was extracted with DCM (2×). The organic layer was washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0% to 60% EtOAc in heptane) to afford the ether XS66 as a colorless oil (2.24 g, 68%).

[0512] 11H 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).

[0513] For C7H 15 O3 + [M + H] + , MS (ESI + ) Calculated: 147.1, Found: 147.0.

[0514] 2 - ((Tetrahydro - 2H - pyran - 2 - yl)oxy)acetaldehyde (XS67)

[0515] To a solution of oxalyl chloride (1.74 mL, 19.9 mmol) in DCM (140 mL) at -78 °C was added dropwise DMSO (2.80 mL, 39.4 mmol). 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 reach RT and stirred for 45 min. Saturated aqueous NaHCO3 was added and the mixture was stirred for 15 min. The organic layer was separated and the aqueous layer was extracted with DCM (2×). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0% to 50% EtOAc in heptane) to afford aldehyde XS67 (1.32 g, 60%) as a colorless oil.

[0516] 1 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).

[0517] Trifluoroolefin (XS71)

[0518]

[0519] (E)-Ethyl 4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-enoate (XS69)

[0520] To a solution of aldehyde XS67 (0.894 g, 6.20 mmol) in THF (4.1 mL) was added a solution of Wittig reagent XS68 (1.78 g, 4.14 mmol, synthesized according to US2007 / 249723) in THF (4.1 mL). The reaction mixture was stirred at 100 °C for 16 h. The reaction mixture was diluted with Et2O, dried over Na2SO4 and filtered through a silica plug. The filtrate was concentrated and the crude product was purified by flash chromatography (silica gel, 0% to 20% EtOAc in heptane) to afford the E-olefin XS69 (0.435 g, 36%) as a colorless oil.

[0521] 1 H 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 = 7.1 Hz, 3H).

[0522] (E)-4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-en-1-ol (XS70)

[0523] The ester XS69 (0.425 g, 1.43 mmol) was purged with N2 and dissolved in THF (7.2 mL). At 0 °C, DIBAL-H (1.0 M in hexanes, 3.16 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 2 h. Next, HCl (1.0 M) was added and the reaction mixture was stirred for 30 min. The aqueous layer was extracted with DCM (3×) and the combined organic layers were washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by flash chromatography (silica gel, 0% to 40% EtOAc in heptane) to afford the alcohol XS70 (0.232 g, 64%) as a pale yellow oil.

[0524] 11H 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).

[0525] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-(2,2,2-trifluoroethyl)but-2-en-1-yl)oxy)silane (XS71)

[0526] To a 0 °C solution of alcohol XS70 (0.230 g, 0.905 mmol), TEA (0.252 mL, 1.81 mmol), and imidazole (68 mg, 1.0 mmol) was added dropwise TBDPSC1 (0.349 mL, 1.36 mmol). The reaction mixture was brought to RT and stirred for 3 h. Water was added and the product was extracted with DCM (3×). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to afford the silyl ether XS71 (0.378 g, 85%) as a colorless oil.

[0527] 1 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).

[0528] Alkyne (XS72)

[0529]

[0530] (E)-tert-Butyldiphenyl((4-((tetrahydro-2H-pyran-2-yl)oxy)-2-((trimethylsilyl)ethynyl)but-2-en-1-yl)oxy)silane (XS72)

[0531] Purge CuI (16 mg, 0.085 mmol) and Pd(PPh3)2Cl2 (60 mg, 0.085 mmol) with N2 (3×) and add a solution of XS60 (0.458 g, 0.854 mmol) in toluene (5.0 mL), followed by trimethylsilylacetylene (0.126 g, 1.28 mmol) and DIPEA (0.298 mL, 1.71 mmol). Stir the reaction mixture at RT for 6 h, concentrate and dissolve in EtOAc. Wash the organic layer with 0.5 M aqueous KHSO4, water and brine, dry over Na2SO4 and concentrate. Purify the crude residue by flash chromatography (silica gel, 0% to 8% EtOAc in heptane) and filter from heptane. Concentrate the filtrate to afford the silyl ether intermediate as an orange oil (0.361 g, 84%). Dissolve the intermediate in MeOH (7.1 mL) and add K2CO3 (49 mg, 0.36 mmol). Stir the reaction mixture at RT for 2 h. Dilute the reaction mixture with EtOAc and wash with saturated aqueous NaHCO3, water and brine, dry over Na2SO4 and concentrate. Purify the crude product by flash chromatography (silica gel, 0% to 10% EtOAc in heptane) to afford the alkyne XS72 as a colorless oil (0.192 g, 62%).

[0532] 1 1H 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).

[0533] Preparation of 3C. Phosphates (XS87 to XS93)

[0534]

[0535] (E)-2-(((tert-Butyldiphenylsilyl)oxy)methyl)-4-hydroxybut-2-enenitrile (XS73)

[0536] React ether XS61 (0.500 g, 1.15 mmol) according to procedure XXK at 45 °C. Use Et2O instead of DC for extraction. Purify the crude product by flash chromatography (silica gel, 0% to 60% Et2O in heptane) to afford the alcohol XS73 (0.261 g, 65%) as a pale yellow oil.

