Ligand-drug-conjugates comprising single molecular weight polysarcosine

Abstract

The present invention relates to a ligand-drug-conjugate (LDC) comprising a single molecular weight homopolymer, in particular a single molecular weight polysarcosine.

Description

[0001] This application is a divisional application of international application No. PCT / EP2018 / 078949 filed on October 23, 2018, entitled “Ligand-drug-conjugates comprising monomolecular weight polysarcosine”, which entered the Chinese national phase on April 22, 2020, and has the application number 201880068932.6. TECHNICAL FIELD

[0002] The present invention relates to monomolecular weight homopolymers, methods of making the homopolymers and their use, especially in conjugation technology.

[0003] The present invention also relates to ligand-drug-conjugates (LDCs) comprising monomolecular weight homopolymers, in particular monomolecular weight polysarcosine. BACKGROUND

[0004] Ligand-drug-conjugates (LDCs) consist of at least one ligand unit which is a polypeptide or protein covalently linked to at least one therapeutic, diagnostic or marker molecule (hereinafter referred to as drug or D) via a synthetic linker. The synthetic linker can comprise one or several bivalent arms for the attachment of the ligand unit and the drug unit, which can be selected from spacers, linkers and cleavable moieties. The linker can also carry any monovalent moiety which can improve the properties of the LDC such as storage stability, plasma stability or pharmacokinetic properties. The protein or polypeptide is usually the targeting unit but can have intrinsic therapeutic properties. The term antibody-drug-conjugates (ADCs) is often used when the ligand unit of the conjugate is an antibody or antibody fragment and is associated with a cytotoxic or chemotherapeutic drug.

[0005] The design of ADCs involves the consideration of numerous different factors: (i) the nature, number, overall hydrophobicity and position of the synthetic linker for conjugation on the ligand; (ii) the nature and mechanism of action of the drug; (iii) the structural elements responsible for drug release after internalization and during intracellular transport; (iv) the properties of the monoclonal antibody (mAb) and the selected antigen target. Recent methodologies have addressed some of the shortcomings of available ADCs such as heterogeneous drug loading (ADC subpopulations with different pharmacological properties), limited mAb-linker or drug-linker stability and suboptimal pharmacokinetic properties (Beck et al., Nat. Rev. Drug. Discov., 2017, 16(5), 315-337).

[0006] Another important factor to consider when designing conjugates is the drug ratio (or drug-antibody-ratio (DAR) of ADCs), which is the average number of drug units conjugated to the antibody. The result of the latest findings is that the actual trend in the field of ADCs is to generate homogeneous conjugates with low to moderate DAR (typically 2-4) and introduce them into the clinic. However, new linker-drug technologies have recently emerged, which aim to overcome the disadvantages of high- loaded ADCs (unfavorable pharmacokinetic properties and tendency to form aggregates, thus complicating the preparation of the conjugate). Such technologies have the potential to introduce the next generation of ADCs with improved efficacy, improved pharmacokinetic properties, improved therapeutic index and are able to target tumors with low target expression, slow internalization or inefficient intracellular processing. In order to achieve such high payload loading without sacrificing the pharmacokinetic properties and formulation stability, new linker-drug design approaches aiming to mask the apparent hydrophobicity of the cytotoxic payload are required.

[0007] In WO2014 / 093394A1, protein-polymer-drug conjugates showing high drug loading and strong binding to the target antigen are reported. The conjugates involve biodegradable and biocompatible poly[1-hydroxymethylethylene hydroxymethylformal] polymer entities that allow conjugation of about 12-25 cytotoxic molecules per mAb and have good pharmacokinetic properties. The main disadvantage of this approach is the extreme polydispersity of the final conjugate due to (i) the polydisperse nature of the linker, (ii) the heterogeneous number of cytotoxic molecules per polymer arm and (iii) the heterogeneous number of polymer arms grafted per mAb.

[0008] In WO2015 / 057699A2 and WO2016 / 059377A1, ADCs loaded with 8-36 drugs are reported by including orthogonal polyethylene glycol (PEG) moieties in the linker design. It is well known that PEG can improve the hydrophilicity, stability and circulation time of small drugs, proteins, bioconjugates and nanoparticles due to its hydrophilicity, biocompatibility and high hydration shell. However, PEG cannot escape disadvantages such as non-biodegradability, possible complement activation leading to hypersensitivity and unclear pharmacokinetics due to anti-PEG antibodies expressed by certain healthy individuals.

[0009] There is a need for ligand-drug-conjugates that combine (i) high drug loading while maintaining good pharmacokinetics and stability, (ii) complete homogeneity of the conjugate at the drug linker level (chemically monodisperse drug linker) and at the conjugate level (homogeneously loaded conjugate) and (iii) biodegradable hydrophilic homopolymers as hydrophobic masking moieties.

[0010] Polysarcosine (poly-N-methylglycine or PSAR) can replace PEG and can be used to design novel protein conjugates with improved properties. PSAR is a highly hydrophilic, biodegradable, non-immunogenic and water-soluble polymer that has been used in several delivery systems for drugs or diagnostics. So far, PSAR has only been provided in the form of a polydispersion, as it is obtained via a condensation ring-opening polymerization of sarcosine N-carboxyanhydride (NCA) or sarcosine N-thiocarboxyanhydride (NTA). Although an acceptable dispersity (gaussian distribution of molecular weights with a polydispersity index > 1) is relatively well defined, these polydisperse PSAR cannot be used in certain fields of application that require the use of shorter homopolymer compounds with a uniform length (uniqueness and specific molecular weight) and thus absolute homogeneity.

[0011] The use of discrete monodisperse PSAR for macromolecular modification is a requirement for the development of conjugates with absolute chemical homogeneity. Such homogenous conjugates have the advantage of sharing exactly the same pharmacological properties (pharmacokinetics and pharmacodynamics), are more straightforward to characterize, allow a better control of the reproducibility of the manufacturing process and meet the requirements of increasingly stringent regulatory requirements for biological conjugates. SUMMARY

[0012] According to the present invention, discrete monodisperse PSAR homopolymers with defined chain length have been obtained using a stepwise submonomer approach on resin. This method is inexpensive, easy to scale up, and can confer acceptable yields and excellent monomer purity to the final product. These monodisperse PSAR homopolymers are used in protein conjugate technology, thus providing ligand-drug-conjugates (LDCs) with improved drug loading capacity, pharmacokinetics and therapeutic efficacy.

[0013] The present invention thus provides monomolecular weight monofunctional homopolymers that meet the above requirements to be used in conjugate technology, in particular in LDCs.

[0014] Such homopolymers have the following formula (I)

[0015] (I)

[0016] wherein

[0017] R1and R2are different and

[0018] one of R1and R2is H or an inert group, the other of R1and R2is a functional reactive group, said group being reactive towards covalent binding with a bondable group under the reaction conditions in which the inert group is non-reactive,

[0019] Z1and Z2, identical or different, are optional spacers, and

[0020] n is 1 or more and k is 2 or more.

[0021] Before the present application is disclosed and described in detail, definitions of terms used in this text are given below.

[0022] Definitions

[0023] According to the present application, any compound (such as reactants, products, monomers, homopolymers, units) can be in the form of a salt, including acid addition salts, base addition salts, metal salts and ammonium and alkylated ammonium salts. Such salts are well known to those skilled in the art. In view of the intended use of the homopolymers of the present application, they are preferably in the form of pharmaceutically acceptable salts.

[0024] Monomeric weight homopolymers By monomolecular weight homopolymer is meant a homopolymer having a unique and specific molecular weight centered around the average molecular weight, as opposed to a mixture of homopolymers of the same nature but having a size and molecular weight distribution. A monomolecular weight homopolymer can be defined with one absolute formula having an absolute number of atoms.

[0025] As opposed to polydisperse homopolymers which are traditionally obtained by one-pot polymerization processes and have a PDI > 1, this monomolecular weight homopolymer can also be referred to as "monodisperse" with a polydispersity index (PDI) equal to 1. It is generally acknowledged in the present specification that the terms "monodisperse" and "disperse" are interchangeable, both defining a homopolymer having a unique and absolute molecular weight, formula and molecular structure, although the term "monodisperse" does not accurately reflect the process of preparation of the product.

[0026] Inert groups or Capping groups By any chemically non-reactive group terminating one end of a homopolymer is meant a group which is non-reactive as compared to a functionalized reactive group terminating the other end of the homopolymer under the defined reaction conditions. The resulting homopolymer is capped in some way by this inert group and is not intended to be covalently linked in use, in particular in LDC technology. In one embodiment, the group only becomes inert after it has been covalently bound to one end of the homopolymer.

[0027] A non-exhaustive list of inert groups includes: acyl groups, in particular acetyl, amido groups, alkyl groups, in particular C 1-20 alkyl, alkyl ether groups, alkyl ester groups, alkyl orthoester groups, alkenyl, alkynyl, aryl, aryl ester groups, tertiary amine groups, hydroxyl, aldehyde groups. Said inert group can also be selected from the same list of groups as the groups defining the functionalized reactive groups (see definition of functionalized reactive groups below).

[0028] A functionalized reactive group means any chemical moiety that is reactive towards covalently binding a conjugable group, said group being reactive when compared to inert groups under defined reaction conditions. In particular, it can bind the following groups: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl; halogen; activated ester such as N-hydroxysuccinimidyl ester, perfluorinated ester, nitrophenyl ester, nitrogen- hetero-benzotriazole and benzotriazole activated ester, acyl urea; alkynyl; alkenyl; azide; isocyanate; isothiocyanate; aldehyde; thiol reactive moiety such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; thiol; acrylate; mesylate; tosylate; triflate; hydroxylamine; chlorosulfonyl; boronic acid -B(OR')2derivative, wherein R' is hydrogen or alkyl.

[0029] A non-exhaustive list of functionalized reactive groups includes: carboxylic acid; primary amine; secondary amine; tertiary amine; hydroxyl; halogen; activated ester such as N-hydroxysuccinimidyl ester, perfluorinated ester, nitrophenyl ester, nitrogen- hetero-benzotriazole and benzotriazole activated ester, acyl urea; alkynyl; alkenyl; azide; isocyanate; isothiocyanate; aldehyde; thiol reactive moiety such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; thiol; acrylate; mesylate; tosylate; triflate; hydroxylamine; chlorosulfonyl; boronic acid -B(OR')2derivative, wherein R' is hydrogen or alkyl.

[0030] It should be mentioned that the terms "inert" and "functionalized reactive" for inert groups and functionalized reactive groups, respectively, are interdependent. This means that under defined reaction conditions of the homopolymer of the application defined by any of formulae (I), (II) and (III), the inert groups will not react, while the functionalized reactive groups will react to covalently bind a reactant. Thus, the inert groups and the functionalized reactive groups in the homopolymer of any of formulae (I), (II) and (III) are different, but they can be selected in their entirety from the same list of groups.

[0031] The term "group" in the context of a functionalized reactive group or inert group according to the present application is to be understood as a group that has no other function than being able to covalently bind a reactant or to remain inert, respectively, under defined reaction conditions.

[0032] For example, an alkyl group used alone or as part of an alkyl ether or alkyl ester means a saturated, straight-chain or branched hydrocarbon group having 1 to 20, preferably 1 to 12, more preferably 1 to 6, in particular 1 to 4 carbon atoms.

[0033] Alkenyl and alkynyl mean at least partially unsaturated, straight-chain or branched hydrocarbon groups having 2 to 20, preferably 2 to 12, more preferably 2 to 6, in particular 2 to 4 carbon atoms.

[0034] For example, aryl used alone or as part of an aryl ester refers to an aromatic group having one or more rings containing 6 to 14, preferably 6 to 10, in particular 6 ring carbon atoms.

[0035] For example, alkylene used alone or as part of an alkylene glycol refers to a divalent saturated straight chain or branched hydrocarbon group having 1 to 20, preferably 1 to 12, more preferably 1 to 6, in particular 1 to 4 carbon atoms.

[0036] Arylene refers to a divalent aryl group as defined above.

[0037] Heteroalkyl refers to a straight chain or branched hydrocarbon chain consisting of 1 to 20 or 1 to 10 carbon atoms and 1 to 10, preferably 1 to 3 heteroatoms selected from the group consisting of: O, N, Si and S, wherein the nitrogen and sulphur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatoms O, N and S can occupy any interior position of the heteroalkyl group or any position where one of the alkyl group is attached to the remainder of the molecule.

[0038] Heteroalkylene refers to a divalent heteroalkyl group as defined above. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.

[0039] C3-C8carbocycle refers to a 3, 4, 5, 6, 7 or 8 membered, monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring.

[0040] C3-C8carbocyclo refers to a divalent C3-C8carbocycle as defined above.

[0041] C3-C8heterocycle refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P or S. One or more N, C or S atoms in the heterocycle can be oxidized. The ring including the heteroatom(s) can be aromatic or non-aromatic. Unless otherwise specified, the heterocycle is attached to its side group at any heteroatom or carbon atom which affords a stable structure.

[0042] C3-C8heterocyclo refers to a divalent C3-C8heterocycle as defined above.

[0043] Furthermore, the terms alkyl, alkenyl, alkynyl, aryl, alkylene, arylene, heteroalkyl, heteroalkylene, C3-C8carbocycle, C3-C8carbocyclo, C3-C8heterocycle, C3-C8heterocyclo refer to groups optionally substituted with one or more substituents selected from the group consisting of: -X, -R’, -O -, -OR', =0, -SR', -S - , -NR'2, -NR'3, =NR', -CX3, -CN, -OCN, -SCN, -N=C=0, -NCS, -NO, -N02, =N2, -N3, -NRC(=0)R', -C(=0)R', -C(=0)NR'2, -SO3 - , -SO3H, -S(=0)2R', -OS(=0)2OR', -S(=0)2NR', -S(=0)R', -OP(=0)(OR')2, -P(=0)(OR')2, -P03 - , -P03H2, -C(=0)R', -C(=0)X, -C(=S)R', -C02R', -C02, -C(=S)OR', C(=0)SR', C(=S)SR', C(=0)NR'2, C(=S)NR'2, and C(=NR')NR'2, wherein each X is independently a halogen: -F, -Cl, -Br, or -I; and each R' is independently -H, -C1-C 20 alkyl, -C6-C 20 aryl, or -C3-C 14 heterocyclic.

[0044] An acyl group refers to a -CO-alkyl group, wherein alkyl is as defined above.

[0045] A monofunctional homopolymer includes a single type of monomer (e.g., N-methyl glycine monomers for poly sarcosine) with one end bearing a functionalized reactive group as defined above and the other end bearing either H or an inert group as defined above.

[0046] A support for solid phase peptide synthesis (SPPS) refers to a support commonly used in SPPS, a well-known method in which peptides anchored to a support (an insoluble polymer) are assembled by successive additions of Fmoc- or Boc-protected amino acids via repeated deprotection-wash-coupling-wash cycles. Each amino acid addition refers to the following cycle: (i) cleavage of the Nα-protecting group, (ii) a washing step, (iii) coupling of the fluorenylmethoxycarbonyl-(Fmoc-) or tert-butyloxycarbonyl-(Boc-) protected amino acid using a coupling agent and a non-nucleophilic base, and (iv) a washing step. Since the growing chain is bound to the support, excess reagents and soluble byproducts can be removed by simple filtration. Because repeated coupling reactions with hindered Fmoc- or Boc-protected N-methylated amino acids are difficult and often suboptimal, low crude purity, difficult purification, and low yields are expected with this technique. Examples of such supports are Wang resin, Rink amide resin, trityl and 2-chlorotrityl resins, PAM resin, PAL resin, Sieber amide resin, MBHA resin, HMPB resin, HMBA resin, which are commercially available and to which the peptide is directly or indirectly bound.

[0047] The term "orthogonal linker" refers to a branched linker unit assembly that connects a ligand to a homopolymer unit and a drug unit so that the homopolymer unit is in a parallel configuration relative to the drug unit (as opposed to a tandem configuration). An orthogonal linker is a scaffold with attachment sites for the components of a ligand-drug-conjugate (i.e., ligand, homopolymer, and drug unit). The term "parallel" is used to indicate the branching of the two components of a ligand-drug-conjugate (LDC), but is not used to indicate that the two components must be in close spatial proximity or have the same distance between them.

[0048] An exemplary graphical representation of an LDC having homopolymer (e.g., polysarcosine) units oriented parallel (i.e., branched) relative to the drug units is as follows:

[0049]

[0050] Wherein (L) is an orthogonal linker unit, w is 1 or greater, typically 1-5, preferably 1-4, more preferably 1-3, and even 1 and 2. This orthogonal structure should not be confused with a linear structure. An exemplary graphical representation of an LDC having homopolymer (e.g., polysarcosine) units oriented continuously (i.e., linearly) relative to the drug units is as follows:

[0051] Ligand-homopolymer-drug

[0052] A non-exhaustive list of orthogonal linkers includes: natural or unnatural amino acids, such as lysine, glutamic acid, aspartic acid, serine, tyrosine, cysteine, selenocysteine, glycine, homoalanine; amino alcohols; amino aldehydes; polyamines or any combination thereof. The person skilled in the art is able to select an orthogonal linker suitable for the intended LDC compound according to his knowledge. Advantageously, L is one or more natural or unnatural amino acids. In one embodiment, L is selected from glutamic acid, lysine and glycine.

[0053] The spacer is a divalent linear arm covalently linking two components of the ligand-drug-conjugate, such as:

[0054] - the ligand unit and the orthogonal linker unit,

[0055] - the orthogonal linker unit and the homopolymer unit,

[0056] - the orthogonal linker and the cleavable moiety,

[0057] - the cleavable moiety and the drug, or

[0058] - the orthogonal linker and the drug.