[0537] 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).

[0538] For C 21 H 26 NO2Si + [M+H] + ,MS (ESI + ) Calcd for: 352.2, found: 352.2.

[0539] (E)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)penta-2,4-dien-1-ol (XS74)

[0540] React ether XS62 (0.284 g, 0.650 mmol) according to procedure XXK at 55 °C. Purify the crude product by flash chromatography (silica gel, 0% to 50% Et2O in heptane) to afford the alcohol XS74 (0.183 g, 80%) as a pale yellow oil and use it as such in the next reaction step.

[0541] (E)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)penta-2-en-1-ol (XS75)

[0542] React ether XS63 (0.193 g, 0.440 mmol) according to procedure XXK at 55 °C.

[0543] Purify the crude product by flash chromatography (silica gel, 0% to 50% Et2O in heptane) to afford the alcohol XS75 (0.112 g, 72%) as a colorless oil.

[0544] 11H 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).

[0545] For C 22 H 30 NaO2Si + [M + Na] + , MS (ESI + ) Calcd: 377.2, Found: 377.3.

[0546] (E)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)hex-2-en-1-ol (XS76)

[0547] The ether XS64 (0.330 g, 0.729 mmol) was reacted according to procedure XXK at 55 °C. The crude product was purified by flash chromatography (silica gel, 0% to 40% Et2O in heptane) to afford the alcohol XS76 (0.241 g, 90%) as a colorless oil.

[0548] 1 1H 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).

[0549] For C 23 H 31 OSi + [M + H - H2O] + , MS (ESI + ) Calcd: 351.2, Found: 351.4.

[0550] (E)-4-((tert-Butyldiphenylsilyl)oxy)-3-cyclobutylbut-2-en-1-ol (XS77)

[0551] React ether XS65 (0.570 g, 1.23 mmol) according to procedure XXK at 50 °C. Purify the crude product by flash chromatography (silica gel, 0% to 50% Et2O in heptane) to afford alcohol XS77 (0.414 g, 89%) as a colorless oil.

[0552] 1 1H NMR (400 MHz, CDCl3) ppm = 7.69 (dd, J = 7.8, 16 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).

[0553] For C 24 H 32 NaO2Si + [M+Na] + , MS (ESI + ) Calcd for: 403.2, Found: 403.3.

[0554] (E)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)-5,5,5-trifluoropent-2-en-1-ol (XS78)

[0555] React ether XS71 (0.375 g, 0.761 mmol) according to procedure XXK at 55 °C. Purify the crude product by flash chromatography (silica gel, 0% to 40% Et2O in heptane) to afford alcohol XS78 (0.272 g, 87%) as a colorless oil.

[0556] 1 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).

[0557] (E)-3-(((tert-Butyldiphenylsilyl)oxy)methyl)penta-2-en-4-yn-1-ol (XS79)

[0558] Ether XS72 (0.190 g, 0.437 mmol) was reacted at 55 °C according to procedure XXK. The crude product was purified by flash chromatography (silica gel, 0% to 50% Et2O in heptane) to give alcohol XS79 (0.123 g, 80%) as a pale yellow oil.

[0559] 1 1H 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).

[0560] For C 22 H 27 O2Si + [M + H] + ,MS (ESI + ) Calcd for: 351.2, Found: 351.3.

[0561] (E)-Bis((9H-fluoren-9-yl)methyl) (4-((tert-butyldiphenylsilyl)oxy)-3-cyanobut-2-en-1-yl) phosphate (XS80)

[0562] 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% to 50% EtOAc in heptane) to give alkyl phosphate XS80 (0.339 g, 58%) as a colorless oil.

[0563] 1 1H 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).

[0564] For C 49 H 47 NO5PSi + [M + H] + ,MS (ESI + ) Calcd for: 788.3, Found: 788.5.

[0565] (E)-Bis((9H-fluoren-9-yl)methyl)(3-(((tert-butyldiphenylsilyl)oxy)methyl)penta-2,4-dien-1-yl)phosphate (XS81)

[0566] The 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% to 30% EtOAc in heptane) to afford the alkyl phosphate XS81 (0.312 g, 76%) as a colorless oil.

[0567] 1 H 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).

[0568] (E)-Bis((9H-fluoren-9-yl)methyl)(3-(((tert-butyldiphenylsilyl)oxy)methyl)penta-2-en-1-yl)phosphate (XS82)

[0569] The 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% to 50% EtOAc in heptane) to afford the alkyl phosphate XS82 (0.195 g, 79%) as a colorless oil.

[0570] 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).

[0571] For C 50 H 51 NaO5PSi + [M+Na] + , MS (ESI + ) Calculated: 813.3, Found: 813.5.

[0572] (E)-Bis((9H-fluoren-9-yl)methyl)(3-(((tert-butyldiphenylsilyl)oxy)methyl)hex-2-en-1-yl) phosphate (XS83)

[0573] The 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% to 40% EtOAc in heptane) to afford the alkyl phosphate XS83 (0.347 g, 67%) as a colorless oil.

[0574] 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).