[0059] For example, the spacer is a divalent linear alkylene, preferably (CH2)4.

[0060] A non-exhaustive list of spacer units includes: alkylene, heteroalkylene (i.e. alkylene interrupted by at least one heteroatom selected from Si, N, O and S); alkoxy; polyether, such as polyalkylene glycol, typically polyethylene glycol; one or more natural or unnatural amino acids, such as glycine, alanine, proline, valine, N-methyl glycine; C3-C8heterocyclyl; C3-C8carbocyclyl; arylene and any combination thereof. The spacer can be linked to one or more drug units when present between the cleavable moiety and the drug unit or between the orthogonal linker and the drug unit. For example, the spacer can be linked to 1-4 drug units, preferably 1-2 drug units. In one embodiment, the spacer between the cleavable moiety and the drug unit is (4-amino-1,3-phenylene)dimethanol.

[0061] In one embodiment, the spacer unit is of formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII),

[0062] (XVII)

[0063] (XVIII)

[0064] (XIX)

[0065] (XX)

[0066] (XXI)

[0067] (XXII)

[0068] wherein the wavy line represents the point of attachment and R6is -C1-C 10 alkylene-, -C1-C 10 heteroalkylene-, -C3-C8carbocyclyl-, -O-(C1-C8alkyl)-, -arylene-, -C1-C 10 alkylene-arylene-, -arylene-C1-C 10 alkylene-, -C1-C 10 alkylene-(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-C1-C 10 alkylene-, -C3-C8heterocyclyl-, -C1-C 10 alkylene-(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-C1-C 10 alkylene-, -C1-C 10 alkylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8carbocyclyl-C(=O)-, -O-(C1-C8alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 alkylene-arylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-(C3-C8carbocyclyl)-C(=O)-, -(C3-C8carbocyclyl)-C1-C 10 alkylene-C(=O)-, -C3-C8heterocyclyl-C(=O)-, -C1-C 10 alkylene-(C3-C8heterocyclyl)-C(=O)-, -(C3-C8heterocyclyl)-C1-C 10 alkylene-C(=O)-, -C1-C 10 alkylene-NH-, -C1-C 10杂 alkylene-NH-, -C3-C8carbocyclyl-NH-, -O-(C1-C8alkyl)-NH-, -arylene-NH-, -C1-C 10 alkylene-arylene-NH-, -arylene-C1-C 10 alkylene-NH-, -C1-C 10alkylene-(C3-C8carbocyclyl)-NH-, -(C3-C8carbocyclyl)-C1-C 10 alkylene-NH-, -C3-C8heterocyclyl-NH-, -C1-C 10 alkylene-(C3-C8heterocyclyl)-NH-, -(C3-C8heterocyclyl)-C1-C 10 alkylene-NH-, -C1-C 10 alkylene-S-, -C1-C 10 heteroalkylene-S-, -C3-C8carbocyclyl-S-, -O-(C1-C8alkyl)-)-S-, -arylene-S-, -C1-C 10 alkylene-arylene-S-, -arylene-C1-C 10 alkylene-S-, -C1-C 10 alkylene-(C3-C8carbocyclyl)-S-, -(C3-C8carbocyclyl)-C1-C 10 alkylene-S-, -C3-C8heterocyclyl-S-, -C1-C 10 alkylene-(C3-C8heterocyclyl)-S-, -(C3-C8heterocyclyl)-C1-C 10 alkylene-S-, -C1-C 10 alkylene-O-C(=O)-, -C3-C8carbocyclyl-O-C(=O)-, -O-(C1-C8alkyl)-O-C(=O)-, -arylene-O-C(=O)-, -C1-C 10 alkylene-arylene-O-C(=O)-, -arylene-C1-C 10 alkylene-O-C(=O)-, -C1-C 10 alkylene-(C3-C8carbocyclyl)-O-C(=O)-,-(C3-C8carbocyclyl)-C1-C 10 alkylene-O-C(=O)-, -C3-C8heterocyclyl-O-C(=O)-, -C1-C 10 alkylene-(C3-C8heterocyclyl)-O-C(=O)-, -(C3-C8heterocyclyl)-C1-C 10 alkylene-O-C(=O)-.

[0069] Any one of the R6groups is optionally substituted with one or more substituents selected from the group consisting of -X, -R’, -O - , -OR’, =O, -SR’, -S - , -NR’2, -NR’3 +, =NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2and C(=NR')NR'2, wherein each X is independently a halogen: -F, -Cl, -Br or -I; each R' is independently -H, -C1-C 20 alkyl, -C6-C 20 aryl or -C3-C 14 heterocyclyl.

[0070] Advantageously, the spacer unit is of formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII),

[0071] (XVII)

[0072] (XVIII)

[0073] (XIX)

[0074] (XX)

[0075] (XXI)

[0076] (XXII)

[0077] wherein the wavy line represents the point of attachment and R6is -C1-C 10 alkylene-, -C1-C 10 heteroalkylene-, -C1-C 10 alkylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -arylene-C1-C 10 alkylene-C(=O)-, -arylene-C1-C 10 alkylene-O-C(=O)-.

[0078] Any one of the R6groups is optionally substituted with one or more =0.

[0079] Ligands refers to any macromolecule (polypeptide, protein, peptide, typically an antibody) that is commonly used in LDC (e.g. antibody drug conjugate) technology, or a small molecule (such as folate or aptamer) that can be covalently conjugated to the synthetic linker or drug-linker of the present work using bioconjugation technology (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally compounds that are selected for their targeting ability. A non-exhaustive list of ligands includes: proteins, polypeptides, peptides, antibodies, full-length antibodies and antigen-binding fragments thereof, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or any other cell-binding molecule or substance. The main class of ligands used to make conjugates is antibodies. As used herein, the term "antibody" is used in the broadest sense and encompasses monoclonal antibodies, polyclonal antibodies, modified monoclonal and polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), antibody fragments, and antibody mimetics (Affibody ® , Affilin ® , Affimer ® , Nanofitin ® , Cell Penetrating Alphabody ® , Anticalin ® , Avimer ® , Fynomer ® , Monobodies or nanoCLAMP ® ). An example of an antibody is trastuzumab. An example of a protein is human serum albumin.

[0080] As used herein, the term "antibody" includes intact antibodies and any antigen binding fragment (i.e., "antigen binding portion") or single chains thereof.

[0081] Naturally occurring "antibodies" are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region is comprised of one domain, CL L . The V H and V LRegions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each V H and V L are composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0082] As used herein, the term "antigen binding portion" of an antibody (or simply "antigen portion") refers to a full-length or one or more fragments of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and CH1 domains of a full-length antibody; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V H and CH1 domains of a full-length antibody; a Fv fragment consisting of the V L and V H domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a V H domain; and an isolated complementarity determining region (CDR) or any fusion proteins containing such antigen binding portions.

[0083] Furthermore, although the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single-chain protein, with V L and V HThe variable regions of the heavy and light chains are also referred to as the VH and VL regions, respectively. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Two antibody domains together, one from each of two polypeptide chains, can be referred to as a single chain Fv (scFv). The term "antibody" should be understood to encompass not only intact antibodies, but also antibody fragments, e.g., scFv, that are capable of binding to an antigen.

[0084] In particular embodiments, the ligand of the LDC is a chimeric antibody, a humanized antibody, or a human antibody.

[0085] As used herein, the term "human antibody" is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences or mutated versions of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al. (2000. J Mol Biol 296, 57-86).

[0086] A human antibody can include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0087] The term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity which has variable regions in which both the framework and CDR regions are derived from human sequences.

[0088] As used herein, "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, such as IgGl or IgG4) that is provided by heavy chain constant region genes.

[0089] The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen".

[0090] Cleavable groups(X), also called "releasable assembly unit", links the drug unit to the rest of the ligand-drug-conjugate. The function of the cleavable group is to release the drug at the site targeted by the ligand. Thus, this unit is able to form a cleavable bond for the release of the drug unit, for example upon enzymatic treatment or disulfide elimination mechanism. Recognition sites for enzymatic treatment are typically dipeptide cleavage sites (e.g. Val-Cit, Val-Ala or Phe-Lys) or sugar cleavage sites (e.g. glucuronide cleavage sites). For example, the cleavable group is a glucuronide group. This technology is well known to the person skilled in the art and, according to his knowledge, he is able to select a cleavable group suitable for the drug of the LDC (e.g. ADC) compound. For example, cleavable groups include disulfide-containing linkers that can be cleaved by disulfide exchange, acid-labile linkers that are cleavable at acidic pH, and linkers that can be cleaved by hydrolytic enzymes (e.g. peptidases, esterases, and glucuronidases). The cleavable group can be selected from the group consisting of

[0091] - one or more natural or non-natural amino acids, for example a cleavable peptide comprising 2-12 amino acids,

[0092] - a sugar moiety linked to a self-elimination group via an oxysaccharide bond,

[0093] - a disulfide linker, and

[0094] - an acid-labile linker that is hydrolysable in the lysosome.

[0095] Advantageously, the cleavable group can be selected from the group consisting of

[0096] - one or more natural or non-natural amino acids, for example a cleavable peptide comprising 2-12 amino acids, and

[0097] - a sugar moiety linked to a self-elimination group via an oxysaccharide bond,

[0098] When a sugar moiety is used, the self-elimination group is considered as part of the cleavable group. A "self-elimination group" is a trifunctional chemical moiety able to covalently link together three spaced chemical moieties, namely a sugar moiety (via a glycosidic bond), a drug D (directly or indirectly via a spacer Z), and an orthogonal linker L (directly or indirectly via a spacer Z). The glycosidic bond can be a glycosidic bond that is cleavable at the target site to initiate a self-elimination reaction sequence leading to the release of the drug.

[0099] When a disulfide linker is used, cleavage occurs between the two sulfur atoms of the disulfide. Various disulfide linkers are known in the art and can be adapted for use in the present disclosure, including for example those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), SMPT (N-succinimidyl-oxycarbonyl)-a-methyl-a-(2-pyridyl-dithio)toluene), and SPP (N-succinimidyl 4-(2-pyridyl-dithio)pentanoate). See, e.g., U.S. Patent No. 4,880,935.

[0100] In some embodiments, the cleavable unit is pH sensitive and, for example, comprises an acid-labile linker (e.g., a hydrazone, hemiketals, a thiosemicarbazone, a cis-aconitate, an orthoester, an acetal, or a ketal group) that is hydrolyzable in a lysosome that can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929). Such linkers are relatively stable under neutral pH conditions, such as in blood, but are unstable at pH 5.5 or 5.0, the approximate pH of a lysosome.

[0101] Ligand drug conjugates (LDC) Refers to any conjugate that binds a ligand and a drug as defined above and involves any means as described above and will be illustrated in the examples of the specification. When the ligand is an antibody, it can refer to an antibody drug conjugate (ADC), which is a preferred embodiment of the present disclosure.

[0102] Conjugable groups Refers to a group that can react with a functionalized reactive group to form a covalent bond. Thus, a conjugatable group comprises a reactive group that reacts with a functionalized reactive group under defined reaction conditions. In particular, a conjugatable group can comprise one of the following groups: a carboxylic acid; a primary amine; a secondary amine; a tertiary amine; a hydroxyl; a halogen; an activated ester such as N-hydroxysuccinimidyl ester, perfluorinated ester, nitrophenyl ester, azido-benzotriazole and benzotriazole activated ester, an acyl urea; an alkyne; an alkenyl; an azide; an isocyanate; an isothiocyanate; an aldehyde; a thiol reactive moiety such as maleimide, halo-maleimide, haloacetyl, pyridyl disulfide; a thiol; an acrylate; a mesylate; a tosylate; a triflate; a hydroxylamine; a chlorosulfonyl; a boronic acid -B(OR’)2derivative, where R’ is hydrogen or an alkyl.

[0103] Drugspharmaceutical or compound of any type, such as a cytotoxic compound, a cytostatic compound, an immunosuppressive compound, an anti-inflammatory compound or an anti-infective compound. Among cytotoxic compounds, one can cite calicheamicin; uncialamycin; auristatin (such as monomethyl auristatin E, known as MMAE); tubulysin analogs; maytansinoid; cryptophycin; benzodiazepine dimer (including pyrrolo[2,1-c][1,4]benzodiazepines known as PBD's); indolobenzodiazepine pseudodimer (IGN); duocarmycin; anthracycline (such as doxorubicin or PNU 159682); camptothecin analog (such as 7-ethyl-10-hydroxy camptothecin known as SN38 or exatecan); Bcl2 and Bcl-xl inhibitors; Thailanstatins; amatoxins (including alpha-amatoxin); kinesin spindle protein (KSP) inhibitors; vinorelbine; cyclin-dependent kinase (CDK) inhibitors; bleomycin; actinomycin or radionuclides and their complexing agents (such as DOTA 177 Lu). Among anti-inflammatory drugs, one can cite corticosteroids such as dexamethasone or fluticasone. Among anti-infective drugs, one can cite antibiotics such as rifampicin or vancomycin.

[0104] The application is now disclosed in more detail. Although it is described in more detail with reference to monomolecular weight poly sarcosine homopolymers, it is recognized that its scope extends to any monomolecular weight encompassed by the above formula (I). Moreover, the benefits of the application are clearly demonstrated in the LDC technology. Of course, its advantages are not limited to this technology and it can exhibit similar or better performance in any field where monomolecular weight, biocompatible, biodegradable homopolymers are needed.

[0105] The application thus more particularly relates to monomolecular weight homopolymers of sarcosine having the formula (II)

[0106] (II)

[0107] wherein

[0108] R1and R2are different and

[0109] one of R1and R2is H or an inert group, the other of R1and R2being a functional reactive group, said group being reactive towards covalent binding of a binding group under the reaction conditions in which the inert group is non-reactive,

[0110] Z1and Z2, identical or different, are optional spacers, and

[0111] k is 2 or more.

[0112] Further features of the homopolymers of formula (I), in particular of formula (II), are given below, used alone or in any combination.

[0113] k is an integer of at least 2, preferably at most 100, more preferably at most 50, in particular 2-30, and more particularly 2-24, 6-24 or 12-24.

[0114] In formula (I) or (II), the functional reactive group R1or R2may be chosen from the following groups:

[0115] - a carboxylic acid group,

[0116] - an amino group NRR" with R and R" each independently chosen from H, a (C1-C6)alkyl group optionally interrupted by at least one heteroatom chosen from O, N and S,

[0117] - a hydroxyl group,

[0118] - a halogen atom,

[0119] - a hydrazine (-NH2-NH2) group,

[0120] - a nitro group,

[0121] - a hydroxylamine group,

[0122] - an azido group,

[0123] - a (C2-C6)alkynyl group,

[0124] - a (C2-C6)alkenyl group,

[0125] - a thiol group,

[0126] - an activated ester group such as N-hydroxysuccinimidyl ester, perfluoroester, nitrophenyl ester, nitrogen- benzotriazole and benzotriazole activated ester, acylurea,

[0127] - a boronic acid - B(OR"")2group with R"" being a hydrogen atom or a C1-C6 alkyl group,

[0128] - a thiol reactive group such as maleimide, halo-maleimide, halo-acetyl, pyridyl disulfide,

[0129] - a mesylate group,

[0130] - a tosylate group,

[0131] - a triflate group,

[0132] - an aldehyde group,

[0133] - an isocyanate or isothiocyanate group,

[0134] - chlorosulfonyl,

[0135] - acrylate.

[0136] As mentioned above, the spacer group Z is optional, both Z1and Z2may be present, only one of Z1and Z2may be present, or both Z1and Z2may be absent. In the latter case, and when the homopolymer of the application is a homopolymer of sarcosine, it has the formula (III)

[0137] (III)

[0138] wherein R1, R2and k are as defined above.

[0139] In formula (I), formula (II) or formula (III), R1may be H or an inert group, and R2may be a functionalized reactive group, or R1may be a functionalized reactive group, and R2may be H or an inert group.

[0140] According to one preferred embodiment, the functionalized reactive group R1or R2is a secondary amine, and the inert group R1or R2is a carboxylic acid which remains unreacted and unbound on the final LDC structure.

[0141] In one preferred embodiment, in formula (I), formula (II) or formula (III), R1is selected from OH and NH2, and

[0142] When R1is OH, R2is COCH3and

[0143] When R1is NH2, R2is CO— G— COOH, G is CH2CH 2、 CH2CH2CH2, CH2CH2CH2CH2, CH2OCH2, CH2SCH2, CH2CH(CH3)CH2, CH2C(CH3)2CH2or CH2N(CH3)CH2.

[0144] The application also relates to a process for preparing a monomolecular weight homopolymer of formula (I), formula (II) or formula (III). Typically, each N-methylglycine monomer is assembled from two sub-monomers, namely a haloacetic acid and a methylamine, on a solid support. Each monomer addition refers to the following cycle: (i) acylation of the resin-bound secondary amine with the haloacetic acid and a carbodiimide or other suitable carboxylate activation method, (ii) a washing step, (iii) nucleophilic substitution of the resin-bound halogen with the methylamine, (iv) a washing step.

[0145] According to the process of the application, comprising the steps of:

[0146] a) reacting a compound of formula (IV) with an acid of formula (V) to obtain a compound of formula (VI)

[0147] (IV)

[0148] wherein R3 is a peptide synthesis solid support and m is 1 or more and less than k,

[0149] (V)

[0150] wherein Hal is halogen,

[0151] (VI)

[0152] wherein R3, m and Hal are as defined above,

[0153] b) reacting said compound of formula (VI) with methylamine

[0154] to obtain a compound of formula (VII)

[0155] (VII)

[0156] wherein R3 and m are as defined above,

[0157] c) repeating steps a) and b) until a compound of formula (VIII) is obtained

[0158] (VIII)

[0159] wherein R3 and m are as defined above,

[0160] d) reacting said compound (VIII) to obtain a compound of formula (IX),

[0161] (IX)

[0162] wherein R2 is an inert group, R3 is as defined above and k is as defined above,

[0163] e) cleavage reaction to obtain a monomolecular weight homopolymer of formula (III) as defined above.

[0164] According to one embodiment of the process, it comprises, in step a), reacting a compound of formula (IV) wherein R3 is a peptide synthesis solid support and m is 3, obtained by Fmoc-solid phase peptide synthesis process. They are well known to the person skilled in the art and, according to his knowledge, he is able to choose any suitable coupling agent, such as N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU).

[0165] In an alternative embodiment of the process of the application, the preparation of a monomolecular weight homopolymer comprises the following steps:

[0166] a) reacting a compound of formula (X) with an acid of formula (V) to obtain a compound of formula (XI)

[0167] (X)

[0168] wherein R3 is a peptide synthesis solid phase and m is 1 or more and less than k,

[0169] (V)

[0170] wherein Hal is a halogen,

[0171] (XI)

[0172] wherein R3, m and Hal are as defined above,

[0173] b) reacting said compound of formula (XI) with methylamine to obtain a compound of formula (XII)

[0174] (XII)

[0175] wherein R3 and m are as defined above,

[0176] c) repeating steps a) and b) until a compound of formula (XIII) is obtained,

[0177] (XIII)

[0178] wherein R3 and k are as defined above,

[0179] d) cleavage reaction to obtain a compound of formula (XIV)

[0180] (XIV)

[0181] wherein k is as defined above,

[0182] e) reacting said compound (XIV) with at least one of succinic anhydride, glutaric anhydride, adipic anhydride, diglycolic anhydride, thiodiglycolic anhydride, 3-methylglutaric anhydride, 3-3-dimethylglutaric anhydride or 4-methylmorpholine-2,6-dione to obtain a monomolecular weight homopolymer of formula (III) as defined above.

[0183] As mentioned before, the homopolymers of the present application can be used in LDC technology, without being limited to this technology.

[0184] The present application therefore also relates to a Ligand-Drug-Conjugate (LDC) having the following formula (XV)

[0185] (XV)

[0186] wherein,

[0187] L is an orthogonal linker allowing the (HP SMW ) to be in an orthogonal orientation relative to (X-D),

[0188] HP SMW is resulting from the covalent binding of a monomolecular weight homopolymer of the present application as described above to said orthogonal linker L,

[0189] D is a drug, in particular a cytotoxic drug, such as monomethyl auristatin E (MMAE) or SN38,

[0190] X is an optional cleavable moiety for the release of D,

[0191] Z is an optional spacer, and

[0192] a is 1 or more, b is 1 or more and m is 1 or more.

[0193] The monomolecular weight homopolymers, in particular the monomolecular weight poly sarcosine, provide efficient hydrophobic masking properties, reduced apparent hydrophobicity, better pharmacokinetic properties and improved in vivo activity of the conjugate when grafted in a parallel, i.e. orthogonal, orientation relative to the drug unit, compared to Ligand-Drug-Conjugates not comprising a monomolecular weight homopolymer grafted in parallel.

[0194] In an alternative embodiment, D is selected from the group consisting of biologically active molecules, therapeutic molecules such as anticancer drugs, imaging agents and fluorophores.

[0195] According to an alternative embodiment of the present application:

[0196] a is an integer of at least 1, preferably at most 6, more preferably at most 3, in particular 2, and more particularly 1, and / or

[0197] b is an integer of at least 1, preferably at most 6, more preferably at most 3, in particular 2, and more particularly 1, and / or

[0198] m is an integer of at least 1, preferably at most 30, more preferably at most 15, in particular 8, and more particularly 4.

[0199] Advantageously, the monomolecular weight homopolymer is a polysarcosine.

[0200] In one embodiment, there is a spacer Z between L and the ligand, and / or between L and HP SMW , and / or between L and X, and / or between X and D.

[0201] Typically, the orthogonal linker connects the releasable assembled drug unit (XD) or drug unit (D) through one or more linker unit assemblies in such a way that the (XD) or (D) unit is in a parallel configuration (as opposed to a tandem configuration) with respect to the homopolymer unit.

[0202] The present application also relates to intermediate compounds of formula (XVI)

[0203] (XVI)

[0204] wherein

[0205] L is an orthogonal linker,

[0206] HP SMW is generated by covalent binding of a monomolecular weight homopolymer of the present application to said orthogonal linker L,

[0207] D is a cytotoxic drug,

[0208] X is an optional cleavable moiety for releasing D,

[0209] Z is an optional spacer, said spacer being capable of binding a ligand, and

[0210] a is 1 or more and b is 0, 1 or more.

[0211] The present disclosure also relates to compounds of formula (XXIII)

[0212] (XXIII)

[0213] wherein, as defined above,

[0214] R6is -C1-C 10alkylene-, -Ci-C 10 heteroalkylene-, -C3-C8carbocyclyl-, -0-(Ci-C8alkyl)-, -arylene-, -Ci-C 10 alkylene-arylene-, -arylene-Ci-C 10 alkylene-, -Ci-C 10 alkylene-(C3-C8carbocyclyl)-, -(C3-C8carbocyclyl)-Ci-C 10 alkylene-, -C3-C8heterocyclyl-, -Ci-C 10 alkylene-(C3-C8heterocyclyl)-, -(C3-C8heterocyclyl)-Ci-C 10 alkylene-, -Ci-C 10 alkylene-C(=0)-, -Ci-C 10 heteroalkylene-C(=0)-, -C3-C8carbocyclyl-C(=0)-, -0-(Ci-C8alkyl)-C(=0)-, -arylene-C(=0)-, -Ci-C 10 alkylene-arylene-C(=0)-, -arylene-Ci-C 10 alkylene-C(=0)-, -Ci-C 10 alkylene-(C3-C8carbocyclyl)-C(=0)-, -(C3-C8carbocyclyl)-Ci-C 10 alkylene-C(=0)-, -C3-C8heterocyclyl-C(=0)-, -Ci-C 10 alkylene-(C3-C8heterocyclyl)-C(=0)-, -(C3-C8heterocyclyl)-Ci-C 10 alkylene-C(=0)-, -Ci-C 10 alkylene-NH-, -Ci-C 10 heteroalkylene-NH-, -C3-C8carbocyclyl-NH-, -0-(Ci-C8alkyl)-NH-, -arylene-NH-, -Ci-C 10 alkylene-arylene-NH-, -arylene-Ci-C 10 alkylene-NH-, -Ci-C 10 alkylene-(C3-C8carbocyclyl)-NH-, -(C3-C8carbocyclyl)-Ci-C 10 alkylene-NH-, -C3-C8heterocyclyl-NH-, -Ci-C 10 alkylene-(C3-C8heterocyclyl)-NH-, -(C3-C8heterocyclyl)-Ci-C 10 alkylene-NH-, -Ci-C 10 alkylene-S-, -Ci-C 10Heteroalkylene-S-, -C3-C8 carbocyclyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-S-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclyl-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-S-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-S-, –C1-C 10 Alkylene-OC(=O)-, -C3-C8 carbocyclyl-OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene-OC(=O)-, -C1-C 10 Alkylene-arylene-OC(=O)-, -arylene-C1-C 10 Alkylene-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-OC(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-OC(=O)-, -C3-C8 heterocyclyl-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-OC(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-OC(=O)-,

[0215] Any of the R6 groups is optionally substituted by one or more substituents selected from: -X, -R', -O - 、-OR'、=O、-SR'、-S - 、-NR'2、-NR'3 + ,=NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 -, -PO3H2, -C(=0)X, -C(=S)R', -CO2R', -CO2", -C(=S)OR', C(=0)SR', C(=S)SR', C(=0)NR'2, C(=S)NR'2and C(=NR')NR'2, wherein each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C1-C 20 alkyl, -C6-C 20 aryl, or -C3-C 14 heterocyclic,

[0216] Z is an optional spacer,

[0217] L is an orthogonal linker,

[0218] X is an optional cleavable moiety for releasing D,

[0219] D is a cytotoxic drug,

[0220] a is 1 or more and b is 0, 1 or more, and

[0221] HP SMW is generated by covalent binding of a monomeric weight homopolymer of the present application to said orthogonal linker L.

[0222] According to a preferred embodiment, HP SMW is generated by covalent binding of a poly-sarcosine homopolymer of the present application to said orthogonal linker L. In this case, in formulae (XV), (XVI) and (XXIII), HP SMW represents

[0223] , or

[0224]

[0225] wherein the wavy bond represents the point of attachment to L or a spacer Z (if present),

[0226] k is 2 or more, preferably k is 2-50, and

[0227] R4represents a capping group.

[0228] Advantageously, R4represents -R', -0 - , -OR', -SR', -S - , -NR'2, -NR'3 + , =NR', -CX3, -CN, -NRC(=0)R', -C(=0)R', -C(=0)NR'2, -SO3 -, -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 or C(=NR')NR'2, wherein each X is independently halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C1-C 20 Alkyl, -C6-C 20 Aryl, or -C3-C 14 Typically, R4 is -OR', -NR'2 or -C(=O)R'.

[0229] In one embodiment, the present invention also relates to a ligand-drug-conjugate (LDC) having the following formula (XV):

[0230] (XV)

[0231] in

[0232] The ligand is an antibody,

[0233] L is the allowed HP SMW an orthogonal linker in an orthogonal orientation relative to (XD) selected from a natural or unnatural amino acid; an amino alcohol; an amino aldehyde; a polyamine or any combination thereof,

[0234] HP SMW represent

[0235] ,or

[0236]

[0237] Wherein, the wavy bond represents the point of attachment to L or the spacer group Z (if present),

[0238] k is 2 or greater, preferably k is 2-50, and

[0239] R4 represents a capping group.

[0240] D is a drug, in particular a cytotoxic drug, such as monomethyl auristatin E (MMAE) or SN38,

[0241] X is an optional cleavable moiety for releasing D, selected from

[0242] o one or more natural or non-natural amino acids, e.g. a cleavable peptide comprising 2-12 amino acids,

[0243] o a sugar moiety linked to a self-elimination group via an oxysaccharide bond,

[0244] o a disulfide linker, and

[0245] o an acid-labile linker hydrolysable in the lysosome,

[0246] Z is an optional spacer, which can also be present between L and X, and / or X and D, and / or L and HP SMW 3- C8carbocyclyl; arylene and any combination thereof,

[0247] a is 1 or more, b is 1 or more, and m is 1 or more.

[0248] The present application also relates to a pharmaceutical composition comprising at least one LDC compound according to the present application and a pharmaceutically acceptable carrier.

[0249] The present disclosure also relates to the use of a LDC compound as described above as a medicament.

[0250] The compound of formula (XXIII) can be used as such without comprising a ligand, as the maleimide moiety can react with a protein (such as serum albumin) in vivo, which then becomes the ligand. Thus, the present disclosure also relates to the use of a compound of formula (XXIII) as described above as a medicament. BRIEF DESCRIPTION OF DRAWINGS

[0251] Figure 1 Representative hydrophobic interaction chromatogram according to Example 12.

[0252] Figure 2 Representative hydrophobic interaction chromatogram according to Example 13.

[0253] Figure 3 Representative pharmacokinetic curve in mice according to Example 14.

[0254] Figure 4A Representative tumor volume over time according to Example 15. Figure 4B Representative percentage survival of mice according to Example 15.

[0255] Figure 5 Representative pharmacokinetic curve in mice according to Example 16.

[0256] Figure 6 ​Representative tumor volume over time according to Example 17. DETAILED DESCRIPTION

[0257] Materials and general methods

[0258] All solvents and reagents were obtained from commercial sources (Sigma-Aldrich, Alfa Aesar, Fluorochem, Thermo Fisher, Carbosynth) and used without further purification unless otherwise stated. Anhydrous DMF and DCM were purchased from Sigma-Aldrich. Fmoc-amino acids, 2-chlorotrityl and Rink amide resin were purchased from Novabiochem. Monomethyl auristatin E (MMAE) and 7-ethyl-10-hydroxy camptothecin (SN38) were purchased from DC Chemicals. PNU159682 was purchased from Kerui Biotechnology Co. Ltd. and exatecan mesylate was purchased from Angene Chemical. Human albumin (cat. no. A3782) was purchased from Sigma-Aldrich. Anti-CD19 and anti-CD22 antibodies were purchased from Euromedex. Trastuzumab (Herceptin®) was purchased from Roche. Synthesis on resin was performed in empty SPE plastic tubes equipped with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All chemical reactions were performed at room temperature under an inert argon atmosphere unless otherwise stated. ® IV) was purchased from Roche. Synthesis on resin was performed in empty SPE plastic tubes equipped with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All chemical reactions were performed at room temperature under an inert argon atmosphere unless otherwise stated.

[0259] Liquid nuclear magnetic resonance spectra were recorded on a Bruker Fourier 300 HD spectrometer, calibrated using the residual solvent peak. Mass spectrometry analysis was performed by the Centre Commun de Spectrométrie de Masse (CCSM) of the UMR 5246 CNRS Institute of Claude Bernard Lyon 1 University.

[0260] Flash® using Interchim (Flash®spherical HP 50 pm) or Biotage ® ZIP ® Flash® using Interchim (Flash®spherical HP 50 pm) or Biotage ® Companion ® Flash® using Interchim (Flash®spherical HP 50 pm) or Biotage ® Flash® using Interchim (Flash®spherical HP 50 pm) or Biotage ®Reversed-phase chromatography was performed on SNAP Ultra C18 (25 pm) columns or Interchim PuriFlash RP-AQ (30 pm) columns. Pre-coated 40-63 pm silica gel (Macherey-Nagel), HPLC-UV (Agilent 1050) or UHPLC-UV / MS (Agilent 1260 HPLC system equipped with a Bruker Impact II Q-ToF mass spectrometer) were used to monitor and analyze chemical reactions and compound characterization by thin-layer chromatography, respectively. TM Thermo UltiMate 3000 UHPLC system with Q-ToF mass spectrometer or Agilent 1260 HPLC system equipped with a Bruker MicrOTOF-QII mass spectrometer) were used to monitor and analyze chemical reactions and compound characterization by thin-layer chromatography, respectively.

[0261] HPLC Method 1 : Agilent 1050 equipped with DAD detector. Mobile phase A was water and mobile phase B was acetonitrile. The chromatographic column was Agilent Zorbax SB-Aq 4.6x150mm 5pm (room temperature). Gradient from 5%B to 95%B in 20 min, then held at 95%B for 5 min. Flow rate was 1.5 mL / min. UV detection was monitored at 214 nm.

[0262] HPLC Method 2: Agilent 1050 equipped with DAD detector. Mobile phase A was water and mobile phase B was acetonitrile. The chromatographic column was Agilent Zorbax SB-Aq 4.6x150mm 5pm (room temperature). Gradient from 0%B to 50%B in 30 min, then held at 50%B for 5 min. Flow rate was 1.0 mL / min. UV detection was monitored at 214 nm.

[0263] HPLC Method 3: Same as HPLC Method 1 but with 0.1% TFA in mobile phase A.

[0264] HPLC Method 4: Same as HPLC Method 2 but with 0.1% TFA in mobile phase A.

[0265] UHPLC Method 5: Thermo UltiMate 3000 UHPLC system + Bruker Impact II Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid and mobile phase B was acetonitrile + 0.1% formic acid. The chromatographic column was Agilent PLRP-S 1000A 2.1x150mm 8pm (80°C). Gradient from 10%B to 50%B in 25 min. Flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The m / z range of the Q-ToF mass spectrometer was 500-3500 (ESI TM UHPLC Method 5: Thermo UltiMate 3000 UHPLC system + Bruker Impact II Q-ToF mass spectrometer. Mobile phase A was water + 0.1% formic acid and mobile phase B was acetonitrile + 0.1% formic acid. The chromatographic column was Agilent PLRP-S 1000A 2.1x150mm 8pm (80°C). Gradient from 10%B to 50%B in 25 min. Flow rate was 0.4 mL / min. UV detection was monitored at 280 nm. The m / z range of the Q-ToF mass spectrometer was 500-3500 (ESI +). The Bruker Compass ® The data were deconvoluted using the MaxEnt algorithm included in the Bruker Compass

[0266] HPLC Method 6: Agilent 1050 equipped with DAD detector. Mobile phase A was water + 5 mM ammonium formate and mobile phase B was acetonitrile. The column was Agilent Poroshell 120 EC-C18 3.0x50mm 2.7pm (room temperature). Gradient from 5%B to 90%B in 10 min, then, hold at 90%B for 2 min. The flow rate was 0.8 mL / min. UV detection was monitored at 214 nm.

[0267] The following examples 1-4 illustrate the synthesis of monomolecular weight poly sarcosine which is part of the present application and involves different solid phase synthesis methods.

[0268] Example 1 : Synthesis of poly-sarcosine compounds (resin on synthesis method 1 )

[0269] The reaction scheme is as follows.

[0270]

[0271] 1.1) General method

[0272] The synthesis on resin was performed in empty SPE plastic tubes equipped with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial theoretical resin loading of 0.63 mmol / g (manufacturer indicated scale). All reactions were performed at room temperature unless otherwise stated.