[0575] (E)-Bis((9H-fluoren-9-yl)methyl)(4-((tert-butyldiphenylsilyl)oxy)-3-cyclobutylbut-2-en-1-yl) phosphate (XS84)

[0576] The 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% to 50% EtOAc in heptane) to afford the alkyl phosphate XS84 (0.519 g, 59%) as a colorless oil.

[0577] 11H 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).

[0578] (E)-Bis((9H-fluoren-9-yl)methyl)(3-(((tert-butyldiphenylsilyl)oxy)methyl)-5,5,5-trifluoropent-2-en-1-yl) phosphate (XS85)

[0579] The 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% to 50% EtOAc in heptane) to afford the alkyl phosphate XS85 (0.179 g, 64%) as a colorless oil.

[0580] 1 1H 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).

[0581] (E)-Bis((9H-fluoren-9-yl)methyl)(3-(((tert-butyldiphenylsilyl)oxy)methyl)penta-2-en-4-yn-1-yl) phosphate (XS86)

[0582] The 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% to 40% EtOAc in heptane) to afford the alkyl phosphate XS86 (0.199 g, 74%) as a colorless oil.

[0583] 1 1H 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).

[0584] For C 50 H 48 O5PSi + [M + H] + , MS (ESI + ) Calculated: 787.3, Found: 787.6.

[0585] Triethylammonium (E)-3-cyano-4-hydroxybut-2-en-1-yl phosphate (XS87)

[0586] Step 1: TBDPS-ether XS80 (0.339 g, 0.430 mmol) was reacted according to General Procedure XXG. The crude intermediate was purified by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to afford the intermediate allyl alcohol as a white solid (0.164 g, 69%). For C 33 H 29 NO5P + [M + H] + , MS (ESI + ) Calculated: 550.2, Found: 550.3.

[0587] Step 2: The intermediate (0.164 g, 0.298 mmol) was reacted according to General Procedure XXH to afford the alkyl phosphate XS87 as the triethylammonium salt in a ratio of phosphate:Et3N of 1:0.9 (79 mg, 94%).

[0588] 1 1H 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).

[0589] For C5H7NO5P - [M - H] - , MS (ESI -)Calculated value: 192.0, measured value: 192.0.

[0590] Triethylamine (E)-3-(hydroxymethyl)penta-2,4-dien-1-yl phosphate (XS88)

[0591] Step 1: React TBDPS-ether XS81 (0.305 g, 0.387 mmol) according to General Procedure XXG. Purify the crude intermediate by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.172 g, 81%) as a white solid.

[0592] 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).

[0593] For C 34 H 32 O5P + [M + H] + , MS (ESI + ) Calculated value: 551.2, measured value: 551.4.

[0594] Step 2: React the intermediate (0.170 g, 0.309 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS88 (74 mg, 85%) as a triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.9.

[0595] 1 H NMR (400 MHz, D2O) ppm = 6.59 (dd, J = 178, 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).

[0596] For C6H 10 O5P - [M - H] -, MS(ESI - ) Calculated value: 193.0, measured value: 193.1.

[0597] Triethylamine (E)-3-(hydroxymethyl)pent-2-en-1-yl phosphate (XS89)

[0598] Step 1: React TBDPS-ether XS82 (0.191 g, 0.241 mmol) according to General Procedure XXG. Purify the crude residue by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.105 g, 79%) as a colorless oil.

[0599] 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).

[0600] For C 34 H 34 O5P + [M+H] + , MS(ESI + ) Calculated value: 553.5, measured value: 553.2.

[0601] Step 2: React the intermediate (0.102 g, 0.185 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS89 (51 mg, 97%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.7.

[0602] 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 (g, J = 7.6 Hz, 2H), 0.92 (t, J = 7.6 Hz, 3H).

[0603] For C6H 12 O5P - [M-H] - , MS(ESI -)Calculated value: 195.0, measured value: 195.1.

[0604] Triethylamine (E)-3-(hydroxymethyl)hex-2-en-1-yl phosphate (XS90)

[0605] Step 1: React TBDPS-ether XS83 (0.345 g, 0.429 mmol) according to General Procedure XXG. Purify the crude residue by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.213 g, 88%) as a colorless oil.

[0606] 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).

[0607] For C 35 H 35 NaO5P + [M+Na] + , MS (ESI + ) Calculated value: 589.2, measured value: 589.5.

[0608] Step 2: React the intermediate (0.211 g, 0.372 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS90 (61 mg, 55%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.7.

[0609] 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).

[0610] For C7H 14 O5P - [M-H] - , MS (ESI- )Calculated value: 209.1, measured value: 209.1.

[0611] Triethylamine (E)-3-cyclobutyl 4-hydroxybut-2-en-1-yl phosphate (XS91)

[0612] Step 1: React TBDPS-ether XS84 (0.515 g, 0.630 mmol) according to General Procedure XXG. Purify the crude intermediate by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.281 g, 77%) as a colorless oil.

[0613] 1 H 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).

[0614] For C 36 H 36 O5P + [M+H] + , MS (ESI + ) Calculated value: 579.2, measured value: 579.5.