[0273] 1.2) Resin loading

[0274] Typically, 500 mg NovaGEL TM Rink Amide beads (0.63 mmol / g, Novabiochem) were swelled in 5 mL DMF for 15 min. The first monomer was added by reacting 10 equivalents of bromoacetic acid with 13 equivalents of diisopropylcarbodiimide (Sigma-Aldrich) in 5 mL DMF for 60 min at room temperature, followed by extensive washing with DMF (5 x 5 mL). The bromoacetylated resin was incubated with 5 mL of 40% (wt) aqueous methylamine (Sigma-Aldrich) for 30 min on a horizontal shaker, followed by extensive washing with DMF (5 x 5 mL) and DCM (5 x 5 mL). The obtained resin was ready for elongation.

[0275] 1.3) Extension of sarcosine compounds

[0276] The extension of the sarcosine oligomer is performed by alternating bromoacetylation and amine displacement steps until the desired length is obtained. The bromoacetylation step is performed by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in 5 mL of DMF. The mixture is stirred for 30 minutes, drained and washed with DMF (4 times 5 mL). For the amine displacement step, 5 mL of a 40% (wt) aqueous methylamine solution (Sigma-Aldrich) is added, the vessel is shaken for 30 minutes, drained and washed with DMF (4 times 5 mL) and DCM (4 times 5 mL).

[0277] 1.4) Cleavage from the resin

[0278] The cleavage of the sarcosine oligomer is performed using 5 mL of a TFA / triisopropylsilane (95:5) solution at room temperature under stirring. The resin is filtered and the obtained solution is evaporated under reduced pressure to obtain a transparent material in oil.

[0279] At this stage, the PSARn-N(CH3)H is dissolved in water for purification (see below) or for final functionalization.

[0280] 1.5) Final functionalization

[0281] To obtain the PSARn-CH2-CH2-COOH compound, the N-terminal of the oligomer is functionalized using 2.5 equivalents of succinic anhydride and 10 equivalents of DIPEA in dry acetonitrile. The mixture is stirred at room temperature for 1 hour and the volatiles are removed under reduced pressure.

[0282] 1.6 Purification

[0283] The PSAR compounds are purified on an Interchim ® The PSAR compounds are purified on an Interchim

[0284] 1.7) Monomolecular weight sarcosine compounds

[0285] The resulting PSAR compounds are listed in the following Table 1.

[0286] Table 1

[0287]

[0288] Example 2: Synthesis of poly-sarcosine compounds (resin on synthesis method 2)

[0289] The reaction scheme is as follows.

[0290]

[0291] 2.1) General method

[0292] Resin synthesis was performed in empty SPE plastic tubes equipped with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All synthesis yields reported are based on an initial resin loading of 1.1 mmol / g (labelled range indicated by the manufacturer). All reactions were performed at room temperature unless otherwise stated.

[0293] 2.2) Synthesis of Fmoc-Sar-Sar-OH

[0294]

[0295] 2.2.1) Synthesis of Fmoc-Sar-Sar-OtBu

[0296] In a round bottom flask, Fmoc-Sar-OH (2000 mg / 6.42 mmol) and HATU (2443 mg / 6.42 mmol) were dissolved in 28 mL of dry DMF. DIPEA (2491 mg / 19.27 mmol) was added and the mixture was stirred at room temperature for 3 minutes. Then, sarcosine tert-butyl ester hydrochloride (1167 mg / 6.42 mmol) was added and the reaction mixture was stirred at room temperature for 90 minutes. Volatiles were removed under vacuum and the residue was diluted with water and extracted 3 times with EtOAc. The organic phase was dried over MgS04, filtered and evaporated under vacuum to give a solid crude. The crude was taken up in EtOAc / DCM 80:20 (v / v) and the white insoluble material was removed by filtration. The filtrate was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 20:80) to give Fmoc-Sar-Sar-OtBu as a white solid (2310 mg / 82%). HRMS m / z (ESI + ) : calc. for [M+H] = 439.2227; found [M+H] = 439.2234; error = -1.5 ppm. HPLC Method 1 retention time = 13.3 min. TLC eluted with 100% EtOAc: Rf= 0.8. + +

[0297] 2.2.2) Removal of the tert-butyl ester

[0298] ​​Fmoc-Sar-Sar-OtBu (2310 mg / 5.27 mmol) was dissolved in 20 mL DCM and 8.5 mL TFA was added slowly. The solution was stirred at room temperature until complete tert-butyl ester deprotection was observed by HPLC (about 2 hours). Then, the volatiles were removed under vacuum and the residue was triturated with diethyl ether to give Fmoc-Sar-Sar-OH as a white solid (1690 mg / 84%). 1 H NMR (500 MHz, DMSO-d6, 100°C) δ (ppm) 2.84 (s, 3H), 2.93 (s, 3H), 4.01(s, 2H), 4.05 (s, 2H), 4.25 (t, J = 4.3 Hz, 1H), 4.34 (d, J = 6.4 Hz, 2H),7.33 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.63 (d, J = 7.4 Hz, 2H),7.85 (d, J = 7.5 Hz, 2H). HRMS m / z (ESI + ): Calcd [M+H] + = 383.1601 ; Found [M+H] + = 383.1602 ; Error = 0.0 ppm. HPLC Method 1 retention time = 6.2 min. TLC eluting with DCM / MeOH 85:15 (v / v): Rf = 0.65.

[0299] 2.3) Resin loading

[0300] Typically, 1000 mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1% DVB, 1.1 mmol / g, Novabiochem) were swelled in 10 mL of DCM for 10 min. Fmoc-Sar-OH (1.2 eq) previously dissolved in 10 mL of dry DCM was added to the resin. DIPEA (5 eq) was added and the reaction vessel was stirred at room temperature for 2 hours. After draining, the resin was washed with DCM (3 times), DMF (2 times), DCM (3 times) and MeOH (2 times). The resin was dried under high vacuum overnight. The level of substitution was evaluated from the weight gain of the resin and / or from Fmoc cleavage test (absorbance measurement at 301 nm) and found to be quasi-quantitative (typically 0.95-1.1 mmol / g). The resin was stored at -20°C until further use.

[0301] 2.4) Fmoc-Sar-Sar-OH coupling procedure

[0302] The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 times of 15 minutes at room temperature. Then, the resin was washed with DMF (4 times) and DCM (4 times). To the resin was added a solution of Fmoc-Sar-Sar-OH (3 equivalents), HATU (2.85 equivalents) and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 2 hours and the resin was thoroughly washed with DMF (5 times) and DCM (5 times). The resin was dried under vacuum and stored at -20°C until further use.

[0303] 2.5) Extension of the poly-sarcosine compound

[0304] The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 times of 15 minutes at room temperature. Then, the resin was washed with DMF (4 times) and DCM (4 times).

[0305] The extension of the poly-sarcosine oligomer was performed by alternating bromoacetylation and amine displacement steps until the desired length was obtained. The bromoacetylation step was performed by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 minutes, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) aqueous methylamine (1.5 mL per 100 mg of resin) was added, the vessel was shaken for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0306] 2.6) Final acetylation

[0307] When the desired oligomer length was obtained, the N-terminal was acetylated using a capping solution made of acetic anhydride / DIPEA / DMF (1 :2:3 v / v) (the vessel was shaken for 30 minutes). The solution was drained and the reaction was repeated once with fresh capping solution. The resin was washed with DMF (4 times) and DCM (4 times).

[0308] 2.7) Resin cleavage

[0309] The poly-sarcosine oligomer was cleaved from the resin with a solution of HFIP / DCM (20:80 v / v) under stirring for 30 minutes. The resin was filtered and the volatiles were removed under reduced pressure to give a solid crude.

[0310] 2.8) Purification

[0311] The Interchim ®RP-AQ (30 pm) columns. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA.

[0312] 2.9) Monomeric weight poly-sarcosine compounds

[0313] The resulting PSAR compounds are listed in Table 2 below

[0314] Table 2

[0315]

[0316] Example 3: Synthesis of poly-sarcosine compounds with one or several azido functionalized orthogonal linkers (resin on synthesis method 3) Example 4: Synthesis of poly-sarcosine compounds with terminal non-orthogonal azido functionalized linkers (resin on synthesis method 4)

[0317] The reaction scheme is as follows.

[0318]

[0319] 3.1) General method

[0320] On-resin synthesis was performed in empty SPE plastic tubes equipped with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial resin loading of 1.1 mmol / g (manufacturer indicated label range). All reactions were performed at room temperature unless otherwise stated. Starting materials were obtained as described in Example 2 above.

[0321] 3.2) Step (1)

[0322] A solution of 3 moles of 2-azidoethan-1 -amine in DMF (1 mL per 100 mg of resin) was added, the vessel was shaken for 45 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0323] 3.3) Step (2)

[0324] To the resin was added a solution of commercially available 2-[4-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)phenyl]acetic acid (5 equivalents), COMU (4.9 equivalents) and DIPEA (4.9 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 90 minutes and the resin was washed with DMF (3 times) and DCM (3 times).

[0325] 3.4) Step (3)

[0326] The target compound was cleaved from the resin using 1% TFA in DCM (v / v) with stirring for 5 minutes (repeated twice). The resin was filtered and the volatiles removed under reduced pressure to give a solid crude which was purified using the protocol described above in Example 2.

[0327] 3.5) Step (4)

[0328] The final bromoacetylation step was performed by the addition of 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times). At this stage the compound was cleaved from the resin (as described in step (3)) and purified using the protocol described above in Example 2.

[0329] 3.6) Step (5)

[0330] The final bromoacetylation step was performed by the addition of 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times). At this stage the compound was cleaved from the resin (as described in step (3)) and purified using the protocol described above in Example 2.

[0331] 3.7) Step (6)

[0332] The bromoacetylation step was performed by the addition of 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 minutes, drained and washed with DMF (4 times). For the amine displacement step, a 40% (wt) aqueous methylamine solution was added (1.5 mL per 100 mg of resin), the vessel was shaken for 30 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0333] 3.8) Step (7)

[0334] The bromoacetylation step was performed by the addition of 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 minutes, drained and washed with DMF (4 times). For the amine displacement step, a 3 molar solution of 2-azidoethan-1-amine in DMF was added (1 mL per 100 mg of resin), the vessel was shaken for 45 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0335] 3.9) Step (8)

[0336] To the resin was added a solution of commercially available 2-[4-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)phenyl]acetic acid (5 eq), COMU (4.9 eq) and DIPEA (4.9 eq) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 90 minutes and the resin washed with DMF (3 times) and DCM (3 times). At this stage the compound was cleaved from the resin (as described in step (3)) and purified using the protocol described above in Example 2.

[0337] 3.10) Monomeric poly sarcosine compounds

[0338] The resulting PSAR compounds are listed in Table 3 below.

[0339] Table 3

[0340]

[0341]

[0342] Example 5: Synthesis of polyethylene glycol (PEG) compounds with azido functionalized orthogonal linkers (resin on synthesis method 5) Example 6: Synthesis of intermediate compounds based on MMAE, SN38, exatecan and PNU159682

[0343] The reaction scheme is as follows.

[0344]

[0345] 4.1) General methods

[0346] Resin synthesis was performed in empty SPE plastic tubes fitted with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All synthesis yields reported are based on an initial theoretical resin loading of 0.47 mmol / g (manufacturer indicated label range). All reactions were performed at room temperature unless otherwise stated.

[0347] 4.2) Resin loading

[0348] Typically, 500 mg Ramage ChemMatrix ®Beads (0.47 mmol / g, Sigma-Aldrich) were swelled in 5 mL DCM for 15 min. The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 times, 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). To the resin was added Fmoc-L-γ-azidohomoalanine-OH (3 equivalents), HATU (2.9 equivalents) and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 1.5 h and the resin was washed thoroughly with DMF (5 times) and DCM (5 times). Unreacted sites were acetylated using a capping solution made of acetic anhydride / DIPEA / DMF (1 :2:3 v / v) (vessel shaken for 30 min). The solution was drained and the resin was washed with DMF (4 times) and DCM (4 times). The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 times, 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times).

[0349] 4.3) Fmoc-Sar-Sar-OH coupling procedure

[0350] To the resin was added Fmoc-Sar-Sar-OH (4 equivalents), HATU (3.9 equivalents) and DIPEA (8 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 2 h and the resin was washed thoroughly with DMF (4 times) and DCM (4 times). The resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for 2 times, 15 min at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times).

[0351] 4.4) Extension of the poly-sarcosine compound

[0352] The extension of the poly-sarcosine oligomer was performed by alternating bromoacetylation and amine displacement steps until the desired length was obtained. The bromoacetylation step was performed by adding 10 equivalents of bromoacetic acid and 13 equivalents of diisopropylcarbodiimide in DMF (2 mL per 100 mg of resin). The mixture was stirred for 30 min, drained and washed with DMF (4 times). For the amine displacement step, 40% (wt) methylamine in water was added (1.5 mL per 100 mg of resin), the vessel was shaken for 30 min, drained and washed with DMF (4 times) and DCM (4 times).

[0353] 4.5) Step (6)

[0354] To the resin was added a solution of commercially available 2-[4-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)phenyl]acetic acid (5 eq), COMU (4.9 eq) and DIPEA (4.9 eq) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 90 minutes and the resin was washed with DMF (3 times) and DCM (3 times).

[0355] 4.5) Cleavage from resin and purification

[0356] The oligomers were cleaved from the resin using 5 mL of TFA / DCM (50:50) solution at room temperature with stirring for 30 minutes. This was repeated once and the combined filtrates were evaporated under reduced pressure to give a solid crude which was purified as described above in Example 2.

[0357] 4.6) Monomeric weight poly sarcosine compounds

[0358] The resulting PSAR compounds are listed in Table 4 below.

[0359] Table 4

[0360]

[0361] 6.1 ) Synthesis of the compound Alkyn- glucuronate-MMAE 6.4 Synthesis of the compound Alkyn-SN38

[0362] The reaction scheme is as follows.

[0363]

[0364] Synthesis on resin was performed in empty SPE plastic tubes fitted with 20 pm polyethylene glass frits (Sigma-Aldrich). Stirring was performed using a Titramax 101 horizontal shaker (Heidolph). All reported synthesis yields are based on an initial resin loading of 1.1 mmol / g (manufacturer indicated label range). All reactions were performed at room temperature unless otherwise stated.

[0365] 5.2) Resin loading

[0366] Typically, 200 mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1 % DVB, 1.1 mmol / g, Novabiochem) were swelled in 4 mL of DCM for 10 minutes. Fmoc-PEG 12- CH2CH2COOH (PurePEG™, 1.2 eq) was added to the resin. DIPEA (3 eq) was added and the reaction vessel was stirred at room temperature for 1 hour. 300 μL of MeOH was added to quench unreacted resin. After shaking for 10 minutes, the solution was drained and the resin was washed with DMF (3 times) and DCM (3 times). The resin was dried under high vacuum.

[0367] 5.2) Step (1)

[0368] The resin was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2 x 15 minutes at room temperature. The resin was then washed with DMF (4 times) and DCM (4 times). The bromoacetylation step was performed by adding 10 eq of bromoacetic acid and 13 eq of diisopropylcarbodiimide (2 mL per 100 mg resin) in DMF. The mixture was stirred for 30 minutes, drained and washed with DMF (4 times). For the amine displacement step, 3 moles of 2-azidoeth-1-amine in DMF (1 mL per 100 mg resin) was added, the vessel was shaken for 45 minutes, drained and washed with DMF (4 times) and DCM (4 times).

[0369] 5.3) Step (2)

[0370] The coupling step of 2-[4-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)phenyl]acetic acid was performed as described above in Example 3 and the compound was cleaved from the resin. The purification of the compound was performed as described above in Example 2.

[0371] 5.4) Mono-molecular weight PEG compounds

[0372] The resulting PEG compounds are listed in Table 5 below.

[0373] Table 5

[0374]

[0375] 6.6 Synthesis of the compound Alkyn-glucuronate-exatecan

[0376] 6.7 Synthesis of the compound Alkyn-PNU159682

[0377]

[0378] The starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427-3433) 110.8 mg (0.087 mmol) was dissolved in MeOH (10 mL) at 0°C. LiOH monohydrate (36.7 mg / 0.87 mmol) was dissolved in water (1 mL) and added slowly to the reaction vessel. After stirring for 70 min at 0°C, the mixture was neutralized with acetic acid (68.2 mg / 1.14 mmol) and concentrated under reduced pressure. The resulting material was taken up in a water / MeOH / DMF solution (1 : 1 : 1 v / v) and purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A: water + 0.05% TFA, mobile phase B: acetonitrile + 0.05% TFA. Gradient range from 10% to 60% B.

[0379] The compound Alkyne-glucuronide-MMAE (mixture of two diastereoisomers) was obtained as a white solid (95 mg / 96%). LC-HRMS m / z (ESI + ): calcd for [M+H] + = 1127.5758; found [M+H] + = 1127.5757; error = 0.1 ppm. HPLC Method 3 retention time = 10.3 min.

[0380] 6.2) Synthesis of the compound Alkyne-glucuronide-(MMAE)2

[0381]

[0382] 6.2.1) Synthesis of the compound Alkyne-glucuronide-(PNP)2

[0383] The starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427-3433) 165 mg (0.240 mmol), 64.2 mg (0.419 mmol) of the commercial 4-amino-3-(hydroxymethyl)phenylmethanol and 40.7 mg (0.300 mmol) of HOBt were dissolved in dry DMF. After stirring for 3 h at 50°C, the volatiles were evaporated and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 40:60 to 0:100) to give the intermediate diol compound as a yellow foam.

[0384] Anhydrous pyridine (4 molar equivalents) was added dropwise to a cooled solution of 4-nitrophenyl chloroformate (4 molar equivalents) in anhydrous DCM (0°C). The mixture was stirred at 0°C for 15 min. A solution of the previous intermediate diol compound (1 molar equivalent) in DCM was added and the mixture was stirred at room temperature for 1 h. The reaction was quenched with a saturated NaCl solution and extracted 3 times with DCM. The organic phase was dried over MgS04, filtered and evaporated in vacuo to give a solid crude which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 30:70) to give compound Alkyne-glucuronide-(PNP)2as a white solid (52 mg / 21% over two steps). 1 H NMR (300 MHz, CDCl3) δ (ppm) 2.04 (s, 3H), 2.06 (s, 3H), 2.11 (d, J = 2.0 Hz, 3H), 2.71-2.92 (m, 2H), 3.72 (s, 3H), 4.11 (q, J = 7.1 Hz, 1H),4.22 (d, J = 8.6 Hz, 1H), 5.18-5.41 (m, 8H), 5.87 (t, J = 6.5 Hz, 1H), 7.31-7.41 (m, 5H), 7.45-7.55 (m, 2H), 7.56-7.71 (m, 2H), 7.83 (d, J = 8.2 Hz, 1H),7.89 (s, 1H), 8.21-8.32 (m, 4H). HRMS m / z (ESI + ): calculated [M+Na] + = 1055.1925; found [M+Na] + = 1055.1955; error = -2.9 ppm.

[0385] 6.2.2) Synthesis of compound Alkyne-glucuronide-(MMAE)2

[0386] 52 mg (0.050 mmol) of the previous compound alkyne-glucuronide-(PNP) 2, 13.7 mg (0.100 mmol) of HOBt and 74.1 mg (0.103 mmol) of monomethyl auristatin E (MMAE) were dissolved in 1 mL of an 8:2 (v / v) mixture of anhydrous DMF / pyridine. The reaction was stirred at room temperature for 24 hours, and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 97:3 to 90:10) to obtain 77 mg (70%) of the intermediate compound, which can be directly used in the deprotection step without extensive characterization. At 0 ° C, 77 mg (0.035 mmol) of the compound was dissolved in MeOH (7 mL). LiOH monohydrate (14.7 mg / 0.350 mmol) was dissolved in water (0.7 mL) and slowly added to the reaction vessel. After stirring at 0°C for 60 minutes, the mixture was neutralized with acetic acid (27.4 mg / 0.457 mmol) and concentrated under reduced pressure. The resulting material was taken up in a water / MeOH / DMF solution (1:1:1 v / v) and added to 30 g of Biotage ® Purification was performed on a SNAP Ultra C18 column. Mobile phase A was water + 0.05% TFA, and mobile phase B was acetonitrile + 0.05% TFA.

[0387] The compound alkyne-glucuronide-(MMAE)2 (a mixture of two diastereomers) (40 mg / 56%) was obtained as a white solid. LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 1025.5623 ; Exp. value [M+2H] 2+ =1025.5599; Error = 2.4 ppm. HPLC Method 3 Retention time = 13.0 min.

[0388] 6.3) Synthesis of compound alkyne-val-cit-PAB-MMAE

[0389]

[0390] To a solution of 58 mg (0.052 mmol) of the starting material (synthesized as described in Tang et al., Org. Biomol. Chem., 2016, 14(40), 9501-9518) and 15 mg (0.077 mmol) of 4-pentanoic acid succinimidyl ester in 3 mL of dry DCM was added 16.7 mg (0.129 mmol) of DIPEA and the reaction was stirred at room temperature under argon atmosphere for 16 hours. Then, the volatiles were removed under vacuum, the resulting material was taken up in DMF solution and purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 25% to 70% B.

[0391] The compound alkyne-val-cit-PAB-MMAE was obtained as a white solid (21 mg / 34%). ESI + [M+Na] + = 1225.7. HPLC Method 3 retention time = 9.0 min.

[0392] 6.7.2) Synthesis of Alkyn-PNU159682

[0393]

[0394] To a solution of 156 mg (0.308 mmol) of the starting material TBDMS-SN38 (synthesized as described in Moon et al., J. Med. Chem., 2008, 51(21), 6916-6926), 113 mg (0.924 mmol) of 4-(dimethylamino)pyridine and 75 mg (0.369 mmol) of 4-nitrophenyl chloroformate in 8 mL of dry DCM was stirred at room temperature for 90 minutes, diluted with 5% acetic acid in water and extracted with DCM 3 times. The organic phase was dried over MgS04, filtered and evaporated under vacuum to give a yellow solid which was used in the next step without further purification.

[0395] To a solution of 175 mg (0.261 mmol) of the yellow solid in 4 mL of dry DMF was slowly added 43 mg (0.782 mmol) of propargylamine. The reaction was stirred at room temperature under argon atmosphere for 16 hours. The volatiles were removed under vacuum and the residue was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 40:60 to 0:100) to give the compound alkyne-SN38 as a bright yellow solid (71 mg / 56%). HRMS m / z (ESI + ): calculated [M+H]+ = 474.1660; found [M+H] + = 474.1664; error = -0.9 ppm. HPLC Method 3 retention time = 9.7 min. TLC eluted with 100% EtOAc: Rf= 0.15.

[0396] 6.5 Synthesis of the compound alkyne-glucuronide-SN38

[0397]

[0398] In a reaction vessel, 50 mg (0.074 mmol) of the starting material TBDMS-SN38-OPNP (synthesized as described previously in section 6.4) and 5 mg (0.037 mmol) of HOBt were weighed. 58.5 (0.223 mmol) of tert-butyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate (synthesized as described in WO2011 / 133039) previously dissolved in 1 mL of anhydrous DMF / pyridine 8:2 (v / v) mixture were added to the reaction vessel. The reaction was stirred at room temperature for 16 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (gradient of DCM / MeOH from 98:2 to 90:10) to give 48 mg (95%) of the intermediate compound (yellow solid) which was directly used in the deprotection step. HRMS m / z (ESI + ): calculated [M+H] + = 681.3130; found [M+H] + = 681.3113; error = 2.5 ppm.

[0399] The 48 mg (0.071 mmol) of this compound were dissolved in 2 mL of DCM, then 500 µL of TFA were added. The solution was stirred at room temperature for 90 minutes and the volatiles were removed under reduced pressure. The crude residue was purified by silica gel chromatography (gradient of DCM / MeOH from 94:6 to 80:20) to give 39.8 mg (96%) of the compound SN38-methylamine as a yellow solid. HRMS m / z (ESI + ): calculated [M+H] + = 581.2606; found [M+H] + = 581.2601; error = 0.8 ppm. HPLC Method 3 retention time = 7.7 min.

[0400] Forty-eight mg (0.069 mmol) of starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427-3433), 39.8 mg (0.069 mmol) of the previous compound SN38-methylamine and 9.3 mg (0.069 mmol) of HOBt were dissolved in 1.5 mL of anhydrous DMF / pyridine 8:2 (v / v) mixture. The reaction was stirred at room temperature for 16 hours and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 98:2 to 95:5) to give 57 mg (74%) of the intermediate compound (yellow solid) which was directly used in the deprotection step. ESI + [M+H] + = 1130.4.

[0401] The 57 mg (0.050 mmol) of the compound were dissolved in MeOH (6 mL) at 0°C. LiOH monohydrate (21.2 mg / 0.504 mmol) was dissolved in water (0.6 mL) and added slowly to the reaction vessel. After 70 minutes of stirring at 0°C, the mixture was neutralized with acetic acid (39.4 mg / 0.656 mmol) and concentrated under reduced pressure. The resulting material was taken up in a water / MeOH / DMF solution (1 : 1 : 1 v / v) and purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0402] The compound alkyne-glucuronide-SN38 was obtained as a yellow solid (20.5 mg / 42%). LC-HRMS m / z (ESI + ): calcd for [M+H] + = 990.3251 ; found [M+H] + = 990.3210 ; error = 4.1 ppm. HPLC Method 3 retention time = 8.2 min.

[0403] Example 7: Synthesis of poly-sarcosine based drug conjugate linkers using glutamic acid as orthogonal moiety

[0404]

[0405] 132.1 mg (0.192 mmol) of starting material (synthesized as described in Renoux et al., Chem. Sci., 2017, 8(5), 3427-3433), 102 mg (0.192 mmol) of isotecan mesylate and 26 mg (0.192 mmol) of HOBt were dissolved in 1 mL of an 8:2 (v / v) mixture of anhydrous DMF / pyridine. The reaction was stirred at room temperature for 16 hours, and the volatiles were evaporated under reduced pressure. The crude residue was purified by silica gel chromatography (DCM / MeOH gradient from 98:2 to 90:10) to give 165 mg (87%) of the intermediate compound (yellow solid), which was used directly in the deprotection step. ESI + [M+H] + = 985.3.

[0406] At 0 ° C, 165 mg (0.168 mmol) of the compound was dissolved in MeOH / THF 1: 1 v / v (16 mL). LiOH monohydrate (70.3 mg / 1.675 mmol) was dissolved in water (1.6 mL) and slowly added to the reaction vessel. After stirring at 0 ° C for 70 minutes, the mixture was neutralized with acetic acid (131 mg / 2.18 mmol) and concentrated under reduced pressure. The resulting material was absorbed in a water / MeOH / DMF solution (1: 1: 1 v / v) and added to 30 g Biotage ® Purification was performed on a SNAP Ultra C18 (25µm) column. Mobile phase A consisted of water with 0.05% TFA, and mobile phase B consisted of acetonitrile with 0.05% TFA. The gradient range was from 10% to 50% B.

[0407] The yellow solid compound alkyne-glucuronide-ixitecan (98 mg / 69%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M+H] + = 845.2312 ; experimental value [M+H] + = 845.2360; Error = -4.8 ppm. HPLC Method 3 Retention time = 8.0 min.

[0408] Example 8: Synthesis of poly-sarcosine based drug conjugate linkers using lysine as orthogonal moiety

[0409]

[0410] 6.7.1) Synthesis of N-(2-((2-aminoethyl)amino)-2-oxoethyl)propiolamide

[0411] glycine tert-butyl ester hydrochloride, 318.3 mg (4.54 mmol) of propionic acid and 61.4 mg (0.454 mmol) of HOBt were dissolved in 5 mL of dry DMF. 1103 mg (10.9 mmol) of DIPEA were added and the solution was stirred on ice (0°C) for 10 minutes. 871 mg (4.54 mmol) of EDC hydrochloride were suspended in 12 mL of dry DMF and added to the reaction vessel. The mixture was stirred in the dark at room temperature for 16 hours. Then, the volatiles were removed under reduced pressure. A saturated NH4CI solution was added and extracted 3 times with DCM. The organic phase was dried over MgS04, filtered and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient from 80:20 to 50:50) to give 255 mg (31%) of propiolyl glycine tert-butyl ester as a clear oil. MS (ESI + ): [M+H] + = 184.0. Elution with petroleum ether / EtOAc (40:60 v / v) and TLC staining with KMn04: Rf = 0.75.

[0412] 255 mg (1.39 mmol) of propiolyl glycine tert-butyl ester were dissolved in 5 mL of DCM / TFA (1:1 v / v) solution. The deprotection reaction was assessed by TLC analysis and was complete after 1 hour of stirring at room temperature. The volatiles were removed under reduced pressure to give 188 mg (105%) of propiolyl glycine as an oily residue which was used without purification in the next step.

[0413] 178 mg (1.40 mmol) of propiolyl glycine and 504.8 mg (1.33 mmol) of HATU were dissolved in 3 mL of dry DMF. 180.6 mg (1.40 mmol) of DIPEA were added and the reaction was stirred at room temperature for 5 minutes. Then, 291 mg (1.82 mmol) of N-Boc-ethylenediamine previously dissolved in 1 mL of dry DMF were added and the reaction mixture was stirred in the dark at room temperature for 30 minutes. Then, the volatiles were removed under reduced pressure. A saturated NH4CI solution was added and extracted 3 times with DCM. The organic phase was dried over MgS04, filtered and evaporated. The crude residue was purified by silica gel chromatography (petroleum ether / EtOAc gradient from 20:80 to 0:100) to give 204 mg (54%) of tert-butyl (2-(2-propiolamidoacetamido)ethyl)carbamate as a pale yellow oil. MS (ESI + ): [M+H]+ = 270.1 ; TLC eluting with 100% EtOAc and stained with KMnO4: Rf = 0.35.

[0414] tert-Butyl (2-(2-propynylaminoacetylamino)ethyl)carbamate, 204 mg (0.758 mmol) was taken up in 5 mL of DCM / TFA (7:3 v / v) solution. The deprotection reaction was assessed by TLC analysis and was complete after stirring at room temperature for 45 min. The volatiles were removed under high vacuum overnight to give 196 mg (92%) of N-(2-((2- aminoethyl)amino)-2-oxoethyl)propynylamide TFA salt as a light yellow thick wax. 1 H NMR (300 MHz, DMSO-d6) δ (ppm) 2.84 (q, J = 6.2 Hz, 2H), 3.29 (q, J = 6.3 Hz, 2H), 3.72 (d, J = 6.0 Hz, 2H), 4.20 (s, 1H), 7.78 (br. s, 3H), 8.12 (t, J = 5.6 Hz, 1H), 8.94 (t, J = 5.9 Hz, 1H). MS (ESI + ): [M+H] + = 170.0.

[0415] Example 9: Synthesis of poly-sarcosine or polyethylene glycol based drug conjugate linkers using glycine as orthogonal moiety

[0416] In a round bottom flask, 25 mg (0.040 mmol) of PNU159692 carboxylic acid derivative (purple solid synthesized as described in WO / 2016 / 040825, see chemical structure above) and 15.1 mg (0.040 mmol) of HATU were dissolved in 1 mL of dry DMF. 10.2 mg (0.080 mmol) of DIPEA were added and the mixture was stirred at room temperature for 2 min. 13.4 mg (0.047 mmol) of N-(2-((2- aminoethyl)amino)-2-oxoethyl)propynylamide TFA salt (previously dissolved in 500 μΐ^of dry DMF) were added and the reaction mixture was stirred at room temperature for 5 min. The volatiles were removed under high vacuum and the residue was purified by silica gel chromatography (DCM / MeOH, gradient from 99:1 to 90:10) to give 14.7 mg (49%) of Alkynyl-PNU159682 as a red solid. HRMS m / z (ESI + ): [M+H] + = 779.2770 ; found [M+H] += 779.2758; Error = 1.5 ppm. HPLC Method 6 Ret Time = 5.4 min.

[0417] Example 10: Synthesis of negative control drug conjugate linkers MAL-glucuronate MMAE, MAL-phenyl-triazole-glucuronate MMAE and MAL-phenyl-PSARn-triazole-glucuronate MMAE

[0418] The reaction scheme is as follows.

[0419]

[0420] 7.1) Resin loading with ethylenediamine

[0421] Five hundred mg of 2-chlorotrityl chloride resin beads (100-200 mesh, 1% DVB, 1.1 mmol / g, 0.55 mmol scale, Novabiochem) were swelled in 5 mL of DCM for 10 minutes. Five equivalents (2.75 mmol, 165.3 mg) of ethylenediamine (Sigma-Aldrich) were added and the mixture was shaken at room temperature for 4 hours, then washed thoroughly with DCM (5 times 5 mL). Unreacted sites on the resin were capped using a DCM / MeOH / DIPEA (17:2:1 v / v) solution (20 minute treatment). The resin was washed thoroughly with DCM (5 x 5 mL) and MeOH (5 x 5 mL), dried under vacuum and stored at -20 °C until further use.

[0422] 7.2) Fmoc-Glu(OAll)-OH coupling

[0423] To the resin containing the deprotected N-terminus (1 equivalent) was added a solution of Fmoc-Glu(OAll)-OH (3 equivalents), HATU (2.85 equivalents) and DIPEA (6 equivalents) in DMF (1 mL per 100 mg of resin). The reaction vessel was stirred for 2 hours and the resin was washed thoroughly with DMF (5 x 3 mL) and DCM (5 x 3 mL). The reaction was confirmed to be complete by a negative Kaiser test. The resin was dried under vacuum and stored at -20 °C until further use.

[0424] 7.3) Alloc protecting group removal

[0425] The resin was suspended in DCM (4 mL per 100 mg of resin) and the mixture was gently stirred by a stream of argon introduced from below the glass frit. Phenylsilane (20 eq) was added and stirring was continued for 5 minutes before Pd(PPh3)4(0.25 eq) was added. The mixture was continued to stir under an argon stream at room temperature for 30 minutes in the dark, before the solution was drained. The treatment with phenylsilane and Pd(PPh3)4was repeated once and the resin was thoroughly washed with DCM (5 x 5 mL), DMF (5 x 5 mL) and MeOH (5 x 5 mL). The resin was dried under vacuum and stored at -20 °C until further use. The resin loading was assessed (Fmoc cleavage test, absorbance measurement at 301 nm) and was typically 0.70-0.80 mmol / g.

[0426] 7.4 (2R,3R,4R,5S,6R)-6-(2-(3-aminopropionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidinyl-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradodecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (NH2-glycoside acid-MMAE) coupling step

[0427] To the resin containing the deprotected carboxylic acid group (1 eq) was added a solution of HATU (4 eq) and DIPEA (4.2 eq) in DMF. The reaction vessel was stirred for 25 minutes, drained and the resin washed with DMF (4 times 5 mL). Then, to the resin was added 1.5 eq of the compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid ("NH2-glucuronide-MMAE"; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831-840) and DIPEA (3.2 eq) in DMF. The reaction vessel was stirred for 3 hours, drained and washed with DMF (5 times 3 mL) and DCM (5 times 3 mL). The resin was dried under vacuum and stored at -20 °C until further use.