[0615] Step 2: React the intermediate (0.275 g, 0.475 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS91 (0.113 g, 77%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.9.

[0616] 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), 312 (q, J = 7.3 Hz, 6H), 2.07 - 1.90 (m, 5H), 189 - 1.56 (m, 3H), 1.20 (t, J = 7.3 Hz, 8H).

[0617] For C8H 14 O5P - [M-H] - , MS(ESI - ): calculated value: 221.1, measured value: 221.2.

[0618] Triethylamine (E)-5,5,5-trifluoro-3-(hydroxymethyl)pent-2-en-1-yl phosphate (XS92)

[0619] Step 1: React TBDPS-ether XS85 (0.117 g, 0.209 mmol) according to General Procedure XXG. The crude intermediate was suspended in heptane and filtered to give the intermediate allyl alcohol (0.103 g, 75%) as a white solid.

[0620] 1 1H 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).

[0621] For C 34 H 31 F3O5P + [M+H] + , MS(ESI + ): calculated value: 607.2, measured value: 607.4.

[0622] Step 2: React the intermediate (0.119 g, 0.196 mmol) according to General Procedure XXH to give the alkyl phosphate XS92 (27 mg, 41%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.6.

[0623] 1 1H 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).

[0624] For C6H9F3O5P - [M-H] -, MS(ESI - ) Calculated value: 249.0, measured value: 249.0.

[0625] Triethylamine (E)-3-(hydroxymethyl)pent-2-en-4-yn-1-yl phosphate (XS93)

[0626] Step 1: React TBDPS-ether XS86 (0.196 g, 0.249 mmol) according to General Procedure XXG. Purify the crude intermediate by flash chromatography (silica gel, 0% to 100% EtOAc in heptane) to obtain the intermediate allyl alcohol (0.103 g, 75%) as a white solid.

[0627] 1 1H 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).

[0628] For C 34 H 30 O5P + [M+H] + , MS(ESI + ) Calculated value: 549.2, measured value: 549.4.

[0629] Step 2: React the intermediate (0.100 g, 0.182 mmol) according to General Procedure XXH to obtain the alkyl phosphate XS93 (43 mg, 84%) as the triethylamine salt in a ratio of phosphate ester:Et3N of 1:0.9.

[0630] 1 1H 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).

[0631] For C6H8O5P - [M-H] - , MS(ESI - ) Calculated value: 191.0, measured value: 191.0.

[0632] Preparation of 3D. Linker-Drug (XS100 to XS105)

[0633]

[0634] Pyrophosphate XS94

[0635] Alkyl phosphate XD36 (79 mg, 0.11 mmol) was reacted with phosphate XS87 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to afford pyrophosphate XS94 (41.0 mg, 45%) as a white solid after lyophilization.

[0636] 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), 366 - 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).

[0637] For C 29 H 44 N7O 14 P2 - [M - H] - ,MS (ESI - ) Calcd: 776.2, Found: 776.6.

[0638] Pyrophosphate XS95

[0639] Alkyl phosphate XD36 (79 mg, 0.11 mmol) was reacted with phosphate XS88 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to afford pyrophosphate XS95 (50.1 mg, 55%) as a white solid after lyophilization.

[0640] 11H 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 = 6.6 Hz, 3H).

[0641] For C 30 H 47 N6O 14 P2 - [M - H] - , MS (ESI - ) calculated value: 777.3, found value: 777.4.

[0642] Pyrophosphate XS96

[0643] Alkyl phosphate XD36 (100 mg, 0.142 mmol) was reacted with phosphate XS89 according to General Procedure XXD. The crude product was purified by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to give pyrophosphate XS96 (48.6 mg, 42%) as a white solid after lyophilization.

[0644] 1 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).

[0645] For C 30 H 49 N6O 14 P2 - [M-H] - , MS(ESI - ) calculated value: 779.3, measured value: 779.5.

[0646] Pyrophosphate XS97

[0647] React alkyl phosphate XD36 (54 mg, 0.077 mmol) with phosphate XS90 according to general procedure XXD. Purify the crude product by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to obtain pyrophosphate XS97 (37.3 mg, 59%) as a white solid after lyophilization.

[0648] 1 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).

[0649] For C 31 H 51 N6O 14 P2 - [M-H] - , MS(ESI - ) calculated value: 793.3, measured value: 793.5.

[0650] Pyrophosphate XS98

[0651] Alkyl phosphate XD36 (105 mg, 0.149 mmol) was reacted with phosphate XS91 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to afford pyrophosphate XS98 (64.1 mg, 51%) as a white solid after lyophilization.

[0652] 1 H 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).

[0653] For C 32 H 51 N6O 14 P2 - [M - H] - , MS (ESI - ) calculated: 805.3, found: 805.5.

[0654] Pyrophosphate XS99

[0655] Alkyl phosphate XD36 (43 mg, 0.061 mmol) was reacted with phosphate XS92 according to General Procedure XXD. The crude product was purified by preparative RP-HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to afford pyrophosphate XS99 (12.3 mg, 23%) as a white solid after lyophilization.

[0656] 11H 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).

[0657] For C 30 H 46 F3N6O 14 P2 - [M - H] - , MS (ESI - ) calculated: 833.3, found: 833.6.

[0658] Linker - Drug XS100

[0659] The azide XS94 (36 mg, 0.044 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave the pyrophosphate XS100 (15.5 mg, 33%) as a white solid after lyophilization.

[0660] 11H 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).

[0661] For C 41 H 58 N9O 19 P2 - [M - H] - , MS (ESI - ) Calcd: 1042.3, Found: 1042.8.

[0662] Linker - drug XS101

[0663] The azide XS95 (44 mg, 0.054 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave the pyrophosphate XS101 (19.8 mg, 34%) as a white solid after lyophilization.

[0664] 11H 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).

[0665] For C 42 H 61 N8O 19 P2 - [M - H] - , MS (ESI - ) Calculated: 1043.4, Found: 1043.6.

[0666] Linker - Drug XS102

[0667] Azide XS96 (42 mg, 0.052 mmol) was reacted with alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave pyrophosphate XS102 (8.9 mg, 16%) as a white solid after lyophilization.

[0668] 11H 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 - 346 (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).

[0669] For C 42 H 63 N8O 19 P2 - [M - H] - , MS (ESI - ) calculated value: 1045.4, found value: 1045.6.

[0670] Linker - drug XS103

[0671] The azide XS97 (32 mg, 0.038 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave the pyrophosphate XS103 (9.0 mg, 22%) as a white solid after lyophilization.

[0672] 11H 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).

[0673] For C 43 H 65 N8O 19 P2 - [M - H] - , MS (ESI - ) Calculated: 1059.4, Found: 1059.8.

[0674] Linker - Drug XS104

[0675] The azide XS98 (56 mg, 0.067 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) afforded the pyrophosphate XS104 as a white solid (22.7 mg, 31%) after lyophilization.

[0676] 11H 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 - 346 (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).

[0677] For C 44 H 65 N8O 19 P2 - [M - H] - , MS (ESI - ) calculated: 1071.4, found: 1071.7.

[0678] Linker - Drug XS105

[0679] The azide XS99 (11 mg, 0.012 mmol) was reacted with the alkyne XD43 according to General Procedure XXE. Purification by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) gave the pyrophosphate XS105 (3.3 mg, 24%) as a white solid after lyophilization.

[0680] 11H 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).

[0681] For C 42 H 60 F3N8O 19 P2 - [M - H] - , MS (ESI - ) calculated: 1099.3, found: 1099.7.

[0682] Preparation of linker - drug (XS107)

[0683]

[0684] Pyrophosphate XS106

[0685] Alkyl phosphate XD36 (90 mg, 0.128 mmol) was reacted with phosphate XS93 according to General Procedure XXD. The crude product was purified by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to give pyrophosphate XS106 (50.1 mg, 48%) as a white solid after lyophilization.

[0686] 11H 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).

[0687] For C 30 H 45 N6O 14 P2 - [M - H] - , MS (ESI - ): calculated value: 775.2, found value: 775.4.

[0688] Linker - drug XS107

[0689] The 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 RT for 45 minutes and then purified by preparative RP - HPLC (25 mM NH4HCO3 in MilliQ / MeCN, gradient: 90:10 to 50:50) to give the pyrophosphate XS107 (14.1 mg, 46%) as a white solid after lyophilization. For C 62 H 79 N 10 O 21 P2 - [M - H] - , MS (ESI - ): calculated value: 1361.5, found value: 1361.9.

[0690] Example 4: Synthesis of conjugates of rituximab with linker - drug compounds XD73, XS54 to XS58, XS100 to XS105, and XS107 with a DAR of 2.

[0691] To the antibody solution (10 to 12 mg / mL), add TRIS (1% v / v, 1 M, pH 8), EDTA (4% v / v, 25 mM), and TCEP (5 mM in water). Incubate the resulting solution at RT for 2 h. After incubation, the reduced antibody was re-buffered to 4.2 mM histidine, 50 mM trehalose (pH 6) and treated with dimethylacetamide (DMA) and linker-drug compound (LD) (10 mM in DMA, >1.5 eq / SH). The final DMA content was about 10% v / v. The resulting mixture was roller-mixed at RT in the dark overnight. Activated carbon was added and the suspension was roller-mixed in the dark for 1 h, filtered and washed with 4.2 mM histidine, 50 mM trehalose (pH 6). The solution was re-buffered to 4.2 mM histidine, 50 mM trehalose (pH 6) and sterile filtered.

[0692] To bring the DAR (drug-to-antibody ratio) of the conjugate close to the target DAR of 2, substitution conjugation was performed with a hydrophobic seco-DUBA payload (SYD980, described in a.o. WO2015 / 177360), which enabled easy DAR determination by HIC. The resulting average DAR of the conjugate with a target DAR of 2 is shown in Table 1.