[0428] 7.5) Fmoc deprotection procedure

[0429] The resin containing the Fmoc protected amino acid was treated with 20% piperidine in DMF (1 mL per 100 mg resin) for 2 times, 15 minutes at room temperature. Then, the resin was washed with DMF (5 times 5 mL) and DCM (5 times 5 mL). The resin was dried under vacuum and stored at -20 °C until further use.

[0430] 7.6) Polyglutamic acid coupling procedure

[0431] To the resin containing the deprotected primary amine group (1 eq) was added polyglutamic acid-CH2-CH2-COOH (2.2 eq), HATU (2 eq) and DIPEA (6 eq) in DMF. The reaction vessel was stirred for 2.5 hours, drained and the resin washed with DMF (3 times 5 mL) and DCM (3 times 5 mL). The resin was dried under vacuum and stored at -20 °C until further use.

[0432] 7.7) Cleavage from resin

[0433] Final cleavage from 2-chlorotrityl resin was performed using a solution of DCM with 20% (v / v) HFIP (2 mL per 100 mg of resin) under stirring at room temperature. The reaction time was 60 minutes. The resin was filtered and the resulting solution was evaporated under a stream of argon. The final residue was dried under high vacuum and used directly in the next step.

[0434] 7.8) Coupling procedure of 3-(maleimidyl)propionic acid N-hydroxysuccinimidyl ester

[0435] To the residue dissolved in dry DMF was added 3-(maleimidyl)propionic acid N- hydroxysuccinimidyl ester (8 eq). DIPEA (10 eq) was added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was quenched with water / TFA (99.5:0.5 v / v) and purified on a 30 g Biotage® SNAP Ultra C18 (25 pm) column. The mobile phase A was water + 0.05% TFA and the mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0436] Compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR6 was obtained as a clear oil (5.8 mg / 14% yield based on initial resin loading). LC-HRMS m / z (ESI + ): Calculated for [M+2H] C 2+ = 989.5064; Found [M+2H] C 2+ 989.5023; Error = 4.2 ppm. HPLC Method 1 retention time = 6.7 minutes.

[0437] Compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR12 was obtained as a clear oil (4.6 mg / 20% yield based on initial resin loading). LC-HRMS m / z (ESI + ): Calculated for [M+2H] C 2+ = 1202.6178; Found [M+2H] C 2+ 1202.6178; Error = 0.0 ppm. HPLC Method 1 retention time = 6.9 minutes.

[0438] Compound MAL-Glu (glucuronide MMAE)-CH2-CH2-PSAR18 was obtained as a clear oil (2.4 mg / 14% yield based on initial resin loading). LC-HRMS m / z (ESI + ): Calculated for [M+2H] C 2+= 1415.7291 ; Experimental value [M+2H] 2+ 1415.7282 ; Error = 0.7 ppm. HPLC Method 1 Ret Time = 6.8 min.

[0439] Example 11 : Preparation of LDC compounds of the invention

[0440] The reaction scheme is as follows.

[0441]

[0442] 8.1) Synthesis of Fmoc-D-Lys(Glucuronide MMAE)-NH2

[0443]

[0444] Compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropionamido)-4-((5S,8S,11S,12R)-11- ((S)-sec-butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1- methoxy-2-methyl-3-oxopropyl)pyrrolidinyl-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10- dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradodecyl)phenoxy)-3,4,5-trihydroxytetrahydro- 2H-pyran-2-carboxylic acid (“NH2-Glucuronide-MMAE”; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831-840) and Fmoc-D-Lys(Boc)-OSu (35 mg / 0.062 mmol) were dissolved in 1.2 mL dry DMF. DIPEA (24.0 mg / 0.186 mmol) was added and the mixture was stirred at room temperature for 20 hours. The volatiles were removed in vacuo. The flask containing the light yellow crude was placed on an ice bath (0°C) and 7 mL of a DCM / TFA (7:3 v / v) solution was slowly added. The solution was stirred over ice until complete Boc deprotection was observed by HPLC (about 2 hours). Then, the volatiles were removed in vacuo and the residue was taken up in DMF to be purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B. Compound Fmoc-D-Lys(Glucuronide MMAE)-NH2 was obtained as a white solid (59 mg / 65%). LC-HRMS m / z (ESI+ ): calculated [M+H] + = 1480.7862; found [M+H] + = 1480.7890; error = -1.9 ppm. HPLC Method 1 retention time = 10.5 min

[0445] 8.2) Fmoc-D-Lys (Glucuronide MMAE)-PSAR n Synthesis of

[0446]

[0447] Compound PSARn-COOH (2 eq; obtained as described in Example 2 and previously dissolved in anhydrous DMF at 0.15 M stock) was added to HATU (1.8 eq) in a vial. DIPEA (5 eq) was added and the mixture was stirred at room temperature for 3 min. Then, compound Fmoc-D-Lys (Glucuronide MMAE)-NH2 (1 eq; previously dissolved in anhydrous DMF at 0.05 M stock) was added. The mixture was stirred at room temperature for 1 h and then injected in a Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 60% B.

[0448] Compound Fmoc-D-Lys (Glucuronide MMAE)-PSAR6 was obtained as a white solid (9.8 mg / 38%). LC-HRMS m / z (ESI + ): calculated [M+2H] 2+ = 975.0133; found [M+2H] 2+ = 975.0088; error = 4.6 ppm. HPLC Method 1 retention time = 7.5 min.

[0449] Compound Fmoc-D-Lys (Glucuronide MMAE)-PSAR12 was obtained as a white solid (3.6 mg / 28%). LC-HRMS m / z (ESI + ): calculated [M+2H] 2+ = 1188.1247; found [M+2H] 2+ = 1188.1233; error = 1.1 ppm. HPLC Method 1 retention time = 7.6 min.

[0450] 8.3) Coupling procedure of 6-(maleimidocaproyl) N-hydroxysuccinimidyl ester

[0451] Compound Fmoc-D-Lys(glucuronide MMAE)-PSARn from previous step was treated with 20% piperidine in DMF for 5 min at room temperature. Volatiles were removed under high vacuum and the dry residue was dissolved in anhydrous DMF. Then, 6-(maleimidocaproyl) N-hydroxysuccinimidyl ester (8 eq) and DIPEA (10 eq) were added and the mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with water / TFA (99.5:0.5 v / v) and purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) cartridge column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The target compound was eluted with 40% B isocratically after an isocratic hold of 10 min (5% B).

[0452]

[0453] Compound MAL-Lys(glucuronide MMAE)-PSAR6 was obtained as a clear oil (5.0 mg / 52%). LC-HRMS m / z (ESI + ): Calcd for [M+2H] C 2+ = 960.5163; Found [M+2H] C 2+ = 960.5167; Error = -0.5 ppm. HPLC Method 1 retention time = 7.1 min.

[0454] Compound MAL-Lys(glucuronide MMAE)-PSAR12 was obtained as a clear oil (1.8 mg / 51%). LC-HRMS m / z (ESI + ): Calcd for [M+2H] C 2+ = 1173.6276; Found [M+2H] C 2+ 1173.6229; Error = 4.0 ppm. HPLC Method 1 retention time = 7.0 min.

[0455] Reversed phase liquid chromatography-mass spectrometry (RPLC-MS): Size exclusion chromatography (SEC):

[0456] 9.1) Compound bromoacetamide-Ngly(triazole-glucuronide MMAE)-PSARn

[0457]

[0458] Alkyne-glucuronide-MMAE (1 eq; obtained as described in Example 6), PSARn-N3- bromoacetamide (1.1 eq; obtained as described in Example 3) and tetrakis(acetonitrile) copper(I) hexafluorophosphate (3 eq) were mixed in a reaction vessel. A solution of DCM / acetonitrile 1 :1 (v / v) was added to give a final concentration of 12 pmol / mL of alkyne-glucuronide-MMAE. The reaction was stirred in the dark at room temperature under argon for 16-20 hours. After removal of volatiles under reduced pressure, the residue was taken up in DMF and purified on a 30 g Biotage ® SNAP Ultra C18 (25 pm) column. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10% to 50% B.

[0459] The compound bromoacetamide-Ngly(triazole-glucuronide MMAE)-PSAR12 was obtained as a white solid (8.5 mg / 51%). LC-HRMS m / z (ESI + ): calculated [M+2H] 2+ = 1151.0179; found [M+2H] 2+ = 1151.0188; error = -0.8 ppm. HPLC Method 3 retention time = 8.5 min.

[0460] 9.2) Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSARn

[0461]

[0462] Alkyne-glucuronide-MMAE (1 eq; obtained as described in Example 6), PSARn-N3- bromoacetamide (1.1 eq; obtained as described in Example 3) and tetrakis(acetonitrile) copper(I) hexafluorophosphate (3 eq) were mixed in a reaction vessel. A solution of DCM / acetonitrile 1 :1 (v / v) was added to give a final concentration of 12 pmol / mL of alkyne-glucuronide-MMAE. The reaction was stirred in the dark at room temperature under argon for 16-20 hours. After removal of volatiles under reduced pressure, the residue was taken up in DMF and purified on a 30 g Biotage

[0463] The compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR6 was obtained as a white solid (3.3 mg / 20%). LC-HRMS m / z (ESI + ): calculated [M+2H] 2+ = 955.9566; found [M+2H] 2+ = 955.9533; error = 3.4 ppm. HPLC Method 3 retention time = 9.2 min.

[0464] Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR12 was obtained as a white solid (9.0 mg / 33%). LC-HRMS m / z (ESI + ): Calculated for [M+2H] 2+ = 1169.0679; Found [M+2H] 2+ = 1169.0621 ; Error = 4.9 ppm. HPLC Method 3 retention time = 8.7 min.

[0465] Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 was obtained as a white solid (11.5 mg / 40%). LC-HRMS m / z (ESI + ): Calculated for [M+2H] 2+ = 1382.1792; Found [M+2H] 2+ = 1382.1803; Error = -0.7 ppm. HPLC Method 3 retention time = 8.6 min.

[0466] Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 was obtained as a white solid (15 mg / 44%). LC-HRMS m / z (ESI + ): Calculated for [M+4Na] 4+ = 820.1309; Found [M+4Na] 4+ = 820.1324; Error = -1.8 ppm. HPLC Method 3 retention time = 8.4 min.

[0467] 9.3) Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEGn

[0468]

[0469] Alkyne-glucuronide-MMAE (obtained as described in Example 6) and PEGn-N3-phenyl-MAL (obtained as described in Example 5) were reacted and purified as described in Section 9.1 above using NMP / DCM 2:1 (v / v) as reaction solvent.

[0470] Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 was obtained as a pale yellow oil (10.4 mg / 45%). LC-HRMS m / z (ESI + ): Calculated for [M+2H] 2+ = 1042.5211; Found [M+2H]2+ = 1042.5218; Error = -0.7 ppm. HPLC Method 3 Ret Time = 8.0 min.

[0471] 9.4) Compound MAL-Phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole- glucuronide MMAE)-PSARn

[0472]

[0473] Alkyne-glucuronide-MMAE (3 eq; obtained as described in Example 6), PSARn-N3-N3-phenyl-MAL (1 eq; obtained as described in Example 3), and tetra(acetonitrile)copper(I) hexafluorophosphate (5 eq) were mixed in a reaction vessel. DCM was added and the reaction was stirred at room temperature under argon in the dark for 16-20 hours. After removal of volatiles under reduced pressure, the residue was taken up in DMF and purified on a 30g Biotage ® SNAP Ultra C18 (25µm) column. Mobile phase A was water + 0.1% TFA and mobile phase B was acetonitrile + 0.1% TFA. The gradient ranged from 10% to 50% B.

[0474] Compound MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)-PSAR18 was obtained as a white solid (10.1 mg / 44%). LC-HRMS m / z (ESI + ): Calculated [M+4H] 4+ = 1022.5082; Found [M+4H] 4+ = 1022.5093; Error = -1.0 ppm. HPLC Method 3 Ret Time = 9.5 min.

[0475] 9.5) Compound MAL-phenyl-Ngly [triazole-glucuronide (MMAE)2]-PSARn

[0476]

[0477] Alkyne-glucuronide-(MMAE)2 (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described above in Section 9.1 using DCM as the reaction solvent.

[0478] Compound MAL-phenyl-Ngly [triazole-glucuronide (MMAE)2]-PSAR24 was obtained as a white solid (12.5 mg / 39%). LC-HRMS m / z (ESI+ ): calculated [M+3H] 3+ = 1371.3767; experimental [M+3H] 3+ = 1371.3818; error = -3.8 ppm. HPLC Method 3 retention time = 10.0 min.

[0479] 9.6) Compound MAL-Phenyl-Ngly [Triazole-galactoside (MMAE)2] -PSARn

[0480]

[0481] Alkyne-galactoside-(MMAE)2(as synthesized in Alsarraf et al., Chem. Commun., 2015, 51(87), 15792-15795) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted as described in Section 9.1 above using DCM as the reaction solvent and purified.

[0482] Compound MAL-phenyl-Ngly [Triazole-galactoside (MMAE)2]-PSAR24was obtained as a white solid (6.5 mg / 55 %). LC-HRMS m / z (ESI + ): calculated [M+4H] 4+ = 1022.7895; experimental [M+4H] 4+ = 1022.7903; error = 0.8 ppm. HPLC Method 3 retention time = 9.2 min.

[0483] 9.7) Compound MAL-phenyl-Ngly (Triazole-val-cit-PAB-MMAE)-PSARn

[0484]

[0485] Alkyne-val-cit-PAB-MMAE (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted as described in Section 9.1 above using NMP as the reaction solvent and purified.

[0486] Compound MAL-phenyl-Ngly (Triazole-val-cit-PAB-MMAE)-PSAR12was obtained as a white solid (4.5 mg / 12 %). LC-HRMS m / z (ESI + ): calculated [M+2H] 2+ = 1207.1494; experimental [M+2H]2+ = 1207.1535; Error = -3.5 ppm. HPLC Method 3 Retention time = 8.6 minutes.

[0487] 9.8) Compound MAL-phenyl-Ngly(triazole-SN38)-PSARn

[0488]

[0489] Alkyne-SN38 (obtained as described in Example 6) and PSARn-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described above in Section 9.1 using DCM / DMF 2:1 (v / v) as the reaction solvent.

[0490] The compound MAL-phenyl-Ngly(triazole-SN38)-PSAR18 (4.0 mg / 20%) was obtained as a bright yellow solid. LC-HRMS m / z (ESI + ): Calculated value [M+2Na] 2+ = 1077.4562 ; Experimental value [M+2Na] 2+ = 1077.4588; Error = -2.4 ppm. HPLC Method 3 Retention time = 7.5 minutes.

[0491] 9.9) Compound MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSARn

[0492]

[0493] Alkyne-SN38 (obtained as described in Example 6) and PSARn-N3-N3-phenyl-MAL (obtained as described in Example 3) were reacted and purified as described above in Section 9.4.

[0494] The yellow solid compound MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18 (5.1 mg / 43%) was obtained. LC-HRMS m / z (ESI + ): Calculated value [M+2Na] 2+ = 1412.5812 ; Experimental value [M+2Na] 2+ = 1412.5852; Error = -3.8 ppm. HPLC Method 3 Retention time = 8.4 minutes.

[0495] 9.10) Compound MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSARn

[0496]

[0497] Reaction of Alkynyl-glucuronide acid-SN38 (obtained as described in Example 6) and PSARn-N3-N3-Phenyl-MAL (obtained as described in Example 3) was performed as described in section 9.4 above using DCM / MeOH 8:2 (v / v) as reaction solvent and purified.

[0498] Compound MAL-Phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)-PSAR18 was obtained as a yellow solid (7.0 mg / 30 %). LC-HRMS m / z (ESI + ): Calculated for [M+2H] = 1271.5080; Found [M+2H] = 1271.5103; Error = -1.8 ppm. HPLC Method 3 Ret Time = 7.8 min. 2+ 2+

[0499] 9.11) Compound MAL-Phenyl-Ngly(triazole-glucuronide-Exetecan)-PSARn

[0500]

[0501] Reaction of Alkynyl-glucuronide acid-Exetecan (obtained as described in Example 6) and PSARn-N3-Phenyl-MAL (obtained as described in Example 3) was performed as described in section 9.1 above using DCM as reaction solvent and purified.

[0502] Compound MAL-Phenyl-Ngly(triazole-glucuronide-Exetecan)-PSAR18 was obtained as a yellow solid (13.2 mg / 64 %). LC-HRMS m / z (ESI + ): Calculated for [M+2H] = 1241.0069; Found [M+2H] = 1241.0088; Error = -1.1 ppm. HPLC Method 3 Ret Time = 7.1 min. 2+ 2+

[0503] 9.12) Compound MAL-Phenyl-Ngly(triazole-PNU159682)-PSARn

[0504]

[0505] ​​​​Reaction of Alkyne-PNU159682 (obtained as described in Example 6) and PSARn-N3-Phenyl-MAL (obtained as described in Example 3) was carried out as described in Section 9.1 above using DCM as reaction solvent and replacing the 0.1% TFA additive in the mobile phase with 0.1% formic acid during the reverse phase purification.

[0506] Compound MAL-Phenyl-Ngly(triazole-PNU159682)-PSAR12 (4.8 mg / 40%) was obtained as a red solid. LC-HRMS m / z (ESI + ): Calcd for C8H6N10O6P [M+2H] 2+ = 994.9185; Found [M+2H] 2+ = 994.9184; Error = 0.1 ppm. HPLC Method 6 Ret Time = 5.0 min.

[0507] Compound MAL-Phenyl-Ngly(triazole-PNU159682)-PSAR18 (7.2 mg / 33%) was obtained as a red solid. LC-HRMS m / z (ESI + ): Calcd for C8H6N10O6P [M+2H] 2+ = 1208.0298; Found [M+2H] 2+ = 1208.0295; Error = 0.3 ppm. HPLC Method 6 Ret Time = 4.9 min.