[0693] Table 1: Synthetic conjugates with antibodies and linker-drug (LD) compounds used in the synthesis

[0694]

[0695]

[0696]

[0697]

[0698]

[0699] Example 5: Activity of phosphoantigen (pAg) conjugates (ADCs) against γδ T cells

[0700] Multiple synthetic linker-drug compounds were conjugated to rituximab (anti-CD20), and the ability of the resulting ADCs to bind to Raji cells and to activate Vδ2γδ T cells after overnight incubation with CD20-positive Raji cells was tested. The linker-drug compounds conjugated to rituximab and the resulting ADCs are within the list shown in Table 1. Pretreated Raji cells were co-cultured with peripheral blood mononuclear cells (PBMC), and the activation (IFNγ production) and degranulation (CD107a) of Vδ2γδ T cells and NK cells were determined using multicolor flow cytometry.

[0701] Materials and methods

[0702] Cell binding

[0703] In vitro experiments were performed using the CD20-positive Burkitt lymphoma human tumor cell line Raji (DSMZ, German collection of Microorganisms and cell cultures GmbH). 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 at 37 °C in a humidified incubator containing 5% CO2 and passaged twice a week. For cell binding in 96-well plates, 100,000 Raji cells / well were washed twice with ice-cold FACS buffer (PBS 1×, 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 (such as rituximab), or non-binding isotype control ADC diluted in ice-cold FACS buffer at a range of concentrations. 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 determined by flow cytometry using NovoCyte (Agilent) and determined as the median fluorescence intensity (MFI). In GraphPad Prism version 9, curves were fitted by non-linear regression with variable slope (four parameters). In GraphPad Prism, the EC 50 value was calculated as the concentration in μg / mL that gave half the response between the bottom and top of the curve. Binding experiments were performed in three independent experiments.

[0704] Functional assay (determination of γδ T cell activity induced by different pAg conjugates)

[0705] For stimulation with the ADC according to the invention, Raji cells were harvested and diluted to 5×10 6at a concentration of cells / mL, and inoculate 50 μL (equivalent to 250,000 cells / well) of this cell suspension into a 96-well plate. Prepare 2-fold concentrated, 5-fold serial dilutions of the ADC or rituximab in complete growth medium (CGM, RPMI-1640 (Lonza)) supplemented with 10% HI FBS (Gibco) and 80 U / mL penicillin-streptomycin solution (Lonza). Incubate the plate-inoculated Raji cells overnight at 37 °C in a humidified incubator with 5% CO2 with 50 μL / well of the serial diluted compound (total 100 μL / well). The next day, wash the 96-well plate with Raji cells and ADC or rituximab by adding 100 μL / well CGM, centrifuge at 300 × g for 3 minutes at room temperature, and remove the supernatant to remove excess unbound compound. As a source of immune cells, thaw cryopreserved PBMCs from healthy human donors, resuspend them in CGM, and place them overnight at 37 °C in a humidified incubator with 5% CO2 to allow the cells to recover. Harvest the recovered PBMCs, count them and dilute them in CGM to 10 × 10 6 cells / mL concentration, and add 50 μL / well (equivalent to 0.5 × 10 6Cells / well). Prepare a 2-fold concentrated anti-CD107a BV421 (BioLegend) solution in CGM containing GolgiStop (Monensin) and GolgiPlug (Brefeldin A) (BD Biosciences), and add 50 μL / well to the Raji-PBMC co-culture. Incubate the samples in a humidified incubator with 5% CO2 at 37 °C for 6 hours. For staining of immune cell subsets, prepare a multicolor antibody staining mixture in Brilliant Staining 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-TCR Vδ2 APC (BioLegend). After the 6-hour incubation period, centrifuge the plate at 300 × g for 3 minutes at room temperature and discard the supernatant. Resuspend the pellet in 50 μL of the antibody mixture and incubate in the dark on ice for 30 minutes. Wash the plate twice by adding 100 μL of ice-cold FACS buffer (PBS 1×, 0.1% v / w BSA, 0.02% v / v sodium azide), then centrifuge at 300 × g for 3 minutes and discard the supernatant. Fix and permeabilize the cells using 100 μL / well of Cytofix / Cytoperm solution (BD Bioscience) and incubate in the dark on ice for 20 minutes. Wash the cells three times by adding 150 μL of BD Perm / wash solution (diluted to 10× BD Perm / Wash buffer in distilled H2O to make a 1× solution before use), then centrifuge at 300 × g for 3 minutes and discard the supernatant. Finally, resuspend the cells in FACS buffer and store them overnight in the refrigerator at 4 °C in the dark. On the third day, wash the stained PBMC / Raji cells once in 150 μL of BD Perm / wash solution, then centrifuge at 300 × g for 3 minutes and discard the supernatant. Resuspend the pellet in a mixture of 50 μL of anti-IFNγ PE-Cy7 (BioLegend) diluted in Perm / Wash solution and incubate in the dark on ice for 30 minutes. After incubation, wash the plate once with 150 μL of ice-cold FACS buffer, then centrifuge at 300 × g for 3 minutes and discard the supernatant. Then, resuspend the cell pellet in 100 μL of FACS buffer and analyze the samples using NovoCyte (Agilent). In GraphPad Prism version 9, fit the curve by nonlinear regression with variable slope (four parameters).In GraphPad Prism, the EC. 50 value was calculated as the concentration in μg / mL that gave half the response between the bottom and top of the curve. Each compound was tested in at least two independent experiments with different donors.

[0706] Results / Conclusions

[0707] Multiple rituximab ADCs and non-conjugated controls were generated, with a drug-to-antibody ratio (DAR) of approximately 2. Its binding to Raji cells was comparable to that of naked rituximab (Table 2), while the non-conjugated isotype control did not show binding (data not shown).

[0708] After overnight incubation with Raji cells followed by co-culture with PBMCs containing Vδ2γδ T cells for 6 hours, the ability of the generated ADCs to induce Vδ2γδ T cell activation was tested. Dose-response curves for Vδ2γδ T cell degranulation (CD107a) and IFNγ production were generated. The linker drug XD18 when conjugated to rituximab (ADC-XD18-r) was earlier described in co-pending patent application number WO2023 / 275025 filed in the name of Byondis B.V. and showed better efficacy and potency than rituximab. The linker drug XD73 was conjugated to rituximab (ADC-XD73-r) or to a non-conjugated isotype control (ADC-XD73-i) and compared with ADC-XD18-r ( Figure 1A 、B and Table 3). The non-conjugated isotype control ADC activated Vδ2γδ T cells with low efficacy and the EC 50 value could not be reliably calculated. ADC-XD73-r activated γδ T cells with efficacy and potency similar to ADC-XD18-r. The linker drug XD45 when conjugated to rituximab (generating ADC-XD45-r) was earlier described in WO2023 / 275025 and ADC-XD45-r showed better efficacy and potency than rituximab (WO2023 / 275025). The linker drugs XS58, XS56, XS54 and XS57 were conjugated to rituximab or to a non-conjugated isotype control to generate the corresponding ADCs and compared with ADC-XD45-r ( Figure 1A 、B and Table 3). The non-conjugated isotype control ADC activated Vδ2γδ T cells with low efficacy and the EC 50Values. ADC-XS56-r and ADC-XS-57-r activate γδ T cells with efficacy and potency similar to those of ADC-XD45-r. ADC-XS58-r and ADC-XS54-r are also capable of activating γδ T cells. Compared to rituximab-induced γδ T cell activation, γδ T cell activation induced by ADC-XS58-r and ADC-XS54-r is more effective ( Figure 2 ).

[0709] In WO2023 / 275025, it was shown that rituximab and rituximab-ADC induce the activation of NK cells, most likely via the well-known FcγRs expressed by NK cells (WO2023 / 275025). Consistently, all of ADC-XD18-r, ADC-XD73-r, ADC-XD45-r, ADC-XS58-r, ADC-XS56-r, ADC-XS54-r, and ADC-XS57-r induce NK cell degranulation (i.e., CD107a) in a similar manner ( Figure 1C ).

[0710] These results indicate that pretreatment of tumor cells with the CD20-binding ADCs leads to dose-dependent IFNγ induction and degranulation of Vδ2 γδ T cells. The ADCs have an active Fc tail, which most likely activates NK cells through well-defined FcγR interactions.

[0711] Table 2: Binding of rituximab pAg ADCs to Raji cells compared to rituximab. EC 50 values (μg / mL) represent the concentration to achieve 50% activation. A 2-fold difference in EC 50 is within the normal variation range.

[0712] Compound <![CDATA[EC 50 (μg / mL)]]> Rituximab 3.30 ADC-XD18-r 3.96 ADC-XD73-r 2.87 ADC-XS58-r 3.23 ADC-XS56-r 2.91 ADC-XS54-r 3.83 ADC-XS57-r 1.88 ADC-XD45-r 2.39

[0713] Table 3: IFNγ or CD107a production by Vδ2 population induced by Raji cells pretreated with rituximab pAg ADCs. EC 5o values (μg / mL) represent the concentration to achieve 50% activation.

[0714]

[0715] NT = not tested; = incomplete saturation curve (EC 50 not calculable); = low number of IFNγ-positive cells, EC 50 not reliably calculable

[0716] Example 6: Activity of other phosphoantigen (pAg) conjugates (ADCs) against γδ T cells.

[0717] More synthetic linker-drug compounds were conjugated to rituximab (anti-CD20) or an unconjugated isotype control. The linker-drug compounds conjugated to rituximab were XS55, XS100 to XS105, and XS107. A variety of rituximab ADCs were generated at a drug-to-antibody ratio (DAR) of approximately 2, as described in the previous examples. The linker-drug compounds and the resulting ADCs are shown in Table 1.

[0718] The ability of these ADCs to bind to Raji cells (using a binding assay as described in Example 5) and to activate Vδ2 γδ T cells after overnight incubation with CD20-positive Raji cells was tested. Pretreated Raji cells were co-cultured with peripheral blood mononuclear cells (PBMCs), and the activation (IFNγ production) and degranulation (CD107a) of Vδ2 γδ T cells and NK cells were determined using polychromatic flow cytometry, as described in the functional assay of Example 5.