[0508] Hydrophobic interaction chromatography (HIC): Trastuzumab-Glu (glucuronate MMAE)-CH2-CH2-PSAR6 (DAR 8)

[0509] 10.1) Synthesis of Compound MAL-Glucuronide MMAE

[0510]

[0511] The starting compound (2R,3R,4R,5S,6R)-6-(2-(3-aminopropionamido)-4-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-(2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (“NH2-glucuronide-MMAE”; synthesized as described in Jeffrey SC et al., Bioconjug. Chem., 2006, 17(3), 831-840) (6.2 mg / 5 µmol) and 3-(maleimido)propionic acid N-hydroxysuccinimidyl ester (14.6 mg / 55 µmol) were weighed and dissolved in 200 µL of dry DMF. DIPEA (8.5 mg / 66 µmol) was added and the mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with 1.5 mL of water / TFA (99:1 v / v) and purified on a 30 g Biotage ® SNAP Ultra C18 (25µm) column. Mobile phase A was water + 0.05% TFA and mobile phase B was acetonitrile + 0.05% TFA. The gradient ranged from 10% to 70% B.

[0512] The title compound MAL-glucuronide-MMAE was obtained as a white solid (4.1 mg / 59%). LC-HRMS m / z (ESI + ): calcd for [M+H] + = 1281.6501; found [M+H] + = 1281.6489; error = 0.9 ppm. HPLC Method 1 retention time = 7.1 min.

[0513] 10.2) Synthesis of compound MAL-phenyl-triazole-glucuronide-MMAE

[0514] 10.2.1) Synthesis of perfluorophenyl 2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetate

[0515]

[0516] In a reaction vessel, commercially available 2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl]acetic acid (299 mg / 1.29 mmol), N,N'-dicyclohexylcarbodiimide (267 mg / 1.29 mmol) and pentafluorophenol (238 mg / 1.29 mmol) were dissolved in 15 mL of dry 1,2-dimethoxyethane. After stirring at room temperature for 2 hours, the insoluble material was removed by filtration and the filtrate was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 80:20 to 20:80) to give the title compound as a white solid (400 mg / 78%). 1 H NMR (300 MHz, CDC13) δ (ppm) 4.01 (s, 2H), 6.87 (s, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H). HRMS m / z (ESI + ): calculated [M+H] + = 398.0446; found [M+H] + = 398.0448; error = -0.4 ppm.

[0517] 10.2.2) Synthesis of N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)phenyl)acetamide

[0518]

[0519] In a reaction vessel, the previous compound perfluorophenyl 2-(4-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)phenyl)acetate (78 mg / 0.20 mmol) was dissolved in 1 mL of dry DCM. 2-azidoethan-1-amine (33.8 mg / 0.40 mmol) was added and the reaction was stirred at room temperature for 1 hour. Then, a 1 N HCI solution was added and the mixture was extracted 3 times with DCM. The organic phase was dried over MgS04, filtered and evaporated under vacuum to give a solid crude which was purified by silica gel chromatography (petroleum ether / EtOAc, gradient from 60:40 to 0:100) to give the title compound as a white solid (18 mg / 31%). MS (ESI + ): [M+H] + = 300.1; HPLC Method 1 retention time = 8.4 min. TLC eluted with 100% EtOAc: Rf= 0.65.

[0520] 10.2.3) Synthesis of Compound MAL-phenyl-triazole-glucuronide MMAE

[0521]

[0522] Compound 6, alkyne-glucuronide MMAE (17 mg / 15.1 μmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (11.2 mg / 30 μmol), and N-(2-azidoethyl)-2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamide (6.3 mg / 21 μmol) from the previous step were mixed in an HPLC vial. 800 μL of anhydrous DCM / acetonitrile / NMP 1:1:1 (v / v / v) solution was added, and the reaction was stirred at room temperature under argon for 16 hours. After removing the volatiles under reduced pressure, the residue was taken up in DMF and concentrated on 30 g of Biotage ® Purification was performed on a SNAP Ultra C18 (25µm) column. Mobile phase A consisted of water with 0.1% TFA, and mobile phase B consisted of acetonitrile with 0.1% TFA. The gradient ranged from 10% to 60% B.

[0523] The title compound MAL-phenyl-triazole-glucuronide MMAE (10.3 mg / 48%) was obtained as an off-white solid. LC-HRMS m / z (ESI + ): Calculated value [M+2H] 2+ = 713.8425 ; Exp. value [M+2H] 2+ = 713.8415; Error = 1.3 ppm. HPLC Method 3 Retention time = 10.2 minutes.

[0524] 10.3) Synthesis of Compound MAL-phenyl-PSARn-triazole-glucuronide MMAE

[0525]

[0526] Compound 6, alkyne-glucuronide MMAE (20 mg / 17.7 μmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate (13.2 mg / 35 μmol) and N3-PSARn-phenyl-MAL (34.3 mg / 28 μmol) from Example 4 were mixed in an HPLC vial. 900 μL of NMP / DCM 2:1 (v / v) solution was added and the reaction was stirred at room temperature under argon for 16 hours. After removing the volatiles under reduced pressure, the residue was taken up in DMF and concentrated on 30 g Biotage ®Purification on a SNAP Ultra C18 (25 pm) column. Mobile phase A is water + 0.1% TFA, mobile phase B is acetonitrile + 0.1% TFA. Gradient range from 10% to 60% B.

[0527] The title compound, MAL-Phenyl-PSARn-triazole-glucuronide MMAE, was obtained as a white solid (16.0 mg / 39%). LC-HRMS m / z (ESI + ): calcd for [M+2H] 2+ = 1168.5759; found [M+2H] 2+ = 1168.5792; error = -2.8 ppm. HPLC Method 3 retention time = 6.8 min.

[0528] Trastuzumab-Glu (glucuronate MMAE)-CH2-CH2-PSAR12 (DAR 8):

[0529] The following LDC compounds were prepared and characterized:

[0530] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR6

[0531] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR12

[0532] Trastuzumab-Glu (glucuronide MMAE)-CH2-CH2-PSAR18

[0533] Trastuzumab-Lys (glucuronide MMAE)-PSAR6

[0534] Trastuzumab-Lys (glucuronide MMAE)-PSAR12

[0535] Trastuzumab-BAC-Ngly (triazole-glucuronide MMAE)-PSAR12

[0536] Trastuzumab-MAL-Phenyl-Ngly (triazole-glucuronide MMAE)-PSAR6

[0537] Trastuzumab-MAL-Phenyl-Ngly (triazole-glucuronide MMAE)-PSAR12

[0538] Trastuzumab-MAL-Phenyl-Ngly (triazole-val-cit-PAB-MMAE)-PSAR12

[0539] Trastuzumab-MAL-Phenyl-Ngly (triazole-glucuronide MMAE)-PSAR18

[0540] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18

[0541] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide-Exatecan)-PSAR18

[0542] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12

[0543] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18

[0544] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0545] CD19-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0546] CD22-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0547] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole- glucuronide MMAE)-PSAR18

[0548] Trastuzumab-MAL-phenyl-Ngly [triazole-glucuronide (MMAE)2] -PSAR24

[0549] Trastuzumab-MAL-phenyl-Ngly [triazole-galactoside (MMAE)2] -PSAR24

[0550] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18

[0551] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole- glucuronide SN38)-PSAR18

[0552] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12

[0553] Trastuzumab-glucuronide MMAE

[0554] Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE

[0555] Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE

[0556] Human albumin-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24

[0557] Their structures are described in Table 6 below.

[0558] Table 6

[0559]

[0560]

[0561]

[0562]

[0563] 11.1) Preparation of conjugates

[0564] 11.1.1) Preparation of antibody-drug conjugates

[0565] Antibody solutions (10 mg / mL in PBS 7.4 + 1 mM EDTA) were treated with 14 molar equivalents of tris(2-carboxyethyl)phosphine (TCEP) for 2 hours at 37°C. For maleimide-based conjugation, fully reduced antibody was buffer exchanged with 100 mM potassium phosphate pH 7.4 + 1 mM EDTA by three rounds of dilution / centrifugation using Amicon 30K centrifugal filter devices (Merck Millipore). 10-12 molar equivalents of drug-linker (from 12 mM DMSO stock) were added to the antibody (residual DMSO <10% v / v). The solution was incubated for 30 minutes at room temperature. For bromoacetamide-based conjugation, fully reduced antibody was buffer exchanged with 50 mM borate buffer pH 8.1 + 1 mM EDTA and conjugation was achieved using 16 molar equivalents of drug-linker over 24 hours at 37°C in the dark. The conjugate was buffer exchanged / purified with PBS 7.4 by four rounds of dilution / centrifugation using Amicon 30K centrifugal filter devices. Alternatively, the conjugate was buffer exchanged / purified using a PD MiniTrap G-25 chromatography column (GE Healthcare) and sterile filtered (0.20 pm PES filter). Conjugates incorporating self-hydrolysable maleimide (MAL-phenyl) groups were incubated at 5 mg / mL in PBS 7.4 at 37°C for 48h to ensure complete hydrolysis of the succinimidyl moiety. Final protein concentration was assessed spectrophotometrically at 280 nm using a Colibri microspectrophotometer device (Titertek Berthold).

[0566] 11.1.2) Preparation of human albumin-drug conjugates

[0567] To a human albumin solution (10 mg / mL in potassium phosphate 100 mM pH 7.4 + 1 mM EDTA), 2 molar equivalents of drug-linker (from a 12 mM DMSO stock solution) were added. Residual DMSO <10% (v / v). The solution was incubated at room temperature for 4 hours. The conjugate was buffer exchanged / purified with PBS 7.4 by four rounds of dilution / centrifugation using an Amicon 30K centrifugal filter device. Alternatively, the conjugate was buffer exchanged / purified using a PD MiniTrap G-25 column (GE Healthcare) and sterile filtered (0.20 μm PES filter). The conjugate was incubated at 37 ° C for 48 h at 5 mg / mL in PBS 7.4 to ensure complete hydrolysis of the succinimidyl moiety. Final protein concentration was assessed spectrophotometrically at 280 nm using a Colibri microspectrophotometer device (Titertek Berthold).

[0568] 11.2) Characterization of Conjugates

[0569] The characteristics of the resulting conjugate are as follows:

[0570] Trastuzumab-Glu (glucuronate MMAE)-CH2-CH2-PSAR18 (DAR 8):

[0571] Denaturing RPLC-QToF analysis was performed using the above-described UHPLC method 5. Briefly, the conjugate was eluted using a mobile phase gradient of water / acetonitrile + 0.1% formic acid (0.4 mL / min) on an Agilent PLRP-S1000Å 2.1x150mm8μm (80°C) and quantified using a Bruker Impact II TM The Q-ToF mass spectrometer scanned the 500-3500 m / z range (ESI + ) to detect the conjugate. ® The MaxEnt algorithm included in the software deconvolved the data.

[0572] Trastuzumab-Lys (glucuronate MMAE)-PSAR6 (DAR 8):

[0573] SEC was performed on an Agilent 1050 HPLC system with an extra-column volume of less than 15 µL (equipped with 0.12 mm id PEEK tubing and a low-volume UV flow cell). The column was a Waters AcquityUPLC ®Protein BEH SEC 200 Å 4.6 x 150 mm 1.7 µm (kept at room temperature) or Agilent AdvanceBio SEC 300 Å 4.6 x 150 mm 2.7 µm (kept at room temperature). The mobile phase was 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). 10% acetonitrile (v / v) was added to the mobile phase to minimize secondary hydrophobic interactions with the stationary phase and to prevent bacterial growth. The flow rate was 0.35 mL / min. UV detection was monitored at 280 nm.

[0574] Trastuzumab-Lys (glucuronate MMAE)-PSAR12 (DAR 8):

[0575] Hydrophobic interaction chromatography (HIC) was performed on an Agilent 1050 HPLC system. The chromatographic column was Tosoh TSK-GEL BUTYL-NPR 4.6 x 35 mm 2.5 µm (25 °C). The mobile phase A was 1.5 M (NH4)2SO4 + 25 mM potassium phosphate pH 7.0. The mobile phase B was 25 mM potassium phosphate pH 7.0 + 15% isopropanol (v / v). The linear gradient was from 0% B to 100% B in 10 minutes, then 3 minutes at 100% B. The flow rate was 0.75 mL / min. UV detection was monitored at 220 and 280 nm.

[0576] 11.3) Conjugate characterization summary

[0577] The conjugate showed one LC-1d (light chain with 1 drug-linker) and one HC-3d (heavy chain with 3 drug-linkers) absorbance peak on its denaturing RPLC chromatogram (DAR 8 conjugate). For mass spectrometric analysis of the heavy chain, the main glycoform (G0F of trastuzumab) was reported. The conjugate showed a single absorbance peak on its HIC chromatogram.

[0578] Trastuzumab-BAC-Ngly (triazole-glucuronate MMAE)-PSAR12 (DAR 8) :

[0579] Deconvoluted LC-1d calculated: 25416; observed: 25417 / Deconvoluted HC-3d calculated: 56529; Obs: 56528

[0580] Monomer purity: 97.2%

[0581] HIC retention time: 8.8 minutes

[0582] Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronate MMAE)-PSAR6 (DAR 8) (= ADC-PSAR6)

[0583] Deconvoluted LC-1d calculated: 25844; observed: 25844 / Deconvoluted HC-3d calculated: 57805; Obs: 57805

[0584] Monomer purity: 99.0%

[0585] HIC retention time: 8.8 minutes

[0586] Trastuzumab-MAL-phenyl-Ngly (triazole-glucuronate MMAE)-PSAR12 (DAR 8) (= ADC- PSAR12)

[0587] Deconvolution LC-1d calculated: 26270; observed: 26270 / Deconvolution HC-3d calculated: 59086; observed: 59086

[0588] Monomer purity: 96.5%

[0589] HIC retention time: 8.8 minutes

[0590] ​

[0591] Deconvolution LC-1d calculated: 25360; observed: 25360 / Deconvolution HC-3d calculated: 56352; observed: 56353

[0592] Monomer purity: 99+%

[0593] HIC retention time: 7.6 minutes

[0594] ​

[0595] Deconvolution LC-1d calculated: 25786; observed: 25786 / Deconvolution HC-3d calculated: 57634; observed: 57632

[0596] Monomer purity: 99+%

[0597] HIC retention time: 7.5 minutes

[0598] ​

[0599] Deconvolution LC-1d calculated: 25661; observed: 25662 / Deconvolution HC-3d calculated: 57264; observed: 57262

[0600] Monomer purity: 99+%

[0601] HIC retention time: 7.0 minutes (DAR 8 conjugate). As observed in the HIC chromatogram, the ADC is a heterogeneous mixture containing ~20% DAR 6, ~20% DAR 7, and ~60% DAR 8 conjugates.

[0602] ​

[0603] Deconv LC-1d calculated: 25794; observed: 25794 / Deconv HC-3d calculated: 57660; observed: 57660.

[0604] Monomer purity: 99+%

[0605] HIC retention time: 7.1 minutes

[0606] ​ ​

[0607] Deconv LC-1d calculated: 25794; observed: 25794 / Deconv HC-3d calculated: 57660; observed: 57660.

[0608] Monomer purity: 99+%

[0609] HIC retention time: 7.1 minutes

[0610] Trastuzumab-MAL-phenyl-Ngly(triazole-val-cit-PAB-MMAE)-PSAR12 (DAR 8)

[0611] Deconv LC-1d calculated: 25871; observed: 25870 / Deconv HC-3d calculated: 57889; observed: 57888

[0612] Monomer purity: 99+%

[0613] HIC retention time: 9.2 minutes

[0614] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR18 (DAR 8) (= ADC-PSAR18) Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-PSAR18 (DAR 8)

[0615] Deconv LC-1d calculated: 26221; observed: 26221 / Deconv HC-3d calculated: 58939; observed: 58939

[0616] Monomer purity: 99 +%

[0617] HIC retention time: 6.8 minutes

[0618] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide-irinotecan)-PSAR18 (DAR 8) )

[0619] Deconv LC-1d calculated: 25567; observed: 25567 / Deconv HC-3d calculated: 56979; observed: 56977

[0620] Monomer purity: 99+%

[0621] HIC retention time: 5.1 minutes

[0622] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR12 (DAR 8)

[0623] Deconv LC-1d calculated: 25938; observed: 25937 / Deconv HC-3d calculated: 58092; observed: 58089

[0624] Monomer purity: 99+%

[0625] HIC retention time: 5.7 minutes

[0626] Trastuzumab-MAL-phenyl-Ngly(triazole-PNU159682)-PSAR18 (DAR 8)

[0627] Deconv LC-1d calculated: 25446; observed: 25446 / Deconv HC-3d calculated: 56614; observed: 56612

[0628] Monomer purity: 99+%

[0629] HIC retention time: 5.2 minutes

[0630] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR 8) (= ADC-PSAR24)

[0631] Deconv LC-1d calculated: 25872; observed: 25872 / Deconv HC-3d calculated: 57894; observed: 57892

[0632] Monomer purity: 99+%

[0633] HIC retention time: 5.1 minutes

[0634] CD19-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR 8)

[0635] Deconv LC-1d calculated: 26647; observed: 26674 / Deconv HC-3d calculated: 60218; observed: 60218

[0636] Monomer purity: 99+%

[0637] HIC retention time: 6.7 minutes

[0638] CD22-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR 8)

[0639] Deconv LC-1d calculated: 27347; observed: 27347 / Deconv HC-3d calculated: 60137; observed: 60132

[0640] Monomer purity: 92.6%

[0641] HIC retention time: 6.8 minutes

[0642] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-Ngly(triazole-glucuronide MMAE)- PSAR18 (DAR 16)

[0643] Deconvoluted LC-1 d calculated: 27569; observed: 27570 / Deconvoluted HC-3d calculated: 62985; observed: 62983

[0644] Monomer purity: 98.5%

[0645] HIC retention time: 8.7 minutes

[0646] Trastuzumab-MAL-phenyl-Ngly [triazole-glucuronide (MMAE)2] -PSAR24 (DAR 16) Trastuzumab-MAL-phenyl-Ngly [triazole-galacturonide (MMAE)2] -PSAR24 (DAR 16)

[0647] Deconvoluted LC-1 d calculated: 27569; observed: 27570 / Deconvoluted HC-3d calculated: 62985; observed: 62983

[0648] Monomer purity: 98.5%

[0649] HIC retention time: 8.7 minutes

[0650] Trastuzumab-MAL-phenyl-Ngly(triazole-SN38)-Ngly(triazole-SN38)-PSAR18 (DAR 16)

[0651] Deconvoluted LC-1 d calculated: 27569; observed: 27570 / Deconvoluted HC-3d calculated: 62985; observed: 62983

[0652] Monomer purity: 98.2%

[0653] HIC retention time: 9.9 minutes

[0654] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide SN38)-Ngly(triazole-glucuronide SN38)- PSAR18 (DAR 16)

[0655] Deconvoluted LC-1 d calculated: 27569; observed: 27570 / Deconvoluted HC-3d calculated: 62985; observed: 62983

[0656] Monomer purity: 99+%

[0657] HIC retention time: 10.2 minutes

[0658] Trastuzumab-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PEG12 (DAR 8) (= ADC-PEG12)

[0659] Deconvoluted LC-1 d calculated: 27569; observed: 27570 / Deconvoluted HC-3d calculated: 62985; observed: 62983

[0660] Monomer purity: 99+%

[0661] HIC retention time: 6.6 minutes

[0662] Trastuzumab-glucuronide MMAE (DAR 8): Trastuzumab-MAL-phenyl-triazole-glucuronide MMAE (DAR 8) (= ADC-PSAR0):

[0663] Deconvolution LC-1d calculated: 27270; observed: 27270 / Deconvolution HC-3d calculated: 62086; observed: 62087

[0664] Monomer purity: 99+%

[0665] HIC retention time: 5.7 minutes

[0666] Trastuzumab-MAL-phenyl-PSAR12-triazole-glucuronide MMAE (DAR 8) (= ADC-PSAR12L):

[0667] Deconvolution LC-1d calculated: 25541; observed: 25541 / Deconvolution HC-3d calculated: 56901; observed: 56900

[0668] Monomer purity: 99+%

[0669] HIC retention time: 7.4 minutes

[0670] Human albumin-MAL-phenyl-Ngly(triazole-glucuronide MMAE)-PSAR24 (DAR 1):

[0671] Deconvolution LC-1d calculated: 24721; observed: 24720 / Deconvolution HC-3d calculated: 54439; observed: 54438

[0672] Monomer purity: 95.2%

[0673] HIC retention time: 9.2 minutes

[0674] Trastuzumab:

[0675] Deconvolution LC-1d calculated: 24884; observed: 24884 / Deconvolution HC-3d calculated: 54926; observed: 54926

[0676] Monomer purity: 98.5%

[0677] HIC retention time: 8.4 minutes

[0678] Anti-CD19 antibody:

[0679] Deconvolution LC-1d calculated: 25793; observed: 25793 / Deconvolution HC-3d calculated: 57657; observed: 57657

[0680] Monomer purity: 99+%

[0681] HIC retention time: 8.5 minutes

[0682] Anti-CD22 antibody:

[0683] Deconvolution calculated: 69765; observed: 69645​​​​​​​​​​​​​​​​​​​​​​​

[0684] Monomer purity: 90.8%

[0685] HIC retention time: 3.9 minutes

[0686] Human albumin

[0687] Deconvoluted LC observations: 23439 / Deconvoluted HC observations: 50595

[0688] Monomer purity: 99+%

[0689] HIC retention time: 4.7 minutes

[0690] Example 12: Non-polymeric based antibody-drug-conjugates (ADC-PSAR0), base

[0691] Deconvoluted LC observations: 24139 / Deconvoluted HC observations: 50517

[0692] Monomer purity: 93.2%

[0693] HIC retention time: 4.7 minutes

[0694] ​

[0695] Deconvoluted LC observations: 24133 / Deconvoluted HC observations: 50692

[0696] Monomer purity: 99+%

[0697] HIC retention time: 4.8 minutes

[0698] ​ :

[0699] Deconvoluted observations: 66556

[0700] Monomer purity: 92.4%

[0701] HIC retention time: 2.5 minutes

[0702] ​ Hydrophobic interaction chromatography (HIC) profiles of an antibody-drug-conjugate (ADC-PSAR12) based on polysarcosine and an antibody-drug-conjugate (ADC-PSAR12L) based on polysarcosine with linear configuration. Figure 1

[0703] The relative exposure of the conjugated payload to the bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR 8 ADC were assessed by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR chromatographic column as described in Example 11. The results are shown in Table 6 below. Example 13: Hydrophobic interaction chromatography (HIC) profiles of antibody-drug-conjugates based on polysarcosine and polyethylene glycolFigure 6. Apparent hydrophobicity of ADC-PSAR12L (linear) and ADC-PSAR12 (isometric) as determined by HIC. The apparent hydrophobicity of ADC-PSAR12L (linear) and ADC-PSAR12 (isometric) was evaluated by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column as described in Example 11. The results are shown in Figure 6. When the polysarcines are grafted in a parallel (i.e. orthogonal) orientation relative to the drug unit, an effective hydrophobic masking property can be provided and the apparent hydrophobicity of the conjugate (ADC-PSAR12) is reduced. However, when the polysarcines are in a linear (i.e. contiguous) configuration, no reduction in the apparent hydrophobicity of the conjugate (ADC-PSAR12L) is observed.

[0704] Figure 2 Example 14: Pharmacokinetic profiles (total antibody concentration over time) in mice after a single intravenous injection of a 3 mg / kg dose of a non-polysarcosine-based antibody-drug-conjugate (ADC-PSAR0) and a polysarcosine-based antibody-drug-conjugate (ADC-PSAR12)

[0705] The relative exposure of the conjugated payload to the bulk solvent and the apparent hydrophobicity of the trastuzumab-based DAR8 ADC was evaluated by hydrophobic interaction chromatography (HIC) on a Tosoh TSK-GEL BUTYL-NPR column as described in Example 11. The results are shown in Figure 6. In the case of equal length (n = 12 monomer units), the polysarcines have a better hydrophobic masking property (shorter retention time) than polyethylene glycol. Figure 3

[0706] Example 15: Pharmacokinetic profiles (total antibody concentration over time) in mice after a single intravenous injection of a 3 mg / kg dose of a non-polysarcosine-based antibody-drug-conjugate (ADC-PSAR0) and a polysarcosine-based antibody-drug-conjugate (ADC-PSAR12) Figure 4A Example 16: Pharmacokinetic profiles (total antibody concentration over time) in mice after a single intravenous injection of a 3 mg / kg dose of a polysarcosine-based antibody-drug-conjugate (ADC-PSAR12) and a poly(ethylene glycol)-based antibody-drug-conjugate (ADC-PEG12)

[0707] Male SCID mice (4-6 weeks old) were injected via the tail vein at a dose of 3 mg / kg of ADC (five animals per dose group, randomly assigned). Blood was drawn via retro-orbital bleeds into citrate tubes at different time points and processed into plasma. The total concentration of ADC was evaluated using a human IgG ELISA kit (Stemcell TM Technologies) according to the manufacturer’s protocol. A standard curve of trastuzumab was used for quantification. Pharmacokinetic parameters (clearance and AUC) were calculated by non-compartmental analysis using Microsoft ® Excel ® software (developed by Usansky et al., Department of Pharmacokinetics and Drug Metabolism, Allergan, Irvine, USA) incorporating PK functions. The results are shown in Figure 7. ADCs comprising polysarcines exhibit favorable pharmacokinetics compared to ADCs not containing polysarcines. Figure 5

[0708] Pharmacokinetic profiles (total antibody concentration over time) in mice after a single intravenous injection of a 2.5 mg / kg dose of polysarcosine-based antibody-drug-conjugates (ADC-PSAR6, ADC-PSAR12, ADC-PSAR18, ADC-PSAR24) with different PSAR lengths in the orthogonal direction; a poly(ethylene glycol)-based antibody-drug-conjugate (ADC-PEG12) and a linear polysarcosine-based antibody-drug-conjugate (ADC-PSAR12L) Tumor volumes (mm3) of ADC of sarcosine (ADC-PSAR0) and poly-sarcosine-based ADC (ADC-PSAR12) 3 ) and survival curves Figure 6

[0709] Female SCID mice (4 weeks old) were subcutaneously implanted with BT-474 breast cancer cells. When the tumors grew to approximately 150 mm 3 ​​At this time, the ADC of Example 14 above was administered as a single i.v. dose at 3 mg / kg (Day 20, 5 animals per group to minimize differences in initial tumor volume between groups). Results are shown in ​ and 4B Tumor volume was measured every 3-5 days by caliper and calculated using the formula (L x W 2 ) / 2. Mice were sacrificed when tumor volume exceeded 1000 mm 3 . ADCs containing poly(methionine) had improved in vivo activity compared to ADCs containing poly(ethylene glycol). No significant body weight changes were observed in treated mice.

[0710] ​ ​ ​

[0711] Experiments were performed in male CD-1 mice (4-6 weeks old) according to the methods described in Example 14. Results are shown in ​ ADCs containing poly(methionine) had improved pharmacokinetic parameters compared to ADCs containing poly(ethylene glycol).

[0712] Example 17: Tumor volume (mm3) in BT-474 breast cancer xenograft model, the model was administered a single intravenous dose of 0.1 mg / kg of Compound 1 or 0.1 mg / kg of Compound 2. 3 Example 18: Tumor volume (mm3) in MDA-MB-231 breast cancer xenograft model, ​ ​ ​

[0713] Experiments were performed as described in Example 15. BT-474 breast cancer cells were implanted subcutaneously in female SCID mice (4 weeks old). When tumors reached approximately 150 mm 3 , ADCs were administered as a single i.v. dose at 2.5 mg / kg (Day 13, 6 animals per group to minimize differences in initial tumor volume between groups). Results are shown in ​ No significant body weight changes were observed in treated mice.

Claims

1. A ligand-drug-conjugate (LDC) having the following formula (XV): (XV) in, L is allowed (HP SMW ) is an orthogonal connector in an orthogonal direction relative to (XD), HP SMW is produced by covalently bonding a single molecular weight homopolymer having formula (I) to the orthogonal linker L (I) in R1 and R2 are different, and One of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group that is reactive toward a covalently bondable group under reaction conditions in which the inert group is non-reactive, Z1 and Z2, which are the same or different, are optional spacers, and n is 1 or greater and k is 2 or greater; D is a cytotoxic drug, X is an optional cleavable moiety for releasing D, Z is an optional spacer, and a is 1 or greater, b is 1 or greater, and m is 1 or greater.

2. The LDC compound of claim 1, wherein the single molecular weight homopolymer is polysarcosine.

3. The LDC compound of claim 1 or 2, wherein the HP SMW is produced by covalently bonding a single molecular weight homopolymer having formula (II) to the orthogonal linker L (II) in R1, R2, Z1 and Z2 are as defined in claim 1, and K is 2-100, preferably 2-50.

4. The LDC compound of any one of claims 1 to 3, wherein R1 or R2 is a functionalized reactive group selected from the group consisting of: - carboxylic acid groups, - amino NRR'', wherein R and R'' are each independently selected from H, (C1-C6)alkyl optionally interrupted by at least one heteroatom selected from O, N and S, - hydroxyl groups, - halogen atoms, - hydrazine (-NH2-NH2) group, - Nitro, - Hydroxylamine group, - Azide, - (C2-C6) alkynyl, - (C2-C6) alkenyl, - thiol groups, - activated ester groups, such as N-hydroxysuccinimide esters, perfluoroesters, nitrophenyl esters, aza-benzotriazole and benzotriazole activated esters, ureides, - Boric acid – B(OR'''')2 group, where R'''' is a hydrogen atom or a C1-C6 alkyl group, - thiol-reactive groups such as maleimide, halomaleimide, haloacetyl, pyridyl disulfide, - mesylate group, - Tosylate group, - trifluoromethanesulfonate group, - aldehyde group, - isocyanate or isothiocyanate groups, - chlorosulfonyl, - Acrylate based.

5. The LDC compound of any one of claims 1 to 4, wherein the ligand is selected from the group consisting of a polypeptide, a protein, an antibody, and an antibody fragment.

6. The LDC compound of any one of claims 1 to 5, wherein D is selected from the group consisting of: biologically active molecules; therapeutic molecules, such as anticancer drugs; imaging agents and fluorophores.

7. The LDC compound of any one of claims 1 to 6, wherein L is one or more natural or unnatural amino acids.

8. The LDC compound of any one of claims 1 to 7, wherein L is selected from the group consisting of glutamic acid, lysine, and glycine.

9. The LDC compound of any one of claims 1 to 8, wherein X is selected from - one or more natural or unnatural amino acids, - a sugar moiety linked to a self-eliminating group via an oxygen glycosidic bond, - a disulfide linker, and - Acid-labile linker that is hydrolyzed in lysosomes.

10. The LDC compound of any one of claims 1 to 9, wherein X is selected from - one or more natural or unnatural amino acids, and - A sugar moiety linked to a self-eliminating group via an oxygen glycosidic bond.

11. The LDC compound of any one of claims 1 to 10, wherein Z is selected from the group consisting of alkylene, heteroalkylene, alkoxy, polyether, one or more natural or unnatural amino acids, C3-C8 heterocyclyl, C3-C8 carbocyclyl, arylene, and any combination thereof.

12. The LDC compound of any one of claims 1 to 11, wherein Z is formula (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII), (XVII) (XVIII) (XIX) (XX) (XXI) (XXII) where the wavy bond represents the connection point and R6 is –C1-C 10 Alkylene-, –C1-C 10 Heteroalkylene-, –C1-C 10 Alkylene-C(=O)-, –C1-C 10 Heteroalkylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -arylene-C1-C 10 Alkylene-OC(=O)-, and Any of the R6 groups is optionally substituted with one or more =0.

13. Intermediate compound having formula (XVI) (XVI) in L is an orthogonal connector, HP SMW is produced by covalently bonding a single molecular weight homopolymer having formula (I) to the orthogonal linker L (I) in R1 and R2 are different, and One of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group that is reactive toward a covalently bondable group under reaction conditions in which the inert group is non-reactive, Z1 and Z2, which are the same or different, are optional spacers, and n is 1 or greater and k is 2 or greater; D is a cytotoxic drug, X is an optional cleavable moiety for releasing D, Z is an optional spacer, and a is 1 or greater and b is 0, 1 or greater.

14. The intermediate compound of claim 13, wherein the single molecular weight homopolymer is polysarcosine.

15. The LDC according to any one of claims 1 to 12 for use as a medicament.

16. Compounds having formula (XXIII) (XXIII) in R6 is –C1-C 10 Alkylene-, –C1-C 10 Heteroalkylene-, -C3-C8 carbocyclyl-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, –C1-C 10 Alkylene-(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-, -C3-C8 heterocyclic-, –C1-C 10 Alkylene-(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-, –C1-C 10 Alkylene-C(=O)-, –C1-C 10 Heteroalkylene-C(=O)-, -C3-C8 carbocyclyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-C(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclyl-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-C(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C3-C8 carbocyclyl-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-NH-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclyl-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-NH-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-S-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclyl-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-S-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-S-, –C1-C 10 Alkylene-OC(=O)-, -C3-C8 carbocyclyl-OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene-OC(=O)-, -C1-C 10 Alkylene-arylene-OC(=O)-, -arylene-C1-C 10 Alkylene-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-OC(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-OC(=O)-, -C3-C8 heterocyclyl-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-OC(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-OC(=O)-, Any one of the R6 groups is optionally substituted by one or more substituents selected from: -X, -R', -O - 、-OR'、=O、-SR'、-S - 、-NR'2、-NR'3 + ,=NR', -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR'C(=O)R', -C(=O)R', -C(=O)NR'2, -SO3 - , -SO3H, -S(=O)2R', -OS(=O)2OR', -S(=O)2NR', -S(=O)R', -OP(=O)(OR')2, -P(=O)(OR')2, -PO3 - , -PO3H2, -C(=O)X, -C(=S)R', -CO2R', -CO2, -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR'2, C(=S)NR'2 and C(=NR')NR'2, wherein each X is independently a halogen: -F, -CI, -Br, or -I; and each R' is independently -H, -C1-C 20 Alkyl, -C6-C 20 Aryl, or -C3-C 14 Heterocyclic group, Z is an optional spacer, L is an orthogonal connector, X is an optional cleavable moiety for releasing D, D is a cytotoxic drug, a is 1 or greater and b is 0, 1 or greater, and HP SMW is produced by covalently bonding a single molecular weight homopolymer having formula (I) to the orthogonal linker L (I) in R1 and R2 are different, and One of R1 and R2 is H or an inert group, and the other of R1 and R2 is a functionalized reactive group that is reactive toward a covalently bondable group under reaction conditions in which the inert group is non-reactive, Z1 and Z2, which are the same or different, are optional spacers, and n is 1 or greater and k is 2 or greater.

17. The compound of claim 16, wherein the single molecular weight homopolymer is polysarcosine.

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