[0719] Results / Conclusions

[0720] The binding of rituximab ADCs to Raji cells was in Figure 3As shown, and all compounds showed binding. The ability of the resulting ADCs to induce Vδ2γδ T cell activation was tested after overnight incubation with Raji cells followed by co-culture with PBMCs containing Vδ2γδ T cells for 6 hours. Dose-response curves for Vδ2γδ 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 better potency than rituximab. The corresponding non-binding isotype control ADCs ADC-XS55-i, ADC-XS100-i, ADC-XS101-i, ADC-XS102-i, and ADC-XS107-i activated Vδ2γδ T cells with low efficacy. ADC-XS103-r, ADC-XS104-r, and ADC-XS105-r showed potency similar to rituximab. Consistently, the corresponding non-binding isotype control ADCs ADC-XS103-i, ADC-XS105-i, and ADC-XS104-I also did not induce Vδ2γδ cell activation. All tested rituximab-ADCs (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 activation of NK cells ( Figure 2 C), most likely via the well-known FcγRs expressed by NK cells (WO2023 / 275025). 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 led to more IFNγ production and degranulation of Vδ2γδ T cells compared to unconjugated rituximab. All tested ADCs had an active Fc tail, which most likely activated NK cells through well-defined FcγR interactions.

Claims

1. A linker-drug compound having the general structure shown in formula (I), 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 does not exist, or is O or NH; X 4a至 Each of the 4d's is independently selected from O and S; X 5 is -H, halogen (F, Cl, Br, I) or nitrile (CN), or -vinyl, ethynyl, ethyl, which is optionally substituted by one or more of the following groups: F, CH3, CH2F, CHF2, CF3, or -C3 to C4 cycloalkyl, C3 to C4 cycloalkenyl, allyl, propargyl, O-CH3, S-CH3, which is optionally substituted by one or more fluorine substituents, or -CHR 1 OR 2 、CHR 1 SR 2 、CHO、CO2R 1 、CONR 1 R 2 wherein R 1 and R 2 are independently selected from H, CH3, CH2F, CHF2, CF3, CH2CH3; x is an integer from 1 to 5; m is 1, 2 or 3; n is 0, 1 or 2; R 1 is H or a connecting part with the connecting part (L), or a prodrug part; R 2 is H or a linking moiety to the linking portion (L), or is Cat+ or a prodrug moiety; R 3 is H or a linking moiety with the linking portion (L), or is Cat+ or a prodrug moiety; R 4 is H or a linking moiety with the linking portion (L), or is Cat+ or a prodrug moiety; Alternatively, when n is 0, R 3 and R 2 are linked via C 1-6 (hetero)alkyl, or When n is 1 or 2, R 3 and R 4 are linked by C 1-6 (hetero)alkyl linkage.

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

3. The linker-drug compound according to claim 1 or 2, wherein W 1 is CH, and R 1 is H or a connecting portion with the connecting portion (L).

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

5. The linker-drug compound according to any one of claims 1 to 4, wherein W 2 is CH2 and m is 1.

6. The linker-drug compound according to any one of claims 1 to 5, wherein X 1 is CH2.

7. The linker-drug compound according to any one of claims 1 to 6, wherein X 3 is O, R 3 is the connecting portion to the linking part, W 1 is CH, R 1 is H, W 2 is CH2 and m is 1, and X 1 is CH2.

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

9. The linker-drug compound according to claim 1, wherein R 2 , R 3 and R 4 , when present, are prodrug moieties selected from the following: -neopentyloxycarbonylmethyl (POM) group and isopropyloxycarbonyloxymethyl (POC) group, -substituted or unsubstituted (hetero)aryl, and -a structure according to formula IV or V: wherein: R a and R a’ are independently selected from H, an optionally substituted amino acid side chain, and a nonpolar side chain comprising an optionally substituted C 1-14 alkyl chain R b is H, benzyl, or a substituted or unsubstituted (C 1-8 ) alkyl group, R c and R c’ are independently selected from H and optionally substituted C1-C6 alkyl, C3-C6 cycloalkyl, aryl or heteroaryl.

10. The linker-drug compound according to claim 9, wherein R 2 , R 3 and R 4 are independently selected from POM groups and POC groups when present.

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

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

13. The linker-drug compound according to any one of claims 1 to 12, wherein the linking moiety (L) comprises a structure according to formula VI or VII: wherein: m is an integer from 1 to 10, preferably 5; AA is an amino acid, preferably a natural amino acid; and p is 0, 1, 2, 3 or 4; q is an integer from 1 to 12, preferably 2; ES is absent or is an extended spacer selected from: wherein R 5 is 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 wherein V is H, ethyl, -(CH2CH2O) p -OMe, CH2CH2SO2Me or CH2CH2N(Me)2, and wherein p is an integer from 1 to 12.

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

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

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

17. The conjugate according to any one of the preceding claims 15 or 16, wherein the linking moiety comprises a cleavable linker.

18. The conjugate according to any one of claims 15 to 17, which is used as a drug.

19. The conjugate according to claim 18, which is used for the treatment of cancer, autoimmune diseases or infections.

20. A pharmaceutical composition comprising a conjugate according to any one of claims 15 to 19, and one or more pharmaceutical excipients.

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